AACC Annual Meeting - Anaheim, CA - August 6, 2019

National Institute of Diabetes and Digestive and Kidney Diseases National Kidney Disease Education Program Laboratory Working Group, Joint Meeting with the International Federation of Clinical Chemistry and Laboratory Medicine Working Group for Standardization of Albumin in Urine. Hilton Anaheim, Huntington Rooms, Anaheim, CA August 6, 2019 12:00 p.m. – 2:00 p.m. PDT

Welcome and Introductions

W. Greg Miller, Virginia Commonwealth University (VCU)
Lorin Bachmann, VCU, Chair, International Federation of Clinical Chemistry (IFCC) Working Group for Standardization of Albumin in Urine (WG-SAU)

Dr. W. Greg Miller, Chair of the National Kidney Disease Education Program (NKDEP) Laboratory Working Group (LWG), welcomed the participants to the meeting and recognized Dr. Lorin Bachmann as Chair of the IFCC WG-SAU. Participants were reminded that the LWG was formed in 2003 with the aim of standardizing the serum creatinine measurement used to determine estimated glomerular filtration rates (eGFRs), which they accomplished in 2010. Subsequently, the LWG, in collaboration with the IFCC WG-SAU, began working to standardize urine albumin (UA) measurements and the progress of this effort has been significant. Initially, the UA measurement problem was defined and confounding factors were addressed. The focus then shifted to developing the infrastructure needed for reference measurement procedures and certified reference materials for UA measurements. The National Institute of Standards and Technology (NIST) has been attentive to establishing a UA reference materials program and developing a reference measurement procedure (RMPs).

To satisfy the requirements of the Joint Committee for Traceability in Laboratory Medicine (JCTLM), the LWG also is collaborating with Drs. John Lieske and Patrick Quint at the Mayo Clinic Renal Testing Laboratory (RTL) and Dr. Jesse Seegmiller at the University of Minnesota (UMN) Advanced Research and Diagnostic Laboratory (ARDL) to develop reference measurement procedures for UA. Dr. Miller expressed appreciation to the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and the NKDEP for their support in this endeavor. Upon completion of this project, the reference system components will be submitted to the JCTLM for listing and then will be available for use by manufacturers for calibration traceability. Planned activities for this meeting were to review the status of the reference materials, reference measurement procedures, and recommendations for UA measurement.

The participants were invited to introduce themselves; a list of participants is provided as Attachment A.

NIST Reference Materials for UA

Karen Phinney, NIST

Standard Reference Material (SRM) 2925 Pure Albumin and SRM 3666 in Frozen Human Urine

Dr. Karen Phinney reminded the LWG of the development background of the work on certifying NIST candidate SRM 2925 as a suitable calibrator for the UA reference measurement procedures and SRM 3666 urine matrix secondary reference materials for UA. Dr. Phinney told participants that the documentation for SRM 2925 is under review by the NIST Office of Reference Materials and the SRM is expected to be issued within the next 3 months. The candidate material for SRM 3666 has been secured. Four concentrations of pooled native human urine samples have completed the acquisition phase: Pool 1, 5–10 mg/L; Pool 2, 20–50 mg/L; Pool 3, 60–180 mg/L; and Pool 4, 275–375 mg/L. The sample certifications for UA and creatinine will begin soon, and the process generally takes 1 year to complete. Dr. Phinney thanked Drs. Miller and Bachmann for their support in helping to obtain the urine samples.

Update on Current NIST Creatinine SRMs

Karen Phinney, NIST

Dr. Phinney presented this update on behalf of Dr. Johanna Camara, who was unable to attend the meeting. NIST material SRM 914a (pure creatinine) was superseded by SRM 914b in November 2018, and, for the first time, its identity, purity, and homogeneity were determined by quantitative nuclear magnetic resonance. The accuracy of the certified creatinine mass fraction is 99.1 percent ± 0.1 percent, and the expiration date is May 31, 2028. Approximately 2 years of sales remain of the current SRM 967a (creatinine in frozen human serum) at the two certified values: Level 1: 0.847 mg/dL ± 0.018 mg/dL and Level 2: 3.877 mg/dL ± 0.082 mg/dL. Prior LWG recommendations were to have low-level concentrations, and NIST is actively obtaining the next batch of material, SRM 967b. The plan is to certify creatinine values with the NIST isotope dilution liquid chromatography mass spectrometry (IDLC-MS) candidate RMP calibrated with SRM 914b. The target values are: Level 1: 0.4 mg/dL (pooled serum with no dilution/stripping) and Level 2: 4.0 mg/dL (pooled serum spiked with creatinine). The current SRM 909c (certified value: 0.824 mg/dL ± 0.018 mg/dL) will serve as the mid-level value. NIST is in the process of awarding a new contract to assist in the full range of services for the development of SRM 967b.

Discussion

The following points were made:

• Dr. Miller asked about the plan to develop the Level 1, 0.4 mg/dL standard. Dr. Phinney pointed out that the goal is to produce the standard without having to incorporate any dilutions. Multiple vendors are interested and have indicated that they are capable of producing the standard. The vendors will need to propose their specific methodologies, and the request for applications has been issued. Dr. Miller offered the LWG services to review the proposals for technical merit.
• A participant asked if the 0.4 mg/dL concentration had to be exact. Dr. Phinney indicated that a range is often given as the criterion to meet. Typically, the contractors have been successful in reaching a target concentration within an acceptable error limit.

Update on RMPs for UA

Karen Phinney, NIST
John Lieske (by phone) and Patrick Quint, Mayo Clinic
Jesse Seegmiller, UMN

NIST

Dr. Phinney presented the NIST report on behalf of Dr. Ashley Beasley-Green, who was to join the meeting later by WebEx. The NIST candidate RMP for UA is based on the isotope dilution mass spectrometry (IDMS) approach involving the use of a nitrogen 15 (¹⁵N)-labeled full-length human serum albumin (HSA) internal standard that is added to the urine specimens. Recent focus primarily has been on the reference material. Because the NIST-Mayo UA method comparison and validation studies analyzing reference materials used three peptides unique to human albumin rather than the 10 peptides that span the amino acid sequence of HSA, the next steps have been to normalize these data. Dr. Beasley-Green is working with NIST statisticians to complete the calculations. Requirements for the JTCLM approvals are being considered, and validation studies with the Mayo Clinic and UMN are ongoing.

Mayo Clinic

Dr. Quint reported on the progress of the albumin reference method. Since the last update, the RTL transitioned to using a new tris (2-carboxyethyl) phosphine (TCEP) protocol for digestion, developed a new synthetic urine solution (i.e., purified ¹⁵N-labeled HSA), began normalizing calibrators with SRM 2925, and installed a new SCIEX Triple Quad 6500+ LC-tandem MS (MS/MS) instrument. Switching to TCEP as a reducing agent has improved the performance of the albumin reference method and enables all steps (e.g., dilution and room-temperature incubation), with the exception of the 37°C incubation and final quench, to be performed by a robotic system. Optimizations and validations are ongoing, and the robotic system is being developed. The RTL purchased ¹⁵N-labeled HSA from Albumin Bioscience (cat#9805) and dissolved in a synthetic matrix consisting of potassium phosphate buffer (10 mM, pH 6.8), sodium chloride (2.3 g/L), urea (12 g/L), and ¹⁵N-labeled albumin (50 mg/L). The synthetic matrix and the calibrators are compatible; the protocol concentration range and linearity are maintained.

Dr. Quint described data generated in the Mayo Clinic RTL-UMN ARDL random urine assay study. Ten samples from the Mayo urine bank were tested at both laboratories using calibrators prepared at each facility. The relative and absolute percentage differences in sample analysis between the two laboratories were assessed by Bland-Altman plots. The results showed relative differences that were within 20 percent for low-mass peptides, suggesting the right direction toward an acceptable correlation, which is promising. The absolute percentage difference, however, was more than 20 percent and may reflect the respective laboratory calibrations. The coefficient of variation (CV) of the overall comparison of the 10 samples was low for a single run and was replicated across laboratories. The higher combined CVs were likely attributable to calibrator differences, which are being addressed. A non-random bias was observed for the high calibration concentrations but can be rectified. The next steps will be to continue the Mayo Clinic method validation, improve the CVs by increasing the number of runs, and continue the Mayo RTL-UMN ARDL method comparisons.

Discussion

• Dr. Andrew Narva wondered about the process for arriving at the synthetic urine formulation and suggested that it be referred to as a calibration matrix. Dr. Quint explained that the final formula is similar to existing combinations used by other investigators, including Dr. Seegmiller. The formula to prepare the buffer can be reproduced with high confidence and is intended to be used as a highly consistent calibrator matrix.
• Dr. Miller sought clarity about deriving CVs from one run. Dr. Quint clarified that the values were derived from the nine different peptides quantified over 10 repetitions; the parent/daughter ion ratios were less than 10 percent CV.
• Dr. Quint anticipated that the Mayo RTL and UMN ARDL calibrator bias will be rectified with the use of SRM 2925 for normalizations. In response to a query by Dr. Phinney on calibrator concentrations, Dr. Quint noted that the RTL and ARDL calibrator concentrations will have values that are traceable to the NIST SRM 2925 standard.
• The LWG discussed the availability of SRM 2925 as the calibrator for the RMPs and agreed that using the material should be coordinated between NIST, Mayo RTL and UMN ARDL. Increasing the albumin concentration in the next batch of SRM 2925 will make it more suitable to prepare adequate volumes of calibrators to value-transfer to working calibrators used for the IDMS reference measurement procedures, The NIST SRM 2925 is intended for use as the primary reference for each laboratory to establish traceability for their respective calibrators.
• Dr. Bachmann asked whether the precision based on using the nine peptides for quantitation had been evaluated. Dr. Quint indicated that those analyses had been completed using the previous reducing agent and are in progress with TCEP; only one of the nine peptides appears to be performing poorly.

University of Minnesota

Dr. Seegmiller provided an update on the UA standardization project. The objectives are threefold: (1) develop a reference measurement procedure for UA; (2) validate the measurement procedure and evaluate precision; and (3) compare the accuracy of results to results with calibration traceable to NIST SRM 2925. In March 2019, the funding contract to support the objectives was finalized, the Mayo Clinic and UMN reached consensus on the calibration matrix, and the supplies were ordered. In addition, method development began and calibrators made using the extinction coefficient method until the NIST SRM 2925 became available in July 2019. Validations were performed from mid-March 2019 to July 2019.

The LC-MS/MS method consists of three steps: specimen preparation incorporating an internal ¹⁵N labeled HSA standard, reversed-phase chromatography, and multiple reaction monitoring (MRM) of six HSA peptides (LVNEVTEFAK, DLGEENFK, YLYEIAR, AEFAEVSK, LVTDLTK, and QTALVELVK), which were verified by the Basic Local Alignment Search Tool (BLAST). Three HSA isoforms were identified in the BLAST from each of the six peptides, but their frequencies vary.

In a 21-day interlaboratory (Mayo Clinic RTL and UMN ARDL) results comparison, the mean CV was 3.96%, ranging from 1.23% to 8.04% for all peptides, all calibrators over the concentration interval 3.13– 200 mg/L. For the human urine control samples, the mean CV was 4.63%, ranging from 3.20% to 6.35%. In conclusion, the LC-MS/MS analysis of the six peptides appears promising for the UA quantitation. Future work will focus on reaching a consensus on the RMP protocol regarding additional replications, aligning the calibration between the Mayo Clinic RTL, UMN ARDL and NIST; and proceeding with the RMP validation using calibration traceable to the NIST SRM 2925 standard.

Discussion

• Participants commented on the frequency and significance of HSA isoforms. Dr. Miller noted that previous studies using the LC-MS/MS method to quantify UA in more than 300 patient urine samples showed no differences in the UA concentrations regardless of the peptide used for quantification suggesting that unusual isoforms are not commonly encountered.
• Dr. Miller highlighted the recommendations on performance goals for bias and imprecision of UA measurements for manufacturers that the LWG and the IFCC WG-SAU compiled and published in the November 2017 issue of the Journal of Applied Laboratory Medicine. The LWG will review the literature on the fraction of total error allotted for the reference measurement procedure steps; and discuss in a follow-up conference call the requirements on uncertainty for the RMPs, goals for accuracy, and the fraction of error that can be contributed at the RMP step in the calibration hierarchy for end-user urine albumin measurement procedures.

National Kidney Foundation (NKF) Kidney Profile Laboratory Engagement

Andrew Narva, NKDEP Director, NIDDK

Dr. Narva provided an update on the NKF Kidney Profile (eGFR and urine albumin-to-creatinine ratio [uACR] testing in one order) laboratory engagement initiative. This engagement is a culmination of several years of collaborative efforts across the nation’s clinical laboratories and clinical societies (e.g., American Society of Clinical Pathologists and American Association of Clinical Chemistry [AACC]) to harmonize reporting units and terminologies. Dr. Narva remarked that although eGFR is widely used and reported routinely for chronic kidney disease (CKD) across the clinical community, UA tests are underutilized. This underutilization is an issue that the Kidney Profile is anticipated to address.

The LWG meetings are held in parallel with the AACC Annual meetings, and, for this 2019 meeting, the session titled “Removing Laboratory Barriers to Improve Kidney Disease Testing and Diagnosis” was planned to discuss the Kidney Profile and describe the clinical and cost implications of recognizing CKD early, as well as the specific actions that each laboratory can take to remove barriers to CKD assessment in primary care. The session also was intended to increase understanding of how clinical laboratory testing can be applied in addressing CKD population health. Dr. Miller planned to join the NKF chief medical officer, Dr. Joseph Vassalotti, and other speakers on the panel.

The NKF recommends that laboratories promote ordering the Kidney Profile as a quality measure, report patient eGFR values, report measurements in uACR in mg/g, and confirm elevated uACR with a first-morning urine sample. Dr. Narva noted that reporting cutoffs will probably be revised once the urine albumin test is standardized. Consideration of a lower cut-off than 30 mg/g, reporting by gender or other characteristics will warrant further discussion in the renal community. Current work analyzing CRIC data may assist in these decisions by providing data on the utility of various cut-off values of uACR in disease prediction.

Discussion

• Dr. Miller pointed out that, based on previous efforts to standardize the units, use of gender-specific lower decision values have depended on obtaining low-level reference standards. Dr. Narva added that educating clinicians on the significance of reporting uACR will need to be reinforced.
• The NKF is working with the Centers for Medicare and Medicaid Services to establish a Current Procedural Terminology (CPT) code, and a Medicare reimbursement code for the Kidney Profile is then likely to follow. Currently, the Kidney Profile is not reimbursable as a panel but the individual measured components are reimbursable. Calculated values are not reimbursable.

Review RMPs’ Validation Data Versus the JCTLM Submission Requirements

W. Greg Miller, VCU

Dr. Miller reminded participants of the JCTLM Database Working Group process for materials and methods submissions and reviewed the guidance documents and data requirements. Following the last meeting, the LWG conducted a series of conference calls with the three reference laboratories—NIST, Mayo Clinic, and UMN—to discuss the submission procedures. The JCTLM requirement for any submitter is to demonstrate the extent of equivalence if one or more RMP is already listed. Because no RMP for UA is currently listed in the JCTLM, reference laboratories for this project will have to (1) perform comparison with an existing method used by a routine measurement laboratory, and (2) participate in an appropriate external quality assessment (EQA) that uses commutable materials. The Mayo Clinic has used a Roche routine measurement procedure, and the College of American Pathologists (CAP) has an EQA program for UA with commutable urine samples that would be applicable. Dr. Miller emphasized that both steps should be easy for the three reference laboratories from this point forward, and that comparisons between the RPMs—a requirement that is in the project’s current workplan—also must be included in the submission.

Dr. Miller informed participants that the Singapore National Metrology Institute, has an RMP for UA in progress—which may be the one that is closest to being submitted to the JCTLM—and is willing to participate in the sample comparison measurements. Each laboratory developing an RMP must make a separate submission to the JCTLM, which occurs annually in May. Whether planning for a 2020 submission for this project is too ambitious is something the LWG will need to decide.

Dr. Miller next led participants in a review of the JCTLM submission guidance documents (e.g., reference standard and compliance templates) and highlighted key points. A peer-reviewed publication of the method, as well as the method’s uncertainty, is required prior to a submission. Reference laboratories should ensure that a quality control check standard is included in each measurement run for uncertainty calculations. The details on compliance will take time to complete and are a recapitulation of the International Organization for Standardization 15193 requirements for RMP content for in vitro diagnostic medical devices and medical laboratories. Detailed standard operating procedures (SOPs) will be necessary. Dr. Miller emphasized identifying areas that may still need to be addressed and ensuring that the minimum required data are collected.

Discussion

• Dr. Phinney commented that the manuscript form of the method often can satisfy the publication requirement if it is submitted to a peer-reviewed journal at the same time that it is submitted to the JCTLM.
• Dr. Bachmann asked whether any of the NIST measurement procedures are close to being finalized. Dr. Phinney explained that the sample preparation, peptides, and methodology used have met the laboratory’s expectations, but the uncertainty calculations have not been completed. The next phase will focus on generating those data and finalizing the SOP.
• Dr. Graham Jones noted that the World Health Organization recently released the essential list of in vitro diagnostics for developing countries that includes the UA and creatinine assays, but the quality of the assays is not addressed. Dr. Jones thinks that the opportunity exists for the clinical community to comment on the impact of assay quality to global health, which the LWG could facilitate.

Other Business

Dr. Narva, Director of NKDEP, announced that he would be retiring from the NIDDK at the end of August 2019 and that this would be his last LWG meeting. He noted that Ms. Jenna Norton, Associate Director, National Kidney and Urologic Science Translation Program, NIDDK, and Dr. Afshin Parsa, Program Director, Division of Kidney, Urologic, and Hematologic Diseases, NIDDK, will continue this effort. Dr. Parsa also is a program officer for the Chronic Renal Insufficiency Cohort Study (commonly called CRIC), which is collecting patient data on UA.

Dr. Miller expressed appreciation to Dr. Narva for his leadership, under which the NKDEP LWG has thrived.

Adjournment

Dr. Miller thanked participants for attending and adjourned the meeting at 1:43 p.m. PDT.

Action Items

• The LWG will review the literature on the fraction of total error allotted for the reference measurement procedure steps and begin discussions on the uncertainty requirements for the RMPs.
• Dr. Miller will schedule a conference call with the NIST, Mayo Clinic, and UMN reference laboratory teams to derive the goals concerning RMP uncertainty.
• The three reference laboratories will need to participate in the CAP EQA for UA that uses commutable materials to support the JCTLM submission requirements.
• The LWG will need to evaluate the data and set a JCTLM submission date.

Participants

Lorin Bachmann, Ph.D., DABCC
Chair, IFCC Working Group for Standardization of Albumin in Urine
Associate Professor, Department of Pathology
Co-Director, Clinical Chemistry Laboratory
Virginia Commonwealth University
lorin.bachmann@vcuhealth.org

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

Jian Dai, Ph.D., FACB, FCACB
Senior Director
Siemens Healthcare Diagnostics
j.dai@siemens.com

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

Horst Glima, Ph.D.
Manager, Research and Development
Roche Diagnostics
horst.klima@roche.com

Lars-Olaf Hansson, M.D., Ph.D.
Associate Professor
Karolinska Institutet
lasse.hansson@me.com

Jack Harwood, M.S., M.B.A.
Siemens Healthcare Diagnostics
j.hawood@siemens.com

Graham Jones, M.D.
Associate Professor, Department of Chemical Pathology
St. Vincent’s Hospital Sydney
gjones@stvincents.com.au

Harvey Kaufman, M.D. (by phone)
Medical Director, Business Development Quest Diagnostics
harvey.w.kaufman@questdiagnostics.com

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

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

Kiyomasa Machida
Scientific and Technical Affairs Specialist
Nittobo Medical Co., LTD.
kiyomasa@nittobogrp.com

Yuri Matsuki
Scientific and Technical Affairs
Nittobo Medical Co., LTD.
yuri@nittobogrp.com

W. 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

Andrew Narva, M.D., FACP
Director, National Kidney Disease Education Program
National Institute of Diabetes and Digestive and Kidney Diseases
National Institutes of Health
narvaa@niddk.nih.gov

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

Patrick Quint, Ph.D.
Technical Specialist II Mayo Clinic
quint.patrick@mayo.edu

Jesse Seegmiller, Ph.D.
Assistant Professor, Department of Laboratory
Medicine and Pathology University of Minnesota
jseegmil@umn.edu

Clark Xu, Ph.D.
Staff Scientist
Instrumentation Laboratory
cxxu@ilww.com