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Objectives: This paper evaluates 16 year results of the Allergy EQA program shared by EQA organisers in Belgium, Finland, Portugal, and The Netherlands., Methods: The performance of Thermo Fisher and Siemens user groups (in terms of concordance between both groups, between laboratory CV, prevalence of clinically significant errors) and suitability of samples (stability and validity of dilution of patient samples) are evaluated using data of 192 samples in the EQA programs from 2007 to 2022. Measurands covered are total IgE, screens and mixes, specific IgE extracts and allergen components., Results: There is perfect (53 %), acceptable (40 %) and poor (6 %) concordance between both method groups. In case of poor concordance the best fit with clinical data is seen for Thermo Fisher (56 %) and Siemens (26 %) respectively. The between laboratory CV evolves from 7.8 to 6.6 % (Thermo Fisher) and 7.3 to 7.7 % (Siemens). The prevalence of blunders by individual laboratories is stable for Siemens (0.4 %) and drops from 0.4 to 0.2 % for Thermo Fisher users. For IgE, the between year CV of the mean of both user groups is 1 %, and a fifteen-fold dilution of a patient sample has an impact of 2 and 4 % on the recovery of Thermo Fisher and Siemens user groups., Conclusions: The analytical performance of Thermo Fisher is slightly better than that of Siemens users but the clinical impact of this difference is limited. Stability of the sample and the low impact of dilution on the recovery of measurands demonstrates the suitability for purpose of the EQA program., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

Objectives: Hemoglobin A 1c (HbA 1c ) is a valuable parameter in the monitoring of diabetic patients and increasingly in diagnosis of diabetes. Manufacturers continuously optimize instruments, currently the main focus is to achieve faster turnaround times. It is important that performance specifications remain of high enough standard, which is evaluated in this study for the new ARKRAY HA-8190V instrument., Methods: The Clinical and Laboratory Standards Institute (CLSI) protocols EP-5, EP-9 and EP-10 were applied to investigate imprecision, bias and linearity. In addition potential interferences, performance in External Quality Assessment (EQA) and performance against the HA-8180V instrument in 220 clinical samples was evaluated., Results: The HA-8190V demonstrates a CV of ≤0.8% in IFCC SI units (≤0.6% National Glycohemoglobin Standardization Program [NGSP]) at 34 and 102 mmol/mol levels (5.3 and 11.5% NGSP) and a bias of -0.1 mmol/mol (-0.01% NGSP) at a concentration of 50 mmol/mol (6.7% NGSP), but with a significant slope as compared to target values. This results in a bias of -1.0 and 0.9 mmol/mol (-2.0 and 0.9% NGSP) at the 30 and 70 mmol/mol (4.9 and 8.6% NGSP) concentration level. Simulation of participation in the IFCC certification programme results in a Silver score (bias -0.1 mmol/mol, CV 1.1%). Interference in the presence of the most important Hb variants (AS, AC, AE, AD) and elevated HbA 2 and HbF concentrations is less than 3 mmol/mol (0.3% NGSP) at a concentration of 50 mmol/mol (6.7% NGSP)., Conclusions: Analytical performance of the HA-8190V is very good, especially with respect to precision and HbA 1c quantification in the presence of the most common Hb variants., (© 2020 Walter de Gruyter GmbH, Berlin/Boston.)

Objectives: Hepcidin measurement advances insights in pathophysiology, diagnosis, and treatment of iron disorders, but requires analytically sound and standardized measurement procedures (MPs). Recent development of a two-level secondary reference material (sRM) for hepcidin assays allows worldwide standardization. However, no proficiency testing (PT) schemes to ensure external quality assurance (EQA) exist and the absence of a high calibrator in the sRM set precludes optimal standardization., Methods: We developed a pilot PT together with the Dutch EQA organization Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek (SKML) that included 16 international hepcidin MPs. The design included 12 human serum samples that allowed us to evaluate accuracy, linearity, precision and standardization potential. We manufactured, value-assigned, and validated a high-level calibrator in a similar manner to the existing low- and middle-level sRM., Results: The pilot PT confirmed logistical feasibility of an annual scheme. Most MPs demonstrated linearity (R2>0.99) and precision (duplicate CV>12.2%), although the need for EQA was shown by large variability in accuracy. The high-level calibrator proved effective, reducing the inter-assay CV from 42.0% (unstandardized) to 14.0%, compared to 17.6% with the two-leveled set. The calibrator passed international homogeneity criteria and was assigned a value of 9.07±0.24 nmol/L., Conclusions: We established a framework for future PT to enable laboratory accreditation, which is essential to ensure quality of hepcidin measurement and its use in patient care. Additionally, we showed optimized standardization is possible by extending the current sRM with a third high calibrator, although international implementation of the sRM is a prerequisite for its success.

Analysis of blood phenylalanine is central to the monitoring of patients with phenylketonuria (PKU) and age-related phenylalanine target treatment-ranges (0-12 years; 120-360 μmol/L, and >12 years; 120-600 μmol/L) are recommended in order to prevent adverse neurological outcomes. These target treatment-ranges are based upon plasma phenylalanine concentrations. However, patients are routinely monitored using dried bloodspot (DBS) specimens due to the convenience of collection. Significant differences exist between phenylalanine concentrations in plasma and DBS, with phenylalanine concentrations in DBS specimens analyzed by flow-injection analysis tandem mass spectrometry reported to be 18% to 28% lower than paired plasma concentrations analyzed using ion-exchange chromatography. DBS specimens with phenylalanine concentrations of 360 and 600 μmol/L, at the critical upper-target treatment-range thresholds would be plasma equivalents of 461 and 768 μmol/L, respectively, when a reported difference of 28% is taken into account. Furthermore, analytical test imprecision and bias in conjunction with pre-analytical factors such as volume and quality of blood applied to filter paper collection devices to produce DBS specimens affect the final test results. Reporting of inaccurate patient results when comparing DBS results to target treatment-ranges based on plasma concentrations, together with inter-laboratory imprecision could have a significant impact on patient management resulting in inappropriate dietary change and potentially adverse patient outcomes. This review is intended to provide perspective on the issues related to the measurement of phenylalanine in blood specimens and to provide direction for the future needs of PKU patients to ensure reliable monitoring of metabolic control using the target treatment-ranges., (© 2019 SSIEM.)

Background Hepcidin concentrations measured by various methods differ considerably, complicating interpretation. Here, a previously identified plasma-based candidate secondary reference material (csRM) was modified into a serum-based two-leveled sRM. We validated its functionality to increase the equivalence between methods for international standardization. Methods We applied technical procedures developed by the International Consortium for Harmonization of Clinical Laboratory Results. The sRM, consisting of lyophilized serum with cryolyoprotectant, appeared commutable among nine different measurement procedures using 16 native human serum samples in a first round robin (RR1). Harmonization potential of the sRM was simulated in RR1 and evaluated in practice in RR2 among 11 measurement procedures using three native human plasma samples. Comprehensive purity analysis of a candidate primary RM (cpRM) was performed by state of the art procedures. The sRM was value assigned with an isotope dilution mass spectrometry-based candidate reference method calibrated using the certified pRM. Results The inter-assay CV without harmonization was 42.1% and 52.8% in RR1 and RR2, respectively. In RR1, simulation of harmonization with sRM resulted in an inter-assay CV of 11.0%, whereas in RR2 calibration with the material resulted in an inter-assay CV of 19.1%. Both the sRM and pRM passed international homogeneity criteria and showed long-term stability. We assigned values to the low (0.95±0.11 nmol/L) and middle concentration (3.75±0.17 nmol/L) calibrators of the sRM. Conclusions Standardization of hepcidin is possible with our sRM, which value is assigned by a pRM. We propose the implementation of this material as an international calibrator for hepcidin.

Background: To provide its participants with an external quality assessment system (EQAS) that can be used to check trueness, the Dutch EQAS organizer, Organization for Quality Assessment of Laboratory Diagnostics (SKML), has innovated its general chemistry scheme over the last decade by introducing fresh frozen commutable samples whose values were assigned by Joint Committee for Traceability in Laboratory Medicine (JCTLM)-listed reference laboratories using reference methods where possible. Here we present some important innovations in our feedback reports that allow participants to judge whether their trueness and imprecision meet predefined analytical performance specifications., Methods: Sigma metrics are used to calculate performance indicators named 'sigma values'. Tolerance intervals are based on both Total Error allowable (TEa) according to biological variation data and state of the art (SA) in line with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Milan consensus., Results: The existing SKML feedback reports that express trueness as the agreement between the regression line through the results of the last 12 months and the values obtained from reference laboratories and calculate imprecision from the residuals of the regression line are now enriched with sigma values calculated from the degree to which the combination of trueness and imprecision are within tolerance limits. The information and its conclusion to a simple two-point scoring system are also graphically represented in addition to the existing difference plot., Conclusions: By adding sigma metrics-based performance evaluation in relation to both TEa and SA tolerance intervals to its EQAS schemes, SKML provides its participants with a powerful and actionable check on accuracy.

Background: Absolute plasma hepcidin concentrations measured by various procedures differ substantially, complicating interpretation of results and rendering reference intervals method dependent. We investigated the degree of equivalence achievable by harmonization and the identification of a commutable secondary reference material to accomplish this goal., Methods: We applied technical procedures to achieve harmonization developed by the Consortium for Harmonization of Clinical Laboratory Results. Eleven plasma hepcidin measurement procedures (5 mass spectrometry based and 6 immunochemical based) quantified native individual plasma samples (n = 32) and native plasma pools (n = 8) to assess analytical performance and current and achievable equivalence. In addition, 8 types of candidate reference materials (3 concentrations each, n = 24) were assessed for their suitability, most notably in terms of commutability, to serve as secondary reference material., Results: Absolute hepcidin values and reproducibility (intrameasurement procedure CVs 2.9%-8.7%) differed substantially between measurement procedures, but all were linear and correlated well. The current equivalence (intermeasurement procedure CV 28.6%) between the methods was mainly attributable to differences in calibration and could thus be improved by harmonization with a common calibrator. Linear regression analysis and standardized residuals showed that a candidate reference material consisting of native lyophilized plasma with cryolyoprotectant was commutable for all measurement procedures. Mathematically simulated harmonization with this calibrator resulted in a maximum achievable equivalence of 7.7%., Conclusions: The secondary reference material identified in this study has the potential to substantially improve equivalence between hepcidin measurement procedures and contributes to the establishment of a traceability chain that will ultimately allow standardization of hepcidin measurement results., (© 2016 American Association for Clinical Chemistry.)

Background: The aim of this study was to compare different analytical methods that are currently in use in the Netherlands for the measurement of whole blood vitamin B6., Methods: This method comparison study consisted of two separate parts. (1) Four laboratories participated in a pilot study in which the commercial Chromsystems and INstruchemie method, and a laboratory developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and HPLC method were compared. Sixty-nine frozen whole blood samples and six lyophilized whole blood samples were used for comparison. (2) In the nationwide part of the study, 49 laboratories participated in the analysis of three identical sets of two whole blood samples of which one set was freshly analyzed, one set was analyzed after storage at -20 °C and one set was analyzed after lyophilization., Results: In both parts of the study, the HPLC and LC-MS/MS methods showed equivalent results for all sample types tested. The Chromsystems method showed a positive bias of 45% (pilot study) and 30% (nationwide study) towards the LC-MS/MS method when fresh or frozen samples were used. The measurement of lyophilized samples showed no differences between the methods. The results of the INstruchemie method were inconclusive due to the low number of participants., Conclusions: The different analytical methods for measuring vitamin B6 produce different results when whole blood patient samples are measured. The recognition of a reference method or the development of suitable reference materials and quality control materials might serve as a first step towards improved standardization or harmonization of the whole blood vitamin B6 assay.

Background: Haemoglobin (Hb) variants are well-known factors interfering with accurate HbA1c testing. This report describes two novel Hb variants leading to inappropriate quantification of HbA1c by ion-exchange chromatography., Methods: Glycated forms of novel Hb variants were recognised in the blood of two patients with diabetes mellitus screened by HbA1c ion-exchange chromatography. Dedicated high-resolution cation-exchange chromatography and subsequent DNA sequencing revealed the exact nature of the variants. Other common techniques for quantifying HbA1c were applied on both samples and haematological parameters were determined to judge possible pathology associated with the novel Hb variants., Results: A fraction of 15% of abnormal Hb was observed in a 37-year-old female. DNA sequencing revealed a heterozygous mutation in the α1-globin gene, resulting in a leucine-to-phenylalanine amino-acid substitution (HBA1: c.301C>T, p.Leu101Phe). We named this variant Hb Weesp. The other novel variant, Hb Haelen, presented as a 40% fraction in a 63-year-old male and resulted from a heterozygous amino acid substitution in the β-globin gene (HBB: c.335T>C, p.Val112Gly). The presence of both Hb variants resulted in aberrant separation of the Hb components, leading to an inadequate quantification of HbA1c., Conclusions: Close examination of HbA1c chromatograms revealed two novel, clinically silent Hb variants that interfere with HbA1c quantification. Healthcare providers need to be aware of the potential of such Hb variants when interpreting HbA1c results.

Background: Carbamylated hemoglobin (carbHb) is reported to interfere with measurement and interpretation of HbA(1c) in diabetic patients with chronic renal failure (CRF). There is also concern that HbA1c may give low results in these patients due to shortened erythrocyte survival., Methods: We evaluated the effect of carbHb on HbA(1c) measurements and compared HbA(1c) with glycated albumin (GA) in patients with and without renal disease to test if CRF causes clinically significant bias in HbA(1c) results by using 11 assay methods. Subjects included those with and without renal failure and diabetes. Each subject's estimated glomerular filtration rate (eGFR) was used to determine the presence and degree of the renal disease. A multiple regression model was used to determine if the relationship between HbA(1c) results obtained from each test method and the comparative method was significantly (p<0.05) affected by eGFR. These methods were further evaluated for clinical significance by using the difference between the eGRF quartiles of >7% at 6 or 9% HbA(1c). The relationship between HbA(1c) and glycated albumin (GA) in patients with and without renal failure was also compared., Results: Some methods showed small but statistically significant effects of eGFR; none of these differences were clinically significant. If GA is assumed to better reflect glycemic control, then HbA(1c) was approximately 1.5% HbA(1c) lower in patients with renal failure., Conclusions: Although most methods can measure HbA(1c) accurately in patients with renal failure, healthcare providers must interpret these test results cautiously in these patients due to the propensity for shortened erythrocyte survival in renal failure., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Accurate and precise bilirubin and albumin measurements are essential for proper management of jaundiced neonates. Data hereon are lacking for Dutch laboratories. We aimed to determine variability of measurements of bilirubin and albumin concentrations typical for (preterm) neonates. Aqueous, human serum albumin-based samples with different concentrations of bilirubin (100, 200, 300, 400, and 500 μmol/L) and albumin (0, 10, 15, 20, 25, and 30 g/L) were sent to laboratories of all Dutch neonatal intensive care units (n = 10). Bilirubin and albumin recoveries of the specimens were measured using locally available routine analytical methods. The mean, standard deviation, and coefficients of variations (CV) were calculated per sample. Bilirubin concentrations were underestimated in the absence of albumin (maximal CV 26.0%). When the albumin concentration was 10 or 20 g/L, the bilirubin concentrations of the samples were overestimated (maximal CV 14.1% and 9.2%, respectively). Variability increased with higher weighed-in bilirubin concentrations. Measured albumin levels were ~10% lower than albumin levels of manufactured samples. Bilirubin concentration did not influence albumin measurements. The maximal CV was 6.8%. In conclusion, interlaboratory variability of bilirubin and albumin measurements is high. Recalibration and introduction of a specific quality assessment scheme for neonatal samples is recommended to ensure exchangeability of bilirubin and albumin measurements among laboratories and to control the observed large variability.

Background: The literature regarding the nonspecificity of applications of the Jaffe (alkaline picrate) reaction for creatinine is generally outdated. We conducted a specificity study to update the nonspecificity information for current Jaffe and enzymatic creatinine assays., Methods: Two serum pools with creatinine concentrations within the pediatric reference interval were spiked with albumin, IgG, unconjugated bilirubin, adult hemoglobin (Hb A), and fetal hemoglobin (Hb F) to produce 1 unspiked and 5 spiked samples per pool. The 35 laboratories participating in the survey used a total of 14 method-analyzer combinations. Measurements were performed in triplicate in a single run in accordance with manufacturer instructions. Absolute differences in creatinine concentration between spiked and unspiked samples were calculated per laboratory. Mixed ANOVA was used to quantify the interferent-related CV component for the Jaffe and enzymatic methods., Results: The interference by bilirubin and Hb A on serum creatinine measurements was <10% for most of the Jaffe and enzymatic methods. Obvious interference was observed among the Jaffe methods in samples spiked with Hb F, albumin, and IgG, but not among the enzymatic methods. The within-laboratory interferent-related CVs for the Jaffe method-analyzer combinations ranged from 8.0%-27% at a creatinine concentration of 40.4 micromol/L (0.46 mg/dL) and from 5.4%-15% at 73.4 micromol/L (0.83 mg/dL). Enzymatic methods had within-laboratory interferent-related CVs of <4% at both concentrations., Conclusions: Albumin, IgG, and Hb F interfered with Jaffe creatinine assays, leading to inaccuracies in estimated glomerular filtration rates that are clinically important, especially in children and neonates. Because protein error and Hb F interference do not occur with any of the enzymatic methods tested, we conclude that enzymatic creatinine methods are preferred for evaluation of kidney function in pediatric cases.

Background: Trace element external quality assessment schemes monitor laboratory performance and provide a stimulus for improvement in accuracy. However, monitoring of participant performance varies according to the scheme and can lead to conflicting conclusions., Methods: Quality specifications based on biological intra- and interindividual variability were calculated and compared to those currently used by various trace element external quality assessment schemes for plasma or serum copper, zinc, and selenium concentrations. For this purpose, we evaluated results reported by participating laboratories in different schemes, at key concentrations, using z scores., Results: Minimal quality specifications developed from the biological intra- and interindividual variability were, for Cu, +/-0.84 micromol/L or 12% of the assigned target concentration, whichever is greater; for Zn, +/-1.20 micromol/L or 15% of the assigned target concentration, whichever is greater; and for Se, +/-0.072 micromol/L or 12% of the assigned target concentration, whichever is greater. Reported performance of the participating laboratories depended on analyte, concentration, and the selected quality specification. In addition, the most commonly used methods for the determination of Cu, Zn, and Se may give different results., Conclusions: The proposed minimal quality specifications based on biological variation are generally slightly less stringent than those currently in use, although they do not drastically change the performance evaluation in the different schemes. These specifications are a first step in the harmonization of practices among the schemes and remain to be evaluated.

Background: Glycohemoglobin (GHB), reported as hemoglobin (Hb) A(1c), is a marker of long-term glycemic control in patients with diabetes and is directly related to risk for diabetic complications. HbE and HbD are the second and fourth most common Hb variants worldwide. We investigated the accuracy of HbA(1c) measurement in the presence of HbE and/or HbD traits., Methods: We evaluated 23 HbA(1c) methods; 9 were immunoassay methods, 10 were ion-exchange HPLC methods, and 4 were capillary electrophoresis, affinity chromatography, or enzymatic methods. An overall test of coincidence of 2 least-squares linear regression lines was performed to determine whether the presence of HbE or HbD traits caused a statistically significant difference from HbAA results relative to the boronate affinity HPLC comparative method. Deming regression analysis was performed to determine whether the presence of these traits produced a clinically significant effect on HbA(1c) results with the use of +/-10% relative bias at 6% and 9% HbA(1c) as evaluation limits., Results: Statistically significant differences were found in more than half of the methods tested. Only 22% and 13% showed clinically significant interference for HbE and HbD traits, respectively., Conclusions: Some current HbA(1c) methods show clinically significant interferences with samples containing HbE or HbD traits. To avoid reporting of inaccurate results, ion-exchange chromatograms must be carefully examined to identify possible interference from these Hb variants. For some methods, manufacturers' instructions do not provide adequate information for making correct decisions about reporting results.

Background: Hemoglobin variants (Hb(VAR)) are not uncommon in the Korean population, with Hb G-Coushatta and Hb Queens being the 2 most common Hb(VAR). Hb G-Coushatta is also the most common Hb(VAR) in Chinese people from the Silk Road region, as well as in some North American Indian tribes. However, data are scarce on the effect of these Hb(VAR) on the different methods used for analyzing HbA(1c)., Methods: Specimens from 24 individuals with 7 Hb(VAR) (Hb G-Coushatta, Hb Queens, Hb G-Hsi-Tsou, Hb Ube-4, Hb G-Waimanalo, Hb Inglewood, and Hb Bologna-St.Orsola) were collected and tested using the International Federation of Clinical Chemistry primary reference method as well as 14 routine HbA(1c) assay methods., Results: Hb G-Coushatta showed a clinically significant effect on the measured HbA(1c), particularly when analysis was performed with ion-exchange HPLC methods with short elution times. This interference could be resolved by measuring the HbA(1c) using other methods such as HPLC with a long elution time, immunoassay, boronate affinity chromatography, and enzymatic assay. Hb Queens showed a clinically significant difference, defined as a >10% deviation from regression lines, in results from the 2 HPLC methods but not in the other methods. The remaining 5 rare Hb(VAR) showed different HbA(1c) results in the different assays., Conclusion: Hb G-Coushatta, Hb Queens, and other rare Hb(VAR) can interfere with glycohemoglobin assays, including ion-exchange HPLC methods with short elution times, but the interference can be resolved using other unaffected methods. It is important to identify these Hb(VAR) through a careful inspection of the chromatograms and apply other noninterfering methods for accurate measurements of the HbA(1c).

The measurement of glycated hemoglobin is central in the monitoring of glycemic control in patients with diabetes. There are at least 30 different laboratory assays commercially available to measure the proportion of HbA1c in blood. In 1995 the IFCC established a Working Group (IFCC WG-HbA1c) to achieve international standardization of HbA1c measurement. The main achievements can be summarized as follows: a) a reference measurement procedure has been established with purified primary calibrators; b) a network of reference laboratories has been developed worldwide; and c) work has begun on implementation of traceability to the IFCC reference system. The IFCC WG-HbA1c recognizes the recommendation of the IFCC-IUPAC Committee on Nomenclature, Properties and Units that the analyte measured by the IFCC reference measurement procedure has been defined as betaN1-deoxyfructosyl-hemoglobin and that the recommended measurement units are mmol/mol. The IFCC WG-HbA1c recommends maintaining the use of the name HbA1c in clinical practice.

Inborn errors of metabolism are rare and laboratories performing diagnostic tests in this field must participate in external quality assurance (EQA) schemes to demonstrate their competence and also to maintain sufficient experience with patient material. EQA schemes for metabolite analyses are available (ERNDIM), but corresponding EQA schemes for enzyme analyses are nonexistent. In this paper we describe a pilot study on lysosomal enzyme testing by four centres in The Netherlands. Quantitative aspects of EQA were studied by interlaboratory comparison of activities of six lysosomal enzymes in a series of buffy coat samples. Interlaboratory variance was enormous. To reduce variance caused by methodological differences, participants reported enzyme activities relative to mean normal values. Beta-D-Galactosidase activities compared well between the participating laboratories (average interlaboratory CV 13%), but for other enzymes large differences were observed, e.g. sphingomyelinase (average CV 38%). Diagnostic proficiency was tested with cultured fibroblasts. In 45 out of a total of 48 tests (12 cell lines, 4 participants) the correct diagnosis was accomplished on the basis of merely biochemical investigations, i.e. without clinical data of the patients. In a survey using blood of a late-onset Pompe disease patient, less conclusive results were obtained. A stable enzyme source was developed for easy distribution. Most lysosomal enzymes were stable upon lyophilization of leukocyte homogenates and during subsequent storage of the freeze-dried material at room temperature, in particular when cryolyoprotectant was added. Shipment of such lyophilized samples is simple and cheap and ideal for an EQA scheme. Our study shows that an EQA programme for enzymatic testing of lysosomal storage diseases is necessary to accomplish reliable diagnostic procedures for lysosomal storage diseases. We recommend that EQA for lysosomal enzymes be implemented through ERNDIM.

Background: We evaluated a quality control scheme for the measurement of urinary uroporphyrin, coproporphyrin, total urinary porphyrins and precursors of urinary porphyrins, delta-aminolevulinic acid and porphobilinogen that was performed in The Netherlands during a period of 5 years., Methods: Six quality control samples were distributed each year to the participating laboratories. Mean concentrations and the corresponding coefficients of variation were calculated., Results: Coefficients of variation varied widely and were very high in the concentration ranges that can be found in patients with low-grade porphyria., Conclusion: Commutable calibrators are needed to improve the laboratory diagnosis of porphyria.

Clinical chemistry laboratory results from different laboratories often show large between-laboratory variation due to factors such as differences in method principles, method applications, calibration procedures or the application of different instrument factor settings within the same calibration procedure. We have examined the possible use of common calibrators to reduce this variation. Three different calibrators were compared: A, freeze-dried preparations of pooled patients' serum samples, spiked to give three concentration levels; B, freeze-dried preparations of pooled patients' serum samples selected on the basis of elevated enzyme activities at three levels; C, a single calibrator consisting of frozen pooled serum samples. These calibrators were sent to 11 participating laboratories together with 14 fresh patients' serum samples. We report the variation of the results of 21 general clinical chemistry analytes obtained in the patients' serum samples before and after recalculation on the basis of the results of the calibrators. For most analytes the use of a multiple point linear regression calibration function is able to reduce the between-laboratory variation considerably from more than 30% (enzymes) to values well within the bias limits set by European quality specifications, when the necessary conditions are met. These conditions include the commutability of the calibrator(s) with fresh patients' material. For the enzymes, calibrator material originating from selectively pooled patients' samples appeared to be necessary, whereas for the substrates selectively pooled serum calibrators spiked with exogenous supplements may be used. For harmonization to be effective in practice, calibrators need to be stable over time and to carry assigned values set by certified reference laboratories, and the quality performance of participating laboratories should be appropriately monitored.

Background: Standardization of HDL-cholesterol is needed for risk assessment. We assessed for the first time the accuracy of HDL-cholesterol testing in The Netherlands and evaluated 11 candidate reference materials (CRMs)., Methods: The total error (TE) of HDL-cholesterol measurements was assessed in native human sera by 25 Dutch clinical chemistry laboratories. Concomitantly, the suitability of lyophilized, saccharose-containing CRMs (n = 11) for HDL-cholesterol was evaluated., Results: In the precipitation method group, which included 25 laboratories and four methods, the mean (minimum-maximum) TE was 11.5% (2.7-25.2%), signifying that 18 of 25 laboratories satisfied the TE goal of

Hb A1c is the analyte of choice for monitoring metabolic control in patients with diabetes mellitus. Here we present a new analytical technique for measuring Hb A1c, capillary electrophoresis. The Hb A1c determination is not influenced by the labile Hb A1c fraction or by carbamylated or acetylated hemoglobin derivatives. Also, hemoglobin variants (Hb F, Hb S, and Hb C) do not interfere. This new application of capillary electrophoresis seems to be a valuable analytical tool for measuring Hb A1c in the clinical laboratory.

The use of lyophilized blood as a reference material for glycated haemoglobin was investigated with respect to IFCC criteria for calibrators and control materials. Ninety-two laboratories, using 11 methods, detected no changes in glycated haemoglobin content when the lyophilizate was stored for one year at 4 degrees C. Affinity chromatography, HPLC, electrophoresis and immunoassay detected no changes following 18 months storage at -84 and -20 degrees C. Samples for HPLC are stable at 4 degrees C for one year, and 5 years at -20 degrees C. For the other three methods, samples are stable for 5 years at 4 degrees C. At 4 degrees C, reconstituted samples are stable for 2 days (HPLC) and 7 days (other three). Lyophilization does not cause matrix effects and inhomogeneity, since mean glycated haemoglobin and reproducibility for lyophilized samples and whole blood were similar. The coefficient of variation for vial filling precision was 0.59%. We conclude that lyophilized blood samples can be used as calibrators and control materials. Their use as calibrators, following assignment of the HbA(1c) value by HPLC, may contribute, in the interim, to the standardized interpretation of long term diabetic control.

External quality assessment schemes (EQAS) for trace elements in body fluids are organized in The Netherlands by the SKZL (Foundation for Quality Assessment in Clinical Laboratories). Medical laboratories participate on a voluntary base. The EQAS is designed to evaluate laboratory performance in terms of accuracy, precision and linearity. Laboratory-made frozen samples of whole blood, serum and urine, enriched with the elements of interest, i.e. Tl, Cd, Co, Hg, Se, Pb, Mg, Li, Al, Cu, Zn and As, are used. The present state of art shows an acceptable mean recovery, intra-laboratory variation and linearity. Interlaboratory variation, comparability of reference ranges and criteria of interpretation used in different laboratories are less satisfying and require improvement.

Three groups of 10 age- and sex-matched nondiabetic volunteers took 0, 750, or 1500 mg of vitamin C each day for 12 weeks. Glycohemoglobin (GHb) was measured by HPLC, electrophoresis, affinity chromatography, and immunoassay at baseline (-4 weeks and -1 day), during supplementation (6 weeks and 12 weeks), and after supplementation ended (6 and 12 weeks). Plasma vitamin C increased twofold during supplementation but, in contrast with the results of Davie et al. (Diabetes 1992; 41:167-73), there were no between-group differences in GHb, glucose, and fructosamine concentrations. Fructosamine may have increased with storage time. The net effects of vitamin C on absolute GHb at 12 weeks vs -1 day (and at 12 weeks vs 12 weeks after) in % GHb amounted to: HPLC -0.035 (-0.050); electrophoresis +0.005 (+0.035); affinity chromatography -0.070 (+0.015); and immunoassay -0.110 (+0.025). We conclude that supplementation of nondiabetics with 750 or 1500 mg of vitamin C daily for 12 weeks does not cause interference in GHb determinations by HPLC, electrophoresis, affinity chromatography, or immunoassay, and does not reduce in vivo Hb glycation.

We investigated the effect of calibration with lyophilized calibrators on whole-blood glycohemoglobin (glyHb) results. One hundred three laboratories, using 20 different methods, determined glyHb in two lyophilized calibrators and two whole-blood samples. For whole-blood samples with low (5%) and high (9%) glyHb percentages, respectively, calibration decreased overall interlaboratory variation (CV) from 16% to 9% and from 11% to 6% and decreased intermethod variation from 14% to 6% and from 12% to 5%. Forty-seven laboratories, using 14 different methods, determined mean glyHb percentages in self-selected groups of 10 nondiabetic volunteers each. With calibration their overall mean (2SD) was 5.0% (0.5%), very close to the 5.0% (0.3%) derived from the reference method used in the Diabetes Control and Complications Trial. In both experiments the Abbott IMx and Vision showed deviating results. We conclude that, irrespective of the analytical method used, calibration enables standardization of glyHb results, reference values, and interpretation criteria.

Glycohemoglobin (gly-Hb) reference ranges of non-diabetic adults with HbAA (n = 17), HbAS (n = 37), HbAC (n = 22), HbSC (n = 8), HbSS (n = 6) and HbCC (n = 3) were determined by 13 methods, based on affinity chromatography, HPLC, electrophoresis and immunoassay. Gly-Hb of subjects with HbAS and HbAC can be measured without major difficulties by most methods. Some give rise to absolute gly-Hb differences > or = 1% compared with subjects with HbAA. Measurement of HbA1c/total Hb cannot be recommended. Some HPLC and immunoassay methods cannot measure gly-Hb in subjects with HbSC, HbSS and HbCC, whereas others may suffer from interference. Most methods showed low gly-Hb, reflecting increased erythrocyte turnover. Use of special reference ranges requires previous knowledge of the condition (affinity chromatography and immunoassay) or separation of gly-Hb and its precursor Hb (HPLC and electrophoresis). Interpretation is, however, not recommended because of the numerous factors that determine erythrocyte turnover.

One hundred eleven laboratories, using 21 different methods based on five different principles, determined glycohemoglobin (GHb) percentages in two identical series of six lyophilized hemolysates and three similarly processed calibrators, distributed 3 months apart. To assign GHb percentages to calibrators, we used HbA1c results from nine participants who used the Bio-Rad Diamat high-performance liquid chromatographic method. Three-point calibration with assigned values improved mean intralaboratory variation (CV) from 6.6% to 3.5%. For samples with low (5.5%) and high (14.1%) GHb percentages, respectively, calibration decreased interlaboratory variation per method (from 10% to 4% and from 6% to 3%), inter-method variation (from 18% to 4% and from 16% to 3%), and overall interlaboratory variation (from 25% to 7% and from 15% to 4%). Without calibration, 71% of the laboratories did not meet the clinically desirable intralaboratory CV of 3.5%; calibration reduced this proportion to 39%. We conclude that, irrespective of the analytical method used, calibration greatly reduces all sources of GHb variation.

Influences of hemoglobin (Hb) variants (HbSS, HbCC, beta-thalassemia, HbAE, HbAS, HbAC, hereditary persistent HbF) and Hb derivatives (carbamylated- and acetylated-Hbs, Schiff base, and those formed in stored blood) on results of glyco-Hb assays by 102 laboratories using 16 different methods were investigated. Affinity chromatography shows deviating results only with homozygous Hb S and C. Correct interpretation of results from patients with decreased erythrocyte half-lives requires previous knowledge on this condition. Measurements of HbA1c by HPLC and electrophoresis are obviously unsuitable for homozygous hemoglobinopathies; for heterozygous hemoglobinopathies and Hb synthesis variants, HbA1c should be expressed as percentage of HbA0 + HbA1c; abnormal Hbs are usually recognized; both carbamylated- and acetylated-Hbs interfere and Schiff base must be eliminated. Except for stored blood, all Hb variants and derivatives gave erroneous results with disposable ion-exchange columns. Dako's immunoassay is not affected by Hb derivatives; glycated Hb variants are not recognized as glyco-Hb and percentages are consequently too low. Glyco-Hb by the immunoassay of Bayer (performed by one laboratory) is not affected by Hb variants and derivatives.

Stable lyophilized ethylenediaminetetra-acetic acid (EDTA)-blood haemolysates were applied in an external quality assurance programme (SKZL, The Netherlands) for glycohaemoglobin assays in 101 laboratories using 12 methods. The mean intralaboratory day-to-day coefficient of variation (CV), calculated from the assay of 12 unidentified pairs over a period of 1 year, was 5.2% (range: 0.2-28.7). Forty-seven per cent of laboratories did not meet the criterion of CV < 5%, whereas 68% did not meet the clinically more desirable 3.3-3.6%. Linearity, as derived from the analysis of five combinations of two haemolysates with low and high glycohaemoglobin percentages over 6 months, was excellent (mean correlation coefficient 0.9953; range: 0.9188-0.9999). Analysis of two samples with high and low glycohaemoglobin percentages gave mean interlaboratory coefficients of variation of 10% for one method performed by several laboratories and 22% for all methods performed by all laboratories. It is concluded that the majority of laboratories do not meet the clinically desirable intralaboratory precision and that an unacceptably high interlaboratory precision exists.

In vitro-synthesized carbamylated and acetylated hemoglobins interfered in assays of glycohemoglobin by HPLC and electrophoresis but had no effects on results obtained by affinity chromatography and enzyme immunoassay. Correlations between long-term serum urea concentrations and glycohemoglobin percentages revealed that, in vivo, carbamylated hemoglobin equivalent to 0.063% of total hemoglobin is formed for every 1 mmol/L of serum urea. The use of acetylsalicylate, either chronically in small doses (200-300 mg/day) or for 1 week at 2000 mg/day, did not cause significant interference from acetylhemoglobin, formed in vivo. We conclude that interference from carbamylated hemoglobin explains only a small part of existing discrepancies between results of glycohemoglobin assays in current use. The interfering effect of acetylhemoglobin formed in vivo with acetyl-CoA as substrate is as yet unknown.

Extraction of steroids from urine with C18 solid-phase extraction cartridges results in an extract containing impurities. If, during the extraction of hydrolyzed urine, an amino (NH2) column is placed in series with the C18 column, then one obtains a sample that is sufficiently clean for gas-chromatographic analysis. Analytical recovery of dehydroepiandrosterone from urine is considerably decreased by the use of increasing amounts of Helix pomatia enzyme preparation. Extraction of the steroid conjugates from urine with C18 columns before the hydrolysis stage is essential for hydrolysis with an amount of enzyme preparation that suffices for complete splitting of the polar steroid conjugates but not so much as to cause insufficient analytical recovery of dehydroepiandrosterone.

The mechanism of the reactions between haemoglobin and glucose in the erythrocyte was investigated in vitro using 14C-labelled glucose. Reaction speed constants were found for the formation of HbA1c (4.2 . 10(-3) and the formation and degradation of the Schiff Base (9.22 . 10(-4) and 0.435, respectively) for concentrations in mmol/l and time in hours at 37 degrees C. An equilibrium constant of 2.12 . 10(-3) was found for the reversible formation of the Schiff Base. The results were included in a mathematical model with which a number of clinically relevant situations were simulated. From the results the conclusion was drawn that during the measurement of fast haemoglobins, the intermediate Schiff Base causes a variation dependent on the glucose concentration at the moment of blood sampling. The model demonstrates that this disturbance can be eliminated by incubating erythrocytes with physiological saline for 10 h at 37 degrees C.

We describe a project, participated in by 24 institutions in The Netherlands and Belgium, to determine normal reference values for steroids in urine by capillary gas chromatography. Urine samples from 288 healthy volunteers were analyzed in triplicate. Reference values, expressed in mumol/24 h, were determined for androsterone, etiocholanolone, dehydroepiandrosterone, 11-keto-androsterone, 11-keto-etiocholanolone, 11-hydroxyandrosterone, 11-hydroxyetiocholanolone, pregnanediol, pregnanetriol, 11-desoxytetrahydrocortisol, tetrahydrocortisone, tetrahydrocortisol, allo-tetrahydrocortisol, and 17-keto- and 17-hydroxysteroids. We also determined reference ratios for etiocholanolone/androsterone, tetrahydrocortisone/tetrahydrocortisol, and tetrahydrocortisol/allo-tetrahydrocortisol; an upper limit of a discriminant function to establish polycystic ovarian disease; and reference values for 24-h urine volume and creatinine excretion. Reference values were determined separately for men and women, each in six age categories: 0-3 months, 4 months-12 years, 13-16 years, 17-50 years, 51-70 years, and older than 70 years. We conclude that these reference values are reliable and form a basis for quantitative interpretation of steroid profiles.