AACC Annual Meeting – Los Angeles, CA – July 19, 2012

National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)

National Kidney Disease Education Program (NKDEP)

Laboratory Working Group (LWG) Meeting

AACC Annual Meeting – Los Angeles, CA

A joint meeting with the International Federation of Clinical Chemistry (IFCC) Working Group for Standardization of Albumin in Urine (WG-SAU)

July 19, 2012
8:00 a.m. to 11:00 a.m.

Participants: Greg Miller, Flavio Alcantera, Harald Althaus, Dave Armbruster, Lori Bachmann, David Bunk, Christa Cobbaert, Joris Delanghe, John Eckfeldt, James Fleming, Neil Greenberg, Lars-Olaf Hansson, Glen Hortin, Yoshihisa Itoh, Graham Jones, Dave Koch, David Lacher, John Lieske, Gary Myers, Andrew Narva, Mauro Panteghini, Karen Phinney, James Ritchie, Heinz Schimmel, David Seccombe, Jack Zakowski

Meeting Minutes

Summary of Action Items:

• G. Miller to initiate conversations with the Food and Drug Administration (FDA) regarding guidance for recalibrating cystatin C assays to be traceable to the new international reference material.
• LWG to begin discussing educational needs for use of eGFR equations and cystatin C recalibration, which may include creating a list of assays that are calibrated to ERM standard and equations that are based on standardized assays.
• G. Miller to facilitate discussions between IFCC (L. Hansson) and CKD-EPI (J. Eckfeldt) working groups so they can investigate a single cystatin C equation that can be used worldwide.
• N. Greenberg to add variable levels of protein to the list of substances that manufacturers need to test for creatinine specificity to the draft guidelines.
• LWG to assist the National Institute of Standards and Technology (NIST) with specifications for the development of urine creatinine and albumin reference material.

Educating Clinicians About Uncertainty in Laboratory Assessment of Kidney Disease

Andrew Narva, NKDEP Director

• NKDEP’s goal is to reduce the burden of kidney disease; the goal of the working group is to translate its work so that clinicians on the front line can understand the issues.
• It is important to understand the P30 as it applies to eGFR equations; P30 is the percent chance that the eGFR is within 30% of the measured GFR. For example, the P30 for the MDRD equation is 80%, which means that there is an 80% chance that the eGFR is within 30% of the measured GFR, i.e., a patient with eGFR of 59 has an 80% chance that the actual GFR is in the range of 42–77.
• eGFR could be understood as an estimate of risk of having decreased kidney function and is not the actual GFR.
• The word “disease” should be used with caution, especially with the elderly with mild decrease in kidney function without other evidence of kidney disease. Kidney function decreases with age.
• NKDEP promotes eGFR as the best tool for evaluating kidney function in the outpatient setting, but notes that it is only one variable that should be considered when assessing the status of an individual patient.
• Urine albumin is the second variable that should be evaluated when diagnosing kidney disease. A recent NHANES study showed that a first void sample confirmed abnormal albuminuria on a random specimen in less than half of individuals. The first void urine is thought to provide more reliable results than a random collection for estimating kidney damage when using urine albumin-to-creatinine ratio (UACR).
• The US Preventative Services Task Force performed an evidence review about the benefits and harms of screening for, monitoring, and treating CKD. It found no evidence of benefit of routine screening of asymptomatic individuals. However, lack of evidence does not preclude a benefit. This study suggests the need for better science to support potential population-based interventions to reduce the burden of CKD.
• NKDEP uses a conservative approach to kidney disease education. All information and tools on its web site are evidence-based and do not promote outside guidelines, do not promote staging based on eGFR, and do not focus on referrals based on eGFR alone. NKDEP promotes awareness and collaborates with public health organizations such as the Centers for Disease Control and Prevention (CDC).

Cystatin C-based eGFR Equations

IFCC Working Group for Standardization of Cystatin C Update
(Lars-Olof Hansson, MD, PhD, Karolinska Hospital, Sweden and Harald Althaus, PhD, Siemens Healthcare Diagnostics Products GmbH)

• Goals of the working group are to produce an international cystatin C calibrator, which was released in 2010, and produce an assay-independent, cystatin C-based, GFR-prediction equation.
• ERM-DA 471/IFCC calibrator is commutable for three assays: Siemens, Gentian, and Sentinel. Roche’s assay became commutable with changes made by the manufacturer.
• Suggestions for ways to achieve minimal batch-to-batch variation and stable eGFR results include:
  • Use 10 pooled samples stored at -70 for lot-to-lot comparisons.
  • Focus on optimal assay performance at lower cystatin C levels, e.g. 0.8 – 2.6 mg/L, to minimize eGFR error using cystatin C equation.
• Three different models for eGFR equations using cystatin C are being evaluated: CYS simple, CYS 1, and CYS knot at 1.6 mg/L; P30 for each equation is 80, 83, and 85%, respectively. More children are needed in the study to determine if a single equation for all ages can be developed. The population being used for development and validation is predominantly Swedish.
• Next Steps:
  • Finalize development of a common equation.
  • Validate an assay-independent equation.
  • Characterize if and by how much P30 can be improved by adding other variables.
  • Determine how much eGFR will be improved by combining and comparing optimal cystatin C and creatinine-based equations.
• Comments:
  • G. Miller stated that it will be highly desirable to develop a single cystatin C equation that can be promoted worldwide; J. Eckfeldt works with the CKD-EPI Working Group and thinks they would be interested in collaborating to develop a common equation.
  • J. Eckfeldt cautions that the iohexol “gold” standard may not give the same result in all laboratories, which should be evaluated as the eGFR equation is developed.
  • Siemens has a problem in recalibrating their assay to the international standard because it requires a full 510K application with the FDA, which is costly. G. Miller and A. Narva suggested that NKDEP engage FDA in discussions about this issue.

CKD-EPI Cystatin C Equation

(Andrew Narva on behalf of Lesley Inker, Tufts Medical Center)

• CKD-EPI Cystatin C Equation group published their work in July 2012 in the New England Journal of Medicine. Its goals were to report a new equation using standardized cystatin C and report a second equation using both cystatin C and creatinine. Variables for the cystatin C equation included age, gender, and cystatin C.
• Results included:
  • Improved performance for the combined equation.
  • P30 was most effective using the combined equation and no real improvement vs. the creatinine equation was seen using the cystatin C equation alone.
  • Cystatin C equation did not perform well at either very high or low eGFRs.
  • Some patients in the subgroup aged 45–75 were reclassified to a more correct GFR classification.
• The cystatin C only equation was not more accurate than the combined equation, but may be useful when race cannot be specified. Also, it may more accurately predict the risk of kidney damage.
• The combined equation was more accurate for eGFR across the range and may be useful for better classifying patients with eGFRs around 60.
• Comments:
  • G. Miller suggested that the LWG develop a task force to work on educational materials about best practices for using the eGFR equations.
  • G. Jones suggested creating a list of assays that are calibrated to ERM standard and equations that are based on standardized assays.
  • It was suggested that, in practice, using the creatinine-only and cystatin C-only equations would be better than using the combined equation.
  • A. Narva commented that the underlying message is the need to look at more than eGFR, which is difficult to convey to clinicians.

Creatinine Specificity Study Education Recommendations

(Neil Greenberg, PhD, Neil Greenberg Consulting and Chair, former IFCC WG-GFRA)

• Conclusions about the creatinine method specificity study included: 1.) There are differences in magnitude and direction of bias for both enzymatic and Jaffe methods when various interfering conditions were present; 2.) Influence of interfering substances was less frequent with enzymatic assays; and 3.) Details of implementation of a method principle influenced its susceptibility to interfering substances.
• Recommendations for Clinical Labs:
  • Each lab should review and understand interference claims published in product labeling.
  • Users should review other literature explaining strengths and limitation of their creatinine assay (A list of selected references will be included in recommendations).
  • Each lab should take into account characteristics of the population it serves and understand potential for interference and non-specificity to affect the creatinine results (Examples will be provided in recommendations).
  • When creatinine results are not consistent with the clinical situation, labs should consider using a different procedure or use another biomarker for kidney function, e.g. cystatin C or iothalamate clearance.
• Recommendations for Manufacturers:
  • Add an explanatory statement to product instructions for use (IFU) clarifying that non-specificity with an individual patient’s sample may still be an important limitation.
  • Acknowledge that calibration traceability to higher order reference materials and reference measurement procedures does not alter the influence of potential interfering substances on a result for an individual patient sample.
  • In the IFU, state the criteria used to determine “bias” or “no bias” from potentially interfering substances.
  • Include the concentrations of interfering substances and creatinine levels that were tested.
  • Concentrations of interfering substances tested should be at least what is typically encountered in diseased patient populations (a minimum list of substances that should be evaluated will be provided in the recommendations).
• The recommendations will be published on the NKDEP web site.
• Comments:
  • G. Hortin suggested that variable protein concentrations be included and N. Greenberg agreed to add it to the list.

IFCC/WASPaLM Task Force Update

(Graham Jones, MBBS, BSc, DPhil, FRCPA, FAACB, St. Vincent’s Hospital)

• KDOQI 2002 guidelines changed renal medicine in Australia and perhaps the world. eGFR has been incorporated into routine practice and has led to collaborative work between laboratory medicine and clinical groups. The third edition of Australian eGFR guidelines and the first one on albumin and proteinuria will be released in August 2012.
• Outcomes of the task force include creatinine assays, units, eGFR reporting, equations, and standard clinical response.
• National CKD programs exist throughout the world. In Mexico, a new organization has been formed that includes lab medicine and clinical organizations.
• In Guyana, there has been work on creatinine standardization and eGFR reporting. A two minute You Tube video explaining GFR, eGFR, and creatinine standardization was created to help educate healthcare professionals.
• The task force is also working on issues of proficiency testing (PT) and creatinine assay standardization.
• Brazil is working towards a national program on eGFR reporting.
• Suggested Next Steps:
  • Survey of activities on national CKD testing programs around the world; LWG can help identify people in different countries to contact.
  • Conduct presentations on GFR and eGFR at international and national meetings.
  • Publish work to provide guidance on procedures for implementing a CKD testing program.
  • Work with manufacturers to determine where they are selling to identify locations that may be using non-IDMS traceable assays (some countries are using assays that are not IDMS-traceable).
  • Solicit communication and support for CKD program organizations.
  • KDIGO guidelines have been very powerful; new guidelines are to be released soon from KDIGO and can be used as a stimulus for national activities.
• Comments:
  • A. Narva commented that NKDEP has initiated two new working groups -- a pharmacy working group to educate pharmacists about CKD and a health information technology working group to improve electronic data related to kidney health. The groups will collaborate to recommend operational and management changes, such as routine reporting of eGFR with e-prescriptions.
  • D. Seccombe stated that nations often lack the infrastructure needed to implement programs to look at chronic disease. He recommended that the task force’s scope be expanded to look at more chronic diseases in these countries with limited resources.

Update on Other Urine Albumin Standardization Activities

Urine Albumin and Creatinine Reference Materials

(Karen Phinney, PhD, National Institute of Standards and Technology)

• SRM 2925 Human Serum Albumin Solution is a primary certified reference material for use with higher order reference measurement procedures for albumin. It is an aqueous solution (1 g/L) that will be characterized over the next several months and will be available in early 2013.
• SRM 3667 Creatinine in Frozen Human Urine is a pool from a minimum of 10 male and female donors. The concentration is ~61 mg/dL and it will be released at the end of 2012.
• LWG guidance was requested to develop specifications for albumin and creatinine reference material development.
• Comments:
  • N. Greenberg asked about the albumin concentration of SRM 3667. K. Phinney commented that there probably is albumin in the material but the concentration has not yet been determined.
  • J. Eckfeldt recommended deleting “serum” from the title of the SRM 2925 material so that it will read “Human Albumin Solution”.
  • G. Miller suggested a three concentration set be developed in order to reduce errors introduced by diluting.

Urine Albumin Reference Materials

(Y. Itoh, MD, DrSci, Asahikawa Medical College)

• The JSCC has developed a secondary reference material for urine albumin. The assigned value is 225.1+/- 9.11 mg/L based on value transfer from ERM-DA470. This is a monomeric human serum albumin that can be used for standardization of urinary albumin and total protein assays, and protein in dipstick tests. It will be submitted to the Joint Committee for Traceability in Laboratory Medicine (JCTLM) within one year (Q3 2013).
• Comment:
  • N. Greenberg commented that ISO 15194 is the standard that supports reference materials within the JCTLM process and that this standard was revised in 2009. Many existing reference materials listed with JCTLM were documented against requirements of ISO 15194-2003.

Urine Albumin Reference Measurement Procedures

(John Leiske, MD, Mayo Clinic and David Bunk, PhD, National Institute of Standards and Technology)

• NIST is working on development of 15N-labeled HSA CRM material, which is designed for use as an internal standard for reference measurement procedures for albumin in urine.
• The urine albumin reference measurement procedure under development at Mayo is a multiplexed LC-MS/MS assay that quantitatively and qualitatively measures 8 peptides. The inter-assay CV is 3-4% and the intra-assay CV is 2-4%. Adding peptides and/or transitions increases precision. Two peptides and two transitions seem to be optimal.
• Next Steps:
  • Method manuscript is under review by Clinical Chemistry and Laboratory Medicine (CCLM).
  • Perform a comparison study with NIST and Mayo methods.
  • Obtain JCTLM certification.
• Comments:
  • The NIST method has similar performance to Mayo’s and differs in that it uses more peptides/transitions. Because it uses more peptides, the NIST method may be useful in evaluating the albumin in samples that disagreed in the harmonization study.
  • H. Schimmel commented that Institute for Reference Materials and Measurements (IRMM) observed a difference when comparing patient samples between routine protein methods and LC-MS/MS methods. Caution should be taken when using a LC-MS/MS method in the standardization of protein assays. J. Leiske responded that the Mayo assay has good correlation with immunoassays for patient samples.

Adsorption to Containers

(Greg Miller)

• Work is completed and the manuscript is almost ready to submit.
• There is less than 1% influence of absorption of albumin to containers.

Urine Albumin Harmonization Status Investigation

(Lori Bachmann, Virginia Commonwealth University Health System)

• The project objectives were to: 1.) Assess current status of harmonization of routine measurement procedures versus ID-LCMS candidate reference measurement procedure using native patient samples; 2.) Evaluate analytical performance characteristics of urine albumin measurement procedures; 3.) Evaluate commutability characteristics of the Japan Society for Clinical Chemistry (JSCC) and diluted IRMM ERM-DA470k/IFCC reference materials; and 4.) Assess utility of candidate reference materials for use in standardization of routine measurement procedures.
• Difference range in agreement among medians was ~40%.
• Bias of routine methods vs. candidate reference measurement procedure expressed as slope in a regression analysis was 0.82 – 1.34.
• Dilution introduced bias in some routine measurement procedures and many manufacturers do not suggest a dilution protocol or dilution solution in their instructions.
• Freeze/thaw effect was small.
• Imprecision study: 9 assays had CV less than 5%, 2 assays plus LCMS had CV between 6-10%, and 5 assays had CV greater than 10%.
• Sample-specific effects were found to be 4-8% of the total imprecision for the different measurement procedures.
• Next Steps:
  • Harmonization manuscript will be submitted in the fall of 2012.
  • Assess commutability characteristics of IRMM and JSCC candidate reference materials.
  • Evaluate the ability of mathematical recalibration (based on reference materials) to achieve method standardization.
• Comments:
  • N. Greenberg asked if manufacturers of assays with performance issues have been notified. L. Bachmann confirmed that notification of manufacturers is planned prior to data publication.
  • H. Schimmel and D. Bunk commented that it would be helpful to correlate the routine measurement procedures to each other.
  • G. Jones suggested looking at the total protein concentration as an explanation for samples that do not correlate.

Physiologic Variability Investigation

(Jim Ritchie, PhD, Emory University Hospital)

• Project is still in the planning stage. The patients will come from Emory University Hospital.

Other items, next steps (Greg Miller)

• G. Miller indicated that the development of general performance recommendations for measuring low concentration creatinine in pediatric samples is a short-term goal of the LWG. Reference materials available to manufacturers for calibration traceability do not have low enough concentrations.
• M. Panteghini suggested contacting people using non-IDMS standardized creatinine results for calculating eGFR.
• G. Miller said that he will be convening several task forces to work on developing additional draft proposals; those interested in contributing should contact him.