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U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Improving the understanding, detection, and management of kidney disease.

AACC Annual Meeting – Anaheim, CA – July 28, 2010

Participants: Greg Miller, Lorin Bachmann, David Bruns, David Bunk, Joris Delanghe, John Eckfeldt, James Fleming, Mary Lou Gantzer, Neil Greenberg, Anders Grubb, Glen Hortin, Yoshihisa Itoh, Chandra Jain, Graham Jones, Harvey Kaufman, Mary Kimberly, Anthony Kileen, David Koch, Hans-Joachim Kytzia, John Lieske, Gary Myers, PhD, Andrew Narva, Eileen Newman, Mauro Panteghini, Karen Phinney, James Ritchie, Max Robinowitz, Heinz Schimmel, David Seccombe, Jill Tate, Linda Thienpont, Jack Zakowski, Ingrid Zegers

Summary of Action Items:

Meeting Minutes

1. Welcome and Introductions, Greg Miller (Chair, LWG), Virginia Commonwealth University

Several projects discussed last year did not make much progress due to lack of funding. Now that we understand the NIDDK funding process and cycles, we hope to have data to review at the meeting next year.

2. Cystatin C Standardization, Anders Grubb (Chair, IFCC WG-SCC, Lund University, Sweden) and Ingrid Zegers (Director, Institute for Reference Materials and Methods, Belgium)

Traceability for the cystatin C reference material is based on SI units (mg/L). Development of specifications, production, and certification of the material took six years. The material is stored at IRMM in Belgium and was produced by adding recombinant cystatin C that is identical to the human protein into a serum pool from blood donors. The final product is lyophilized. The value was assigned against the primary reference material of isolated cystatin C, whose concentration was established by dry mass analysis. Three different immunochemical techniques were used to transfer the value from the secondary serum calibrator to the first reference preparations: enzyme amplified single radial immunodiffusion (two studies), turbidimitry (three studies), and nephelometry (two studies). Based on these studies, the assigned value and uncertainty is 5.48 +/- 0.15 mg/L.

John Eckfeldt asked how the calibrator can be used since the assigned value is high and what is known about the commutability. For his studies of eGFR, the range of interest is ½ mg/L; he asked how the material should be diluted. Anders Grubb answered that the level is high because manufacturers need to produce a standard curve to test their products. The certificate of analysis does not discuss commutability because the larger scale commutability study is still ongoing. Preliminary commutability results should be published in the next six months.

Preliminary commutability studies have been performed with dilutions of the calibrator and patient samples demonstrating a range of cystatin C results. The calibrator was diluted in saline for most of the methods or the N-diluent for the Siemens method. For the Siemens, Gentian, and Sentinel (Abbott) assays, the patient samples and diluted calibrator showed the same dose-response curve indicating commutability. Continuing studies of a larger group of methods indicate that there is acceptable commutability for 11 of the 14 methods tested. All manufacturers should be able to use the calibrator; Roche has already made changes to their method to achieve commutability between the reference material and patient samples. The calibrator is available from IRMM in Belgium and the following publication describes a method for calibrating assays: Blirup-Jensen S, Johnson AM, Larsen M. Protein standardization V: Value transfer. A practical protocol for the assignment of serum protein values from a reference material to a target material. Clin Chem Lab Med 2008;46:1470-9.

Greg Miller asked when the material will be listed by the Joint Committee for Traceability in Laboratory Medicine and Ingrid Zegers replied that the deadline for 2011 was just missed so it will be listed in 2012. Greg Miller asked if all manufacturers are working toward standardization with this material and Ingrid Zegers replied that all manufacturers that have been contacted are interested in participating in the commutability study and most are planning to recalibrate their assays.

Heinz Schimmel commented that the current material has shown good commutability at 60% and greater dilution of the material. This dilution will allow calibration to approximately 3 mg/L which will cover the 1-2 mg/L range of interest. The material should not be used undiluted to calibrate methods at the higher range unless commutability with patient samples has been confirmed.

3. Urine Albumin Adsorption, Mary Kimberly (for Mary Robinson), Centers for Disease Control and Prevention (CDC)

The first phase of this project involved evaluation of albumin absorption onto various materials and the study design and summary results were presented last year. Since then, all of the experiments have been repeated with the addition of one more material and using urine rather than buffered human serum albumin (HSA). More extensive experiments have been performed to demonstrate the efficiency of the rinsing strategy using 125I-labeled HSA. Also, tube-to-tube transfer experiments were performed to determine if adsorption can be directly detected using the albumin assay.

Tube-to-tube transfer experiment over 5 hours: Urine was transferred to another container every 30 minutes for a total of 12 transfers. Using low concentration baseline urine at pH 6.6 that was spiked to both 10 and 30 mg/L with HSA, there was no measurable loss of albumin after 5 hours at room temperature. When the baseline urine was adjusted to pH 4.8 and spiked to 30 mg/L with HSA, the hydrophilic-coated vials appear to bind less albumin. Creatinine remains the same in each cup. Albumin depends on the pH of the urine even though the assay reagent is prepared in a TRIS buffer at pH 8.0.

The rinsing strategy was shown to be efficient by using 125I-labeled albumin applied to a circular disk and rinsed several times. The counts per minute for each rinse and for the disc after six rinses shows that essentially all albumin had been removed after the second rinse and it was decided to rinse each material five times with water in all future experiments. The amount of albumin retained in solution and the amount adsorbed onto discs after five hours at room temperature was evaluated for each of the 18 materials at pH 5.0 and 8.0 and also for several urine samples with a range of albumin concentrations in polystyrene sample cups and hydrophilic vials.

Conclusions: For all materials and urine samples tested, greater than 99% of albumin remained in solution after five hours at room temperature; adsorption in sample cups was lower for polystyrene than polyethylene, and hydrophilic-coated sample vials adsorbed less albumin than polystyrene sample cups. This was also detected in transfer experiments, but only when the urine pH was below 5.0; albumin measurements using the immunoturbidimetric Roche Tina-Quant Assay are lower when urine sample pH values are lower than 5.0 even though the assay buffer is pH 8.0. The effect of urine pH on other methods is unknown. Albumin concentration and pH are the most important factors affecting adsorption. It will be necessary to determine whether pH adjustment is necessary for the samples collected for the Biologic/Pathologic Variability Study.

Max Robinowitz asked if labs typically measure pH before assaying albumin and Greg Miller replied that they do not. Greg Miller summarized that adsorption is not an important problem in the clinical interpretation of urine albumin results.

4. Urine Albumin Method Harmonization, Lori Bachmann, Virginia Commonwealth University

Project objective: Use native human urine to evaluate the agreement among manufacturers' albumin methods and also compare against the isotope dilution LC-MS/MS candidate reference procedure being developed at Mayo by John Lieske's group. These urine samples will also be used to compare analytical harmonization of total protein and creatinine methods. Another objective is to evaluate the commutability of the Japan Society of Clinical Chemistry candidate reference material developed by Yoshi Itoh's group in Japan. The study includes a comprehensive representation of manufacturers (N=5) and platforms (N=11).

Study Design: Collect 340 fresh, residual native samples submitted for routine urine albumin measurement and which represent a wide variety of disease pathologies. Samples will be collected over eight to 10 weeks by three sites. Fresh aliquots of each sample will be shipped overnight to each manufacturer and frozen aliquots will be sent for LC-MS/MS. Shipments will include spiked matrix samples (salts, reducing substances, and drugs) to evaluate the influence of common matrix components on the methods.

Analysis: Imprecision will be evaluated by ANOVA, the bias for each method will be calculated versus LC-MS/MS method, unusual or discrepant results will be evaluated for analyte-specific matrix influences. An aliquot will also be sent to the National Institute of Standards and Technology (NIST) where the candidate reference method procedure is being expanded. Because these samples represent a wide variety of disease processes, the albumin molecular forms may contain disease-related post-translational modifications or truncations that will allow NIST to better define the measurand. Aliquots of the candidate reference material will be included in each manufacturer's assay run so the commutability of the reference material can be evaluated, as well as its ability to harmonize method results.

Funding has been secured for this project and sub-award processing for all three collection sites is nearing completion. Confirmation of participation has been received from all manufacturers, and the target start date is the week of January 10, 2011.

John Eckfeldt asked for clarification that the aliquots will never be frozen before the manufacturers assay them. Lori Bachmann confirmed that this is true and added that the researchers plan to include a subset of samples that have gone through a single freeze-thaw cycle to be sure that there is not a freeze-thaw effect on the reference method.

Graham Jones asked if the pH will be determined on the samples, and Lori Bachmann confirmed that a dipstick analysis will be performed on each sample.

5. Urine Albumin Reference Measurement Procedure John Lieske, Mayo Clinic

Aims: Validate the LC-MS/MS as a reference measurement procedure to quantify intact albumin in urine, compare clinical sensitivity and specificity of the LC-MS/MS assay performed at Mayo and NIST, as well as with other immunoassays, using a population of patients with or without overt diabetic nephropathy, and develop the LC-MS/MS methodology to characterize the urine albumin measurand in normal and disease states.

The method involves trypsin digestion of both the human sample and an internal standard that is a labeled, recombinant protein. There are three trypsin fragments that can be reliably detected; two near the beginning of the albumin protein and one at the end. Three QC materials are used to monitor precision; the CVs ranges from 4-8% if one high intensity transition is used for each peptide and 3-7% if all transitions for each peptide are used. For each of the three peptides, intra-assay CVs range from 1-4% and inter-assay CVs range from 3-6%. The bias between fragments has been found to be 1-3% and assay of the several hundred human urine samples will confirm these biases between fragments. There may be some biology with pieces of the albumin being chewed off or other modifications that change the way it is detected.

In order to evaluate if there are native fragments in albumin that change the way we are able to quantify it, ten urine samples that had already been assayed were filtered through a 10 kDa filter; what was retained was re-suspended, measured, and compared to the pre-filtration result. Overall a slight 4% loss of albumin was found, but it correlated exactly with all three fragments so this was interpreted as simply loss caused by the filtration procedure. If native fragments were present, they should affect each of the trypsinized fragments differently, especially the ones at the beginning and end of albumin.

David Bunk, National Institute of Standards and Technology

It is important to characterize albumin in urine and determine what types of structures are represented in normal and disease states. In order to look at the protein structure, NIST will quantify many fragments, including the three fragments measured in the Mayo method. The isotope-labeled intact albumin that is used for an internal standard in the Mayo method can also act as an internal standard for the structural characterization because digestion of this intact protein yields a ratio of peptides that reflects the intactness of the protein. When compared to the ratio of peptides in the endogenous albumin in urine, one can see if it is intact or if there are post-translational modifications.

Glen Hortin commented that there is a map showing the important glycines for glycation of albumin; in glycated albumin the main site of glycation is in the lysine side chain so this may explain a slight difference in the ratios, especially in a diabetic population. David Bunk agreed that it is a good idea to look back at the old data to locate the preferred sites and determine if they fall in any of the tryptic peptides.

Heinz Schimmel asked if the three peptides are found in a 1:1:1 ratio in the sample, how one would explain that they come from an intact protein rather than from fragments. John Lieske explained that it is unlikely they come from fragments because the filtration experiment did not change results significantly.

6. IFCC Task Force — Chronic Kidney Disease, Graham Jones, St. Vincent's Hospital, New South Wales

The IFCC has created a task force to promote quality CKD testing worldwide, which is a collaboration between the clinical chemistry and pathology communities. An electronic survey designed to help understand the current status of testing was sent to the 90-member organizations and 22 responses were received, with a bias towards the more developed parts of the world. Most countries did not have official published policies regarding reporting of eGFR with requests for creatinine, and most laboratories did not routinely report eGFR with a creatinine result. Most countries reported using creatinine assays that are standardized to the IDMS reference method, but four European countries reported that only some laboratories use IDMS-standardized assays.

A poster from Greece presented at AACC stated that the assay variability was too high to consider eGFR reporting. An IFCC member from Uruguay, where a standardization program was set up by their QAP, reported that there are many providers of creatinine assays from small local businesses or other parts of the world where the assays have not been IDMS standardized. Other countries have not made IDMS alignment of creatinine methods a priority. Further activities include gathering additional global information, promoting assay standardization through serum-based QAPs and education, and supporting program development.

Because all methods are IDMS standardized in the U.S., Greg Miller asked for discussion about removing the older eGFR equations from the website. Nancy Accetta commented that most of the website traffic comes from inside the U.S. The potential concern is for people from other countries that are not yet IDMS aligned, but this needs to be weighed against the problem that many people in the U.S. do not know the difference between the two calibration schemes. Neil Greenberg suggested that most major manufacturers sell only IDMS standardized creatinine assays worldwide. Jim Fleming suggested and Greg Miller agreed with removing the non-standardized equation and adding a statement that the remaining equation is only for use with IDMS-traceable creatinine methods. John Eckfeldt stated that it is important to facilitate other worldwide manufacturers to establish IDMS-traceable methods, especially if there is only one equation. Mary Lou Gantzer commented that Siemens offers both standardized and non-standardized methods worldwide.

Joris Delanghe commented that the updated Schwartz formula is only valid for enzymatic IDMS-standardized methods. Greg Miller agreed that pediatrics is an interesting problem because IDMS-traceable Jaffe methods may give lower results for methods that are sensitive to protein, making it even more important to remove the old Schwartz equation. Graham Jones offered the opinion that removing the old equations is an improvement because it will make it simpler. Another comment was that clinicians do not have control over where samples are tested. Greg Miller added that all U.S. laboratories are using standardized methods and the Siemens Dimension Jaffe non-IDMS-traceable method has been shown to produce results in close agreement with IDMS values in the CAP accuracy based LN24 Survey. Andrew Narva commented that most clinicians never knew there was a problem with creatinine methods, do not know that it has been fixed, and that less detail on the website meant for clinicians is better. It may confuse clinicians to add a comment that the equation should be used with creatinine results from IDMS-traceable methods. Greg Miller agreed with Graham Jones' comment that if the equation is used with a creatinine result from a non-IDMS-traceable method, it will only cause a 5% error in eGFR calculation. No one gave any reasons to keep the older equations.

7. Informational Posting on CKD-EPI Equation, Andrew Narva (Director) NKDEP, NIDDK

The website will add a short educational posting explaining the CKD-EPI equation, including a brief description of the equation and an introductory paragraph that states "The CKD-EPI equation is a new equation published in 2009 to estimate GFR from serum creatinine, age, sex, and race for adults greater than 18 years of age. NKDEP has not made a recommendation on general implementation of this equation. The equation is still being validated and while offering some improvement for eGFR between 60 and 120 mL/min/1.73m2, it is not clear that implementing CKD-EPI in place of the MDRD Study equation would alter clinical detection or management of patients with CKD." The CKD-EPI equation is marginally better than the MDRD Study equation and mostly beneficial in the range of 60-120 and thus of less consequence in the management of CKD. Other equations are being evaluated and it is not an efficient use of resources to change the recommendation every time a new equation is published. If anything, a more important change is educating clinicians in the use of urine albumin in assessing patients. Also, NKDEP wants to remain consistent with other agencies.

Graham Jones commented that it is likely that the use of the CKD-EPI equation will be recommended in Australia because reporting up to 90 is the current practice and this equation improves eGFR results in the 60 to 90 range.

David Seccombe commented that in Canada it was decided not to recommend the CKD-EPI equation because it would lead to further confusion for clinicians even though they report results up to 90. He asked if the U.S. is planning on recommending reporting of results between 60 and 90 because one issue is detection of early disease; and he stated that although the MDRD is not quite as accurate above 60, it can serve to show downward trends. Andrew Narva answered that NKDEP will not change its current reporting recommendations because the limitations of the MDRD equation for eGFR > 60 mL/min/1.73m2 are unchanged.

8. List of Literature Citations to Post (distributed with agenda), Greg Miller

Last year it was identified that a list of some key references posted on the website may be useful. He suggested that the group have a discussion about the philosophy regarding the kinds of papers that should be listed and noted that it is not the intention to become a compendium of publications. A list of possible publications that have been submitted by members was circulated to everyone for consideration. Greg Miller asked for comments about the papers on the list.

Greg Miller summarized that there is not much enthusiasm for a list of publications other than as needed to support our statements.

9. Serum Creatinine Method Specificity (Interactive Workshop), Neil Greenberg (Chair, IFCC WG-GFRA), Ortho-Clinical Diagnostics, Inc.

Much progress has been made on this project since the meeting last year when the data collection had just been completed. Study overview: Twenty healthy control subjects and about 20 unspiked patients samples were collected in each of 19 pathological groupings similar to groups in which eGFR is measured. Additionally, in order to test several endogenous substances, spiked pools were created. A normal serum pool with creatinine 88 µmol/L (1.0 mg/dL) and a supplemented normal serum with creatinine 336 µmol/L (3.8 mg/dL) were each spiked with four levels of acetone, pyruvate, acetoacetate, and ascorbate. For each spiked interferent, the two pools were assayed on four enzymatic and three Jaffe creatinine commercial methods and an LC/MS reference method (Neil Dalton's Lab, London). NIST SRM 967 reference material was used on all methods to verify trueness. The methods used were Siemens DIMENSION Jaffe and enzymatic, Beckman-Coulter SYNCHRON DxC 800 Jaffe (Rate CREm modular method) and enzymatic (CR-E cartridge method), ROCHE INTEGRA 800 Jaffe (Gen2 Compensated) and enzymatic (Creatinine Plus Version 2), and Ortho VITROS enzymatic (IDMS-traceable slides).

Data for endogenous substance interference was shown as interferographs and a summary table that looked at bias in the range of 10-20% and >20% at the highest concentration of interferent. It is difficult to draw any generalized conclusions with respect to Jaffe or enzymatic method performance in the presence of these substances. It appears that all of the Jaffe methods are rate methods, but some are rate-blanked and some are not. Details about the methods are still being compiled. Pyruvate was included in the study because it is known to accumulate in serum upon standing and it is interesting to note that the normal creatinine pool showed the most interference with pyruvate while this interference disappeared in the higher creatinine pool.

Data for the specificity study was captured in a table that displayed the number of samples in the groups that were biased by more than 0.10 mg/dL (88 µmol/L) or 10%, whichever is greater (based on the data for the non-diseased control group), either positively or negatively for each of four enzymatic and three Jaffe methods; cells were highlighted with beige if there were four to five biased samples and with purple if there were greater than six biased samples. Looking at the group of 59 diabetes patient samples known to have elevated levels of ß-hydroxybutyrate (N=19), glucose (N=20), and HbA1c (N=20), the Jaffe 3 method had a large number of positively biased samples in all three patient subgroups and the Jaffe 2 method only showed a positive bias in the patient group with elevated glucose. It is important to note that we do not know the basis for a given bias, e.g., HbA1c probably is not the actual reason for bias and it is something else in that group of patients causing the bias. In the cardiovascular disease group only the Jaffe 3 method showed a positive bias. Looking at cardiovascular disease patients on specific drugs, patients taking lidocaine showed a positive bias and literature reports support that this bias is caused by metabolites of lidocaine in enzymatic methods that use sarcosine oxidase as the coupling enzyme. The three Jaffe methods showed both negative and positive biases with this same group of patients, but the mechanism for bias with these methods is unknown. In the high bilirubin patient group (all but one were >19 mg/dL and most of these samples were collected from only three patients), three of the four enzymatic methods and one of three Jaffe methods had a large number of samples with a positive bias. For the group of patients with low albumin, only the Jaffe 3 method showed a positive bias; and in the group with total protein values between 8 and 18 g/L, one enzymatic and two Jaffe methods showed positive bias. There was essentially no bias seen in the kidney disease patient group that had higher concentrations of creatinine.

In general there were more problems seen with the Jaffe methods, but no method was trouble-free. There were significant differences in both amount and direction of bias among methods within family groups indicating that not all Jaffe nor enzymatic methods were alike. Spiked interferents were more significant at normal creatinine concentrations. Diabetic patient panels showed frequent bias with Jaffe methods and patients with known kidney disease under clinical management had few problems with all methods examined. A manuscript describing this study should be ready for publication later this year.

Discussion to develop recommendations that can be made based on the information learned from this study:

John Eckfeldt asked if the conclusion that the spiked interferences were more significant at normal creatinine levels was based on expressing the interference as a percentage or in absolute terms. Neil Greenberg agreed that the results were expressed as percent recovery, but a 50% change at 1.0 mg/dL creatinine will result in a 50% change in the eGFR levels.

Dave Bruns pointed out that the slide with the raw numbers shows that there were more problems at normal creatinine levels. Neil Greenberg emphasized there were more methods with >20% bias in the normal creatinine pool than in the elevated pool.

Harvey Kaufman asked if the manufacturers' package inserts make statements about these interferences. Neil Greenberg stated that one manufacturer indicated no bilirubin interference in their enzymatic method, but this study showed dramatic bilirubin interference. However, published findings show that conjugated bilirubin has a more dramatic interference than unconjugated bilirubin. Neil Greenberg stated that Ortho uses unconjugated bilirubin to test interference with their methods and suspects that the high bilirubin samples used in this study had significant levels of conjugated bilirubin. This could explain the different information found in the manufacturers' labeling versus the experimental data. Manufacturers' labeling should be reviewed before making conclusions.

Tony Killeen suggested that it is possible that something other than bilirubin is the real cause of the interference seen in that patient panel. Neil Greenberg agrees that it is not known what is driving the biases found in the patient panels.

Dave Seccombe commented about their recently completed cycle of a proficiency survey which uses human serum. The creatinine is shifted by a process of dialysis so many of these interfering substances are not present. The performance criterion was changed from a target value +/-15% to +/-7.6%. The three challenges sent had values of approximately 60, 90, and 120 µmol/L. The overall failure rate was 30% across almost all platforms. At the low creatinine level, all methods performed poorly; and as creatinine concentration increased, the performance improved.

Neil Greenberg asked Joris Delanghe about a study that was done regarding bilirubin interference with creatinine. However, the focus of that paper was on fetal and neonatal bilirubin, which contains mostly unconjugated bilirubin.

Mauro Panteghini asked if correlations have been done to test linearity of bilirubin interference with the enzymatic methods. Neil Greenberg stated that this has not been done. Although the bilirubin levels of the samples were known, they were very high at approximately 20-40 mg/dL (300-600 µmol/L), and there was not a range of concentrations that would allow this type of analysis. A linear series of spikes was not performed.

Glen Horton suggested that a panel of components be developed for which all manufacturers check interference with their methods.

Greg Miller suggested a statement that a method should not have a bias/interference greater or less than "XX" across a series of classifications of patient type. Neil Greenberg commented that in this study, bias was defined as +/- 0.1 mg/dL or 10% whichever is greater and was based on the distribution observed in the control group.

Graham Jones commented that in Australia they are trying to decide whether or not to make a recommendation for enzymatic methods. The data do not indicate the extent of the problem (e.g., failures just outside of the range versus way outside range). There may be a class/method statement that can be made regarding reproducibility. Is there a class difference that can be detected by further analysis of these data, or is each assay so individual that a statement cannot be made by method class? Neil Greenberg agreed that while this presentation does not reflect information about the magnitude of the biases, it is intended to have all the data available for review once it is published. There was a difference in magnitude of the bias; and from sample to sample, the variability was huge in some of the cases. Because we do not know the quantity of the interferent (e.g., lidocaine metabolite), we have to make a broad brush view of a very complex problem and the conclusions drawn have to be limited. Simplistic recommendations cannot be made.

Tony Killeen commented that these data reiterate that linking creatinine to eGFR is for stable patients and not for those in diabetic ketoacidosis. Neil Greenberg agreed that we need to be cautious about doing GFR calculations in complex patient groups.

Jack Zakowski commented that the study achieved the goal to disprove/prove the belief that Jaffe methods have much interference and enzymatic methods do not have any interference. Enzymatic versus Jaffe method decisions may have to be made based on the patient population. Anders Grubb suggested that cystatin C should be used because it does not have these interferences. Jack Zakowski countered that cystatin C may have interferences from other components.

Joris Delanghe asked if we can take into account the prevalence of these interferents in a clinical situation to develop a more objective method for weighing the pros and cons. Neil Greenberg agreed with this approach.

Neil Greenberg invited participants to email him with further comments or ideas.

10. Urine Albumin Physiological Variability, David Koch, Emory University

The objective of the study is to determine which urine collection approach is optimal: 24 hour, first morning void, second morning void, or random collection with ratio to creatinine. Knowledge of physiologic variability is also needed to assist with measurement procedure performance requirements. Two institutions, Emory University and Grady Memorial Hospital, have submitted for IRB approval. Diabetic subjects with stable CKD and elevated urine albumin will be recruited. Samples will be collected based on a very difficult collection protocol and shipped to Mary Robinson at CDC for albumin and creatinine testing. The 12-week collection plan for each subject is as follows:

Subjects will be paid half at the beginning and half at the end of the study to encourage completing the study.

Max Robinowitz asked about the relevance of the first morning void in subjects that have to void during the night and thus do not have a concentrated first morning void. David Koch answered that this is another variable; all urine excreted will be collected during weeks 1, 6, and 12, which may help answer this question.

Another comment was made that physical activity is a more important confounder that the difference between first and second morning void. David Koch commented that the study is not designed to answer this question. However, a survey can be used for subjects to record significant physical activity.

Another question was asked about how subjects who become unstable during the study will be handled. David Koch answered that subjects recruited will have had stable levels of microalbumin prior to the start of the study and that an endocrinologist will assist to identify subjects that become unstable. The goal is to have 15 subjects at each site complete the two cycles for a total of 30 and they will start with 25 subjects at each site, knowing that some subjects with be lost due to becoming unstable or dropping out.

11. New Business - Serum Albumin and Dialysis Center Requirements, David Bruns, University of Virginia

Low serum albumin is a strong predictor of mortality in renal dialysis patients and there are national guidelines that call for monitoring of serum albumin in the dialysis population with target values of 3.5 g/dL, or more preferably 4.0 g/dL. This is now a Centers for Medicare & Medicaid Services (CMS) quality parameter which is likely to translate into dollars since there is pay-for-performance in the treatment of dialysis patients. However, BCG assays are not equal. After the University of Virginia (UVa) lab changed to a new BCG assay in 2005, the proportion of patients meeting the 4.0 g/dL albumin target improved significantly from 22% to 70%. After the assay was changed again in 2009, the percentage of patients meeting the 4.0 g/dL target went from 60% to 17%. Because of this, sample comparisons were performed with several laboratories. Serum samples were measured at several laboratories using several different methods. The results had differences similar to what had been experienced when changing methods at UVa. Dave Bruns asked for recommendations for addressing the problem.

Glen Hortin asked if the problem is isolated to renal patients. Dave Bruns answered that the exchange samples were a mixture of renal and non-renal patients and that there was no obvious difference in discrepancy for different patient types.

Joris Delanghe suggested that the exchange study include turbidimitry and nephelometry methods.

Andrew Narva commented that CMS has been monitoring serum albumin for awhile and they recognize that there are two methods, BCP and BCG, so perhaps they can also deal with analyzer-specific reference ranges. Dave Bruns suggested that these would probably have to be updated each time there is a tweak in the method.

John Eckfeldt commented that this same problem was seen at University of Minnesota Medical Center, Fairview, when they compared nephelometry, electrophoresis, and dye binding methods. They had the same conversation with their dialysis units. This is clearly a problem and there is an issue of traceability that leads to method-specific biases that are likely patient group specific.

Graham Jones commented that this problem provides the basis for need for a quality standard for bias for albumin. It also goes back to traceability and how wide is the uncertainty of value assignment from manufacturers. IMEP-17 (2002) harmonization assignment for albumin had wide discrepancies among methods.

David Seccombe asked what method was used to derive these treatment guidelines of 3.5 and 4.0 g/dL as the answer is unknown at this time.

David Bruns commented that he would like to see the methods harmonized. It would be great if the LWG would see this problem to be within its purview. Greg Miller agreed that serum albumin standardization needs improvement and that there are significant issues between BCG and BCP routine assays.

Note: The ERM-DA 470k/IFCC Human Serum Proteins reference material includes albumin in the list of certified plasma proteins. Furthermore, JCTLM recognizes immunochemical methods (turbidimetry/nephelometry principles) as reference measurement methods for serum albumin.

12. Closing Comments

Max Robinowitz asked about reporting urine albumin as the albumin/creatinine ratio since he has not heard this discussed. Greg Miller replied that the use of the albumin/creatinine ratio came from the conference held in 2007. He added that the proceedings and recommendations from this conference were published in 2009 and that studies published since then have supported this recommendation.

Mauro Panteghini asked about the project to standardize urine creatinine measurement and produce a urine creatinine reference material. Greg Miller replied that while this project was not on the agenda for this meeting, NIST is working on preparation of a reference material for urine albumin and creatinine but is waiting to better define the measurand for albumin. David Bunk suggested that NIST prepare the urine creatinine reference material now and wait to prepare the urine albumin reference material after the measurand is better defined. Greg Miller suggested that everyone would support this approach.

Andrew Narva thanked everyone for a great deal of effort to advance lab measurements in CKD and especially Greg Miller for leading the group.

Page last updated: March 1, 2012