Volume 45, Issue 4

The cerebrospinal fluid (CSF) is a key sample in the research for novel molecular biomarkers of neurodegenerative disorders. CSF represents a repertoire of neuro-secreted, biosynthesised and metabolised molecular products of the central nervous system (CNS). Diffusion of macromolecules from the peripheral circulatory system to the CSF is highly regulated by the blood-brain barrier, which prevents uncontrolled distribution of proteins in the CNS. The development of reproducible high resolution separations of proteins in 2-D electrophoresis methods by the advent of immobilised pH gradient has opened the route to multivariate holistic protein pattern investigation of CSF into neurodegenerative disorders. Moreover, the introduction of pre-fractionation techniques such as free flow electrophoresis is currently increasing the dynamic depth of proteome analysis. Alzheimer's disease (AD) and other forms of dementia, demyelinating diseases, Parkinson's disease (PD), and Creutzfeldt-Jakob disease (CJD) have been evaluated for biomarker discovery by CSF investigation in multiple studies. However, the statistical design of these clinical cross-sectional investigations remains a limited factor given the strong statistical power required for complex multivariate analysis. These initial evidences are of particular interest in dissecting specific molecular mechanisms. The development of fast and economic profiling of CSF by linear matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) is providing a new ancillary technology to assess sample quality and pre-analytical requirements. In the following we take into account all these issues in the CSF proteomics investigation, especially highlighting the possible application in the development of clinical molecular biomarkers. Clin Chem Lab Med 2007;45:437–49.

Haemophilia A is the most common inherited bleeding disorder caused by defects in the F8C gene that encodes coagulation factor VIII. This X-linked recessive disorder occurs in approximately 1:5000 males. Haemophilia A is diagnosed based on normal prothrombin time, altered activated partial thromboplastin time and reduced factor VIII activity in plasma. Carrier females are usually asymptomatic and can be identified only by molecular analysis. The most frequent mutations in F8C are intron 22 and 1 inversions, which occur in approximately 50% and 5% of patients, respectively, with a severe phenotype. Large gene deletions are observed in approximately 5% of alleles from patients with severe haemophilia A. The remaining severe cases and all moderate and mild cases result from numerous point mutations and small insertions/deletions, which are de novo mutations in one-third of cases. Thus, molecular diagnosis of carrier status and prenatal diagnosis in families without intron 22 or 1 inversions is based on scanning techniques or gene sequencing. When the disease-causing mutation cannot be identified, molecular diagnosis is performed by linkage analysis of several DNA polymorphic markers linked to F8C . Given the clinical heterogeneity among haemophilic patients, many groups, including our own, have examined the relationships between prothrombotic gene variants and haemophilic phenotype to investigate whether prothrombotic gene variants modify clinical expression of the disease. Clin Chem Lab Med 2007;45:450–61.

Analytical quality specifications play a key role in assuring and continuously improving high-quality laboratory services. However, I believe, that there are two “missing links” in the effective management of quality specifications in the delivery of laboratory services. The first is the evidence that pre-analytical variation and related problems are not taken into great consideration by laboratory professionals. The second missing link is the communication of quality specifications to clinicians and other possible stakeholders. If quality specifications represent “the level of performance required to facilitate clinical decision-making”, they cannot be used only for internal quality management procedures but must be communicated to facilitate clinical reasoning, decision-making and patient management. A consensus should be achieved in the scientific community on these issues to assure better utilization of laboratory data and, ultimately, improved clinical outcomes. Clin Chem Lab Med 2007;45:462–6.

Background : Impaired function of calcium ion transporter sarco(endo)plasmic reticulum Ca 2+ -ATPase2 (SERCA2), encoded by ATP2A2 gene, was observed in hypertension. The aim of this study was to screen for mutations in the ATP2A2 gene, in hypertensive patients compared to healthy controls. Methods : The frequency of a novel mutation in exon 15 of ATP2A2 , coding SERCA2, was studied in 107 hypertensive patients and a control group of 50 healthy volunteers. 24-h ambulatory blood-pressure monitoring (ABPM) was carried out. ATP2A2 genotyping was performed by denaturing HPLC and sequencing. Results : In exon 15 of the ATP2A2 gene, a novel c.2171G>A polymorphism was identified that does not change the amino acid sequence. The frequency of the A allele was significantly higher in normotensive controls than in hypertensive patients (p=0.017). GA genotype carriers demonstrated a tendency towards lower blood pressure values in the doctor's office (p=0.367 systolic, p=0.439 diastolic blood pressure) and measured by ABPM. Conclusions : Our results suggest a protective role of the A724A (c.2171G>A) polymorphism of ATP2A2 in subjects without hypertension. Clin Chem Lab Med 2007;45:467–70.

Background : Folate is important in purine and thymidylate synthesis and, via homocysteine remethylation, facilitates S-adenosylmethionine-dependent transmethylation. Low folate availability leads to hyperhomocysteinemia, which is a risk factor for arterial vascular disease and venous thrombosis. Genetic variation in folate-metabolizing genes may affect folate availability and hence confer a greater risk of venous thrombosis. Methods : We genotyped the thymidylate synthase ( TYMS ) 28-bp repeat and 6-bp deletion, and the reduced folate carrier ( RFC1 ) 80G>A and AICAR transformylase/inosine monophosphate (IMP) cyclohydrolase ( ATIC ) 346C>G polymorphisms in population-based controls (n=431), and assessed their effect on plasma total homocysteine (tHcy), and serum and red blood cell (RBC) folate. We investigated the associations between these variants and disease risk in a retrospective case-control study on recurrent venous thrombosis (n=173) as well. Results : None of the genotypes, alone or in combination, were associated with major changes in tHcy. However, the TYMS 28-bp repeat was associated with serum and RBC folate levels. We found no evidence that the genetic variants studied were associated with recurrent venous thrombosis risk. Conclusions : The TYM S 28-bp repeat and 6-bp deletion, and RFC1 80G>A and ATIC 346C>G polymorphisms are not associated with tHcy, but we did observe an association between the TYMS 28-bp repeat and serum and RBC folate in a general population. None of the polymorphisms was associated with recurrent venous thrombosis risk. Clin Chem Lab Med 2007;45:471–6.

Background : The Pro12Ala polymorphism of the peroxisome proliferator-activated receptor-γ2 ( PPARγ-2 ) gene has been variably associated with insulin resistance, obesity and type 2 diabetes in several populations. However, this association has not been studied in Iranian subjects and we hypothesized that this variation might be associated with insulin resistance, type 2 diabetes and related metabolic traits in this population. Methods : The Pro12Ala genotypes were determined by PCR-restriction fragment length polymorphism in 696 unrelated subjects including 412 non-diabetic controls and 284 type 2 diabetic patients. Results : The frequency of the Ala allele was 9.4% and 5.9% in controls and type 2 diabetic subjects, respectively [adjusted odds ratio (OR) 0.457, p=0.005]. The Ala allele did not show a significant effect on anthropometric and biochemical parameters in the type 2 diabetic group, whereas in non-diabetic subjects, carriers of the Ala allele had significantly lower fasting insulin (p=0.007) and homeostasis model assessment of insulin resistance (HOMA-IR) (p=0.009) levels compared to Pro/Pro subjects. Multivariate logistic regression analysis showed that Pro12Ala polymorphism was an independent determinant of type 2 diabetes in this population. Conclusions : Our results for a sample of Iranian type 2 diabetes cases and controls provide evidence that the Pro/Ala genotype of the PPARγ-2 gene is associated with insulin sensitivity and may also have protective role against type 2 diabetes. Clin Chem Lab Med 2007;45:477–82.

Background: The lecithin:cholesterol acyltransferase ( LCAT ) gene is located on the long arm of chromosome 16 and encodes a highly conserved enzyme that catalyzes the formation of most plasma lipoprotein cholesteryl esters. Two autosomal recessive disorders, familial LCAT deficiency (FLD) and fish eye disease, are associated with germline LCAT mutations. Patients with FLD and fish-eye disease frequently present with corneal opacity, anemia and renal failure with proteinuria. Methods : We clinically and biochemically characterized a German patient with classical FLD and used molecular genetic analysis to identify a novel homozygous LCAT mutation within codon 178. Results : The insertion of adenine identified is located in one of the two motifs that resemble sequences found in several lipases, and results in a frameshift with a stop codon at residue 214. Therefore, the mutation alters a large portion of the LCAT enzyme, including both protein regions with putative lipase activity. Clinically, the female patient presented with corneal opacity, mild anemia and a slow deterioration in kidney function that led to a requirement for hemodialysis until she received a renal transplant. Conclusions : The present data provide additional insights into the genotype/phenotype correlations of FLD and thus may improve the genetic diagnosis of this interesting inborn error of metabolism. Clin Chem Lab Med 2007;45:483–6.

Background : Reticulocyte-type 15-lipoxygenase-1 (ALOX15) has anti-inflammatory and inflammatory effects and is implicated in the development of asthma, arthritis and atherosclerosis. Previously, we screened the human ALOX15 gene for variations because genetic variability in ALOX15 might influence these diseases. We found a C>T substitution at position c.–292 in the ALOX15 promoter that created a novel binding site for the transcription factor SPI1 and increased ALOX15 mRNA levels in monocytes from c.–292CT heterozygous volunteers. Methods : To test whether the higher mRNA levels led to higher ALOX15 activity, we performed an activity assay and measured the arachidonic acid metabolite 15(S)-hydroxy-eicosatetraenoic acid [15(S)-HETE] by HPLC analysis. To test whether this polymorphism was associated with coronary artery disease (CAD), we investigated its association in a case-control study involving 498 Caucasians. Results : The c.–292C>T polymorphism was associated with higher enzyme activity in heterozygous carriers. Intriguingly, this polymorphism also showed a tendency to be protective against atherosclerosis. Conclusions : These results suggest that increased ALOX15 activity may attenuate inflammation, which could be caused by an increase in 15(S)-HETE and eventually by its metabolites, the lipoxins. Clin Chem Lab Med 2007;45:487–92.

Background : Genetic polymorphisms of β3-adrenergic receptor (AR) Trp64Arg and β2-AR Gln27Glu may result in significant change in the functions of these receptors. The aims of the present study were to investigate the association between Trp64Arg , Arg16Gly and Gln27Glu polymorphisms and the susceptibility to obesity and hypertension in a Chinese population. Methods : A total of 437 Chinese subjects including 149 obese hypertensive patients, 139 non-obese essential hypertensive patients, and 149 non-obese normotensive healthy controls were enrolled in the study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and allele-specific (AS)-PCR assays were used to identify Trp64Arg , Arg16Gly , and Gln27Glu genotypes. Results : The allele frequencies of 64Arg and 27Glu in the obese hypertensive group were 0.178 and 0.128, respectively. Both were significantly higher than in the non-obese hypertensive and the control groups (p<0.05). Further analysis showed that this association existed only in male hypertensive patients. Conclusions: These data reveal that frequencies of β3-AR 64Arg and β2-AR 27Glu were significantly higher in our obese hypertensive patients than in the non-obese hypertensive population and healthy controls. β3-AR Trp64Arg and β2-AR Gln27Glu genetic polymorphisms are associated with obesity in Chinese male hypertensive patients. Clin Chem Lab Med 2007;45:493–8.

Background : Urinalysis comprises three techniques: urinary flow cytometry, test strip analysis and determination of specific urinary proteins. We investigated the diagnostic possibilities of combining these methods for a cohort of patients with a variety of well-documented urological and nephrological pathology. Methods : Urinary samples from 407 in- and out-patients with nephrological or urological pathology were retrospectively included in our study. Test strip analysis (URISYS 2400), urinary flow cytometry (UF-100) and urinary protein analysis [albumin, total protein, α 1 -microglobulin (A1M), α 2 -macroglobulin (A2M)] were performed. Results : In discriminating upper and lower urinary tract infections, A1M and A1M/log(white blood cells) can be used, whereas pathological casts only give poor discrimination. The ratio A2M/log(red blood cells; RBC) allows differentiation between cystitis and pyelonephritis, while glomerular diseases can be recognised by the log(RBC×urinary total protein). Combining A2M and urinary albumin allows the determination of acute prostatitis. Conclusions : Using combined parameters provided by various urinalysis techniques, ratios can be proposed with diagnostic value for classifying renal and urological conditions. Possible integration in computer-based knowledge systems may offer valuable information for clinicians dealing with these pathologies. Clin Chem Lab Med 2007;45:499–504.

Background : Anti-extractable nuclear antigen antibodies (ENA) are markers of connective tissue diseases (CTDs). Methods : We compared FIDIS reagents in the multiplex fluorescent microsphere immunodetection system to INNO-LIA and immunodiffusion for 174 antinuclear antibody-positive patients, 102 with well-defined CTDs and 72 disease controls. Results : No significant differences were found in sensitivity or specificity between FIDIS and immunodiffusion, or between FIDIS and INNO-LIA for all anti-ENA in all CTD patients; nor were any differences found for individual anti-ENAs within distinct CTDs. The FIDIS sensitivity was 41% (anti-SSA) and 17% (anti-SSB) in lupus erythematosus (LE) or primary Sjögren's syndrome; 5% (anti-ribosome and anti-Sm) in LE; 17% (anti-RNP) in LE or mixed CTD; 21% (anti-Scl70) in systemic sclerosis; and 61% (anti-centromere) in limited systemic sclerosis. The specificity reached 88%–100%. Receiver operating characteristic curve areas did not differ between FIDIS and INNO-LIA. Agreement ranged from 91% (anti-SSB) to 99% (anti-Jo1) between FIDIS and INNO-LIA, and from 95% (anti-Scl70) to 100% (anti-Sm) between FIDIS and immunodiffusion. Samples scored positive with all techniques in 83% (anti-centromere), 70% (anti-RNP), 67% (anti-Jo1), 60% (anti-SSA), 40% (anti-SSB), 33% (anti-ribosome), 25% (anti-Sm) and 13% (anti-Scl70) of cases. Conclusions : The diagnostic performance of FIDIS anti-ENA reagents is comparable to immunodiffusion and INNO-LIA. Clin Chem Lab Med 2007;45:505–12.

Background : Selective serotonin reuptake inhibitors (SSRIs) in general, and citalopram/escitalopram in particular, are widely used to treat clinical depression. However, SSRI bioavailability and non-compliance represent major issues, especially in the clinical trials setting. In this context, frequent drug-level measurements for compliance monitoring would be a desirable tool to improve clinical outcomes with SSRIs. However, the liquid chromatography techniques available are expensive, requiring excessive sample preparation, and suffer from high complexity. We sought to develop a rapid method for the measurement of citalopram/escitalopram levels in human plasma by fluorimetry. Methods : A total of 34 frozen human plasma samples were thawed at room temperature and repeatedly centrifuged in cellulose to remove aggregates, proteins and solids. Fluorescence spectra were measured in the range 270–450 nm with excitation at 240 nm on a FluoroMax 3 spectrofluorimeter. Control samples contained known concentrations of SSRIs. Results : SSRI absorbance spectra were recorded in the range 230–320 nm. The shape of the spectra and the absorbance of citalopram and escitalopram were very similar, with UV maximum absorbance at 239 nm. The maximum extinction coefficient was ε 239 =15,930 M –1 cm –1 for citalopram and ε 239 = 13,630 M –1 cm –1 for escitalopram. The fluorescence spectra of SSRIs are unique and are characterized by the presence of two well-defined conjugated spectra with maxima at 300 and 382 nm. Conclusions : Fluorimetry is very suitable for assessment of plasma SSRI levels. This inexpensive and efficient technique can objectively and reliably quantify drug levels in biological fluids, thereby directly determining the level of patient adherence to the prescribed drug regimen. This method will be useful in a broad spectrum of applications, from compliance/bioavailability assessments in animal and human experiments to utilization in large-scale clinical trials. Clin Chem Lab Med 2007;45:513–20.

Background : Quantification of bioactive, free serum cortisol concentrations can characterize adrenocortical function more appropriately compared to total serum cortisol measurement. Ultrafiltration or equilibrium dialysis of serum samples allow direct measurement of free serum cortisol concentrations but respective methods have poorly been validated so far. The aim of our study was to investigate the analytical performance of free serum cortisol measurement employing equilibrium dialysis and ultrafiltration. Methods : Two commercially available ultrafiltration devices and self-assembled dialysis cells, respectively, were studied. Cortisol was quantified in filtrate or dialysate using an automated immunoassay system. Using two serum pools, the inter-assay coefficient of variation was determined for the three methods and a method comparison was performed. Results : Inter-assay coefficients of variation (n=10) between 3.2% and 14.8% were observed in the imprecision study. Method comparison demonstrated close agreement between free serum cortisol results obtained by ultrafiltration and equilibrium dialysis, respectively (equilibrium dialysis=1.2×ultrafiltration+3.9 nmol/L; r=0.99; n=35). Conclusions : Direct quantification of free serum cortisol after equilibrium dialysis or ultrafiltration of the samples offers acceptable reproducibility and results in close agreement can be obtained. Both methods can potentially be introduced into a routine laboratory setting. Clin Chem Lab Med 2007;45:521–5.

Background : During percutaneous coronary intervention, the technique of stent implantation (both direct and complementary stenting) is guided using both clinical and angiographic features. We assessed potential relationships between procedural parameters and angioplasty-induced variations in inflammatory parameters in patients treated by these two different techniques. Methods : A total of 85 consecutive patients due to undergo stent implantation were prospectively enrolled. Inflammation was assessed in terms of C-reactive protein, fibrinogen, erythrocyte sedimentation rate and leukocyte count in samples taken before and 24 h after angioplasty. Patients were classified based on whether they underwent complementary (n=47) or direct stenting (n=38). Results : Inflammation after complementary stenting was related to the duration of inflation (r=0.59; p<0.001), whereas inflammation after direct stenting was related to the inflation pressure (r=0.61; p=0.007), as assessed by C-reactive protein variation. None of the other parameters influenced the inflammatory response. Conclusions : The inflammatory response after stent implantation depends on the mode of stenting. We therefore hypothesize that the inflammatory response after stenting might be related to the histological composition of the atherosclerotic plaques involved. Clin Chem Lab Med 2007;45:526–30.

Background : The National Kidney Disease Education Program recommends that clinical laboratories, when asked for an estimation of glomerular filtration rate in a patient by means of the “four-variable” Modification of Diet in Renal Disease (MDRD) Study equation, also provide the measurement result for creatininium concentration in plasma and the appropriate reference interval. On the other hand, clinical laboratories seeking accreditation for compliance with ISO 15189:2003 need to demonstrate that the physiological reference intervals communicated to all users of laboratory services are appropriate for the patient population served, and for their measurement systems. Methods : Ten clinical laboratories in different regions of Spain collaborated in identifying reference individuals and producing reference values for the concentration of creatininium in plasma using RD/Hitachi Modular Analytics analysers, and for the volume rate of glomerular filtrate in kidneys (glomerular filtration rate), estimated with the “four-variable” MDRD Study equation. All the logistic work was carried out in co-operation with the supplier of the reagents and analysers (Roche Diagnostics España, S.L., Sant Cugat del Vallès, Catalonia, Spain). Using all the reference values obtained by each laboratory, multicentre reference limits were estimated non-parametrically. Results and conclusions : Reference intervals estimated in this study for concentrations of plasma creatininium are 52–85 μmol/L for women and 64–106 μmol/L for men. The diagnostic specificity of the estimated glomerular filtration rate is 99,2% when applied to healthy persons to screen for chronic kidney disease. Clin Chem Lab Med 2007;45:531–4.

Background : Ischemia-modified albumin (IMA) is an emerging marker of ischemia. To investigate the applicability of IMA for the diagnosis of skeletal muscle ischemia, we examined IMA changes as measured by the albumin-cobalt binding test, in a group of healthy volunteers after standardized exercise-induced calf muscle ischemia. Methods : A total of 12 healthy volunteers underwent standardized exercise on a plantar flexion pedal. Ischemic conditions were achieved by inflating a femoral blood pressure cuff at incremental pressures of 0, 60, 90, 120 and 150 mm Hg. Calf muscle ischemia was identified by synchronous 31 P magnetic resonance spectroscopy, measuring intracellular concentrations of phosphocreatine (PCr) and inorganic phosphate (Pi). In addition, IMA, serum albumin, lactate, troponin T (TnT) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) were measured at baseline and at 5, 10, 30, 360 and 720 min after cuff release. Results : Magnetic resonance spectroscopy showed calf muscle ischemia in all participants upon exercise and cuff inflation. Circulating IMA concentrations increased significantly after cuff release (p=0.03) and returned to baseline within 30 min. While we found a significant negative correlation with albumin, there was no association of IMA levels with lactate or intracellular levels of PCr or Pi in samples obtained at baseline and post-ischemia. TnT and NT-proBNP remained within the normal range throughout the observation period in all participants. Conclusions : IMA may represent a clinical marker for skeletal muscle ischemia, although its lack of specificity requires careful clinical interpretation of data. The short period of IMA elevation after ischemic exercise requires standardized conditions for use as a diagnostic tool and hints at IMA applicability as a marker of prolonged or chronic ischemia. Clin Chem Lab Med 2007;45:535–40.

We measured serum follicle-stimulating hormone (FSH), luteinizing hormone (LH) and prolactin concentrations on a bioMérieux Mini Vidas system in a pediatric population ranging in age from 1 to 19 years. Reference intervals were established separately for females and males, with stratification by age group and by Tanner's pubertal stage. FSH values were higher in females than in males, and were lowest in both sexes of age class 2 (4–8 years), increasing thereafter to the upper limit for stage PIV (females) and stage PV (males). LH values showed a similar pattern of change: concentrations were lowest for class 1 (1–3 years) and class 2 (4–8 years), and highest for stage PII (females) and stage PV (males). No significant difference was observed according to gender. Prolactin values did not differ markedly according to gender or pubertal status. Clin Chem Lab Med 2007;45:541–5.

Background : After maternal alcohol consumption during pregnancy, many neonates affected by less apparent forms of fetal alcohol syndrome disorder (FASD) do not receive proper diagnosis or treatment. There is thus a need for laboratory markers for early detection of alcohol-exposed neonates. The aim of our study was to assess the efficiency of the usual alcohol biomarkers measured in cord blood to identify alcohol-exposed neonates immediately after birth. Methods : A 1-year study was conducted in the labor wards of the maternity units of the Auvergne, Central France at the time of term delivery. The patients answered an anonymous self-completion survey concerning alcohol use (Alcohol Use Disorder Identification Test; AUDIT) during their pregnancy. Aspartate aminotransferase, alanine aminotransferase, and γ-glutamyltransferase concentrations and the percentage of carbohydrate-deficient transferrin were measured in maternal and cord blood. Results : We collected 870 maternal-fetal sample pairs. Two cases (0.2%) of typical FASD were detected. We report a non-significant correlation between maternal and cord blood biomarkers. None of the cord blood biomarkers differed significantly between newborns of alcohol-exposed and unexposed mothers. Conclusions : We demonstrate that the usual alcohol biomarkers are not effective in cord blood for identifying alcohol-exposed neonates. Clin Chem Lab Med 2007;45:546–8.

Background : Analytical performance of clinical laboratory testing should be evaluated against objective quality specifications. The Stockholm Conference consensus recommendation states that from a hierarchy of quality models, the highest-ranking model should be used. This study reports the performance of a routine clinical laboratory using these quality recommendations. Quality standards recommended for use in manufacturers' package inserts are also compared against the objective criteria. Methods : Imprecision of 22 analytes was monitored by analyzing three levels of commercial quality control (QC) material daily over a 6-month period. The performance for each analyte was evaluated against defined quality specifications based on biological variation and the National Cholesterol Education Panel (NCEP) criteria. Results : Overall, objective quality specifications were met by the laboratory for 18/22 (82%) analytes. The performance for analytes not meeting the criteria was assessed against quality targets based on a model lower in the hierarchy, i.e., interlaboratory imprecision data derived from an accredited EQA program. All analytes met these quality targets. When quality targets set by the manufacturer were applied to biological variation and NCEP criteria, results for only 8/22 (36 %) analytes met the targets. Conclusions : Objective quality specifications can be consistently achieved in a routine laboratory. The hierarchy model of the Stockholm Conference should be promoted for global adoption. Clin Chem Lab Med 2007;45:549–;52.

An alcohol-associated change in the serum transferrin glycoform pattern, carbohydrate-deficient transferrin (CDT), is used as a biomarker of chronic moderate to heavy alcohol consumption. A current limitation in CDT analysis is the lack of standardization, which hampers clinical and analytical comparison between studies. This situation prompted initiation of a Working Group (WG) on CDT Standardization under the auspices of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). The standardization work aims to define and validate the analyte, select a reference method, work out procedures for the production of reference materials, and make suggestions for the clinical usage of CDT. The first recommendation of the WG is that disialotransferrin should be the primary target molecule for CDT measurement and the single analyte on which CDT standardization is based. It is further recommended that HPLC should be the analytical principle considered as the basis of an interim reference method until a suitable mass spectrometric reference method is established. In clinical use, CDT should be expressed in a relative amount (% CDT), to compensate for variations in the total transferrin concentration. Clin Chem Lab Med 2007;45:558–62.