Virtual Annual Meeting - January 20, 2023

Welcome and Introductions
Greg Miller, Virginia Commonwealth University (VCU), Jesse Seegmiller, University of Minnesota (UMN)

Dr. Greg Miller, Chair of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Laboratory Working Group (LWG), welcomed the participants to the meeting. Dr. Jesse Seegmiller is Chair of the International Federation of Clinical Chemistry (IFCC) Working Group for Standardization of Albumin in Urine (WG-SAU). The LWG and IFCC-WG-SAU are collaborating to standardize urine albumin (UA) measurements. A reference measurement system is being developed to standardize results, and updates were provided at this meeting. The participants were invited to introduce themselves; a list of participants is provided as Attachment A.

On this 20th anniversary of the launch of the LWG, Dr. Miller took the opportunity to provide some background and history. In 2002, the National Kidney Foundation (NKF), through the Kidney Disease: Improving Global Outcomes organization, recommended reporting estimated glomerular filtration rate (eGFR) along with serum creatinine using the recently developed Modification of Diet in Renal Disease (MDRD) Study equation. The newly formed NIDDK National Kidney Disease Education Program (NKDEP) met with representatives from the Clinical Chemistry Committee of the College of American Pathologists (CAP) to request assistance addressing ways for clinical laboratories to report eGFR. The Clinical Chemistry Committee’s first recommendation was to standardize creatinine measurement across clinical laboratories to enable consistent results for eGFR reporting across clinical laboratories.

In 2003, the NKDEP LWG was established with two major goals: promote reporting eGFR and standardize creatinine results. Accomplishing these goals would reduce the variability in creatinine and eGFR results and improve the quality of the eGFR reporting. NKDEP LWG recommendations for improving serum creatinine measurements were published in the January 2006 issue of Clinical Chemistry (Myers et al., 2006). A 2013 report in the Archives of Pathology & Laboratory Medicine (Killen et al., 2013) reviewed creatinine measurements using commercially available in vitro diagnostic (IVD) assays from 2003 to 2011. The data demonstrated that creatinine calibration was standardized for measurement procedures from large global IVD manufacturers, demonstrating the success of the LWG’s creatinine standardization project.

Following the creatinine standardization, the LWG shifted its focus to developing reference measurement procedures (RMPs) and reference materials for UA measurements and combined efforts with the IFCC-WG-SAU. A review published in the Archives of Pathology & Laboratory Medicine (Miller et al., 2009) highlighted the issues related to measurement and reporting of UA excretion. In 2021, the NKDEP LWG was renamed the NIDDK LWG.

Overview of the UA Standardization Program
Greg Miller, VCU

Dr. Miller provided an update on the UA end-user measurement procedures, beginning with an assessment of analytical precision. A seminal study reported in the March 2014 issue of Clinical Chemistry (Bachmann et al., 2014) documented the state-of-the art for measurement of UA in routine procedures and was the starting point of the LWG UA standardization project. The study evaluated 16 commercially available UA measurement procedures from major IVD manufacturers. The results showed that the coefficient of variation (CV) ranged from 1.5 to 5.8 percent for 14 of 16 procedures examined. The CV contribution from specimen-specific influences (e.g., urine matrix or albumin molecular forms) was minimal, ranging from 3.1 to 6.6 percent for 12 of the 16 UA procedures. These findings thus provided new insight into a long-time controversy related to this type of interference with clinical laboratory measurement procedures. Of the 16 commercial procedures evaluated, 4 had experimental design challenges and data on those procedures could not be interpreted appropriately.

In this same study, UA measurements were made on 332 unfrozen patient samples (i.e., samples stored at 2–8 °C) and a candidate reference procedure developed by the Mayo Clinic Renal Testing Laboratory. The data showed that at low, medium, and high UA concentrations, the median values among the end-user clinical laboratory procedures differed by approximately 40 percent. This difference meant that interpretation of results was compromised when using fixed decision values, which has been a common practice for interpreting UA. Recent data from the CAP Accuracy-Based Survey ABU-B-2022 examining similar UA concentrations assessed with procedures commonly used in the North American market showed similar differences in median values, suggesting a continuing need for this standardization program. Collectively, these findings indicate bias as the dominant source of disagreement or variability between the manufacturers.

In 2017, the LWG reported clinical performance goals for UA measurement using a biological variability model derived from reasonable estimate of within individual CVs of 20 to 25. The results suggest that the total allowable error for an acceptable clinical laboratory performance CVs between day repeatability and specimen-specific effects are well under the goal of 6 percent, with bias goals of 7 to 13 percent as goals for the standardization program. In 2015, Braga et al proposed that the fraction of uncertainty for the various traceability steps in a calibration hierarchy could be partitioned; this model has been widely accepted in the metrological community. This model takes into account that the total uncertainty or total allowable error at the patient result level represents 100 percent of the allowable uncertainty; 50 percent is attributable to system calibration, and 30 percent could be due to uncertainty of the reference material. The LWG is developing performance goals for the RMPs leveraging the Braga partitioning model of uncertainty and presented preliminary (unpublished) calculations. Using this model, an RMP with a CV of 4 percent is fit for purpose.

Additionally, in 2017, the LWG published a report (Seegmiller et al., 2017) describing the metrological traceability for calibration of UA measurement procedures that was based on the ISO 17511standard for IVD medical devices. The National Institute of Standards and Technology (NIST) produced standard reference material (SRM) 2925, which is now commercially available, and can be used to prepare a calibrator for the liquid chromatography–tandem mass spectrometry (LC-MS/MS) RMPs. A Reference Laboratory Network, consisting of four groups—Health Sciences Authority (HSA), Singapore; Mayo Clinic; NIST; and University of Minnesota is in development. All four laboratories have developed RMPs and provided updates later in the meeting. One strategy is to collect a panel of patient samples that is value assigned by a RMP and can be used by the manufacturers to calibrate their internal procedures used by clinical laboratories. A second strategy is to develop another SRM of albumin in frozen human serum, which is in process at NIST and has been labeled as SRM 3666. When available, manufacturers will be able to use SRM 3666 in their calibration hierarchies. Dr. Miller commented that either process should work successfully, and both processes likely could run in parallel.

Dr. Miller summarized that, in terms of developing a reference system for UA, the IVD measurement procedures have acceptable precision (i.e., repeatability) performance in the clinical laboratory and are adequately selective for albumin. The remaining challenge is bias, which this reference system will address to achieving standardized results. The main goals are to reduce misclassifications near the clinical decision values and enable lower gender-stratified decision values. He encouraged manufacturers to maintain their current calibration hierarchies and plan to adopt the NIDDK higher-order reference system when available. The COVID-19 pandemic significantly slowed the work of the LWG, but efforts have since resumed across the participating reference laboratories. An extent of equivalence (or Round Robin) among the four candidate RMPs will launch soon and will support Joint Committee for Traceability in Laboratory Medicine (JCTLM) submissions to credential the RMPs. Dr. Miller touched on next steps, including the timeline of activities, which he revisited at the end of the meeting.

NIST SRMs for UA
Ashley Beasley-Green, NIST, Karen Phinney, NIST

Dr. Ashley Beasley-Green updated the LWG on the NIST SRMs and acknowledged other members of the team: Drs. Johanna Camara and Karen Phinney.

SRM 2925—Recombinant Human Serum Albumin Solution (Primary Reference Calibrator for Urine Albumin)

The NIST SRM 2925 is a highly purified recombinant human serum albumin protein stored in a buffer solution. Its intended use is as an unlabeled calibrator for the NIST LC-MS/MS measurement procedure for albumin in human urine and for value assignment for other calibrators. The certified value assignment for SRM 2925 was performed using amino acid analysis, and the standard has molar and mass concentration values of albumin. In addition to the certificate of analysis, NIST Special Publication 260 199 provides extensive analyses of this material. These include quantitative analyses of the amount of albumin present and qualitative assessments, such as the disulfide profile and peptide sequencing to verify that albumin is the analyte. Released in February 2020, SRM 2925 is listed in the JCTLM database and is available through NIST.

SRM 3666—Albumin in Frozen Human Urine

The SRM 3666 is albumin and creatinine in frozen human urine. Its intended use is to support the calibration of clinical laboratory measurement procedures for albumin and creatinine in human urine. SRM 3666 is a four-level material composed of endogenous albumin acquired from pooled patient samples. The SRM 3666 will contain certified values for albumin, creatinine, and the albumin-to-creatinine ratio.

Dr. Beasley-Green reminded participants that NIST distributed aliquots of SRM 3666 material to VCU Clinical Chemistry Laboratory (Drs. Bachmann and Miller) for preliminary assessment of albumin and creatinine using routine clinical assays, including the Abbott Architect assay. The four concentration ranges are: Level 1, 5–10 mg/L; Level 2, 20–50 mg/L; Level 3, 60–180 mg/L; and Level 4, 200–600 mg/L. The results showed that the values for albumin were within the expected target ranges and that the creatinine levels were within the normal ranges. In addition, preliminary assessment at NIST and among clinical laboratories using the NIST isotope dilution (ID) LC-MS/MS method showed similar results to the VCU assay. NIST next moved to certification of SRM 3666, which Dr. Beasley-Green described, explaining that the final certification values are still under review. With the exception of the Level 1 concentration of approximately 8 mg/L, the uncertainty was at or less than the required limit of 4.1 percent. The certified values for creatinine using a modified NIST ID-LC-MS/MS method for creatinine in urine were consistent with the VCU results.

Dr. Beasley-Green announced that NIST is planning to initiate an interlaboratory study to evaluate the computability of SRM 3666 for use as a calibrator and as a trueness control for standardizing clinical measurement procedures. The identification platforms will include IVD measurement devices for UA and creatinine that represent the major global market share. Study participants must agree to use of their data, and the measuring system will be identified in published reports and manuscripts. Samples will include the SRM 3666 four-level material and 44 single-donor urine samples (11 in each concentration level) that bracket SRM 3666 albumin concentrations, with creatinine levels within the normal range. The reference procedure for albumin will be the NIST ID-LC-MS/MS and NIST ID-LC-MS/MS RMP for creatinine in serum adapted for human urine.

Reference Measurement Procedures for UA

NIST
Ashley Beasley-Green, NIST

Dr. Beasley-Green provided a brief update of the NIST RMP for UA, which was published in 2014 in the Journal of Proteome Research (Beasley-Green et al., 2014). The ID-LC-MS/MS is a multiplexed method that supports quantitative and qualitative assessment of UA. The assay was developed using 11 peptides that span the human serum albumin amino acid sequence, with 2 transitions per peptide. Five transitions are being used for quantitative assessments of specific peptides and span the three domains of albumin, and 18 transitions are being used for qualitative assessment.

The ID-LC-MS/MS method uses intact nitrogen-15 (¹⁵N)-labeled full-length human serum albumin as the internal standard, which is spiked into samples (specimens and quality control material) prior to any processing. Sample handling is kept to a minimum to alleviate loss of material and reduce the measurement uncertainty. Samples are subjected to trypsin digestion, analyzed, and assessed as previously described. The detection range is 5 mg/L to 500 mg/L. The next steps will be to complete the SRM 3666 certification, release the standard and complete the extent of equivalence study. Documentation and other published information about this effort can be accessed from the NIST website.

Discussion

• Dr. Bachmann asked whether a dilution protocol for this measurement procedure had been validated for analyzing specimens higher than the current range. Dr. Beasley-Green noted that goal was to use the NIST method for value assignment of the NIST material, which currently does not extend past the target range of 500 mg/L, but a validated dilution protocol could be considered in the future when the need arises.

UMN
Jesse Seegmiller, UMN

Dr. Seegmiller provided a brief overview of and update on the UMN Advanced Research and Diagnostics Laboratory (ARDL) UA RMP. Six human serum albumin peptides for the candidate UMN LC-MS/MS RMP have been identified: DLGEENFK, LVNEVTEFAK (L-K), YLYEIAR, AEFAEVSK, LVTDLTK, and QTALVELVK. Three albumin isoforms from each of the six peptides, and potential variants, are documented in the literature. Twenty-four multiple reaction monitoring interactions are used in the RMP, each with unlabeled albumin followed by the 15N-labeled full-length human serum albumin standard transition. The validated UMN ARDL LC-MS/MS RMP for UA is operational, and each calibrator, a control, and a specimen are analyzed monthly. Since the last update, the UMN in house study comparing immunoassays—specifically the Roche amino acid assay—received institutional review board approval, and results are being reviewed. The next step will be to prepare for the extent of equivalence study that was described later in the meeting.

Mayo Clinic
John Lieske, Mayo Clinic

Dr. John Lieske noted that the Mayo Clinic and UMN RMPs for UA are similar, and he plans to work with Dr. Seegmiller and UMN to determine next steps. Dr. Lieske explained that the Mayo Clinic Renal Testing Laboratory currently does not perform its routine quality control of samples, and he noted that operations have significantly slowed during COVID-19.

HSA, Singapore
Qinde Liu, HSA

Dr. Qinde Liu provided an overview and update of the Chemical Metrology Laboratory (CML) ID LC MS/MS RPM for UA measurement, which is similar to the NIST method. Samples are digested using trypsin and spiked with the ¹⁵N-labeled full-length human serum albumin internal standard yielding eight peptides; these peptides were quantified simultaneously. The National Metrology Institute of Japan (NMIJ) CRM 6202a was used as the calibrator, and the sample size ranged from 30 mg to 300 mg. The CML LC-ID-MS/MS procedure was published in 2021 in Clinical Chemistry and Laboratory Medicine (Chen et al., 2021) and is being used to certify the CRM HRM-3004a albumin and creatinine in human urine at a low (40 mg/L) and high (226 mg/L) values. Albumin values using CRM 6202a and SRM 2925 calibrators were similar, and the relative deviation was well within the expected uncertainty of the CRMs. Commutability testing of the CRMs across five models of clinical analyzers available in Singapore showed satisfactory results, which now are published in Analytical and Bioanalytical Chemistry (Hong et al., 2022).

NIDDK Extent of Equivalence Study (Round Robin) to Compare Results from Four Reference Measurement Procedures
Greg Miller, VCU, Qinde Liu, HSA

Dr. Miller noted that the LWG has met and discussed an approach to conducting this round robin and has developed a statistical model to support the study.

Dr. Liu detailed the process. Eight urine samples (two vials of each sample) and two units of HRM-3004 CRMs will be shipped to the four laboratories on January 26, 2023, with expected delivery on January 30 or 31, 2023. The accompanying documentation will consist of study protocol, product specification, certificate of analysis, safety data sheets for urine samples, sample receipt form, and the result form. Laboratories should return the sample receipt form to Dr. Miller.

Discussion

• Dr. Miller asked about the timeline to complete the study. Dr. Liu pointed out that the manual weighing procedure will be extensive and can take upwards of 2 months to complete the cycles with two CML analysts supporting the work.
• Dr. Beasley-Green noted that 2 months is a reasonable time for NIST to complete the analysis. Coordinating and scheduling instrument time to complete the runs in one sequence will be critical.
• Dr. Seegmiller agreed with the 2-month timeframe. Dr. Lieske could not commit to this schedule at this meeting but will begin discussions with his colleagues at Mayo Clinic.
• Dr. Miller explained that the LWG will need to submit a publication by mid-year demonstrating the extent of equivalence prior to the JCTLM submission and can proceed with the other three laboratories if Mayo Clinic will need more time.
• Dr. Mauro Panteghini asked whether the intent is to submit one RMP for the four laboratories. Dr. Miller replied that the intention is that each laboratory would nominate its RMP as an independent submission because the assays are not identical.

Updates from NIDDK
Afshin Parsa, NIDDK

Dr. Afshin Parsa, Program Director, Division of Kidney, Urologic, and Hematologic Diseases (KUH), NIDDK, on behalf of NIH, expressed appreciation to the LWG for its continued efforts on this project. NIDDK is starting to plan approaches to promote timely adoption of this reference standard as the project is being finalized. NIDDK also is continually seeking various opportunities to facilitate buy-in from other clinical laboratories, the renal community, and NKF, including interactions at scientific meetings and conferences. Drs. Parsa and Jenna Norton, Program Director, KUH, NIDDK, have been discussing the possibility of using some of the data—or lack thereof—collected regarding the variability in albumin measurements related to the platforms and reference standards. These data then will be used to estimate the positive impact of using the new reference standard on disease classification and prognosis for renal, cardiovascular, and general mortality-based outcomes. Albuminuria has been one of the most potentially powerful predictors for adverse outcomes.

Dr. Parsa highlighted that NIDDK recently updated its eGFR-based calculator on the NIDDK website to reflect the Chronic Kidney Disease Epidemiology Collaboration 2021 race-free equation and also plans to update the professional laboratory pages. NIDDK is continuing to promote use of cystatin C along with creatinine to derive eGFR measures when the eGFR value is close to a critical decision point (e.g., drug dosing, kidney transplant eligibility). The combined serum creatinine-cystatin C equation yields slightly higher accuracy and less bias than equations using serum creatinine alone.

Dr. Norton noted that because many changes in positions and affiliations have occurred during the COVID-19 pandemic, NIDDK is in the process of refreshing the participants list for these meetings and will be sending emails to better gauge the interest to remain on the list.

Summary and Follow-up Items
Greg Miller, VCU

Dr. Miller reviewed the LWG’s next steps and timeline.

• July 2023—The LWG will plan to meet during the American Association for Clinical Chemistry meeting in Anaheim, California, to discuss implementation procedures.
• May 2024—The LWG will work with the laboratories to prepare for RMP nominations to JCTLM.
• May 2025—The LWG anticipates RMPs and SRM 3666 to be listed by JCTLM.
• Date is TBD—NIDDK will conduct an assessment of the clinical impact of UA standardization.

Other Comments

In response to a question from Dr. Panteghini about plans to submit CRM HRM-3004a as secondary calibrator, Dr. Liu appreciated the suggestion but explained that those discussions have not occurred in the HSA. Participation in the Round Robin could provide some preliminary comparison data that will be necessary for such a submission. Dr. Phinney mentioned the various comparisons that would be needed, and Dr. Miller added that convening a meeting to further discuss a formal assessment of this material can be considered.

Dr. David Seecombe asked whether samples analyzed in the Round Robin would be sufficient to support a broader study in other clinical laboratories. Dr. Liu noted that the samples being evaluated were collected from a single donor to support the HSA External Quality Assessment program and quantities are limited.

Adjournment

Dr. Miller expressed appreciation to the reference laboratories for their support and thanked participants for attending. He adjourned the meeting at 12:36 p.m. EST.

Action Items

• Dr. Beasley-Green will check with NIST about providing SRM 2925 for the Round Robin study.
• NIDDK and SCG will update the participants list.

Lorin Bachmann, Ph.D., DABCC
Associate Professor, Department of Pathology
Co-Director, Clinical Chemistry Laboratory
Virginia Commonwealth University
lorin.bachmann@vcuhealth.org

Ashley Beasley-Green, Ph.D.
Staff Scientist, Biomolecular Measurement Division
National Institute of Standards and Technology
ashley.beasley@nist.gov

Steven Breeze, Ph.D.
Abbott Point of Care
steven.breeze@apoc.abbott.com

Johanna Camara, Ph.D.
Research Chemist, Chemical Sciences Division
National Institute of Standards and Technology
johanna.camara@nist.gov

Jian Dai, Ph.D., FACB, FCACB
Managing Director
Mindray
j.dai@mindray.com

Joris Delanghe, M.D., Ph.D.
Professor, Department of Clinical Chemistry
University Hospital Ghent
joris.delanghe@ugent.be

Diane Ellis-Edwards, Ph.D.
Director of Chemistry and Immunoassay
Medica Corporation
dellisedwards@medicacorp.com

James Fleming, Ph.D., FACB
Vice President and Vice Chair, Scientific Affairs
Department of Science Technology
Laboratory Corporation of America
jim_fleming@labcorp.com

Yoshihisa Itoh, M.D., Dr.Sci.
Professor and Chairman, Department of Laboratory Medicine
Asahikawa Medical University Repository
yoshiito@d8.dion.ne.jp

Anthony Killeen, M.D., Ph.D.
Ellis S. Benson Professor, Department of Laboratory Medicine and Pathology
Laboratory Director, Advanced Research and Diagnostics Laboratory
University of Minnesota Medical Center
kille001@umn.edu

John Lieske, M.D.
Professor of Medicine and Director
Mayo Clinic Renal Testing Laboratory
lieske.john@mayo.edu

Qinde Liu, Ph.D.
Consultant Scientist and Team Lead
Chemical Metrology Division
Health Sciences Authority
liu_qinde@HSA.gov.sa

Greg Miller, Ph.D.
Chair, Laboratory Working Group
Professor, Department of Pathology
Co-Director, Clinical Chemistry Laboratory
Director, Pathology Information Systems
Virginia Commonwealth University
greg.miller@vcuhealth.org

Jenna Norton, Ph.D., M.P.H.
Program Director
National Institute of Diabetes and Digestive and Kidney Diseases
National Institutes of Health
jenna.norton@nih.gov

Mauro Panteghini, M.D.
Clinical Pathologist
Cattedra di Biochimica Clinica e Biologia Molecolare Clinica
University of Milan
mauro.panteghini@unimi.it

Afshin Parsa, M.D., M.P.H.
Program Director
National Institute of Diabetes and Digestive and Kidney Diseases
National Institutes of Health
afshin.parsa@nih.gov

Pinak Patel
Product Manager
Medica Corporation
ppatel@medicacorp.com

Karen Phinney, Ph.D.
Research Chemist, Analytical Chemistry Division
National Institute of Standards and Technology
karen.phinney@nist.gov

David W. Seccombe, M.D., Ph.D., FRCPC
Managing Director
Canadian Eternal Quality Assessment Laboratory
dseccombe@ceqal.com

Jesse Seegmiller, Ph.D.
Chair, IFCC Working Group for Standardization of Albumin in Urine
Assistant Professor, Department of Laboratory Medicine and Pathology
University of Minnesota
jseegmil@umn.edu