UNITED STATES OF AMERICA
	DEPARTMENT OF HEALTH AND HUMAN SERVICES
	FOOD AND DRUG ADMINISTRATION

	

	
	TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES
	ADVISORY COMMITTEE MEETING
	  This transcript has not been edited
	or corrected, but appears as received
	from the commercial transcribing
	service.  Accordingly the Food and
	drug Administration makes no
	representation as to its accuracy.
	Gaithersburg, Maryland
	Wednesday Morning, June 26, 2002
	COMMITTEE MEMBERS PRESENT:
	
	DAVID C. BOLTON, Ph.D., Chair
	New York State Institute for Basic Research
	
	JOHN C. BAILAR III, M.D., Ph.D.
	University of Chicago
	
	ERMIAS D. BELAY III, M.D., Ph.D.
	Centers for Disease Control and Prevention
	
	STEPHEN J. DeARMOND, M.D., Ph.D.
	University of California San Francisco
	
	SAMUEL H. DOPPELT, M.D.
	The Cambridge Hospital, Cambridge, Massachusetts
	
	LISA A. FERGUSON, D.V.M.
	United States Department of Agriculture
	
	PIERLUIGI GAMBETTI
	Case Western Reserve University
	
	KATHARINE E. KNOWLES
	Health Information Network
	
	JEANNE V. LINDEN, M.D.
	New York State Department of Health
	
	JEFFREY J. McCULLOUGH, M.D.
	University of Minnesota
	
	STEPHEN R. PETTEWAY JR., Ph.D.
	Bayer Corporation
	
	PEDRO PICCARDO, M.D.
	Indiana University
	
	SUZETTE A. PRIOLA, Ph.D.
	Rocky Mountain Laboratories
	COMMITTEE MEMBERS PRESENT (CONT'D):
	
	ELIZABETH S. WILLIAMS, D.V.M., Ph.D.
	University of Wyoming
	
	SIDNEY M. WOLFE, M.D.
	Public Citizen
	
	ALSO PRESENT:
	
	DAVID ASHER, Ph.D.
	Office of Blood Research and Review
	FDA Center for Biologics Evaluation and Research
	
	JAY S. EPSTEIN, M.D.
	Office of Blood Research and Review
	FDA Center for Biologics Evaluation and Research
	
	MAHMOOD FARSHID, Ph.D.
	Office of Blood Research and Review
	FDA Center for Biologics Evaluation and Research
	
	WILLIAM FREAS, Ph.D.
	Committee Executive Secretary
	
	ELLEN HECK
	Eye Bank Association of America
	
	RICHARD HURWITZ, M.D., F.A.C.S.
	LifeNet
	
	DAVID KORROCH
	Lions Medical Eye Bank of Eastern Virginia
	
	C. RANDALL MILLS, Ph.D.
	Regeneration Technologies, Inc.
	
	P.J. PARDO
	Tutogen Medical, Inc
	
	P. ROBERT RIGNEY JR., J.D.
	American Association of Tissue Banks
	ALSO PRESENT (CONT'D):
	
	ROBERT ROHWER, Ph.D.
	University of Maryland VA Medical Center
	
	RICHARD RUSSO
	International Osteotech, Inc.
	
	RUTH SOLOMON, M.D.
	Office of Blood Research and Review
	FDA Center for Biologics Evaluation and Research
	
	ALAN E. WILLIAMS, Ph.D.
	Office of Blood Research and Review
	FDA Center for Biologics Evaluation and Research

	

	

	*  *  *  *  *

	C O N T E N T S
	SPEAKER/SESSION:                           PAGE
	Dr. Solomon                               17
	
	Dr. Taffs                                 33
	
	Dr. Hogan                                 62
	
	Dr. Brown                                 99
	
	Dr. Eshkol                               131
	
	Dr. Lynch                                151
	
	Ms. Wilson                               176
	
	Ms. Heck                                 205
	
	Dr. Rohwer                               229
	
	Dr. Farshid                              259
	
	Dr. Hurwitz                              280
	
	Mr. Russo                                299
	
	Dr. Mills                                321
	
	Dr. Rohwer                               351
	
	Open Public Hearing                      372
	
	Questions to the Committee               401
	
	Dr. Greenwald                            501
	
	Dr. Williams                             510
	
	Committee Discussion                     530
	
	*  *  *  *  *
	P R O C E E D I N G S
	(8:00 a.m.)
	DR. FREAS:  Mr. Chairman, members of the committee, invited guests, members of the audience, I would like to welcome you to our 12th meeting of the Transmissible Spongiform Encephalopathies Advisory Committee.  Both days of this meeting will be open to the public and you are welcome to the entire meeting.
	At this time I would like to go around and introduce the members at the head table and I will wait just one second until I have a chance to sit down.  After I introduce the members of the head table I will be reading to you the conflict of interests statement for this meeting.
	When I call out the names if you would raise your hand I would like to go around and introduce the members at this time.  In the first seat at the table right in front of the podium at the end of the table is Dr. Pierluigi Gambetti, Professor and Director, Division of Neuropathology, Case Western Reserve University.
	The next committee member is Dr. Lisa Ferguson, Senior Staff Veterinarian, US Department of Agriculture.
	In the empty chair we will soon have Dr. DeArmond, Professor, Department of Pathology, University of California, San Francisco.
	Our next committee member present is Dr. John Bailar, Professor Emeritus, Department of Health Studies, University of Chicago.
	The next committee member is Dr. Pedro Piccardo, Associate Professor, Indiana University School of Medicine.
	Around the corner of the table is Dr. Elizabeth Williams, Professor, Department of Veterinary Service, University of Wyoming.
	Next we have a temporary voting member from the Blood Products Advisory Committee.  He is a full member of the Blood Products Advisory Committee and a temporary member here today, Dr. Samuel Doppelt, Chief, Department of Orthopedic Surgery, The Cambridge Hospital, Cambridge, Massachusetts.
	Next is the Chairman of this advisory committee, Dr. David Bolton, head of the Laboratory of Molecular Structure and Function, New York State Institute for Basic Research.
	Next is another temporary voting member and acting consumer representative for today, Katharine Knowles, Executive Director, Health Information Network, Seattle, Washington.
	Going around the corner of the table is a committee member, Dr. Ermias Belay, Medical Epidemiologist, Centers for Disease Control and Prevention.
	In the next chair is Dr. Suzette Priola, Investigator, Laboratory of Persistent and Viral Diseases, Rocky Mountain Laboratories.
	Next we have Dr. Jeffrey McCullough, Professor, Department of Laboratory Medicine and Pathology, University of Minnesota.
	Next I would like to welcome back a former member of this committee who is serving a split term on this committee.  I would like to welcome back Dr. Sidney Wolfe, Director, Public Citizen Health Research Group, Public Citizen.
	Next we have a temporary voting member, Dr. Jeanne Linden, Director, Blood and Tissue Resources, New York State Department of Health.
	Next is our nonvoting industry representative, Dr. Stephen Petteway, Director of Pathogen Safety and Research, Bayer Corporation.
	Two committee members could not join us today.  They are Dr. Richard Johnson and Ms. Shirley Walker.
	Now I would like to read the conflict of interests statement for this meeting:
	"The following announcement is made part of the public record to preclude even the appearance of a conflict of interests at this meeting.
	"Pursuant to the authority granted under the committee charter, the Director, Center for Biologics, Evaluation, and Research, has appointed Dr. Sam Doppelt, Ms. Katharine Knowles, and Dr. Jeanne Linden as temporary voting members for this meeting.
	"Based on the agenda it has been determined that the committee will not be providing advice on specific firms or specific products at this meeting.  Topics being discussed by the committee in open session are considered general matters issues.
	"To determine if any conflicts of interest exist the agency reviewed the agenda and all relevant financial interests reported by the meeting participants.  In accordance with 18 USC 208 Dr. Sam Doppelt has been granted a waiver that permits him to participate fully in the committee discussions.
	"We would like to note for the record that Dr. Stephen Petteway is serving as a nonvoting industry member for this committee.  He is employed by Bayer and thus has interest in his employer and other similar firms.
	"With regards to the invited guests the agency has determined that the services of these guests are essential.  The following reported interests are being made public to allow participants to objectively evaluate any presentations and/or comments made by the invited speakers:
	"Major Ronny Alford is employed by the Armed Services Blood Program, United States Air Force.
	"Dr. Larisa Cervenakova is employed by the American Red Cross.
	"Dr. Aliza Eshkol is a senior scientific advisor for Serono International.
	"Dr. Luisa Gregori is employed at the Medical Research Service, VA Medical Center.  She is a researcher and has contracts with firms in the blood industry and firms developing TSE removal products.
	"Ms. Ellen Heck is Director Transplant Services Center, University of Texas, Southwestern Medical Center, Dallas.
	"Dr. Thomas Lynch is Senior Vice President for Regulatory and Quality for Clearant.  He consults with companies engaged in the production of human tissues and tissue-based products.
	"Dr. Robert Rohwer is principal investigator on contracts for applied research supported by various blood companies and companies involved in the development of TSE removal products.  He also consults with blood companies and companies involved in TSE removal.
	"Dr. Diane Wilson is chief operating officer, Community Tissue Services, Dayton, Ohio.
	"Listed on the agenda are speakers making industry presentations and speakers giving committee updates on regulated industry and outside organizations.  Since these industry and update speakers have financial interests associated with their employer and other regulated firms they were not screened for these conflicts of interest.
	"All other meeting participants are aware of the need to exclude themselves from discussions involving specific products or firms for which they have not been screened for conflict of interests.  Their exclusion will be noted for the public record.
	"With respect to all other meeting participants we ask in the interest of fairness that they address any current or previous financial involvement with any firm whose product they wish to comment upon."
	So ends the reading of the Conflict of Interest statement.  Dr. Bolton, I turn the microphone over to you.
	DR. BOLTON:  Thank you, Bill.  We will now have an announcement by Jill Warner on behalf of Dr. Zimmerman.
	MS. WARNER:  Good morning.  I have just a brief announcement to make on behalf of CBER.  As many of you on the committee and those in attendance to day are aware, one of the most dynamic and growing areas of product development in biological products is the area of tissues, cellular and gene therapies.
	In order to facilitate the coordinated development of these products, the research, review, and policy, we are announcing the formation of a new Office of Cellular, Tissue, and Gene Therapy Products.
	This new office will consolidate the staff and functions from our current Division of Cellular and Gene Therapies in the Office of Therapeutics and Research and Review currently and also the human tissue program staff from the Office of Blood Research and Review.
	Our target date for the new office is October of this year and the kinds of products that will be regulated in this new office include cellular therapies, tissue- based products, tissues, also gene therapies, transplantation products, and we welcome your comments as we go forward.  We look forward to working with this committee on the TSE issues that affect these and other FDA-regulated products.
	If you do have comments that you would like to submit Dr. Cheryl Lard Whitesford, who is our CBER ombudsman, would be happy to take your call or your e-mail.
	Thanks.
	DR. BOLTON:  Thank you, Jill.  This morning I would like to welcome you all, especially the new members or new returning members of the committee.  We have a rather full agenda today so I won't say much.
	We are going to be discussing many issues related to tissues, cellular-based and tissue-based products for trans- plantation.  I think at this point we should just move right on with this.
	I understand that one of our speakers, Dr. Paul Brown, may or may not be able to attend this morning.  Paul was feeling a little under the weather so he is scheduled to speak at two different times, at 9:30 and somewhat later than that, 10:45. If Dr. Brown is able to make it he will combine both of his talks in one segment and present them both at 9:30.  I just want everybody to be aware of that.
	At this time without further ado I will open up our first topic for discussion which is the Validation of Procedures to Prevent Contamination and Cross- Contamination with TSE Agents of Human Tissue Intended for Transplantation.
	The first presentation in this is the introduction and background, which will be presented by Dr. Ruth Solomon.  The title of this is Current and Proposed FDA Regulations and Guidance pertaining to TSE and Human Cells, Tissues, and Cellular and Tissue-Based Products Intended for Transplantation.  Dr. Solomon.
	DR. SOLOMON:  Good morning.  The first topic today is a discussion of the validation of procedures to prevent contamination and cross-contamination with TSE agencies of human cells and tissues intended for cross-contamination.
	These human cells and tissues would include cellular therapies, hematopoietic stem cells, reproductive cells and tissue, musculoskeletal tissue, skin, dura mater, heart valve allografts, and others.
	FDA requests advice from the TSE Advisory Committee on the measures for donor screening, measures for tissue recovery and processing, and design of a clearance study appropriate to prevent contamination and cross-contamination of human cells and tissues intended for transplantation by TSE agents.  The new term for these products are human cells, tissues, and cellular and tissue-based products or HCT/Ps.
	There are three approaches to reduce the risk of TSE transmission by cell and tissue transplantation.  The first is careful screening of the donor for TSE and risk factors of TSE and testing if and when validated.
	The second approach would be control of recovery and processing of cells and tissues to prevent contamination and cross-contamination and the third would be the use of steps during manufacturing of cells and tissues to remove or inactivate TSE agents.
	The talks today will discuss each of these three approaches in more detail. We know that unlike blood transfusions given to humans there have been documented cases of iatrogenic transmission of CJD to the recipients of human cells and tissue products.  Dr. Brown will discuss this further but, just briefly, there have been transmissions of CJD through human dura mater transplantation.  Most of these are from dura mater from different donors that had been commingled during processing.
	There have also been transmissions of CJD through corneal transplantation. There has also been transmission through human pituitary derived growth hormone administration and this will be discussed by Dr. Eshkol.
	Dr. Brown will also discuss the potential transmission of vCJD and CJD by other human cells and tissues and will give the experimental evidence in animals.
	Now I would like to give you a background on FDA's regulatory approach to TSE transmission, actual and potential, by human cells and tissues.  I will review the current tissue regulations, the current recommendations that are in the form of guidance to industry, and then the proposed cell and tissue regulations and the proposed recommendations and guidance.
	Since FDA is in the process of finalizing the proposed regulations and issuing draft guidance we thought that this was an appropriate time to discuss TSEs and relate them to cell and tissue transplantation.
	The current tissue regulations were finalized in 1997 and they can be found in 21 CFR Part 1270.  These regulations cover screening and testing of potential donors for HIV, HBV, and HCV.  TSE is not included in this final rule.
	There are also requirements for written procedures and record keeping.  One of the requirements in the section on written procedures says that a tissue establishment is required to have validated procedures to prevent infectious disease contamination or cross-contamination during processing and this is contained in 1270.31(d).  This final rule also contains information about inspection and enforcement.
	Along with the final rule we issued a guidance for industry called "Screening and Testing of Donors of Human Tissue Intended for Transplantation."
	This guidance document did recommend screening for CJD and recommended to defer donors with a diagnosis of CJD, a family history in a blood relative of CJD, a history of receiving dura mater transplant, a history of receiving human pituitary growth hormone.
	Recently this past March we also issued another final guidance for industry called "Validation of Procedures for Processing of Human Tissues Intended for Transplantation."  The purpose of this guidance was to clarify Section 1270.31 of the rule.  This guidance explains that infectious disease contamination includes viral, bacterial, fungal, and TSE agents and we would expect that tissue establishments have validated methods to prevent contamination by viruses, bacteria, and fungi at this time.  Dr. Farshid from FDA and several tissue processors will discuss process validation later.
	We also said in this guidance that validated methods to prevent contamination by TSE agents would be expected later if and when such methods are agreed upon by scientific experts such as the members of this committee and become available.
	CDRH, which currently regulates dura mater, issued a guidance document in October '99 entitled "Guidance for the Preparation of a Pre-Market Notification Application for Processed Human Dura Mater." This guidance contains all of the above donor suitability recommendations and also recommends that a potential donor of dura mater with any degenerative or demyelinating disease of the CNS or who has died in a neurologic or psychiatric hospital be excluded.
	This guidance also recommends growth and histologic exam of the full brain, disinfection by a method validated to reduce CJD infectivity, and the prohibition of batch processing.
	Now for the proposed regulations and guidance.  In September 1999 FDA proposed a rule entitled "Suitability Determination for Donors of Human Cellular and Tissue-Based Products."  This proposed rule would require screening of all cell and tissue donors, including a medical history interview, for risk factors and clinical evidence of HIV, HBV, HCV, human TSEs including CJD, and also there would be additional screening for particular human cells and tissues.  It also would require testing of all cell and tissue donors for HIV, HBV, HCV, and syphilis and additional testing for particular human cells and tissues.  We received comments on this proposed rule and we are in the process of finalizing it.
	In addition we proposed a rule on January 8, 2001, entitled "Current Good Tissue Practice for Manufacturers of HCTPs Inspection and Enforcement."  This proposed rule would provide controls over facilities, personnel, equipment, environment, incoming materials, labeling, storage, process controls, process validation, record keeping, adverse reactions, and product deviation reporting and tracking and would also contain information about inspection and enforcement.  We have received comments on this proposed rule and it is also being finalized.
	I would like to point out that this GTP proposed rule would prohibit the pooling of cells and tissues from different donors during any part of manufacturing.  By "pooling" we mean placing in physical contact or mixed in a single receptacle; however, this proposed GTP rule would also provide an exemption or alternative from any GTP requirement.  A firm could submit a request for an exemption or alternative accompanied with valid data.
	For instance, a request might be submitted for an exemption to the pooling prohibition.  FDA would weigh the potential increased risk of contamination and cross- contamination with emerging infectious disease agents such as TSE agents against the potential benefit of improved elimination of conventional infectious disease agents such as viruses, bacteria, and fungi.  Dr. Taffs will be discussing a risk assessment.  There will be several talks about single donor processing versus batch processing from Dr. Lynch, Dr. Brown, Ms. Wilson, and Ms. Heck.
	Now for the proposed guidances. Two weeks ago we issued a draft guidance for industry entitled "Preventive Measures to Reduce the Possible Risk of Transmission of CJD and vCJD by human cells, tissues, and cellular and tissue-based products.  This is the subject of Topic #2.  Dr. Greenwald will discuss this draft guidance further but as a background for my topic, for this topic, the draft guidance briefly would have certain recommendations for donor screening for CJD and would recommend deferral for CJD or variant CJD diagnosis, dementia, degenerative or demyelinating disease of the CNS, or other neurologic disease of unknown etiology, deferral for risk factors for CJD such as a blood relative with CJD, receipt of dura mater or human pituitary growth hormone, and it would also include risk factors deferral for variant CJD such as the cumulative time spend in the UK, Europe.  It addresses military people stationed in Europe and their dependents.  It also addresses blood transfusion in the UK and receipt of bovine insulin manufactured from cattle in the UK.  Basically it is the same recommendation that all of you are familiar with in the blood donor guidance except there is an exception made to HLA-matched hematopoietic stem cells.
	In addition to the screening measures that I just mentioned in the draft guidance that Dr. Greenwald will discuss several other screening measures have been suggested.  One of them is to have an upper age limit for donors of cells and tissues. The thinking behind this is that the median age at death from CJD is 68 years or in the older population; however, in considering such a recommendation we would be concerned about the long incubation period and the probability that the donor would be infectious during some portion of the incubation period.
	An upper age limit was proposed for blood donors by the blood industry but was not implemented.  You should realize that proposing an upper age limit may seriously reduce tissue supply.  For instance, 50 percent of US donors of ocular tissue are age 61 or older.
	The tissue bank and eye bank standards do not recommend any upper age limit.  This is left to the discretion of the eye or tissue bank director to set donor age limits in the firm's SOP; however, there are age limits in industry standards for semen donors.  They have to be less than 40 years old, oocyte donors less than 35 years old, donors of cardiovascular tissue less than 61 years old.
	A second possible additional donor-screening measure would be to exclude donors for head trauma.  The rationale here is to avoid possible CNS contamination of the cells and tissues that you retrieve. Again, head trauma is not addressed in the industry standards and we have to realize that such an exclusion may reduce the tissue supply.  For instance, 13 percent of eye donors cause of death is trauma.
	Then we could consider some additional donor testing measures such as a brain autopsy and that should be found negative in order to accept the donor. Dr. Hogan is going to discuss the brain biopsy and the brain autopsy.
	With the brain autopsy we should remember that there could be a delay in distributing time-sensitive human cells and tissues such as cornea.  The brain biopsy if we were to use that would need to be validated to show that it is predictive of autopsy diagnosis of TSE.
	So we would like the committee to evaluate the appropriateness of each of the measures and the controls that will be discussed today or others that you recommend to prevent TSE transmission to the recipients of human cells and tissues.
	Again, we are dividing this into additional donor screening and testing criteria, then specified methods of recovery and processing of cells and tissues, and under that specific methods of decontamination of surgical instruments and work surfaces and the removal and/or inactivation of TSE agents.  Both of the latter topics will be discussed by Dr. Rohwer.
	In addition we would like you to consider this question.  Should pooling or commingling of human cells from different donors during manufacturing ever be permitted?  If so what controls should FDA require in assessing whether a request for an exemption from the proposed pooling prohibition should be granted?
	Finally, the questions that will be asked at the end of this topic.  Question number one, which of the following measures and controls is or are appropriate to prevent TSE agent transmission to recipients of human cells and tissues?  Additional donor screening and testing measures to determine donor eligibility or exclusion such as upper age limit, head trauma exclusion, negative brain biopsy, or autopsy?  Specific methods of human cell and tissue recovery and/or processing to prevent contamination and cross-contamination such as the decontamination of instruments and surfaces?  We will be asking you how should this be accomplished.
	The methods for removal and/or activation of TSE agents during manufacturing and single donor aseptic processing versus permit pooled processing under circumstances, which circumstances and with adequate controls, which controls? Also, if you had other appropriate measures and controls we would appreciate hearing them.
	The second question will ask you to comment of the design of a satisfactory TSE agent clearing study for human cells and tissues in terms of the following criteria. What would be a suitable TSE agent strain and animal model?  Can we accept measurement of abnormal forms of prion protein alone or would we require infectivity bioassays?  Can we accept substantial reduction and how much should that be or require complete elimination of detectable prion protein and/or infectivity?  Should we accept a single validated method or require that more than one validated method be included in the study and any other suggestions for the design of a clearance study that you may have?
	Thank you.
	DR. BOLTON:  Thank you, Dr. Solomon.  We have a little time if there are some questions from the committee at this point for Dr. Solomon.
	Seeing none, then we will move on. Our next speaker is Dr. Rolfe Taffs.  He will tell us about the risk assessment models for estimating the risk of transmitting TSE by human tissue intended for transplantation.  Dr. Taffs.
	DR. TAFFS:  Good morning.  I'm very pleased to have an opportunity today to present information on risk assessment models after the transmission of TSEs by human tissues intended for transplantation.
	During my talk I ask you to keep in mind two important points.  The first point is that this talk represents the views of the author and is not the official position of the Food and Drug Administration.  Now, that having been said, the second point is that you will note later in the talk that many assumptions are made in risk assessments.  Not all of these assumptions are established scientific fact, nor is there complete agreement on some of the numerical values used in the models that I will present but my goal this morning is to describe the development and application of probabilistic risk assessments in estimating TSE exposure risks associated with the use of human tissues.
	Efforts have been made in the past to quantitate the risk of TSEs associated with the use of pharmaceutical products. The first citation shown here is often called the German Model.
	The second citation is a paper that appeared in the PhRMA Association Journal BioPharm a few years ago.
	The third citation is a more recent report on harmonization of risk assessments for the scientific committees of the European Commission outlining the essential elements of quantitative risk assessment.
	While no detailed guidance on risk assessment for tissues is currently available from the FDA the agency attempts to harmonize with other regulatory authorities wherever possible.  The EC risk assessment publications are available on the Internet and they provide a reasonable framework for harmonizing approaches to science-based risk assessment and for developing risk-assessment models.
	The last citation is a recent risk assessment model for cornea transplantation providing one example of the various approaches that might be taken to assess TSE risks associated with the use of allograft tissues.
	Risk analysis can be thought of as a comprehensive, structured approach to dealing with risk.  It is comprised of risk assessment, risk management, and risk communication.  This presentation focuses on risk assessment.
	The elements of risk assessment include hazard identification, exposure to assessment, hazard characterization, and risk characterization.  Hazard identification examines the source of a risk capable of producing an adverse effect together with a quantitative description of that adverse effect.  Exposure assessment evaluates the levels and the duration of exposure to the risk.  Hazard characterization seeks to determine the dose-response relationship and the mechanisms of those critical effects.  Risk characterization estimates the probability of the occurrence and the severity of the adverse effects along with attendant uncertainties.
	I think it is important to consider that variability and uncertainty in the risk model should be described.  Risk assessment and risk assessors need to explore the scientific basis for the risk estimation and explicitly state the assumptions made in modeling risk in order to avoid any false sense of precision.  The assumptions and constraints of the model should also be described.
	Now, the principles outlined in this slide are really not so foreign.  All of us are risk assessors.  We evaluate the risks that we encounter every day.  I have an anecdote to illustrate the components of this slide.  I have a risk mitigation strategy so that I know I will arrive at meetings such as this on time so I always leave early.
	I had a little problem coming to the last advisory committee meeting.  It had to do with a flat tire.  Now, the risk mitigation strategy that I had in place at the time was to leave early.  It turned out not be quite fully effective so I performed an additional risk assessment that involved the likelihood that I would encounter a nail in the road and that I would have an opportunity to change a tire or have my tire repaired so that I would still arrive at the meeting on time.
	Other factors that were incorporated in that risk assessment included the age of my tires.  So I instituted a new mitigation strategy that involved buying new tires and I made this meeting on time. That's an example.
	Probabilistic risk assessment provides a means to obtain specific objectives.  Risk modeling lets us quantitate the relative contributions of the parameters in the risk model, identify critical elements for further research, and ultimately to obtain accurate information to aid in making regulatory decisions.  In the case of TSE risks associated with the use of human tissues this third objective has not yet been completed although certainty considerable progress has been made.
	Sensitivity analysis, also known as importance analysis, lets us examine the relative contributions of different parameters in the risk model.  By "parameters" I mean all of the elements that can be incorporated in the model and could affect the outcome of the risk assessment calculations.
	The objectives of sensitivity analysis are to evaluate the effects of changes in the model parameters and identify those parameters that have the greatest impact on the magnitude of risk. Sensitivity analysis lets us examine the assumptions, the variability, and the uncertainty in the model and quantify the impact of each of the parameters on the outcome of the risk assessment.
	I must point out that to my knowledge this important feature of risk assessment has not been addressed in previously published models of the risk of TSE in the use of human tissues or pharmaceutical products.
	Efforts are ongoing in the Center for Biologics, Evaluation, and Research to develop quantitative science-based risk assessment models, including a generalized model for tissues that could be applied in specific circumstances as adequate data become available.  Tissue risk assessment models for CJD have several components. Some of these are listed here:  Disease prevalence, donor availability and utilization, sources of uncertainty, and the potential impact of infection within the donor pool are incorporated in the model. The model includes diagnosed cases of CJD, undiagnosed symptomatic cases, and asymptomatic cases during the incubation period of the disease.
	Data inputs for the model include a population age distribution, age-specific all-cause deaths, age-specific rates of CJD deaths, and age-specific donor utilization for a given tissue.  The model also incorporates parameters for donor screening, processing, and possibilities for cross-contamination.  The results I will present shortly were obtained from this generalized model and it is important to note that different tissues vary with regard to their procurement, tissue processing, and implantation procedures.  These unique features need to be taken into account in a risk assessment for any given tissue.
	Some sources of information are readily available to develop risk assessments for CJD in tissues.  Four sources that I have used for modeling are shown here, including published estimates of CJD incidents, age-specific mortality, population estimates from the US Bureau of the Census and, as an example of data available for a specific tissue, information on cornea donations available from the Eye Bank Association of America.
	At the same time I should note that for many tissues comprehensive and accurate information on donor utilization and tissue processing are not readily available and such information would be needed to provide reliable estimates of the risk.  Efforts to compile such information for different tissues should be encouraged and acknowledged.
	As one simple example of the information available for CJD-risk models this figure shows the age distributions for CJD incidents in the United States and the United Kingdom adjusted for the nearly five-fold difference in population between the two nations.  The distributions are remarkably similar as has been noted in a number of publications.
	As an example of the information available for tissue utilization this slide compares the age distribution for cornea donors and for all caused deaths in the US. All caused deaths are shown in hundreds adjusting the scale and the figure so that the difference between the two distributions is readily apparent.  Note the observed reduction in cornea use from potential donors over the age of 70 attributable to cornea procurement and implantation practices at different locations within the United States.
	The point is that the age distribution for the utilization of a specific tissue may not correspond with the potential donor pool reflected in age- specific all-cause death rates so reliable information is needed for specific tissues if we desire to model CJD risk accurately.
	For the generalized risk model the variables listed here were identified as important parameters.  These include the number of symptomatic cases that are undiagnosed or not detected by current screening methods, the number of asymptomatic cases, and CJD prevalence. Additional screening procedures or donor exclusion criteria can also be incorporated in the model.
	Other important variables include decontamination, that is, reduction in infectivity that may result from processing steps, the effect of cross-contamination or commingling, batch size, and the numbers of donors, recipients, and graft materials that may be used in a given transplant procedure. These topics will also be discussed in detail by other speakers later today.
	To illustrate how these parameters fit in the overall strategy for risk assessment this diagram places the CJD risk model in a framework consistent with the risk assessment elements shown in an earlier slide.  The underlying distribution for donors, disease, and population are considered as a part of the exposure assessment.
	Recovery, processing, and transplant procedures are also captured under exposure assessment.  The risk characterization includes several parameters shown on the next slide.  Risk characterization includes the proportion of missed cases, incubation period, symptomatic period prior to diagnosis, and actual disease prevalence.  The parameters captured under exposure assessment are also listed here.
	Two important points must be made. The first is that except for the number of donors in the model these parameters are not regarded as single-point estimates in this model.  Instead the calculation of the risk depends on the underlying distribution of each parameter.  This is probabilistic risk assessment.  Each input distribution reflects the variability and uncertainty for the individual parameter.  For example, the estimate of the proportion of missed cases is modeled as a range of values from .5 to 10 percent using a triangular distribution whose most probable value in the model is 1 percent.
	The asymptomatic period is modeled on a range of values from 5 to 40 years with a median of 10 years.  Other components include prevalence, number of donors in a batch, number of transplant items per donor and recipient, medical history review, cross-contamination, and reduction in infectivity by tissue processing, and these are all components of the probabilistic risk model.
	In calculating the risk the distributions for each of these parameters are repetitively sampled at random using the median as the most probable value for the distribution.  Each iteration computes the number of exposures that would occur and following 10,000 iterations an output distribution can then be calculated showing the mean number of exposures and its variability given input distributions.  This method is often referred to as Monte Carlo analysis.  The number of donors used in this illustration was fixed at 25,000 per year. These distributions are easily adjusted in the model to coincide with the values to be expected for a given tissue procurement process and transplant procedure.
	The next step was to quantitate the number of exposures to CJD-infected tissue that might occur in one year under different model assumptions.  The two parameters that were varied in the comparisons that I am about to show were medical history review and commingling of tissues during processing.  The 75-percent value indicated here for medical history review actually is modeled by a distribution of 60 to 85 percent expressing the variability or uncertainty in a proportion of cases where we assume that the medical history is complete, accurate, available for review, and used without error to detect infected donors.  The 100 percent value assumes that the review is effective in every case.  The distribution for commingling assumes that commingling occurs in half of the process tissues and results in cross-contamination if infected material is present.
	The likelihood of the presence of infectious material was based on the other parameters in the model and those parameters were not varied for these comparisons. Results are shown in the next four slides expressed as means and probability distributions for the number of exposures per year based on an input of 25,000 donors.
	These comparisons are meant as examples of the application of the model and are not intended to represent a given tissue or process.  This output distribution shows the mean number of exposures per year and its probability distribution when medical history review is effective in approximately 75 percent of the cases and where commingling may occur in half of the processed tissues.
	Under the assumptions of this model the estimate of the number of exposures is 8.4 per year for 25,000 donors.  The fifth and the 95 percentiles are indicated below the histogram.
	When the same model is run in the absence of any commingling the mean number of exposures per year per 25,000 donors declines from 8.4 to 1.7.  As shown here, assuming 100 percent medical history review, the model estimates that the number of exposures would be 5.5 per year when commingling occurs.  The same model estimates that 1.4 exposures would occur in the absence of any commingling.
	The results of these four models are summarized in this table.  The variability of the means is expressed as a 5 to 95 percent confidence interval for the mean number of exposures per year.  One advantage of this modeling approach is that it allows other parameters to be evaluated such as additional donor exclusion criteria, age, for example.  Under this set of model assumptions, using 8.4 as the baseline number of annual exposures, exclusion of donors over age 60 or 65 would be expected to reduce annual exposures to 1.1 or 2.6 respectively.  At the same time a large proportion of otherwise suitable donors would be excluded by these criteria as shown in this table.
	A most useful outcome of the model is the ability to compare the model parameters to determine which of them have the greatest importance.  Sensitivity analysis was conducted to see which parameters are driving the risk in the model.  The two examples I will show are for the models incorporating 100 percent medical history review in the presence or absence of commingling.
	This figure is called a tornado diagram.  The length of the horizontal bars indicates the relative impact of the parameters on the estimated risk.  In the presence of commingling and batch size are the greatest contributors to the risk.  The other parameters in the model are shown in order of magnitude of their impact on exposure risk.  Note that the number of transplanted tissues is a significant contributor to the risk model but it is less important than commingling and batch size under the assumptions in this model.
	In the absence of commingling, the same model estimates that the number of transplanted tissues is the largest risk driver, having moved up from 4th place in the previous diagram.  As the input parameters are modified, for example, by imposing additional screening or mitigation procedures, the magnitude and order of the risk drivers can change.
	Sensitivity analysis allows us to identify significant parameters in the risk model and consider risk mitigation strategies or focus on the parameters where better scientific parameters where better scientific information is most needed.
	Several parameters in the models I've described are limited by lack of good information.  These so-called data gaps are areas in which better information would improve the accuracy of the risk model. More data are needed on the amount of CJD agent that is present in or could contaminate tissue during procurement from a CJD-infected donor, the progression of CJD and the infectivity of different tissues during the course of the disease, the extent of reduction of CJD agent that might occur during processing of tissues from a CJD-infected donor, the donor utilization and allograft implantation practices for specific tissues, and the extent of cross- contamination by instruments or equipment that might occur during processing.
	In conclusion I hope I have shown in these examples that probabilistic risk assessment allows detailed evaluation of exposure under differing model assumptions. It should be recognized that different tissues and processing methods have unique models and should be assessed separately.
	The modeling indicates that commingling is potentially a major driver of risk.  Other parameters such as the number of transplanted tissues are also significant drivers.  Finally, additional data are needed to provide accurate estimates of exposure of allograft recipients to TSE- infected tissues.
	I must thank my CBER collaborators in this work for their priceless expertise and enduring support, Dr. David Asher in the Office of Blood Research and Review and Dr. Steven Anderson in the Office of Biostatistics and Epidemiology.  As a final comment, other presenters at this meeting will discuss important topics highly relevant to these risk assessment models, including possibilities for additional testing, the impact of batch processing, cross-contamination, disinfection, and clearance.
	I anticipate that the models I have discussed this morning will be further developed as an outcome of those presentations.  I also hope that we might consider the information shared today in the context of developing quantitative risk assessments that improve our ability to make good decisions regarding the safe use of human tissues.
	Thank you.
	DR. BOLTON:  Thank you, Dr. Taff. Are there questions from the committee?
	Yes, John.
	DR. BAILAR:  You start your risk assessment model with what's becoming a standard four-step kind of thing, hazard identification and so forth.  That model was originally elaborated for carcinogens and I'm not sure that it really applies in full force here.
	You've shown that it can be useful but as you went on you slid off into a multiplicative model and I wonder if the analysis might be simplified and the need for data clarified if you just began with a straight-through multiplicative model.  Here is the probability the potential donor is infected, here's the probability that if it is infected the screening measures will remove that donor from the pool, here's the probability that the infection will survive the processing, and so forth.  If you take it step by step like that I think the whole thing might become a good bit more clear. I'm not sure the bottom line would change, but I think also the data needs would stand out in starker contrast.
	DR. TAFFS:  I agree.  The efforts of the committee that met to harmonize risk assessments in the European Commission efforts attempted to identify the elements of risk assessment that are in common between all of the different applications of risk assessment and identify those elements that were unique and they attempted to construct a framework that might be useful for risk assessors to evaluate a particular problem and develop a model.
	Now, in fact the risk assessment computations are conducted in exactly the process that you indicate; that is, all of the parameters are incorporated into a single model and evaluated sequentially in order to come up with the probabilistic computations that I showed today.  So in practice the actually computations and evaluation of risk proceed in the fashion that you describe.
	The point to having such a framework in place that uses the four components of risk assessment is to be sure that someone developing a model for the first time thinks about all of the different components that are necessary in order to be certain that all the parameters are in place when the calculations are conducted.
	DR. BOLTON:  Dr. McCullough?
	DR. McCULLOUGH:  My question has to do with the medical history review.  I'm sorry if I missed this explanation but you use the term "medical history reviewed." Does this mean the history was obtained but was not medically reviewed or that there were parts of the history that were not known or not obtained?  Can you elaborate on what you mean by that?
	DR. TAFFS:  Yes, in the model we allow for several possibilities.  One is that the review is unavailable.  Another is that the review is not used, for example, in the cases of legislative consent.  Another is that the medical history is incomplete or inaccurate, and another is the possibility that the medical review leads to some error. So those are captured under the distribution 60 percent to 85 percent that was used in this generalized model.
	DR. BOLTON:  I have a question. Have you taken the parameters, say, that may be available from dura mater transplants and tried to plug that into the model to see what results you get since we have known transmissions with a certain period of time that you might be able to evaluate the usefulness of the model?  Have you done that?
	DR. TAFFS:  I think that is a very interesting prospect.  In any case a risk assessment model should be verified against available data when the date become available and are sufficient in order to provide a good evaluation of the model.  In that regard I think it might be possible at some point in the near future to examine the reliability of the model as applied to specific tissues, for example, cornea transplants.
	At the same time I think we all have to recognize that a great deal of information is yet needed in order to generate a model that's capable of producing accurate estimates.  Even so, the utility of the model in identifying the parameters that are the greatest drivers of risk makes it a very useful tool in assessing risk drivers and coming to some conclusions about good strategies for reducing or eliminating risk.
	DR. BOLTON:  I would agree.  In my opinion one of the best uses of these models is to identify the gaps in the information that are most critical because when you begin to plug in the parameters and see how the model behaves sometimes those areas will stand out very clearly.
	This committee and the FDA obviously are very concerned about those caps in knowledge that lead us either to be unable to make decisions or to be unable to make rational scientifically-based decisions.
	DR. TAFFS:  I agree completely.
	DR. BOLTON:  Are there other questions?  Dr. Bailar?
	MR. BAILAR:  One other comment on a minor note, the probability bounds that you show are based solely on what a statistician calls sampling error.  They do not reflect bias in the data.  They do not reflect problems in the model.  The real bounds of uncertainty could be wider, maybe a great deal wider.  You might want to comment on that on further analysis.
	DR. TAFFS:  I think as the model develops we will be able to represent the distributions for the individual parameters much more accurately.  That depends on the availability of solid scientific information.
	As just one example, a mitigation strategy such as decontamination during processing, to the extent that that decontamination is validated then the distribution for the impact of this decontamination on the outcome risk assessment becomes much more precise and its impact on the total risk is quantifiable.
	Until that information becomes available we are in a position of having to assign distributions for the individual parameters that reflect current understanding but are not necessarily based on established scientific fact.
	DR. BOLTON:  Further questions or comment?
	Thank you, Dr. Taff.
	Our next speaker is Dr. Nick Hogan, who will be presenting additional testing measures, the potential value of post-mortem transorbital frontal lobe needle biopsy. Nick.
	DR. HOGAN:  Good morning.  Well, I'm glad to hear that batch and commingling are a big problem because there is no batch or commingling for corneal donors.
	I was asked today to come and talk with you about possible alternatives should testing for CJD be required.  We have come up with some interesting issues that we need to discuss.
	This really stems from the meeting in January of 2001 of this committee where after the voting it was commented on that there would be a recommendation that when a test for TSE-associated prion protein is validated such a test should be applied to tissue donors.  It comes from the charge statement today, of course, which this committee might also consider that an autopsy or failing that a brain biopsy validated to show that it is predictive of an autopsy diagnosis of TSE be performed on some or all cadaveric donors.
	Well, any of these that applied, be they autopsy or brain biopsy, you have to know that there are greater than 45,000 corneal donors per year and based on the way this is done for corneal donors there is first a medical history exclusion and then the corneas are obtained.  This would have to be applied to all 45,000 corneal donors regardless of the fact that subsequent testing for, say, hepatitis or for viability of endothelial cells might exclude that tissue.
	This just shows a corneal transplant, essentially functionally blind and then after the transplant.  Now, if an autopsy is required what impact would this have on the system?  Well, the current autopsy rate in the United States is somewhere between 10 to 13 percent.  That translates in major centers to between four and ten per week.
	Now, the corneal donor procurement in major centers is about the same, eight to ten per week, so if autopsies were required on these this would double the number of autopsies per week that would be required. So there are essentially 45,000 additional autopsies per year or 124 per day nationwide.  Obviously this is going to require additional staff in terms of taking care of and interpreting the autopsy.
	Additionally, the cost of an autopsy ranges between $1,500 and $3,000. The cost of a brain-limited autopsy, I found a bargain, $750 to $2,000, and who is going to pay for this?  Under current Medicare rules Medicare will not pay for autopsies and most insurance companies have gone along with that.  So the cost for an additional autopsy would likely be translated to the family's responsibility.
	The biggest issue about autopsy is the biological time constraint.  For a cornea there is a limit of four to eight days after death in which the tissue is viable for transplant.  Most centers use four.  The reason for that is that the cornea endothelium, which is at the base of the cornea, which is at the base of the cornea -- this is the surface; this is the anterior chamber side -- is responsible for keeping the cornea detergessed.
	If water gets in the cornea it becomes cloudy and it will fail as a graft. This is a non-replacing tissue.  It does not mitose.  It starts dying immediately after death and by the end of four days roughly 50 percent of the tissue can be gone.  So this is the reason why time is such a big issue here.
	Now, the standard time for a full autopsy based on the College of American Anatomic Pathologists is six weeks.  That's a full autopsy.  It's unclear in the way the FDA charge statement reads whether they mean that it is going to be just a neuro- pathologic exam.  I must assume that that is what they mean.  But even if just the brain were to be opened the logistical issues would still be in place, that is, a standard neuropathologic examination with fixing of the tissue, evaluation of the slides for status spongiosis, is on the order of somewhere, quickly, two weeks to three weeks, as Dr. Gambetti and DeArmond and others know, and this is clearly beyond the viability time.
	Now, what if the brain were to be opened but just a portion of the brain to be taken, a brain biopsy with the cranium open? Well, of course, doing any frozen section analysis should be able to be done within that four-day time period, actually within 24 hours.  That is about what we need in order to get a usable cornea to the recipient.
	Obviously in the other diagnostic tests, Western Blot, et cetera, that might be validated by this committee they could also be done in that time period.  But have we looked at other tissues as well?  I would be interested in knowing what the committee thinks about the urine and CSF testing that is also being utilized and looked at.
	But even with these, again, you have to think about the biological time constraints.  So after the meeting in January a year ago we were talking about these issues and what would happen if exactly this question came up and we struck the idea that perhaps since the eye is going to be removed from these cadavers anyway what about going into the brain through a transorbital approach?  Is that feasible?
	The problem is this is not currently a neurosurgical site that is used. There are obviously issues.  If the eye is in place in a living patient it's difficult to get past the eye to the orbital roof and into the brain.  There's limited flexibility and limited space.
	There's only one technique that uses a transorbital craniotomy approach. That has recently been put forward by Shanno at the University of Pennsylvania to get at skull-based tumors but even that utilizes removal of a bone flap on the frontal bone.
	So we are really treading on virgin territory here and we need to totally evaluate whether trans-orbital approach would be feasible and if testing could be validated.  So we need to look at whether the biopsy site is adequate, if tissue could be procured, what diagnostic tests would be used with that material, what kind of analysis would be required, what personnel and financial issues might be involved with that.  I will discuss some of the issues.
	Now, obviously once the eye is out the orbit is left and everything is still there, including the orbital fat, extraocular muscles, vessels, and nerves. And This is what a post-enucleation eye looks like.  The eye has been removed but the conjunctiva is still present, the fornices, the base of the conjunctiva, is present.  This is the stump of the optic nerve.  And the big problem, all the orbital fat and extraocular muscles are still present.  You have to get through that to get to the orbital roof but if you can do that, the orbital roof is an excellent candidate because it is the second thinnest bone in the orbit, the first being the medial.
	The orbital apex, which would get you to the temporal lobe, is a little bit thicker, as is shown in this slide.  Here is the orbital apex at the back of the eye, here is the orbit, is thick.  If you went through this you could get to the temporal lobe but going through the orbital roof, which should be approachable once the eye is gone, could get you to the frontal lobe.
	As shown here in this coronal section, going up in through the orbital roof to the frontal lobe gets you to the frontal cortex.  A little bit further laterally you can get more cortex and less white matter but the further you get laterally the thicker the bone becomes.
	The frontal cortex obviously is a good site for looking for agent.  As has been proved in many animal models, most brain biopsies that are done {IOn}in vivo{IOff} are done from frontal lobe and, as Paul Brown has shown, looking at infectious agents in the frontal cortex versus other regions in a scale of 1 to 4, 4 being the most frontal lobe, is a relatively good site for looking for agent, representative, at least.
	So how can we get to the brain? Well, you have to reflect the superior orbital tissue.  You can do that with cutting and a periosteal elevator that would expose the orbital roof.  Then using a chisel and a rongeur, as seen in this CT 3-D reconstruction, you could break through the orbital roof and expose the under side of the brain.  The dura could then be excised and the brain accessed.
	Then the question is how do you get the tissue out?  You still have a relatively tight window.  We have been looking at some issues of using a trocar.  This is an old test type that has a sleeve on it like a cork borer, if you would, going up through this hole and getting a core sample of brain.  A needle is too small.  Large-bore needles are too small to utilize for most testing, I think, or even some of these thoracic trocars which have disposable sleeves you can use to remove brain.
	An issue for any family is can you if you do this reconstruct the site and the answer is yes, you just replace the orbital tissue with a placode and cotton, you put a cornea sterile shell over the top which has little hooks on it, pull the eyelid down, and, as is shown, this is pre-enucleation on the same patient and post-enucleation. There should not be an issue regarding that.
	Well, the tissue pull-out and the condition of it go hand in hand with whatever validated tests you might use; that is, if you want to use morphological analysis obviously you can't pull out brain soup or you won't be able to analyze for the protein.
	Biochemical assays on the other hand don't have to be quite so intact.  The volume also is an issue and perhaps a smaller biopsy might be usable.  But the issue here really is the test and whatever test you use validation is going to be the key.
	Then the question is does it need to be 100-percent sensitive and specific? Well, we've addressed this before and I would have to say yes, it has to be rapid and it has to be within 24 hours, 36 at the outside.  It has to be reproducible and not only in the lab but also in the procurement situation.  The techs have to take from the same place, be able to get it to the labs in the same condition from case to case.  There are numerous guidelines available for that.
	Now, for the case of corneas there are 45,000 tests so even if you had a 95- percent sensitivity specificity you are introducing a huge number of false positives and negatives.  For 98 percent it's still 900 so it really must be very close to 100- percent specific and sensitive.
	As everyone knows, sensitivity declines, false negatives increase, and the risk of missing a case as specificity declines, the false positive increases, and you risk of throwing away good tissue.
	Then the question is would this test adequately diagnose pre-clinical disease?  We have already excluded a good majority of risk by the medical history, so could this pick up disease which is not prevalent by clinical examination or is this just an indication for sub-testing, you only test those that don't have adequate history, of some other decision?
	Once you have the tissue how do you test it?  Do you test it at every eye bank or is there a regional center that you send it to to be analyzed?  Obviously a regional center is going to have greater viability between tests but it is going to take some time to get the sample to them, which will again decrease your window.
	There is going to be a lot more need for personnel both in terms of the procurement and the testing.  You have to train them to do the tests and handle the results, administrative requirements in terms of oversight, reading, reporting, archiving, and this all comes down to the bottom line of cost.  Cost will be for the instruments, if they are not disposable how you maintain them, the technician time for the procurements, sample transporting, cost of testing, assay costs, tech costs, and, of course, the administrative costs that I just discussed with you.
	Really, that is a big issue because if these costs are too huge these are going to be passed on.  They presumably would be assumed by an eye bank, would be passed on to the consumer in terms of a more expensive cornea, and unless Medicare reimbursement were to go up coincidentally this could really exclude a lot of eligible recipients from having this procedure.
	But the bottom line is can this approach to be used?  Yes, the tissue procedure can be performed.  We have done it in two cases.  Tissue can be procured in various conditions and the testing probably can be performed in a timely manner for use in accessing should that be required. However, the costs will be high, as I have discussed, and validation will remain a problem.  What test are we going to use and can it be validated under field conditions, that is, from the time the tissue is obtained in the field?
	None of this says anything about what effect on donor availability might be. If you introduce this sort of requirement families are very concerned about the appearance of the body and the additional time that this might take is certainly unclear.
	We are trying to get together a reasonable questionnaire that we can provide as an analysis of this to ask family members after we have gotten through it all would you have donated this if an autopsy were required.  We have to be careful because obviously you don't want to say, "Well, thanks for the corneas.  That's going to cost you $1500.  Would you still do it?"  So I think it is reasonable to ask those questions.
	But still the major barrier to corneal transmission remains the historical exclusion.  These criteria are continually updated.  They were recently updated to exclude dementia of any sort, including Alzheimer's Disease except that clearly caused by train tumor, head trauma, or stroke, and there has not been a single case of corneal transmission in this country since institution of the medical exclusionary criteria.  The 1974 case, the only positive case in the world, occurred before these exclusionary criteria were implemented.
	So I would ask you that while determining what additional safeguards might be put in place to protect the population from CJD, a rare disease, we must remain vigilant in our pursuit against the devastating blindness caused by corneal disease which affects thousands of people in this country.
	Thank you.  Any questions?
	DR. BOLTON:  Thank you, Dr. Hogan. Questions from the committee?
	DR. DeARMOND:  Well, Nick, so why should we do it if the historical approach has been 100 percent safe so far?
	DR. HOGAN:  Well, and that's the issue.  Obviously there is a risk of something getting through.  That happened in the United Kingdom.  It may have happened elsewhere.  So with that risk of getting through, do you test?  Do you test all patients?  That's the question.
	We are talking essentially here about availability.  If you are going to reduce the age of donors 61 and over that gets rid of 50 percent of corneal donors, head trauma another 13.  You're at 63 percent.  Then the issue has been brought up on some of the papers that were discussed here by Dr. Solomon if variant CJD were to come to this country and we had to exclude donors that were younger than 50 because they would be at the greatest risk we don't have any corneas left.  So the issue here would be testing, to be sure, but I ask that same question myself.
	DR. DeARMOND:  Another question: As you know, the great majority of pathologists won't do an autopsy on a CJD patient.
	DR. HOGAN:  Nor will surgeons do brain biopsies.
	DR. DeARMOND:  No.  We have been lucky that they will do it but they are very reluctant because for every one of those they have to close the operating suite for many hours with multiple personnel to decontaminate it so they don't like doing that.
	But this is also risky.  It's actually much more complicated, actually, than removing the brain at first sight.  It may be ultimately very simple but cracking the skull and pulling out the brain is actually a very simple procedure.
	Here you have to rongeur your way up and then put a trocar up inside, the CSF is going to pour out, so the general pathologists who are doing these 45,000 corneal extracts are going to panic.  Is there a simpler way or how do you deal with this contamination and the complexity of the procedure or is it too complex?  Maybe that's the question.
	DR. HOGAN:  Well, I think either procedure is complex in terms of the containment issues but, again, the risk of those 45,000 having CJD is very low.  You still have to assume that they all do.  But we are working on some way to devise perhaps a trocar that could go through the orbital roof and get this biopsy in one fell swoop. It certainly would have CSF licked but your issues are well taken.  We still have CSF and there will still be containment problems.
	DR. BOLTON:  John?
	DR. BAILAR:  What's the imbalance between supply and demand?  How many more corneas per year do we need?
	DR. HOGAN:  Well, I have a slide on that.  I didn't bring it.  In the United States we are pretty much close to what we need.  There still are people who have to wait three or four weeks to get a cornea. But eventually the majority, I would say, 90 percent of patients in this country, are able to get a cornea within a short time frame.
	That's why corneas in this country are transported, are shipped, worldwide.  So we do supply some corneas to other parts of the world.  Some of our other speakers might be able to address that issue also.
	DR. BOLTON:  Please use the microphone and introduce yourself.
	MS. HECK:  Ellen Heck for EBAA and UT Southwestern Eye Bank.  Your question about the brain biopsies is of particular concern to me because Nick in his examples said the technician could do this.  I think your risk of contamination from a technician is much greater than your risk of contamination with a pathologist and although Dr. Hogan, who works in Dallas where I work, is used to a metroplex where pathologists and neuro-pathologists are readily available I caution you to remember that at least 30 percent if not greater of the corneal procurements occur in rural areas where you will not have the access to the technology and the control of contamination that you have in a large university setting.
	I think if you start trying to say that you are going to train eye bank technicians to do interorbital biopsies that our contamination concern is maybe greater than our risk concern in the cases that they might open up in a hospital room because not all corneal procurements are done in an operating room.  Many of them are done in a patient's room or in some other site which would be difficult to control.
	DR. BOLTON:  Pedro?
	DR. PICCARDO:  You are aiming to have 100 percent certainty.  I mean, if I understand correctly you want 100 percent certainty?
	DR. HOGAN:  Of course.
	DR. PICCARDO:  Well, with this technique, which to me looks a little bit cumbersome, I mean, with the kind of work you put into getting a little piece of brain from a patient like this you could cut the whole brain out.  I think if you cut the whole brain out you have a better chance of going to whatever you want and analyzing whatever you want because a good amount of tissue well taken, relatively well preserved, you can freeze, you can fix, you can do a number of things.  With these technicians I don't think you can.
	The other thing is this is sometimes patchy so something negative here will not say anything about the rest of the brain.  I think if you create a sense of security when in reality I don't think you have it.
	DR. HOGAN:  I agree.  The issue here, the reason why it was brought up at all, was the issue of containment and we have already discussed that.
	DR. DeARMOND:  Well, Nick, I do think the idea is very good and validating it is going to be hard because you have to do the procedure and then do the autopsy and test.  The problem is we usually get people at the end stage of the disease.  We never get a person who is unsuspected of CJD.  At least I've never seen on show up yet but maybe I missed it.  Maybe I'm just not good enough to see it.
	DR. HOGAN:  I doubt that.
	DR. DeARMOND:  So at end stage disease we've even had some problems with the cortical biopsy two to three months before the patient died being negative and then when we do the autopsy it is clearly positive.  So validation of the technique, I think, is going to be tough for those unsuspected cases of CJD but the idea, I think, is still a valid one and certainly could add some degree of confidence about the cornea.  Maybe a different approach or some other mechanism because of this fear of pathologists.
	And it really irritates me that they have this fear.  It's unethical, it's immoral.  The other physicians take care of the patient, the family lives with the patient, and the lousy pathologist won't even go in and do his job.  I've even told them they should get out of this business and get into the restaurant business or something that's safer.
	But accepting that they are chicken we have to come up with some simple method that would be easy, maybe a way of going laterally, a burr hole that would be very simple with a needle biopsy or a fairly large needle biopsy might be another way where they will feel a little more comfortable about what they are doing and they can see what they are doing.
	I'd like to talk to you more about how we could think through such a thing because I think it's an interesting approach and it could give some more security about other transplants as well as corneal.
	DR. HOGAN:  Yes, and that is certainly a key here.  The problem, I think, as you said, is personnel and time.  So whether you go through the orbit or through the brain I think that is irrelevant.  It's how you validate it in terms of what tests you are going to do, is it going to be worthwhile to do 45,000 tests to detect one case, and is it worth the cost?
	DR. BOLTON:  From a na‹ve biochemist's point of view let me ask our neuropathologists their opinion.  What about looking at the optic nerve since it is right there and we are looking for infectivity in the cornea, which is downstream from that? Is that feasible?  If there was a test that could detect very small amounts of abnormal PRP what is your opinion?
	DR. GAMBETTI:  I have never tested the optic nerve so I have no idea how rich the optic nerve is.
	DR. HOGAN:  I have some slides with data on that if you would put up the last two.
	DR. GAMBETTI:  The retina would be eventually probably a much better area to test than the optic nerve.
	DR. BOLTON:  Would that simplify the procedure and would it do so if it does without compromising the reliability of the result?
	DR. GAMBETTI:  Yes, to the retina would simplify the procedure.  I don't know the reliability of the retina vis-a-vis the cerebral cortex.
	DR. BOLTON:  Pedro, you are shaking your head no?
	DR. HOGAN:  Could we put on the last couple of slides?  Go ahead.  If you would allow me.
	DR. BOLTON:  Yes, do you have data on this?
	DR. HOGAN:  Yes.  As you know, when I was back with Stan we did some regional data on looking at the titer of the agent in the scrapie model inoculated.  The amount of agent in the retina is about that of brain. In cornea is five logs less than in brain and that's in the IC route.
	The optic nerve is a little bit somewhere in between.  There is a paper out from Collins' group using the ELC Western Blot technique that looked at regional titers in eyes of variant CJD and sporadic CJD patients and compared to the amount of agent in the brain that present invariant CJD optic nerve is 25 percent, that in brain.  That present invariant CJD retina is 2.5 percent, that in brain, and there is none detectible, that is, less than .00025 percent, that in brain, none detectible in sporadic CJD optic nerve or retina.
	Now, we know it's there.  We have done work on the animal models and we know that the PRP res is present in retina and there is a retinal destruction that comes from it.  But we are working.  Actually, I have a small grant to see if whether we can get the agent titered in the retina in human cases.  Cases are tough to come by, as Dr. Belay knows.
	DR. BOLTON:  Pedro and then Suzette.
	DR. PICCARDO:  I'm going to repeat something I said already.  Let's start all over again.  In multiple cases a patient that comes with a diagnosis of CJD the pathology is fairly clear.  A neuro- pathology of the brain is fairly clear to make a diagnosis; however, we will come to cases, and I have experience in that, in which it becomes difficult.
	Sometimes there is the association between the pattern of immuno-stoichiometry and PRP risk.  In some cases I saw a significant amount of immunopositivity on the brain; however, on the contralateral side which we have frozen in some areas we couldn't see any PRP risk by Western Blotting, vice versa, all the permutations that you want, et cetera.
	Once again in most of the cases it is clear; however, in some of the cases it is not clear even having the whole brain in your hand.  So what I am afraid of is that with this system the aim is to have 100 percent certainty and it will create an appearance of something that is very well controlled when I'm not sure it will be very well controlled.
	The other issue is coming to what Steve DeArmond mentioned.  I think that the neuropathologists and the pathologists in general should be trained to do the autopsy and to go through the procedures.  Provided you use common sense it is not dangerous.  I don't see why not.  Money is an issue but here we are dealing with a disease that has no cure and is fatal so I don't see any other way around it.
	DR. BOLTON:  Sue.
	DR. PRIOLA:  You stole my question earlier but I have one more.  You said there were no cases of corneal transmission after 1974 when you instituted this patient background.  How many corneal transplants have been done since 1974?
	DR. HOGAN:  In the last ten years there have been 250,000.  The data has not been collected prior to that.
	DR. PRIOLA:  So given that the risk of sporadic CJD is one per one to two million people would you expect to even pick one up?
	DR. HOGAN:  Well, that's a big question.  That is a whole talk in itself. There are issues beyond the issue of prevalence in the population, especially since these are all dead.  It is probably more like one in 100,000 because they are all dead people, not living people.  The one per million is for living people.
	There are some biological issues about the cornea, anyway.  What is the titer of the agent in the cornea?  Extremely low, maybe undetectable.  How long does it have to be in someone in order to produce the disease?  If it does produce the disease how long is the incubation period?  Could it extend beyond a patient's lifetime?  Most of the corneal donations that are done are in older patients so it may not be detectible.
	DR. PRIOLA:  Yes, my point is that the fact that you don't have any cases since 1974 may not be due to --
	DR. HOGAN:  Somebody didn't report them.
	DR. PRIOLA:  Right, yes.
	DR. BOLTON:  Pierluigi?
	DR. GAMBETTI:  Going back in time to have a general view of the problem, I think there are several difficulties that are not very different from those the surveillance centers are dealing with.  And one is that, as I think Steve DeArmond already underlined, it is really very difficult to get any procedure done on any case that is even vaguely suspected of having CJD or just to rule out CJD, just the fact that the result worked.
	So one way to get around that but again will increase the costs is to have identified centers that will perform that procedure for a reimbursement.  So that means transporting.  I don't know what percentage of cases which will be started that will be tested that will be tested in a major center and which will be those that are tested in rural areas where it will be very difficult to get.  I don't know what is this percent.  But one has to keep in mind that eventually one has to designate certain centers for the procedure to be performed.
	Second, having said that, once the donor already is in that center then we also examine.  University Hospital at Case Western Reserve University has examined because they were again reluctant to remove the brain.  They have examined these possibilities, the burr hole, the biopsy, transorbital biopsy, and so on and at the end they came to the conclusion that removal of the brain was the most practical thing to do because you had then much more tissue to examine and the time involved was actually less.  Maybe the risk of contamination might have been a little bit more but I think you are in a relatively large center and they have the septic room, a special room, so that does not play a role.
	Now, concerning the fact that how many you may miss, in our case, as Steve DeArmond way saying, we see generally cases at the end of the disease.  What happens if you look at early cases?  Presymptomatic was mentioned.
	In our experience with brain biopsy, again, a population in which the disease may be there, is likely to be there, but at a very early stage we miss about 20 percent of the cases just on the fact that the tissue is very small rather than being representative of the whole brain like we do in autopsy because we take it from different areas.
	So there is the possibility of missing cases, especially if one uses, for example, immuno-staining.  So certainly one should use Western Blot or other diagnostic tests rather than histology but by limiting yourself to small areas there is the possibility of the order of 10 to 20 percent missing, really, the case.
	So major centers probably should be involved if one wants to do it and then this implies increasing costs.  And once that you are in a major center it looks like probably the most practical thing to remove the brain.
	DR. BOLTON:  Nick, if you have your slide?
	DR. HOGAN:  I apologize.  This was actually not supposed to be in there.  This was supposed to be put on at the end.  I wasn't finished with this slide.  Go back two.
	This is the data we had from hamster study.  We looked at hamsters both presymptomatically at seven weeks after IC inoculation and ten weeks after inoculation. These are the titers in ID50 units per gram, in cornea about five logs less than that in brain, optic nerve pretty close to brain, retina even closer.  Again, that's IC inoculation.
	This is what came from Wadsworth Collins' group where in sporadic CJD there was nothing detected based on this ELC Western Blot analysis.  In variant CJD 2.5 percent in retina.  This is compared to brain.  That means if brain is 100 percent then in retina it's 2.5 and optic nerve 25. So that is the data that you were looking for.
	DR. BOLTON:  Thank you.  I think we will move on now.  I see Dr. Brown in the back of the room there.  Paul, I'm sorry we kept you.  I hope you are feeling a little better.
	Our next speaker will be Dr. Paul Brown.  Everybody on this committee is very familiar with Dr. Brown.  He is former chairman of this committee.  He will be telling us two things.  As I said before, we are going to combine his talks into one session.  The first will be "Experience with Human Dura Mater Allograft and Pituitary- Derived Hormones:  Lessons for Other Human Tissues," and we will take whatever questions the committee has for Dr. Brown.
	Then we will move into his second talk, which will be "Potential for Cross- Contamination of Bone and Soft Tissue with Higher-Risk Tissues During Recovery."  As soon as Dr. Brown is mic'd we will give him the floor.
	Paul.
	DR. BROWN:  Good morning.  Can you hear me all right?  Well, I stand before you as testimony of the fact that high-dose steroids don't do much for dermatitis but they certainly are mood brighteners.
	This will be very short in part because there is not a whole lot of data bearing on the issue that you have been asked to address and, second, because if there were it probably wouldn't help you too much.  But let's reverse the order in which you thought you were going to hear these things because it's a more logical approach to give you what we have in terms of tissue infectivity and then to tell you what we have in terms of the consequences of that, which would be the tally of the iatrogenic 0transmission of cases of CJD.
	So in the first slide if we could focus that, please, and lower the lights, this is the slide that I usually show for popular consumption.  It's colorful and it gives the principal message which is that in human beings affected with Creutzfeldt-Jakob disease infectivity is indeed widespread in the body and not just limited to tissues of the central nervous system.
	These represent data accumulated over 20 to 30 years of inoculating tissues from human patients with Creutzfeldt-Jakob Disease into primates, which are far and away the most sensitive and susceptible bioassay animal for human tissues.  Most of this data come from inoculation of squirrel monkeys.  Some of it comes from inoculation of chimpanzees.  They both have rates of transmission when inoculated with brain tissue of something like 95 percent so they are an extremely sensitive host.
	What you see is that brain and eye contain infectivity more often, the brain, as I say, 95 percent, the eye a lower percentage but still very high.  You will see the numbers in a bit.
	Dura mater and pituitary gland in fact were not inoculated.  I have included them in the slide simply because we know they are infectious in humans from the fact that they are a transmitted disease in humans.  Spinal cord, spinal fluid.
	But then a variety of peripheral organs are also infectious in a small proportion of patients that were tested, lung, liver, kidney, spleen, lymph nodes. Our experience in primates indicates that human blood is not infectious but I think it is arguable in view of the established fact that blood is infectious in a number of experimental models, including rodents, sheep, and monkeys.
	The muscles which were tested that had no infectivity were heart, skeletal muscle, adipose tissue, testis and prostate, and none of the excretions or secretions yielded infectious material.
	Now, as I say, that is a pretty generalized slide but when I show you the numbers on which this data are based you will see that this is perhaps a more optimistic judgment than might be made.
	Brain was inoculated into 259 patients.  This data, by the way, you may say well, why don't you give us some titers? We would all be interested to know what the titer of spleen is in a human being compared to brain.
	This alone represents experiments that probably consumed 1,000 squirrel monkeys and it is just not done, especially today.  So what data there is is likely to be the only data that there ever will be in primates.  Whether humanized transgenic mice turn out to be as good a bioassay as primates remains to be seen.  On the other hand humanized transgenic mice if they over-express may actually be unrealistic. You may get, for example, a transmission and an over-expressing humanized mouse that has no basis in reality at all.  So beware of transgenic bioassay animals until it is established that they represent realistic results.
	You can see that most of the other tissues with the exception of liver, kidney, and spleen were not inoculated into or they did not come from very many patients.  These denominators represent the donors, that is, the number of humans that were tested, not the number of animals that came down.
	So lung, for example, four humans were tested, two of them were positive, but that involved somewhere between 8 and 12 monkeys.  We did not achieve transmission from any of the other tissues shown, marrow, whole blood, leukocyte, serum, the muscles that were shown on the previous one, but, you see, adipose tissue was only inoculated from one patient, gingiva and intestine from a single patient.
	So you cannot really be on the basis of this data in any way confident that infectivity would not be present in peripheral tissues were 10, 15, or, better yet, 100 patients used for bioassay experiments.
	As I say, the data is very meager with respect to peripheral tissues.  Such as it is it's primarily negative but I wouldn't necessarily go home thinking that we have shown that there is no infectivity, for example, in peripheral nerve.  Inoculated five patients, that's not bad.  No transmissions.  What we can say from this for sure is that if infectivity does exist in peripheral tissues it exists at very low levels.  Otherwise we would have a transmission every time we inoculated a tissue.
	That really is all we know from primate experiments about infectivity in human tissues.  I did want to show you one other slide, however, which is a very interesting slide because it illustrates the fact that these are all patients with Creutzfeldt-Jakob disease on the left.  The species inoculated, spider monkeys, squirrel monkeys, chimpanzees, all very susceptible hosts, all transmitted in short order when the brain was inoculated by the intra- cerebral route.  But the incubation periods following peripheral routes of infection were typically longer, sometimes much longer, and sometimes apparently long enough so that a transmission never occurred and these were animals that were observed for up to ten to fifteen years.
	Point:  When you inoculate material directly into the brain you will have transmissions far more often than when you give the same material by a peripheral route of infection.
	So even when you have tissue that is infectious it is not a guaranteed transmissible tissue and most products that are used by humans are not inoculated directly into the brain.
	Now we go to iatrogenic disease.
	DR. BELAY:  May I ask a question?
	DR. BOLTON:  Paul, would you like to entertain some questions on this part first?
	DR. BROWN:  This is so quick I think it will --
	DR. BOLTON:  You want to move on? We'll hold them then.
	DR. BELAY:  So what are the consequences of infectivity in human tissues?  Well, here they are.  Growth hormone has accounted for almost 160 cases of CJD worldwide, dura mater has accounted for what is probably now about 90 cases of CJD worldwide, and there are, as you know, very rare and now getting increasingly old cases of cross-contamination of instruments at corneas in which transmission has occurred.
	When you break this down country by country it looks like this.  So Japan is the overwhelming champion of dura mater graft transmissions.  France is a comparable champion of growth hormone transmissions. And the US and the United Kingdom together have had about 60-odd transmissions between us.
	The other sources of iatrogenic disease, cross-contamination, surgical instruments, corneal transplants, gonadotropin in Australia, are really trivial compared with these three major sources and these three major sources will not have escaped your attention that they are all from tissues developmentally or in close proximity to the brain.
	You may want to ask why Japan has such an overwhelming number of dura mater graft transmissions and why France should have had such a large number of growth hormone transmissions.  We don't and never will know the answer with confidence to either of those questions but Japan probably did not import a greater proportion of contaminated dura mater than any other country.  What it did do was import enormous numbers of dura mater grafts relative to other countries.
	If you use the estimated denominator for risk of iatrogenic CJD from Japan you get a risk that is something like 0.03.  So compared with growth hormone, which, for example, in France is now running along at the rate of about one in 18 recipients, you can see that even with dura mater, pooled as it was, inadequately sterilized as it wasn't, it has not been relative to the number of dura mater grafts used a very florid source of iatrogenic disease.
	So I think the answer to Japan probably is the fact that the Japanese used 20,000 dura mater grafts a year in its heyday and that represents a multiple.  We don't know what multiple, but a multiple, probably 10 to 20 times more frequently used in Japan than elsewhere.
	With respect to growth hormone, that is a very interesting story, I think, and not to bore you with the reasons for the conclusion it is likely that France ran into trouble from a combination of difficulties.
	My reading of the French experience is that their primary problem was the fact that in France autopsies of the brain typically were only done on patients with neurologic disease; that is, unlike the US where a complete autopsy is specified and you do the brain and you do a complete autopsy in someone who has died of a myocardial infarction in France this was not typical.
	In France when you had a neurologic disease you often had the brain examined. If you didn't die from a neurologic disease you didn't even if an autopsy was done.  It didn't include the brain.
	The consequence of that is that fully one-third of brains and brain autopsies came from hospitals with large neurologic services, which is a huge bias because the pituitaries were only able to be removed, obviously, from patients who had had autopsies of the brain.  So one-third of the autopsies in France came from hospitals that were primarily neurology hospitals. That's bad.  It was obviously a very wonderful source for pituitaries, the only source that was available, but in retrospect a bad idea.
	A second element of the French story is that every one of the patients in France so far has shared a treatment period that is relatively constricted.  Unlike the US and the United Kingdom where there has been a random incidence of CJD starting from about 1967 all the way up through 1977 in France all of the cases of CJD shared a window of treatment between the beginning of 1984 and the middle of 1985, a period of 18 months.
	So it appears that this large ever-expanding number of autopsies that were done and pituitaries that were collected as growth hormone use became more prevalent combined with a rather bad, risky source combined with what is probably either both bad luck in terms of having a few patients with CJD in the pot, so to speak, and probable massive disregard of cross- contamination during that period at some point.
	In France, as elsewhere, the pools of pituitaries varied from several hundred to 10- to 20,000 and in the period at issue here in the mid and early 1980s batch pools typically ran to at least 10,000.  So there was an opportunity for a greater dispersion of contamination.
	The final issue is that the method of processing, and I haven't got the details on this, the final chromatographic step for the purification of growth hormone gave you the opportunity to take either a large number of fractions or a smaller number of fractions and although I've never seen hard data to support the contention Dr. Parlow, who in this country made the growth hormone after 1977 and whose hormone evidently has remained and was then free from infectivity because no US patient who has only received the Parlow treatment has come down with CJD.
	So that the Parlow method of chromatography and the cuts he took, which he says were very narrow, may have made a big difference in terms of the amount of infectivity that might have gotten through. So this is a combination of factors.  Again, I don't think we will ever work them out with precision, although the French Inspector General and the Director of INCERN once again want to rehash the whole thing and go over it and find out if they can a little more precisely what the cause was because they have a dozen lawsuits on the table in France.
	Finally, here are four anecdotes. They represent cases of Creutzfeldt-Jakob disease in recipients of 57, 63, 54, and 55 years.  The fourth column, the organs donated were liver, bone, pericardium, and kidney.
	The diagnosis of the donor is known in three instances, 42 cerebral aneurysm, 46 myocardial infarct.  The other ages aren't known but one gentleman died of an infarct and the final patient nothing is known about.
	So in no case was the donor visibly suffering symptoms that could possibly been Creutzfeldt-Jakob disease.  Autopsies were not done on them so we have no idea whether they had incipient Creutzfeldt-Jakob disease.
	The age of the recipients is very satisfactory for sporadic Creutzfeldt-Jakob disease and the organs that were donated, to the best of our knowledge, two of the organs, bone and pericardium, are unlikely to have contained infectivity even if the donor had CJD.  The liver and the kidney, as you have seen, could have been infectious.
	My reading of this is that all four cases were probably sporadic Creutzfeldt-Jakob disease but we just don't know because we don't have enough data on the donors.  But I thought you would like to see it, anyway, to know that there are a few cases, a handful, in which iatrogenic transmission from peripheral tissues might have occurred.  And that will conclude it.
	DR. BOLTON:  Thank you, Paul.
	Questions?
	DR. BELAY:  I just wanted a clarification about the infectivity data for the various organ tissues that you provided in the previous tables, I believe three or four slides back.
	DR. BROWN:  Shall we put that on?
	DR. BELAY:  The question is were all the data derived from primate models or was it a combination?
	DR. BROWN:  No primate models. Well, I don't know what you mean.  The tissue that was inoculated was all human; that is to say, these were tissues from humans with CJD completely.
	The recipients were either squirrel monkeys or chimpanzees.  That is the bioassay animal.
	DR. BELAY:  All the recipient animals were primates?
	DR. BROWN:  The bioassay animals were invariably squirrel monkeys or chimpanzees.  While I think of it let me point out something.  If I'd known this was going to be an issue, it has nothing to do with we had to I've just said, but there was this whole question raised in previous discussions about if you did a biopsy would you stand to miss the diagnosis because it wouldn't have been an area that was either neuropathologically or immunologically involved.
	We do have information from our review of cases in France long ago from about 1965 through 1975, when it was habitual in France as a routine measure for anybody coming in who was suspected of CJD to have a biopsy and who in many cases went on to have an autopsy.  Somewhere between 50 and 100 of those cases had both biopsies and autopsies done.
	Our analysis indicated that no matter where the biopsy was done in France, at least, in the way that they did it, and they were usually frontal temporal biopsies or parietal temporal biopsies, the rate of positivity relative to the autopsy was 95 percent.  So you may miss some but you aren't going to miss many.
	I think this concern about doing a biopsy and missing is a legitimate concern but you have to understand that if you get a piece of frontal temporal cortex you have an overwhelming likelihood of finding tissue that is pathological.
	DR. BOLTON:  A follow-up?
	DR. BELAY:  I think Dr. Brown would agree with me that it might be appropriate to add that the dura mater graft associated with CJD cases worldwide, well, actually, a vast majority of them were associated with a single brand of dura mater or Lyodura, which was produced by a single manufacturer, which I believe is very, very significant because a vast majority of the worldwide dura mater-associated CJD cases actually received one brand of dura.
	At that most of those dura, which is Lyodura, those grafts were produced before 1987.  I think that is worth mentioning.
	DR. BROWN:  Yes, I think it is worth mentioning too, thanks.  Lyodura is bad news.  I don't know why Brown and ÄÄÄÄ are still in business.  I would have thought they would have been sued out of existence a long time ago but they are.
	In 1987 they introduced urea exposure to their proceedings.  Before that they hadn't and to the best of my knowledge there are only two cases of dura mater transmissions using dura mater that was provided by other sources.  One was in Italy in which a university produced dura mater and one was in this country in which a probable, almost certain, dura mater transmission occurred from a different brand.
	DR. BOLTON:  Dr. Gambetti?
	DR. GAMBETTI:  Another point of information concerning the infectivity of the cerebral spinal fluid which is important because nowadays CSF is shipped all around many countries.
	DR. BROWN:  Most of it comes to you, Pierluigi.
	DR. GAMBETTI:  You had in one slide two classes, I believe, concerning infectivity.
	DR. BROWN:  Let's go on because the numbers are better.
	DR. GAMBETTI:  Then you have a number 3 out of 26.
	DR. BROWN:  Yes, that's the number, something like that.  Let's go back one more.
	DR. GAMBETTI:  If I remember correctly in that paper among these 26 cases there were also Kuru cases?
	DR. BROWN:  That's correct.
	DR. GAMBETTI:  Kuru cases.  Do you know.  Is there a way to know whether --
	DR. BROWN:  Well, I can tell you that some of them, one or two.  No, I don't. I can't tell you whether one, two, or three were from CJD or Kuru.  My guess is that probably all three were from CJD.
	I have to go back on my word.  I don't think very much cerebral spinal fluid was ever collected in New Guinea.  I think the likelihood is that these are probably CJD.
	I can go back and later today tell you exactly if you would like or you can give me a call.  In the slide as a whole Kuru is represented but it is not disproportionate; that is, some of them are Kuru.  Most of them are CJD for the whole slide.
	DR. GAMBETTI:  So you would consider CSF infectious?
	DR. BROWN:  I would.  I do.  It is. I mean, I don't think anybody can argue that CSF would not be infectious.  The nice thing about this number and CSF is the Kretchmar's technique, the so-called SIFT, screening for intensely fluorescent objects to determine the presence of pathological PRP, his numbers match these numbers.  You get the same rough percentage of positives as this. That's a point that was not picked up by many readers in the paper, but it made me quite sanguine about the reliability of that particular test.
	DR. BOLTON:  Are there other questions of comments?
	DR. DeARMOND:  Nobody can duplicate the squirrel monkey experiments that I know of, again, and the transgenic mice present a possibility.  What would have to be done to convince you that one of the transgenic mouse models, the MHU2M or a pure humanized or whatever would be valid?  Because the monkey is a gold standard.
	DR. BROWN:  Ideally it seems to me the most convincing proof would be to take archived specimens of these same tissues and put them in the transgenic mice but I don't know whether that would ever be possible. If it were God knows when we go to our archives, which have suffered 40 years of various problems and misfilings and label losses, whether we could retrieve enough tissue to make that possible I don't know.
	Alternatively, less ideal, I suppose we could certainly find a handful of these tissues to titer, for example, in parallel.  John Collins (?) and I have already done an experiment which has never been published and probably never will be in which the same three human tissue specimens from three different patients with CJD he titered in his transgenic mouse assay and we titered in squirrel monkeys.  From that it appeared that the transgenic mouse assay was as good or better in terms of the end point of the infectivity level as were the primates.
	So what news there is in that regard is good news.  I am just worried in transgenics whether, as I pointed out, they might not risk being too sensitive.
	DR. DeARMOND:  There can be spontaneous disease and you worry about things like that.  Another question perhaps related to a future topic this morning is what sort of a decrease in infectivity titer, say with decontamination, do you think is safe?  One hundred percent loss would be ideal but it seems unlikely today and still preserving an expensive instrument.
	From your studies with the monkeys and other studies do you have a sense of if you have ten to the ninth (109) in brain and you were able to decontaminate it what level below would you have to go to to feel pretty comfortable that it wouldn't transmit?
	DR. BROWN:  Some people in the audience are going to think I have seeded these questions from you because that's a question that I love to answer.  The answer is obviously it depends on the likely burden of infectivity in whatever it is you wish to decontaminate.
	If it is a surgical instrument used on the brain from a CJD patient I think you would want to decontaminate the works.  I don't think you would probably accept anything short of full sterility.
	If it is a plasma product and you could guarantee a one log drop in infectivity 100 percent of the time it would be enough because there is no way in the world a plasma product is ever going to contain as much as one log of infectivity per mil.  It just won't happen.  If it had happened we would see hundreds of patients already who had gotten CJD from plasma products.
	So it has to be balanced against what you expect from tissue.  As a rule I would think viewing these experiments and data from other laboratories if it's a question of non-central nervous system tissue or tissues which are developmentally associated with the nervous system like the eye or in proximity like the dura mater if you are outside the central nervous system and its associated tissues the amount of decontamination that you ought to require shouldn't be massive.
	Let's just carry it through one more step.  It looks very much as though in patients with variant CJD there may be regularly infectivity in the spleen and maybe the tonsil and the rest of the lympho- reticular system.  We don't know about the peripheral organs yet, and incidentally we do have titer information on human brain from sporadic CJD and on average it is a little lower than five logs per gram.  It is not like a hamster.  Even in the most infectious tissue in human beings titered in squirrel monkeys and in chimpanzees the average end point dilution was about ten to the five (105).  It was a little less than ten to the five (105).
	I would suppose based on an analogy with other diseases like scrapie, which has been very well studied, that the spleen titer is not going to be likely to be much more than ten to the three (103) if that. It might very well be down to ten to the two (102) or ten to the one (101)
	So you would still want, I think, if you were a general surgeon operating, taking a spleen out of a patient that you knew had variant CJD, you would want full sterility.  I don't think the same is true for sporadic CJD in general surgery.  It seems to me that you could undoubtedly get away with less sterility and be happy.
	The other thing is you don't always have to go to autoclaving at 134 and two normal sodium hydroxides.  There is a tendency for people to think that well, either this works or it doesn't, "works" meaning full sterility or nothing, but the fact is that the tenth normal sodium hydroxide is a damn good decontaminant.  It is not as good as normal but it's damn good.
	Diluted bleach is good, urea is good, and there are a couple of other chemicals that have been looked at by you, your group, I think, and a couple of others. They work and they work to the tune of three or four logs as opposed to seven or eight logs.  These things ought to be considered for decontamination where they might be more appropriate than the really harsh methods.
	DR. BOLTON:  Dr. Epstein?
	DR. EPSTEIN:  In the earlier discussion today it was suggested that screening might be done on the ocular tissue directly rather than trying to access the brain.  In you studies I think you had four out of five patients had a positive ocular tissue.
	Could you just comment on how the eye tissue was sampled?  In other words were these whole eyes, were these retinas?  What did you actually culture?
	DR. BROWN:  They aren't specified, Jay, because they typically were a pool, lens, cornea, retina, and so we have no data indicating which of the components of the eye produced the transmission, the infectivity that transmitted.
	Based on studies that Nick has done and Collins the presumption is, and I think it's a plausible presumption, that most of the infectivity in the eye itself is in the retina and progressively less in the tissues forward of the retina in a very orderly way so that by the time you reach the conjunctiva and the cornea you're not talking about much infectivity.
	Of course, the problem is by definition one infectious unit transmits disease so you don't need much infectivity, particularly in a graft that is going to sit there for the duration instead of a plasma product which is going to be metabolized quickly.
	DR. BOLTON:  Dr. Doppelt?
	DR. DOPPELT:  On this list of tissues that can be infective do you have any data on bone or other peripheral soft tissues like ligaments and tendons?
	DR. BROWN:  I think bone was inoculated from just one patient.  It was negative for what that's worth.  Bone, however, is a tissue that has been looked at.  I don't think we need to bring the slide up again.
	DR. BOLTON:  It's on our handout.
	DR. BROWN:  Well, I think both were at some point inoculated.  I think it's the second slide or the one after that that we might as well leave on, bone marrow from two patients and I don't see bone at all.  So we don't have any data on bone.  Two bone marrows were negative.
	Bone, however, has been inoculated in several studies on experimental and natural scrapie and on experimental BSE and it has been negative.  These are good studies.  This is cattle to cattle, taking bone and inoculating a gemisch (?) of bone into the brains of other cattle.  So by analogy with other TSEs bone apart from marrow is not likely to be infectious.
	DR. BOLTON:  Any further questions? Thank you, Paul.  Thank you for coming in even when you are under the weather.  We always appreciate your input.
	I think what we will do is we will take our next speaker, Dr. Aliza Eshkol, and then we will take the break, okay?  We are a little bit behind schedule, but really because we took Paul out of order.
	Dr. Eshkol will speak on the experience of a commercial manufacturer of a pituitary-derived hormone.  This is really the Serono experience.
	Dr. Eshkol.
	DR. ESHKOL:  Thank you for inviting me to share with you some of our own experience on pituitary-derived growth hormone, which is not available any more for distribution in any case, but one can still learn from the history of it and of the various validation studies which were done in the past.
	Pituitary growth hormone was started to be prepared from pituitary glands in the early '50s.  Initially the pituitaries were collected and they were put into cold acetone and kept there until they were processed and the process was quite a harsh process of extraction, of generally ÄÄÄÄ things and subsequently ÄÄÄÄ by either a strong acid or a strong alkaline procedure, mainly the Wilhelmy procedure. These products were only semi-purified and they contained a lot of aggregates and dimers which are frequently antigenic.
	From the '70s on extraction and purification were done on frozen glands by significantly milder methodologies and they included some size exclusion chromatographies in order to isolate them on a ÄÄÄÄ and exclude the dimers and the aggregates.
	Now, regarding the safety, as you all know better than me, probably, that it depends on several issues.  It depends on the raw material.  Obviously pituitary glands can become contaminated.
	It depends on the process, on the capacity of the process to eliminate the agent, if present, on the decontamination procedures, on avoiding cross-contaminations on the back sides and eventually, if available, final quality control.
	Now, the first issue is the source. The pituitary glands are collected post-mortem.  The majority of the donors come from the older population.  Donors of nervous system disease or mental illness were excluded but that obviously has its limitations and mostly no brain histology was done.  So the conclusion is there is absolutely no certainty that the donors are free of CJD, actually the opposite. Everybody is sure that they are very included between the respondents who harbor the CJD or who had the disease.
	In March of 1985 the NIH learned that a 22-year old man died of CJD.  He received growth hormone from 1966 to 1976, namely growth hormone produced by the harsh old methodologies and in spite of that it was transmitting the disease.  In April '85 two additional deaths were reported.
	Now, factually it was known in principle to some investigators and particularly the concern was is the transition to the milder methodologies better able to exclude such agents during the process of the purification and a validation, a clearance study, was initiated in the UK in '79.  It was published in August '85 by Taylor in Lancet and actually they showed that the process which was used and the process is called the Lumley-Jones (?) process was capable of eliminating about five logs of infectivity.
	The process includes an aqueous phase protein extraction, a selective isoelectric precipitation of the growth hormone at pH 4.9 subsequent solubilization, concentration, chromatography.  On various types of columns we use sephacryl in order to isolate finally the monomeric growth hormone.
	In '85 after the occurrence of the first CJD cases it was recommended to introduce two additional steps, mainly a millipo filtration with a cap of 100,000 daltons or a nanofiltration with a filter with a cutoff of 20 nanometers or six more of urea treatment.  These two were introduced in 1985 only.
	In view of the introduction of these two steps we first initiated a clearance validation of those two steps selectively.  We analyzed two filters, a 100,000 cutoff filter and a 25-nanometer filtration step, each of them followed with urea treatment subsequently.
	The control of the strain which was used for the challenge had a 9-log infectivity.  After the filtration there was about a 2-log reduction in infectivity with both filters.  It seemed, however, that the urea was more effective in further reducing the infectivity following the molecular weight filtration rather than the ÄÄÄÄnonomole filtration by reducing by more than five logs of infectivity or about 4.4 logs the infectivity.
	In spite of the fact that it was clear that growth hormone from pituitary glands is going to be discontinued and the recombinant growth hormone was available at that stage we decided to continue our investigation in order to know even in retrospect what was the risk, actual, with the growth hormone which we produced so we proceeded to do more extensive validation studies looking at the whole process.
	In that study we first of all checked whether there is any kind of background contamination in the equipment which was used.  So the AGH purification procedure without a challenge was done up to the Sephacryl chromatography step.  The monomeric growth hormone was concentrated and the whole product in its entirety was inoculated into 66 weaning hamsters and none developed scrapie, indicating that they did not have any kind of a background contamination.
	Then the process was done in three separate steps.  One, starting material was from the same batch for all the three events.  This is the original purification methodology and that is the material which was prepared up to '85 and there was a reduction of about four logs of infectivity which is quite similar to the one that was initially described by the Taylor in the Lancet '85 paper.
	Now, this material when subsequently subjected to an additional filtration and the six mole of urea there was a further reduction and a total elimination of nine logs of infectivity so quite effective.
	Subsequently all the equipment which was used during this clearance study was decontaminated by either steam autoclaving or by one normal NaOH.  Then the same equipment was used again for the production of a scaled-down process of growth hormone and aliquots of the monomeric after the Sephacryl stage of growth hormone were inoculated into 12 hamsters and the residual material was again ultrafiltered and urea treated and the entire product again was inoculated into 135 hamsters. None showed clinical signs of disease, no histological lesions of the brain, indicating that the decontamination process apparently was effective.
	The next issue is cross- contamination, obviously, so what we did is we divided the whole process into four risk categories.  Each step was performed in a separate isolated area from the other.
	The flow of the process goes from gland handling, first extraction, crude extract, semi-purified protein to purified protein whereas the traffic of people and of equipment can go only in the reverse direction so in order to avoid any kind of potential cross-contamination from one risk category to the other one.
	The effect of batch sizes:  Based on the assumption that there might be one out of 10,000 CJD-bearing donors in donors of the pituitary glands, if the batch size would have been 5,000 it would mean that about one out of two batches could be contaminated and that would translate into 25,000 vials which could potentially be contaminated.
	We have opted actually from the very beginning of using small batches, between 5- to 600 glands per batch, which means that about one-tenth of that would be potentially contaminated and, as you heard earlier from Paul Brown, some people actually use even much higher batch sizes of 10,000 glands or even more.
	Based on all these issues, based on the batch size, based on the clearance levels, and based on the incidence of potential inclusion of contaminated glands the calculated worst case and also best case, which is not on this slide, of contamination of growth hormone produced with and without the added ultra-filtration and urea treatment steps and the bottom line is that the worst case, the old process, it is one in 50,000 vials which would have been contaminated and which means that about one in 64 patients or children could have been exposed to such a contaminated vial.
	With the new purification process with the added filtration and urea treatment the risk obviously is significantly lower in view of the additional five-log clearance. The best case would have been about 1,000 times lower.
	The problem is that we cannot know whether in effect we did have ever any case or we will have ever any case.  Up until now we have no reported case of CJD with the product which we have distributed but we know that the incubation periods can be extremely long.  Here we have up to 37 years, 38 years reported recently, and the incubation with the gonadotropins in Australia, the four cases between 13 and 16 years, a little bit shorter, but with the growth hormone the incubation periods in some cases are extremely long.
	So therefore we cannot exclude today that we did not have ultimately probably some contamination, but if it was it was very low and our worst case calculation is definitely wrong because if it would be true then we would have had already a case so we are somewhere between the worst and the best calculation in reality.
	What it shows is that growth hormone, which is a single-chain protein and non-glycosylated, can be purified with a large safety margin provided that the procedures do include steps which can eliminate and inactivate CJD or any other TSE agent and the process is carried out under strict conditions and includes effective decontamination procedures.
	As Paul Brown alluded earlier, in France they had actually exactly a similar process, almost 90 cases of CJD up to the years of '87 where only this kind of procedure was used, but my suspicion told me that there were very severe problems of cross-contamination and I even had a hunch at which step.
	Now, the question is can total elimination of potential infectivity be guaranteed?  Well, the total elimination is already a loaded term.  What does it mean? We have no CJD case reported but in view of the long incubation periods we are still watching out whether there is going to be something.  What is sure, that if there was any residual infectivity in some batches it must have been extremely low.
	Total absence of risk would need assurance of the safety of the whole material which is particularly important in the case of older age group donors and in view of the fact that this is produced by pooling of the pituitary glands but unfortunately the currently available test metals do not have the sensitivity which would make this feasible.
	Thank you very much.
	DR. BOLTON:  Thank you, Dr. Eshkol. Are there questions or comments from the committee?  Steve?
	DR. DeARMOND:  When I was a medical student we took endocrinology.  I remember one of the endocrinologists said to us there was a great benefit from using hormones extracted from the pituitary because they were never purely the hormone; there were other factors associated with it which were beneficial.  But this was a long time ago before synthetics were available so I don't know how they could have tested that hypothesis.
	Is there any place today in medical practice for hormones or products derived from human pituitaries or do we rely entirely on synthetics?
	DR. ESHKOL:  Today we rely totally on the recombinant growth hormone but actually even before that already with the modified purification methodology which was employed using the chromatographic methodologies actually all the other pituitary hormones they're already excluded, which remained the TSH, LH, and FSH.
	What people were thinking was that a little bit of FSH and a little bit of LH might be useful for these children but actually in practice they have not been exposed to it already from the '70s even with the extractive growth hormone, which did not contain these hormones.  So what did not change was the transition to the recombinant anything in that sense.
	DR. BOLTON:  Dr. Brown?
	DR. BROWN:  Another data point on that, an experimental study was done once in our lab to see if we could transmit disease from archived aliquots of various batches in growth hormone.  The results fit very nicely into the concept that there was only a scattered transmissible dose here and there in hormone batches that were given to humans.
	The data were that out of 70-odd different lots of growth hormone, which represented about half the lots of growth hormone distributed in this country, only one lot transmitted disease and in that lot only one of three monkeys that were inoculated.  So that would give you a little idea of how little infectivity there was.
	DR. ESHKOL:  That is obviously true for what has been done in the USA and what raises the big question is the experience in France.  Why did they have so many cases? So is it really, as you have pointed out, because they had a higher number of pituitary glands which came from CJD patients?  I think it is a combination. That is definitely one of the contributing factors but I think that also the procedures, particularly cross-contamination and decontamination, which were probably not appropriately respected, could have played a bigger role in that.  But it shows that pituitary derived growth hormone does have the potential of transmitting in large numbers of patients the disease.
	DR. BAILAR:  I have been a little puzzled here as a newcomer to this field by the concern about older donors when we seem to know so little about when potential donors might acquire the disease and what their titers are during all the years it is lurking around unrecognized.
	Could you comment on that?  Does anybody know about these things?
	DR. ESHKOL:  I am not an expert on that and I think that Paul Brown could probably answer that much better if he is ready to do that.
	DR. BROWN:  Yes, there is no information whatsoever apart from a single observation in a patient with variant CJD by which I mean there simply is no information to know whether during the incubation period of disease in human beings there is infectivity and if so where.  That's in humans.
	The one exception that I know about is the demonstration of pathological PRP.  I think it was in the appendix of a patient who was operated on about 13 months before the first symptoms appeared.  So that is probably the single data point that I know of in human beings.
	In experimental models there is no question that infectivity typically occurs in a rising curve throughout at least the mid to late incubation period of the disease.  So the gist or the thrust of your question is is there a risk of transmitting disease from pre-clinical CJD patients.  The answer is almost certainly yes, there is but we can't put a number on it.
	DR. DeARMOND:  Our experience, of course, with the mouse and hamster models is that clinical signs begin in the last 10 or 20 percent of the incubation period.  So over a four-month period, say, in a mouse it is the very last stage.  But we can follow the protein and infectivity going up to that stage.
	In fact the brain is very filled with abnormal protein and shows lots of vacuolation when they finally get sick.  But as you go back further you see bits of that already occurring.  So, extrapolating to a human, if there is a similar event, say the disease begins spontaneously in one location in the brain and from the start of symptoms to death it is about four to six months, that could mean that it has been percolating in the brain for two to three years, maybe two years, before it manifests itself with clinical signs if we can extrapolate from hamster and mouse to human.
	DR. BOLTON:  Ermias?
	DR. BELAY:  I just wanted to make one point.  As you know, the human growth hormone associated with CJD, the outbreak of human growth hormone associated with CJD is an ongoing outbreak.  It is not over yet. We still continue to have cases pop up almost every year.
	So I think it's too early to say that the patients who received a purified form of human growth hormone would not develop CJD in the future because these patients received a human growth hormone after 1977 and close to 1985 and it's possible that the purification would actually reduce infectivity and possibly increase the incubation period.
	So we need to observe this outbreak for some period of time in the future before we say that the purification would probably have prevented the CJD in the human growth hormone recipients.
	DR. ESHKOL:  I full agree with you and this is why I had actually on my last slide that we can not exclude that we are not going still to see some cases which might have very long incubation periods because of very low residual infectivity in the product.  This is not excluded.
	DR. BOLTON:  Additional comments and questions?
	Okay, what I would like to do now is to shorten our break slightly to ten minutes.  So I have 10:36.  Let's meet back here at 10:46.
	(Recess)
	DR. BOLTON:  Our next presentation is by Dr. Tom Lynch from Clearant.  He will be telling us about limiting batch size, the effects of batch size on risk of contamination with infectious agents, and I believe this is work that Dr. Lynch actually did while he was at CBER.  Tom.
	DR. LYNCH:  Good morning.  Yes, the slides are coming up.  I can certainly talk to the donor standards but I think Ms. Wilson would be upset if I did.  If I could have the right slides I can begin.
	The remarks I am going to make this morning are in fact based on a study we did some years ago at CBER in the context of human plasma-derived proteins, including a product referred to as pooled plasma solvent/detergent treated.
	Our experience with hepatitis and HIV has taught us that the dissemination of an infectious agent into a population contribute can be far more efficient to the use of administration of pooled therapeutic products than through the use of products derived from single donors.  We performed an analysis in the context of these two product categories, pooled plasma-derived proteins and pooled plasma, in order to get a quantitative measure of this risk.
	We were specifically asking two questions.  One was whether limiting the size of manufacturing pools from which plasma derivatives were made would enhance the safety of these products.  This is question isn't really relevant to today's proceedings but the second question may be, which was should any single donor product be converted to a pooled product for any reason.
	Our approach at the time was a far less sophisticated mathematical model than the one presented by Dr. Taffs this morning. We simply related the prevalence of an infectious agent in a donor population the size of the pool to the risk of the recipients of that product made from those pools.
	Prevalence would include for an agent for which no screening or testing is performed the natural prevalence in that population whereas for an agent such as HIV for which testing is performed prevalence would represent the residual risk of a false negative test.  The size of the pool was measured in donors rather than donations simply because the number of donors would reflect more accurately this underlying prevalence risk.
	Finally, the risk to the recipients was something we struggled in defining.  We finally settled on a very conservative measure.  We defined risk as the probability of exposing a recipient of a given product to a product made from a pool that included a donation, a unit, and material from an infected donor.  Now, this definition of risk was actually fairly flexible, another virtue it had.  It also was a measure of the proportion of the total product made derived from these "contaminated pools" and the risk of contaminating a given manufacturing pool was also equivalent to this definition of risk.
	What we did not attempt to assess was the risk that such a product would pose to a recipient of that product of actual infection.  The relationship between exposure and infection was recognized as a very complex relationship driven by a number of factors shown on this slide.  But certainly the risk of infection is no greater than the risk of exposure.  It could be somewhat less depending on these factors so the use of exposure as a measure of risk was taken to be a conservative or worst case estimate of risk.
	This is an example of data from that analysis.  It shows on a three- dimensional plot the risk at any given combination of parameters.  Pool size is shown here.  You will notice it goes up to very large numbers relevant to plasma derivatives and it encompasses a range of prevalences within the donor population from one in a million up to one in 600,000.  What this slide illustrates is that the risk of contamination or exposing the recipient of the product to the infectious agent increases as pool size goes up and as the prevalence of the agent in the population goes up.
	A number of things needs to be changed in order to make an analysis like this relevant to your deliberations.  Pool size, for example, is one.  A pool size of 600,000 is simply not relevant, I think, to the tissue industry.
	Another issue that is of little or no importance is the risk that a chronic user of a therapeutic product faces a cumulative task.  Transplants of allografts, for example, tend to be single episodes in a patient's life so I have made no attempt to calculate the cumulative risk associated with repeated exposure.
	There were a couple of other assumptions that we made that are relevant to the model I'll show you today.  First of all, we assumed that there was no dilution effect.  This idea that somehow by taking a contaminated unit and diluting it with a far greater number of uncontaminated units you could reduce the titers of infectivity below the level of infectivity, we assumed that that would not be effective and we also discounted any effect of antibodies that may be present in the pool that might potentially neutralize an agent.
	The reason for that is that obviously not all infectious agents are neutralized by antibodies effectively and, secondly, for an emerging agent, a new agent, the antibody titers in the donor population may not be sufficient to protect the recipients of a product.
	So this is a scaled down tissue-based version of the model I just showed you.  Again, the pool size in donors is shown along this horizontal axis.  The risk is shown on the vertical axis.  Note that it goes up to one percent here because the pool size is smaller and therefore the amplification of the primary risk associated with the prevalence of an infectious agent is also less.
	But the same relationship exists on this slide.  The risk increases as the prevalence increases and as the pool size increases as well.  So clearly increasing pool size is associated with an increased risk, although here that risk according to these parameters would be below one percent.
	What I did want to elaborate on a little bit is this large green area.  I don't want to give the impression that this is a flat or neutral area or that there is no influence of the parameters there so I've replotted some of this data in the next slide.
	This is simply converting the vertical axis to a log and you can show that even at very low levels of risk the influence of pool size and prevalence are the same.
	This slide illustrates that the unitary risk, the pool size of one, this is the unitary risk reflected by the prevalence of the infectious agent and you amplify that risk almost as intuition would predict in proportion to the increased size of the manufacturing pool.
	Now, the actual numbers on which these graphs are based I think were provided to the committee in tabular form.  There are lots of numbers there, so it is a little bit more difficult to grasp than the graphical presentation but they are available for your use.
	Also, the model that I have just presented is a simple binomial model.  The rationale for using it was elaborated in a publication of the original analysis and a copy of that publication has also been provided to you.  In the interest of historical accuracy, your past chairman, Dr. Brown, performed a similar analysis, specifically in the context of TSE agents.
	This is just a replot of the same data, next slide in linear form.  So the conclusions from analyses such as these are as follow:  For the conversion of a single donor product to a pool product the theoretical risk of an exposure to a recipient of this product increases nearly in proportion to the size of the pool from which those products are made.  This is especially true for small or moderately sized pools and especially true for agents with relatively low prevalence.
	Now, we recognized originally at the time that this risk is one factor and it may be mitigated or offset or outweighed by other factors.  This is where judgment comes in and where this committee is charged to operate.  For example, the incorporation of a broadly effective pathogen and activation step might justify pooling where the inactivation requires the pooling process and there are numerous cases of therapeutic products that cannot be made from single donor source materials.  They must start from pooled material in order for the manufacturing process to be carried out.
	The classic example of that are the plasma derivatives themselves.  Here the risk-benefit, however, can be evaluated quantitatively only when you know what the risk associated with the given infectious agent is.
	I think I'll conclude there and take any questions that you may have.
	DR. BOLTON:  Thank you, Dr. Lynch. Questions?  Steve?
	DR. DeARMOND:  It seems it gets down to, as I see it and I'm not in business or in industry and we don't worry about money in academics because we don't have any, but the cost of doing all of this has to be weighed, I guess, to some degree.
	Ultimately it seems to me the best test is whether the material is infective and to date we don't have an idea test but bioassay is by far the most sensitive but that would be expensive to do bioassays. You have to take the animals and it takes a while for them to get sick.  Especially if you have low titer it could be two years or a year and a half and your time lines are probably not in that length.  You make a batch of plasma and you would expect to have it on the market, I guess, after processing, within the order of months.  What sort of time lines do you deal with?
	DR. LYNCH:  You are right about the time to market for most allograft tissues. My familiarity is primarily with musculoskeletal tissues and that certainly fits with the sequence that you just mentioned.  But the products can be on the market for quite some time.  Current dating puts a fairly substantial shelf life on some of these products.
	With regard to testing you are absolutely right.  The impact of testing is going to be on what I refer to as unitary risk of prevalence.  You can start with a relatively high prevalence of an infectious agent in a donor population and drive the effective prevalence down substantially by introducing effective testing.
	To the best of my knowledge there is no perfect test so that risk has never been driven to zero but it can be driven to very small numbers.  So in using these three-dimensional plots what you would be doing by introducing a test is driving that Y-axis back toward the lower numbers and you can assess the impact of risk from there.
	I should point out that one other distinguishing feature relevant to your question and relevant to the application of these numbers that distinguishes the tissue industry from the plasma derivatives industry is that a fairly large number of products can be derived from a single donor so that unitary risk when you view it from the number of patients that are exposed to material derived from an individual donor can be amplified by the number of products made.  But to the extent that this practice is uniform across the tissue industry the proportions of risk would not be altered but the absolute values might be increased.
	DR. DeARMOND:  One of the questions I was asking Paul Brown was how low does the amount of, say, prion protein have to be before it's no longer a problem?  Obviously, we don't know the answer but there is with each product perhaps a different threshold for creating disease in a recipient.
	DR. LYNCH:  And I made no attempt to distinguish between the risk levels associated with the material such as dura mater where tests show it can have a substantial risk associated with it to a product like a bone dowel where absent contamination with blood or marrow we think the risk would be substantially lower than that.  I haven't tried to distinguish between those types of products because one would really need to know what proportion of each would derive from an individual donor.
	Perhaps it's best to think of these numbers in terms of the discrete products and again we are talking about exposure so the levels of infectivity don't enter in but ultimately that's what you are trying to get to, what is the true public health risk in terms of transmitting these diseases by these various practices.
	DR. DeARMOND:  Could I ask one more?
	DR. BOLTON:  Sure.
	DR. DeARMOND:  This is has to do more with the people who are donors.  There are in some cases with plasma derivatives from what I read in the material that is sent to us some are repeated donors of blood for plasma derivatives.  What proportion are repeat donors?
	DR. LYNCH:  Well, as I understand it a cadaveric donation is a once in a lifetime event.
	DR. DeARMOND:  Yes, that you can only do once.  My family doesn't get any money for it.
	DR. LYNCH:  I assume that the idea of a repeat donor, which is quite prevalent in the source plasma sector and somewhat less common in the recovered plasma sector, is of little significance in the tissue and organ donor population but I stand ready to be corrected on that.
	DR. BOLTON:  Additional questions? Yes.  Please introduce yourself.
	DR. SOLOMON:  Ruth Solomon, FDA. That's is correct but you have to remember we will be overseeing reproductive cells in tissues and the semen donor can donate almost as often as the source plasma donor.
	DR. LYNCH:  Excellent point, yes.
	DR. BOLTON:  Other questions?  I would like to bring up one issue and get comments especially from our epidemiologists and that is the difference between the prevalence of disease which we talk about a lot with sporadic CJD, which is somewhere between one-half and one per million population per year, and the prevalence of infection, which is going to depend, as we discussed a little bit earlier, on the incubation time and if that is a 20-year incubation time in sporadic CJD then the prevalence of infection may be 20 times higher.  Instead of one in 1,000,000 it may be one in 50,000.  So comments?
	DR. BELAY:  I think another way of looking at it would be what's a lifetime risk of acquiring CJD for a person.  That would be obviously higher than one per million.  One per million would be for each year.  Accumulated over time it could be right about one in 10,000 is the best estimate that I have of potential lifetime risk of CJD.
	DR. BOLTON:  John?
	DR. BAILAR:  I agree.  There is a potentially big difference between the lifetime incidence of infection and the lifetime incidence of disease.  It need not be all related to the length of the incubation period.
	Some people may carry the infection asymptomatically forever, never come down with the disease if they live to 150.  I have been puzzled by the numbers I have read in this package and some I have heard this morning by what does seem to be a discrepancy between the size of the lifetime risk and the size of the risk in the donated products.  The latter seems to be generally bigger so there may be a lot of this stuff lurking around out there that we just don't know about.
	DR. BOLTON:  Other comments? Steve, did you want to say something?
	DR. DeARMOND:  I did but I didn't know how to do it.  First of all, I presume you were talking about sporadic CJD and the idea that it may be caused by an infection. There was an implication that carrying infection is not the way I think of sporadic CJD, that it is like Alzheimer's, a change in the protein configuration that leads to the disease that occurs spontaneously for some reason.
	It seems like once that happens, although we have no evidence that there can be CJD that percolates along without producing disease although a person can have an auto accident and die before it manifests itself, but it seems once it takes hold it goes to completion.  You could argue that we haven't proven that so I am having some trouble with the one per million but that is really based on the number of people who die.  Is there really something much more prevalent in the population?  We have no evidence that there is something more prevalent that could transmit the disease to somebody else.
	DR. BOLTON:  Yes, let me frame my question or hypothesis a little better. First of all, I think it was stated earlier that we have no way of knowing what the incubation time, and I use that term in the sense of from the point of either infection of spontaneous conversion of the initial PRP molecule from PRP-C to PRP-SC, what that time is to the onset of clinical disease in sporadic CJD.
	It could be six months, which would be a good extrapolation from infection by iatrogenic transmission in the cases of direct intracranial inoculation, or it could be 20 years.  So it's probably somewhere between that, six months and maybe 20 or 40 or 50 years, but it's in there somewhere.
	Now, during that time once that event has begun those individuals are going to have some abnormal PRP in their brains and our experience with transmission of sporadic CJD would indicate that they are infectious and therefore they are infected in that sense and whether they initially got the disease through a rare infection or whether it was spontaneous conversion of PRP is really immaterial at that point.
	So then the question of the prevalence of infected individuals becomes important because that number is going to be much higher.  If the incubation time of the average sporadic CJD case is 20 years the prevalence of infected individuals is at least 20 times higher.
	As John said, if there are people who have the ongoing process in place but will never come down with clinical disease then the number could be much, much higher. Of course, we just don't have a way at this time to assess those numbers.
	So I just wanted to bring that out and get some discussion on that because clearly when we start talking about issues of donor screening, tissue screening, and what have you, the prevalence of infection or disease, whatever it is, is very important as we begin to deliberate on how to best regulate that or how best to increase the safety factor.
	DR. BELAY:  I think it's going to be very, very difficult to come up with a specific number for the kind of data that you are looking for.  That is why I say it is probably better to look at a lifetime risk of acquiring CJD wherever it is coming from, sporadic, iatrogenic, or otherwise, but I would think that the number that you are looking for would probably be anywhere between one per 10,000 to one per 1,000,000 but it will be very difficult to come up with a specific number.
	DR. BOLTON:  And as we look at different age demographics how does that change?  I mean, my inclination would be to think that the older you get the closer to the one in 10,000 that number becomes but that may not be correct.
	DR. BELAY:  I agree with you.  The older you are your chance of potentially incubating CJD would definitely be higher or at least sporadic CJD.
	DR. DeARMOND:  In terms of just looking at the deaths of CJD certainly over the age of 50 it's almost up to four to five per million compared to the overall population because they are all concentrated essentially over the age of 50.  Ninety, ninety-five percent occur over the age of 50 and drop off after the age of 72.
	DR. BOLTON:  Yes, that was always my recollection, that there is a drop with age over 70 or so.  How dramatic is that drop?  I don't know if this will ever come into play with respect to tissue donations because at that point the tissues that could be donated might not be optimal.
	But is that something that we should be considering as well?  It doesn't seem like it because, as you say, they prefer younger tissue.
	DR. BELAY:  But I think you brought up a very good point because if you look at the data from CJD it tends to rise in the 5th decade and then the 6th decade and all of a sudden tampers down when you reach the 8th decade.
	So the incidence or the death rate from CJD in persons in their 80s is almost similar to persons in their 50s so there is some difference as people age.  Particularly after 78 or 79 the incidence of CJD drops down.
	DR. BOLTON:  So we will have to keep that in the back of our minds here as we discuss this further.
	DR. DeARMOND:  Do Pierluigi and my lab and Nick Hogan's lab have to start a study to try to find PRP scrapie of infectivity in every autopsy brain that we do?
	DR. BOLTON:  Sure, I think that sounds great.  You can do that for free, right?
	We will move on now.  We are going to pick up part of the section that Paul Brown has already presented.  This is Topic #1-3, "Single Donor Aseptic Recovery and Processing of Human Tissue," and our next presenter will be Diane Wilson, R.N., from Tissue Services and she will be talking again on the issue of cross-contamination of bone and soft tissue with higher risk tissues during the recover of the tissue.
	Diane.
	MS. WILSON:  It's a pleasure to be here.  What my main topic today is talking about, what the AATB standards are and how they apply to donor processing and donor procurement in a single donor procurement.
	Some of my slides are graphic and I apologize for that but I didn't really know what level of understanding that the advisory committee had about donor procurement and processing so I wanted you to get a general overview of the entire process.
	I wanted to start real quickly on donor screening just to give you an idea of how we go through the process.  The first thing that happens is a call from the hospital, the evaluation of the hospital records, then a consent from the next of kin and a total medical-social history.  This does include all high-risk activities and neurological function of the potential donor, past history, past surgeries, hospitalizations, anything that we can paint into a picture to make a total puzzle to make sure that our donor tissue will be safe.
	Serological and infectious disease screening, the first five tests listed there are mandatory by AATB:  Hepatitis-B core, HIV by DNA, DNA by PCR, and HIV Antigen are optional tests.  Most AATB-accredited banks are doing those but they are optional.
	The physical exam is a mandatory screening test and they are done on all categoric donors.  Then you will find tissue banks with a little bit of variety.  We use a physician letter to the family physician to find out anything else about the donor that we may not have picked up in the hospital records.  And because we are part of a blood center we will look at the blood donor registry for past deferrals but those are also optional.  You will find tissue banks will vary a little bit on the amount that they do above and beyond the standards. Then we have a medical director review.
	Informed consent is extremely important to us and AATB puts a lot of emphasis on this.  We have to make sure that no coercion has been applied, that all of the information that we give the next of kin is accurate and not misleading, that everybody is done in understandable terms, that infectious disease consent has been obtained in order to test them for hepatitis and HIV and the other testing, and that we have access to those medical records because we will be getting a copy of an autopsy if it has been performed and we need a total copy of the medical records from the hospital or at least be able to summarize those records.
	The new things that have come up in the last year, AATB has added to the standard saying that we should, not a "shall" at this time, discuss with the families whether they agree to profit versus non-profit use of the tissue, cosmetic use, use of tissue outside the United States, and in order to use tissue for research we need to have consent for that.  And then always give the families the right to ask questions because this is the most devastating time of their life and at the time you are going to be talking about consent you want to make sure that they have a full understanding of what you are talking about, whether tissue to them means eye tissue, bone tissue, or a piece of bone marrow.
	Donor identification, the first thing that you'll do when you get to the hospital is they must identify the donor and it will be assigned a unique ID number for the tracing of all the tissues and this number will then follow that donor through the entire process.
	Prior to retrieval at least one of the staff members shall identify the potential donor's ID against the consent for tissue donation knowing that they are retrieving the proper tissues and they have the accurate donor.  Donor identity will also be documented in each and every chart as to how you identified, whether it was a toe tag, a wristband, or whatever source of identification was used.
	We do have time constraints written into AATB.  Up in the top corner of the slides I have written the standard for AATB in case anyone wants to see more detail on these standards.  If a donor has been refrigerated or placed in a refrigerated cooler prior to 12 hours we have 24 hours to begin the retrieval of that tissue.  If they have not been refrigerated within 12 hours then we must begin tissue procurement within 15.
	The environment must be documented. We must do all donors in an aseptic fashion. We are going to use the standard surgical prep, the same that would be used on any other surgical procedure in a hospital, sterile packs, sterile instrumentation, and the technique must be trained upon, documented, and according to standards.
	The general site where the tissue was removed has to be documented.  This means was it removed in an operating room or a morgue or a procedure room or a facility room that the tissue bank may own.  All working surfaces before and after retrieval will be scrubbed using bactericidal and antimicrobial agents for cross- contamination.
	The preparation of the donor, we'll start with cleansing, preparing and draping the skin as well as soon as the technician will do a gowning and gloving as any surgeon or scrub tech would do on any other surgical procedure and this shall be accomplished with the same diligence as used routinely for all operative procedures and this is the standard.  Whether or not you are going to terminally sterilize the tissue this is the standard.  The aseptic technique that will be followed and the one we train on is the American Association of Operating Room Nurses or the AORN standards.
	The surgical staff shall perform the scrub of their hands and their forearms prior to retrieval of any tissues.  They will wear head coverings, eye shields, mask, and scrubs at the time that they do these procedures.  This is whether they are in a funeral home or in a morgue or any other place and sterile gowns and gloves shall be donned after they scrub and all tissue recovered, again, will be used under aseptic technique.
	Here are just a few slides of scrubbing the forearms and gowning in a proper procedure.  The gloving technique is usually closed glove because the first person to glove is alone.  We don't have the luxury of a scrub person to help us to begin a proper surgical technique.
	In prepping the donor they will shave first any areas where an incision site will be.  We also will stay away from any abraded areas or open wounds.  Here we begin our surgical dissection, usually start at the hip, go down to the ankle, go through the fascia lata, which is one of the grafts that now are used in place of dura mater since we don't have any dura.
	Here they are removing the femur. The same surgical technique is used for every part of this process.  They are disarticulating the knee at the knee joint. This is the femur and the tibia with the deflected patella.
	Going down to the tibia to the ankle, just making a clean dissection.  The rest of the muscle and all will be left with the donor for reconstruction purposes.  And removing of the tibia down at the ankle and here is the Achilles tendon coming around the backside of the calcaneus there.
	The last graft that is usually removed from the donor will be the ileum. They will go back up to the hip and remove the ileum.  In case of contamination this would be your number one place to get contamination would be by nicking the bowel at this time.
	Pre-processing cultures are mandated by AATB.  There has been a lot of information out recently about some recent infections and preprocessing cultures must be done on all tissues retrieved.  Every individually recovered and packaged tissue shall have a pre-processing culture and this is prior to the treatment of any antibiotics or sterilizing of that tissue.
	Even if the tissue is going to be sterilized you must have a culture taken to give you an idea of what bio-burden the donor may have had at the time.  It can be used in the evaluation by the medical directors to see if possibly there was some systemic infection.  Those results will be maintained in the medical chart and the M.D. will review those prior to release.
	Immediately after removing the tissue from the donor they will be wrapped individually in aseptic containers.  These will be labeled with the donor ID and the type of tissue.  This is just to keep the tracking of the tissue number throughout the process.
	Here she is doing a swab culture on a tibia.  They will swab the entire graft top to bottom or any cut surfaces prior to putting it into the package.  After each tissue is removed it is individually wrapped prior to the removal of the next tissue.  So if you have a contamination problem your problem will probably be picked up as you go through the case so each graft is wrapped individually as it comes out.
	Here she is wrapping it in one single barrier and then she is going to wrap it this way.  After this point then it will be labeled with the identification.
	The transportation of the tissue from the procurement site to the freezer, it will be packaged in a manner to permit environmental conditions to be maintained during that transport time.  The containers do not require monitoring during the transport time, according to AATB, as long as the containers have been validated.  If they have not been validated containers then they would need to put a temp tail in there or something to show that they had been kept at refrigerated or frozen temperatures.  The maximum time that AATB allows on wet ice or refrigerated temperatures prior to freezing or processing is 72 hours.
	The transport receptacles must contain the following labels and this is even if you are coming from a coroner's office back to your own facility.  Any time it is carried in a car or shipped on an airplane it must say "Human Tissue Enclosed," the name and address of a retrieval agency and processing center if different than the retrieval agency, and it must state, "Quarantine, Not Suitable for Transplant in its Current Form."
	Reconstruction of the donor is mandated on all tissue donors no matter whether they will be cremated or not.  We do follow whatever funeral home guidelines they have.  Usually we use dowels, rods, PVC piping, and our closure technique will either be usually a baseball stitch or a running stitch.  Here is some muscle being sewn back up on the thigh around the internal brace and then a closure to the donor's skin after that time.
	Most tissue banks will tell you that this is their signature as to their care of the donor; therefore, much care is taken during this process in cleaning the donor up.
	Single donor tissue processing, processing methods, all tissue shall be processed by methods known to be validated to prevent contamination and cross- contamination.  Some tissue banks will use AORN draping such as you were use in the operating room.  If they are using any process other than draping of their back tables they must have validated their surfaces and processes to show that there is no cross-contamination on their work surfaces and before and after processing they will be scrubbed again and wiped down with bactericidal and antimicrobial agents.
	And this is just a typical clean room on a draped surface.  You will see other banks that use stainless steel counters and process directly on those. Those should have been validated prior to use if that is what they are using.
	A typical central supply area where instruments are wrapped after each donor whether being processed or procured and then sterilized with the use of steam or gas sterilizers.  Reagents and supplies according to AATB standards used in processing and preservation shall be of appropriate grade for the intended use and sterile if indicated.  On the receipt of all supplies we do record all reagents and supplies, including the type of agent, manufacturer, lot number, date of receipt, expiration date, and inspection of that reagent.  This is also in case there is a recall on any type of agent that we may have used on a donor; therefore, we will know what tissue to quarantine.
	Tissue ID numbers, in processing each tissue shall be assigned that unique ID number which still will follow it through the process.  All tissue units shall be assigned the same ID number as if they are identical in a lot.  So once the donor has been given a number that becomes your lot.
	In pooling AATB does address this issue and it is not allowed.  Tissue from multiple donors shall not be pooled during retrieval processing, preservation, or storage at any time.  Cross-contamination, written procedures shall be prepared, validated, and followed for prevention of infectious disease contamination or cross-contamination by tissue during the processing.  So it is very important to us that when a donor tissue is brought into a room no other donor tissue is there and no two tissues ever touch at any time during that process.
	Here is a processing lab.  What you see on that right hand is a metal glove that is actually on top of his other gloves. This is just for safety.  Frequently you will have a lot of sharps in your hands so this is a safety glove.
	This is a typical cutting with the use of a sterile band saw.  All of this is autoclavable.  The entire band saw is taken. That is an ileum he has in his hand and that is the acetabulum of the ileum he is cutting there, just as a reference.
	Small saws may be used.  These, of course, too, have to be able to be autoclaved and sterile at all times.  These are little cancellous blocks that you may see used in a cervical fusion or upper neck.
	Some people will centrifuge tissue. It depends on what their validated process is or their process is for tissue banking but this is a centrifuge cup.  It will be centrifuged to bring out all of the lipids and marrow.  You will see a lot more of that in the other speakers that are talking about processing.
	Each graft is individually packaged and sealed in the room that it was processed in or in the packaging room to make sure that, again, there is no cross-contamination.
	Ultrasonic baths are used on some donors.  This is again to bring out fat and lipids, you will see.  It just depends on what type of methods the tissue banks are using.
	These are patella ligaments.  I just put a couple of pictures in here of different grafts that you might hear about over the years.  When you talk about young donors you are talking about patella ligaments, tri-cortical blocks, and some of the weight-bearing grafts that are extremely important in tissue banking.  You will hear tissue bankers say that young donors are of extreme importance because of the type of grafts you can get.
	The older donors also are because the young donors are going to be used for more of these structural grafts and the older donors for more of a ground type of material that may be used as a packing material on a hip surgery.  So you will see the need for both types of donors.
	Femoral heads and short distal femurs or femoral condyles, cortical struts used in hip surgeries, back surgeries, tri cortical blocks, it's an ileum with a tri cortical block.  You will hear about three sides of the cortical there with a trabecular bone in the middle.  These have to be from young donors.  It is very important.  These are the grafts, the teller ligaments, femoral grafts, and tri cortical blocks, that you will always see massive back orders for, surgeons waiting for these grafts in very high demand.
	Fibular segments may be used in a back surgery.  Crushed cancellous can come from a younger donor or an older donor.  It is used a great deal in hip surgeries, knee surgeries, packing materials, defects in bone, but this is not a weight-bearing structure; therefore, it can come from some of those older donors.
	Final tissue culturing, once the tissue has been processed terminal sterilization by a radiation or ethylene oxide on those lots of tissue.  You must do 10 percent destructive sterility testing to show that you have no contamination after the process or an equally validated procedure such as 100 percent swab testing.
	If no terminal sterilization has been done and you are calling the tissue aseptically processed, you are not going to terminally sterilize, then you would do 100 percent swab culturing according to standards.
	Tissue release, the final release after all the grafts are finished, you put the whole puzzle together and quality control will review all of the following areas again prior to release.  That will be the procurement and processing records, the medical director approval.  AATB does require that a physician release the donor suitability, the serology review again, the culture review again, meaning the procurement cultures and the processing cultures, any water residuals from the freeze-dried tissue, irradiation results will all be put together prior to the release of that.
	Then once your tissue has been released it is an AATB standard that the record shall include the name and address of the tissue bank, the type, quantity, and unique ID number of each tissue graft, the recipient name and hospital number or Social Security number of the recipient, the transplantation site, the date and the time, the ordering physician, the dispensing physician, and the tissue preparation person if applicable.
	Most tissue banks will have some type of a tissue log that they will give to the hospitals to help them in filling out these papers.  It's not mandatory that they return all of these.  We strongly encourage it and most tissue banks will have a way to go back and look at those that have not been returned because in case of a recall or any problems we certainly need to know where the tissue has been transplanted.
	And that's it.
	DR. BOLTON:  Thank you, Diane.  I particularly appreciate that last slide. I'm going on vacation next week to go fly-fishing, so I'm ready.
	Questions?  Ermias.
	DR. BELAY:  You say you don't allow cross-contamination of the products.  I was curious about some of the instruments you showed us on the slides, particularly the saws, the different saws you use to cut different bone parts.  Are those instruments sterilized in between donors after processing between one donor from the next?
	MS. WILSON:  Yes, they are. According to AATB standards you must sterilize in between donors.  The saws, a lot of them that have any type of a rubber cord cannot be steam-sterilized but they can be gas-sterilized.
	You would then validate them in your gas sterilizer to show that you do not have any residual tissue left in them or growth on the grafts but even the large band saw can be steam-sterilized and the other things with the cords are gas-sterilized.
	DR. BELAY:  The other question I had was do you have any demand and supply problems with bone in the United States?
	MS. WILSON:  Yes, we do.  The demand far exceeds the supply, especially of weight-bearing grafts, the patella ligaments, the femoral grafts, Achilles tendon, tri cortical blocks.
	DR. BOLTON:  With respect to the band saw, aside from the sterilization you said that can be autoclaved.  Is it scrubbed?  Is the saw itself scrubbed in any way to remove tissue or particulates?
	MS. WILSON:  The saw is.  The saw will have a large blade that goes around the wheels.  It will not only be cleaned but blown out with an air hose.  Then it will also be soaked with a disinfectant and then it will be sterilized after that period of time.
	The blades on the saw usually are a one-time use only.  They can be re-autoclaved but because of just cuts in handling the blades to the personnel we usually just discard those.
	DR. BOLTON:  So the blade itself is used for one patient?
	MS. WILSON:  The blade is for one-time use but the saw itself that has to be autoclaved, too, has to be cleaned and you have to get out the other residual bone powder that it will create.
	MS. KNOWLES:  And I have a question.  When you blow it out with the air hose is that connected up to a vacuum?
	MS. WILSON:  Yes.  Most people will have that, yes.
	DR. BOLTON:  Yes, Dr. Wolfe?
	DR. WOLFE:  You mentioned that since you don't harvest dura mater that you have been using fascia lata.  Certainly autologous fascia lata is one of the alternatives to dura mater.  Is it your experience that there has been an increased amount of fascia lata request or demand as the use of dura mater has decreased?  Could you just comment on that?
	MS. WILSON:  From our experience there was initially when we stopped doing dura mater.
	DR. WOLFE:  When did you stop doing dura mater?
	MS. WILSON:  1996, our bank did and most banks stopped very shortly thereafter. We had an increase for fascia for probably about a year.  I believe most places now are using more synthetic or bovine.
	DR. WOLFE:  Stopped in '96 for what reasons or what was the context?
	MS. WILSON:  We had done dura probably since 1987 and when the new pre-market approval, 510(k)s were required on dura mater, we did a very small amount of dura mater and elected to not do dura.
	DR. PETTEWAY:  As far as the sterilization of the tissue, the ethylene oxide irradiation, what types of organisms have been validated for sterilization with these types of tissues, just to give us an idea of how effective they may be?
	MS. WILSON:  I probably can't answer for all banks.  I know that we use bacillus pumilus as an impregnated strip that goes in every lot of irradiation as our control.  We can have no growth coming back out of that lot.
	There are some papers that have been written by the Michigan Tissue Bank on the dosage that's required for chlostridium and other bacterial forms.
	DR. PETTEWAY:  It's mostly bacterial?
	MS. WILSON:  Exactly right, bacterial.
	DR. BOLTON:  Any other questions? Yes.
	DR. SOLOMON:  Thank you very much, Diane.  I don't know if you will know the answer or someone in the audience.  Could you comment on of all the tissue banks that are out there that we know of how many are accredited by AATB and what percent are not accredited but still follow the AATB standards?
	MS. WILSON:  Ellen Heck actually might be a little better at that as chairman of the accreditation committee.  I believe there are approximately 60 accredited banks. How many other banks there, Bob Rigney, do you have an answer?
	MR. RIGNEY:  Ruth could probably give us the number that had registered with FDA at this point under the new registration requirements.  We have 74 banks that are currently credited by the AATB involved in either retrieval, storage, processing and distribution, one or more of those functions.
	How many other banks are out there, it depends on how you define what a tissue bank is.  Our members who are accredited by us retrieve and process and distribute the majority of the tissue in the United States. Does that answer the question?
	DR. DOPPELT:  That brought up some more questions.
	DR. BOLTON:  Go ahead.
	DR. DOPPELT:  I was just going to say, as Bob Rigney said, probably 90, 95 percent of the tissue distributed in the United States comes from AATB-accredited banks.  If there are 73 or 74 accredited banks there is a large discrepancy between that number and the number that the FDA has as registered organizations that in one way or another deal with tissue.
	But if you ask the question how many banks are there functioning in a similar fashion to the accredited banks, that is, they are still distributing tissue, that are not accredited, just as a guess I would probably say there are maybe another ten or twelve that are functioning in that capacity.  The discrepancies are other organizations that are doing cells and so forth.
	DR. BOLTON:  Dr. McCulloch.
	DR. McCULLOUGH:  This is a question about the irradiation and sterilization. Are there any issues about whether all of the parts of a particular piece of tissue are adequately irradiated?  I know in irradiating blood, for instance, there are different doses of radiation that actually apply to different parts of the blood bag and you have to be sure that all the dose you want actually gets to all the blood.  It seems like this might be more of a problem for large pieces of bone or things like that.
	MS. WILSON:  Right, AATB standards state that the minimum dose is 1.5 megarads and that process should be validated as to the dosimetry of the boxes.  The irradiation company that you work with you should have worked with to make sure that your boxes are equally penetrated and that your bone is penetrated.
	DR. BOLTON:  Any other questions? Good, we'll move on.
	Our next speaker is Ellen Heck from the Transplant Services, University of Texas and she will talk about the Eye Bank Association of America.  I also understand that it is her birthday today so happy birthday, Ellen.
	MS. HECK:  Who gave you that information?
	DR. BOLTON:  We have our sources.
	MS. HECK:  Thank you.  There are many similarities in eye banking and tissue banking but also some differences but like with tissue banking we are concerned about screening, sterility, and asepsis to arrive at what we hope is a safe yet effective product.
	We really believe that the screening process begins with public and professional education because we are using a second person historian.  Unlike the blood industry where you can interview the donor themselves we are getting our information secondhand, if you will, so we do want to have an educated historian from whom we are going to be gathering the screening information as well as an expert resource in our professional and medical staff to know the sorts of questions and answers that we are looking for when we begin to do our screening process.
	The consent process can never get to that point because in our initial screening we will talk with the hospital about things such as the admitting diagnosis, the temperatures, the fluid volumes that were administered so we can do hemodilution calculations, the white blood cell counts, any high risk behaviors or infections that might be present in a potential donor before we even proceed to take a consent.
	Then, of course, the consent may be declined.  After that, however, if it is accepted we have some 45 in-depth questions that we will be asking the historian about the person's medical history and their social history.  Now, these comply with the EBAA guidelines that help us to reduce transmission of disease.
	These include things that are applicable to the CJD.  When we talk about death with neurological disease of unestablished etiology certainly we don't know to what extent we are going to get the correct answers based on the stage that an individual might be as we have heard repeatedly this morning but nevertheless we feel that these questions have helped us with both recipient safety and with technician safety as we ask these questions.
	The standard goes on to specifically reference dementia.  Unless it can be attributed to cardiovascular disease, brain tumor or head trauma donors with metabolic-induced dementia may be acceptable under certain consultations and the medical director may decide to use those.
	Our concern is that we are excluding a number of cases that we don't truly believe are infective such as Alzheimer's Disease but we believe in doing so, because we are doing this in the field without the ability to do long-term studies and without the ability to know when someone says well, we think that grandmother may have had Alzheimer's we don't know what that's based upon in terms of a diagnosis and what extent that was so we just go ahead and rule those patients out of the pool initially.
	These are the things we are asking about, loss of memory, inappropriate responses, confusion, gait changes, human- derived pituitary hormone, or dura mater. We can prescribe the questions that are going to be asked in the field but we also need to remember that there is a certain skill involved in eliciting this information so we do a lot of training trying to get our people to a point where they can ask these questions and know what subsequent questions to ask to get the information that they are truly seeking in order to screen this.
	Again, we don't know whether they are valid until you get to late in the progression of the disease with CJD but if we do it can prevent an inappropriate retrieval.  Once again, we are not confident in the diagnosis of Alzheimer's so we do rule those out in the field.
	In addition to screening we do a physical assessment of the donor which has been very helpful to us in diagnosing other diseases, particularly some of the high-risk behaviors, but we don't know that it has any value for us in the CJD situation.
	Following that, probably the most relied-upon screening tool that we have is serologic testing.  Now, that testing may occur before procurement but most frequently is going to occur after procurement.  Those are the tests that Diane mentioned earlier that are prescribed by the FDA or prescribed by the EBAA and that may remove donors. Unfortunately, at this moment we have no such readily applicable screening test for CJD.  I wish we did.  But those rule out a number of other donors.
	Finally, we get into the processing and we may have rejections occur here.  We have more medical history review.  We are looking at whether anyone else participated in collecting donor tissue and getting information from them, trying to construct a complete donor pool.  So we have again a lot of different opportunities to reject a donor as we go through this process.
	Finally, by the time we get to final labeling and release we get to this point and hopefully we never get to the second point right down here but we have all been familiar with it because of FDA regulations and certainly sometimes it does happen.
	But if we have done everything right here we have had seven opportunities to reject this tissue from transplant before we ever get to actual release.  There is the initial review with the hospital personnel where we go over the first five or six questions.  Then there is the family- obtained history where I told you that we go over at least 45 questions.  There is the medical chart review.  There is the testing that we do.  There is the procurement review which includes really a review of all of the information that we have, the processing review, which again is brought in-house and allows several people who didn't go over it in the initial stage to go over it again, and then finally the final release of the tissue review.  So we have gone through a number of steps.
	The second step that we believe is necessary to provide safety is sterility or sterilization.  Sterility is an issue that has been a great deal of concern to us. About six or eight months ago Paul Brown and Nick Hogan talked to the Eye Bank Association meeting and we talked a great deal about sterilization of instruments.
	So we want to reiterate something that Diane said about the other tissues. This is a single set of instruments per donor.  We do it a little bit differently in eye banking because we do what we call an inside and outside set of instruments.  Here are the outside instruments that come in contact with the conjunctiva and the outer portion of the eye and the inside instruments and I want to particularly call your attention, if I may, to these inside instruments to these tiny little scissors right here.  These are very sensitive instruments which require a great deal of sharpness and proficiency to do the proper type of surgical technique.  Now, this isolation of inside and outside instruments has been very helpful to us in controlling bacterial contamination.  I don't know that it helps us with CJD.
	Many eye banks after Dr. Brown's talk went to additional sterilization, decontamination stage with their instruments and what this really amounts to is that we rinse the instruments immediately following the procurement and we bring the instruments back in a moist environment, keep them moist all the time, and then we sterilize them at 135 degrees for 30 minutes.  This is the first decontamination step with the instruments.
	After that we go through a cleaning process where we scrub the instruments, we check them for function, and we place them in Cidex for 30 minutes.  Following the Cidex 30-minute deactivation we rinse them and put them into a milk bath and we rinse them with 70-percent isopropyl alcohol.  We dry them, we lubricate them, and then we sterilize them a second time.  That is before they would then be ready to use on an additional donor.
	These instruments are already showing some corrosion and deterioration in function based on this high sterility time but we felt it was worth the extra effort and the extra sterility exposure based on what Dr. Brown had told us.  He felt we were reducing our risk significantly with this step and it doesn't as yet cause us great additional cost.
	We are certainly looking into disposable instruments but at the moment don't have the kind of scissors that I told you in the beginning were so important to us because the surgical removal of the tissue is a very important step in whether the tissue is functional.  If you put too much stress and strain on the tissue in removing it because you don't have the appropriate instrumentation then you may indeed damage the corneal endothelial cells that you are actually trying to transplant.
	The third step is asepsis and we do aseptic procurements.  Just like in the tissue bank we establish sterile fields, we use personal protective apparel, we use sterile instrument kits which we just talked about, and we try to do this in an aseptic manner, OR technique, et cetera although we do not need an OR for the retrieval of corneas.
	We can set up this environment in a very limited space because obviously we are removing a very limited tissue that is fairly surface in its nature.  This has been very effective in controlling contamination of bacteria and fungus and in limiting environmental exposure and technician exposure.
	In the processing of corneal tissue, which really does not under go a great deal of processing, but if we have removed a whole globe and come back to the laboratory to excise a cornea we do that with the same procedures as we would do the removal except, of course, here now we have a hood cleaning, which is a step we would not have if we were doing the procurement in situ.  We do the hood cleaning, the personal protective apparel, the sterile field, the sterile instruments and the aseptic technique.
	We generally clean our hoods with a bleach solution and an alcohol solution. Hoods are required to be cleaned before and after each use and are usually cleaned at regular intervals in addition to that.  The majority of our hoods undergo culturing processes on monthly intervals to validate that the cleaning processes are indeed working.
	We do establish a sterile field. Notice we have a sterile instrument kit that's used for each procedure.  When we come back and do the decontamination steps of these instruments they are kept in one single container so that there is no batching or mixing of instruments during the sterilization process as well.
	This just shows you what happens, our personal protective apparel.  Here you see a corneal scleral rim being removed. The things that are important to us here are that we have a nice even rim, that we don't put undue tension on this as we separate it, causing loss of cells on the endothelium or damage to the tissue.
	Well, our final step in processing to assure safety is labeling.  All of our tissue is labeled "Single Patient Application Only."  I think it is important to consider the number of applications this might represent from a single donor because it is dramatically different from some of the things that you have heard today.
	Single patient application from a corneal donor, assuming that we had both eyes and were acceptable for donation, you would have two corneal grafts.  If it was a whole eye donation you would have two scleral grafts which could result in either two grafts or up to eight grafts so that you have a maximum potential here from one corneal donor or approximately ten applications.
	Finally, the graft is labeled "Not Sterile" and again for single patient use only.  It is sealed with a tamper-proof seal and an individual number and labeling process so that the graft can be tracked.
	Finally, when FDA speaks eye banks are used to jumping and we certainly are willing to do anything that makes our grafts more safe.  But I think it's important also for you to realize that we could not complete our mission if we were not concerned for safety.  We have always been eager to do what it took to be sure that the recipients received a safe graft.
	Finally, our mission affects thousands of individuals each year, both those who receive the transplants and those who give the transplants, and we are conscious of this and are concerned for both.  Now, these two children, this little girl is the recipient of a corneal graft. This young lady's sister died in an automobile accident and was the donor of a corneal graft.  So when you are dealing with recipients and donor families like this you can't but take your mission seriously and I assure you that our commitment to safety is very serious.  Thank you.
	DR. BOLTON:  Thank you, Ellen.  Are there questions from the committee or comments?  Ermias?
	DR. BELAY:  One of the donor screening parameters that you describe is getting historical information from the families, historical information on whether that donor received human growth hormone?
	MS. HECK:  Yes.
	DR. BELAY:  I always have difficulty understanding how this historical information is collected.  If you take cornea as an example, the donor is dead at the time of the donation, one, and, two, the next of kin may not be available to provide the appropriate data.
	In addition a good chunk of the corneas, my understanding is, are collected under what is known as legislative consent, which would mean the family is not even available to provide that kind of information.
	Now, do you have any information on what proportion of, let's say, the corneal donors we actually collect that type of information on?
	MS. HECK:  Although at one time that was a very high percentage of the donor population, and I think back in the '70s it may have accounted for as much as 50 percent of the donor population, it is probably down now to something closer to 15 or 20 percent.
	The experience that I gave you is our experience and we collect none of our tissue without family consent and family medical-social history interview.  I think you are seeing that becoming the more common trend where back in the '70s, perhaps, the medical examiner or legislative consent was more prevalent but it certainly has diminished.
	In terms of getting that information from the family, if I may, we get it from the best historian available at the time.  That may be a family member.  It may be a close friend.  It may be both a family member and a close friend.  We look at multiple sources to try to do that.  We also may incorporate an interview with the primary care physician if there is a primary care physician.  This is done by either nursing personnel or trained transplant personnel.
	DR. BOLTON:  When you say we collect none without family consent is that "we" the Eye Bank Association of America or is that your local facility?
	MS. HECK:  That's our local facility.  The Eye Bank Association of America does at this moment permit legislative consent tissue but, again, I think the majority of members have moved away from that.
	DR. BELAY:  This has a direct bearing on the questions that we are considering because most of the additional criteria in the FDA guidance relies heavily on historical information provided by family members.
	MS. HECK:  Yes.
	DR. BOLTON:  Other questions?  I'll just make a comment on that and that is that again when you look at those potential donors that are removed via donor exclusion criteria through historical information it still will only get those that are clinical and a very small percentage of those that would be pre-clinical like those that are removed for variant CJD risk or other iatrogenic CJD.  But those that are incubating disease will still not be picked up by that so we go back to our neuropathologists to save us in that case.
	Yes, Dr. McCullough.
	DR. McCULLOUGH:  Can I pursue the legislative consent donors?  You say roughly 15 to 20 percent of all donations would be so that essentially means that 15 to 20 percent of corneal donations would be obtained without a medical history?
	MS. HECK:  Certainly without a family interview.  I won't say completely without a medical history because the medical examiner will be getting information and providing information to you which is helpful in constructing your donor profile. But you will be absent that family interview piece.  That's correct.
	DR. McCULLOUGH:  That would always occur just at the medical examiner's venue and everything else does involve a family history?
	MS. HECK:  The laws vary somewhat state to state.  It is called the Medical Examiner and Coroner Law.  If it's a coroner that releases the tissue then you would not have that advantage of a medical examiner.
	I think it is worth pointing out, however, that the majority of these cases, certainly not all of them but the majority of these cases, fall into that category under age 50 that you talked about which are less likely to be manifesting the disease or have symptoms that we could track.
	I know after you said the instance I might get it after 50 I thought maybe I should just go home and shoot myself because I have, as you heard today, passed 50 but then you said if I could hang out until 72 maybe I'd be all right so I'm thinking that one over.
	But those ME cases are usually traffic accidents, violent deaths, unexpected deaths of some sort, and do fall primarily towards the lower end.
	DR. McCULLOUGH:  What percentage of those would have experienced head trauma?
	MS. HECK:  A lot of them experience head trauma.  That is an interesting question if I may step from EBAA to AATB because our bank does both bone, skin, and ocular tissue.  In ocular tissue sometimes the head trauma will preclude you from getting the cornea simply because there's enough edema that you can't get them.
	But that certainly doesn't preclude me from getting bone and skin because it doesn't affect that.  So head trauma, although reduced by helmet laws and other things, it's still a major source of donor tissue in this country.
	DR. DeARMOND:  What level of head trauma?  Everybody bumps their head.  What do you consider serious?
	MS. HECK:  Well, I'm thinking primarily of motorcycle and MVAs, motor vehicle accidents.  That's the head trauma that we see mostly that's caused the fatality and there's a significant amount of that.
	DR. BOLTON:  David?
	MR. ASHER:  I'd like to clarify something about the medical standards.  As I understand it, the screening questions if they detect a history of loss of memory, inappropriate response, confusion, or gait changes, those would be deferral factors but a medical director has the option of reinstating if in the medical director's opinion those things are due to cerebral vascular disease or other known causes?
	MS. HECK:  That's correct.
	MR. ASHER:  The reason I'm concerned is that's an exact description of our last dura associated case.
	MS. HECK:  I'm sorry.  I didn't hear the end of your statement.
	MR. ASHER:  That is an exact description of what happened with our last dura mater associated case.  It had all those things and the person doing the medical review attributed them to cerebral vascular disease, which the donor also had.
	MS. HECK:  I think that over the last few months, several months and maybe the last couple of years, you find that if you get a composite of those things they are much more likely to be rejected even if you think you know what they are from.
	I know certainly we have moved that way and I believe that the majority of eye banks have, too.  It's when you may have one of those but not the composite of the three that the medical director might be likely to say I know what this is from.
	MR. ASHER:  Since these cases are almost certainly not autopsied on what basis would the medical director make that decision to reclassify such a donor?
	MS. HECK:  Those would primarily be made on findings in the medical chart, previous diagnosis by consultants, things that we would review by calling primary care physicians or getting information from attending physicians in the hospital if there's some specific documentation present in the chart.
	DR. BOLTON:  Other questions? Okay, very good, Ellen.  Happy birthday again.
	We will move on.  I think what we're going to do is we are going to take the next two presentations.  We are running about 45 minutes behind schedule.  What I'd like to do is take the next two, which have to do with equipment and instrument sterilization and what have you and process validation.
	Then we'll break for lunch before the industry presentations on process validation so we can get all of those in without everybody feeling the pangs of hunger and we'll come back after lunch and have those.
	So our next speaker is Dr. Robert Rohwer from the VA Medical Center at the University of Maryland, Baltimore.  Bob will talk on "Equipment and Instruments:  TSE Agent Disinfection in Routine and Exceptional Situations."  Bob.
	DR. ROHWER:  I was disappointed that nobody asked Ms. Heck what the milk bath was for.  I'm curious and I'll have to ask her myself.
	I had the liberty of reengineering this title to suit myself so this is what we are going to discuss, disinfection and sterilization of TSE-contaminated surgical instruments.  On the other hand I know of no systematic study of this issue and as a consequence we're going to have to beat around the edges of this subject a little bit.
	What I'm going to do is review some of the principles of inactivation of these agents which I think are important and we will look at their application to the inactivation by sodium hydroxide and heat, which are the only two really effective inactivants that we know of.  I'll mention bleach and then I'll go over the WHO recommendations for disinfection and also tell you how we go about this job in our laboratory and give you some anecdotal evidence from the laboratory that suggests that it does in fact work.
	However, I'll finish by telling you about an incident that occurred in Windsor, Canada, in which they attempted to apply these methods on a very large scale to disastrous effect so they are still things we have to do and learn about this process.
	The main point I'm going to make in the beginning of this talk is that the susceptibility to an activation of TSE agents if they are looked at in a kinetic fashion are not that different from what you would expect from normal viruses, the more resistant, from the virus families or spores.  On the other hand even though this is true, TSE agents are still very difficult to disinfect.  The reason for that, I'll try to convince you, is not really due to the failure of the inactivants themselves but rather due to inadequate exposure of the agents to these inactivants.
	What I'm going to tell you is based on some old work of myself and these references and I highly recommend this more recent document from the WHO laying out many of the issues involved with disinfection and infection control for these agents.  This document was put together as a consensus document.  It involved myself, David Taylor, Paul Brown, a bunch of people, and I think it really does represent the best current thinking on this.
	Now, I want to go over just the inactivation process itself so that we are all on the same wavelength when I discuss the actual data for the TSE agents.  I am going to be discussing curves like this in which the TSE agent is exposed to some process, heat or chemical, for a period of time and we have the surviving fraction over here.
	What's going on in an activation curve like this is that if we started with this many units of infectivity everything is happening very fast and very early in the inactivation process.  By the time we are here on this curve we have only got 10 percent of the population left and by the time we are here we only have 1 percent of the population left.
	Another way of looking at it is if you look on this axis down here 90 percent of the effect occurs very quickly, 10 percent in the next tiny little time interval here, 1 percent in this time interval here, .1 percent of the effect here, .01 percent here, et cetera.
	This initial rate of inactivation describes the way 99.99 percent of the infectivity is behaving in this curve; however, in this curve right here we are describing how only one part per thousand is behaving.  It is a bimodal distribution of effect.
	The properties intrinsic to the agent are reflected in the initial rate of inactivation.  This is basic chemistry.  The vast majority is being inactivated and the interpretation is much less complex than for the residual fraction, which is a complex function of environmental parameters much more complex but depends on the context of the agent, the milieu in which it finds itself to some extent.
	Here's an example of this type of inactivation using bleach at the concentration in which it is recommended to be used on the bottle.  This is not the highly effective concentration which is recommended from David Taylor's work which is 5 percent, ten times this concentration, or undiluted bleach.  Nevertheless we see that we have a very rapid destruction of infectivity on contact with bleach and then a residual survival from that point forward.
	We get the same kind of thing when we put conventional viruses into the same brain homogenate milieu showing you that there is a protective effect of the bioburden of the brain homogenate itself. These same viruses in highly purified form are killed to undetectable levels almost instantly.
	The inactivant of choice in our hands has been sodium hydroxide.  This is some work that Paul Brown and I did many years ago comparing Creutzfeldt-Jakob disease and 263K scrapie which can be challenged at higher titers to these various concentrations of sodium hydroxide.
	The main thing I want to point out is that at one normal we had inactivation to undetectable levels for both CJD and scrapie after 60 minutes of exposure but almost as good an effect from 10th normal; however it falls off dramatically when you go below this concentration.  Somewhere between here and here we are losing efficacy.  Also, even after 15 minutes of exposure we have done most of the work.
	On the other hand this is not a consistent finding and the reason for the inconsistencies probably has to do with the details of the experimental approach.  So where it has been highlighted in yellow these are all experiments from the literature in which complete, and by "complete" I mean inactivation to no survivors given the size of the challenge that was actually assayed, these experiments represent that experience but in the same chart we also have a number of instances in which very high levels of inactivation were reached but some residual infectivity was nevertheless recovered.
	A similar picture pertains for heat except the initial activation is even much more dramatic.  This was a kinetic experiment looking at what used to be the standard autoclave temperature of 121 degrees centigrade.  This was set up in an oil bath so samples could be taken very rapidly.
	Once they came to temperature and then cooled and then assayed what we see here is that by the time the sample got to temperature, and the ramp time here was about 30 seconds, we had 99.999 percent of the population destroyed.  The surviving fraction is at the level of parts per million but it does survive and it drags out here for another 15 minutes or so.
	Similar data have been developed by David Taylor using a somewhat different system in which brain macerates rather than homogenates were statically exposed to these temperature regimes here for these times and temperatures, giving the result here.
	This is somewhat shocking.  At first glance this looks like a shocking result in the sense that survivors, and by "survivors" I mean infectivity survived this treatment to the tune of four infections out of 13 animals inoculated under these conditions, et cetera, but if you look at this data and plot it the same way that I plotted my previous curve these points would all fall down on this part of the curve.
	The reason for that is that these are only partially effective in killing the infectivity.  We are at limiting dilution and when you put it back on a chart like this it would be in the same range.  So these data are entirely consistent with each other.
	Well, of course, what we would like to know is what gives rise to this residual surviving population.  This is where our public health problem actually is, in this material that we can't get rid of completely.  A clue is these dry heat inactivation experiments that were conducted by Paul Brown about ten years ago.
	Here again we have even higher temperatures than I showed you just a moment ago for ten 60-minute exposures, 160 degrees centigrade, and only getting two to three logs of inactivation where nine logs is possible.  What this is telling us is that dry heat inactivation is much, much less effective than wet heat inactivation for the destruction of these agents.  Well, this is not a big surprise because there are a number of sporulating bacteria that can survive to about these levels under these same kinds of conditions.
	I think it gives us a clue as to what may be going on with these surviving populations.  Now, 132 degrees is a very significantly higher temperature than 121 degrees for a steam sterilization where an activation takes place in just seconds, really.  On the other hand 132 degrees is only incrementally more effective than 121 degrees under dry heat sterilization conditions where an activation could take days under these particular conditions.
	And the thing that we have to remember is that the surviving population is very small.  This is a very small fraction of the starting infectivity.  This is parts per million of what we began with.
	So I'm going to offer you this model which is that in this case there is a horizon line here.  This is fluid down here. What I'm trying to show you here is that in a tube or a bottle or in the circumstances under which these experiments were done in the case of homogenate there is an opportunity for the material to boil as it comes to temperature and throw the infectivity on the glass where it might dry.
	In the case of the macerates the macerate is forced into the tube and leaves a streak behind it which can dry as the tube heats up on the way to inactivating temperatures.  And it's important to remember that in brain we have about 50 percent fat, which can form actually a layer over this which could be quite impenetrable to steam and water, especially if you think that this is happening only at the level of parts per million.
	This leads to the next slide.  What this data show is that the infectivity is not intrinsically resistant to steam sterilization.  The problem is with delivery of the inactivant and it is for that reason that we make the following recommendations. It's very important to prevent drying. We're very careful in the laboratory when we are done with an instrument to immerse it in water or sodium hydroxide, typically sodium hydroxide in our case, and to do that prior and during steam sterilization to make sure everything is wet and nothing can dry on the instrument.
	It's very effective to combine two or more methods.  This is has been demonstrated in a number of publications now, either pre-treating with sodium hydroxide followed by heat or, of course, an even more stringent situation is to put the instrument in sodium hydroxide and then sterilize it.
	These factors also contribute, we feel, to an effective sterilization. Surfactants, homogenization, good dispersion of the material, agitation if it's possible to eliminate sanctuaries in the vessel itself.  And in our experience refinement, and this is strictly anecdotal, seems to reduce the potential for protective associations and as the material becomes more and more refined it becomes more susceptible to inactivation.
	Well, all of this is reflected in this WHO document which I recommended to you earlier.  Basically the recommendations there are that instruments should be kept moist until cleaned and decontaminated and they should be cleaned as soon as possible. Avoid mixing these kinds of things.  I'm going to leave this for you to discover on your own because it is available on the Web.
	Then there is this hierarchy of recommendations for inactivation which were recommended.  For disposable instruments, materials, and these types of things, they should be incinerated if at all possible. And the recommendation was even made for instruments exposed to high infectivity tissues, for example, known surgery to a CJD brain, be destroyed in this way.  I'm not myself in agreement with this but it certainly is the most absolute way to go about it.
	In terms of the hierarchy of heat and chemical combinations the most stringent is to immerse in sodium hydroxide, heat in an autoclave for 121 degrees for 30 minutes, clean rinse in water, and subject to routine sterilization.  If the instrument won't take this then use sodium hydroxide or sodium hypochlorite for at least an hour and then transfer to water and do the same thing.
	Third on the list is to immerse in sodium hydroxide or sodium hypochlorite for an hour, remove and rinse in water, and then transfer to an open pan, heat and gravity displacement or porous load autoclave.  And then finally immerse and boil in sodium hydroxide.  This has been shown to be effective by David Taylor.  Hypochlorite can also be used in this way though there are a lot more problems associated with hypochlorite in our opinion than sodium hydroxide with corrosion, et cetera.
	In our laboratory we use the following regime.  We start with a sterile instrument.  We use it on infected material. We keep it wet.  If it's convenient or possible to clean it or we are cleaning it anyway before we put it in the wet environment and we do that with a ChemWipe (?) or whatever, we keep it wet.  We make sure it's kept immersed before it's put in sodium hydroxide overnight.
	It then goes to an autoclave where it is cooked for 132 degrees for an hour under sodium hydroxide or for some instruments in water if they can't take this process.  Then from this position, and I'm trying to indicate the loss of infectivity here by the diminution of the red in this scheme, we take it to our cleaning bath where it gets sonicated again in a detergent cleaner that is about one-tenth normal sodium hydroxide at 60 degrees and it's probably inactivating in and of its own right.  Once it comes out of the cleaner it is packaged and then goes through a standard autoclave sterilization before it goes into our sterile pool and is reused.
	We have some evidence that this works because we have been doing these experiments in the laboratory for the last several years looking at very low levels of infectivity associated with blood-borne TSE infectivity.  In particular I'm going to show you this experiment just because I think you will be interested in it as well. This is new data where we inoculated a large cohort of animals, hamsters in this case, at a low concentration of infectivity and at three-week intervals we pooled the blood from 20 animals and then we inoculated 100 recipients with each pool of blood.
	We have the data on the next slide. This is the incubation time of these animals, of the animals got sick.  These were the times in which we created the pools over here on the side.  So what we have here are two pools containing 100 animals each with no infections at all in them.  There's really no distinction between the instruments that were used here and used here.  Here, just as we had expect, we started seeing cases as the disease progresses.  And in fact on the next slide we get the status plotted and we see these two points without cases here and about mid-clinical disease we start seeing effect and an increase in effect as the disease progresses.
	One more example of this is we have done plasma fractionations on endogenously infected hamster blood where we fractionated all the fractions given here out of the plasma by the cone fractionation and again reinoculated 100 animals or more in this case with each one of these fractions.  And the data here, all the little dots are animals that contracted scrapie in the course of this experiment according to their incubation time here and the actual fraction that was inoculated here.  But it is important that as we got farther and farther into the fractionation we see fewer and fewer cases and we never did see cases in the case of fraction 2 and fraction 5.
	Well, that slide didn't make it in here.  Anyway, I was just going to point out that the actual schema for fraction 2 and fraction 5 are the end products of two branches of the cone fractionation scheme and it would make sense that the removal would be highest for those two fractions but, again, it shows you that we can actually do these experiments without cross- contamination into these types of materials.
	This method that we use contrasts very strongly with the method that is typically used in a hospital setting where this part of the scheme is missing.  In fact what happens is you start with a sterile instrument, you use it for surgery or whatever, and then it goes to a cleaner. This is done in a specialized washer using heat and detergents.  From there it goes to a package and is sterilized in an autoclave under conventional conditions and then goes back into circulation.
	So this is a far less stringent procedure; nevertheless, it is likely to be highly effective, I think, in terms of obtaining sterile instruments for reuse. The one thing that bothers me about it is this step right here where the cleaning step occurs before a disinfection step, which is what we have tried to achieve in the laboratory over here.
	From my perspective this cleaning apparatus overwhelms all other contamination issues.  It creates a secondary decontamination problem.  If contaminated instruments are actually introduced into this machine you have a problem of the contamination of the washer and contamination of the waste stream from that washer.
	This, again, has not been done in any systematic way but talking with the people who are responsible for this type of process they are adamant that this has to be done, it can't be done any other way, and these machines are adequate to the task.  On the other hand I don't believe that they should be presumed to be adequate and this should be investigated carefully.
	I'm going to finish with an example in which a CJD case was identified after neurosurgery.  This happened in the Hotel Dieu Grace in Windsor, Ontario, last year about this time.  What happened is there was a neurosurgery on a patient.  I'm not sure what sex it was.  They were subsequently diagnosed with dementia on subsequent observation.  A 14-3-3 CSF  sample was taken and it turned out to be positive.
	This patient did eventually have Creutzfeldt-Jakob disease, as you will see. That, at least, is a relief.  But on this discovery right here the hospital staff decided that they had to pull all of their instruments and sterilize them.
	Now, they had already been through this wash cycle and sterilization cycle and the instruments that were used in the neurosurgery had not been tracked and so they had been mixed with the general pool and so the hospital felt that they had to sterilize everything.  They sterilized the entire pool at once overnight.  They didn't do any test samples and they used autoclaving and one normal sodium hydroxide as recommended in the WHO guideline for this.
	The next morning they had a lot of electrolysis corrosion and destruction of these instruments.  There was a lot of complaining about fumes.  I'm not sure where that came from but there was some chemistry that we don't experience going on in this process.  In the end they estimate they destroyed about $10 million worth of instruments.
	The complete details from this incident haven't been released yet so it's hard to evaluate exactly what happened. There were obviously chemical incompatibilities in these mixtures that were autoclaved and I would have to point out that the WHO guidelines are based on laboratory experience and they were our best good faith recommendation for how to deal with these agents in that type of setting.
	Clearly we need to develop and validate procedures that will work for a hospital setting as well and sort out the issues.  This incident could be of great value to us in terms of sorting out the factors that led to this wholesale destruction.
	My concluding remark would be that the instrument washer was not considered a source of vulnerability even during the debriefing on this incident which I was invited to attend, getting very cold stares from lots of people.
	Sterilization of the instruments is pointless without sterilization of the washer from my point of view and if the washer itself is sterilizing then re- sterilization of the instruments was unnecessary.
	I think there is one final slide. I want to make the following point, that in terms of dealing with this episode in particular I think we can't lose track of this feature, that this was a case of Creutzfeldt-Jakob disease which they identified.  They might not have.  He might have had the surgery.  He might have died in the surgery, whatever, in which case the instruments would have been processed the way they always would have been and we would never have known the difference.
	We can really only identify a minor proportion of the potential exposure to this disease and that is these cases that we defer.  We have no way of identifying incubating sporadic cases, at least not yet.
	For that reason I feel it's pointless to implement measures that attempt to reduce the risk from known cases to below the irreducible risk from unidentifiable cases.  It's important to find out what that irreducible risk actually is by modeling and risk assessment but we also have to maintain some realism in terms of what we are attempting to do.
	The only caveat here is that this is true unless we apply a uniform higher standard to the whole process and not just apply it to point instances of contamination.
	DR. BOLTON:  Thank you, Bob.  I think in that last slide you made the same point that I had made earlier about not being able to identify these incubating sporadic cases.  There are questions.  We need to hurry along so I'll try to make these brief.
	Steve?
	DR. DeARMOND:  How do you test for residual infectivity on surgical instruments?  Is there any way to do that in a practical way?
	DR. ROHWER:  I think we need a paradigm because that's going to be tricky. We don't usually do neurosurgery on hamsters and mice, at least not large-scale neurosurgeries, which you would want to do in a case like this.
	A paradigm has been advanced by the Weissmann group.  They are using these stainless steel sutures which they then expose to infectivity and then implant in the brains of animals.  We have looked at that a bit and I think it has quite a bit of promise, actually, as a way to go.
	They are rods, not canulas, which means that you can control exactly what happens to the entire surface that's exposed.  The animals do tolerate it quite well.  So it is a way to go, I think.
	I know that there is some funding for this in Europe that comes out of the EC program, TSE Program, but I haven't heard any updates on it recently as to whether there is new data on that.
	DR. BOLTON:  Sue?
	DR. PRIOLA:  You hypothesize that this small fraction of agent which is protected in your inactivation studies might be due to protection by the high-fat content of the brain.  It perhaps forms a varnish over the agent.
	Have you done inactivation kinetics with infectivity from other tissues such as spleen or lymph node and do you see a similar kinetics?
	DR. ROHWER:  We haven't.  We have not.  We are looking for high levels of infectivity when we do these experiments because we are showing such high levels of inactivation.  But there are lots of things like that that could be done.  I mean, you could delipidate (?) the brain and do that experiment as well or you could look at spleen.  That's a good suggestion.  I think there is a huge parameter space associated with inactivation which has not been explored and should be.
	DR. BOLTON:  Briefly.
	DR. DeARMOND:  I was just going to say that have you tried sonication with lipid solvents, chloroform, methanol, acetone, something that wouldn't really corrode the instruments, and then go through the process?
	DR. ROHWER:  We have done sonication with sodium hydroxide but that doesn't count.  It's very effective.
	DR. BOLTON:  Dr. Solomon.
	DR. SOLOMON:  This is a very na‹ve question and we might want to save it for the discussion but if you were a professional organization and wanted to develop some standards now what would you recommend?
	DR. ROHWER:  My feeling is that making those recommendations in the vacuum of not having, obviously, a full appreciation of the compatibility issues with the recommendations that were made in the WHO guidelines would be difficult.  But in terms of looking at compatibility those experiments are much easier to do than infectivity experiments.  It's a matter of putting various instruments in various combinations and various alloy combinations and that kind of thing in a pot and trying it and seeing whether you get corrosion.
	That kind of thing, I think, should be done to see if we can get a handle on just what the issues actually are.  We know that when we use our cheap -- not all of them -- we have a survival of the fittest program in the laboratory for surgical instruments.  But we only buy cheap Pakistani stainless and some of it is very, very resistant to sodium hydroxide in heat and some of it isn't.
	DR. BOLTON:  David, the last question before our next presentation.
	DR. DeARMOND:  You will be pleased to know that investigators in the FDA Center for Devices are conducting exactly the kinds of experiments with instruments that you've mentioned.
	DR. ROHWER:  Very good.
	DR. BOLTON:  We will anxiously await those results.
	Our final presentation before lunch will be "Process Validation for Conventional Agents" presented by Dr. Mahmood Farshid.  I hope, Mahmood, I didn't butcher your name too badly.
	DR. FARSHID:  "Mahmood" was very close.  It is like saying "my mood."  My mood, your mood, good mood, bad mood.  I hope everybody is in a good mood.
	My presentation will be a brief overview of viral validation studies and also the approach that we take in evaluating such studies.  The product which currently requires viral validation studies falls loosely into three different categories: Monoclonal antibodies and recombinant products produced in cell culture.  These are highly characterized products and extensively tested and they have an excellent viral safety record.  Blood and blood products and other human blood products, probably tissue and soft tissue and bones also fall in these categories. But my main focus would be in the plasma- derived product where I draw most of my experience.
	Also, animal-derived product which these are, for example, lymphocyte produced by rabbits or antivenin produced in horses. So it would depend on what kind of a starting material you are using the approach to the viral validation study will be somehow different.
	There are some complimentary approaches in basically reducing the risk of viral infection, so viral inactivation essentially is only one component of this multi-faceted approach which includes donor screening, donor history assessment, and written and oral questionnaire.
	Donor testing, which in the case of whole blood includes testing for antibodies to HIV-1 and 2 and p24 antigen and serological tests for HCV and HBSAG and anti-HBC and anti-HTLV-1 and 2, and syphilis.  For plasma, for instance, HTLV and Anti-HBC is not included in the test. Pharmaco-vigilance and finally in case of the pooled and plasma-derived product the last line of defense will be viral inactivation and removal and basically validating the manufacturing process for removal of this virus.
	So basically the aim of viral validation is to provide evidence that their production process will effectively inactivate or remove viruses which could potentially be transmitted by this product. So here we are talking about a relevant pathogen which may be present in a starting material.
	Also to provide indirect evidence that the production process has the capability to inactivate or remove novel or yet undetermined viruses.  Basically here we use an array of viruses to cover also those who are undetermined or emerging viruses because not all viruses are screened for. For example, HGV and TTV and SLV and other viruses also will be covered by the inactivation or removal process.
	This is basically a common virus clearance method.  This list is by no means complete and basically is drawn, as I mentioned, from my experience with plasma- derived products.  There are other methodologies which are being used.  It can be divided.  The viral clearance basically includes viral inactivation and removal which inactivation could be a chemical inactivation like, for example, using a solvent detergent, which is very well known or physical inactivation like heat which we just heard about.  The removal includes also chromatography or precipitation or using nanofiltration.
	In evaluating the viral validation studies there are different components in the study that we need to evaluate.  One will be the scale down process step.  The viral validation studies not done in actual manufacturing settings and for very obvious reasons because it is not desirable from a standpoint of G&P and also is not practical because you need a huge amount of virus to do that.  Therefore the laboratory model or the scaled down model of the manufacturing process is being basically designed and it will be used for the validation studies.
	The other will be spiking, to do that deliberately, the one step which needs to be validated will be spiked with high titer virus.
	Finally, the reduction of that virus, either inactivation or removal, will be determined in subsequent steps.  If the number of steps being validated then the reduction of clearance from these steps will be summed up and that will be basically total log reduction value for that particular process.
	In evaluating the studies we look at the choice of viruses, what kind of viruses may have been used and if it is appropriate.  And the design of the validation study, which is essentially validity of the scaled-down process, this is very important in order to determine that what is obtained is relevant to the actual manufacturing process.
	The study should provide evidence that the scaled-down model actually is relevant and basically mimic the actual manufacturing process.  The kinetics of inactivation also needs to be shown as we saw when Dr. Rohwer showed some of the kinetics of some heating activation.
	And if it is removal we need to know the whereabouts the virus was removed. Basically the mass balance needs to be shown and determined.  Also, the robustness of the process as a whole also needs to be determined and that maybe by introducing some deliberate changes in the process to see if the process as a whole is robust. Also, the limits of sensitivity of the assay used for infectivity also needs to be determined and finally the log reduction of how much log reduction is achieved in the whole process.
	In terms of viral selection the viruses that can potentially be transmitted by product, basically we refer to them as a relevant virus.  These are pathogenic viruses.  So the first choice will be to get the virus which is relevant.  Of course, this is not always possible because some of the relevant virus do not grow in tissue culture such as Hepatitis B and Hepatitis C.
	In that case we use a specific model viruses which basically are viruses which physically and chemically are similar to the relevant viruses as close as possible.  In addition to that non-specific model virus also will be used.  These are simply to show basically the overall capacity of the step inactivating viruses and for this purpose usually viruses which are highly resistant and small ÄÄÄÄ viruses will be included in the panel of the viruses.
	Therefore the selection of viruses basically is dependent on the nature of the starting material, if it is cell-derived or human-derived or animal-derived and for the reason that I stated because we need to be close to the relevant viruses in this product.  Also we need to consider the practicality of using these viruses, for example, availability of suitable culture system and availability of high titer stock which is necessary to do this kind of experiment.  Also the availability of reliable methods for quantitation of this virus.
	These are a panel of viruses that are used for doing viral validation studies in a plasma and plasma-derived product and probably can be applied to, I would say, all human-derived product if one wants to do their viral validation study.
	HIV-1 is being used as a model virus for HIV-1 and 2 and also HTLV and this is required for any kind of studies which are done and any type of viral validation studies.
	For Hepatitis B basically it is not modeled directly.  There are some model viruses like ÄÄÄÄ Hepatitis B or ÄÄÄÄ Hepatitis B which could be used as model viruses; however, in revalidation studies we do not require any of these viruses to be used and the capacity of a system to clear Hepatitis B basically would be extrapolated from looking at the panel as a whole.
	For Hepatitis C there are a number of specific model viruses.  Some of them are used more often.  The bovine viral diarrhea virus is one which in terms of its size and genomic structure is very close to Hepatitis C and being used in the validation studies.
	CMV also which is a ÄÄÄÄ virus is larger than HCV, is more resistant in terms of inactivation, and is probably a better choice if one wants to do the inactivation studies.  We encouraged the manufacturer that they can use both of them because BVDV because of its size would be better in removal and validation of removal studies and CMV is because of high resistance probably would be better to be used in inactivations.
	HAV, there is a laboratory strain of HAV which can basically be used as a relevant model for Hepatitis A and this is also because of small non-envelope viruses which can also qualify as basically being used to show the rigor of the overall capacity of the step in inactivating viruses.
	Because of the presence of a number of herpes viruses inclusion of one of them in the validation studies is desirable, for example, like PRV, and that is also qualified as having one DNA virus in the panel which basically covers maybe for Hepatitis B as well, although it is a larger virus.
	For B19 PPV can be used for human parvovirus B19.  This is a small highly resistant ÄÄÄÄ virus and it basically is a good virus to show overall capacity of the inactivation or removal step and also it can be basically used as a surrogate or as a model for B19.
	This is a panel of the viruses which are used for the cell line driver. Basically the panel which we use, as I mentioned, is dependent on what kind of starting material basically is used in the manufacturing.  Here the required virus is the retrovirus because the presence of this virus is endogenous in mice and hamster cell line.  Also the PRV may cause latent infection in some of the cell line and that need to be there.
	And ÄÄÄÄ virus is present in a number of different cell cultures and that is included in a panel and MVM, which is also parvovirus, is a highly resistant virus and again it will show basically to determine the overall capacity of the system in clearing the viruses.
	So if we look at the selection of viruses it is intended to include the DNA and RNA viruses, both single and double stranded, lipid and non-lipid viruses should be included and in terms of their sizes it should include large, intermediate, and small size.
	In term of their resistance it would be from highly resistant to inactivation to very easily inactivated.  By doing so basically we cover the viruses which are undetermined or emerging viruses and that will basically increase the assurance of the overall capacity of the system to remove the viruses in general.
	The other component for looking at the viral validation, as I mentioned, is the scaled down purification process which usually is one-tenth to one-hundred of the full scale.  In this, as I mentioned, some data should be provided to indicate the relevancy of the small downscale to the actual manufacturing process.  That is the only way that we can determine that the result that obtains is really relevant to the actual manufacturing process.
	For example, the buffers, the pH, protein concentration, and the product should be the same as full-scale manufacturing.  The test material which I'll use for this kind of testing should come from actual manufacturing process.  The intermediate material should come from the actual manufacturing process and put it through their scale down.
	All the critical operation parameters should be kept as that has full scale, for example, bed height, flow rate, and so on and the absolute values, which I mentioned, temperature, pH, should also be kept as that of the actual manufacture.
	Also, make sure that in term of product specification that the product is identical to the production scale.  So basically the scaled-down models should be substantially equivalent to the actual manufacturing process if you want to basically extrapolate from the result obtained in a scaled- down model to that of actual manufacture.
	So the overall criteria for effective virus should produce significant viral kill and should be reproducible and controllable at the process scale and modelable at the laboratory scale because some processes are difficult to model; therefore, it would be difficult to determine the actual capacity of that step in clearing the virus.  So modelability of that method, basically this step will be important.
	And it should have minimum impact on the product yield and activity.  It basically should not affect the product.  It is intended to kill the viruses and should not kill or remove the product itself.  It should not generate new antigens or leave any toxic residue.  It should not be mutagenic or carcinogenic.
	The manufacturing process for blood-derived products should contain at least two effective steps for removal and activation of viruses and "effective" refers to one which basically produces significant viral removal or inactivation.  At least one step should be effective against non-envelope viruses.
	At least one stage in the production process must inactivate rather than remove viruses.  So total reliance or removal may not be sufficient.  The removal process is very difficult to basically model in a lab and inactivation are more robust; therefore, if total reliance on removal we ask that one inactivation step also be included.
	In evaluating the result if one single step having a large effect gives more assurance of viral safety than several steps having the same overall effect.  You may get studies which show the same level of overall viral reduction.  One of them may be obtained by two single steps and one may be obtained by five or six different steps. The one which is obtained by two different steps will definitely provide more assurance, that is, more effective in basically clearing of virus.
	Even under the best of circumstances there are limitations.  One needs to realize that viral validation study just provides an estimate and assurance of how the system will work and they are not absolute.  The limitations include that laboratory strain may behave differently than native viruses because most of the viruses, even the relevant ones, are laboratory adopted viruses and they may behave differently as the one that they are present and why.
	The source of plasma or immunoglobulin may have neutralizing antibodies so that may affect the overall viral kill and overestimate the viral so the presence of neutralizing antibodies is a variable which needs to be considered when doing inactivation or removal.  For example, in case of Hepatitis A that anti-A is usually present and in many cases is the result of over-estimation of the capacity of that particular step in killing the viruses where the killing may be as a result of the presence of a neutralizing antibody and not because of the effectiveness of that particular step.  And there may exist in any virus population a fraction that is resistant to inactivation.  I think this has been mentioned this morning.
	A scaled-down process may be different from full scale.  Sometimes it is difficult to basically model the actual manufacturing process and there will be some differences and it is difficult to determine how those differences are going to affect the overall clearance.  So that also needs to be considered, that what we see is done in the lab and is not an actual manufacturing process.  That will be another limitation.
	The total virus reduction may be overestimated because of repeated and similar process steps.  The different steps that will be validated should be orthogonal. They should work by independent mechanisms in order to be acceptable to add the total viral reduction from this different step, basically to sum them up.
	The ability of a step to remove viruses after repeated use may vary.  This is probably true for chromatography, which the residents sometimes use repeatedly, so what you get in the beginning in the course of the validation may not be after a number of years.
	That concludes my presentation. Thank you.
	DR. BOLTON:  Thank you.  Briefly, questions or comments from the committee?
	DR. EPSTEIN:  I just wanted to make two quick comments.  Thank you, Mahmood.
	The first is that we also care about the absence of log removal and we try to develop a standard that is applicable to what we think is the pathogen burden in the product.  So there's the idea of overkill relative to some upper limit of potential contamination.
	Then you touched on this indirectly but when you have a series of processes it's true that we look at the summation of logs clearance.  But it's also true that we more or less routinely will ask for some thru-put experimentation to show that at least for the critical steps it's valid to sum the logs reduction.
	DR. BOLTON:  That was a good point, Jay.  I would have made that except I'm too hungry to think about it so an excellent point.
	So here's what we will do.  I want to cut lunch short from an hour to 45 minutes.  We'll meet back here at 2:00.
	(Whereupon, at 1:17 p.m., a luncheon recess was taken.)
	A F T E R N O O N   S E S S I O N
	(2:05 p.m.)
	DR. BOLTON:  Our first presentation for the afternoon part of the session, which is "Process Validation - Industry Presentations," will be from Dr. Richard Hurwitz.  He is the Interim President and CEO and Medical Director of LifeNet.
	Dr. Hurwitz.
	DR. HURWITZ:  Good afternoon. Thank you very much for inviting me to speak.  I would like to first give a little bit of an overview of the impact of tissue banking in the United States and talk a little bit about validation but first a little bit of history.  LifeNet, which is located in Virginia Beach, was established as a tissue bank in 1982 to provide allograft skin for the local burn center. When it became apparent --
	DR. BOLTON:  Dr. Hurwitz, let me just interrupt you for a second.  I didn't get a chance to speak with you beforehand but I have asked each of the other industry representatives to please be as brief as possible.  I would like to keep your presentation to 10 to 12 minutes so we'll have time for questions.
	So anything that is not necessary you can bypass quickly.  Thank you.
	DR. HURWITZ:  How do I decide? LifeNet has grown to be the largest nonprofit full-service tissue bank in the United States.  It was one of the first banks to be accredited by the AATB and is ISO 9001-certified.
	LifeNet's donors come from many areas of the country largely through relationships with organ procurement organizations which in many states function as the appropriate stewards for donated human organs and tissues.  The ethical issues surrounding tissue donation, care of the donor family, and profit making are separate but important ones.
	In addition to musculoskeletal banking LifeNet is the second largest provider of cryo-preserved human heart valves.  My comments today, however, will be related to musculoskeletal banking and nothing from the central nervous system.
	To show you the overall impact of tissue banking this slide shows the increasing number of tissue donors in the United States to meet the growing demand. In 2000 almost 20,000 tissue donors were processed.  LifeNet, for example, accepts donors up to the age of 70.
	This slide indicates the number of grafts by type distributed by LifeNet in 2001.  Allograft bone is used in the majority of spinal fusion operations. Autografting, which is the alternative to allografting, significantly increases patient morbidity and prolongs operating times.  Demineralized bone particles enhance the outcome in thousands of dental procedures yearly and the ability to use allograft tendons for sports medicine knee construction procedures also greatly facilitates the operative procedure.
	The next two slides indicate the present and projected needs for allograft tissue.  In 2001 more than 400,000 different procedures involving bone graft were performed and the projections are for continued increase.
	For soft tissue grafts, which includes skin, fascia, tendons, and pericardium, the projections are for continued increase over the 90,000 implants or transplants that were performed in 2001. It is important to note that to preserve the functional integrity of tendons a processing methodology somewhat different from the one used for bone grafts is utilized; therefore, it is important that antimicrobial process validation be done for each separate tissue type application.
	This slide illustrates some examples of the application of tissue engineering in the design of bone grafts which better serve the clinical need such as these machined allografts for spinal fusion procedures, demineralized bone powder used in dental applications, and you have already seen pictures of a patella ligament used for sports medicine.  Hundreds of individual grafts therefore can be made from a single donor.  The actual and predicted clinical utilization of a human tissue is an indicator of beneficial outcome; however, outcome studies are limited and expensive to perform in a controlled fashion, especially in a rapidly changing arena of new innovation.
	Historically tissue banking is an extension of organ donation and transplantation.  Tissue donors are screened, tested, and aseptically procured by LifeNet like organ donors.  In fact many organ donors are also tissue donors. Properly performed, tissue transplantation should be as safe as organ transplantation. The question still needs to be asked whether the expectation is for a zero infection rate from human tissue.  What about for organs or eyelet cells or other human tissue?  Clearly the risk-benefit must be considered.
	The approach to tissue banking changed in 1991 when LifeNet became aware that a 1985 donor had transmitted HIV to recipients of organs and tissues.  The case was reported to and thoroughly investigated by the CDC and subsequently published in the New England Journal of Medicine.
	Recipients of tissue which contained residual bone marrow, fresh frozen grafts, became HIV-positive while recipients of processed bone grafts did not; hence, the new era of tissue banking at LifeNet unfolded with the development of procedures for removing bone marrow and for disinfecting bones and soft tissues. LifeNet was alarmed at the recent reporting of tissue allograft-related infections from clostridia and other pathogens by the CDC. It has been subsequently determined by Dr. Kiner that 12 out of 14 of the clostridia cases were from one bank, which was not AATB accredited.
	It also has become apparent from the CDC investigation that grafts which are processed differently to preserve cell viability or graft function such as ÄÄÄÄ condyles, tendons, and menisci have more often been implicated in patient infection.
	I have summarized in this slide all of the complaints received by LifeNet concerning possible allograft-related infections or graft contamination during the past four years.  Each case has been investigated and to date no serious allograft-related infection documented. Many of these reported positive cultures were done in the operating room prior to implantation.  This has been recommended against by the orthopedic societies because of frequent environmental contaminants.
	This last case is significant in that tendon tissue from a donor processed by one bank known as tissue processor A in the MMWR resulted in a clostridial infection and other tendon tissue from the same donor processed by LifeNet was used in four recipients who have been followed without evidence for infection.
	The HIV case notwithstanding, the risk of disease transmission from allograft tissue has always been considered to be extremely low.  Many but not all banks have been inspected by the AATB and those conforming to standards have been accredited.
	At last count the CDC has gathered information on 54 cases of allograft-related infection over the past four and a half years.  Twenty-five of these were from one bank.  With hundreds of thousands of tissue transplants each year is there a significant public health risk or can the reported allografted infections be explained by deviation from excepted AATB standards?
	There are multiple steps to assure safety.  This information has been shown to you before.  I would like to note that all of the data concerning screening information and the circumstances leading to death and the initiation of procurement must be evaluated by the medical director, who needs to do all of the research to draw a conclusion that any particular donor is safe.
	The donor screening is performed to reduce or eliminate the donor who may be at risk for transmitting malignancy, bacterial disease, parasitic disease, viral disease, and, of course, prion-associated disease. You have already been shown the standard screening questions that are asked which concern high-risk behavior and specifically symptoms, signs, or past history that would suggest CJD-related disease.
	Aseptic procurement and processing are essential for infectious disease safety and to preserve the functional intent of the allograft tissue.  Following procurement all musculoskeletal tissues are held in quarantine until qualified for release.  A swab surface culture of each tissue is performed.
	Tissue cultured positive for a list of pathogens is irradiated prior to processing with 15 to 25 kilorad.  Tissues cultured positive for clostridia, fungi, or yeast are discarded.  In fact 10 percent of tissue is discarded and never enters the clean room for processing.
	Many other potential tissue donors are rejected just on the basis of screening and they are not procured.  The reasons for discard include not only procurement cultures but positive serologies, medical history, autopsy findings, and hemodilution. Therefore by utilizing strict donor screening, aseptic procurement, procurement culturing, final medical director review, and appropriate pre-processing discard the risk of an infectious agent being present in tissues sent for processing is minimized and the likelihood of significant bio-burden similarly reduced.
	LifeNet uses clean room technology to control contamination.  Each room is tested to Class 100 at rest and environmentally monitored.  Processing suites are decontaminated between procedures using commercially available reagents and all surgical instruments are sterilized following AAMI guidelines.
	Based on our own HIV transmission case in other transmissions this MMWR admonition was published in 1993 which said it is prudent to process bone and bone fragments and carefully evacuate all marrow components from whole bone whenever feasible.
	The Allowash process implemented in 1995 was developed to facilitate the removal of bone marrow and the introduction of antimicrobial agents into the allograft tissue without altering function.  Allowash itself is a combination of biological detergents used in combination with isopropyl alcohol and hydrogen peroxide to remove bone marrow, blood elements, and lipids.
	Centrifugation and ultrasonification are also employed to achieve near total cleaning of the bone. LifeNet has always employed single-donor processing and strongly objects to the pooling of donors per AATB standards.
	In addition to bacterial and viral testing a number of other parameters were validated, including measurement of detergent residuals, osteoinductivity, and inflammatory response in a mouse model, measurement of protein residuals, light in electron microscopy.  Biomechanical testing included compression strength and tensile strength measurement.
	Time kill studies using Allowash solutions for the six USP organisms depicted by the asterisk and others tested as well, including clostridium sordelli, which is the organism that led to the death of the tendon recipient, demonstrate greater than ten to the sixth (106) log kill.  These studies were done in the presence of bone marrow and cortical bone to mimic the processing environment.
	The time kill studies of the allowash components were also performed with model viruses for HIV, HTLV, and Hepatitis-B and C.  LifeNet is completing additional studies looking specifically at measurement of sporicidal activity.  A definitive reference lab protocol for quantitating activity against anaerobic as well as aerobic spores has been difficult to find. We believe it is important to have a validation model which incorporates the cleaning process as well as the disinfection process.
	We have therefore begun a validation protocol utilizing uniform size cubes of cancellous bone impregnated with bone marrow and known bacteria.  Each step of the Allowash process will be examined by an independent lab to determine reagent consumption, protein removal, and bacterial log reduction.
	All production equipment is qualified using standard qualification practice.  LifeNet continues to evaluate other methods which might be considered for terminal sterilization such as low temperature irradiation, plasma phase hydrogen peroxide, and decellurization methods.
	Post processing cultures are performed using BacTAlert, a very sensitive, automated microbial detection system based on carbon dioxide and other metabolite measurement.  A rinse of the final graft or a co-process sample inoculated into media for the BacTAlert system in a seven-day incubation protocol has been validated.  If any post-processing culture is positive the entire donor is discarded except when skin organisms are detected, in which case tissue is reprocessed and retested.
	AATB inspects and accredits tissue banks and has continued to work with the FDA and CDC to develop and update standards. AATB accreditation should be required with the governmental financial support to help cover the expense of comprehensive inspection.
	Single donor processing should be mandatory to prevent the possibility of cross-contamination and to prevent the need for a greatly expanded recall if required for a possible disease transmission.  Dura should not be procured for transplantation as only dura and chorda (?) have been implicated in a TSE transmission. Validation standards should be uniform for all banks and not proprietary and presented in a scientific form for general agreement and they should be specific for each tissue-type process.
	Additionally, with regard to TSE tissue donors are not presently screened for travel or residence in Europe or for receipt of bovine insulin.  It is unclear how such screening would affect the number of tissue donors.  Once would guess that in areas near military bases it would have a significant effect.
	Also recommend investigation of all potential allograft infections with central reporting and investigation to determine more thoroughly the incidence and cause of disease transmission through route cause analysis.
	In conclusion human tissue transplantation is widely used and safe. Although there may be a temptation to consider a final sterilization process for human tissue this should not be a recommendation until a process which does not interfere with the biological properties of the tissue can be validated.
	The doctor is saying, "No, I wouldn't call you a mad cow exactly.  I would say you are a cow with issues."
	Thank you.
	DR. BOLTON:  Thank you, Dr. Hurwitz.  Questions from the committee? Dr. Wolfe.
	DR. WOLFE:  Again, one of our favorite topics, dura mater.  The policy that you mentioned in the recommendations, do not procure or distribute dura, is this the policy of LifeNet or is this what you would recommend more generally for all the tissue banks?  Is this is a policy of AATB or what?  Just what is the origin of this and what are your own views about the use of cadaveric dura in this country now?
	DR. HURWITZ:  Well, it certainly is a LifeNet policy.  I don't know if AATB has a specific stand on dura.
	DR. WOLFE:  When did LifeNet adopt this?  Is it recent in the wake of the alternative tissues or what?  When did you come out with it?
	DR. HURWITZ:  I don't remember. For several years.
	DR. BOLTON:  Any questions?
	DR. DOPPELT:  Rich, in terms of the Allowash could you clarify how different tissues are processed, for example, bone versus the soft tissue, bone, tendon, bone or fascia lata versus the cancellous cubes or struts?
	DR. HURWITZ:  Bone tissue undergoes processing with all of the Allowash steps, which include the detergents in association with centrifugation or ultra- sonification, isopropyl alcohol, and hydrogen peroxide. Soft tissues do not tolerate the hydrogen peroxide step very well and that is not done with most soft tissues.  Fascia is generally irradiated, tendons are not, and they are not treated with the peroxide.
	DR. DeARMOND:  Is there any residual skeletal muscle attached to any of these tissues?
	DR. HURWITZ:  No.
	DR. DeARMOND:  And as far as we know there has never been a case of CJD that could be linked to these grafts.  Is that correct or have I missed something which I do all the time?
	DR. BOLTON:  I just missed your questions.
	DR. DeARMOND:  The bone and tendon grafts, has anyone ever reported a case of CJD that could be linked to it?
	DR. HURWITZ:  Not that I'm aware of.
	DR. BELAY:  Not as far as I know.
	DR. BOLTON:  Thank you very much.
	Our next presenter is Richard Russo.  He is Executive Vice President and General Manager of International Osteotech, Inc., from Eatontown, New Jersey, which is near where I live, actually.
	MR. RUSSO:  We are going to keep this moving along quickly.  We have already touched on several of the issues that I was going to present today.  Other speakers have already touched on them so I am going to move quickly here.
	The important thing I want to make with this slide is that risk reduction is a multifaceted activity and cannot rely on some processing step all by itself.  So in order to have an effective risk program not only do you screen donors.  You have to have adequate donor deferral and tissue discard policies, single donor processing most likely, adequate cleaning and disinfection be batches, and we will come back to that later on, and certain processing standards to actually either remove or inactivate agents of infection.
	Even though we have three different types of pathogens that can cause disease transmission in tissue we are going to focus today in my talk on viruses and standard non-demineralized grafts and viruses of concern are these.
	Before we go right into inactivation and removal it's important to remember why a surgeon is doing a bone graft and why we are providing that surgeon with allograft tissue.  The primary purpose is to support bone formation across a defect.  Not all processing has the same effect on bone graft performance.  Essentially the bone graft can overwhelm the patient's ability to form bone at times and we have to make sure that we tip the balance in the favor of bone formation when we are processing.
	Failure of a bone graft typically leads to revision surgery.  So a failure of a bone graft is not without risk to the patient.  It has direct morbidity and risk of disease transmission in the second procedure.  So we have to be careful when we are thinking about processing for safety if that processing for safety is done in such a way it also limits the capacity of the graft to support bone formation.
	Here are some of the types of treatments that can be used in bone processing or tissue processing which will have at least an effect on bone formation. So, as you can see, a large number of things can impact on an allograft bone graft negatively.  This is just an example of the way that you might process a certain type of tissue, demineralized tissue, with three different treatment programs or processing programs.  This information was presented at the North America Spine Society and was conducted by another firm and they looked at the three different types of demineralized grafts from the perspective of osteoinduction and they found dramatic differences in the graft performance in the validated animal model.  So I'm making a point that processing matters.
	Well, when you start looking at risk reduction you need to start thinking about the tissues that you are going to process.  We are speaking here specifically at Osteotech about musculoskeletal tissues, both soft tissues and hard tissues and with hard tissues we are looking at cancellous and cortical tissues.  Today's presentation is focusing on the hard tissue.
	Now, we need to start thinking about the tissue that we are actually processing.  So we can realize that bone tissue has two general phases, organic and inorganic.  The inorganic phase is the largest phase of the bone.  It's approximately 70 percent by mass so the bone is 70 percent mineral by mass.  That means 30 percent is organic.  So we have to begin to think about what diseases will be in the organic side.  Since we are thinking today about blood-borne viruses we are going to especially focus on the blood supply.
	Well, this is a nice picture of a bone.  I know everyone is very familiar with it, but you need to put your hat on and say well, if you are processing bone what are you facing?  You are facing a tissue which has both a long diatheses here with a large intramedullary canal as well as cancellous bone up the metaphyseal area here.
	And bone is heavily vascularized even though there is a large mineral component.  Approximately 95 percent of the blood supply in bone is going to be in the intramedullary canal and about another five percent is going to be in the haversian and Volksmann canal systems.  This is a picture of cortical bone and you can see even though it's very dense there's a lot of vascularity to this tissue so virus can in be a lot of different places in bone tissue.
	Trabecular bone and cancellous bone, on the other hand, has a very different structure and is very open and this can be viewed as an extension of the intramedullary canal and that is how I categorized it before.
	So now we know where the blood is and blood is being the primary vector here for these viruses of concern.  We need to start thinking about the maximum viral burden in bone before we start thinking about log reductions.
	If we don't know how much is there, as was asked before about TSE, it means nothing to say I've reduced so many logs because that begins to become misleading and you think you are doing something good when maybe it is inadequate.  So there are no published reports that quantitate viral burden in bone but you can extrapolate from blood because that is the vector.
	We know that bone is divided into discrete compartments and we remind you again we are talking about primarily the medullary canal, the Volksmann, and haversian canal.  The osteo-sites themselves are not directly connected to the blood system so that's not where we are going to find a lot of virus.  But we are looking primarily at the Volksmann and haversians system and the medullary canal.
	We had to decide what was a dose of bone.  What would a patient be exposed to? Looking at the records, we decided to be conservative.  The virologists were telling us this constantly so we decided that 90 cc of nondemineralized tissue was probably a good dose to work with.  It is probably more than most patients would see.
	Occasionally a patient will have a whole bone replaced and then we are talking about hundreds of cc but for the most part the vast majority of patients are going to have less than 90 cc so we used 90 cc as a conservative marker.
	We calculated the blood volumes by, to be honest with you, hiring a consultant who is a specialist in bone architecture, Dr. David Burdine at Indiana University Medical School, who calculated the blood volumes in the various compartments.
	Then we were able to go with the virologist to calculate the theoretical maximum viral burdens so now we have arrived at what we have to remove.
	So how do you go about this then? We'll show you some numbers in a minute.  As was suggested by the FDA speaker right before lunch, we followed pretty much his protocol.  We built a model facility in the lab which was qualified for this.  You cannot introduce it into your factory, obviously.
	We had a relevant panel of five viruses.  We did not use the woodchuck virus.  We used the duck hepatitis and bovine diarrhea viruses to get at the hepatitis issues along with HIV, CNV, and polio, which we used as an exceptionally resistant and stable virus.  So we had a complete panel with the biophysical characteristics of the RNA and DNA.
	As was suggested, we demonstrated quantitative recovery so we could know how much we could claim.  That was very important.  You may not be able to recover any more bone but most likely you put more virus into the bone tissue than you could recover.  You are limited to the amount that you could recover.
	We go through a process step.  We quantitate the viral log reduction and we calculate the difference between the maximum viral burden and the log reduction.  We don't give any value to a step that reduces less than one log of virus and the viral clearance in total has to be at least in our opinion three logs greater than the maximal viral burden to provide a significant or acceptable level of safety.
	We had first started working on demineralized bone.  Demineralized bone is a type of bone which goes through a primary process first and then after it's finished, like most of the normal pieces of bone that Dr. Hurwitz showed you, most of them, the larger mineralized pieces, it has been ground up, thoroughly demineralized, and in our case there's less than .5 percent residual calcium and the particle sizes are about 100 to 500 microns in size.  So there has been a lot of processing.  There is virtually no blood, cellular elements, or lipids left.
	So the maximum viral burden we calculated for these three viruses, and these were the only ones we could get at that time information on from the blood values, was almost low three logs, 3.18 logs.
	Then we did it for mineralized tissue and we looked at both log values for active and inactive marrow.  Younger patients will have active marrow.  They will have more virus in that marrow or at least can have more virus in that marrow so we went with the higher, more challenging numbers.  So we decided that this was going to be the basis in nondemineralized bone for the maximum viral load or theoretical maximum viral burden.
	When we evaluated several steps in the demineralization process to take a look at what we could do there with that type of tissue we were able to achieve these log values for a demineralization step and, as was suggested before, we actually had two demineralization steps in this process but because they operate by the same mechanism you can only claim one.  We then looked at ethanol, which also has two steps in there, but we only claimed one.  These marks here indicate that no virus could be recovered after the treatment, but this was the maximum amount that we had demonstrated recovery of so we are limited to that number.
	Interestingly enough, we found out that lyophilization had virtually no effect on HIV or CMV and it was not done for the duck hepatitis virus, so that actually was a very minimalistic step.  But the total log reduction values are at this line here.  We take the maximum theoretical viral burdens and we subtract those and we come up to a safety assurance level or SAL.  So these were the log reductions and this is the viral burden.
	For mineralized tissue that had not gone through the demineralization step but was actually finished before that we used the surfactant and alcohol and removal as our three major steps and we were able to achieve these inactivation steps with alcohol.  That was with surfactant.  So total inactivation values were down here. Removal was also done and we then were able to get, and that is stated over here, got total clearance, clearance being the aggregate of removal and inactivation.
	We compared to the theoretical maximum viral burden, which we see here and we used a higher value, obviously, because we were being conservative.  You see the safety assurance level.
	So the take-away here is that current validation guidance documents and virology perspectives can be successfully adapted to the processing of bone tissue but not all tissue.  I would not know how to do this for heart valves.  I would not know how to do this for other forms of tissue.  This does not apply to ligaments and tendons. That allograft performance can be maintained while producing a significant SAL using conservatively calculated theoretical maximal viral burdens.  These are some of the guidance documents that we worked with. These are the old standbys.  They were not designed with bone in mind but they can be adapted and here are some additionales.
	I want to talk to you a little bit about TSE and then conclude.  What we did was look at prion inactivation by our existent or current cleaning procedures in an engineering study.  I myself was not part of this study team, so I'm giving you the summary results of this.
	They did spike samples using the hamster scrapie homogenate.  They evaluated by the prion western blot assay and they looked at the solid surface cleaning procedures we used for tables and saws and things like that but mostly just on tables.
	We used two different agents as disinfectants.  One is Sporklens, which is a commercially available product that has peracetic acid, hydrogen peroxide-acetic acid, and has a pH down in the 1.5 to 2.0 at the level of use, and Expor, which contains chlorine dioxide and has a pH down at about 2.7.  They did three runs, one with Sporklens, one with Expor, and were able to show significant value log reductions but the question is what's the burden.  We don't know.  We don't know how safe this makes the surface after it has been cleaned yet.  We need to come up with a maximum TSE or prion burden.
	But we can adapt this validation process to this task and that's the important thing.  Now, I also want to take a look at one other thing and that has to do with post-cleaning residuals of tissue on hard surfaces.
	We were able to develop some methods for evaluating down to the limit of detectability of how much lipid, bone, and blood would be left on a table, a bandsaw surface, or a MedClean after it had just been physically cleaned.  What we got were levels of lipids down here.  What we're talking about is micrograms per square feet. As you can see, you can do a very effective job on blood, eventually.  We had to develop some new assays to get down to that level. There weren't any commercially available.
	But the important point here I want to make is that cleaning and the standard GNPs are just as important as the use of any more advanced or high-tech cleaning procedure or processing agent.  So with that I think I've kept my promise.  I think we are 12 minutes and we are done.
	DR. BOLTON:  Thank you.  Questions or comments from the committee?
	DR. DeARMOND:  What was the cleaning?  How did you clean again?
	MR. RUSSO:  Well, what we did is we have a written SOP that has been validated before for disinfection and inactivation of viruses.  We took that same SOP which I can share with you at some point in time.  It was really a simple SOP that is not so different than what should be done most places.
	We used the Expor and the Sporklens and we were able to get the tissue burden, if I could call it that, the residual tissue down to those levels.  We were able to achieve those log reduction levels in the prions.
	DR. GAMBETTI:  You mentioned here that you Western blot.  I guess it was used also to see how really the process of cleaning, how successful, so could you expand on that, on how you used the Western blot to prove that the tissue had been cleaned of prion?
	MR. RUSSO:  Well, these two sets of data were generated independently.  So we had done a series of cleaning validations prior to that using different assay methods to detect the present of residual lipid calcium phosphate for bone and hemoglobin for blood.
	Then separately or recently we did this engineering study on the log reduction. So we didn't use the western blot assay for the detection of the -- well, we may have in one of those.  I'd have to get back to you on that to make sure that I don't misspeak myself to let you know which actual test was used to determine the residual organic material.  I will do that, okay?
	DR. BELAY:  I was just curious about how you actually spiked the different viruses into the bone.  Were they injected in a vessel?  And what kind of detection methods did you use for viral load?
	MR. RUSSO:  Well, I believe that actually two different methods were used. In the demineralized tissue, of course, we are dealing with something that's approximately one to 500 microns in size and that is going through these solutions.  So essentially what is happening is that the tissue is spiked but then put into the solution and the whole bone, the non- demineralized bone, they create a cavity and then actually put in a stopper.
	I might have to go back and review that protocol because it has been a while, but it is actually a physical mechanism in which after a lot of validation to figure out how we could demonstrate recovery we actually drilled a hole and inserted a little piece of material containing the virus and then closed it back up.
	So it's not the same thing as natively infected tissue which is going to be infected throughout the bone marrow cavity and throughout the haversian system but was the best that could be approximated using a three-dimensional object.
	DR. BELAY:  Did you use cell cultures to detect the viral load?  What was the detection method?
	MR. RUSSO:  Pardon me?  Jim, can you help?  There were cell cultures that were used but I don't know precisely which. The experiments were done by Quality Biotech, which is now a division of ViroMed.
	OsteoTech has an R&D Department but we're focused on bone formation and we have to go outside for issues on safety and things like that.
	DR. BOLTON:  Other questions?
	DR. PETTEWAY:  You showed the potential to remove viruses using the system with several viruses so the potential may exist that you could remove prions.  Have you thought about how you would do those experiments or if they are worth doing?
	MR. RUSSO:  No, to be quite honest with you.  One of the things we did was come here today to learn about the state of the art so we could figure out if such a thing was possible.  If you look at the data that I presented you'll notice that in one of the studies for the mineralized or the non- demineralized tissue we did not use HIV as the marker.  We used murine leukemia virus.
	That was because the laboratory became a little bit upset about us spiking bone and then using high pressure to force it through the channels of the bone in producing a liquid spray in their laboratory.  They felt that spraying HIV wasn't the best thing.  At the moment, given what we don't know about TSE, we are little concerned about how to safely conduct such studies.
	DR. BOLTON:  A question from the audience?
	DR. FARSHID:  Farshid, FDA.  I just would like to say that the estimate of viral clearance should always consider the worst-case scenario.  The worst-case scenario would be the highest estimate of viral load in that given donor.  That is how we look at it.
	I think looking at the tissue separately and trying to determine the viral burden in the tissue will introduce a variable which is very difficult to control. Therefore in order to have a high degree of assurance that the system works you need to consider the highest level of the virus and that probably for most of the virus will be the viral load in ÄÄÄÄ period.
	MR. RUSSO:  We can talk about this later but we can possibly pick up the highest viral load that would exist outside of bone at the highest inspection period and use that.  Then we can possibly compare the two and that would be an interesting thing to do.  That is an interesting suggestion.
	DR. BOLTON:  I think we will move on to our next presentation.  Our next presentation is by Dr. Randall Mills.  He is Vice President of Operations for Regeneration Technologies and he also will be talking about process validation.
	Dr. Mills.
	DR. MILLS:  So we don't cover too much of what has already been covered I'll try to skip as much of this that the other speakers have already touched on so we can go on to the next slide.
	As the person responsible for producing tissue at our facility, and we are an extremely large producer of human allograft, processing over 5,000 donors a year and about 200,000 allografts, I spend a lot of time worrying about the types of issues that are associated with allograft safety and making sure our recipients receive the safest allografts possible.
	To that end we developed a process to actually sterilize tissue.  There's a number of reasons why we did this but I want to point out there are really three typical reasons why tissue that would be used in allograft transplantation would not be sterile.
	The first and probably the most significant out of these three is that these tissues are recovered cadaverically.  So after a person dies there is an opportunity for bacteria that normally reside in the gut to cross over the gut lumen into the blood stream and contaminate the tissue.  In actuality this is happens quite frequently even though we recover tissues with inside the FDA, State of New York, and AATB standards it is not uncommon.  As a matter of fact it is actually more common to see contaminants on tissue depending on the extent you culture them.
	Now, some important issues about these contaminants where contamination occurs in this way is that these contaminants are typically pathogens and very often may be spore formers and you have heard about the clostridial transmissions that have occurred recently that the CDC is very interested in, too.  Most likely occurred via clostridial spores.
	The next type of contamination that occurs is during tissues recovery.  Now, two points about this type of contamination. It's typically a very low bioburden and the second thing is it's typically a non-pathogenic bioburden, either coagulate ÄÄÄÄ staph propionic bacterium or the bacillus species.
	Then the last type of contamination that could occur would be due to a screening failure.  This is primarily for viruses and without a doubt out of these HCV represents the greatest risk to tissue banking right now, at least as best as we can estimate this.  This is based primarily on the very high level of sero-prevalence we see for HCV among tissue donors being somewhere north of 1 percent confirmed RIBA positive HCV patient.
	So with this in mind we developed a tissue sterilization process that would allow us to transition from the aseptically processed model to what we call the BioCleanse model.  As occurs in the aseptic processing model, we use donor screening first.  We follow that than by a sterilization process.  We just thought that sterilization, although lofty, should be accomplished.  We are then able to conduct a sterilization review and then lastly we have a culturing scheme.
	For conventional tissue, and this has been talked about, this is our viral screening panel.  Before we process any tissue at RTI we do a significant amount of up-front screening to make sure that the tissue is safe and this is our viral testing panel.
	For conventional agents on the back end we also do USP sterility culturing, 14-day destructive culture, aerobic and anaerobic, two-temperature, two-media culture.  We think one used in conjunction with appropriate bacteria stasis and fungus stasis testing represents the most sensitive method for determining contamination.  We also monitor the environment.  We evaluate the strength of our tissues, residual moisture, and again, process run records.
	With regards to TSEs we have talked about this for some time with regard to donor screening.  We do all of the typical TSE exclusionary criteria.  We have also instituted the European donor exclusion criteria voluntarily.  I can tell you as a point of interest we have not seeing this affect donation dramatically at all.  We are quite comfortable with the decision.
	We also do tissue exclusion.  Now, we don't process dura mater but we also don't process vertebral bodies and obviously we don't process corneas.  This is all because of the potential risk of contamination of cerebral-spinal fluid or brain tissue.
	We have line clearance and tissue segregation and we have also, and what I'll talk the majority of the rest of this presentation about, processing measures aimed at removing different types of disease reservoirs.
	So this is the process that we developed to do this, BioCleanse.  It's a low-temperature chemical sterilization process that is fully automated configured similarly to an autoclave.  We load the tissue into this device, we seal it up, and it uses very rapid fluctuations between pressure and vacuum to fully penetrate and fuse the tissue with different cleaning solutions.
	Also, during this process we use multiple fluid exchanges and that leads to massive serial dilution.  So we end up with a very large reduction of organic material left in the tissue and then lastly we follow that up with rinse cycles to remove all the different chemicals that we use to sterilize the tissue.
	A note about the process, it does not use any excessive heat, does not use any irradiation, and does not use ethylene oxide.  This is an example.  This is actually the BioCleanse system at our facility in Alachua, Florida.  What you are looking at, that's actually a two-story configuration that sits inside an 85,000 square foot manufacturing facility.  It's, again, configured like a pass-through autoclave so what you are looking at is tissue would come in one side, be sealed into the chamber, be exposed to the process, and then be removed from the other side so we have unidirectional flow of tissue throughout this process.  It's a very complex process, obviously, by this three-dimensional ÄÄÄÄ it is also a very expensive process.
	This is just an example of some of the support equipment that is required to run this process.  Again, this is that second floor.  This is where the different chemicals and solutions are prepared and delivered to the sterilization chambers below.
	This is what a sterilization chamber looks like.  There are four in each bank.  We have two banks of four.  Each chamber is a totally a separate and independent unit in and unto itself.
	This is an example of a technician loading tissue into the process.  After he is done putting the tissue into the process he seals the chamber up and enters the donor ID.  And then one of the nice things about this process is that it is completely automated.  One of the reasons we were able to validate this process so successfully is because it is an automated process that is not technician-dependent.  So the process is run and actually controlled and monitored from a remote location so we can avoid bringing contamination into our tissue-processing facility.
	When we did the validation for this, unfortunately at the time we were doing the validation for this process, there was no road map so we really had to cast a very wide net.  So we thought long and hard about the types of parameters that we thought were necessary for tissue sterilization process.
	The first we think is essential is to have complete matrix penetration and then followed by removal of potential disease reservoirs, particularly blood and bone marrow and lipids.  Dr. Rohwer spoke earlier about it and he hit it dead on.  You cannot sterilize what you cannot touch.  So unless you get complete penetration and removal of these elements it is very, very difficult, if not impossible, to actually sterilize tissue.
	Obviously we wanted to have a process that could eliminate bacterial and fungal contamination, including spores. That became important here recently with the recent reports of infection and a death. Enveloped and non-enveloped viruses have a process that we can remove the germicides that we use and then lastly have tissue that is functional.
	Now, as we were doing all of these we thought there were a couple of overriding principles that we had to keep in mind.  One is we had to do all of these under worst case conditions depending on the type of study we are doing that factually defines worst-case conditions.  We also wanted a validation that would account for both process variability as well as tissue variability so for all of these studies we had to look and consider whether it is a younger donor, a middle-aged donor, or older aged donor, male and female, and other types of tissue variabilities, the different types of tissues that we see.
	We wanted the process to be, obviously, very repeatable and whenever possible we conducted all of our validation studies in full-scale production equipment using the technicians that actually run it as opposed to scaled-down laboratory studies which for some agents obviously were important.
	Now, what we ended up with at the end of this was a massive validation.  We have over 10,000 pages of validation data on this BioCleanse process and I can tell you after having gone through a lengthy review of our validation studies with the FDA although it was a painful experience, and I'm not going to tell you it wasn't, it was a very constructive experience at the end. We think we came out on the other side of that process much better because of it.
	I would touch on a couple of key studies we think are important.  Obviously, with over 10,000 pages now I can't go into all of it but a couple of key studies that we think were pretty significant.  The first is a tissue penetration study.  In this study we added a tracing dye to our cleaning solutions.  We actually complexed in this study Fitzi (?) to a 65 kilo ÄÄÄÄ protein. We loaded tissues into the chamber and we ran the cycle for only five minutes.  Now, a typical full run of BioCleanse is eight hours long, so this is five minutes out of eight hours.
	We removed the tissues and examined them histologically.  As was alluded to in the previous presentation, this is what the haversian system or a vascular system of cortical bone looks like.  So we evaluated tissue histologically and looked to see where dye had penetrated and in fact at a five-minute time point we were able to completely penetrate the deepest aspects of not just cancellous but cortical bone and in fact tendon sample size 59 times in a row.
	In this study we were looking at the same thing but using endogenous substances as the surrogate marker.  We evaluated histologically tissue for the presence of blood and marrow elements.  Now, obviously on the left is a proximal femur that has been sectioned open and inside is what you would typically find, blood and bone marrow.  On the right is a BioCleansed femur that was processed whole and intact. There were no cuts, holes, or other manipulation done to the tissue before it was processed and it was sectioned afterwards.
	Macroscopically it's very clear to see that the process does a very good job of removing blood and bone marrow from the medullary canal and from the cancellous bone.  When we looked histologically we saw the same thing was also happening. Obviously on the left-hand side we can see haversian systems with blood elements.  On the right side same haversian systems have been completely evacuated of blood and bone marrow.
	From this study we actually have started doing, and I have some preliminary results but not in slide form, a prion removal study using this concept.  What we did was we spiked scrapies into the marrow cavities of these long bones, created a scaled-down model for this, obviously, and ran it through the process and then did a mass balance to see where the prion had gone off and obviously at the end completely homogenized the tissue and evaluated that by western blot.
	Now, I will tell you with all the caveats this was a pilot study.  This was to determine feasibility of a larger scale study but out of that we actually got very positive log reduction.  It seems that log reduction of at least three logs is going to be very possible with this system.
	This study actually combined two what we think are very important factors. One is being able to penetrate the tissue but the second is being able to penetrate the tissue and actually penetrate it sufficient enough concentration and quality of germicide where you can achieve sterility inside the cortical bone.
	So what we did in this model was we drilled small holes inside the densest part of cortical bone, again being part of this worst-case model.  We took the densest, thickest part of cortical bone, drilled small holes, and we took Bacillus Stearothermophilus, which for this process is the most resistant biological indicator, and we had trapped it inside the bone.
	We sealed it up with a self-tapping titanium screw and we ran this at one-quarter of the total processing time, about two hours of contact time in the process.  At the end of that we removed the biological indicator.  We cultured the biological indicator as well as the construct, using a test that would detect the bacilli stearothermophilus if it was there.
	We ran positive, negative, and recovery controls and actually had to spend a significant amount of time validating this model.  The results at the end of that were 26 out of 26 times we ran this process we were able to sterilize without failure the biological indicator seated within the densest part of this cortical bone.
	Now, that was a very good construct model that we prepared because it was using a very resistant organism in the most difficult part of tissue to reach.  In this model we went with a little bit more of a relevant testing.  We actually went out and recovered donors with premortem septicemia. They had multiple bacterial pathogens both gram-negative and gram- positive, aerobic and anaerobic organisms.
	We took the bioburdens of those greater than 1900 CFU on Donor One per gram and greater than 1400 CFU per gram on Donor Two.  We processed these tissues within BioCleanse.  Then we took the tissue out and we destructively cultured the tissue again in the two media, two temperature, culturing scheme recommended by FDA.  Obviously we had all the necessary bacteria stasis and fungal stasis testing.  What we ended up with was all of the tissues for this model ended up sterilized and completely free of contamination.
	This is a broad range of all the different viruses and bacteria.  Obviously here we were able to completely cure enveloped and non-enveloped viruses, RNA, DNA, small, large, resistant, and easy to kill.  The different types of vegetative bacteria and fungi we established this process can kill are the typical types of things we either see contaminating the tissue at recovery or associated with orthopedic infection.
	Then lastly we validated the system against spores, both clostridium spores and bacillus stearothermophilus spores.  None of this clearance took more than one quarter of the process time.  So again we have a great amount of overkill and a tremendous amount of redundancy built into the process.
	Really quick to go through strength testing obviously for our constructs this was alluded to earlier.  If you sterilize the process but you damage the tissue during the process you really haven't done anything beneficial to the patient.  So we needed to make sure that the tissue was functional so we tested it in a number of different applications.
	This is axial compression.  This is very good for spinal applications which the majority of our tissue is used in.  Compared BioCleansed tissue to untreated tissue, untreated was not processed in any way, BioCleansed again for worst case, to make this as difficult as possible, we actually ran it through the process four times so we would exaggerate the conditions the tissue was exposed to and noticed no difference.
	This is by diametral compression. It is the most sensitive method for testing. Again, no difference.
	Shear testing, this is good for torque.  A lot of our tissues are actually machined in this shape that gets screwed into the patient.  No difference again in shear testing.
	Three-point bend testing for the most part is actually for bone.  It is really a meaningless test but it is the one that is most often referenced in the literature so we did that and there was no difference there.
	This is actual product testing. This is probably the most relevant thing we did because this not only picks up differences for the process but also looks and sees if there's any downstream effect of either freeze-drying or freeze-drying followed by reconstitution and again no difference.
	From the biocompatibility standpoint we needed to make sure that this tissue was functional when it was implanted. The first thing we did with regards to biocompatibility was we validated the process to completely remove all of the different germicides that we use.
	So our starting point for these tests was essentially sterile, clean bone. We ran it through this biocompatibility problem and did not have any reactions going through it.
	So looking back on what we were able to do, we were able to completely sterilize the tissue because we were able to completely penetrate the tissue and remove all of the blood and bone marrow.  Our process is not just bactericidal and spungicidal but it's also sporacidal.
	We can kill enveloped and nonenveloped viruses.  The tissue is functional and biocompatible but, most importantly, because this is a completely automated process it is a validated process. It is one that is not dependent on technician interaction for its success but in fact is reproducible.
	So just to conclude there is a residual risk of disease transmission when only screening and testing are used but clearly the things that pose the greatest risks are HCV and bacteria.  We believe that both tissue exclusion, not processing things like dura mater, vertebral bodies, and removal of diseased reservoirs reduce the risk of both conventional and emerging pathogens and our initial prion data seems to support that.
	This process has been validated to sterilize tissues without altering its biocompatibility or its biomechanical integrity.  The process is completely automated which allows us to validate it. We have also implemented this process in anticipation of FDA's GTP requirements that are coming out which we think are going to be exceptionally important for the industry.
	Then lastly we have had a tremendous amount of clinical success with this.  We have had over 200,000 of these grafts implanted, we have had good acceptance by the surgical community, and we have not had a single infection since we have used this process.
	So I'll take any questions.  Thank you.
	DR. BOLTON:  Thank you.
	Questions?  Yes.
	DR. LINDEN:  If you are concerned about HCV why are you doing that only for HIV and not HCV?
	DR. MILLS:  That's a really good question.  The answer, and we're working hard, actually, to do NAT for HCV.  The problem is our samples are collected cadaverically.  The problem we have had is sample stability because the blood sample gets drawn from a cadaver.  If any of your are familiar with what that looks like it very frequently can be associated with a tremendous amount of hemolysis.  That can be as late as 24 hours post-death.
	Then the sample would need to get spun down and sent to our testing facility. Now, we could test it at our testing facility almost immediately but a lot of these tissues are being transported across the country so realistically there is at least a 48-hour period before we could actually get the samples up on test.
	And what we have seen so far is we just have some sample stability issues. Now, we are trying to work on ways of stabilizing the HCV nucleic acids but we are not there yet.
	DR. BOLTON:  Other questions?
	DR. DOPPELT:  You said a couple of times that in the BioCleanse process you are using chemical solutions and germicides. What chemicals and solutions are you using?
	DR. MILLS:  We use three fundamental types of solutions.  We use alcohols, peroxides, and detergents.  Now, how we mix that is a proprietary cocktail but we use those three in different forms.
	DR. DOPPELT:  Is that in any way different from what other people are using?
	DR. MILLS:  The cocktails are, absolutely.
	DR. DOPPELT:  But, I mean, they are all using detergents and --
	DR. MILLS:  I can tell you the order is important.  Obviously the most fundamental thing that's important is you have to completely penetrate the tissue. Dr. Rohwer said if you can't penetrate it, you can't kill it.  So there's a big difference between taking a piece of tissue and soaking it in isopropyl alcohol versus taking a tissue and completely perfusing it with isopropyl alcohol.
	DR. BOLTON:  Ermias?
	DR. BELAY:  You would say you had already implemented the European deferral policy in your company?
	DR. MILLS:  Correct.
	DR. BELAY:  I was wondering if you could tell us a little bit more about the kind of policy that you implemented and your experience because there is a lot of data on the impact of that policy in cells and tissues and your experience potentially would be helpful for us.
	DR. MILLS:  The European exclusion, we have had that in place, I believe, for over six months now.  I think we are obviously changing to the new guidelines now, but I think the one we had was European travel over the last six months.  We have had no impact, almost no impact.  We have excluded some donors but there have been very, very few.
	A point that should be considered is that we don't necessarily draw from donor pools that are around military bases and that is something worth considering.  Just because we haven't had an impact doesn't mean the industry as a whole wouldn't have an impact.
	DR. DOPPELT:  I may have misunderstood.  You said something about a log reduction of three for something.  That was scrapie?
	DR. MILLS:  Yes.
	DR. DOPPELT:  What is your log reduction for the other viruses like HIV and HCV?
	DR. MILLS:  Well, starting out, the spores are greater than six log kill of the most resistant spores at one-quarter cycle. Everything else we did we did with less than one-quarter cycle.
	So, for example, with HIV we actually only tested two compounds against HIV.  Both of them had greater than four log.  We ended up with I think 8.8 log reduction for HIV just on two chemicals. All three compounds would have obviously inactivated the HIV.
	In all cases all viruses were brought to non-detectable limits so it was really just a matter of what we could spike into the tissue, not on the tissue but actually into the tissue and then what we could meaningfully recover.  For HCV it was something like 13 logs.  They get absurd they get so high but the viruses actually are pretty easy.
	DR. BOLTON:  Maybe I misunderstood. Do you have a scaled-down version of your BioCleanse unit in your system?
	DR. MILLS:  Yes.
	DR. BOLTON:  And that's what you conducted the prion studies in?
	DR. MILLS:  Yes.  We actually have a couple of scaled-down versions depending on the types of studies we are performing.
	DR. BOLTON:  Other questions?  Let me ask you this.  Your full-scale version is for processing larger amounts of tissue in a maybe more efficient means.  Is a scaled-down version something that could be used on a smaller scale by other tissue manufacturers?
	DR. MILLS:  The reason we went to the large, it's one of those machines, that enormous complex, actually processes a little chamber one at a time.  It's one donor's worth of tissue through that at a time.  So actually it's almost staggering to say that mammoth machine we put up there is pretty small scale already.
	When we scaled them down to the laboratory standpoint they do get to the point where the reaction chamber could fit on a table and you would have a series of other chemical banks around you and the like.
	The problem runs into this.  If what you are trying to do is anticipate GTPs then you need equipment that's validatable. You need software that's validatable.  There are just, like, a number of considerations like no threaded pipe, sanitary valves.  The whole thing has to be sterilizable itself.
	So you run into facility costs that I can tell you ran us about $25 million.  It would be difficult to make, like, an autoclave version of this that you could just sell as a unit.  It would be very difficult.
	DR. BOLTON:  Thank you.  We will now move on and back to Bob Rohwer, who's going to tell us about the experience with TSE agent clearance studies in experimental models.
	Bob, I'm going to ask you if it is possible to go less than 30 minutes?  Thank you.
	DR. ROHWER:  Over the last ten years we have done quite a large number of clearance studies on a commercial basis and some of them we did out of our own interest as well.  That's what informs the presentation I am going to give you now.
	These are some of the things we have looked at over the years.  We have done a lot of these studies using blood and blood products both from bovine and human origin. A number of studies were done using the cone fractionation, but we have also looked at the Kissler-Nietschman fractionation System.
	We have done quite a bit of work with various manufacturing methods for bovine collagen and then we have just recently have completed on bovine gelatin. You will hear tomorrow from Whizzer Gregori about a series of experiments that were done characterizing the Asahi Planov filters with these agents.
	So the types of manufacturing steps that we have looked at are given here, depth filtration, membrane filtrations, phase separations of various sorts, extractions, precipitations, column chromatography, thermal inactivations, irradiation, chemical inactivations, and others.  What I'm going to focus on is rather than describe individual experiments which would go on and on and on I'm just going to give you the highlights of what we've learned from this experience and we have learned as we've gone along.
	I think we always did design good experiments but we're designing even better ones these days with this experience behind us.  The key elements in these types of studies are the scaled-down process itself, the choice of agent and host that you're testing, the spike modality and how it's introduced into the material, and the processing itself and how you do that.  We are just going to go through these things one at a time.
	The scale down is usually left to the client.  Quite frequently what we are brought is an existing virus validation procedure which they've used in the past for HIV, porcine parvo, et cetera, to look at conventional viruses and it is relatively easy to adapt that to the study of the TSE agents.
	On the other hand we have learned some things as we've gone along.  Some of these scale-downs are really scaled down and our preference has become, even though it would seem to be more convenient to work on the 100 mill or 50 mill scale, what often happens as you work through a process is your sampling starts removing volume and material and by the time you get to the end you are working with very little material. You have very little room for flexibility in sampling an assay.  If you want to take an extra pH sample it gets a difficult and, quite frankly, we prefer to work on a slightly bigger scale.  The other thing is as you get to smaller scale surface effects begin to dominate the separations and we worry about that in the case of these very adherent agents.
	Well, the agent is a real issue. We can choose from the various mouse strains of scrapie, the hamster scrapie strain, which is the one we prefer.  There several strains of CJD out there out there that we can use and we have used some of them, the Fukuoka strain in particular.  And we within the last couple of years brought the BSE variant CJD model into our lab using the model that was developed by Moira Bruce at the MPU.
	The only one that is completely unambiguous is the BSE variant CJD model because it is fairly immutable in any animal system you put it in.  If that is what you're looking at it is the relevant strain for that particular agent.
	Otherwise our feeling is that the choice is pretty arbitrary.  For example, it's not clear to me in particular whether the variation we've seen between these various models within strains is any greater than the variation between scrapie and CJD and because there is so much variation it's arbitrary and we use that as a justification for working with the hamster, which is our preferred model.
	One you pick the strain you have to pick a host and there are lots of arguments out there.  Various inbred mouse strains can be used with virtually any of mouse-adapted strains and the hamster strain, which is convenient and well characterized, and then we have the transgenic mice models.
	If the PRP molecule is the whole story then this is a model of choice, definitely, for doing these studies; however, there are some caveats attached to using these strains.  That is that the most effective transgenic models are not real clean representations of the prion gene in the mouse.
	The ones that work best are chimeras, mixtures between the host, either cow or a human, and the mouse gene.  A lot of these vectors carry the doppel (?) gene in with them.  The ones that have the short incubation times usually have many random and multiple insertions and the expression of the gene is actually aberrant compared to its normal context.
	If we really wanted to get to something that was close to a true humanized mouse, for example, we would use one of the replacement gene replacement models where it's a one to one replacement of the model gene with the mouse gene.  The problem with these is that they are not necessarily as convenient to use as some of the others.
	Let's go on since we are pressing for time.  Another example of the things you have to consider here is that what we've noticed is that as a background it's important to remember that BSE, even though it seems to be a very stable strain in terms of its re-isolation from various animal models, nevertheless presents very differently in cattle and humans.  You would never confuse variant CJD with BSE clinically.
	On a laboratory scale this gives you something of a conundrum because we really feel that the hamster 263k scrapie model is clinically much more similar to BSE in cattle than the BSE strain itself is in the VM mouse.
	On the other hand BSE in the VM mouse gives us a disease is clinically more similar to variant CJD in humans.  So the point here is that even at the level of selecting a host strain to work with it's somewhat arbitrary.
	Let's talk about spiking.  The central problem with TSE spikes is that TSE infectivity is poly-dispersed.  It has a wide spectrum of physical and chemical properties because it is typically in an aggregated state associated with other cellular components and these things affect the way in which it fractionates and partitions.
	As a consequence to that it is quite a different situation than you would have compared to working with something like porcine parvo virus where you can make a very highly purified, highly uniform monodispersed agent which regardless of where you introduced it into a process stream you would pretty much have what would be there anyway even if you had brought it all the way from the beginning of the process to that point.
	In the case of these agents you may be selectively removing certain components of the distribution in earlier steps which then don't get tested in later steps or the converse of that is if you introduce the agent at a later step you are removing something that's already been removed by an earlier step.
	Another problem with spikes is that the only source of high titer infectivity in these diseases is CNS tissue of the brain in particular.  And brain drive spikes give you this tremendous advantage.  In the hamster we have almost ten to the tenth (1010) infectious doses per gram but when they're used to spike something like blood or a low titer tissue that has nothing to do with the central nervous system the relevance of these spikes is questionable.
	One way to get around that is to take some fraction of the brain, and a number of people have experimented with microsomes, liposomes, detergent lipid protein complexes.  Aventis has been working with caveola domains, fibrils.  The problem with these things is I'm not sure that they really represent the true {IOn}in vivo{IOff} situation, either.  They behave better in terms of the fact that they are more homogenous when you put them in as a spike but they are not necessarily more relevant.
	The one place where this is not a problem is if the source tissue actually is a brain, for example, if you were doing an experiment on pituitaries, or if your major concern is cross-contamination of the tissue you are collecting by CNS tissues, in which case a brain-derived spike is exactly the right thing.
	The alternative is to use endogenous infectivity.  Here the relevance is not questioned but the problem is the clearance potential is very low because the titers are typically low for non-CNS tissues.
	On the other hand in the case of blood we have demonstrated now that we can actually get very accurate measurements out of these low titer tissues and I will show you in a moment how this can work to your advantage in getting an accurate representation of what you're trying to treat.  Another problem, however, is blood is a little bit unique in the sense that its physical state is the same in large animals and small animals whereas something like organs are quite another story.  It's pretty hard to compare the heart of a hamster to the heart of a cow.
	Heino Diringer ran into this when he did his dura mater validation tests using hamster dura.  The hamster dura is so fragile that it disintegrates under these conditions whereas the human dura would not.
	Another issue is the introduction of the spike.  There's been some mention of that in the last couple of talks but it really is a fundamental issue.  Every tissue is going to have some intrinsic infectivity in it in proportion to the amount of blood that's in that tissue because we know that there is infectivity in blood and at least in clinically affected animals it's about ten infectious doses per mill in the rodents.
	But there may be other sources of intrinsic infectivity and it's very difficult to mimic this intrinsic association of infectivity with solid tissues like dura mater, tendons, hides, bones simply because it's not clear how to introduce the infectivity into the tissue itself in a realistic way.
	And if you fail to do it, on the other hand, when you do this type of test you are measuring the way brain homogenate is inactivated in the presence of these other materials rather than how well the process actually can seek out and destroy the infectivity in those materials.
	The converse of this is extrinsic infectivity, which comes from cross- contamination, and because the brain has such high levels of infectivity and the central nervous system has such high levels of infectivity cross-contamination is fairly likely during the collection of other non-CNS tissues.  And in many cases it maybe the most significant source of infectivity, in which case again a brain drive spike is appropriate.
	There are other issues we could deal with, sampling, sample preparation for titration, assay methods, cross- contamination issues, logistics, but I'm going to go on now and talk about experimental design next.  What we've learned and what I'm beginning to feel more and more strongly about is that wherever possible it's best to test endogenous infectivity first even if there's virtually nothing there.  The reason for that is that you should at least do the test and carry it as far as you can until you run out of infectivity because sometimes it goes farther than you think and there's no question about its relevance.
	We have the methods now for doing this.  The end result is that if you take this approach it really supersedes any evidence that you develop from a spike that claims higher levels of infectivity if you can't develop the same level of infectivity with the intrinsic infectivity.
	I'll give you an example.  Here, this is the example I gave earlier.  Let's go on.  Here is where this fractionation diagram was that I was looking for in the last talk.  Again, this time I'm going to focus not on these two fractions, albumin and IVIG, but rather on the fraction four where after this extensive cone fractionation we still had two infections in this fraction.  This is two infections out of the equivalent of 50 ml of blood inoculated.
	These infections come, as you follow the cone fractionation, on this side they come very close to the end.  The next thing beyond here is fraction five or albumin itself.  So it has carried through all of these steps, one plus two plus, well, that is the soup, actually, all the way to fraction four and the fraction four pellet, which means that without any spiking at all, starting with ten infectious doses per ml and a significant volume of blood, we are able to carry it all the way here and demonstrate about two and a half logs of removal.  That's really the only removal I think we can reasonably claim in that case.
	When you can't use endogenous infectivity we have come to realize that another very important aspect of TSE experimentation is this concept that we are calling conditioning of the spike.  What I'm getting at here is that when you have a process that goes from step A, B, C, D, E, F, G, et cetera, to make sure that when you are testing step C that you are not just retesting and re-removing the same subfraction that you would have removed at step A it's important to run step A, possibly step B, and then take your value from step C.
	In an ideal way the way you would run an experiment is you would spike here, collect and measure until you run out of infectivity, and then spike at B and do the same thing over again, spike at C and do the same thing over again, et cetera.  Now, this would be an extraordinarily extravagant thing to do.  It would be lovely if we had the resources to do an experiment this way but we usually have to make compromises.
	Nevertheless it is important to keep this concept in mind because, for example, in a scheme like this where in the very early steps we might have a solvent detergent treatment of cryo, for example, and a filtration step following it if we then come down here and have to re-spike it, step D, and we haven't gone through this solvent detergent step and we haven't done this filtration we may essentially remove the same material that we removed here again at spike D.
	This secondary spike needs to be treated in some way to condition this spike for what's preceded it in this process diagram.  Of course, the other corollary here is you want to carry the process as far as you can on a single spike before re-spiking.
	The same thing would be true here. If this filtration is equivalent to this one then you really can't count the removal from this filtration because you probably already removed it there unless you establish, as you'll see in the next slide, an experiment and you do the experiment this way where you show that the removal by the filtration is completely stochastic.
	In other words what we're doing here is this is the actually process and this has worked very well for us on a number of occasions.  Again, there will be an example of this in these Asahi studies that will be presented tomorrow.  But basically what we are saying here is if you spike at A and you get a certain level of removal and you're selecting a sub-fraction by so doing it if you then rerun that exact same step over again you wouldn't expect any removal at all.
	On the other hand if you do that removal and it's on the same level as you got here then you can say that the limitation of this step is a statistical one, it is not a selective one, and as a consequence it is valid to count this in multiple iterations through the process.
	How am I doing here?  Not too good. I'm almost done.
	One of the last points I want to make here is another thing that we have learned, very importantly, is that you always want to track the product stream directly for removal and not get this information by some surrogate method.  And I give you an example of a column chromatography here where, for example, if you were to challenge with ten to the eighth (108) infectious doses you might find when you assayed the flow-through that you had ten to the eighth (108) infectious doses recovered in the flow-through.
	You might find the same thing in the first wash.  It might go down in the second wash or the third wash, but it is absolutely essential to look at the eluate because you could also have ten to the eighth (108) infectious doses here.
	And to pass this just to another step without making this measurement is not valid and the reason for that is that the precision of the measurement is only about a half log and when you look at this you really can't distinguish these three numbers significantly so you can see these kinds of things and we have seen them.
	A couple of comments about the evaluation of total clearance, in my opinion endogenous studies take precedence over any type of spiking study.  The continuous processing takes precedence over stepwise values.  In other words the data you develop from a continuous process takes precedence over stepwise values and you have to use great caution in interpreting cumulative removal from similar stepwise steps.
	When you start evaluating total clearance, I think it can be summed up this way.  The exercise is worthwhile because high values are definitely better than low ones but I think it's a big mistake to make too much of this type of data and the actual values should not be interpreted too literally.
	In the end the thing that gives us the greatest confidence in these studies is that as more and more of them are being performed using a greater number of models and modalities we can start to compare them and as the data accumulate for multiple agents, spikes, assays, scale downs, et cetera, the convergence of diverse approaches on the same result provides the greatest security for the ultimate outcome and interpretation of that outcome.
	So there's great value in doing it more than one way in more than one laboratory and with more than one model.
	DR. BOLTON:  Thank you, Bob.  I'm sure we must have questions for Bob from the committee.  No, you are going to stun me. Nobody has any questions?
	DR. ROHWER:  We're all getting tired.
	DR. BOLTON:  Well, I thank you.  I think that you have made these points before to most of the people who have been on the committee in past years and you have made them well, I think, so I think it's all beginning to sink in.
	Well, at this time we will move to the open public hearing and Dr. Freas will take over.
	DR. FREAS:  As part of FDA's advisory committee procedure we hold open public hearings for members of the public who are not on the agenda and would like to make a statement concerning matters pending before the committee.
	Mr. Chairman, at this time we have received three requests to speak at today's open public hearing.  They are from Margie Baker, Moira Kennedy, and David Korroch. Margie Baker, would you please come forward? You can either use the microphone there or come to the podium.
	While you are approaching the microphone we are asking that you address any financial interest that you may have with any products that you may wish to comment upon.  These presentations will be timed for six minutes.  A yellow light will go on at the end of five minutes.  Go ahead.
	MS. BAKER:  I don't know if Dorothy Scott is here but I would like to thank her because she advised me to attend this meeting and she and I both acknowledge that no one has ever caught CJD from beef insulin or from blood transfusion yet people who have used UK beef insulin are deferred from donating blood.  I am a type 1 diabetic survivor for 58 years since age 53 with basically no complications.  My only complications were caused by three weeks on synthetic human insulins, the only insulin that is readily available in the US now. Like many thousands of our fellow citizens, I cannot medically tolerate the synthetic insulins.  Pork insulin is only a little better, very difficult to find.
	And I will tell you why these insulin guidelines must be changed.  I have no financial interest in CP Pharmaceuticals from Wales, UK.  My interest is caused by my need for their beef insulin, which I import, which should be readily available in the open US market because as I was here I broke my one bottom of isophane insulin and I am on the verge of being very sick because I can't get it.
	I called for pork insulin at CV Pharmaceuticals and they don't think they can even get the pork so you might be saying goodbye to me.
	Many suffer or have been maimed or die because it is no longer sold in the United States.  Please don't patronize us by saying import.  Only 50 people in the United States know how to import.  The rest aren't even aware that there is such a thing as bovine insulin, much less find out how to import it, that their health will improve if they use it.
	If they do happen to read the FDA CDER beef insulin site they are scared away by the mad cow warning, which is very inaccurate, so they go to the local pharmacy and buy what is available as the people with diabetes have always done.  Fortunately, I found CP's website, asked my doctor to sign a letter of necessity for my nonprescription drug for the FDA, paid the USDA for my import permit.  It now costs $95.  I typed my personal use letter for the FDA and then filled out and faxed the order.
	That may sound very easy to you but it isn't.  It's harder than paying taxes and the extra $145 permit and shipping is an expensive hoax.  Beef insulin should again be available in our local pharmacies without prescription as it has been since 1921.
	The tradeoff of a theoretical risk of mad cow disease for a real human need is a disadvantage to the diabetic and not a credit the FDA.  The guidelines need to be changed.  I understand that the FDA refuses to allow CP to market their 25-year proven beef insulin here, requiring tests as though it were a new drug and requiring CP to provide that its bovine insulin contains no BSE prions.
	BSE in beef insulin have not even been proven by you, the scientists, but I can prove to you that many have died for lack of what I'm here to talk about.  CP's insulin has already been proven safe over decades by tens of thousands in many countries.  This proven insulin has been approved for market by the UK Medicines Control Agency and regulatory bodies in 12 other countries.
	The governments don't impose suffering on their needy diabetics, shielding them from a remote possibility of CJD.  Is compassion not considered?  As your committee ponders the issues before it today please review the risk-benefit equation as it relates to the diabetic and society.
	Remember the polio vaccine?  That had real risks but was allowed because of a desperate need.  Remember, CJD is not contagious.  Don't block the one manufacturer who is ready, willing, and able to supply our desperate need and involves no BSE risk.  You will eliminate such suffering and expense.
	Please change your guidelines on bovine insulin.  CP has made application for it, I believe, in '98 or '99 and withdrew it because of the FDA's total bogus reasoning. CP's pancreas crystals come from US cattle. Can BSE prions stick to stainless steel equipment for nine years?  Beef pancreas crystals don't carry BSE so how can they stick to said equipment?  Even if the cow had mad cow, well, anyhow.
	Dr. Asher asked me, he said, "Mad cow prions might splatter on the pancreas at slaughter."  Then are there still mad cow parts allowed to be sold in the United States?  Think about it.  The only insulins available in the United States are defective for many.  If I am the one in a hundred million years of treatment that might get CJD from beef insulin then that risk is better that the agony of taking FDA-approved synthetic insulins.
	The FDA is adamant about protecting diabetics from the theoretical risk of CJD if we take beef insulin.  The USDA is concerned about our insulin- contaminating animals.  I hope you can see reality.  We do not need protection from theory.
	The FDA and USDA should change their regulations on the UK beef insulin. The possibility of transmission of BSE through beef insulin to humans has never been seen.  It is an unproven theory.  We ask that you remove the warnings of BSE threat from all beef insulin guides, including the Internet, and allow it to once again be marketed in the United States.  No one has ever caught CJD from beef insulin but many have sickened and died from your guidelines of withholding on UK beef insulins.
	Remember, as doctors you are required to do no harm and to save lives. Thank you, and a copy of my speech is out on the table and I've also put a copy around to all the committee members.  Thank you.
	DR. FREAS:  Thank you, Margie. Thank you for coming today and making this presentation and sharing your personal experience with us.  Your hand-out will be posted on the FDA web site and we do appreciate your taking the time to come up here.
	Our next speaker in the open public hearing is Moira Kennedy from San Francisco, California.
	MS. KENNEDY:  First of all, congratulations for pronouncing my name correctly.  That's very rare.  I actually come from Santa Rosa, not San Francisco, though.
	I was very interested in the speeches this morning because I myself am an expert in transplant from a different point of view from the distinguished speakers.  I myself had a transplant six years ago and I'm very glad that all of those very stringent methods and restrictions were not in place then or I would probably have died waiting for my kidney.
	I would like to thank the speakers, especially Dr. Hogan and several members of the committee, and I think you are one of them, who actually spoke for the patient and spoke for the needs of the patients rather than just for the scientific considerations about transmissible spongiform encephalopathies because our needs with the shortage of organs from my point of view that is the most important.
	Like I say, I'm very thankful that I've got my kidney.  I'd further like to add that it would be too easy to bring in restrictions that would cost more lives than actually save them by these methods that are supposed to make organs and tissues safer.
	I came to address the advisory committee on a related issue which has already been referred to by several speakers.  That again is bovine insulin. The FDA's web page actually devotes more words to warning against bovine insulin because of BSE than it does to giving information about reporting which is what it is supposed to be doing.
	We need bovine insulin available because the biosynthetics or recombinant DNA insulins are not the same as the insulin that your healthy pancreases secrete.  They are chemically different even if they do have recombinant DNA.
	Firstly, once a bio-synthetic human insulin is injected ÄÄÄÄ molecules fold over.  This is does not happen when insulin is naturally secreted.  Secondly, all injected insulins, no matter what they are, follow a different path to the blood stream than a naturally secreted insulin.
	Thirdly, as the previous speaker already mentioned, beef insulin has a greater ability to warn people of impending hypoglycemia.  What has been established and in fact the biosynthetic insulin, Humulin, was placed on the FDA's ten most wanted lists of most reported drugs some years back because so many people have died through hypoglycemic unawareness.
	For those of you that don't know what that is a person who injects insulin can tell if they've got too much in their blood and they need to go and get some sugar or get something to eat.  Mostly you get symptoms through your nervous system and you know to do that.
	With the biosynthetic insulins this often doesn't happen so you might be speaking at a microphone and suddenly you keel over and black out.  It's not really good if you are driving a car and that happens but that does happen.
	This is why some of those people that Margie was talking about need to import at great expense from the United Kingdom bovine insulin because they know that they can't survive without it, especially if they live alone.  They could go unconscious. They could die.  This happens.  This is recorded as happening.  A lot of people live in fear of that.
	A study by the British Diabetes Association found that 47 percent of its members experienced some different problems when they were transferred from bovine insulin to the biosynthetic human insulins during the mid to late '90s.
	I have chosen to use beef insulin despite these unproven possibilities of BSE, TSE, CJD.  Here's my story.  I was diagnosed with type 1 diabetes or what I have renamed as pancreas failure -- that's the new "in" word þ -- when I was a young child and I spent 35 years on bovine insulin without any complications at all.  After I was switched to the biosynthetics I began to experience really wild blood swings from blood sugars up to 600 down to 40, up and down.
	Any of you who are doctors who deal with people who have diabetes know what I'm talking about.  Almost immediately I began to experience memory loss, confusion, and circulatory problems and muscular problems like severe cramping.
	Within four years I was diagnosed with kidney failure.  I already mentioned that I had a transplant.  Now I had to live with the side effects of imminosuppressants which is like another disease of its own which makes diabetes quite simple in comparison.
	These wild swings of high and low blood sugar, especially when you go low after having been very high, and this happens up and down constantly, are implicated in the damage to the small blood vessels of the retina and the kidneys.  I believe that my kidney failure was because of or caused by bio-synthetic human insulins, which is all that is available commercially in this country any more.
	I had no idea that insulin itself could be a problem until I had a chance posting on the Diabetes Forum web site and I began to do some research of my own and also to come in contact mostly through the Internet with other people with diabetes who were complaining about some of the same stuff that I was.
	This was pretty new to me and they weren't making it up.  I wasn't making it up.  Hey, something is going on here. Anyway, six months ago I resumed beef insulin imported from the United Kingdom.  I figured I've got nothing to lose.  I spent 35 years on this stuff.  It's not going to hurt me.
	The first thing I noticed was a much more leveling out of my blood sugars and a return of my memory and it's nice. What was it one of your politicians said, something about losing your mind?  Dan Quale, you know the one.
	Other problems like my circulatory system maybe is improving.  Certainly my muscle cramps and things have improved completely.  I know that I'm doing a lot better on the bovine insulin than I was on the bio-synthetic.  You can't really do much worse than lose your kidneys.
	From my contacts I know that there are more people than these 50 who are importing from Britain who would benefit but many people are scared off by the BSE warnings on the FDA's web site and others are skeptical because it appears that the FDA is only very grudgingly supporting it or, sorry, not supporting it at all but is only grudgingly approving it.
	I hope that this advisory committee will recommend to the FDA that they remove the restrictions on a medication that is essential to those of us who depend on it. I will just close by saying that one size does not fit all and one insulin does not fit all.
	Thank you.
	DR. FREAS:  Thank you, Moira, and we appreciate your coming this long distance to make this presentation before the committee.  Thank you very much.
	Our next speaker in the open public hearing is David Korroch.  He is Executive Director of the Norfolk Eye Bank.
	MR. KORROCH:  Thank you for the opportunity to speak today.  I would like to compliment the committee on your efforts towards diligently ensuring the safety of tissue in the United States.
	My name is Dave Korroch.  I am Executive Director of the Lion's Medical Eye Bank and Research Center of Eastern Virginia located in Norfolk, Virginia.  The Lion's Medical Eye Bank recovers and provides for transplant over 500 corneas per year, restoring sight to citizens of Virginia, the United States, and other countries.
	My comments today are to call to your attention the potential effects of the implementation of the draft guidance for industry to screen out potential tissue donors who may pose a risk of transmitting variant CJD or any other form of CJD. Specifically I would like to address Section 4, Parts 4, 5, 6 and 8.
	Section 4, part 4 will rule out for corneal donation those who have traveled to the UK and have spent months or more cumulatively in the UK from 1980 through '96.  Part 5 will rule out current or former US military members, civilian military employees, or other dependents who reside at US military bases in northern Europe for six months or more from 1980 through 1990 or else were in Europe for six months or more from 1980 to '96.  And part 6, as you are aware, excludes those who lived cumulatively for five years or more in Europe from 1980 until the present.
	While the intention of these guidelines is to only remove from the donor pool those who fall into these categories they effective exclude all military and their dependents.  It will not be feasible for the transplant agencies to access military travel records in a timely manner to determine whether the potential donor's travel history falls within these guidelines.
	Specifically the sensitive nature of corneal tissue requires that it be transplanted as soon as possible after recovery to retain the viability of the corneal endothelium.  Every eye bank in the United States that is accredited by the Eye Bank Association of America asks families consenting to donation questions about their potential donor's behavior and medical history that are designed to reveal any potential presence of CJD.
	The Lion's Medical Eye Bank averages between 20 and 30 cornea donors or 10 percent of our donor base from military personnel and their dependents based at Oceana Naval Air Station in Virginia Beach, Langley Air Force Base, and the Norfolk Navy Base.
	This is a loss of at least sixty corneas that could have restored sight to people suffering from corneal trauma, degenerative disease, congenital malformation, or infection.  On its face that number doesn't seem like much but when you add our eye bank's numbers to the numbers of other eye banks serving military areas the potential loss of corneal tissue could be significant to the corneal blind in the United States.
	Section 4, part 8 excludes from donation those potential donors that have used certain bovine-derived insulin to control their diabetes.  Again, while the intention may be to rule out those specific donors who have used such products the effect is to exclude all insulin-dependent diabetics as potential cornea donors.
	There is no reliable way for transplant agencies to confirm that patients with IDDM are not using certain types of insulin.  Since 1999 the Lion's Medical Eye Bank has had 165 donors with a history of IDDM representing over 300 pieces of potentially sight-saving tissue.  That represents a little over 11 percent of our donor base.  Between those two categories we are talking about 20 to 25 percent of our cornea donor base being excluded.
	In closing I would like to remind you all that corneal transplants are among the most successful of all tissue and organ transplants and that 46,000 patients a year rely on the steady supply of corneas to help them regain their sight.  Also as a note an upper age limit of 61 would exclude 27 percent of eye donors or 10,000 transplantable corneas in the United States. The proposed guidelines could put in jeopardy the hopes of thousands who may need their sight restored through corneal transplantation.
	Thank you for your time.
	DR. FREAS:  Thank you, David, for your informative presentation.
	Is there anyone else in the audience who at this time would like to address the committee on topics relating to this meeting?
	Seeing none, I will now close the open public having and turn the microphone over to the chairman.
	DR. BOLTON:  Very good.  Thank you, Bill, and thanks to those three speakers from the public that contributed to our discussion today.  What you said is not always easy for us to hear but it's important that we hear it and it's important that the FDA hear it as well.
	At this time I would like to open up to committee discussion in general before we have the questions presented to us.  We shouldn't spend too much time in general discussion because I think our directed discussion after the questions are presented will be more productive.
	So if anyone had anything they would like to say now, questions, comments regarding the presentations that we have had this morning.
	Steve?
	DR. DeARMOND:  This morning or could we say something about later?
	DR. BOLTON:  Oh, this morning and this afternoon, whenever.
	DR. DeARMOND:  The question I have is what were the reasons for the bovine insulin ban?  I don't remember reading it in all the tome of things we received.  Could that be concisely presented?  Is it just because they came from cows in Great Britain?
	DR. BOLTON:  Would somebody from FDA like to present that information?  The question is what is the basis of the banning of bovine insulin.
	DR. DeARMOND:  Well, that might be the wrong term.  The inability for it to be marketed over here in large quantities. Maybe that's better.
	MS. CHU:  Bovine insulin was available in this country in the past.  We had two manufacturers, Nova ÄÄÄÄ and Eli Lilly.  A few years ago Nova ÄÄÄÄ decided not to market bovine insulin and then in 1999 Lilly also withdrew marketing of bovine insulin.  So therefore the two approved bovine insulin now became not available in this country.  We can only approve a product when a manufacturer submitted an NDA to us and then we evaluate the data and find the quality and the safety and the efficacy information adequate.  Then we will approve the product.
	DR. DeARMOND:  Well, did they stop manufacturing it because of the FDA --
	MS. CHU:  No, it has nothing to do with FDA.  It is completely due to the manufacturers' own strategy, i.e., marketing products.  They decided not to do that because it is much easier for them to have limited product lines.  When firms market multiple products it became very complicated, manufacturing, distribution.
	DR. DeARMOND:  Well, how do we respond to these comments?  It doesn't seem to make sense.  There's a market for this. I can't understand.  Is it just too expensive to manufacture from US cattle pancreases?  If there is a market for it and these people are paying premium price for it it seems like the companies would want to do that.  And if it is a better product than synthetic human I'm lost in all of this.
	MS. CHU:  We would not know the strategy of the firms; however, I do know that bovine insulin was sold much cheaper than human synthetic insulin.
	DR. BOLTON:  I don't want to pursue this too much longer because it is really off the subject of our meeting today. Perhaps we can take this up with FDA and have a presentation on it another time.  My interpretation is that it is basically market forces that caused cessation of production of bovine insulin in the US and if market forces dictated it could be reinstituted but it doesn't seem like there's call for that right now.
	It may be that the patient population that desires bovine insulin is too small to make it cost effective.  I don't know.
	MS. KNOWLES:  I don't know if this is a correct interpretation but I think that what these two women were trying to share with us was that the decision we make today and tomorrow may impact them further.  That is how I interpreted their comments.
	DR. BOLTON:  No, I don't think so. I don't think our deliberations today have anything to do with insulin.
	DR. GAMBETTI:  As a point of clarification my understanding is that really FDA bans only bovine insulin imported from the UK.  That is the only FDA limitation.  Am I right?
	MS. CHU:  No, that's not true.  Our policy is we will not import drugs derived from bovine manufacturer in Europe as long as the source material is not from BSE country.  So we do have bovine drugs on the market in this country that are manufactured in Europe but bovine material will come from non-BSE countries.
	So therefore our policy is that we ban bovine material derived from bovine countries not the manufacturing site, from BSE countries.
	DR. BOLTON:  There are questions regarding the tissues and the tissue-based products aspect of our discussions.  Lisa?
	DR. FERGUSON:  I have a real basic question.  Could the FDA clarify exactly what list of tissues would be affected?  I have heard semen and oocytes.  Are those included?  Is there something else beyond that that we are not thinking of that would also be included in the guidelines?
	DR. SOLOMON:  Ruth Solomon, FDA. The proposed rules when finalized would include the tissues that we currently regulate, that is, musculoskeletal tissue, occular tissue, and skin.
	It would also include reproductive cells and tissues, hematopoietic stem cells, and dura mater and heart valves would become tissues instead of devices.  It would also include cell therapies.
	DR. PETTEWAY:  Just a point of clarification, from what we have seen the processing of different tissue types is really different and probably carries different risk based on how it's processed so the response to these questions is not meant to be generic, is it?  Or should the committee advise based on one tissue class versus another?
	DR. BOLTON:  You are referring probably to part B and I have a clarification for that when we get there, but it has more to do with recommending whether FDA should recommend or require specific types of things rather than us recommending specific methodologies and what have you.  We will get into that discussion at the time.
	I sense that there are not very any overwhelming general questions or discussions.  Maybe we should move right to the questions at hand and then we can discuss them in a more relevant way.
	Ruth, are you going to present the questions for us?  Very good.  It takes the job away from me.  It's nice to see that technology has not taken over everywhere. We're going to have overheads.
	The questions, I'll just tell you up front, A and B, we are asked to vote on. The voting for A is going to be relatively straightforward.  The voting for B is what becomes a little bit more complicated because we are going to vote on a somewhat more abstract issue than what it appears there.
	Ruth, you have the floor.
	DR. SOLOMON:  The first question is, "Which of the following measures and controls is (are) appropriate to prevent TSE agent transmission to recipients of human cells and tissues"?
	1A says recommend additional donor screening and testing measures such as upper age limit, a head trauma exclusion, or a negative brain autopsy or biopsy.
	DR. BOLTON:  Actually, I'd just like to stop right there at 1A and have discussion on that and vote before we come to 1B because it is otherwise going to get too complicated.  The question is we are going to be asked to vote whether we believe that FDA should recommend additional donor criteria and those are criteria in addition to a negative history for CJD or familial CJD, the variant CJD risk factors that have already been suggested to be incorporated into the preliminary draft of the guidance, is that right?
	DR. SOLOMON:  Draft, the draft guidance.
	DR. BOLTON:  And these additional criteria are being considered are, first of all, an upper age limit, not specified but the concept, head trauma exclusion, or a negative brain autopsy or biopsy, which implies then that we would require that brain autopsies or biopsies be done.  So I will open this up for discussion now. Ermias?
	DR. BELAY:  I have a question.  I think I understand the rationale for upper age but I'm struggling with the rationale for head trauma exclusion.  What's the rationale behind that?
	DR. BOLTON:  Well, I had that same question in the conference call before this meeting.  The rationale is that with head trauma if the person were incubating CJD it might release brain tissue into the circulatory system and contaminate other tissues.
	I'll give you my personal feeling on this.  I think the likelihood of the coincidence of major head trauma in an incubating CJD case is pretty rare and I don't know that we can use that as a means to exclude tissue donations.  So in my opinion I think that's not a good additional criterion to recommend but I think you have to each form your own opinions.
	Steve?
	DR. DeARMOND:  Yes, I would agree. What if it's a 35-year old who runs his motorcycle into a tree?  You wouldn't expect him to have CJD so it doesn't really make much sense.  What if they had the head trauma 20 years before they die?  If they had CJD at some point it should have already presented itself.  So it doesn't make sense.
	DR. BOLTON:  Well, I think the head trauma is coincident with death so it has to do with that and it's a major feature of the deceased.
	Yes, Pierluigi.
	DR. GAMBETTI:  A more basic question, it looks like we are talking about A; 1 and 2 are debatable, as we can see. The only one it would really be very helpful to be, number 3, but we heard that there are problems of feasibility.
	So what prompts these additional exclusion criteria?  Is there really any factual event that prompts us now to discuss additional criteria, because that's my understanding, to those that already exist? In other words something bad happened that we have to add criteria to whatever was available before?
	DR. BOLTON:  Ruth, would you like to address that?
	DR. SOLOMON:  No, nothing bad happened.  These were suggestions that we have received from others and we are not saying that we support these.  We just wanted to put them on the table.
	DR. BOLTON:  My sense is that it's an attempt to be all-encompassing.  If we're considering these issues let's consider all of the things they could come up with that might have some benefit.  Again, I think you are right.  The first two are very questionable.  The third one is concrete but may not be implementable.
	Again, we also heard discussions from different tissue processors saying that they do not process central nervous system tissue and then during the collection process we may be proposing to open the cranium and take out the brain and I assume that maybe would happen after the other tissues are taken but who knows and now we have a potential for contaminating the very tissue that we are trying not to contaminate in the process of determining whether it is safe by doing a brain biopsy or an autopsy.
	Pedro?
	DR. PICCARDO:  I agree with what Pierluigi Gambetti said.  I think the question is restricted to a negative brain autopsy or biopsy.  Now, the issue is about percentage of certainty.  So if the question to the committee or the question generally is we want 100 percent certainty I don't think we can get away from the autopsy if a label or whatever you consider is appropriate and then there is the risk- benefit factor included, et cetera, et cetera.  Then because of what we heard today, then the autopsy could be waived.
	But I think the issue is I cannot separate the "c" part from the percentage of certain things which we are asked to talk about.
	DR. BOLTON:  Dr. Doppelt.
	DR. DOPPELT:  I just have a question regarding that.  Someone, I think it may have been you, pointed out that in terms of brain biopsy there are results and the confidence you have in the results varies from one site to another.  So a single biopsy isn't going to give you what you are looking, 100 percent certainty, so it doesn't sound like it is achievable, number one, and, number two, getting biopsies on all these people just isn't going to happen.  I mean, it's not workable.
	DR. PICCARDO:  Yes, I think I made a comment on that, but I think Pierluigi Gambetti also brings forth the same concept. Now, there is some work that we are doing right now as we speak and this is not related to CJD.  This work has not been presented yet.  It will be presented soon. But I think it is pertinent to this discussion.  I am going to enforce this not related to CJD, but it is related to a genetic form of prion disorders in which we could analyze by autopsy three patients that we knew had the mutation because we sequenced the PLV gene.
	At the same time these three people died of accidental cardiac arrest in their 40s, three of them, and it's interesting to see.  So we had their full brain, which we analyzed, we had the genotype, and we had a lot of tissue ÄÄÄÄ chemistry.  In two of those cases, after extensive pathologic analysis of the whole brain, meaning cerebral and cerebellum, in two of those cases there was one area of the cerebellum that had an equivocal PLP deposition and plaques and all the rest was all negative.
	So to give an idea of the complexity of the issue that's why I started saying I think from that lesson and from the lessons that we heard before, I mean, if you don't have the full brain for 100 percent certainty I don't think you can say that.
	DR. BOLTON:  Yes, that's what I was going to say.  I think, summarizing what Pierluigi and Steve had said earlier, is that it doesn't seem to make sense to talk about doing a biopsy when in fact it may be cheaper and faster and safer to take out the entire brain.
	So then I think you are really considering whether we want to suggest that FDA recommend that that be done on each patient for donation, again recognizing the possibility, although rare, that if an individual did have incubating CJD that you are now opening the cranium and taking out the brain, which may be infectious, and at the same time or at some point with the same individual that is going to be possibly donating the tissue.
	Yes, Beth?
	DR. WILLIAMS:  I was just going to say I think there are some differences in risk here.  Obviously dura mater material is going to carry much greater risk and that might be a tissue where you might want to require brain examination as compared to bone or the ligaments.  But there are some differences there.
	DR. DeARMOND:  Yes, for the cornea. This was related more to the cornea, which has a high possibility in a CJD patient of being contaminated, but from the reluctance of pathologists who will take out, what did we see, 45,000 corneas are removed?  If they had to do a brain biopsy at this stage I think that number of corneas would drop dramatically because they are reluctant to do this until we work out a high throughput system of some sort that's easy to do.
	So I think these questions should be put to the future.  We don't have a high throughput system.  Systems are being developed but they are not necessarily easy to do at this stage, but it's possible to have a very simple system in the next couple of years, maybe in the next six months.
	DR. BOLTON:  Lisa?
	DR. FERGUSON:  Well, is it also possible for things like dura mater or cornea that are a risk to maybe combine some of these and not necessarily say you have to do a brain biopsy on each and every one?  If you have a 20-year old is that useful?  But if you have a 55-year old perhaps it might be.  Can you combine it and say well, okay, if you are less than this age, no, but if you are over this age, perhaps?
	DR. BOLTON:  With two comments, I'll punt that to the FDA, but I think that they would prefer to harmonize the guideline so that it is more uniform and in fact you may have a single donor that is donating various tissues which then, of course, you have to go to the most restrictive set of conditions and that may be difficult to implement.  So what's your feeling on that?
	DR. SOLOMON:  Yes, that's correct. The tissue approach is trying to create a minimum floor of donor screening, testing, and GTPs for all cell and tissue donors.  We brought the question of which tissues should we apply these things to TSEAC in January of 2001.
	We didn't really get a definitive answer.  Basically you just said that dura mater and cornea are the most risky, but we didn't get any other feedback.  So we would prefer to even though it may not be scientifically valid to try to harmonize, as you said, the requirements or recommendations, certain basic requirements for all tissue donors and then other requirements for other tissues.
	DR. BOLTON:  So you would prefer that in A here we talk about the general case, the floor, if you will, for donors, not to say that in the case of corneas and dura mater, although there is a question but it seems like dura mater is becoming increasingly unpopular in terms of being produced, but those might have additional recommendations.
	DR. SOLOMON:  Yes, possibly.
	DR. BOLTON:  Is there additional discussion?
	DR. DeARMOND:  There might be and I would guess there is data that's already available if the tissue banks or the eye banks are able to plow through it.  The analogy here would be to cord blood banking. This has been done probably because cord blood banking is newer than these other things but it is possible to go through and look at the point at the donation and collection process where the donor gets excluded.  Is it because of transmissible disease testing?  Is it because of the medical history that was obtained from the chart?  Was it because of an interview with a family member?  That data would all be there if someone could dissect it and that would really form a solid base of information to decide which of these steps really gives the key information that causes donor deferral.
	You could presume that this would apply to CJD or the kind of issue we are talking about here.  This is pretty well known for cord blood and I would guess that the eye banks and the tissue banks could look at their donor information and pretty easily figure out what steps cause them to identify and defer potentially infectious donors and that would really form a basis for information to make these decisions.
	DR. BOLTON:  Are there additional questions or discussion?
	MS. HECK:  I'm speaking to that issue that the gentleman just raised.  Ellen Heck from UT Southwestern, EBAA.  I think by far the largest percentage of our donors are deferred pre-retrieval and that is in the questioning and medical history portion of our deferment.
	A range of between eight and eleven percent are deferred because of serological testing but by far the largest number of deferrals comes in the front end of the process.  Of course, there are other tests that come in in the physical condition of the tissue, but if you are looking for exclusionary criteria for risk factors they are primarily going to be at the front end.
	DR. BOLTON:  But I'm looking for more detail on the front end.  For instance, with the analogy with cord blood banking you take a history from the mother, you look at the baby's chart, and you can also talk to the father.
	Which of those three steps really gives the key information on which the decision is based?  I would guess at least with tissues there is a medical record and there is some sort of interview with the next of kin.  There might be other people or other sources of information and you can tell from that where you learn what you need to know in order to make a decision about that donor.
	MS. HECK:  That's not different for our tissues.  It's the same place, the medical chart and the interview.
	DR. BOLTON:  What I'm saying is the data are there if you want to look and see which of those is the thing that really tells you what you want to know.
	DR. DeARMOND:  This is very complicated.  I can see all the risk-benefit issues coming up here.  Certainly there's going to be a big loss of tissues if we are too stringent on it.
	The big problem comes after the age of 50.  The problem, I shouldn't say "big." The problem begins there.  Looking at those curves this morning, the donation of curves go up to about 60, I think, and then they stopped.  The tissues are not taken very much after the age of 60, just as the CJD curve.
	DR. BOLTON:  Is that not true? What did the curves --
	DR. DeARMOND:  It depends on the tissue.
	DR. BOLTON:  To what age, 61 to 80?
	MR. KORROCH:  Anywhere from 25 percent to 33 percent of corneas transplanted in the United States are taken from age 61 to 80.
	DR. DeARMOND:  So right in the CJD area.
	DR. BOLTON:  Yes, I think you have to weigh the prevalence in its many forms of sporadic CJD versus losing 25 to 30 percent of corneal transplants, which I think would be devastating.
	Is there other questions or discussion?  What I would like to do is to go through and vote on A, those three criteria, with respect to the floor level of all tissue donors.  Then we can come back and revote on them with respect to neurological tissues or high-risk tissues like corneas and dura mater.  Does that make sense?  We'll make it two votes.  It will be easier to make the decision that way.
	DR. DOPPELT:  I just wanted to ask one quick question about the brain biopsies. You mentioned if you take out the entire brain you run the risk of contaminating the tissue that you are trying to protect but what about contaminating the environment, also?  Two weeks later or three weeks later you find out that the brain was positive. What happens to all those instruments in the room and the equipment that came in contact with the tissue?
	DR. BOLTON:  Well, presumably all of that is going to be decontaminated every time it's used anyway but it's a risk if in fact the decontamination procedures are not successful.  Of course, I realized as I was saying that that if you take the brain out and it is positive you should find it and those tissues would not be used anyway.  But it is a risk for those who are doing the procedure, I suppose, taking out the brain.
	DR. GAMBETTI:  That's why I was saying that probably an operation like that has to end up being done in specialized institutions, not just in any possible institution, exactly for that reason that you mentioned.
	DR. BOLTON:  And that then means really regionalizing the tissue collection centers because you are not going to ship the body to collect a cornea, I don't think.
	DR. EPSTEIN:  Yes, I just wanted to add a third stage of voting.  I think if a committee member has a proposition to put on the table for use of a combined criterion such as the brain autopsy only for donors with age over X it would be very useful to vote on that as well.  Now, if there is no such proposition, well, okay.
	DR. BOLTON:  We'll take that as stage 3.
	MR. RUSSO:  Richard Russo, Osteotech.  I thought I'd give you one specific fact that I managed to collect prior to coming to the meeting.  I called two OPOs and asked them about the percentage of donors that they recovered that would be above 60 years of age.
	One OPO located here on the east coast in the Maryland area said that they get 50 percent of their donors above the age of 70.  One that was located in the State of Indiana said 50 percent above the age of 60. Now, these were tissue donors as a whole.
	I would like to give you then one other perspective in addition to that fact is that, of course, with the informed consent products people are going to have to explain to the donor's family that they are going to have a total removal of the brain in order to do an autopsy.
	Most of these donors at the moment have families that prefer to do an open casket funeral and the bodies are reconstructed to allow for that.  In certain parts of the country getting the donors to approve the collection of tissue from the upper extremities is difficult because in the warm weather in the South, for example, people are set out in half-sleeve shirts and things like this.
	I think that might have a dramatic effect on donation if you said you were going to have to open the skull.  I think that needs to be considered.  I don't have any information for you but I just wanted to present that as something to consider.
	DR. BOLTON:  Maybe our neuropathologist can comment.
	My experience, which is extremely limited, is that you can actually remove the brain and reconstruct the face without any obvious sign.
	DR. GAMBETTI:  I personally think that there is more risk to disfigure involved in biopsy, transÄÄÄÄ biopsy, than by removal of the brain with a regular craniotomy.  That does not disfigure at all. Everybody who does autopsy has a tremendous experience with that whereas with the other system you may create in fact a much more visible scar.
	DR. BOLTON:  Yes, a comment from the floor.  Introduce yourself, please.
	MR. PARDO:  P.J. Pardo, Tutogen Medical.  The guidance document by CDRH for the processing of dura addresses already the brain autopsy and biopsy as well as some of the other issues that the committee has brought.  Currently dura is regulated as a medical device, so therefore it falls under the CDRH purview at this time.  Thank you.
	DR. FERGUSON:  I just want to make sure I understand the question and exactly what we are voting on.  I'm sorry.  I'm a bureaucrat.  I should be able to do this. But if we vote yes on this baseline thing essentially we would be saying that we believe that extra criteria are necessary for all of this list of tissues given earlier.  Is that correct?
	DR. BOLTON:  Yes, the first vote and we will take them one by one.  The first vote will be do we recommend that FDA add this criterion as an additional donor eligibility criterion for all tissue donations.  That will be the first vote.  So if there are not any other questions I think we can take that and I believe we'll take a voice vote for this.
	So the first part of the question A will be do you believe that the committee should recommend an upper age limit as an additional donor eligibility criterion for all tissue donors?
	DR. FREAS:  Dr. Gambetti?
	DR. GAMBETTI:  No.
	DR. FREAS:  Dr. Ferguson?
	DR. FERGUSON:  No.
	DR. FREAS:  Dr. DeArmond?
	DR. DeARMOND:  No.
	DR. FREAS:  Dr. Bailar?
	DR. BAILAR:  No.
	DR. FREAS:  Dr. Piccardo?
	DR. PICCARDO:  No.
	DR. FREAS:  Dr. Williams?
	DR. WILLIAMS:  No.
	DR. FREAS:  Dr. Doppelt?
	DR. DOPPELT:  No.
	DR. FREAS:  Dr. Bolton?
	DR. BOLTON:  No.
	DR. FREAS:  Ms. Knowles?
	MS. KNOWLES:  No.
	DR. FREAS:  Dr. Belay?
	DR. BELAY:  No.
	DR. FREAS:  Dr. Priola?
	DR. PRIOLA:  No.
	DR. FREAS:  Dr. McCullough?
	DR. McCULLOUGH:  No.
	DR. FREAS:  Dr. Wolfe?
	DR. WOLFE:  No.
	DR. FREAS:  Dr. Linden?
	DR. LINDEN:  No.
	DR. BOLTON:  We would like to get the industry's perspective.
	DR. PETTEWAY:  No.
	DR. BOLTON:  That was pretty clear.
	Now, the next one, do you believe that the committee should recommend a head trauma exclusion donor eligibility criterion for all tissue donors?
	DR. FREAS:  Dr. Gambetti?
	DR. GAMBETTI:  No.
	DR. FREAS:  Dr. Ferguson?
	DR. FERGUSON:  No.
	DR. FREAS:  Dr. DeArmond?
	DR. DeARMOND:  No.
	DR. FREAS:  Dr. Bailar?
	DR. BAILAR:  No.
	DR. FREAS:  Dr. Piccardo?
	DR. PICCARDO:  No.
	DR. FREAS:  Dr. Williams?
	DR. WILLIAMS:  No.
	DR. FREAS:  Dr. Doppelt?
	DR. DOPPELT:  No.
	DR. FREAS:  Dr. Bolton?
	DR. BOLTON:  No.
	DR. FREAS:  Ms. Knowles?
	MS. KNOWLES:  No.
	DR. FREAS:  Dr. Belay?
	DR. BELAY:  No.
	DR. FREAS:  Dr. Priola?
	DR. PRIOLA:  No.
	DR. FREAS:  Dr. McCullough?
	DR. McCULLOUGH:  No.
	DR. FREAS:  Dr. Wolfe?
	DR. WOLFE:  No.
	DR. FREAS:  Dr. Linden?
	DR. LINDEN:  No.
	DR. FREAS:  The industry's opinion?
	DR. PETTEWAY:  No.
	DR. BOLTON:  Moving right along now, do you believe that the committee should recommend a negative brain autopsy or biopsy as an additional donor eligibility criterion for all tissue donors?
	DR. FREAS:  Dr. Gambetti?
	DR. GAMBETTI:  No.
	DR. FREAS:  Dr. Ferguson?
	DR. FERGUSON:  No.
	DR. FREAS:  Dr. DeArmond?
	DR. DeARMOND:  No.
	DR. FREAS:  Dr. Bailar?
	DR. BAILAR:  No.
	DR. FREAS:  Dr. Piccardo?
	DR. PICCARDO:  No.
	DR. FREAS:  Dr. Williams?
	DR. WILLIAMS:  No.
	DR. FREAS:  Dr. Doppelt?
	DR. DOPPELT:  No.
	DR. FREAS:  Dr. Bolton?
	DR. BOLTON:  No.
	DR. FREAS:  Ms. Knowles?
	MS. KNOWLES:  No.
	DR. FREAS:  Dr. Belay?
	DR. BELAY:  No, but I would like to make the dura mater as an exception.
	DR. BOLTON:  We'll come to that next.
	DR. FREAS:  Dr. Priola?
	DR. PRIOLA:  No.
	DR. FREAS:  Dr. McCullough?
	DR. McCULLOUGH:  No.
	DR. FREAS:  Dr. Wolfe?
	DR. WOLFE:  No.
	DR. FREAS:  Dr. Linden?
	DR. LINDEN:  No.
	DR. FREAS:  The industry's opinion?
	DR. PETTEWAY:  No.
	DR. BOLTON:  Question 1, Part 2A, do you believe that the committee should recommend an upper age limit as an additional donor eligibility criterion for special cases, high-risk tissue such as cornea or dura mater?
	DR. FREAS:  Dr. Gambetti?
	DR. GAMBETTI:  I need some more information here.  If I'm correct concerning numbers, if I understand correctly, if we require an autopsy or a biopsy we are dealing with a number of 20,000 cases of cornea transplant a year?
	DR. BOLTON:  Forty-five thousand, but first we are just talking about an upper age limit.  You have two more to go before you get to the autopsies.
	DR. GAMBETTI:  Oh, I'm sorry.  No.
	DR. BOLTON:  Should I repeat the question?  Do you believe that the committee should recommend to the FDA that an upper age limit be used as an additional donor eligibility criterion for high-risk tissues?
	DR. FERGUSON:  Now, is this separate from what we might be voting on as combining some of these things, perhaps?
	DR. BOLTON:  Yes, we'll ask for combinations later.
	DR. FERGUSON:  Okay, no.
	DR. FREAS:  Dr. Piccardo?
	DR. PICCARDO:  No.
	DR. FREAS:  Dr. Williams?
	DR. WILLIAMS:  No.
	DR. FREAS:  Dr. Doppelt?
	DR. DOPPELT:  No.
	DR. FREAS:  Dr. Bolton?
	DR. BOLTON:  No.
	DR. FREAS:  Ms. Knowles?
	MS. KNOWLES:  No.
	DR. FREAS:  Dr. Belay?
	DR. BELAY:  No.
	DR. FREAS:  Dr. Priola?
	DR. PRIOLA:  No.
	DR. FREAS:  Dr. McCullough?
	DR. McCULLOUGH:  No.
	DR. FREAS:  Dr. Wolfe?
	DR. WOLFE:  No.
	DR. FREAS:  Dr. Linden?
	DR. LINDEN:  No.
	DR. FREAS:  The industry rep?
	DR. PETTEWAY:  No.
	DR. BOLTON:  Next, do you believe that the committee should recommend head trauma exclusion as an additional donor eligibility criterion for high-risk tissues?
	DR. FREAS:  Dr. Gambetti?
	DR. GAMBETTI:  No.
	DR. FREAS:  Dr. Ferguson?
	DR. FERGUSON:  No.
	DR. FREAS:  Dr. DeArmond?
	DR. DeARMOND:  No.
	DR. FREAS:  Dr. Bailar?
	DR. BAILAR:  No.
	DR. FREAS:  Dr. Piccardo?
	DR. PICCARDO:  No.
	DR. FREAS:  Dr. Williams?
	DR. WILLIAMS:  No.
	DR. FREAS:  Dr. Doppelt?
	DR. DOPPELT:  No.
	DR. FREAS:  Dr. Bolton?
	DR. BOLTON:  No.
	DR. FREAS:  Ms. Knowles?
	MS. KNOWLES:  No.
	DR. FREAS:  Dr. Belay?
	DR. BELAY:  No.
	DR. FREAS:  Dr. Priola?
	DR. PRIOLA:  No.
	DR. FREAS:  Dr. McCullough?
	DR. McCULLOUGH:  No.
	DR. FREAS:  Dr. Wolfe?
	DR. WOLFE:  No.
	DR. FREAS:  Dr. Linden?
	DR. LINDEN:  No.
	DR. FREAS:  The industry opinion?
	DR. PETTEWAY:  No.
	DR. BOLTON:  Now it gets complicated.  Do you believe that the committee should recommend to the FDA a negative brain autopsy or biopsy as an additional donor eligibility criterion for high-risk tissues?
	DR. DeARMOND:  If we say that by "recommend" you mean it's not being forced, this is not law, this is a recommendation?
	DR. BOLTON:  Good question.  We would be recommending that they recommend. I suppose that would be in guidance rather than in regulation.  So it is not being proposed as the force of law, I suppose.  It would be the recommended procedure.
	Jay, do you want to comment on that?  Am I getting this right?
	DR. EPSTEIN:  I don't think we have completely decided that because we have yet to publish a final rule and then there will also be a guidance and where we draw the line between what's in the rule and what's in the guidance may not be clear at this point in time.  But I think our general intent, whether it ends up recommendation or guidance, is if the committee feels it is an important additional donor eligibility or exclusion criterion we would seek to make that an enforceable standard by whatever mechanism.
	DR. BOLTON:  Dr. Linden?
	MR. LINDEN:  Jay or somebody else from FDA, can you please clarify the existing rules or recommendations for dura? My understanding is the same as the gentleman from Tutogen, that it is already there for dura.
	DR. EPSTEIN:  That's correct.  The current regulatory status of dura mater is that it is a device.  We do have guidance in place which is the basis for device approvals for dura.  That includes a criterion for examination of the brain. Now, as Dr. Solomon said earlier, under the proposed rulemaking dura mater and also heart valves would have their status removed from device approval as pre-market approval into regulation under the provisions for control of communicable disease as a cell or tissue or cell- or tissue-derived product.
	So therefore they would then be captured under the more general scheme. Now, this does not mean that we might not repromulgate a specific guidance for a specific tissue.  I think it would be our expectation that in whatever final guidance we do for cellular and tissue products we would indicate that the existing guidance for dura should still be practiced but it would no longer be a pre-market approval process.  It would be subject to verification on inspection, basically.
	DR. BOLTON:  Ruth, do you want to add to that?
	DR. SOLOMON:  The current CDRH guidance into any draft guidance that we would issue under the tissue scheme.
	DR. BOLTON:  In any case I guess our recommendation would be useful in guiding the FDA however this ends up being placed in whatever guidance and/or regulation so we will vote on it with that assumption.
	DR. DOPPELT:  I just want to make one point.  In terms of whether it is binding by law it's a recommendation, it's nice.  In the real world if the FDA says I think this is nice everybody says we have to do it.  That's the way the world works.
	DR. WILLIAMS:  I guess I had a question about high risk.  Are we talking about both cornea and dura, both of them together, or are we going to split it?
	DR. BOLTON:  It is up to us.  We are freewheeling at this point so what is your pleasure?
	DR. WILLIAMS:  I think they should be different.  I think one carries a much higher risk than the other.
	DR. BOLTON:  Ermias?
	DR. BELAY:  I agree with Beth.  I think they should be different.  I would also like to point out that the recommendation to do an autopsy for dura mater donors was actually recommended by this committee.
	DR. GAMBETTI:  Can I know the number that we are talking about?  We said 45,000 for cornea.  How about dura?  How many cases are we talking about?
	DR. BOLTON:  Dr. Wolfe, would you like to comment on that?
	DR. WOLFE:  I think it's down to several thousand a year at the most.  It's continuing to drop as people wise up.
	MR. PARDO:  As a former producer of dura I do not know of anyone in the industry that is processing dura under a 510(k) today.  They might be doing it under the prior to 1976 guidelines but I do not know of anyone producing dura.
	So again the suggestion that you made that the two tissues should be separated is probably a very wise suggestion since the regulations for dura are already in place as recommended by this committee several years ago.
	DR. WOLFE:  Isn't that Miami organization doing dura processing still? Do you think they are doing it under the old regulation?  Is that it?
	MR. PARDO:  I'm not sure.  There was only two 510(K) approveds.  We had one and I don't know who had the other one and we are no longer producing dura, either.
	DR. WOLFE:  When did you stop producing dura?
	MR. PARDO:  In 1999, when the new regulations came into effect.  Basically it makes it almost impossible to produce dura under those regulations.
	DR. BOLTON:  Dr. Bailar?
	DR. BAILAR:  If I understand the harvesting process you are not going to get dura without opening up the head anyway.
	DR. BOLTON:  That certainly would be the easiest way to get it.
	DR. GAMBETTI:  That was exactly the point I was going to make.  Dura mater is different from corneas in this regard because you have to do a corneatomy anyway. And the committee when they made the recommendation actually made the point that since you are already opening the brain why don't you take the brain tissue as a sample.
	The other relevant issue is dura mater is a little bit different from corneas because for dura mater there are the alternatives, fascia and also animal products, whereas for cornea you have to use human corneas.
	DR. BOLTON:  It's a sure sign that we have sat through too many presentations when we miss something like that but that is a good point.
	Should we then vote on these individually and take them now so that the question would be, taking dura mater first, do you believe that the committee should recommend to the FDA that a negative brain autopsy or biopsy be an additional donor eligibility criterion for dura mater donors?
	DR. GAMBETTI:  Start with the dura mater.  Yes.
	DR. FREAS:  Dr. Ferguson?
	DR. FERGUSON:  Yes.
	DR. DeARMOND:  Yes.
	DR. BAILAR:  Yes.
	DR. PICCARDO:  Yes.
	DR. DOPPELT:  Yes.
	DR. BOLTON:  Yes.
	We'll keep voting yes until somebody says no.
	MS. KNOWLES:  Yes.
	DR. BELAY:  Yes.
	DR. PRIOLA:  Yes.
	DR. McCULLOUGH:  Yes.
	DR. WOLFE:  Yes, until they are banned and then it won't be necessary.
	DR. LINDEN:  Yes.
	DR. PETTEWAY:  Yes.
	DR. FREAS:  And the industry opinion is yes so it is 14 yes votes, unanimous.
	DR. BOLTON:  So in the final part of something Part of 1A it will be do you believe that the committee should recommend to the FDA that a negative brain autopsy or biopsy be an additional donor eligibility criterion for corneal donors?
	DR. GAMBETTI:  No.
	DR. FREAS:  Dr. Ferguson?
	DR. FERGUSON:  No.
	DR. FREAS:  Dr. DeArmond?
	DR. DeARMOND:  I think I have to say no, also, until it's specified better.
	DR. FREAS:  Dr. Bailar?
	DR. BAILAR:  No.
	DR. FREAS:  Dr. Piccardo?
	DR. PICCARDO:  No, but I think the label should be something to the effect of the risk-benefit.  Something should be on the label.
	DR. FREAS:  Dr. Williams?
	DR. WILLIAMS:  No.
	DR. FREAS:  Dr. Doppelt?
	DR. DOPPELT:  No.
	DR. FREAS:  Dr. Bolton?
	DR. BOLTON:  No.
	DR. FREAS:  Ms. Knowles.
	MS. KNOWLES:  No.
	DR. FREAS:  Dr. Belay?
	DR. BELAY:  No.
	DR. FREAS:  Dr. Priola?
	DR. PRIOLA:  No.
	DR. FREAS:  Dr. McCullough?
	DR. McCULLOUGH:  No.
	DR. FREAS:  Dr. Wolfe?
	DR. WOLFE:  No.
	DR. FREAS:  Dr. Linden?
	DR. LINDEN:  No.
	DR. FREAS:  Industry opinion?
	DR. PETTEWAY:  No.
	DR. BOLTON:  Now, I would like to entertain suggestions for combinations of upper age limit, head trauma, and/or negative brain autopsy or biopsy for either all donors or for selected higher risk donors.  Is there any such combination that someone would like to suggest?  I see none.
	DR. DeARMOND:  Would you please state that over again?
	DR. BOLTON:  Would you like to suggest a combination of the three criteria? In other words let's say a negative brain autopsy or biopsy for corneal donors over age 70, for example, some combination like that, over age 70 with head trauma?
	The one that I can think of logically is an elderly donor that's donating high-risk tissue like a cornea or dura mater.  Well, dura mater we're already recommending a negative brain autopsy or biopsy result so that is a mute point.
	But in the case of corneal donors you might suggest that.  Now, I just put that out there, hearing that maybe 50 percent of the donors are over age 60 or over age 70, again, you are talking about maybe 20-some thousand autopsies a year. Oh, that's right, it wasn't corneas; it was all donors.  So even 20 percent would still be a significant number of autopsies or brain biopsies.
	Is there any inclination towards voting on that?
	DR. DeARMOND:  My dementia must be kicking in but didn't we just vote that for cornea and dura, the age limit, and said there was no restriction?
	DR. BOLTON:  Well, for both we did individually.  Whether you would want to put them in combination.  Jay was asking that we ask for that.  I'm not sure if it makes any sense, either, at this point in the day.
	DR. FERGUSON:  Well, I can see the logic for it but I don't feel like I have enough information at this point to make a good recommendation as far as the combination of age and everything else and to do the risk-benefit ratio there.
	DR. BOLTON:  That's our usual state of affairs here.  I don't sense that there is any particular interest into going into that so I think we should then move on to Part B of this question which now Ruth can present to us.
	DR. SOLOMON:  1B says specified methods of recovery and/or processing to prevent contamination and cross- contamination by TSE agents and the first bullet is decontamination of instruments and surfaces.
	I'll read the introductory part of Question 1.  "Which of the following measures and controls is (are) appropriate to prevent TSE agent transmission to recipients of human cells and tissues?"  And 1B has to do with recovery and processing methods and the first bullet would be decontamination of instruments and surfaces and we would be interested in a discussion of how that would be accomplished.
	DR. BOLTON:  Now this is where to me it got very confusing but I think after talking with Dr. Asher and Dr. Epstein that what they would like us to do is to basically answer the question should the FDA either require or recommend and, as we just heard, recommending is almost the same as requiring, specific decontamination procedures, specific methods of decontaminating instruments and surfaces used for the recovery and processing of tissues.
	Now, we are not necessarily at this point in time suggesting which one should be recommended but the concept is should they identify specific types of treatment, normal sodium hydroxide for an hour, autoclaving at 135 for an hour, whatever, much like I think Dr. Rohwer presented in his slide that represented the WHO recommendations.  Should the FDA recommend specific procedures?  In other words if they don't recommend specific procedures it will be left more general.  I think we can have some discussion on that.
	The same thing for methods of removal or inactivation of TSE agents, should they recommend specific methods?  Is there enough known about specific methods and the last question is more straightforward, should single donor processing be mandated or should pooling ever be allowed?
	So let's take all of those in terms of opening for discussion.
	Sue?
	DR. PRIOLA:  With the second one, methods for removal and/or inactivation from accidentally contaminated, we don't have a way to tell if something's been accidentally contaminated much less a means of sucking it out if we know what's in there, so that seems to me that's not even viable to consider, that second one.
	DR. BOLTON:  Yes, Jay?
	DR. EPSTEIN:  Perhaps I could help explain how we are hoping the committee would deal with this set of questions.  It's at two stages.  On the one hand we are asking should we incorporate into a regulation or guidance a statement such as there shall be validated procedures for decontamination of instruments and surfaces. That's yes or no and we are hoping you will vote; however, at that point, assuming there is a vote yes, we then seek a discussion well, what kinds of things might we recommend in guidance.  In other words, where is the science?
	So, for instance, you could say well, FDA really ought to recommend that people adhere to WHO guidance in this area or you could say well, we think nothing short of the following procedures should be a bare minimum.  So there is partly a vote needed and then there's partly an essay question.
	I think that the issue with accidentally contaminated we don't really mean an accident.  What we mean is that it is unintentional.  It may be there, it may not be there, but it's inadvertent.  It's something that may happen.
	What we are really saying is that the second stage is validated procedures for clearance of TSE agents.  In other words should we promulgate a requirement that in effect obligates the industry to do validation studies on TSE clearance from tissues?  Once again, if the answer is yes then a regulation might say you shall have validated procedures demonstrating clearance of TSEs from tissues and then the guidance would then need to say what exactly do we think is a sufficient demonstration.
	So I hope that helps.
	DR. BOLTON:  Sue?
	DR. PRIOLA:  So then I want to backtrack.  I don't know the WHO regulations.  What do they apply to, just in general decontamination for TSE or are they specifically for organ collection?  What are those guidelines aimed at?
	DR. SOLOMON:  They are aimed at all of the tissue and cells that I mentioned before.  Organs are not on the table.
	DR. PRIOLA:  So they recommend that upon any tissue collection you decontaminate with one mol of sodium hydroxide for --
	DR. BOLTON:  It's for the instruments and the surfaces.
	DR. PRIOLA:  That's what I want to know.
	DR. BOLTON:  It is decontamination of the environment and any instruments used.
	DR. PRIOLA:  For any tissue collection?
	DR. BOLTON:  I think that's right. And if you recall it's a sixth or seventh step in descending order of desirability. If you can do the first one, do the first one.  If you can't do that, do the second one.  If you can't do that, then do the third one, and they descend, I think at least presumably, in descending order of effectiveness as well.
	David?
	MR. ASHER:  Yes, we should be clear that the WHO guidelines originally were mainly intended to address safety in surgery; however, they are clearly relevant to the issue being discussed here.  They would be a framework to consider decontamination procedures in such a setting and in general.  They do list six measures in descending order of confidence.
	DR. BOLTON:  Pierluigi?
	DR. GAMBETTI:  Yes, just to see if I understand it, could the first question in the "B" part of the question, the contamination, be rephrased as follows, that we recommend that all tissue banks, whatever, all institutional organizations collecting tissue should decontaminate the instruments against the TSE agent?
	I assume they all sterilize their instruments when they have finished using them.  Are we asking here to add to their protocol sodium hydroxide, autoclaving at 154, to include also decontamination against TSE?  Is that rephrasing it?
	DR. BOLTON:  If I understood Jay correctly what he would like is a two-part question.  The first part is do we think that they should recommend or require validated decontamination procedures and, as he said, that's a simple yes or no.
	If we answer yes then what sorts of guidelines or what framework of guidelines might be used?
	DR. GAMBETTI:  I thought that was automatic that they decontaminated the instruments every time, that they used sterile instruments every time.
	DR. BOLTON:  But not validated for TSE inactivation.
	DR. SOLOMON:  You are correct. This is specific additional decontamination just focused on TSE.
	DR. DOPPELT:  If you are going to ask that question it seems to me like in the previous question you have to separate high-risk tissue from the regular tissues, because we don't seem to have much of a problem.  Theoretically there could be but we have not seen a problem for bone ligament and blah, blah, blah, so you have to separate it, I think.
	DR. BOLTON:  Good point.  Other discussion?  Questions?
	DR. BELAY:  I think FDA is concerned that whatever kind of donor default criteria you use it is possible that some infected donors could slip through and could we recommend to prevent that from contaminating other grafts by recommending additional sterilization methods.
	DR. BOLTON:  Yes, that's another issue.  You might have an individual incubating sporadic CJD slip-through but if you are only taking low-risk tissues would you recommend these specific decontamination procedures or would you only recommend them if they were taking both low-risk and high- risk tissues?  The problem here is really a logistical one for the collection agency because they are then going to have to separate their surgical instruments between low risk and high risk.
	It may very we well be worth doing that.  On the other hand if you get a mix-up and you don't have proper decontamination- sterilization features, then you have defeated the purpose of segregating those two sets of instruments and possibly sites. I don't know if site would be different.
	So additional discussion with that thought in mind?
	DR. DOPPELT:  I don't think the issue of separating instruments is that big of a deal.  If you take dura there are only a few places that are doing it and probably pretty soon nobody is going to be doing it. That is the reality.
	Corneas versus the other tissues, they are in point of fact using different types of instruments.  If you want to separate them that's not an issue.
	DR. DeARMOND:  I presume and, again, you couldn't write a test this way. The medical students would lynch you.  I guess the idea is that we would like for all of these tissues in addition to disinfecting or sterilization of bacteria fungi and viruses we would like to add the TSE component to it.
	DR. BOLTON:  That's correct.
	DR. DeARMOND:  That doesn't seem to come out in here very well because what they ask is specific methods and those are evolving and there's a series already out there that seem to be fairly effective.
	DR. BOLTON:  Right, and I believe that in the highly likely event that we would vote yes on the first question there that we could simply recommend that specific procedures like those recommended by the WHO should be considered.
	I'm sure that they will come back to us at some point and ask us again to talk about this.  I think that that would get across the appropriate thinking of the committee.
	Ermias.
	DR. BELAY:  It might be appropriate, as you propose, to look at the issue by high-risk tissues and the other tissues.
	DR. BOLTON:  It's easy enough to do that and it's actually easier to make the vote that way so why don't we do that again? Is there additional discussion at this point?
	DR. DeARMOND:  Just one other point.  It turns out a lot of the decontamination methods that are even used for bacteria and fungi work to some degree with prions, also, and the same thing could be true with sodium hydroxide and high temperature, probably destroy an awful lot of the other material.  So ultimately one method may be useful in the future but we don't know that yet.
	DR. BOLTON:  Lisa?
	DR. FERGUSON:  Just one question on the last point about the pooled processing. Are there any of these tissues that are actually being processed as a pool?  I mean, all the presentations talked about single-donor processing.  Is there something that we're missing that would be in a pool?
	DR. BOLTON:  Jay, would you like to address that?
	DR. EPSTEIN:  At the present time we believe that there are no pool-processed tissues although historically there were and the question is whether we should put in the final rule what we put in the proposed rule, which is a prohibition against commingling in processing.
	Just to be sure there's clarity, the proposed rule offered the possibility of request for waiver against that prohibition.
	DR. DeARMOND:  So is that true even now for plasma products?
	DR. EPSTEIN:  No, no, I'm just talking about tissue.  Plasma products, plasma derivatives are pooled products of necessity.
	DR. DeARMOND:  I see.
	DR. BOLTON:  It's understandable that this committee immediately reverts to blood and blood products because we have talked about it for so long.
	DR. DeARMOND:  They are a tissue, also.
	DR. BOLTON:  Yes, but not being considered here.  I would just like to say I think it's wise to always keep that option open for a company to apply under special conditions, to produce a product from pooled tissue, although it might not be desirable now but we might not foresee some point at which case that would be appropriate.  So I think that's a good idea to keep that open.
	DR. DOPPELT:  I just want to add one point about pooling.  It was just said, and I think that's correct, that right as of now there isn't anybody, any tissue banks, that are pooling.
	But there had been a few up until recently that were and they changed their practice but if you don't have a restriction against pooling there very well might be some banks that would revert to pool processing simply because it's more economical to do so and, as has been pointed out by many members, they may actually have a little bit more confidence in their procedures than perhaps there should be for the TSEs.
	DR. BOLTON:  Lisa?
	DR. FERGUSON:  Well, can I ask FDA a question?  And this might get into compromises with your rulemaking.  If we're voting on this process and we make a recommendation for a single-donor processing is that based solely on TSE concerns or are you guys going to go ahead and do that anyway based on other concerns?  Am I making sense?
	DR. EPSTEIN:  I think the proposal for single-donor processing was in fact based on TSE concerns primarily.  That's not to say that there are not risks from other agents that we don't have methods to control but the lead concern in the proposed rule really was TSE.
	DR. BOLTON:  Additional discussion, questions?
	First Dr. Linden.
	MR. LINDEN:  Well, I just want to agree with you that I would hate to see the pool issue be closed because there may be very valid reasons to do so, as in plasma derivatives, which are in fact safer than FFP.  So if it's linked with some sort of inactivation process that can only be done in a pooled fashion you could actually be safer than if you are not.  So I wouldn't want to close that option out.
	DR. BOLTON:  Yes.
	MR. RUSSO:  Richard Russo, OsteoTech.  I just wanted to support Dr. Linden's comment.  I do think that there will be additional technologies in the future.  If the FDA were to adopt a rule that would give a blanket prohibition against anything without any opportunity to make a variance submission you would have to move heaven and earth to change that particular rule.  I just think it forecloses progress in the future.
	It should be right now that there shouldn't be any multiple donor processing without an explicit approval from FDA.
	DR. BOLTON:  Thank you.
	Yes, Dr. Doppelt?
	DR. DOPPELT:  I would just like to ask if anybody has had any experience sterilizing equipment using the World Health Organization criteria because the one example that we heard one hospital presumably did what they were supposed to do and somehow ruined all of their equipment.
	I'm not sure how that happened but if that is a common event hospitals can't afford $10 million on every case.
	DR. BOLTON:  My laboratory routinely does the following, which is a variation on the WHO.  Everything, surgical instruments and what have you, immediately go into one normal sodium hydroxide at room temperature.  They soak usually overnight but at least for one hour.
	Then to that is added sodium dodecyl sulphate, SDS, to a one percent concentration.  That then is usually diluted five to ten-fold and autoclaved.  Then the instruments are taken from that and washed and rinsed and then set up and sterilized by conventional autoclaving.
	I think, as Dr. Rohwer said, many stainless steel instruments hold up to this beautifully well.  We do have some very nice German stainless steel instruments that have gone through this many, many times.
	And then occasionally you will get a set of instruments that just falls apart on you and I don't know what the difference is.  They are all supposed to be stainless steel.  It just seems to be somewhat unpredictable.  I assume it has to do with the quality of the steel but I don't know exactly why.
	Clearly, instruments that are not stainless steel will suffer terribly.  You do get an electrolytic reaction going on in one normal sodium hydroxide and you can get all kinds of interesting things happening.
	Steve?
	DR. DeARMOND:  And we don't know what went on at that hospital, whether they got the instruments from one surgical suite and then mixed them all together so they had to decontaminate everything because that seems excessive and whether they decontaminated materials that really weren't in contact with tissues and destroyed those in the process.
	There is a hierarchy of decontamination that you go through beginning with the disposable for the highest infectivity probability, including very good surgical scissors because it's cheaper to replace a $500 or $1,000 pair of scissors than to have a problem with the patient.
	DR. BOLTON:  And there is another level of concern here and that is that you need to decontaminate the instruments before anybody touches them to try to scrub them and clean them.  That is our primary concern is that I don't want anybody trying to scrub an instrument, especially a sharp pair of scissors, unless they have already been what we consider terminally sterilized so that's a major concern.
	DR. DOPPELT:  So one message here is if you buy cheap instruments, crummy instruments, and they fall apart you shouldn't have bought them in the first place.  Is that right?
	DR. BOLTON:  Yes, Dr. McCullough first.
	DR. McCULLOUGH:  I think while it's relatively straightforward to vote on the first part of whether the FDA should require demonstration of effective decontamination process I don't feel comfortable voting on some particular process.  I mean, we had a nice show and tell from several different groups or manufacturers and at least a couple of those processors were proprietary so we don't know details.
	As far as I know the committee has never really seen exactly what the WHO recommendations are.  I don't see how I could vote to recommend a particular method to the FDA at this point.  So it seems to me this discussion might not be so difficult.
	How would we know what to recommend?  We didn't have a crisp presentation of here are seven options; do you want to recommend one of these?  Then I could vote on it.  I can't now because I don't know what they are.
	DR. BOLTON:  What we might do is recommend that we look at this again at our next meeting, at specific decontamination and sterilization procedures.  Now, I'm still somewhat focused on instruments and environment.  When we get to actual validation of processing steps for tissue as far as I'm concerned we are so far away from that that I don't even know where to start.
	Every single tissue and product is almost going to be different and I'm not sure exactly where we start with that discussion so aside from suggesting that the FDA should look into it I don't know what else we can do with that.
	Ermias?
	DR. BELAY:  I was just going to comment on the Canadian situation to just set the record straight.  We had been consulting with the Canadians at the time they had that problem and by no means would that hospital situation be representative.
	It was a very unique situation.  It was a panic kind of situation.  They rounded up practically every surgical instrument in the hospital, which was up to 5- to 6,000 instruments, and they were trying to autoclave all those instruments within one day.  The volume of sodium hydroxide they used was basically gallons and gallons.  In that kind of situation there are always opportunities for error and accidents and that is exactly what happened.
	DR. BOLTON:  Dr. Wolfe?
	DR. WOLFE:  The suggestion I want to make is both for 1B and 1C when we get to it.  I think it is an extension of what you were just saying, which is I think that people here are probably going to vote yes to let's do something and in both the case of B and C, which is the processing, we would like them to come to us next time with the details because we could stay here all night and say well, this part of what this company presented looked good but it's proprietary; we can't find out what it is.
	So I think not just for the sake of getting out of here but for the sake of having a more enlightened discussion next time we should just vote on the first part of this one and when we get the C the same thing and then the next time we will have a bunch of specific suggestions as to how to implement those.
	DR. SOLOMON:  The proprietary materials were procedures for decontaminating the tissue.  This question is asking about decontamination of the instruments and surfaces, just to clarify that.
	DR. WOLFE:  I understand that but still we don't have enough details.  You were asking us to recommend which ones to do.  I'm just saying it would be much simpler to come to us next time or in the intervening times with some of your suggestions since we will likely vote yes, show us something.
	DR. EPSTEIN:  Again, I think we have not put specific procedures in front of the committee to vote on.  The question we are asking you to vote on is whether we should promulgate requirements or recommendations in these three areas, decontamination of instruments and surfaces, clearance from tissue per se and I think it would be helpful just to strike the word "accidentally" when you vote it, and single-donor processing.
	So those are yes/no votes. Anything beyond that today, I think, is whatever comment you wish to make about specifics and if there are no comments that's fine, too.
	DR. DeARMOND:  Well, Mr. Chairman, will you rewrite the questions so we can actually --
	DR. BOLTON:  I will try and I am going to hold any comments from the audience at this point because I want to get on with it while we have this thought clearly in mind about what this question is.
	(Discussion off the record)
	DR. BOLTON:  I have problems with this because we are really talking about should the FDA recommend specific validated methods for decontamination of instruments and surfaces used for recovery and processing.  Jay, is that correct?
	DR. EPSTEIN:  Yes, again, our current guidance does not specify methods for TSE decontamination.  We have recommendations that there be validated procedures in place for control of contamination and cross-contamination.  We have interpreted that to include TSE validation but there are no standardized methods at this point in time.
	We are asking should the FDA create recommendations for the use of specified methods specific for decontamination of TSE agents (a) from surfaces (b) to clear from tissue, and then in addition should there or should there not be single donor processing?
	I think we are getting caught up in the length of the sentence, which is really recommend specific methods for decontamination of instruments, recommend specific methods for removal and inactivation, recommend single-donor aseptic processing.  That's what we are asking.
	DR. DOPPELT:  This would be recommending decontamination of the instruments for TSE agents used on high-risk tissue.  Is that right or wrong?
	DR. BOLTON:  We hadn't split that yet.  We were going to vote on them separately, as we did before, but, I'll tell you, I think where I'm getting caught up on this is that it's difficult for me to vote to recommend methods when I don't know what methods we are talking about.
	So I think I would prefer to say that the committee recommends that the FDA pursue specific methods for decontamination of instruments, pursue their own recommendations for specific validated methods for decontamination of instruments and surfaces, et cetera, because I don't know how we can recommend that they require these things if we don't know what they are going to be.  We just don't have enough information to deal with that at this point.
	Yes, Dr. Bailar.
	DR. BAILAR:  I agree with Dr. Wolfe, Dr. Bolton, that we don't know enough about the specifics here to vote on whether to adopt specifics and we have heard from FDA about that.
	I am concerned a little bit, too, and I mean a little bit, that we not go too far.  At some point we should begin to rely on the educated professional judgment of the people who are doing these things.
	I would like to at least consider whether we can give them some flexibility in how they meet the basic requirement that these things be decontaminated.
	DR. BOLTON:  Well, I think that would be the area where the requirement would be to use a procedure that is validated to inactivate these agents and the choice of which procedure would be up to the local facilities.
	The problem is there are essentially none of these that are validated.  I'm not sure what you would call a validated procedure for inactivating prions on stainless steel scissors and I'm not even sure how you would go about validating that.
	What we want to recommend to the FDA is that they begin to move in that direction, to move towards a requirement, really.  It is an issuance of guidance that these procedures be defined, at least a list or a set of procedures that can be used, and that at some point it be required that one or more of those procedures be used.
	But we don't know what those procedures are at this point.  I think it would be impossible for us even if we stayed here until midnight to define what those procedures are this time.
	Steve.
	DR. DeARMOND:  Yes, the problem is the lead-in sentence, "Recommend specific methods," that should be struck.  It's just, "Recommend for HCT/P recovery and processing: decontamination of instruments," and I would say that that would be reasonable and I guess that's what they're actually getting at.
	It is not, as was suggested, that we are getting construed by the language. The language is horrible in these sentences and have too much stated in them.  We have to get to clearer language in these questions to answer them yes or no.
	DR. BOLTON:  Yes, Dr. Bailar.
	DR. BAILAR:  After this much discussion I think the message to FDA should be pretty clear and I'm not sure we need to vote on these things.
	MS. KNOWLES:  And there has been more than one time when other committees have rewritten the questions.
	DR. BOLTON:  Oh, we do this regularly.  It's a common occurrence.  We also have very often not voted on something like this because we couldn't really decide. I agree with you, Dr. Bailar, that I think that our message to the FDA is relatively clear.  If there was a way that we could state that and vote on it it would be helpful.  So I would propose that we are recommending that the FDA pursue specific validated inactivation procedures or begin to define specific inactivation procedures for the decontamination of instruments and surfaces used for recovery and processing, that they begin to define validated methods for removal and/or inactivation of TSE agents from HCTPs, those two questions and nothing further.
	We are just recommending that they move in that direction and that we should consider specific methods and processes at a later meeting.  Is that satisfactory?
	DR. DOPPELT:  I second the motion.
	DR. GAMBETTI:  And also should consider separating high-risk from low-risk tissues.
	DR. BOLTON:  And in those considerations should differentiate between high-risk tissue collection and low-risk tissue collection.
	Does the committee sense that they understand the question?  Are you shaking your head no, Steve?  You are not allowed to do that, now.
	DR. DeARMOND:  No, go ahead and state it again clearly because you threw "specific" back in again.  I don't see how the FDA has defined find specific methods. Those are still evolving and there are methods out there that already exist.
	I thought the question was to recommend that the people involved in this decontaminate their surfaces and do these other issues.  The problem with the lead-in sentence, it says that they should use specific methods and we don't know what those are so we have to eliminate that and we are not going to know what they are for another couple of years, probably.
	DR. BOLTON:  I guess that's the difference in philosophy.  Your suggestion is to require the tissue processors to decontaminate things now.
	My suggestion is to recommend to the FDA they begin to define the processes that they should use and not worry about requiring them to do anything yet.  So my suggestion is that we are telling them to begin to define the specific processes that would be used and to differentiate those with respect to high risk and low risk tissues.
	We are not going to say anything about what we are requiring anybody to do yet because we don't know what we should do.
	DR. DeARMOND:  So that's why I say state the question again because the statement now puts the pressure on the FDA.
	DR. BAILAR:  But you are putting the pressure on the FDA, not on the industry now.
	DR. BOLTON:  Yes, that's right. I'm putting the pressure on the FDA because I want them to come back to us with a defined set of methods that we could then discuss and decide upon as recommended validated methods.  Or maybe they're not validated.  Recommended methods that would be used.
	DR. DeARMOND:  Well, maybe one more time.  State your question as you phrased it.
	DR. BOLTON:  Could I have the transcription read back to me?
	DR. LINDEN:  To me it sounds like in the first part it is decontamination of instruments.  All of this discussion stems from the assumption that people are going to vote yes, at least for high-risk tissues on this first issue.
	That issue, we can still vote on that first thing because what you are asking FDA to do then is to say okay, come back to us and give us some more information on what techniques are available and then we can make a recommendation on what should be done if we vote yes on this first issue.
	Is that right?  It seems to me that it all stems from the yes-no vote on that first part.  If you vote yes you want FDA to come back.  I'm lost.
	DR. BOLTON:  I think we are all getting lost.  That is an issue that I really don't want to go in and take a vote on something that we are really not sure about what we are voting on.
	It's not going to do us any good and I don't think we have enough information to vote on anything that has any specifics to it in terms of methodology or anything so I think that what we can do is to convey to the FDA that we need more information, that we need them to develop their proposed methodologies that they would like us to consider, and then come back and discuss that at another time.
	We could, I suppose, vote to say we are interested in having these but we don't know what those things are specifically. Yes, we would like to have decontamination procedures for instruments and surfaces but we don't know exactly what they are.
	DR. GAMBETTI:  We would like to consider the possibility.
	DR. BOLTON:  Dr. Bailar.
	DR. BAILAR:  I still don't see the necessity to vote on anything here.  I'm sure that FDA understands our concerns.  I would like to leave it to FDA whether and when to come back to us with something that we can vote on here.  I don't even want to pressure them into doing that.
	MS. KNOWLES:  But I think we have to say that and then vote on it.
	DR. GAMBETTI:  Yes, I think the message should not be that we are rejecting. We are willing to discuss this if it is presented in a better way, a more understandable way.
	If we don't vote it may look like we are rejecting the whole issue.
	DR. BOLTON:  That is exactly right. The committee recommends that the FDA define specific decontamination procedures for instruments and surfaces used for recovery and processing and define or propose methods for removal and/or inactivation of TSE agents from HCT/Ps for future consideration by this committee as either regulation or recommendations.
	DR. FERGUSON:  With a distinction between low- and high-risk tissues.
	DR. BOLTON:  With a distinction between low- and high-risk tissues.  Could we take a vote on that?
	DR. FREAS:  Dr. Gambetti?
	DR. GAMBETTI:  Yes.
	DR. FREAS:  Dr. Ferguson?
	DR. FERGUSON:  Yes.
	DR. FREAS:  Dr. DeArmond?
	DR. DeARMOND:  Yes.
	DR. FREAS:  Dr. Bailar?
	DR. BAILAR:  Yes.
	DR. FREAS:  Dr. Pardo?
	DR. PARDO:  Yes.
	DR. FREAS:  Dr. Williams?
	DR. WILLIAMS:  Yes.
	DR. FREAS:  Dr. Doppelt?
	DR. DOPPELT:  Yes.
	DR. FREAS:  Dr. Bolton?
	DR. BOLTON:  Yes.
	DR. FREAS:  Ms. Knowles?
	MS. KNOWLES:  Yes.
	DR. FREAS:  Dr. Belay?
	DR. BELAY:  Yes.
	DR. FREAS:  Dr. Priola?
	DR. PRIOLA:  Yes.
	DR. FREAS:  Dr. McCullough?
	DR. McCULLOUGH:  Yes.
	DR. FREAS:  Dr. Wolfe?
	DR. WOLFE:  Yes.
	DR. FREAS:  Dr. Linden?
	DR. LINDEN:  Yes.
	DR. FREAS:  Our industry rep?
	DR. PETTEWAY:  Yes.
	DR. BOLTON:  Now, I'd like you to note that I specifically left out the single donor processing because I would like to consider that separately.  I believe that we can take a more definitive vote on that and I would suggest that the committee recommends that at this time single-donor aseptic processing be the default standard, if you will, and that pooled processing only be considered under a special application to the FDA.
	DR. FREAS:  Dr. Gambetti?
	DR. GAMBETTI:  Yes.
	DR. FREAS:  Dr. Ferguson?
	DR. FERGUSON:  Yes.
	DR. FREAS:  Dr. DeArmond?
	DR. DeARMOND:  Yes.
	DR. FREAS:  Dr. Bailar?
	SPEAKER:  Yes.
	DR. FREAS:  Dr. Pardo?
	DR. PARDO:  Yes.
	DR. FREAS:  Dr. Williams?
	DR. WILLIAMS:  Yes.
	DR. FREAS:  Dr. Doppelt?
	DR. DOPPELT:  Yes.
	DR. FREAS:  Dr. Bolton?
	DR. BOLTON:  Yes.
	DR. FREAS:  Ms. Knowles?
	MS. KNOWLES:  Yes.
	DR. FREAS:  Dr. Belay?
	DR. BELAY:  Yes.
	DR. FREAS:  Dr. Priola?
	DR. PRIOLA:  Yes.
	DR. FREAS:  Dr. McCullough?
	DR. McCULLOUGH:  Yes.
	DR. FREAS:  Dr. Wolfe?
	DR. WOLFE:  Yes.
	DR. FREAS:  Dr. Linden?
	DR. LINDEN:  Yes, provided it's not too restrictive.
	DR. FREAS:  And our industry opinion?
	DR. PETTEWAY:  Yes.
	DR. BOLTON:  We now come to Question 2.  Believe it or not, we're only to Question 2.  This is going to take I think some discussion but perhaps not very long to vote.  Ruth?
	DR. SOLOMON:  Question 2, "Please comment on the design of a satisfactory TSE agent clearance study for HCT/Ps in terms of the following criteria:  (A) suitable TSE agent strain and animal model (B) accept measurement of abnormal forms of prion protein alone or require infectivity assays (C) accept substantial reduction or require complete elimination of detectable prion protein and/or infectivity (D) accept a single validated method or require that more than one validated method for eliminating TSE agents be included in the study.
	DR. BOLTON:  Before I open this up for discussion this is not so much a voting issue but a recommendation and I think that we have heard in Dr. Rowher's presentation today as well as in several presentations in the past that in each case of these questions it really depends on the specific tissue that you're looking at and the specific process.
	So we could spend from here until next Sunday discussing each possibility but I think it's not really worthwhile.  It is clear that there is a variety of suitable agent strains and animal models to be used, each depending, as Dr. Rowher suggested, on what tissue you're looking at, what process you're looking at.
	Either looking at PRP scrapie as a physical marker and/or infectivity as a biological marker can be acceptable or not acceptable depending on which approach one is using and et cetera.  The reduction in titer that's required, again, varies on the type of spiking, the source of the spiking, the type of process, the expected bioload in the tissue, and clearly it would be desirable to have more than one validated method but in some cases a single validated method may be all that's possible.
	So, having said that and given my own opinion on all of those, I'll open it for discussion to get any other information and opinions so that we can communicate those to the FDA.
	Yes, Steve and then Sue.
	DR. DeARMOND:  These are not yes and no answers.  There's no way we can do that.  These are Blue Book answers.  We all should have been handed out our little essay books because the --
	DR. BOLTON:  Well, this is your chance.  It says to please comment on.  So you can --
	DR. DeARMOND:  Yes, please comment on suitable TSE agent strains.  Of course, every strain has really markedly different properties.  A new variant CJD and CJD are really markedly different and they really require different animal models.  The bovinized transgenic mouse does beautiful with variant CJD and the humanized ones don't do very well at all and the ones that respond to CJD itself are quite variable. From the MHU2M model to the HUPRP model they all have different properties and each one of them may be valid under different conditions.
	An overexpression of those may, as Paul Brown brought up, create false positivities.  On the other hand they create short incubation times so that you can get answers quicker.  So this is a very complicated question with many answers to it.
	And there are two components, strain and the model.  It's not answerable at this stage without a blue book.
	DR. BOLTON:  Sue?
	DR. PRIOLA:  David, I completely agreed with what you said to introduce this and I'd just like to add that at least on part B, which is the only one that I feel comfortable even making some proclamation on, and that's that given what Bob Rowher has shown us that there should be a requirement, no matter what is decided, depending upon what tissue you're looking at, what your requirements are, that infectivity should always be assayed for because to my knowledge I've never seen any convincing evidence that you can correlate lack of PRP-SC with lack of infectivity or correlate level of PRP-SC with level of infectivity.  So at least in that Part B infectivity remains the gold standard and should always be.
	DR. DeARMOND:  Could I comment on that?
	DR. BOLTON:  Yes.
	DR. DeARMOND:  There's no question. Infectivity is the ultimate gold standard. The problem is with low titer infectivity on an instrument or in a tissue or one of these processes it could take a year and a half to two years to 500, 600 days in a mouse model to get an answer, which is beyond what most people will wait for, which is why that would be the last resort.
	If the PRP model, if the PRP measurements, which can be done in the order of 24 hours, are negative, if the titre is so low that PRP scrapie doesn't even show up in it, then I think you go to the next model.
	DR. PRIOLA:  Just real quick. That's right where Part C would come in. That's part of your blue book thing.  You'd have to make the decision what constitutes an acceptable or not level of infectivity and what would be a cutoff point for an experiment like that.
	I'm just saying that infectivity I think should be given heavier weight always than PRPSC.
	DR. BOLTON:  Steve?
	DR. PETTEWAY:  I think maybe an analogy to some of the things that we've done with plasma, for instance, that it's really going to end up being a combination of things because you're looking at more than one parameter showing that any of this inactivation or removal that removes infectivity is important.
	Showing that it's valid and reproducible is also important and that may not be feasible with infectivity.  So demonstrating you have a correlation between the process's ability to remove prions in infectivity and in using the prions to demonstrate reproducibility makes a lot of sense.
	So I think that requiring that there are validation studies to demonstrate reduction and then leaving the details of that to whoever is doing the studies to produce a valid study based on what's available makes the most sense rather than trying to dictate it up front.
	DR. BOLTON:  Additional thought and discussion?  John?
	DR. BAILAR:  Is anything known about the infective dose of these things? Is one prion as bad as 100,000 or whatever?
	DR. DeARMOND:  It's something like 5,000 PRP molecules for one infectious unit, something like that or 50,000.
	DR. BOLTON:  It depends again on the model but it's somewhere between 10,000 and a million depending on -- I mean, this is, again, it's as complicated an issue as you can get.  It's different if you look at mouse versus hamster.
	DR. DeARMOND:  And strains within those are all different.
	DR. BOLTON:  That's right so it's very complicated and how you relate that to one infectious dose in humans is completely unknown.  We did not how many PRP molecules are equivalent to one infectious dose in humans and, of course, it also depends on the route of inoculation and everything else.
	DR. DeARMOND:  And, of course, the other complication now is we have this soluble protease-sensitive PRP scrapie, which seems to be in some strains of an agent 50 percent or more of the total PRP scrapie.  So it's getting to be more complicated and the assays that are being developed now look at both the protease- sensitive or the soluble PRP scrapie plus the protease-resistant PRP scrapie.  You get curves that define different strains of agent based on their being able to separate the two.
	DR. BOLTON:  Other comments?  We're not going to vote on anything here.  If there's anything regarding this particular issue that you want to communicate to the FDA now is a good time to do it.
	MS. KNOWLES:  Just from my experience with the BPAC Committee there have been many times when actually there have been people internally at FDA who have developed algorithms that then they brought to the committee for review and suggestions and comments and maybe that's something to think about with this particular issue, too.
	DR. BOLTON:  Dr. Gambetti?
	DR. GAMBETTI:  I think what I would include in the recommendation is that really, as was said already, one has to use probably a different method according to the situation.  So I think we all agree that it would be nice to have.  There should be some clearance study.  But they may not be one study, and there is no way to decide at this point.  One has to compromise time versus sensitivity of the test and that has to be open according to the system.
	So this would be a recommendation. These tests are needed.  These questions actually are a little bit ahead of the time. That is the problem.  But there has to be flexibility on which one to use according to the situation.
	DR. BOLTON:  Yes, I agree.  I think that's basically what Steve was saying as well.
	DR. PETTEWAY:  Just one more follow-up.  I don't think that the committee or even the FDA and certainly I would not have predicted the method that Regeneration Technologies is using to get into the bone, to extract what's in the bone, the pressure. I mean, I think it's important to allow industry to use some of the mother of invention philosophy, which is require that the clearance or removal is demonstrated but leave it up to the people that know the specifics to invent methods to do it, not limit them.
	DR. BOLTON:  With the assumption that they're going to run that by the FDA before they start their studies and they're going to get some agreement that that's going to be acceptable once it's done because nobody's going to launch into one of these several hundred thousand dollar or million dollar clearance studies without knowing that the end result is somehow going to be acceptable to the FDA.
	DR. PETTEWAY:  Just to comment on that just again from all of our experience in the plasma industry we did that up front and the FDA was very open, very receptive, and very helpful in all of the studies in designing the studies as we went through this.  I'm sure they would be the same in this regard.
	DR. BOLTON:  Absolutely.  Now, what I would like to do is take a break for 15 minutes if it's --
	MR. PARDO:  Before you move on to the next subject I have not heard an answer to this question.  Does the committee now support the FDA additional donor questionnaire for tissues related to travel? Previously this committee had voted against it.
	Several of the presenters talked about the impact on the industry, and yet it is almost 6:00, and I still don't know the answer.  Thank you.
	DR. BOLTON:  I don't believe that was on our agenda to consider this morning, today at least.  It was whether we support the European travel restrictions for tissue donors, right?  Jay, I think it's on the agenda for tomorrow.
	DR. EPSTEIN:  No.  I mean, Topic #2 is the draft guidance that we published which contains the travel exclusion recommendations so we're about to discuss that.
	DR. BOLTON:  After the break.  So let's meet back here in 15 minutes.  That's at 10 after 6:00.
	(Recess)
	DR. BOLTON:  We will now begin Topic #2, which is the FDA Draft Guidance on Preventative Measures to Reduce the Possible Risk of Transmission of Creutzfeldt-Jakob Disease and variant Creutzfeldt-Jakob Disease by human cells, tissues, and cellular- and tissue-based products.
	And after having said all that I'll just remind you that this is in fact a draft guidance.  And so the first presentation will be by Dr. Melissa Greenwald, and she will present the Draft Guidance.  And then after that Dr. Allen Williams will present the Possible Effects on Tissue Supply. Dr. Greenwald?
	DR. GREENWALD:  Thank you.  To update the committee and in order to do this I'm going to be presenting background information about how the draft guidance came about and point out the main differences between the tissue draft guidance and the blood final guidance which has been published and present the specific recommendations that are made in the draft guidance.
	The need for guidelines to prevent the transmission of CJD and vCJD was discussed at a January 2001 TSC Advisory Committee meeting.  The committee did vote unanimously that there is a significant risk of transmission of vCJD from HCT/Ps as compared to the risk of vCJD from blood transfusions.
	The committee agreed that the tissues for the greatest risk for transmission are dura mater and cornea and the committee also stated that there is no reason to believe that the risk for transmission of vCJD in tissues is less than that for sporadic CJD.
	There was a majority agreement that the FDA should recommend donor deferral criteria for possible exposure to the BSE agent.  It was also noted that there needs to be a way to make allowances for HLA- matched hemopoietic stem cells.
	There was no advice from the committee regarding specific deferral criteria, including such information as what countries to include, time periods of potential exposure, or duration of exposure. There was no vote regarding whether a donor history interview should be required for all HCT/P donors.
	Because of CBER's concern for the potential public health risk associated with vCJD in tissue and based upon the recommendations of the committee a tissue draft guidance was written and was published this month.  The guidance was modeled after the blood guidance that was published as final guidance in January 2002, using the same donor deferral criteria.  So we used the same criteria.  At this time there is no information available about risk reduction versus supply reduction for tissues like that that is available for blood.
	There are three main differences between the tissue and blood guidances. Since a tissue guidance will not be implemented until after the donor eligibility rule is finalized we're not going to do a two-phase implementation of this.  Also, the tissue guidance has wording that may sound confusing but we will not disallow the collection and use of HLA-matched hematopoietic stem cells in cases of urgent medical need when there's matching issue.
	Also, because little is known about the impact of this guidance that it would have on the tissue supply there is a request for firms to submit data either known or generated assessing the impact of these recommendations on the tissue supply.
	Moving on to the content of the guidance itself, I'm only going to go into detail about the specific recommendations and just give you an overview of the rest of the document.  The background section establishes the regulatory authority for creating this guidance, states the public health concern regarding CJD and vCJD, and also explains the TSE Advisory Committee recommendations that we just discussed. There is also discussion about CJD and vCJD as disease entities giving information about clinical presentation, diagnosis, and epidemiology.
	There is discussion about the basis for making a CJD recommendation, including previous recommendations made in guidance as well as a discussion of known cases of CJD transmission by tissues.  The tissues known to have transmitted CJD include dura mater and cornea.
	Next there was a discussion about the basis for making the vCJD recommendations.  There's a listing of the five currently recognized risks of exposure to BSE agent, and includes supporting information about the exposure risks.
	And finally the recommendations themselves.  It is recommended that firms determine ineligible any donor who has any of the following risk criteria.
	Number one is any donor who has been diagnosed with vCJD or any other form of CJD.
	A donor who has been diagnosed with dementia or any degenerative or demyelinating disease of the central nervous system or any other neurological disease of unknown etiology.  However, a tissue from a donor with dementia may be acceptable based upon an evaluation by the medical director if it is confirmed by gross and microscopic examination that the dementia is caused by cerebrovascular accident, a brain tumor, head trauma, or toxic/metabolic causes and is confirmed not to be caused by a TSE.
	Donors who have an increased risk of classic CJD, those being recipients of dura mater transplants, recipients of human derived growth hormone, or persons with relatives who have CJD.
	Donors who have spent three months or more cumulatively in the UK from 1980 through the end of 1996.
	Donors who are current or former United States military members, civilian military employees, or dependents of military or civilian employees who resided at US military bases in Northern Europe for six months or more from 1980 through 1990 or elsewhere in Europe for six months or more from 1980 through 1996.  And yes, these geographical locations are defined in the document.
	Donors who have lived cumulatively for five years or more in Europe from 1980 until the present.  And this time in Europe will include time spent in the UK from 1980 through 1996, which is one of the other deferral criteria.
	Also donors who have received any transfusion of blood or blood components in the UK between 1980 and the present.
	And the last one is for donors who have injected bovine insulin since 1980 unless it can be confirmed that the insulin was not manufactured from cattle in the UK.
	Some important additional information that is contained in this draft guidance.  As I said, all geographical references in the deferral recommendations are defined.  Recognition is given to HLA-matching issues with hematopoietic stem cells.  If hematopoietic stem cell donor would otherwise be determined ineligible by recommendations 3 through 8 the risks of using the cells may be outweighed by the benefits and in that case HSCs may be collected and stored and their use may be considered an urgent medical need.
	It's also recommended that the CJD/vCJD screening questions be included in the donor's medical history.  Unfamiliarity with the term "CJD" may be taken as a negative response.  Donors who have CJD in blood relatives are excluded unless the diagnosis of CJD was subsequently found to be inaccurate, the CJD was iatrogenic, or lab testing shows that the donor is without the mutation associated with familial CJD. Obviously this is going to be more important for reproductive donations.
	There is provision to allow HCT/Ps from donors considered ineligible to be retained for nonclinical scientific or educational uses with proper labeling and storage and industries affected by this draft guidance are encouraged to submit any data that they have now or could obtain through studies concerning the effect of implementation of these recommendations on the tissue supply.
	And this is currently draft guidance and is not necessary to be implemented at this time.  When final guidance is issued there will not be a two-step phase in period.
	So our charge to TSEAC today is not a vote.  We would just like for you to comment on the recommendations made in this draft guidance.  You directed us to make recommendations for donor deferral and these are recommendations.  We would also like you to please consider how information may be obtained about the effect of implementing these tissue donor deferral criteria on the tissue supply in the United States and the next speaker, Dr. Williams, will be speaking more directly to that issue.
	DR. BOLTON:  Thank you, Dr. Greenwald.  Are there questions now for Dr. Greenwald or would you like to hold them until after our next presentation?  So Dr. Williams will now present on the possible effects on the tissue supply of the draft guidance.
	DR. WILLIAMS:  Thank you.  As most of you are painfully aware, the discussions related to the travel deferrals to protect against the theoretical risk of variant CJD exposure was a carefully constructed balance between this theoretical risk and the estimated loss of blood donors which in itself would have a negative impact if that would exceed certain levels.
	So what I'm going to discuss today is very briefly some of the considerations that went into arriving at the data which allowed those discussions and then some of the implications for the tissue donor pool which for the most part is far less well-defined than the blood donor pool in general.  And I'll end with a few comments as a segue into tomorrow morning's discussion on some of the impact in the blood donor pool of the latest guidance related to variant CJD.
	Very briefly, in early 1999 surveys of donor travel were conducted.  This was a cooperative venture between a number of blood centers and the survey was conducted actually at the request of this committee to provide travel data related to travel in the United Kingdom and we also included Europe in that survey measurement.
	These surveys were done among geographically and demographically representative blood donor populations and ultimately they supported estimates of the national impact of a travel deferral on a donor population with many assumptions inherent, one of the major ones being that in the face of the very limited information a linear response existed between travel in a country that had potential BSE contamination of its meat supply could be equated to potential to transmit variant CJD through a blood donation.
	The estimates that arose from that study ultimately include the level of donor loss that would occur at different time exposure levels, and secondly the estimation of the overall risk burden, again related to time, and a portion of the risk removed by different policy options.
	Going back to June 1999, this was the initial graph shown to this committee, which basically used that time travel information to compute the overall burden of risk and the percentage of donors that would be deferred in any given time period used as a deferral criteria and, as you know, ultimately in that initial policy decision we ended up with a deferral for six months travel in the United Kingdom at an estimated loss of 2.2 percent of the donor supply and a removal of about 86 percent of the theoretical risk based on that risk burden estimate.
	The methods used in that estimate were a random sample of donors at 12 blood center sites in really a very short time period, a two-month time period.  We mailed out 19,000 anonymous scannable surveys in a single mailing with a cover letter, got about half of those surveys back, and the survey asked questions about travel, basic demographics, sex and age, first time or repeat donation status, and educational level.
	Now, there has been a history of conducting surveys within the National Heart, Lung and Blood's REDS study and this really served as the basis for attempting to collect the data by this mechanism.
	Some known advantages from this in prior surveys is that in the setting of a well-defined sampling frame and experienced field sites, surveys are well-established, reproducible data collection tool for the blood donor population and there are now quite a list of published studies from REDS and others showing that this data are reproducible.
	In addition blood donors being a special population they generally provide a favorable response rate.  If you get down under 50 percent you start to wonder about the validity of the survey rate's internal validity and we generally enjoy a 50 to 70 percent response rates depending on the length of the survey.
	Also, the anonymity factor is important, particularly when you're measuring something like donor risk, because if you have information in a linked manner it implies some sort of operational consideration for blood that may have been donated previously.  Also, surveys allow a wide scope of information and simultaneous collection of demographics so it applies real-time data collected that can be stratified against different variables, including demographics, and really very easy to conduct analysis.
	However, surveys do have limitations.  The findings tend to be reproducible but difficult to validate against truth by another mechanism.  Some important sample subsets may be underrepresented in the response.  For instance, we tried to get data about military populations and the extent that they traveled in the UK and Europe.  We got something like a 10 percent response rate. So obviously the data there weren't terribly reliable and in fact that has turned out to be a very important variable in a blood donor setting.
	The Office of Management and Budget under the Paperwork Reduction Act requires review and approval of data collection using federal funding.  Under survey mechanisms this generally takes from six to twelve months to get that approval.  So any sort of rapid response capability that is a hurdle.
	And finally in conducting information like we would need to make these sorts of estimates you have to deal with a matrix of multiple countries visited times in multiple travel time frames and that makes a lot of complexity in the survey document.
	For instance, this is one question out of the 1999 survey, did you live in the UK, and then lists all the countries included in the UK or the Republic of Ireland between '80 and '89 or 1990 to 1996 and then provides a total of nine different categories of time periods.  So it's complex for the donors to answer this as a survey.
	Now, in thinking about how to assess impact on the tissue donor pool the media thinking is well, we'll do a survey and see what the responses are likely to be. The problem is it's a little different population, a little different setting, and even in a donor screening situation it's going to have some different characteristics and I just wanted to point out some of these difficulties and make some suggestions as to how facilities might approach collecting data and then as requested submit this to FDA to document what the impact might be.
	First of all, the tissue donor pool has epidemiologic characteristics but they're really not well-defined compared to the blood donor population.  Even blood donors in the typical blood center of the demographics beyond age, sex, and ZIP code are not too well known and really through the REDS study have we been able to collect extended demographics like educational level and travel by specific survey mechanisms. So in general tissue donors are not well characterized so the sampling frame is a little difficult to establish.
	Sites generally are not experienced in research data collection.  Those of you who have conducted surveys know that if you put a stack of forms on the table and ask someone to fill them out you won't get a very well-controlled data collection and subsequently the data may not be that reliable.  So trained staff is very important in doing something like this.
	In the face of unknown demographics perhaps one way is to assume general population demographics for the tissue donor pool.  That will certainly vary where demographics are known.  In the case of semen or oocyte donors there are age restrictions and those can be factored in.
	Motor vehicle data on organ donation perhaps might be relevant to tissue.  I don't have those data available but perhaps that might get a sense as to what the demographic shifts are in terms of willingness for an individual to identify themselves as organ and tissue donors.
	The tissue donor populations have been described.  This is information provided by Dr. Solomon from musculoskeletal and skin.  We're talking approximately 20,000 donors or 750,000 tissues, ocular tissues, mainly corneas, about 47,000, including 5,000 non-US tissues.
	And in the future potential regulatory oversight of cord and peripheral stem cells, dura mater, semen, oocytes, where again even the donor pool size is as well as the demographics are unknown at this point except for the known age restrictions.
	In the blood donor setting, as presented before, there are several stages of donation and in the blood donor setting I think it's pretty clear that most of the self-deferral done by donors occurs before they ever appear at a blood center to donate the blood.
	That's through information provided through the media, letters transmitted by the blood center, telephone conversations with the blood center at the time they make an appointment, and so forth.  There's also some self-deferral at the actual blood collection site.  Then there's the interview-based deferral data from this you'll be seeing tomorrow from a number of blood organizations.  And the final category, those who fail to appropriately defer.  And this has implications because, one, it's a false negative response, which obviously we try to avoid, and secondarily there are some implications of having post-donation information available on a product that's been previously collected.
	This is a shift that I would predict would occur for tissue donors. Clearly self-deferral before collection might be applicable in some cases but certainly for cadaveric donors that's not a consideration and probably the primary way of collecting data is going to be by secondary interview of family members or others who know the donor well.
	This raises a new issue.  Whereas an individual may know their travel history, complex as the question may be, relatives may have a much more difficult time answering that question and we'll probably have to address the don't-know factor.  I know this individual was in the UK for a period of time.  It might have been two months, it might have been two months.  I just really don't know.  I suspect that's going to be larger in the tissue population.
	Again, there's going to be a failure in some cases to identify a disqualifying factor resulting in false negative responses.  It may result in an ineligible donor or post-donation information and there needs to be considerations as to how to handle that information when that occurs.
	The data from the blood donor surveys were looked at intensely demographically and the age and education variable clearly was the most associated with travel, as one might expect.  There's an age-increasing prevalence of travel to the United Kingdom and this was confounded with the education variable.
	The data shown here are for travel ever to the United Kingdom.  This is not the six-month deferral.  It's travel ever.  And the overall figure for the surveyed population was 22.8 percent.  And you can see the marked increase from those who have less than a high school education less than 1 percent travelled, some college 20 percent, those with post-graduate degrees 36 percent for an overall of 22.8 percent, quite a difference between the range.
	Age also showed a big correlation, and in fact confounded the educational variable.  Educational variable, while useful if the data were available, these are not generally available on most blood donor populations because the centers really have no reason to collect that information.  So to try to stratify that would be difficult. Age, however, is available and you see a similar breakdown from the younger donors, 16.4 percent up to the older plus-65 donors approximately 31 percent.
	Also, first-time repeat status, you see in general for UK travel by sex gender is not that much different, a little higher in males overall but you find if you look for the older population the women tend to travel as they get older and females exceed males in the older population but in general it's an even comparison.
	And then first-time versus repeat. A pretty clear distinction, first-time donors closer to about a 14 percent travel prevalence versus repeat donors, which comprise 80 percent of the donor population, much closer to the mean overall.
	Now, how to assess the travel deferral impact among tissue and cell donors?  Based on the factors that I've outlined, probably the major one being lack of an adequate sampling frame, the survey mechanism to assess tissue donor loss may not be viable.  It may be possible on a local setting and if any sites wish to use the same or similar survey that was used in the blood donor survey we'd be happy to share that and you're welcome to try to collection the information but I think trying to define a well-constructed survey in the absence of a good sampling frame is going to be quite difficult.
	One potential way to approach it is to consider travel deferrals for first-time blood donors at the local blood center in a geographic area where the tissue donors are being collected, particularly if one can do some age adjustment against the two donor populations.
	This may reasonably predict donor travel characteristics of local tissue and cell donors.  Admittedly some donors will have originated from other areas of the country but so do blood donors.  It would be, I think, quite difficult to pinpoint differences in the two populations from that aspect.
	Alternately for centers that implemented the deferral the initial impact of the variant CJD deferral for all donors at the local blood center might be usable. After about the first two months or so of implementation there's a culling effect of repeat donors so after that point one needs to consider first-time donors only.
	So that's one potential way to look at it and if sites could compute that figure and determine how that would impact the necessary tissues that they distribute that would be useful information.
	Finally one might consider a well-designed pilot implementation program. I think this would get not only at the potential impact of donor loss but also could be used to identify operational difficulties, another area where I think FDA would be very interested in having information to produce the final guidance.
	A second consideration here is that, as hopefully you'll hear tomorrow, there are some innovative programs being developed in the blood donor setting, ways to recognize donors who are lost by the deferral and actually provide incentives for them to identify donors to replace themselves.  And as this continues a deferred donor identifies additional donors that continue to be recognized by the blood center for their important contribution to the blood collection process.
	So that's another area that can be used to help offset donor loss and a program started by Stanford is starting to pick up steam in some other blood centers now.
	Finally I want to comment on the ability of our country to assess rapid data related to blood donor loss and tissue donor loss.  The REDS study has been mentioned many times here and it's been an absolutely critical mechanism for data collection over the past 12 or 13 years.  As it undergoes its renewal I'd like to suggest some things for the committee to consider and potentially recommend.
	One is that the REDS collection sites be expanded from their current 6 up to 10 or 12 to provide better representation of the country, including some of the coastal areas not currently represented.  The site already has capable donor and donation data systems.  Perhaps this could be expanded to cover areas like blood collection data, inventory, and distribution so that we better understand the dynamics of how blood is collected and used, which is also relevant to the overall supply.
	NHLBI generally has been very responsive to HHS data needs.  There are some things which would facilitate this further in a new study such as a rapid survey capability, provisions for providing OMB exemptions for critically needed data, having established IRBs which are educated to recognize blood donor-related issues, having experienced staffing on board to address the rapid response need, and then finally where the bureaucratic hurdles can't be surmounted potentially have some of the sites participate in private sector cost sharing to facilitate data collection.
	In the 1999 survey actually the blood centers supported the data collection effort and it wasn't until the data was in hand and in a database that federal funds were then used to analyze it.  So that's one potential way to proceed.
	These are just some thoughts about the tissue donor pool and potential ways to get at the information survey mechanism. Again, we would be happy to share the instrument and the procedures, but it will be a little tougher task in this donor population.  Thank you.
	DR. BOLTON:  Thank you, Dr. Williams.  Now what we should do is take any questions or have any discussion regarding both of these presentations, which really again focus on the draft guidance to reduce the risk of transmission of CJD and vCJD in the human cell tissues and cellular-based tissue products.
	Comments, questions?  Dr. Wolfe.
	DR. WOLFE:  Perhaps this could have been said earlier, but then we would be later here.  I really feel obligated to spend a minute or two on the whole issue of dura mater again.  Dr. Gambetti and colleagues in a paper wrote I guess last year in Neurology, "Even the most stringent donor screening and dura mater processing practices may not totally eliminate the potential for an infectious graft.  Because of this inherent albeit small risk of CJD transmission by dura mater grafts surgeons may want to consider the alternative use of autologous fascia lata, temporalis fascia, or synthetic substitutes, and there's also US-based bovine pericardium as well."
	And I think that what Dr. Gambetti and his colleagues are saying is unlike the issue of the cornea, where there isn't any alternative, I mean, a lot of what we've been talking about today and I think that although a little prolonged it's been a useful conversation, there are tissues that either are not a very high risk or are irreplaceable.  You can't do synthetic cornea or retina or whatever else.
	And the thing that distinguishes dura mater is (a) it is very high risk. It's two-plus, not four-plus, as brain but it's up there next to brain and there is a growing number of substitutes.  When I raised this issue in the first incarnation of this advisory committee five years ago there was a vote essentially saying there was no circumstance anyone could think of where you couldn't use something other than dura mater.
	There was a surgeon then, a neurosurgeon from the UK, who told us about the fact that at that time there had been a ban for I think eight years.  It's now thirteen years it's been banned in the UK, five years it's been banned in Japan, and just last month after a poll of neurosurgeons in Canada it was banned in Canada.
	So we have three countries doing something that's much more intelligent than what has gone on here and I just want to raise this because under this topic of measures to reduce possible risk of transmission of CJD or Creutzfeldt as we were taught in the earlier days this has got to be seriously considered.
	I mean, if it is necessary, which maybe it is, to poll neurosurgeons that should be done.  I did a very informal poll based on neurosurgeons who had been residents when I was 35 or 40 years ago and two neurosurgeons to whom I had referred patients.  One was at UCLA, one was at Hopkins, one was at the University of Virginia, and one was at Medical College of Virginia, and none of them were using dura mater, none of them said their departments had been using it for several years, and one of them, John Jane, who is the editor of the Journal of Neurosurgery, signed the petition that we filed last August to the Device Division.  Tell me why this is regulated as a medical device and I'll be informed.
	So I just want to put this on the table because rather than seeing in Dr. Williams' last slide the future of an expanding number of people using cadaveric dura mater it's going out of style.  You heard from several companies they don't do dura mater any more, to use modern vernacular, and this is an example of where the so-called invisible hand of the market place, the Adam Smith notion, should not be allowed to operate.  This will die of its own accord.  But we would all feel terrible if between now and the time it dies of its own accord without a ban by FDA there is another case in the United States.  It would be a tragedy.
	None of us who had an automobile accident who would wind up unconscious in the emergency room and would need dura, would need some sort of repair to broken or destroyed dura, would like to have dura mater put on our brains.  And if we don't and we don't want it for our families I don't think we would like anyone else.  The minority of neurosurgeons really needs to be taken out of the loop on this.  Enough of that topic.
	DR. BOLTON:  Additional discussion. Lisa?
	DR. FERGUSON:  I had a question for Dr. Williams.  I mean, your slides on the number of corneal donors?  There was a number on there, 5,000 non-US.  Does that mean that they were non-US citizens but the corneas were harvested in the US or does that mean that those were obtained outside the US and brought in?
	DR. SOLOMON:  EBAA could probably answer this better but no, those are corneas that were obtained in the US from US donors that were exported outside of this country.
	DR. BOLTON:  So they're exports.
	Dr. Bailar?
	DR. BAILAR:  I'd like to follow up on Sid Wolfe's comment.  I don't feel like I'm well enough educated about this to come to any decision today.  Is it fair for us to ask FDA to come back at our next meeting with some specific analysis and perhaps a proposal?  I'd like to hear directly from people who still use dura mater to find out why they do so.
	DR. BOLTON:  We're actually not considering anything to do with dura mater at this time.  That's just basically a statement by Dr. --
	DR. WOLFE:  The topic is mentioned in the guidance.  That's all.  I mean, it's one of the topics there.  That's all.
	DR. BOLTON:  Ermias?
	DR. BELAY:  I beg to disagree with the assessment that the gentleman made.  And the reason is I'm not aware of any CJD case that resulted from a dura mater that's processed under the current FDA recommendations.
	In other words I'm not aware of any CJD case that's resulted from a dura mater that's processed under the current approved FDA recommendations.  All the dura mater CJD cases that we've been referring to, almost all of them, the vast majority of the cases received a single brand of dura and that dura was produced before June 1987.
	And that company was a single company producing a single brand of dura, which is Lyodura, so I think we need to make that distinction very, very clear.
	DR. WOLFE:  Most of the cases were, but the last case, and you're technically right, was a process using 10th normal, not 1 normal, sodium hydroxide.  But the literature is replete with both scrapie and other transmissible spongiform encephalopathy causing organisms being resistant to even 1 normal.
	So I think that it's a little bit iffy.  I agree with what Dr. Gambetti has written.  Dr. Brown has said the same thing, that we just aren't sure, even with the modern methods, that we're going to be able to pick this up.
	And if you combine that with the donor selection problem, and we've heard over and over again today that it is entirely possible that there is infective brain tissue in someone that has no pathological or clinical evidence of the disease, so if you combine the defect there with I believe still a residual defect in the processing and the presence of enormous alternatives I just don't see any need for it.
	DR. BOLTON:  I don't want to beat this to death.  If we have discussion on other aspects of the draft guidance other than dura mater?  Yes?
	DR. DOPPELT:  I'd like to turn to the issue of the history of travel in England and Europe and the question is for blood donors, I mean, they're alive and you can ask them their own history.  For tissue donors in general they're not alive and so what kind of accurate information are you going to get from the next of kin in terms of how long somebody was traveling, where they were, the specifics?  I mean, they're probably not going to have that information. It's not retrievable.
	DR. BOLTON:  I agree.  I think this is one of the key issues in terms of our input into the draft guidance in the previous meeting where we made some of these recommendations and now where we're being asked to comment on the draft guidance.
	It's how do we deal with these don't-know questions that Dr. Williams discussed and what impact these may have and how to ascertain what the impact will be in fact on actual tissue donations.
	Because again we're faced with the same problem we had in the blood and blood products area is that we have to weigh the hoped-for increase in safety versus the ultimate loss of some tissue that's going to be donated and the impact on the supply.
	And so I think that's an area that I would like to focus discussion on now, and get any questions out or thoughts about that.  In other words how are we going to or how will the collection sites accurately assess the travel history and how will they handle the uncertainty?  What does an "I don't know" mean?  Did your loved one reside in England for three months or more from 1980 to 1996?  And how is that going to be handled?  Is there discussion?
	DR. WOLFE:  Just a suggestion on that.  I mean, it is possible, obviously, with the consent of relatives, near friends, to check passports.  I mean, it's not quite the same but one can at least get some kind of crude information that has to do with foreign travel and where it is.
	I mean, it's complicated but so is being in the dark and not having a clue because of exactly what was just mentioned about foreign travel, the person's dead. Passports are alive.
	DR. DOPPELT:  I'm on my second passport.  I don't know how long they're good for but usually you pitch them and so that information may not be available. Second of all, if I croaked I don't think anybody would be able to find where I stuck my passport.  I may even have trouble finding it.
	DR. BOLTON:  Lisa?
	DR. FERGUSON:  I have a comment and a question again getting back to how many cells and tissues are covered under this and my focus is on the reproductive tissues, I suppose.
	I'm very familiar with what's been done in the animal world in regards to the lack of transmissibility, especially of BSE, in semen, embryos, and oocytes.  I wonder has similar work even been started or attempted in the human arena?  Is there anything to draw from there?
	And if there is, I mean, do we need to lump those into this type of guidance? Does that even make sense?
	DR. BOLTON:  I'm not aware of any information that would bear directly on infectivity in those tissues in humans. Steve, do you have?  And Paul Brown is not here.  Bob Rohwer, do you have any?
	I don't know that any information exists in that area and extrapolating from hamsters and mice could be very risky in that sense.
	DR. FERGUSON:  Or even from cattle and sheep would also be risky.
	DR. BOLTON:  Cattle and sheep, exactly.
	DR. PRIOLA:  This is Paul Brown's paper that was actually in our packet, thank God, and they had at least one instance where one patient tested semen and two patients vaginal secretion and neither transmitted.  It's a very, very limited data but negative, which probably doesn't really mean much.
	DR. BOLTON:  Very good, Sue.  You get an A-plus.  Sue gets an A-plus for doing her homework.  Dr. Bailar?
	DR. BAILAR:  I have two questions. Maybe we should take them separately.  The first is what is the chance that somebody would still be an unrecognized carrier as a result of exposure, say, 25 years ago? Would most of them have come to the surface now with some kinds of symptoms?
	DR. BOLTON:  For variant CJD?
	DR. BAILAR:  Yes.
	DR. BOLTON:  We don't know that.  I mean, we are in a probably early but impossible to tell apart of the epidemic curve for variant CJD in those individuals who have resided in the UK for their entire lives.  Obviously someone traveling through and living there for three months or six months or a year, has received, if any exposure, a lower exposure and their incubation time clearly could be several decades or longer.
	So I don't think that we can expect that a majority of variant CJD cases have appeared as yet, especially for those who have limited travel and limited exposure to the BSE-contaminated beef or beef products.
	DR. BAILAR:  The other question is why should it make a difference whether somebody was wearing a uniform?  If some constraint is good for people who were in the military while they were there why shouldn't it apply to everybody else and vice versa?
	DR. BOLTON:  You mean in terms of the exact wording of that restriction?
	DR. BAILAR:  The wording of the guidance here.
	DR. BOLTON:  I think it has to do with whether those individuals were eating on base or food supplied on base or whether they would have access to that food.  Now, if you were a civilian living there but not associated with the military, not living on base, you would have had a different food supply.  It has to do with the source of beef either in the northern European theater or the southern European theater.  Much of that was sourced from the UK during the high-risk period and that's why there are the differences in the years that are described but that's basically what the difference is.
	Other questions or discussion? Yes, Dr. Linden.
	DR. LINDEN:  Well, I really just wanted to second Dr. Bailar's comment.  I think the feeling in the industry is that there's a concern about donor loss but in most cases people are not going to know and if the answer is unless you know definitively that's okay that might be okay. If the answer is well, you need to find out or defer then we basically have no tissue, I think.  That would really be a problem.  But perhaps there could be some sort of pilot study to really look at how this would apply in the donor tissue.
	DR. BOLTON:  This is my concern is that when you actually get to the mechanics of implementing this what's going to be the procedure when the answer is I don't know, and what's the time delay and just the basic fact of the close relative not having the information at hand, not perhaps being motivated sufficiently to go out and find that information within a prescribed period of time, is that immediately going to lose us many, many donors for tissue?  And I don't know that there's any way to answer that here except to ask for pilot implementation and to see the effects in those sites.
	Now, the question is then who is selected to be the pilot site and who's going to volunteer to do that if that were the case.  I'm not exactly sure how that would go.  Perhaps, Dr. Doppelt, you could comment on that.
	DR. DOPPELT:  Well, I don't think anybody's going to be very enthusiastic about taking that project on.  I mean, I think it comes down to what Jeanne said. You go through this donor screening form, which is fairly detailed, and it is up to the medical director to decide what's a plus/minus answer and which way do you interpret it but with this situation you may wind up with yes, they were in England but I don't know when and I don't know how long.
	And so then what do you do with that?  You know that there could potentially be a problem.  And so many people may just say well, pitch it, forget it, but that's going to have a negative impact on supply and perhaps unnecessarily so.  So I don't think you're going to get many volunteers.
	DR. BOLTON:  Dr. Bailar?
	DR. BAILAR:  This might be a good topic for a little survey.  You could ask people coming through for, say, blood donation was your spouse in England during this time period and if so for how long and see how many of them say gee, I don't know.
	DR. BOLTON:  I'm trying to think how you would validate the answers.  I mean, you would get responses back and one way to view that would be what percentage of I don't know answers there were.
	But in terms of the affirmative answers or the negative answers you wouldn't have any way of validating whether those affirmative or negative answers were in fact correct.
	DR. BAILAR:  I don't think you could directly validate them but I think there would be a fairly high level of accuracy if somebody says yes, he was stationed in England and I was with him for eight months or no, he was never gone from home as long as three months.  That's pretty specific.
	DR. BOLTON:  I think it's worth considering having something like that done.
	DR. DeARMOND:  So what I'm understanding is we have no data on this at all yet or it's very speculative and so an experiment has to be run.  And the experiment that's being proposed is that we use the same deferrals for blood and blood products and apply it to tissues and if we implement that over the next six months to a year we'll see what the effect is on tissue donation.  Is that one way to think of it?
	DR. BOLTON:  Well, I think we're a step back from that.  This is a draft guidance which is now open for public discussion and the question I think immediately to us is what's our first feedback on this and obviously the comments from the public and the input from the public are being solicited before it goes then to guidance.
	So I think basically what we did previously was we suggested that there was risk for variant CJD in these tissues and that something should be done.  We in fact did not specify countries or times and what the FDA has done is to incorporate the guidance for blood donations.
	DR. DeARMOND:  And do we have any new data on that from the United Kingdom about the true infectivity of each of these possible donated organs?  Do we know more about the cornea?
	DR. BOLTON:  I think we know nothing more than we knew then.
	DR. DeARMOND:  So it's a reasonable concept that the cornea is going to be infected based on CJD itself, on sporadic CJD, but we don't know and we don't know about any of the organs.  The viscera are involved in variant CJD but are any of the other --
	DR. BOLTON:  I note Nick Hogan standing up, probably to remind us that the article from Collin's group he discussed earlier --
	DR. HOGAN:  Nick Hogan.  The infectivity of any of the partitions in sporadic CJD is less than .0025 percent that in brain, a variant CJD.  In variant CJD retina it's 2.5 percent that of brain and in optic nerve 25 percent of brain.  Those are the only good studies.  The stuff that Paul did was a gemisch of stuff that was thrown into chimpanzees and you really can't use that data qualitatively.
	DR. BOLTON:  So in that sense those data provide substantial justification for being concerned about corneal transplants from anyone who has been exposed to variant CJD and that's about the limit of what we have in terms of new information.
	DR. DeARMOND:  So we're to evaluate whether this guidance is a reasonable approach to it or unreasonable.  Is that the bottom line?
	DR. BOLTON:  I guess so, yes, and probably variations in between.
	DR. DeARMOND:  Right.
	DR. BOLTON:  Parts of it may be reasonable but parts of it may be --
	DR. DeARMOND:  Once again we have no concept of the impact at this stage or very little concept.  Like with blood, we have fairly good data on the impact of the deferrals.
	DR. BOLTON:  Well, I suppose we had better estimates of the impact because we had more of the survey information that was done on that actual population because you have repeat donors and it was well known that, for example, there were coastal differences in foreign travel versus the central part of the country.
	So we could recognize that there were regional differences and we had some way to calculate what the impact would be. Unfortunately, with the tissue donors there doesn't seem to be a ready way to get that sort of information.
	I don't know if there's any sort of database on people who have pulled out tissue donor or organ donor cards, for example, and to try to assess that.  I mean, the way that it's been done so far is to try to look at, again, blood donors and see how they fall into this.
	But I don't know that you could make the leap from a blood donor to a tissue donor.  So I don't know exactly how we would get this information except to try maybe to survey existing donors through surveys of their relatives at the time of donation before these are implemented to try to see what percentage of their existing population would be deferred under these guidelines.
	DR. DeARMOND:  So it gets down to an experiment?
	DR. BOLTON:  Essentially, yes.
	DR. GAMBETTI:  Is there a way in which one could get some information concerning foreign travel by tissue donors as compared to the blood donors by comparing the demographics of the two populations?
	For example, if the AIDS subdivision and education and all the other parameters are similar I think what one could assume then the pattern of foreign travel might be comparable.  So that would be just a short cut but I don't know whether it's possible.
	DR. DOPPELT:  Well, if you wanted to do a survey of potential donors, I mean, it may be that we could persuade some of the tissue banks to do that.  But in point of fact the tissue donors are the low risk. It's a low-risk tissue.  The higher risk are the corneas and it may make more sense to do that.
	On the other hand we also heard that 20 percent of the donors it's legislative consent so there's 20 percent right off the top that they're not going to have a clue.  So you're dealing with 80 percent of the 45,000 donors so that may be a place to start but I don't think turning to the standard tissue donors is really the best way to go because that's really a low-risk tissue.
	DR. DeARMOND:  So that's a good suggestion, maybe focus.  If that's for the first one how you implement this is focus on the corneal donors and what information can be gained from them before we go to any other recommendation, before we implement the whole thing.  They might be asked all these questions, if possible could be asked of their loved ones, and see if any responses of any importance to us can be derived.  That would be a good idea, keep it to one tissue, a high-risk tissue.
	DR. BOLTON:  You mean keep the deferrals to high-risk --
	DR. WOLFE:  The experiment to one tissue, the survey or experiment, whatever.
	DR. BOLTON:  That sounds like a reasonable suggestion.  I see a pained look from Ruth there.
	DR. SOLOMON:  I just wanted to comment on what Dr. Doppelt brought up about the I don't know.  The same situation exists when we ask the next of kin these very detailed questions about the sexual habits of the deceased in terms of MSM in the last five years or for a female donor have you ever had sex with someone in the high-risk group in the past 1twelve months.
	Those are also difficult questions to answer and I'm wondering what does the industry do now when they get a I don't know on one of those.
	DR. DOPPELT:  Right.  Actually, I think that's a little bit easier because when you get an I don't know there's no other source of information so probably unless there's something else to indicate in terms of the physical exam that might indicate that there was some high-risk behavior they would probably accept the donor.
	On the other hand for travel in Europe, I mean, it's more likely that somebody will know yes, they did travel but then the details I don't know.  So that actually puts you further out on a limb.
	DR. BOLTON:  Well, the real question is when the answer is I don't know is that interpreted as yes or no because you have to go one way or the other and, I mean, given the fact that these are rare and the risk is very low I suppose you could assume that accepting the donor could be the default but then basically what you'd be saying then is unless you tell me for certain that this individual falls in one of these categories we're going to assume that he or she does not.
	What does the committee feel about that interpretation?
	MS. KNOWLES:  If someone says I don't know I think it's really better to err on the side of caution and you have to also remember in terms of high-risk behaviors that there are still a lot of people in this country who are men having sex with men, do not self-identify, will never tell anyone in their family, and I just think we have to remember the literal translation of the word "assume" and just not assume.
	DR. BOLTON:  Dr. Bailar?
	DR. BAILAR:  I think again it would help to know whether the I don't knows are common or uncommon.  If they're pretty uncommon it isn't going to matter much and I would throw them out.  If it makes up a large part of that 20 percent I'd have to think again.
	DR. WOLFE:  It would seem that with 45,000, if that's the estimate of eye donors, corneal transplant donors, a year that with a survey instrument that was modified somewhat from the one used in the blood area and administered proactively to people as they are signing up or as their families are signing up for donors we could in a very short period of time get answers to the question Dr. Bailar raised, well, what percentage is I don't know, and actually start getting some demographics and in probably six months accumulate enough date to have a much better idea of what we are dealing with with this highest of risks, putting aside dura mater for the moment. Because whatever recommendations there are to be made for the lower risk tissues they're going to be less stringent than this and this is doable, I think.
	DR. BAILAR:  Yes, the people you ask in this survey do not need to be next of kin of potential donors.
	DR. WOLFE:  That's right.
	DR. BAILAR:  They could be any kind of sample from that part of the population, like blood donors.  I'm just trying to think of an easy way to get to enough of them.
	DR. BOLTON:  But I would suggest that with 45,000 corneal donations a year and I'm not sure if that means 45,000 corneas or 22-, but whatever it is you're still talking about in a half a year getting 5- to 10,000, perhaps, responses on a survey that could be fairly easily designed.
	DR. WOLFE:  Then the Eye Bank should be very interested in cooperating with us for reasons we've heard today.
	DR. BOLTON:  Right, so I think that that would be a reasonable recommendation for us to give to the FDA in terms of how to collect information on that.  And that will tell us a lot about what impact this may have on donations if one were to implement this for all donations or only for corneas.
	So I think we can assume that we've effectively communicated that to the FDA. And is there any other discussion with respect to the guideline?  Hearing none, then I will adjourn the meeting.  Move to adjourn the meeting for this evening, and we will resume in the morning.  Do I hear a second?
	DR. WOLFE:  Second.
	DR. BOLTON:  All in favor?  Take a formal vote.  Very good.  We stand adjourned until tomorrow morning at 8:30.  But if you want to come here early you can have coffee.
	(Whereupon, at 7:10 p.m., the PROCEEDINGS were continued.)
	*  *  *  *  *