Meeting Minutes – Manufacturers' Forum
July 28, 2011

National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
National Kidney Disease Education Program (NKDEP) Manufacturers Forum
AACC Annual Meeting — Atlanta, GA
July 28, 2011
11:00 a.m. to 12:00 p.m.

The NKDEP Laboratory Working Group (LWG) met in conjunction with the International Federation of Clinical Chemistry and Laboratory Medicine's Working Groups for Glomerular Filtration Rate Assessment (WG-GFRA) and for Standardization of Albumin in Urine (WG-SAU), as the membership and goals overlap, and many of the scientific activities are conducted as joint efforts.

Participants: Greg Miller, Lori Bachmann, Diana Blanco, David Bruns, Glenn Carlisle, Joris Delanghe, Anne Dawnay, John Eckfeldt, Mary Lou Gantzer, Neil Greenberg, Yoshihisa Itoh, Hans-Joachim Kytzia, Jack Levine, John Lieske, Dave McDiarmic, Anjana Nair, Andrew Narva, Eileen Newman, Karen Phinney, Mary Robinson, Murray Rosenthal, Scott Isbell, Miriam Stella, Dave Torrens, Jack Zakowski

Meeting Minutes

1. Status of Creatinine Calibration Traceability to IDMS, Greg Miller, Virginia Commonwealth University; LWG and WG-SAU Chair
Based on responses from participants in the College of American Pathologists Comprehensive Chemistry Survey, 84% of laboratories report eGFR and, of those, 82% report eGFR with all ordered creatinines, while 11% report it only when it is requested. 39% use the original MDRD equation for use with non-standardized creatinine assays, even though all major manufacturers now provide assays with calibration traceable to IDMS. Manufacturers present agreed with the statement, "essentially all US labs are using methods traceable to IDMS." We need the help of the manufacturing community to assist in educating their customers to use an equation intended for creatinine methods with calibration traceable to IDMS. Only 4% used the CKD-EPI equation although 30% were reporting numeric eGFR values above 60 mL/min/1,73 m² suggesting that many are extending the MDRD equation beyond its appropriate limits. Thus, more education is also needed regarding the limitations of different equations. The remaining 70% of the respondents reported ">60 mL/min/1,73 m²" when the actual numerical value was above 60 mL/min/1,73 m².

2. CKD-EPI and Other Newer Equations, Andrew Narva, NKDEP Director
Creatinine is now standardized and clinicians are receiving estimates of GFR. Another equation, CKD-EPI, was discussed briefly last year. As the field matures, there will be other equations and some that use markers other than creatinine. In the future, NKDEP will not endorse a specific equation, but rather suggest equations that meet certain criteria. At this point, both MDRD and CKD-EPI give acceptable eGFR results and education is needed so that it is understood that if you want to report numeric values above 60, CKD-EPI may be the better equation to use. Another educational issue that will be addressed is assuring that clinicians understand the limitations of creatinine measurement as well as the limitations of GFR estimating equations.

3. Creatinine Specificity Project Report and Recommendations, Neil Greenberg, Chair, IFCC WG-GFRA
Now that creatinine assays have been standardized, we want to investigate the specificity characteristics of the enzymatic and Jaffe methods. We learned several years ago in our work with calibration that bias in creatinine can alter the eGFR and misclassify a patient. Since interfering substances contribute to biases, we wanted to investigate the frequency and magnitude of non-specificity biases in widely used commercial methods. We evaluated 365 individual patient samples representing 19 disease categories and a healthy control group, as well as sera at two creatinine concentrations supplemented with acetoacetate, acetone, ascorbate and pyruvate. Creatinine was measured by four collaborating manufacturers -- Ortho Clinical Diagnostics, Roche, Siemens, and Beckman Coulter -- to evaluate four enzymatic and three Jaffe procedures, as well as an LC-IDMS procedure for reference. A manuscript is in review by Clinical Chemistry and we hope to see it published before the end of the year. Some of the key findings: 1) There were differences in both magnitude and direction of bias among measurement procedures, whether enzymatic or Jaffe; 2) The influence of interfering substances was less frequent with the enzymatic procedures, but no procedure was unaffected; and 3) The details of the specific implementation of a method principle (Jaffe and/or enzymatic) influenced its susceptibility to interfering substances.

4. Urine Albumin Method Harmonization Study Preliminary Report, Lori Bachmann, Virginia Commonwealth University
The objectives of this project are to: 1) Evaluate harmonization among methods for urine albumin using native human urine samples; 2) Assess agreement of routine methods with the IDMS cRMP; 3) Evaluate commutability characteristics of JSCC and diluted ERM-DA470k/IFCC reference materials for urine albumin; 4) Evaluate basic performance characteristics of the urine albumin methods; and 5) Assess harmonization of urine total protein and creatinine methods. Seventeen methods (16 quantitative) from five manufacturers were evaluated. Fresh, non-frozen human urine samples (343) that had been submitted for routine urine albumin measurement were used, and non-frozen aliquots were measured by 17 routine methods. Frozen aliquots of each sample were measured by IDMS at the Mayo Clinic and 41 fresh and frozen paired samples were also assayed to assess freeze-thaw effects. JSCC cRM and diluted ERM DA470k/IFCC RM were prepared by each manufacturer and included in each analytical run along with patient samples. Centrally prepared IRMM reference materials and QC materials were also included. To evaluate the effect of different matrices on the methods, urine samples were spiked with potential interfering substances or adjustments were made to the pH and ionic strength. A urinalysis and urine electrolytes measurements were performed to characterize the matrix of each sample.

Preliminary Data and Observations:
The median for the reference LCMS method and the average median of all methods were very close in the patient sample concentration intervals of 12-20, 20-300 and above 300 mg/L. The range of differences of medians for all of the methods was approximately 40% in each of the concentration intervals. Regression of each method versus the LCMS method showed that some methods had no outliers and others had aberrant results for some individual urine samples. Diluted ERM-DA470K RM appeared commutable with patient samples for most methods. Results for centrally prepared diluted ERM-DA470k/IFCC materials tracked closely with patient samples for 11 of 16 commercial methods.

5. Serum Albumin Standardization Issues, David Bruns, University of Virginia
National guidelines (KDOQI) call for monitoring of serum albumin as a quality indicator for dialysis centers. Reimbursements are affected by how many patients meet/do not meet the target values (BCG assay): 3.5 g/dL and (better) 4.0 g/dL. Unfortunately, BCG assays are not all equal. A change in assay procedure in the method used at University of Virginia (UVA) in 2005 resulted in many more patients meeting the target levels. Prior to the method change, 22% met the goal of 4.0 g/dL and after the change, 74% met that goal. Another method change in 2009 caused a change from 60% of patients meeting the 4.0 target to 17%. This effect was not seen in only dialysis patients because all patient weekly means dropped after the change in 2009. The results of a sample exchange comparing results with three other assays, showed a difference of about 0.4 between the other assays and the UVA assay, suggesting that there is a problem with BCG assays. A survey compiled by B. Doumas of reference values for serum/plasma albumin by method shows that the ranges are extremely varied. David Bruns asked for suggestions from manufacturers about how to resolve this problem.

Jack Levine suggested that D. Bruns talk with Neil Greenberg to create a round robin exchange of samples among manufacturers. This data is an eye opener and most manufacturers will correct a problem when shown that there is clinical significance.

6. Urine Albumin Reference Measurement Procedures, Karen Phinney, National Institute of Standards and Technology and John Lieske, Mayo Clinic

NIST Update:
NIST has prepared SRM 3667 (Creatinine in Human Urine), which is a single level of pooled urine, collected from healthy males/females (minimum of 10 donors). There are 1,000 bottles with 10 mL urine in each. They will perform isotope dilution LC-MS reference measurement procedure to quantify the creatinine. Certification measurements are expected to be finished by fall 2011. In addition, SRM 2925 (Human Serum Albumin Solution), which is a primary certified reference material for use with reference measurement procedures for albumin in urine, has been prepared. It is 1 g/L aqueous solution of recombinant human serum albumin with 1 mL per vial. Quantification by amino acid analysis using isotope dilution LC-MS/MS should be completed by fall 2011. The NIST developed LC-MS/MS reference method has measured eight tryptic peptides of human serum albumin with ~1% CV for replicates. They will validate this procedure by comparison with Mayo's IDMS method.

Mayo Update:
The method uses five tryptic peptides that are consistently seen by LCMS and averages the results for a final value. Over a range of albumin concentrations, the inter-assay CV is about 1% and the intra-assay CV at low, medium, and high concentrations of control material is about 1-2%. There is a bias between the different fragments used suggesting that the N- or C-terminus is cleaved in a small percentage of urines.

Lori Bachmann asked if they are concerned about the fact that the NIST albumin reference material is a recombinant material that would not have post-translational modifications found in clinical samples. Karen Phinney replied that in order to standardize any method, a reasonably stable material is needed, but it is a good idea to look for variation in results for clinical samples.

7. Urine Albumin Adsorption Project: Mary Robinson, Centers for Disease Control and Prevention
G. Miller commented that this project was presented last year and should be published soon. Mary Robinson summarized the container adsorption project by stating that polystyrene is not a good option for urine albumin and that polypropylene is better; the best material is a hydrophilic coated material but it is more expensive. The overall influence of adsorption at clinically important concentrations of urine albumin is being addressed and will be included in the final manuscript.

8. Urine Albumin Reference Materials, Yoshi Itoh, Asahikawa Medical University
The candidate urine albumin reference material is a buffer based purified monomeric human serum albumin in lyophilized form with an inter-vial difference of 1.31%. It is stable at 4-10 ºC for up to 10 hours and -80 ºC for 40 months. The assigned value (mean ± U) by value transfer from ERM-DA470 is 225.1± 9.11 mg/L when reconstituted in 3 mL of purified water. This material is defined as a "working reference material" in the traceability chain for urine albumin. A commutability study using 129 urine samples was performed with seven different methods. Using the manufacturers' calibrators, the between-methods CV was 8.8%; using the new reference material to calibrate the assays, the between-methods CV was 6.6%. NEDO (New Energy and Industrial Technology Development Organization) in collaboration with RECS (Reference Material Institute for Clinical Laboratory Standards) has recently assigned the value for albumin in ERM-DA470 by ID/MS. It has proven to be very close to the existing assigned value.

9. Concluding Statements, Greg Miller
We feel that we have made substantial progress since last year. We have a lot of information that will help to develop educational materials and recommendations for measurement procedure performance. We expect to have new educational material for using eGFR estimating equations available on the web site later this year, and will prepare recommendations for serum creatinine specificity for final review at our meeting in July 2012. Regarding urine albumin, we should have JCTLM listed reference materials and reference measurement procedures for urinary albumin in the next few years.