U.S. Department of Health and Human Services

AACC Annual Meeting - Philadelphia, PA - August 3, 2016

National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
National Kidney Disease Education Program (NKDEP) Laboratory Working Group (LWG)

Joint meeting with the International Federation of Clinical Chemistry and Laboratory Medicine Working Group for Standardization of Albumin in Urine (WG-SAU)

Hampton Inn Philadelphia Center City-Convention Center
William Penn Room
Philadelphia, PA

August 3, 2016
8:00 a.m. to 12:00 p.m.

Final Summary

Welcome and Introductions

Greg Miller, Virginia Commonwealth University

Dr. Greg Miller, Chair of the NKDEP LWG, welcomed the participants to the annual LWG meeting. The participants introduced themselves; a list of participants is provided as Attachment A.

Summary of Workshop on Urine Albumin Standardization

Greg Miller, Virginia Commonwealth University

Summary of Progress

The NKDEP-LWG and IFCC hosted a meeting to review the status of urine albumin measurement in 2007. Recommendations from that meeting were published in Clinical Chemistry 2009;55:24–38. Some standardization recommendations were intended for immediate implementation, most of which have been accomplished. The albumin/creatinine ratio (ACR) should always be reported using units of mg/mmol or mg/g, depending on the country’s standard measurement system. The group reiterated the recommendation to use the first morning urine sample rather than 24-hour collection. It also asked laboratories not to use “microalbumin” in test names because it implies that a small albumin molecule is being measured, although Dr. Miller noted that it is difficult to change the name of a test once it has become established.

The group also made recommendations for research needed to improve standardization of urine albumin (UA) measurements. It is likely that ACR decision thresholds for chronic kidney disease (CKD) risk should be lower than currently understood and that men and women have different thresholds, but laboratories are not able to measure UA adequately to enable use of lower thresholds. The biological variability of UA is not well investigated, although it is generally agreed that within individual CV is approximately 30 to 40 percent. UA and urine albumin/creatinine (UAC) measurement results should be standardized among different measurement procedures, and performance specifications for UA measurement that are satisfactory for medical decision making should be developed.

Dr. Miller reviewed the current status of UA measurement. Adsorption to containers has been shown to be less than 1 percent (Robinson, et al. Clin Chim Acta 2014;431:40-5). Median biases among commercially available immunoassay procedures are approximately 40 percent and, for most procedures, vary with concentration (Bachmann et al. Clin Chem. 2014;60:471-80). The precision of most immunoassay procedures has been deemed adequate, but for some procedures, dilution protocols introduce a bias and nonlinear calibration is a problem. There is reasonable evidence that most immunoassays recognize the common molecular forms of albumin in urine.

Reference materials for UA still are needed. The Bachmann study investigated the commutability of diluted Institute for Reference Materials and Measurements (IRMM) ERM DA470k/IFCC, which is a reference material for serum proteins, including albumin. On dilution to urine concentrations, DA470k/IFCC was not commutable with clinical samples for most UA measurement procedures. This is a challenge because, despite it not having been designed for use for UA measurements, it is currently used by manufacturers because it is the only available certified reference material. Several reference materials have been created or are being created in partnership with the National Institute of Standards and Technology (NIST). NIST SRM 3667, creatinine in frozen human urine, was available in 2013; NIST SRM 2925, pure human albumin to calibrate reference material measurement procedures, and NIST SRM 3666, albumin in frozen human urine, are in development. NIST SRM 3666 is planned for four concentrations and will be commutable for use with immunoassays. Dr. Miller noted that creatinine also will be value assigned in this material when it is available. SRM 3666 has been delayed due to difficulty in identifying urine donors. NIST is contracting with an academic medical center to obtain residual urine specimens to pool to get the base urine materials. Isotope dilution mass spectrometry (IDMS) procedures currently are in development by the Mayo Clinic and NIST that are in the process of comparison and validation. Joint Committee for Traceability in Laboratory Medicine (JCTLM) submission is anticipated in the near future for a reference measurement procedure.

Recommendations for the Measuring Interval (Analytical Measurement Range)

The goal determined at the 2015 NKDEP LWG meeting for the analytical measurement range (AMR) is 2 to 400 mg/L. The lower limit, 2 mg/L, was set as the limit of quantitation based on literature suggesting that the 5 to 10 mg/L range is likely to become clinically useful in the future, when albumin standardization is implemented and lower thresholds become achievable. The current upper value is more flexible and may be limited by technology. The 2 to 400 mg/L AMR is recommended for new measurement procedures or reformulated reagents.

Performance Recommendations for Measurement Procedures

The bias and precision goals defined at the 2015 NKDEP LWG meeting were based on within individual biological variability in the range of 30 to 40 percent. An analytical CV of 6 percent is adequate and achievable. Sample-specific effects were estimated to be CV approximately 7 percent, which is a relatively small contribution to overall variability and uncertainty. A total allowable error of less than 25 percent is adequate. Considering variability from analytical, sample specific and biological sources, a bias less than 13 percent is a reasonable goal for standardization.

Freeze-thaw Experiment Results (Preparation for Commutability Assessment)

Lori Bachmann, Virginia Commonwealth University

As part of a larger project to assess commutability of reference materials for UA measurement in development at NIST, Dr. Lori Bachmann presented preliminary data on an investigation of possible effects of a freeze-thaw cycle on UA values.

The current study was designed to compare the effects of freezing (≥ 66 hours at -70 C) and thawing urine samples on UA measured by 10 different commercial methods from five different manufacturers. Preliminary evaluation suggest that UA results for previously frozen and never-frozen samples were not significantly different. Although rigorous statistical analysis still needs to be completed, after several data anomalies are investigated for transcription or other “blunder” type issues, analysis thus far suggests that thawed, previously frozen at -70 C urine samples can be used in the planned commutability assessment as representative of never-frozen samples.

Initial evaluation of results confirm the inter-manufacturer assay bias found in the 2014 study. Although most samples had a statistically significant, but modest, median bias of about 1 percent compared to reference standards, a few samples showed a larger discordance.

The investigators also assessed measurement imprecision by analyzing variation of UA values in triplicate measurements by each method. Seven of the methods showed CVs less than 5 percent. Three of the methods, however, showed higher CVs.

Future plans include completion of a formal statistical analysis of UA results for frozen and never-frozen samples, investigation of the data outliers, and development of the experimental plan for commutability assessment of the new reference materials.

Discussion

Drs. Bachmann and Miller made the following clarifications regarding the sample handling procedures followed by the manufacturers of the in vitro diagnostics (IVD) UA methods:

  • The manufacturers were instructed to refrigerate the samples after receipt and before measurement, and not to refreeze them. Note that which urine samples had been frozen or not was blinded to the participants.
  • The manufacturers followed their instructions for use (IFUs), which might include centrifuging cloudy samples. The urine samples were centrifuged after pooling and after freezing and thawing prior to aliquotting and shipment, so it is unlikely that they needed centrifugation again.

Drs. Dave Armbruster and Anthony Killeen asked for more detail about the manufacturers’ analytical methods, particularly whether all of the methods were immunochemical or whether any were dye binding. Dr. Bachmann indicated that all methods were immunochemical and she will present details about each manufacturer’s analytical method, based on the manufacturers’ IFUs, with the finalized results of the freeze-thaw experiment.

The outliers in the freeze-thaw experiment results were discussed. The following points were made:

  • Reanalyzing the outlying samples by other methods to investigate possible commonalities, as suggested by Ms. Jillian Tate, is not possible per experimental protocol, whereby sample material was not retained by the manufacturers after analysis. Sample sizes were small, so only limited amounts of sample remained after analysis. Samples were anonymized, so demographic commonalities cannot be assessed.
  • Outlying results showed both positive and negative scatter. Dr. Bachmann suggested interference and precipitation as possible causes.
  • Aliquots might not have been homogeneous. The experimental protocol was designed, however, to produce homogeneous aliquots. Samples were well-mixed and allowed time to equilibrate. The protocol reproduced the sample handling measures that a clinical laboratory would be expected to take.
  • Some outliers might have resulted from transposition issues. The data will be reviewed for such errors when finalized.
  • Dr. Miller stated that it is reasonable to expect some outliers in the commutability assessment and understanding the reason behind all anomalous results is unlikely. The commutability design will need to include a sufficient number of samples that outliers can be discarded and results will still be valid.
  • Urea. Dr. Joris Delanghe proposed that urea might be interfering with albumin measurement. Dr. Bachmann responded that urea was not measured in the samples.
  • Total protein. Dr. George Schwartz suggested analyzing samples for total protein, which might interact nonspecifically with antibodies in the immunoassays.
  • Specific gravity and pH. Dr. John Eckfeldt asked whether specific gravity and pH were measured in the samples. Dr. Bachmann replied that the volume of unpooled samples tends to be severely limited, with typical volumes on the order of 20 mL. Many clinical samples had to be discarded because of insufficient volume. Collaboration with other academic medical institutions on sample collection might result in more samples of sufficient volume.

The following additional tests will be performed on urine samples for the commutability assessment:

  • At Dr. Killeen’s suggestion, future samples will be checked for evidence of urinary tract infections using a urine dipstick analysis. Dr. Killeen proposed further that samples be filter-sterilized if possible.
  • A urine sample aliquot will be saved for further analysis (e.g., total protein or electrophoresis) in case albumin results are anomalous.

Dr. Miller summarized that the preliminary results of the freeze-thaw experiment indicate that frozen samples will be acceptable in the commutability assessment and that additional urine sample qualification will be incorporated into the design for the commutability assessment of new reference materials.

Urine Albumin Reference Measurement Procedures

Ashley Beasley-Green, NIST; John Lieske, Mayo Clinic

Dr. Ashley Beasley-Green reviewed the NIST isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) measurement procedure for UA. She also described the status of an ongoing validation study comparing the results of UA measurements conducted by NIST and the Mayo Clinic Renal Reference Laboratory.

In the NIST assay, a labeled internal albumin standard (intact 15N-labeled, recombinant human serum albumin) is added to a urine specimen. Because the first phase of the study indicated that something in the urine samples interfered with albumin assays, phase 2 now incorporates centrifugation of the specimen but no pH adjustment before further manipulation. After trypsin digestion, 10 peptides are used for qualification of molecular identification and for quantitation of UA concentration.

The Mayo Clinic protocol differs in two ways. Because urine pH can vary widely, from pH 4 to pH 9, the urine samples are diluted and the pH adjusted to be constant from sample to sample. Second, quantitative analysis is based on three peptides, which are also present in the NIST method, whose sequences were confirmed by Basic Local Alignment Search Tool (BLAST) to be unique to human albumin.

Validation of the performance of the two candidate reference measurement procedures used urine samples provided by the Mayo Clinic and calibrant reference standards, ranging from 1 to 150 mg/L, supplied by NIST. Each laboratory processed and analyzed the same materials. Albumin values obtained by immunoassay (carried out by the Mayo Clinic) and by mass spectrometry (MS) (using the Mayo Clinic and NIST protocols) were comparable but not identical.

Dr. Beasley-Green emphasized the impact of incorporating multiple albumin peptides for investigation of albumin protein structure variants; such qualitative information, she observed, can provide useful information about the significance of albumin variability in patient samples.

Discussion

The outliers in the validation study of the reference measurement procedures were discussed. The following points were made:

  • Dr. Beasley-Green cited biological variability as a possible cause of anomalous results. Biological variability, which can cause modifications in some of the peptides measured in the reference measurement procedures, could contribute to the differences observed for some peptide fragments. Such issues will affect the sample size needed for the commutability assessment.
  • Dr. Miller suggested that albumin fragmentation in the samples might contribute to analytical variability.
  • Dr. Jesse Seegmiller proposed that cross-reactivity with peptides from proteins other than albumin with the same charge-to-mass ratios as the NIST method peptides might be a source of analytical variability.
  • Dr. Lori Bachmann suggested to measure a set of samples prepared by the same preparation protocol using both measurement procedures.

The following points were made regarding comparing the data from the different sets of peptides selected for the Mayo Clinic and NIST reference measurement procedures:

  • Dr. Seegmiller stated that during method development, the Mayo Clinic selected for its reference measurement procedure only those albumin peptides that were unique to human albumin and would not overlap with other human protein peptides. Dr. John Lieske added that the peptides selected by the Mayo Clinic also were chosen empirically because they produced a consistent and strong signal.
  • Dr. Beasley-Green indicated that the peptides selected by NIST were chosen to produce data across the amino acid sequence of albumin. She added that one goal of the NIST method is to investigate biological modifications of albumin, such as truncations and post-translational modifications. If such biological modifications occur in an antibody epitope, error might be introduced in the corresponding immunoassay. Some of the peptides observed in the NIST method could be used to qualify any outlier specimens observed in the commutability study for NIST SRM 3666 albumin in frozen human urine.
  • Dr. Miller observed that it is important to ensure that outliers are not derived from proteins other than albumin, because such interference would increase the uncertainty of the data. Dr. Beasley-Green responded that the anomalous peptides were not used in albumin quantification; the three peptides in common between the Mayo Clinic and NIST procedures were used for quantification. The other peptides provide qualitative information about sequence variations that might cause issues with the immunoassays. Including all of the peptides in the coefficient of variation for the measurement increases the observed variability because both biological and analytical variability are incorporated.
  • Dr. Seegmiller noted that the NIST measurement procedure is designed to provide information about immuno-nonreactive albumin. Obtaining this information is a separate goal from harmonizing the results of the Mayo Clinic and NIST procedures.
  • Dr. Beasley-Green commented that none of the peptides were consistent outliers across all of the donor urine specimens.

Dr. Flavio Alcantara asked which epitopes are targeted in the immunoassays and whether their sequences are the same as those of any of the peptides measured in the MS-based assays. Dr. Seegmiller replied that the MS-based assays use a different approach than the immunoassays and are based on the amino acid sequence of albumin. Dr. Beasley-Green added that protein folding can result in different regions of the amino acid sequence of the protein interacting with the antibody, which is difficult to capture using MS-based assays. Dr. Miller commented that epitopes for commercial antibodies are typically proprietary.

Differences in sample handling between the Mayo Clinic’s and NIST’s reference measurement procedures were discussed. The following points were made:

  • Dr. Dror Yahalom observed that the Mayo Clinic’s measurement results generally were higher than those of NIST. Dr. Beasley-Green responded that specimens were processed differently by the Mayo Clinic and NIST. The Mayo Clinic diluted samples and adjusted their pH before digestion, whereas NIST only added the internal standard prior to digestion. Dr. Seegmiller noted that urine pH can be highly variable, ranging from pH 4 to 9. The Mayo Clinic typically performs a 1 to 5 dilution to normalize the pH. The optimal pH for trypsin digestion is pH 8. The pH normalization step might increase efficiency and reduce bias. IVD manufacturers also routinely dilute samples, which normalizes osmolality as well. Salt concentrations affect the trypsin digestion. A participant asked whether the internal standard’s recovery could be used to compensate for non-optimal digestion pH.
  • Dr. Beasley-Green noted that although the pre-digestion protocols differed, the albumin measurement results obtained by the Mayo Clinic and NIST were comparable. Dr. Miller pointed out that although comparable, the results were not in close enough agreement to conclude that the measurement procedures gave equivalent results. Better agreement needs to be achieved to qualify as reference measurement procedures that are suitable for use.
  • Dr. Beasley-Green stated that the NIST measurement procedure seeks to minimize sample handling. Sample handling can cause material loss. Dr. Seegmiller replied that studies have shown that albumin loss to plastic container walls is not generally a concern, however, particularly at the relatively high concentrations being measured.
  • Dr. Bachmann suggested that in an effort to standardize the reference measurement process, the Mayo Clinic and NIST consider using identical sample handling protocols. Performing the trypsin digestion differently is introducing variability into the measurement that is unrelated to variability of the albumin molecule.
  • Dr. Beasley-Green indicated that NIST would evaluate the influence of urine pH on the urine albumin measurement to address the issue of sample processing between the two methods. Once determined, the optimal sample processing conditions would be incorporated into the NIST method.
  • Dr. Seegmiller observed that the bias toward lower results in NIST’s patient samples was not observed in the calibrant samples, all of which had the same pH and osmolality.
  • Dr. Killeen suggested other sample conditions to consider harmonizing for the trypsin digestion, including temperature, digestion duration, trypsin concentration, and the source of trypsin.

Deviation from linearity at high concentrations was discussed. The following points were made:

  • Dr. Bachmann observed that lower and higher concentration samples were nonlinear. Nonlinearity at high concentrations is a concern because the upper limit for the AMR needs to be extended beyond 150 mg/L.
  • Dr. Maria-Magdalena Patru suggested that in the study of extending the AMR to higher upper limits, the data be presented with the lower concentration data enlarged to ensure that any nonlinearity is easy to observe. Dr. Miller proposed expressing future comparison data as a difference plot, which would be more informative than concentrations on both axes.

IVD Manufacturer Reports on Progress to Address Urine Albumin Measurement Procedure Issues Identified in the Assessment Study Reported in Clinical Chemistry 2014;60:471¬80

Dror Yahalom, Abbott Laboratories; Jian Dai, Siemens Healthcare Diagnostics

Dr. Yahalom stated that a positive bias was observed at low concentrations in the Clinical Chemistry assessment study. In response, Abbott Laboratories analyzed internal standards but did not observe such a bias at low concentrations. The samples used in the 2014 study no longer are available. Accordingly, Abbott Laboratories is satisfied that its assay performs accurately.

Dr. Jian Dai noted that in the Clinical Chemistry assessment study, bias was observed for only one of Siemens Healthcare Diagnostics’ platforms. Siemens modified the calibration algorithm and the anchoring levels of the calibration, but neither approach improved the bias. Currently, Siemens is exploring possible effects from the antibody source. When the reference measurement procedures are finalized, improvements to the manufacturers’ methods can be tested.

Discussion

Dr. Dai indicated that expanding the AMR to 400 mg/L is a concern to IVD manufacturers. Measuring high concentrations without losing the sensitivity required to measure low concentrations likely will prove challenging. A possible compromise is automatic reruns of samples. Dr. Miller responded that accuracy at low concentrations will be more critical than extending the AMR to higher concentrations. Performing automatic dilutions is a viable approach to measure samples with high concentrations of albumin.

NIST SRM 3666 Albumin (and Creatinine) in Frozen Human Urine

Greg Miller, Virginia Commonwealth University; Ashley Beasley-Green, NIST

Revised Plan to Obtain Urine Specimens

Dr. Miller discussed the progress of NIST SRM 3666, albumin in frozen human urine. He showed the goal albumin concentrations for each pool (1-10, 20-40, 80-120, 275-325 mg/L). The vendor has been unable to recruit donors at the higher concentrations. In light of this challenge, the material procurement approach will be changed. Using residual urine from academic medical centers is proposed; no restrictions will be placed on donors’ age, sex, body mass index, or health status. Typically, enough urine can be acquired from 50 mL volumes to pool. Samples will be rejected if dipstick tests are positive for blood, nitrites, or leucocyte esterase. The samples will be mixed and centrifuged, and they will be stored at -70 C or colder until 2.5 L of samples at each concentration have been acquired.

The frozen aliquots will be shipped to the vendor, who will thaw, mix, centrifuge, and pool them to obtain the needed 2.5 L at each desired concentration. The mixed pools will stand overnight at 2–5 C; during the mixing process, they will be filtered and dispensed in 1.0 mL aliquots into polypropylene screw-capped vials with NIST labels. The final product will be stored at -70 C or colder and shipped to NIST.

An advantage of this procedure is the ability of a larger number of donors to minimize the impact of any sample specific influences; the disadvantage is that the albumin is obtained from individuals with a wider range of clinical conditions.

Commutability Assessment

The original design retained individual donor vials for the commutability assessment. The revised design will require a commutability assessment using a separate set of individual donor urine samples.

Timeline

The anticipated timeline for this project includes contractual work for the remainder of 2016, donor urine procurement between March and May 2017, and pool and aliquot preparation in June 2017. This timeline has not yet been discussed with the vendor, so the assumption that they can complete the work within this timeframe is unconfirmed. The individual donor set for the commutability assessment will be procured on a flexible schedule, potentially in parallel with the progress of the aggregate pool, so that all samples can be ready at the same time. The commutability assessment with manufacturers’ participation likely will be conducted between August and October of 2017; Dr. Miller asked the manufacturers to make note of this tentative timeframe. He added that the NKDEP LWG probably would meet before the commutability assessment is conducted next year, at which time they could review the protocol and adjust it as needed.

Discussion

The screening tests for urine samples were discussed. The following points were made:

  • Blood. Dr. Schwartz observed that many patients with kidney necrosis will test positive for blood in their urine. A response was made that the levels indicative of infection are substantially below those of necrosis.
  • Blood-borne viruses. Dr. Harvey Kaufman suggested that collecting serum samples to screen for blood-borne viruses be considered. Dr. Phinney responded that in developing other urine reference materials, screening for blood-borne viruses was not performed. Instructions to handle the sample material as potentially infectious will be provided. Dr. Bachmann added that urine generally is not considered infectious, and the samples will be handled by clinical laboratory staff, who are experienced in biohazard precautions. Dr. Eckfeldt noted that testing for reportable diseases would require obtaining informed consent from the donors.

Blood-borne viruses. Dr. Harvey Kaufman suggested that collecting serum samples to screen for blood-borne viruses be considered. Dr. Phinney responded that in developing other urine reference materials, screening for blood-borne viruses was not performed. Instructions to handle the sample material as potentially infectious will be provided. Dr. Bachmann added that urine generally is not considered infectious, and the samples will be handled by clinical laboratory staff, who are experienced in biohazard precautions. Dr. Eckfeldt noted that testing for reportable diseases would require obtaining informed consent from the donors.

Dr. Miller commented that to reduce the level of effort required for recruitment, donors will not be stratified by cause of albuminuria.

In response to a question, Dr. Miller indicated that preservatives will not be added to the samples.

Reference Material for a Low Concentration of Creatinine in Human Serum

Johanna Camara, NIST

Dr. Camara reviewed the status of the development of a reference material for a low concentration of creatinine in human serum. The proposed concentration for the reference material is approximately 0.4 mg/dL. In development of the low-concentration reference material, artificial serum matrices were used to dilute the current frozen serum reference material. Three types of artificial serum were used as diluents. The reference material was analyzed using an ID-LC-MS reference measurement procedure. Background levels of creatinine were detected in two of the artificial sera, but good results were obtained for spiked recoveries. In initial screening tests, the candidate reference materials were diluted with normal serum or spiked with creatinine.

A round-robin test with eight laboratories, 16 analytical platforms, and six samples is planned. The candidate reference material and samples have been prepared. The next step will be to distribute the samples to the laboratories. After the round-robin test is completed, performing a commutability assessment will be considered, depending on the degree of interlaboratory agreement found at low concentrations.

Discussion

Interference issues were discussed. The following points were made:

  • Dr. Dai commented that interference issues with glucose and uric acid are known for creatinine analysis. Corrections for nonspecific interactions are needed; therefore, the matrix should contain glucose and uric acid. Dr. Camara responded that it is unclear whether glucose and uric acid are present at normal levels in SeraFlex synthetic human serum. For SigMatrix Serum Diluent, glucose and uric acid have not been added.
  • Dr. Miller stated that a primary goal is to develop a reference material typical of a pediatric sample to assist with obtaining accurate measurements in children.

Dr. Dai observed that the measurement of creatinine by enzymatic methods is more accurate than the Jaffe reaction but also more expensive, which will be a barrier to clinical laboratories’ switching methods.

Background levels of creatinine in serum diluents was discussed. The following points were made:

  • Dr. Miller asked whether the normal serum used in dilutions of the candidate reference material was treated with creatininase. Dr. Camara responded that it was not.
  • Dr. Delanghe suggested removing background creatinine by a combined treatment with creatininase and creatinase to increase efficiency.

Calibration Uniformity for Iohexol Clearance Measurement Procedures Used for Measured Glomerular Filtration Rate

George Schwartz, University of Rochester; John Eckfeldt, University of Minnesota

Dr. Eckfeldt briefly outlined the rationale for this iohexol calibration study. Although inulin was historically the gold standard for measuring glomerular filtration rate (GFR), other filtration agents also have been introduced over the years. One of the newest agents, iohexol, was developed originally as a contrast agent; more recently, iohexol has been used to measure GFR by assessing rates of disappearance of injected iohexol from plasma. Because incorrect iohexol measurements could generate inaccurate equations for estimating GFR, Drs. Schwartz and Eckfeldt have investigated the consensus of iohexol concentration measurements in four different laboratories in the United States and Sweden.

Dr. Schwartz summarized the results of the study to date. In typical clinical procedures, plasma iohexol samples are analyzed along with a series of quality control samples. Until now, 20 percent variation in iohexol values among the quality control samples has been considered acceptable; Dr. Schwartz observed that more rigorous quality control expectations are needed.

From 2003 to 2014, GE Healthcare was the only provider of iohexol reference standards. Since September 2014, new reference materials have become available through Fluka and other companies. After new iohexol reference material was introduced to the University of Rochester Medical Center Toxicology Laboratory in 2014, researchers noticed a decrease in GFR values. Because Dr. Schwartz was conducting a long-term study of CKD that monitored the GFR of participating patients every 2 years, the possibility of reference standard-related inaccuracies in GFR estimates was a particular concern.

A National Institutes of Health (NIH) administrative supplement has enabled the researchers to conduct a comparative Iohexol Quality Assurance Study in laboratories in Lund, Sweden; Minnesota (Mayo Clinic and the University of Minnesota, Fairview); and the University of Rochester Medical Center in Rochester, New York. Identical samples were sent to all of the laboratories, including reference material from Equalis, a Swedish provider of external quality assessment for clinical laboratories. Although the GFR values of several of the laboratories showed a close match to Equalis consensus values, the pre-2014 University of Rochester values were 10 to 12 percent lower than those values. Pre-2014 GFR values for the University of Rochester study have now been multiplied by 1.12 to reconcile earlier data with current calibration standards.

This study underscores the importance of external standards and external quality assessment to ensure data consistency and accuracy, especially for measurements that directly affect patient care.

Discussion

The Equalis external quality assessment program was described further. Dr. Eckfeldt stated that Equalis’ iohexol test material is available for purchase in the United States. He suggested that all laboratories that perform iohexol measurements consider joining the Equalis external quality assessment program. Thirty-five laboratories, primarily located in Scandinavia, are participating in the Equalis program.

Analytical issues associated with the two optical isomers of iohexol were discussed. The following points were made:

  • Dr. Schwartz noted that the Mayo Clinic’s liquid chromatography (LC) method detects both isomers as a single peak. Powdered iohexol contains different ratios of isomers than iohexol solutions, but solutions prepared from powdered iohexol will equilibrate between the two isomers over time.
  • Dr. Eckfeldt stated that autoclaving an iohexol solution as is done for the pharmaceutical preparation equilibrates the isomer ratio, which remains constant after equilibration.
  • Dr. Eckfeldt observed that although high-performance liquid chromatography (HPLC)-based methods detect the isomers as separate peaks, the two isomers have the same mass in MS-based methods.
  • Dr. Eckfeldt commented that the isomeric ratio in powdered iohexol might depend on the manufacturer, as well as the product lot. Omnipaque might autoclave the material because its powdered product has an equilibrium isomeric ratio. GE Healthcare appears to have little variation in isomeric ratio between product lots.
  • In response to a question, Dr. Seegmiller indicated that studies have shown that the kidney does not alter the isomeric ratio of iohexol.

The following points were made in a discussion of the effects of measurement method on the GFR:

  • Dr. Eckfeldt observed that measuring GFR by urinary iohexol and iothalamate clearance produces different results. The measurement method must therefore be considered in GFR estimation equations.
  • Dr. Seegmiller stated that bias introduced from renal excretion of iothalamate appears to be an issue only at low levels of GFR (i.e., less than 40 mL/min per 1.73 m2). The secretion component of renal excretion does not appear to be a problem for iohexol. Results for measured GFR (mGFR) determined by iothalamate measurement appear to be correlated with mGFR determined by inulin measurement.
  • Dr. Schwartz commented that determining which mGFR method produces the “real” value is difficult.

Dr. Eckfeldt stated that the use of iohexol for mGFR determination is not FDA-approved. Concentrations are five to 10 times lower than for contrast studies, but still might be a concern for renal patients. He added that iohexol clearance for mGFR determination is not reimbursable by Medicare, but it can be billed as a radiopharmaceutical. Radiolabeled tracers no longer are used to measure clearance rates.

Other Business and Next Steps

Greg Miller, Virginia Commonwealth University

No other business items were raised.

Adjournment

The meeting was adjourned at 11:29 a.m.

Action Items

  • Dr. Bachmann will present details about each manufacturer’s analytical method with the finalized results of the freeze-thaw experiment.
  • The Mayo Clinic and NIST will consider harmonizing sample handling in their reference measurement procedures to optimize the pH and osmolality of the trypsin digestion.
  • NIST will decide whether testing donors for blood-borne viruses is necessary in preparing NIST SRM 3666.

Participants

Flavio Alcantara, M.D., Ph.D.
Medical Chief of Section, Central Lab Division
Hospital das Clínicas da Faculdade de Medicina
University of São Paulo
Email: flavio.alcantara@hc.fm.usp.br

Dave Armbruster, Ph.D., DABCC, FACB
Scientific Affairs Manager
Abbott Diagnostics
Email: david.armbruster@abbott.com

Lori Bachmann, Ph.D., DABCC
Associate Professor, Department of Pathology
Virginia Commonwealth University
Email: lbachmann@mcvh-vcu.edu

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

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

Nancy Cataldo, M.B.A.
National Clinical Studies Coordinator
Nova Biomedical

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

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

John Eckfeldt, M.D., Ph.D.
Professor, Department of Laboratory Medicine and Pathology
Assistant Director, Advanced Research and Diagnostic Laboratory
University of Minnesota
Email: eckfe001@umn.edu

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

Neil Greenberg, Ph.D., DABCC
Principal Consultant
Neil Greenberg Consulting, LLC
Email: ngreenbe@frontier.com

Lars-Olof Hansson, M.D., Ph.D.
Associate Professor
Karolinska University Hospital
Email: lasse.hansson@bonetmail.com

Harvey Kaufman, M.D., M.B.A.
Medical Director, Business Development
Quest Diagnostics
Email: harvey.w.kaufman@questdiagnostics.com

Anthony Killeen, M.D., Ph.D.
Professor, Department of Laboratory Medicine and Pathology
Laboratory Director, Advanced Research and Diagnostic Laboratory
University of Minnesota
Email: kille001@umn.edu

Horst Klima
Manager, Research and Development
Roche Diagnostics
Email: horst.klima@roche.com

Hans-Joachim Kytzia
Roche Diagnostics GmbH
Email: hans-joachim.kytzia@roche.com

John Lieske, M.D.
Professor of Medicine
Director, O’Brien Urology Research Center
Mayo Clinic
Email: lieske.john@mayo.edu

W. Greg Miller, Ph.D.
Working Group Chair
Professor of Pathology, Department of Pathology
Director, Pathology Information Systems
Director, Clinical Chemistry Laboratory
Virginia Commonwealth University
Email: gmiller@vcu.edu

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

Maria-Magdalena Patru, M.D., Ph.D.
Senior Manager, Scientific Affairs
Ortho Clinical Diagnostics
Email: mpatru11@its.jnj.com

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

Stella Raymondo-González
Comisión Directiva
Polo Tecnológico de Pando
Email: raystela@gmail.com

George Schwartz, M.D.
Professor, Department of Pediatrics
University of Rochester Medical Center
Email: george_schwartz@urmc.rochester.edu

Jesse Seegmiller, Ph.D.
Fellow, Department of Laboratory Medicine and Pathology
University of Minnesota
Email: jseegmil@umn.edu

Jillian Tate, M.Sc., FFSc, RCPA
Department of Chemical Pathology
Royal Brisbane and Women’s Hospital
Email: jill.tate@health.qld.gov.au

Catherine Townsley
Vice President, Marketing
Gentian Technology AS
Email: catherine.townsley@gentian.no

Heidi Vebø
Product Manager
Gentian Technology AS
Email: heidi@gentian.no

Clarke Xu, Ph.D.
Staff Scientist
Instrumentation Laboratory
Email: cxxu@ilww.com

Dror Yahalom, Ph.D., M.B.A.
Senior Scientist
Abbott Laboratories
Email: dror.yahalom@abbott.com

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