Albumin determined by bromocresol green leads to erroneous results in routine evaluation of patients with chronic kidney disease.

OBJECTIVES: Measurement of plasma albumin is pivotal for clinical decision-making in patients with chronic kidney disease (CKD). Routinely used methods as bromocresol green (BCG) and bromocresol purple (BCP) can suffer from aselectivity, but the impact of aselectivity on the accuracy of plasma albumin results of CKD-patients is still unknown. Therefore, we evaluated the performance of BCG-, BCP- and JCTLM-endorsed immunological methods in patients with various stages of CKD. METHODS: We evaluated the performance of commonly used albumin methods in patients with CKD stages G1 through G5, the latter divided in two groups based on whether they received hemodialysis treatment. In total, 163 patient plasma samples were measured at 14 laboratories, on six different BCG and BCP-platforms, and four different immunological platforms. The results were compared with an ERM-DA-470k-corrected nephelometric assay. The implications on outcome is evaluated by the proportion of patient results <38 g/L for the diagnosis of protein energy wasting. RESULTS: Albumin results determined with BCP- and immunological methods showed the best agreement with the target value (92.7 and 86.2 %, respectively vs. 66.7 % for BCG, namely due to overestimation). The relative agreement of each method with the target value was platform-dependent, with larger variability in agreement between platforms noted for BCG and immunological methods (3.2-4.6 and 2.6-5.3 %) as opposed to BCP (0.7-1.5 %). The stage of CKD had similar effects on the variability in agreement for the three method-groups (0.6-1.8 % vs. 0.7-1.5 % vs. 0.4-1.6 %). The differences between methods cause discrepancies in clinical decision-making, as structurally fewer patients were diagnosed with protein energy wasting upon using BCG-based albumin results. CONCLUSIONS: Our study shows that BCP is fit for the intended use to measure plasma albumin levels in CKD patients from all stages, including patients on hemodialysis. In contrast, most BCG-based platforms falsely overestimate the plasma albumin concentration.

Commutability of proficiency testing material containing amitriptyline and nortriptyline: A study within the framework of the Dutch Calibration 2.000 project.

BACKGROUND: External quality assessment schemes (EQAS) can provide important information regarding accuracy and comparability of different measurement methods if the sample matrices are composed of commutable material. The aim of this study was to assess the commutability of different matrices for the material used in an EQAS for amitriptyline and nortriptyline. METHODS: Proficiency testing material (PTM) and patient samples containing amitriptyline and nortriptyline were prepared, collected, pooled, and distributed to participating laboratories for analysis. Low, medium and high concentrations of both drugs in liquid pooled human, lyophilized human and lyophilized bovine serum were tested in this study. The measurement deviation of the PTM results to the patient serum regression line were normalized by dividing trough the average within-laboratory SD (SDwl) derived from the results reported in the official EQAS, resulting in a relative residual. The commutability decision limit was set at 3 SDwl. RESULTS: With 10 laboratories participating in this study, 45 laboratory couples were formed. All matrix types delivered several relative residuals outside the commutability decision limit. The number and the magnitude of relative residuals for both drugs were lower for liquid human sera as compared to lyophilized human and bovine sera. CONCLUSIONS: The PTM used for amitriptyline and nortriptyline is preferably prepared with human serum, although not all relative residuals are within the commutability decision limit.

Commutability of proficiency testing material containing tobramycin: a study within the framework of the Dutch Calibration 2.000 project.

BACKGROUND: Results from external quality assessment schemes (EQASs) can provide information about accuracy and comparability of different measurement methods, provided that the material used in these schemes behave identical to patient samples among the different methods, a characteristic also known as commutability. The aim of this study was to assess the commutability of different matrices for the material used in an EQAS for tobramycin. METHODS: Proficiency testing material (PTM) and patient samples containing tobramycin were prepared, collected, pooled, and distributed to participating laboratories for analysis. Low, medium, and high tobramycin concentrations in liquid human, liquid bovine and lyophilized bovine serum were tested in this study. The patient serum results of every laboratory were plotted against each of the other laboratories, and the distances of the PTM results to the patient serum regression line were calculated. For comparison, these distances were divided by the average within-laboratory standard deviation (SDwl) of the results reported in the official EQAS for tobramycin, resulting in a relative residual. The commutability decision limit was set at 3 SDwl. RESULTS: With 10 laboratories participating in this study, 45 laboratory couples were formed. For human serum, only one relative residual for high concentrations of tobramycin was found outside the commutability decision limit. For liquid and lyophilized bovine sera, the number of relative residuals outside the decision limit was between 15 and 18 for low, medium, and high tobramycin concentrations. CONCLUSIONS: The PTM used for tobramycin is preferably prepared with human serum.

A Multilaboratory Commutability Evaluation of Proficiency Testing Material for Carbamazepine and Valproic Acid: A Study Within the Framework of the Dutch Calibration 2000 Project.

BACKGROUND: Medical laboratories are required to participate in interlaboratory comparisons of the analyses they perform. The materials used in these comparisons need to be of sufficient quality so that the comparison provides a picture of the performances. One of the main characteristics of the testing material is commutability, which is the ability of a material to yield the same numerical relationships between results of measurements as those relationships obtained when the same procedures are applied to patient samples. The aim of this study was to assess the commutability of 3 different matrices for the preparation of proficiency testing material (PTM) for the analysis of carbamazepine and valproic acid. METHODS: Patient samples and PTM containing various concentrations of carbamazepine and valproic acid were collected, prepared, and shipped to different laboratories for analysis. Reported results for patient samples from each laboratory were plotted against results for patient samples of each of the other laboratories, and the corresponding regression line was calculated. The distance of results from PTM to the regression line is a measure for commutability. The distance is expressed as a multiple of the SDwl (average within-laboratory SD as calculated from external quality assessment scheme results) and referred to as relative residual. A commutability decision limit of 2 SDwl was set. RESULTS: For carbamazepine and valproic acid, a total of 78 and 105 laboratory couples respectively could be formed. The number of relative residuals for liquid human serum outside the commutability decision limit was 1, 4, and 0 for low, medium, and high concentrations of carbamazepine, respectively and 3, 1, and 0 for low, medium, and high concentrations of valproic acid, respectively. In both liquid and lyophilized bovine sera, the number of relative residuals outside the commutability decision limit was between 2 and 15 and between 6 and 21 for carbamazepine and valproic acid, respectively. CONCLUSIONS: Although not all results for PTM with carbamazepine and valproic acid are within the commutability decision limits, a preference for human serum can be seen.

Harmonisation of seven common enzyme results through EQA.

BACKGROUND: Equivalent results between different laboratories enable optimal patient care and can be achieved with harmonisation. We report on EQA-initiated national harmonisation of seven enzymes using commutable samples. METHODS: EQA samples were prepared from human serum spiked with human recombinant enzymes. Target values were assigned with the IFCC Reference Measurement Procedures. The same samples were included at four occasions in the EQA programmes of 2012 and 2013. Laboratories were encouraged to report IFCC traceable results. A parallel study was done to confirm commutability of the samples. RESULTS: Of the 223 participating laboratories, 95% reported IFCC traceable results, ranging from 98% (ASAT) to 87% (amylase). Users of Roche and Siemens (97%) more frequently reported in IFCC traceable results than users of Abbott (91%), Beckman (90%), and Olympus (87%). The success of harmonisation, expressed as the recovery of assigned values and the inter-laboratory CV was: ALAT (recovery 100%; inter-lab CV 4%), ASAT (102%; 4%), LD (98%; 3%), CK (101%; 5%), GGT (98%; 4%), AP (96%; 6%), amylase (99%; 4%). There were no significant differences between the manufacturers. Commutability was demonstrated in the parallel study. Equal results in the same sample in the 2012 and 2013 EQA programmes demonstrated stability of the samples. CONCLUSIONS: The EQA-initiated national harmonisation of seven enzymes, using stable, commutable human serum samples, spiked with human recombinant enzymes, and targeted with the IFCC Reference Measurement Procedures, was successful in terms of implementation of IFCC traceable results (95%), recovery of the target (99%), and inter-laboratory CV (4%).

Systematic monitoring of standardization and harmonization status with commutable EQA-samples--five year experience from the Netherlands.

BACKGROUND: Equivalence of results among laboratories is a major mission for medical laboratories. Monitoring of test equivalence is structurally integrated in the Dutch External Quality Assessment (EQA) scheme since 2005. Commutable poolsera, single donation "spy" sera and biological variance tolerance limits have been introduced in the EQA scheme for evaluation of the degree of test equivalence and its determinants. METHODS: In the annual cycle scheme 24 samples, covering the (patho)physiological measuring range for 17 analytes, are assayed by 220 participating laboratories at biweekly intervals. Test equivalence was evaluated by calculating overall median interlaboratory coefficients of variation (CVs) and its bias and imprecision components. Data from 2005 and 2010 schemes are evaluated to investigate trends in performance and success of standardization efforts. RESULTS: Overall median interlaboratory CVs in 2010 were mostly better than in 2005. Median interlaboratory CVs became <5% for electrolytes and substrates, and <10% for enzymes. Improvement in median interlaboratory CVs over these five years is mainly explained by improved method standardization, especially for enzymes and creatinine. CONCLUSION: The Dutch EQA-program proves to be a powerful instrument to evaluate test equivalence. It allows monitoring standardization efforts in a highly effective way and gives insight into remaining standardization potential.

Harmonization and external quality assessment of antithrombin activity assays.

UNLABELLED: In the framework of a Dutch project named "Calibration 2000" harmonization of antithrombin activity assays was studied. The commutability of potential calibrators for antithrombin was assessed by means of a twin-study design, which is a multicentre, split-patient sample, between-field-methods protocol. The twin-study consisted of simultaneous analysis of fresh-frozen patient plasmas and three potential calibrators for antithrombin by 30 Dutch laboratories forming 15 couples. The state-of-the-art intralaboratory standard deviation (SD(SA)) was used to assess the commutability of the potential calibrators. The regression line residuals for the potential calibrators were normalized by expressing them as multiples of SD(SA). All residuals of the potential calibrators were within the 3×SD(SA) limit. One potential calibrator was used in an attempt to harmonize antithrombin assay results in a Dutch field study. The interlaboratory coefficient of variation (CV) of the antithrombin results for three test samples could be reduced from 6.9 - 13.2% (before harmonization) to 5.6 - 9.8% using the common calibrator. CONCLUSION: The potential calibrators were commutable. Limited harmonization was achieved by using a common calibrator for all participants.

The effect of acidification and oxalate concentration on urine calcium measurements in EQAS materials and patient samples.

BACKGROUND: An increase in urine calcium compared to the consensus value was observed in some urine samples of the Dutch External Quality Assessment Scheme (EQAS). It appeared that the increase was due to the addition of oxalate by the EQAS organizers and preanalytical acidification of the samples by some of the participants. Because of this observation, the effect of urine acidification on urine calcium level in EQAS and patient samples with added oxalate was investigated. METHODS: Twenty-four EQAS urine samples and 20 patient urine samples were subject to recovery measurements of urine calcium before and after addition of sodium oxalate and acidification. RESULTS: Differences in urine calcium between acidified and non-acidified samples up to 30.9% have been observed in EQAS samples with added oxalate. Patient samples show differences up to 80%. Differences between acidified and non-acidified samples are minimal for low calcium oxalate levels but increase with higher levels. Samples without added oxalate show equal urine calcium results between acidified and non-acidified samples. CONCLUSIONS: Urine calcium results are decreased in non-acidified samples with an excess of oxalate. In case of hyperoxaluria, acidification of patient urine collections and EQAS samples is recommended for correct urine calcium values.

Trueness verification and traceability assessment of results from commercial systems for measurement of six enzyme activities in serum: an international study in the EC4 framework of the Calibration 2000 project.

BACKGROUND: The in vitro diagnostics directive of the European Union requires traceability to higher order reference measurement procedures and materials for analytes in assuring the result trueness and comparability of laboratory measurements. Manufacturers must ensure that the systems they market are calibrated against available reference systems. Validation of metrologically traceable calibrations is, however, required. METHODS: A commutable serum-based material was analyzed in three reference laboratories and target values for six enzymes (ALT, AST, CK, GGT, LD, amylase) were assigned using IFCC reference measurement procedures. 70 laboratories in Germany, Italy, and The Netherlands measured the same enzymes in the material using procedures from six commercial companies. A system for maximum allowable error was developed from the biological variation model and the results of the various procedures were tested on their compliance to trueness and between-laboratory and within-laboratory variations relative to the maximum allowable. RESULTS: For ALT results were relatively good. >95% of laboratories using systems from Dade, Olympus, Ortho and Roche are expected to comply traceability within the biologically derived limits, and 94% respectively 89% from Abbott and Beckman. For AST and GGT only Dade respectively Olympus fully complied. For CK all companies showed significant bias. Nevertheless >95% of laboratories applying Abbott, Beckman and Roche systems will comply. Finally, LD and amylase measurements require significant improvement. Some manufacturers continue to sell on the European market assays giving results which are not traceable to the internationally accepted reference systems. CONCLUSIONS: The traceability of enzyme measurements obtained with routine procedures to internationally accepted IFCC reference systems is not yet satisfactorily accomplished in clinical practice.

Harmonisation of factor VIII:C assay results: study within the framework of the Dutch project 'Calibration 2000'.

In a Dutch project for harmonisation of factor VIII coagulant activity (FVIII:C) assays, the commutability of potential calibrators for FVIII:C was assessed by means of a 'twin-study design', which is in essence a multi-centre, split-patient sample, between-field-methods protocol. Commutability was defined as the degree to which a material yielded the same numerical relationships between results of measurements by a given set of measurement procedures as those between the expectations of the relationships for the same procedures applied to those types of material for which the procedures were intended. The study consisted of the simultaneous analysis of fresh frozen patient plasmas and three potential calibrators for FVIII:C by 16 Dutch laboratories forming eight couples. The state-of-the-art intra-laboratory standard deviation was used to assess the commutability of the potential calibrators. One potential calibrator was used to harmonise FVIII:C assay results in a Dutch field study. The inter-laboratory coefficient of variation of two test samples could be reduced significantly, but no significant effect was observed with three other test samples. We recommend that at least three different sample dilutions be used in each FVIII:C assay, in agreement with previous recommendations.

External Quality Assessment in The Netherlands: time to introduce commutable survey specimens. Lessons from the Dutch "Calibration 2000" project.

The performance of suitable secondary reference material for the use of trueness control of six routinely measured clinical enzymes in the Dutch External Quality Assessment (EQA) scheme is described. The reference material of choice was selected using the split-patient-sample between-field method (twin study) design as described in an earlier study of the Calibration 2000 project in The Netherlands. This material, which was proven to be commutable for all wet chemistry systems, was implemented as the national enzyme calibrator. It consisted of a cryo-protected lyophilised serum with additions of recombinant human enzymes. Various batches of the frozen version of this material without cryo-protection additive, called native EQA samples, were used in the general EQA scheme for performance evaluation. The results of Calibration 2000 calibrated and non-Calibration 2000 calibrated laboratories were compared for both the regular (spiked with non-human enzymes) and native EQA samples in terms of precision and bias with established reference method values for the native samples. The regular samples showed mean between-laboratory CV ranges for all six enzymes involved (low-high) of 5.5-10.3% for the non-calibrated users vs. 4.6-10.8% for the calibrated users. For the native samples these respective ranges were 5.2-9.9% vs. 2.2-4.9%. Without exception, the group of Calibration 2000 calibrated users showed the lowest bias against the reference method values. Regular EQA samples (spiked with non-human enzymes) showed poorer performance than native samples and are not suitable for accuracy assessment purposes, the main aim of EQA schemes. Native samples that are commutable should be used for trueness control in current EQA schemes.

Commutability assessment of potential reference materials using a multicenter split-patient-sample between-field-methods (twin-study) design: study within the framework of the Dutch project "Calibration 2000".

BACKGROUND: The Dutch project "Calibration 2000" aims at harmonization of laboratory results via calibration by development of commutable, matrix-based, secondary reference materials. An alternative approach to the NCCLS EP14 protocol for studying commutability of reference materials is presented, the "twin-study design", which in essence is a multicenter, split-patient-sample, between-field-methods protocol. METHODS: The study consisted of the simultaneous analysis of fresh patient sera and potential reference materials (PRMs) for HDL-cholesterol (HDL-C) by 86 laboratories forming 43 laboratory couples. Six subgroups of method combinations were formed. The patient sera were selected and interchanged by each laboratory couple. The PRMs consisted of three types: C37, prepared according to the NCCLS C37 protocol; Fro, frozen selectively pooled human serum; and Lyo, which was the same serum pool as Fro but lyophilized in the presence of sucrose. All PRMs were provided in three HDL-C concentrations. The regression line residuals for the PRMs were normalized by expressing them as multiples of the state-of-the-art within laboratory SD (SD(SA)). In addition, the extra contribution of each PRM to the total measurement uncertainty, CV(Netto), was calculated. RESULTS: Averaged over the three PRM concentrations, 1.6% of the C37 residuals were outside the 3 SD(SA) limit. For the Fro and Lyo PRMs, these values were 2.4% and 11.1%. CV(Netto) values for C37, Fro, and Lyo were 2.9%, 4.3%, and 5.3%, respectively. CONCLUSIONS: The present twin-study design, as a practical alternative to the NCCLS EP14 protocol, is a viable way of studying commutability characteristics of PRMs. The study suggests that the C37 PRMs are the best candidates for a future reference material.

Selection, preparation, and characterization of commutable frozen human serum pools as potential secondary reference materials for lipid and apolipoprotein measurements: study within the framework of the Dutch project "Calibration 2000".

BACKGROUND: The Dutch project "Calibration 2000" aims at harmonization of laboratory results via calibration by development of matrix-based secondary reference materials. We considered the selection, preparation, and characterization of 34 potential reference materials (PRMs). METHODS: Sixteen PRMs were prepared either strictly according to the NCCLS C37-A protocol or in a less stringent and more convenient way. In addition, 18 commercial, so-called human serum-based calibrators or controls were purchased and tested. Lipoprotein integrity was evaluated by examining the physicochemical characteristics of the materials. Commutability of the PRMs was assessed in 86 Dutch clinical laboratories, using a multicenter split-patient-sample between-field-method (twin-study) design. Normalized residuals of the PRMs with respect to the patient regression lines were calculated; in addition, the extra contribution of each PRM to the total measurement uncertainty (CV(Netto)) was calculated. On the basis of these results, the most native PRM was selected to investigate its potential to reduce interlaboratory variation and to improve lipid and apolipoprotein standardization. RESULTS: In general, only the NCCLS C37-A-type materials displayed normalized residuals below the decision limit for commutability and had small CV(Netto) values ranging between 0 and 3.8%. This contrasts with the findings in regularly pooled frozen sera and lyophilized cryoprotected PRMs. In two subsequent external quality assessment surveys, the NCCLS type C37-A materials contributed to reducing the intermethod lipid and (apo)lipoprotein variation to approximately 2-4%. CONCLUSIONS: NCCLS C37-A materials have a strong potential as secondary reference materials, not only for cholesterol but also for HDL-cholesterol, LDL-cholesterol, triglyceride, and apolipoprotein measurements.