Guidance for Industry
For Platelet Testing and Evaluation of Platelet Substitute Products
[PDF version of this document]
Draft Guidance
This guidance document is being distributed for comment purposes only.
Comments and suggestions regarding this draft document should be
submitted within 60 days of publication in the Federal Register
of the notice announcing the availability of the draft guidance.
Submit comments to Dockets Management Branch (HFA-305), Food and
Drug Administration, 5630 Fishers Lane, rm. 1061, Rockville, MD
20852. All comments should be identified with the docket number
listed in the notice of availability that publishes in the Federal
Register.
For questions regarding this draft document contact
Ms. B. Poindexter or J.G. Vostal, M.D., Ph.D., at 301-496-2577.
Additional copies of this draft guidance are available from:
Office of Communication, Training, and Manufacturers Assistance (HFM-40)
1401 Rockville Pike, Rockville, MD 20852-1448
(Phone: 1-800-835-4709 or 301-827-4573),
Internet at http://www.fda.gov/cber/guidelines.htm.
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Biologics Evaluation and Research (CBER)
May 1999
TABLE OF CONTENTS
- INTRODUCTION
- BACKGROUND
- SPECIFIC RECOMMENDATIONS
- In vitro Evaluation of Platelet Biochemistry and Function
- Platelet Survival in Circulation
- Clinical Hemostatic Efficacy
- Evaluation of Platelet Substitutes
- REFERENCES
Guidance for Industry
For Platelet Testing and Evaluation of Platelet Substitute Products
Draft - Not for Implementation
| This guidance document represents FDA's current thinking on
platelet testing and evaluation of platelet substitute products.
It does not create or confer any rights for or on any person and
does not operate to bind FDA or the public. An alternative
approach may be used if such approach satisfies the requirements
of the applicable statute, regulations, or both. |
- INTRODUCTION
The original platelet testing guidelines were issued by the Food
and Drug Administration (FDA) in July 1981 as announced in the
Federal Register of October 2, 1981 (46 FR 48768). Since then,
new instrumentation, such as flow cytometry, and new information
about platelet physiology and biochemistry have altered the way
that platelets can be evaluated. These advances have prompted the
release of updated guidance regarding platelet testing and
evaluation of platelet substitute products. This draft guidance,
when finalized, will update and replace the June 1981 guideline
entitled "Platelet Testing Guidelines" and delineate principles
of general applicability for evaluation of platelets collected
and processed by novel technologies.
Table of Contents
- BACKGROUND
Platelets participate in a number of reactions which contribute to
maintaining thrombotic hemostasis in circulation. When activated
by exposed subendothelium or by circulating agonists, platelets
form aggregates which are incorporated into a platelet plug that
prevents local hemorrhage. In addition, platelets also recruit
neutrophils and monocytes by exposing P-selectin on their surface,
contribute to signal transduction in neutrophils and endothelial
cells by trans-cellular metabolism of released lipid precursors,
serve as a site for activated clotting factor assembly and exert
a physical force to retract clots. Together these diverse aspects
of platelet physiology make up the clinical efficacy of platelets
(1, 2).
The current gold standard of clinical platelet efficacy evaluation
is in vivo survival of transfused radiolabelled platelets. It is
based on the assumption that viable circulating platelets can
participate in the responses which make up the clinical platelet
efficacy. Because in vivo testing of radiolabelled platelets is
associated with a small amount of risk, in vitro tests have been
utilized as a screening process to eliminate procedures which
clearly result in suboptimal platelet products (e.g., cold-stored
platelet concentrates) without requiring human trials. Over the
years no single in vitro test has stood out as a direct surrogate
for platelet efficacy. It has now been recognized that a battery
of tests, which examine different parts of platelet physiology,
can produce a reasonable estimate of platelet efficacy in vivo.
Many recent technological advances have been applied to the
production and storage of platelet concentrates. The effects of
these novel procedures on platelets should be evaluated against
accepted conventional methods used in blood banking practice. The
following tests are a guideline for evaluating platelets subjected
to novel methodologies. Although it may not be feasible or
appropriate to conduct all tests prior to a clinical evaluation
(i.e., prior to submission of an Investigational New Drug
Application (IND)[21 CFR 312]), a reasonable number of tests that
look at different aspects of platelet physiology is desirable.
The evaluation of platelets and platelet substitutes is grouped
into four categories:
- In vitro Evaluation of Platelet Biochemistry and Function
- Platelet Survival in Circulation
- Clinical Hemostatic Efficacy
- Evaluation of Platelet Substitutes
Table of Contents
- SPECIFIC RECOMMENDATIONS
- In vitro Evaluation of Platelet Biochemistry and Function
The objective is to demonstrate that platelets which have been
processed through a new collection procedure or subjected to new
storage conditions can respond to a variety of stimuli equally
well as platelets processed and stored by FDA approved blood
banking practices. Many of these tests are further discussed
and referenced in a review by the Biomedical Excellence for Safer
Transfusion (BEST) task force (3).
The state of platelets before and after procedure:
Morphology:
Platelet morphology should be visually inspected at different
levels of resolution, starting with a discs vs. spheres estimate
(4, 5). The presence of different morphological forms should
be quantitated (6), and finally the platelets should also be
examined by electron microscopy (7).
Biochemical status:
For stored platelets, in vitro tests of platelets have generally
not correlated well with platelet performance in vivo, however,
platelet cellular levels of ATP, glucose, and lactate should
offer some indication of platelet performance (3). A drop in
platelet count with an increase in the level of lactate
dehydrogenase in medium can be used as a measure of cellular
lysis. The pH of platelet suspension above 7.6 and below 6.2
at the end of the storage period has been shown to correlate
with decreased in vivo performance (3, 8).
Markers to determine % of platelets activated by procedure:
Activation of platelets is associated with surface expression
of the following surface antigens: GMP-140 (P-selectin, CD 62),
CD 63, and the active form (fibrinogen-binding) of GPIIb/IIIa
(detected by PAC-1) (9). -thromboglobulin and/or Platelet
Factor 4 released by activated platelets into the medium are
platelet-specific proteins and can be measured as indicators of
platelet activation (10). Platelet Factor 3 activity
(procoagulant surface for binding clotting proteins) also becomes
increased with platelet activation (11).
Physiologic responses:
The functional ability of a platelet can be estimated by their
response to osmotic stress and by the extent of agonist-induced
shape change (12). Aggregation to increasing concentrations of
physiologic agonists such as ADP, collagen, epinephrine, or to
dual agonist combinations of ADP/epinephrine and ADP/collagen
will give an idea of the responsiveness of the platelet (10).
The presence the platelet serotonin uptake and agonist-induced
serotonin secretion and agonist-induced expression of platelet
activation markers such as GMP-140, will also evaluate the
platelet physiologic responses (3).
Quantitation of microparticles:
The physiologic role of microparticles is not clear yet but they
may be involved in thrombosis and/or immunogenic sensitization
against platelets (13). Their quantitation will better
characterize the platelet product.
Comments:
These tests should be run as a paired comparison with platelets
stored in FDA approved storage containers (i.e., stored in gas
permeable plastic bags, containing equal volumes, equal numbers
of platelets and of white cells as the test platelets, on a
rotator at 20-24). If an alternative storage medium other than
plasma is used, the in vitro test conditions should mimic the
conditions encountered by the platelets infused in vivo (i.e.,
resuspension in normal plasma). If non-plasma stored platelets
are resuspended in plasma for in vitro testing and compared to
plasma-stored platelets, the resuspending plasma should be
equivalent to the plasma used for storage. That is, platelets
resuspended in fresh-frozen plasma should not be compared to
platelets resuspended in plasma stored at room temperature for
5 days.
- Platelet Survival in Circulation
Prolonged in vivo circulation survival of transfused platelets
has been taken as a sign of a functional, undamaged platelet.
Any new procedure for platelet collection and storage which does
not demonstrate significant changes in platelet responses on in
vitro tests should be further tested for its effects on platelet
in vivo survival. Recommendations on carrying out such tests
have been published (14). Design of such tests should include
normal volunteers receiving radiolabelled autologous platelets
subjected to the novel treatment. Recent advances in double
labeling of platelets with 1 1 1 Indium and 5 1 Chromium for
a simultaneous comparison in a single recipient of novel vs.
conventional methods of platelet treatment have provided
satisfactory results with less scatter in data points. The
extent of data scatter will determine the number of volunteers
that need to be tested to derive a clear conclusion about the
effects of the test treatment. Filing of an IND [21 CFR 312]
or an IDE [21 CFR 812] as appropriate is required before such
studies are conducted.
- Clinical Hemostatic Efficacy
Platelet efficacy in vivo has proven to be very difficult to
define. Currently there are no adequate clinical tests which
will demonstrate platelet efficacy. In the past, bleeding time
was thought of as the test of platelet efficacy, but numerous
published studies have demonstrated the lack of correlation time
between surgical bleeding and skin bleeding time and the
variability of the bleeding time even with a single patient (15).
Current surrogate end points for platelet efficacy have been a
sufficient number of platelets (often taken as >20,000/ul) that
have demonstrated a normal response to a battery of in vitro
tests and normal in vivo half life. The assumption is that a
sufficient number of circulating and intact platelets will offer
adequate protection against spurious intraorgan bleeding (16, 17).
Thus, the clinical performance (efficacy) of platelets obtained
with novel methodologies should be evaluated by inclusion of
these platelet products in clinical practice. Records of the
hemostatic effectiveness, clinically defined by changes in
epistaxis, hematuria, and/or petechia etc. should be included
as a part of the IND application. Bleeding times may be
submitted as additional data on efficacy of tested platelets.
- Evaluation of Platelet Substitutes
Manufactured platelet substitutes have recently been introduced
as an optional replacement for collected and stored human
platelets. These substitutes often emulate a single aspect of
hemostatic platelet response and thus have been even more
difficult to define in terms of platelet efficacy based on
in vitro tests and in vivo survival. For example, many platelet
substitutes have a very short circulating half-life in vivo which
would place them in the poor platelet efficacy category. Yet,
the platelet substitutes can decrease bleeding time in a
thrombocytopenic animal model (18). Thus, platelet substitutes
may be able to replace one part of the platelet response and be
used in specific clinical situations, such as acute trauma, as
opposed to long term prophylaxis. The aspect of platelet response
that the platelet substitute can replace should be defined and
the clinical benefit to the recipient tested accordingly. In
some instances, experienced investigators using a population of
clinically stable aplastic thrombocytopenic patients have been
able to correlate platelet count with skin bleeding time (19)
or with stool blood loss using radiolabelled red blood cells
(20). These studies suggest that platelet substitute efficacy
could be demonstrated in a similar patient population using such
methodology. Furthermore, an additional set of in vitro animal
tests may be necessary for definition of efficacy and safety of
platelet substitutes. These should include an evaluation of
the following aspects:
Evaluation of prothrombotic potential:
A thrombosis model(s) in normal animals (21, 22) and in animals
with disseminated intravascular coagulation (DIC) should be used
for testing, since these conditions will be encountered
clinically.
Evaluation of immunogenicity:
Infusion of only subcellular parts of the platelet may induce an
immunogenic response. It should be demonstrated in test animals
that the platelet substitute is no more immunogenic than intact
platelets.
Additional toxicity due to platelet additives:
Chemicals that are used to produce platelet substitutes and
remain in the final product should be evaluated for toxicity,
mutagenicity, and carcinogenicity at plasma concentrations
expected to be reached in a recipient of multiple platelet
substitute transfusions, as could occur with a platelet-
refractory patient. It should also be recognized that platelet
substitutes, like platelets, will be given to recipients in
their reproductive years and that reproductive toxicology and
teratology studies on the additives may also need to be done.
It should also be noted whether the additives interfere, such
as by adding color to plasma, with common clinical laboratory
test determinations.
Comments:
FDA has approved a number of current platelet products, collected
and stored by accepted blood banking practices, as safe and
effective therapies for thrombocytopenia. Platelet substitutes
which aim to be alternatives to the current platelet product should
demonstrate a clear benefit-to-risk ratio before they are
considered for clinical trials.
Table of Contents
- REFERENCES
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activation, Blood Rev. 9, 143-153.
- Wu, K.K. (1996) Platelet activation mechanisms and markers in
arterial thrombosis, J. Intern. Med. 239, 17-34.
- Murphy, S., Rebulla, P., Bertolini, F., Holme, S., Moroff, G.,
Snyder, E., and Stromberg, R. (1994) In vitro assessment of
the quality of stored platelet concentrates., Transf. Med.
Rev. 8, 29-36.
- Bertolini, F. and Murphy, S. (1994) A multicenter evaluation
of reproducibility of swirling in platelet concentrates.,
Transfusion 34, 796-801.
- Fratantoni, J.C., Poindexter, B.J., and Bonner, R.F. (1984)
Quantitative assessment of platelet morphology by light
scattering: a potential method for the evaluation of platelets
for transfusion., J. Lab. Clin. Med 103, 620-631.
- Kunicki, T.J., Tucelli, M., Becker, G.A., and Aster, R.H.
(1975) A study of variables affecting the quality of platelets
stored at room temperature., Transfusion 15, 414-421.
- White, J.G., Ultrastructural physiology and cytochemistry of
blood platelets, in The Platelet, K.M. Brinkhous, R.W. Shermer,
and F.K. Mostofi, Editors. 1971, Williams and Wilkins. p.83.
- Murphy, S, Kahn, RA., Holme, S., Phillips GL, Sherwood W,
Davisson W, Buchholz DH (1982) Improved storage of platelets
for transfusion in a new container. Blood 60, 194-200.
- Shattil, S.J., Cunnigham, M., and Hoxie, J.A. (1987) Detection
of activated platelets in whole blood using activation-dependent
monoclonal antibodies and flow cytometry, Blood 70, 307.
- Holmsen, H. (1994) Significance of testing platelet functions
in vitro. Eur J Clin Invest. 24, Suppl.1, 3-8.
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Freyssinet JM (1996) Physiopathological significance of
catalytic phospholipids in the generation of thrombin.
Semin Thromb Hemost 22, 157-164.
- Holme, S., Moroff, G. and Murphy, S. for the Biomedical
Excellence for Safer Transfusion Working Party of the
International Society of Blood Transfusion (1998) A multi-
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for extent of shape change and response to hypotonic shock.
Transfusion 38, 31-40.
- Owens, M.R. (1994) The role of platelet microparticles in
hemostasis, Transf Med Rev 8, 37-44.
- Snyder, E.L., Moroff, G., Simon, T., and Heaton, A.(1986)
Recommended methods for conducting radiolabeled platelet
survival studies, Transfusion 26, 37-42.
- Rodgers RP, Levin J (1990) Semin Thromb Hemost A critical
reappraisal of the bleeding time.16, 1-20.
- Aster, R.H. and members of the Consensus Development Panel
(1987) Platelet transfusion therapy-consensus conference,
JAMA 257, 1777-1780.
- McCullough, J., Steeper, T.A., Conelley, D.P., Jackson, B.,
Huntington, S., and Scott, E.P. (1988) Platelet utilization
in a university hospital, JAMA 259, 2414-2418.
- Rybak, M.E. and Renzulli, L.A. (1993) A liposome based platelet
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Biomater. Artif. Cells Immobilization Biotechnol. 21, 101-108.
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287, 155-159.
- Slichter, S.J. and Harker, L.A. (1978) Thrombocytopenia:
mechanisms and management of defects in platelet production.
Clin Haematol 7, 523-539.
- Kessler, C.M. and Hanson, S.R. (1994) Platelet thromboembolism,
Eur. J. Clin. Invest. 24, 38-41.
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Table of Contents
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