Recommendations on the measurement and use of the alcohol consumption biomarker CDT. A position paper from the IFCC Working Group on CDT standardisation.

BACKGROUND: Carbohydrate deficient transferrin (CDT) is a biomarker for excessive alcohol consumption utilized in clinical and forensic medicine and workplace testing. Previously, many different analytical methods for CDT were used and the measurand varied considerably, making direct comparison of test results difficult. To end this confusion, the IFCC established a working group on CDT standardisation (WG-CDT) which completed its tasks in 2017. METHODS: This IFCC position paper by the WG-CDT summarizes state of the art information about the measurand and the analytical methods and gives concise recommendations for its utilization. RESULTS: The results achieved by the CDT standardisation process led to accuracy improvements in national external quality assessment schemes over the years. A brief review of ROC based comparison studies with the traditional biomarkers (GGT, MCV, ALT and AST) discusses the bias resulting from inadequate study populations. In large groups of the general population the superior diagnostic performance of CDT is confirmed. CONCLUSION: The relationship between alcohol intake versus resulting CDT is discussed as well as the cutoff and measurement uncertainty. Concerning the application in practice, potential pitfalls are considered and recommendations handling both analytical and preanalytical caveats are given. Finally, some examples of serious misunderstandings in publications about CDT are addressed.

External quality assurance programme for enzymatic analysis of lysosomal storage diseases: a pilot study.

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.

Neurotransmitter metabolites in CSF: an external quality control scheme.

We report an international external quality control scheme on neurotransmitter metabolites in cerebrospinal fluid (CSF). The neurotransmitter metabolites homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) are analysed to diagnose inborn errors of neurotransmitter metabolism. HVA is the catabolite of dopamine; 5-HIAA is the catabolite of serotonin; and MHPG is the catabolite of noradrenaline. In the first phase, 12 laboratories from six countries participated in this special quality control scheme to define the present state of the art and achieve harmonization in analytical outcome and interpretation. In the second part, recoveries, dilutions and methods for sample preparation were compared. The results of 3 of 12 laboratories were excluded because of unacceptable intralaboratory coefficients of variations (CV) for HVA and/or 5-HIAA. The inter- and intralaboratory CVs, the linearity and the recovery were acceptable for the other laboratories for both parameters. Unacceptable differences in the reference ranges between laboratories, leading to differences in interpretation of the results, became obvious. There was a significant improvement of the interlaboratory CV for HVA after standardization with a calibrator. The reproducibility of MHPG measurement appeared to be adequately established in only two laboratories and recovery was low in all five measuring this metabolite. The quality control scheme is an invaluable tool for controlling the analytical outcome and providing support to laboratories to improve their quality.

A model for harmonization of routine clinical chemistry results between clinical laboratories.

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.

Survey of total error of precipitation and homogeneous HDL-cholesterol methods and simultaneous evaluation of lyophilized saccharose-containing candidate reference materials for HDL-cholesterol.

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

Preparation of a candidate primary reference material for the international standardisation of HbA1c determinations.

We prepared a candidate primary reference material for the forthcoming international standardisation of beta-N-terminal glycated hemoglobin A measurements. It consists of well-defined mixtures of purified beta-N-terminal glycated hemoglobin A and non-glycated hemoglobin A. First, beta-N-terminal glycated hemoglobin A and non-glycated hemoglobin A were isolated, purified to homogeneity, and characterised. The techniques used were cation exchange and affinity chromatography for the purification, and high performance liquid chromatography, capillary isoelectric focusing, electrospray ionisation mass spectrometry, and peptide mapping for the characterisation. Hemoglobins from blood of healthy, non-diabetic volunteers were obtained with a purity of > 99.5% for non-glycated hemoglobin A and of > 98.5% for beta-N-terminal glycated hemoglobin A. However, results from peptide mapping indicate that the beta-N-terminal glycated hemoglobin A preparations still contain some non-beta-N-terminal glycated hemoglobins, co-eluting with beta-N-terminal glycated hemoglobin A. The exact content of beta-N-terminal glycated hemoglobin A in these preparations could be determined by a procedure consisting of standard addition, enzymatic cleavage and quantification of the resulting beta-N-terminal peptides to be in the range from 95-97.5%. Since the beta-N-terminal glycated hemoglobin A and non-glycated hemoglobin A content could be exactly determined in the materials prepared, mixtures of both components could be successfully used to calibrate the candidate reference methods.

Capillary electrophoresis system for hemoglobin A1c determinations evaluated.

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.

Trace elements in body fluids: external quality assessment scheme in The Netherlands.

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.

Evaluation of a reference material for glycated haemoglobin.

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.

Vitamin C and glycohemoglobin.

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.

Testing the accuracy of total cholesterol assays in an external quality-control program. Effect of adding sucrose to lyophilized control sera compared with use of fresh or frozen sera.

We studied the suitability of various types of human serum preparations to test the accuracy of total cholesterol measurements in the External Quality Assessment scheme in The Netherlands, in which approximately 180 laboratories participate. Checked against the certified Abell/Kendall Reference Method, large reagent-dependent negative biases were observed with lyophilized serum that was insufficiently cryoprotected. The biases for the reagents of Du Pont, Roche, and Beckman averaged -16.7%, -9.2%, and -7.6% respectively; the least bias, -0.4%, was obtained with reagent from Boehringer Mannheim. The beneficial effect of cryoprotection with sucrose was demonstrated by the decrease in interreagent variation from 5.4% to 1.9%, the latter value being comparable with the values for fresh and once-frozen pooled serum (1.3% and 1.7%, respectively). We conclude that the detrimental effect of lyophilization on serum matrix can be minimized by suitable cryoprotection with 200 g/L sucrose.

Standardization of glycohemoglobin results and reference values in whole blood studied in 103 laboratories using 20 methods.

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.

Hemoglobins S and C: reference values for glycohemoglobin in heterozygous, double-heterozygous and homozygous subjects, as established by 13 methods.

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.

Effect of calibration on dispersion of glycohemoglobin values determined by 111 laboratories using 21 methods.

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.

Influence of hemoglobin variants and derivatives on glycohemoglobin determinations, as investigated by 102 laboratories using 16 methods.

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.

Glycohaemoglobin: comparison of 12 analytical methods, applied to lyophilized haemolysates by 101 laboratories in an external quality assurance programme.

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.

Interference of carbamylated and acetylated hemoglobins in assays of glycohemoglobin by HPLC, electrophoresis, affinity chromatography, and enzyme immunoassay.

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.

Steroid profile for urine: reference values.

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.