Analysis of exhaled breath composition in lung disease patients can indirectly point to biochemical changes that occur in the fluid lining airway surfaces. The parameters of redox and acid-base changes, and of inflammatory changes relevant in the pathogenesis of most pulmonary diseases are currently most widely determined in exhaled breath condensate. The collection of exhaled breath condensate is a safe, non-invasive, easy and simple diagnostic procedure that is suitable for longitudinal studies and applicable in patients of all age groups, irrespective of the disease severity. In spite of many scientific studies involving lung disease patients, methodology for exhaled breath condensate collection and analysis has not yet been realized for daily utilization. Additional studies of the exact origin of condensate constituents and standardization of the overall analytical process, including collection, storage, analysis and result interpretation, are needed. Irrespective of these limitations, further investigation of this sample type is fully justified by the fact that classical specimens used in the management of pulmonary disease are either obtained by invasive procedures (e.g., induced sputum, biopsy, bronchoalveolar lavage) or cannot provide appropriate information (e.g., urine, serum). Analysis of exhaled breath condensate in the future might contribute significantly to our understanding of the physiological and pathophysiological processes in lungs, to early detection, diagnosis and follow up of disease progression, and to evaluation of therapeutic response. Clin Chem Lab Med 2007;45:945–52.

August 9, 2007

Biochemical markers of alcoholism

Minna L. Hannuksela, Marja K. Liisanantti, Antti E.T. Nissinen, Markku J. Savolainen

Page range: 953-961

Abstract

Alcohol and alcohol-related diseases have become a major cause of death in Western countries. The most sensitive and specific of the commonly used biomarkers of alcohol intake are carbohydrate-deficient transferrin (CDT), and the combination of γ-glutamyltransferase (GGT) and CDT. Other widely used laboratory markers are GGT, mean corpuscular volume of erythrocytes and the ratio of aspartate aminotransferase to alanine aminotransferase. Blood ethanol levels reveal recent alcohol use. However, more specific and sensitive biomarkers to improve the detection of excessive alcohol use at an early stage are needed. New biomarkers, not yet used in routine clinical work, include phosphatidylethanol, fatty acid ethyl esters, ethyl glucuronide, sialic acid, and acetaldehyde adducts. Clin Chem Lab Med 2007;45:953–61.

August 9, 2007

Polymorphisms of the peroxisome proliferator-activated receptor-γ coactivator-1α gene are associated with hypertrophic cardiomyopathy and not with hypertension hypertrophy

Shuxia Wang, Chunyan Fu, Hu Wang, Yi Shi, Xiqi Xu, Jingzhou Chen, Xiaodong Song, Kai Sun, Jianwei Wang, Xiaohan Fan, Hongjian Wang, Xiaomin Yang, Tujun Huan, Rutai Hui

Page range: 962-967

Abstract

Background : The clinical phenotype of both hypertrophic cardiomyopathy (HCM) and left ventricular hypertrophy (LVH) induced by hypertension is heterogeneous. Genetic factors may contribute to this heterogeneity. Evidence is accumulating that the peroxisome proliferator-activated receptor-γ coactivator-1α ( PGC-1α ) gene plays a role in cardiac hypertrophy. The aim of our study was to identify the association between PGC-1α gene polymorphisms and cardiac hypertrophy. Methods : A total of 270 consecutive HCM patients and 2486 hypertensive patients, comprising 1180 with LVH and 1306 without LVH, as well as 894 healthy controls, were successfully investigated. Polymorphisms of the PGC-1α gene were genotyped by PCR-restriction fragment length polymorphism and confirmed by sequencing. Results : The Ser482 allele (rs8192678 G>A and A>A) and CC genotype of Thr394Thr (rs2970847) conferred increased risk for HCM [odds ratio (OR) 1.52, 95% confidence interval (CI) 1.11–2.11; OR 1.49, 95% CI 1.15–1.98, respectively]. The maximum ventricular thickness was greater in HCM patients carrying the Ser482 risk allele than in carriers of the non-risk allele (20.7±4.1 vs. 19.1±4.3 mm, p<0.05) and for the CC Thr394Thr genotype (20.9±4.6 vs. 19.0±4.2 mm, p<0.05). No association was found between PGC-1α polymorphism and hypertension with or without LVH. Conclusions : Our data indicate that variants of the PGC-1α gene are correlated with increased risk for HCM. Clin Chem Lab Med 2007;45:962–7.

August 9, 2007

Association of interleukin-1 genetic polymorphisms with the risk of rheumatoid arthritis in Chinese population

Chong-ge You, Jian-feng Li, Xiao-dong Xie, Yan Zhu, Pei-qiang Li, Yi-rong Chen

Page range: 968-971

Abstract

Background: Previous studies suggest that a variable number of tandem repeats polymorphism in the second intron of the interleukin-1 receptor antagonist gene and two single nucleotide polymorphisms at positions −511 and +3954 of the interleukin-1β ( IL-1B ) gene are associated with an increased risk of autoimmune diseases. In the present study, we evaluated associations between these genetic factors and an increased risk of rheumatoid arthritis (RA) in a population from Northwest China. Methods: A total of 240 patients with RA and 227 healthy controls from Northwest China were investigated using PCR and PCR-restriction fragment length polymorphism. Genotype and allele distributions and haplotype construction were analyzed. Results: The genotype and allele distributions of IL-1B +3954 and IL-1RN polymorphisms were significantly different in RA patients compared to controls (p<0.001 and p<0.001; p=0.028, p=0.023, respectively). Significant differences were also observed between the RA and control groups for the haplotypes IL-1B –511C/+3954C/IL-1RN *1 , IL-1B –511C/+3954T / IL-1RN *1 and IL-1B –511T/+3954T/IL-1RN *1 [p=0.017, odds ratio (OR) 0.721, 95% confidence interval (CI) 0.551–0.944; p=0.030, OR 2.111, 95% CI 1.060–4.204; and p=0.029, OR 2.909, 95% CI 1.066–7.902, respectively]. Conclusions: These findings suggest that IL-1B +3954 and IL-1RN genetic polymorphisms are associated with a significantly increased risk of RA in this Chinese population. Clin Chem Lab Med 2007;45:968–71.

August 1, 2007

Highest accuracy of combined consensus clinical criteria and SNRPN gene molecular markers in diagnosis of Prader-Willi syndrome in Thai patients

Moltira Promkan, Somporn Teingtat, Atchara Stheinkijkarnchai, Pornswan Wasant, Pimpicha Patmasiriwat

Page range: 972-980

Abstract

Background: Prader-Willi Syndrome (PWS) is a complex human genetic disease arising from a loss of paternal allele expression of imprinting genes on chromosome 15q11–q13. Normally the CpG islands at this site are heavily methylated in the maternal allele, but unmethylated in the paternal allele and therefore activated in gene expression. Only the methylated allele should present in PWS patients when methylation-specific PCR (MSP) is analyzed. Methods: This paper reports an analysis of PWS in Thai patients using consensus diagnostic criteria based on a combination of clinical data, basic G-banding and fluorescence in situ hybridization (FISH) cytogenetics, PCR-based methylation assay, and bisulfite sequencing of the CpG islands of SNRPN to confirm 15q deletion or the methylation pattern of the SNRPN promoter and exon 1. Lack of complete clinical reports or inadequacy of the minimum laboratory support required had made it difficult to diagnose PWS, Angelman syndrome and other microdeletion disorders. Results: Accuracy of 100% was obtained for diagnosis of the PWS study patients using the minimum requirements necessary. A total of 20 patients were diagnosed as PWS based on clinical criteria and the scoring tool for PWS, and the same approach was applied to four separate patients with some unmatched criteria but phenotypic similarity to PWS. Findings showed that 70% of those clinically diagnosed as PWS patients (14/20) had a deletion at 15q11–q13 according to FISH, while all 20 patients showed MSP positive of SNRPN gene. Six cases (30%) without a paternal deletion were confirmed to have maternal uniparental disomy (mUPD) of PWS by MSP and methylation sequencing approaches. Noteworthy, two of the six cases with mUPD were 3.5 year-old twins. None of the five cases with scores lower than the reported consensus criteria showed positive G-band, FISH or MSP results. Conclusions: We demonstrate here the high power of combining clinical findings, FISH and MSP in definitive diagnosis of PWS and in distinguishing between the two major different types of molecular mechanisms. No false positives or false negatives were observed in our analysis. Clin Chem Lab Med 2007;45:972–80.

August 9, 2007

Use of maternal plasma for non-invasive prenatal diagnosis of fetal ABO genotypes

Jin-Lai Meng, Xie-Tong Wang, Yu Wang, Ya-Fei Yue, Xin Wang, Zi-Jiang Chen

Page range: 981-986

Abstract

Background : Measurement of free fetal DNA in maternal plasma opened a door for non-invasive prenatal diagnosis. Prenatal diagnosis of fetal ABO genotypes can provide a basis for the prevention and therapy of maternal-fetal incompatibility. We identified fetal ABO genotypes using fetal DNA in plasma from pregnant women with blood group O. The aim of the study was to investigate the accuracy and feasibility of this method. Methods : A total of 105 blood group O women in middle or late pregnancy were enrolled. Fetal DNA in maternal plasma and genomic DNA in umbilical vein blood from newborns were extracted using a QIAamp DNA Blood Kit. DNA was amplified to identify ABO genotypes by PCR with sequence-specific primers (PCR-SSP). The genotype results were evaluated using serologic tests for ABO phenotyping. Results : Using DNA from umbilical vein blood, ABO genotypes of 105 newborns were successfully identified by PCR-SSP. Using fetal DNA from maternal plasma, 88.6% (93/105) fetal ABO genotypes was correct; 12 false results were from 66 pregnant women with fetuses of type non-O. The accuracy in middle pregnancy was lower than that in late pregnancy, although the difference was not significant (0.05<p<0.10). Conclusions : It is feasible to use measurement of fetal DNA in plasma from pregnant women with blood group O for prenatal diagnosis of fetal ABO genotypes. The method is useful for the diagnosis and therapy of ABO maternal-fetal incompatibility and hemolytic disease of the newborn. Clin Chem Lab Med 2007;45:981–6.

July 8, 2007

Relationship of the CAG repeat polymorphism of the MEF2A gene and coronary artery disease in a Chinese population

Yaling Han, Yong Yang, Xiaolin Zhang, Chenghui Yan, Suya Xi, Jian Kang

Page range: 987-992

Abstract

Background : Recently, a mutation in the human myocyte enhancer factor-2A ( MEF2A ) gene was reported to be responsible for an autosomal dominant form of coronary artery disease (CAD). In addition, missense mutations in sporadic CAD patients were also described. Both results support the disease-causing relationship between MEF2A and CAD/myocardial infarction. On the other hand, conflicting hypotheses have been put forward in other studies. Methods : We screened exons 7 and 11 of MEF2A through single-stranded conformation polymorphism PCR and direct sequencing to clarify the relationship between MEF2A and CAD in an independent case-control study involving 726 individuals in China. Results : Exon 11 showed a high degree of heterogeneity, which was caused by a polyglutamine (CAG) n polymorphism. Frequencies for the different (CAG) n alleles were not the same between patient and control groups. Of note, the distribution frequency of the (CAG) 9 allele was higher in the patient group than in the control group (p<0.001). This effect was independent of age, gender, hypertension, diabetes mellitus, hyperlipidemia and smoking in a logistic regression model (p=0.001, odds ratio 1.245, 95% CI 1.095–1.417). It was also observed that the (CAG) 9 allele was related to the extent of CAD, which was defined as no CAD, or single-, double- or triple-vessel disease (p trend 0.000). Conclusions : Based on our data, we speculate that the CAG repeat polymorphism is associated with coronary heart disease in the Chinese population and the (CAG) 9 allele may be an independent predictive factor for CAD. Clin Chem Lab Med 2007;45:987–92.

August 9, 2007

Specific real-time PCR vs. fluorescent dyes for serum free DNA quantification

Mihelaiti Chiminqgi, Stéphane Moutereau, Pascal Pernet, Marc Conti, Véronique Barbu, Jerôme Lemant, Mory Sacko, Michel Vaubourdolle, Sylvain Loric

Page range: 993-995

Abstract

Background : Detecting and quantifying circulating free DNA in patient serum has become a major challenge. New methods using conventional or automated DNA amplification have been developed. As quantitative real-time PCR (QPCR) remains expensive and requires dedicated automated instrumentation, we questioned whether simple quantification using fluorescent dyes is efficient for determination of free DNA levels in serum. Methods : Serum samples from 180 cancer patients and 58 healthy volunteers were used for DNA quantification according to three methods: (i) using an exonic part of the β-globin gene as the amplifying target; (ii) amplifying a 105-bp intron 1 part of the housekeeping cyclophilin A gene, both referring to specific standard curves; and (iii) using a PicoGreen DNA quantification kit without amplification. Results : The 58 samples from healthy controls showed a reference limit of (95th percentile) <160 cyclophilin gene copies/mL. The 180 cancer samples displayed values ranging between 300 and 215,000 copies/mL. The cyclophilin method showed a high level of correlation with both the β-globin (r=0.911, p<0.0001) and PicoGreen (r=0.915, p<0.0001) methods. Conclusions : Aside from the disadvantage that the QPCR assays can only be used in clinical biochemistry laboratories that possess QPCR apparatus, the use of direct PicoGreen quantification displays major advantages in a routine context: it is less time-consuming and is quite inexpensive, but is still correlated with QPCR. Clin Chem Lab Med 2007;45:993–5.

August 1, 2007

Increased glycation of hemoglobin and plasma proteins in normotensive, non-diabetic obese Indian subjects: putative role of lipid peroxides

Viswanathan Sathiyapriya, Nambiar Selvaraj, Hanumanthappa Nandeesha, Zachariah Bobby, Aparna Agrawal, Magadi Gopalakrishna Sridhar, Purushothaman Pavithran, Nadaradjan Rattina Dasse

Page range: 996-999

Abstract

Background : Glycation and lipid peroxidation are spontaneous reactions believed to contribute to the pathogenesis of many clinical disorders. The purpose of the present study was to evaluate the levels of lipid peroxides and glycated proteins in normotensive, non-diabetic obese Indian subjects and to assess possible associations between them. Methods : A total of 28 obese male subjects and 20 non-obese subjects were included in the present study. Whole blood glycated hemoglobin, plasma lipid peroxides and fructosamine levels were estimated in both groups. Results : Lipid peroxides, glycated hemoglobin and fructosamine levels were significantly higher in obese subjects in comparison with non-obese subjects. We also found a significant association between malondialdehyde and body mass index (r=0.424, p=0.025). Partial correlation analysis revealed that malondialdehyde was significantly correlated with glycated hemoglobin (r=0.590, p=0.01) and fructosamine (r=0.442, p=0.021) after controlling for glucose. Conclusions : Increased glycation of proteins was found in normotensive, non-diabetic obese Indian subjects. These data also support the premise that lipid peroxides per se play a role in the glycation of hemoglobin and plasma proteins. Clin Chem Lab Med 2007;45:996–9.

August 9, 2007

The relationship between red blood cell and reticulocyte indices and serum markers of iron status in the cord blood of newborns

Mari Ervasti, Sanna Kotisaari, Ulla Sankilampi, Seppo Heinonen, Kari Punnonen

Page range: 1000-1003

Abstract

Background : The aims of this study were to assess the relationship between red blood cell and reticulocyte indices and biochemical iron status measurements, and to define reference values of these markers in the cord blood of newborns. Methods : In cord blood samples from 199 full-term newborns, cellular indices were assessed using an ADVIA 120 hematology system and iron status was analyzed by measurement of serum iron, transferrin, transferrin saturation (TfSat), transferrin receptor (TfR) and ferritin. Results : Cellular hemoglobin in red blood cells or reticulocytes was independent of serum iron markers such as TfSat, TfR and ferritin. The percentage of hypochromic red blood cells (%HYPOm) and reticulocytes (%HYPOr) correlated significantly with TfSat and TfR-F index (TfR/log ferritin). Importantly, %HYPOm and %HYPOr were also positively correlated with the high immature reticulocyte fraction (IRF-H) and the mean cell volume of red or reticulocytes. Conclusions : In newborns, accelerated erythropoiesis is a major contributor to red blood cell and reticulocyte indices, which provide conflicting results when compared with serum markers of iron status. Apparently, the serum proteins ferritin, transferrin and TfR are more appropriate tools for the diagnosis of iron status in newborns. Clin Chem Lab Med 2007;45:1000–3.

August 9, 2007

Association of amino-terminal propeptide of type III procollagen and acute myocardial rejection in male patients receiving heart transplantation

Yen-Hung Lin, Chung-Pin Liu, Ron-Bin Hsu, Chi-Ming Lee, Shoei-Shen Wang, Hsien-Li Kao, Chia-Lun Chao, Yu-Chien Shiau, Chi-Sheng Hung, Lin-Chu Liao, Yi-Lwun Ho

Page range: 1004-1008

Abstract

Background : The amino-terminal propeptides of type I and III procollagens (PINP and PIIINP) are markers reflecting the status of collagen turnover. We hypothesized that measurement of these serum procollagen propeptides could be used to non-invasively assess acute rejection in heart transplant recipients. Methods : In heart transplant recipients, endomyocardial biopsy specimens taken at 6 and 12 months after surgery were used for study. PINP and PIIINP were measured postoperatively at 3, 6, and 12 months. Results : A total of 20 male heart transplant patients and seven male control subjects were enrolled. Five patients showed rejection 6 months after transplantation (group 1), while 15 patients showed no rejection (group 2). In group 2 patients, serum PINP and PIIINP levels decreased significantly 6 months after transplantation. In contrast, elevation of serum PINP and PIIINP levels persisted in group 1 patients 6 months after transplantation. At 6 months after transplantation, group 1 patients had significantly higher PIIINP levels than group 2 patients (p=0.025) and controls (p=0.003). After immunosuppressive therapy, all group 1 patients were free of rejection 12 months after transplantation and serial serum PIIINP levels decreased significantly in these patients. Conclusions : Serum PIIINP levels represent a non-invasive method to reflect the occurrence and resolution of acute rejection. Clin Chem Lab Med 2007;45:1004–8.

August 9, 2007

Changes in platelet count and indices in pulmonary tuberculosis

Ergun Tozkoparan, Omer Deniz, Ergun Ucar, Hayati Bilgic, Kudret Ekiz

Page range: 1009-1013

Abstract

Background: Recent studies show that platelets have important roles in the immune system. Little is known about the clinical significance of platelet indices. Changes in platelet indices, including platelet distribution width (PDW), mean platelet volume (MPV) and plateletcrit, in pulmonary tuberculosis were investigated. Methods: Platelet indices were quantified in 82 patients with active tuberculosis and 87 patients with inactive or non-tuberculous disease (controls). Radiological extent of the disease was assessed. Results: There were significantly higher PDW (40.9±23.5% vs. 27.0±14.5%), MPV (10.05±2.36 vs. 8.83±1.47 fL) and plateletcrit (0.330±0166% vs. 0.266±0.128%) values in the active tuberculosis group, which decreased significantly with anti-tuberculous therapy. Erythrocyte sedimentation rate and plateletcrit showed significant correlation (r=0.54 and r=0.66) with radiological extent of tuberculosis, while PDW and MPV correlations with radiological extent of tuberculosis were also significant but weaker (r=0.31 and r=0.23). In a subpopulation of controls with pneumonia, which leads to acute phase reaction, PDW, MPV and plateletcrit values were significantly lower than in the tuberculosis group. Conclusions: We suggest that PDW, MPV and plateletcrit change in tuberculosis and that these changes may not reflect only acute phase reaction and disease activity. The potential role of platelet indices in tuberculosis immunopathogenesis remains to be investigated. Clin Chem Lab Med 2007;45:1009–13.

August 9, 2007

Activity or mass concentration of bone-specific alkaline phosphatase as a marker of bone formation

Ivica Avbersek-Luznik, Tanja Gmeiner Stopar, Janja Marc

Page range: 1014-1018

Abstract

Background : Recently published data identified bone-specific alkaline phosphatase (BALP) as a good marker of bone formation in different bone diseases and osteoporosis. Two methods are available for BALP determination: one measures enzyme activity, the other its mass concentration. We compared results for BALP activity and its mass concentration in a group of 88 healthy pre- and postmenopausal women to identify which is a more useful marker for detecting early menopausal bone remodelling changes. Methods : We measured BALP activity and BALP mass concentration in relation to femoral neck (FN) and lumbar spine (LS) bone mineral density (BMD) and some other widely used bone markers: osteocalcin (OC), procollagen type I N-terminal propeptide (PINP) and serum C-terminal telopeptide cross-links of type I collagen (CTx) in serum samples from 50 premenopausal (age 45.9±4.6 years) and 38 postmenopausal (age 54.4±4.5 years) women. Results : Healthy postmenopausal women exhibited 34.2% (p<0.01) and 27.3% (p=0.000) higher levels of BALP activity and mass concentration than premenopausal women, respectively. At the same time, FN and LS BMD were not significantly different between the groups. CTx values were significantly higher in postmenopausal women (p=0.018), while OC and PINP were not. We observed significant correlation between BALP activity and mass concentration (r=0.85, p<0.01). The correlation between BALP activity and FN BMD or LS BMD was insignificant. BALP mass correlated significantly with LS BMD (r=–0.370, p=0.033) but not with FN BMD. As expected, we proved a significant positive correlation for both BALP methods with the other bone markers measured in our study. Conclusions : Postmenopausal women have slightly higher bone turnover. Since LS and FN BMD were not significantly lower in our group of healthy postmenopausal women, but BALP and CTx were markedly higher, we suggest that measurements of BALP and CTx might be useful as early markers of higher bone turnover. Finally, our results did not show any differences between the clinical utility of BALP activity and BALP mass concentration measurements. Clin Chem Lab Med 2007;45:1014–8.

August 9, 2007

A simple isocratic HPLC assay to determine linezolid concentrations in different biomatrices for in vivo and in vitro studies

Stefanie Swoboda, Michael Ober, Konstantinos Anagnostakos, Heinrich K. Geiss, Markus A. Weigand, Torsten Hoppe-Tichy

Page range: 1019-1022

Abstract

Background : Linezolid is an important therapeutic option for the treatment of infections caused by multiresistant Gram-positive bacteria such as vancomycin-resistant Enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA). However, the clinical benefit of linezolid is threatened by the emergence of resistant strains of MRSA and VRE reported in North America and Europe. For effective antimicrobial treatment, it is extremely important to have exact knowledge of drug concentrations at the site of action. Methods : A simple HPLC method for the rapid and precise determination of linezolid in different biomatrices (e.g., plasma, soft tissue, bone, dialysis fluid and used microbiological broth) was developed and validated. Proteins were precipitated with acetonitrile and separation was performed on a reversed-phase C8 column with a mobile phase consisting of water/acetonitrile (80:20, v/v). UV detection was performed at 251 nm. Results : This method has a lower limit of quantification of 0.3 μg/mL and a linear calibration range of 0.5–40 μg/mL. The method showed excellent reproducibility, with an inter- and intra-day assay precision of <5% (% relative standard deviation), as well as excellent accuracy, with inter- and intra-day assay accuracy ranging from 100.6% to 103.2%. Stability up to 6 months in water and plasma was proven. Quantitative recovery was possible after up to three freeze thaw cycles. Conclusions : The method is useful in the acquisition of in vivo and in vitro data. It is simple, flexible, specific, precise and reproducible, as well as of adequate sensitivity for clinical use. Clin Chem Lab Med 2007;45:1019–22.

August 9, 2007

Pitfalls in measuring the endocannabinoid 2-arachidonoyl glycerol in biological samples

Michael Vogeser, Gustav Schelling

Page range: 1023-1025

Abstract

Background : The endocannabinoid 2-arachidonoyl glycerol (2-AG) undergoes spontaneous isomerization to biologically inactive 1-AG. This effect has not been adequately addressed in previous studies that reported 2-AG concentrations in biological samples. Methods : Liquid chromatography tandem-mass spectrometry (LC-MS/MS) was used for 1-AG and 2-AG analyses. Results : Identical collision-induced disintegration spectra were found for 1-AG and 2-AG. For specific detection of both compounds, which share a common mass transition, baseline chromatographic separation is mandatory, even when applying MS/MS technology with its generally high detection specificity. When using standard chromatographic conditions with the very short run times typically used in LC-MS/MS methods, co-elution of 2-AG with 1-AG, which is present in human serum, causes false 2-AG results. Conclusions : Our data highlight that the analytical specificity of MS/MS can be limited by interference from isobaric isomers with identical disintegration patterns. The specificity of this technology must be carefully evaluated for each individual application. Clin Chem Lab Med 2007;45:1023–5.

August 9, 2007

Partitioning reference values for several subpopulations using cluster analysis

Martin Gellerstedt, Per Hyltoft Petersen

Page range: 1026-1032

Abstract

Background : A crucial question when developing reference intervals is whether different subpopulations need their own reference interval or if a single joint reference interval can be used. It is reasonable to use partitioned reference intervals in situations where a single interval results in considerable variation in sensitivity between subpopulations. The aim of partitioning is to harmonize the sensitivity of the reference intervals, i.e., to make the sensitivity similar for all patients, regardless of patient characteristics. Statistical criteria to identify when partitioning is adequate have been developed over the last two decades. These criteria are applicable when considering two subpopulations, but recently a procedure for considering several subpopulations has been developed. When several subpopulations are considered, there is a possibility that some subpopulations could form a group or cluster that could share a common reference interval. However, there is no formal systematic approach to indicate how to divide these subpopulations into clusters. The aim of this study was to suggest such a systematic approach for clustering. Methods : A clustering technique was applied to data including several subpopulations. The technique is based on measuring the distance between separated reference limits and successively pooling subpopulations divided by short distances. A cluster is defined by a group of subpopulations that are close to each other and that differ from subpopulations in another cluster. A cluster recruits new subpopulations as long as the subpopulations can be pooled without violating a partitioning criterion. Conclusions : We have suggested a procedure for partitioning a number of Gaussian (or Gaussian-transformable) subpopulations into clusters. This is the only formalized procedure indicating how to analyze several subpopulations and identify a suitable number of groups and reference intervals. Using a computer program developed for partitioning issues, the approach was easy to adopt. Clin Chem Lab Med 2007;45:1026–32.

August 1, 2007

A plea for intra-laboratory reference limits. Part 1. General considerations and concepts for determination

Rainer Haeckel, Werner Wosniok, Farhad Arzideh

Page range: 1033-1042

Abstract

Accurate results for quantitative procedures can be useless if the reference limits for the interpretation of laboratory results are unreliable. Recent concepts for quality management systems require that laboratories pay more attention to identification and verification of reference limits. Scientific recommendations often claim that each laboratory should determine intra-laboratory reference limits, which should be reviewed periodically. This recommendation is currently neglected by most laboratories; instead they use reference limits from external sources, despite various problems of transference. Prospective and retrospective methods either using or neglecting disease prevalences (polymodal or unimodal concepts, respectively) and applying different statistical approaches for determining reference limits have been described. The various procedures are reviewed with regard to their diagnostic sensitivity, specificity and (non-)efficiency. The present gold standard is the reference limit concept according to IFCC recommendations (a unimodal prospective approach). This concept, together with trueness-based standardization, is the most useful basis for harmonization of the decision-making process with laboratory results, despite complex problems of traceability and transference. This harmonization is at present only achieved for a limited number of analytes for which SI units and traceability can be technically realized. For the majority of measurands in laboratory medicine, much research is still required and results cannot be expected in the near future. For these measurands, a need remains for internal, efficient and simple identification of population-based reference limits. Therefore, newer retrospective concepts were developed that use large data sets from laboratory information systems to derive intra-laboratory reference limits. These approaches appear promising and should be further developed. Clin Chem Lab Med 2007;45:1033–42.

August 1, 2007

A plea for intra-laboratory reference limits. Part 2. A bimodal retrospective concept for determining reference limits from intra-laboratory databases demonstrated by catalytic activity concentrations of enzymes

Farhad Arzideh, Werner Wosniok, Eberhard Gurr, Wilhelm Hinsch, Gerhard Schumann, Nicodemo Weinstock, Rainer Haeckel

Page range: 1043-1057

Abstract

Background: The current recommendations for establishing intra-laboratory reference limits (RLs) cannot be fulfilled by most laboratories because of the expense involved. In the current study, a bimodal method was developed to derive RLs from data stored in a laboratory information system without any assumption concerning the distribution of the diseased subgroup. Methods: A smoothed kernel density function (D mix ) was estimated for the distribution of combined data for non-diseased and diseased adult subjects. It was assumed that the “central” part of the distribution represents the non-diseased population, which was defined and used to estimate a Gaussian distribution of either the original values or Box-Cox transformed data. This normal distribution was now considered the distribution of the non-diseased subgroup (D nd ). Percentiles were calculated to obtain retrospective RLs. The density function of the diseased subgroup (D d ) was calculated by subtracting the non-diseased density function from D mix (D d =D mix –D nd ). The intersection point of the D nd and D d curves identified the RL with the highest diagnostic efficiency. Results: The model was applied to catalytic activity concentrations of several enzymes with data from different laboratories. The RLs obtained were similar to recently published consensus values. Differences between laboratories were small but significant. Gender stratification was necessary for alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutymaltransferse (γ-GT), not significant for lipase and amylase and inconsistent among the laboratories for alkaline phosphatase (AP) and lactate dehydrogenase (LDH). Age stratification was only tested for two groups (18–49 and ≥50 years) and was significant for AST (females only), γ-GT and lipase, not significant for amylase and inconsistent for AP, LDH and ALT. For γ-GT, further stratification for age in decades was necessary for males. Creatine kinase MB (CK-MB) values were not stratified owing to the low number of data available. Conclusions: Retrospective RLs derived from intra-laboratory data pools for the catalytic activity concentration of enzymes using a modified procedure plausibly agreed with published consensus values. However, most RLs varied significantly among laboratories, thus supporting the “old” plea for intra-laboratory RLs. Clin Chem Lab Med 2007;45:1043–57.

August 9, 2007

Biological variation of thyroid autoantibodies and thyroglobulin

Esther Jensen, Per Hyltoft Petersen, Ole Blaabjerg, Laszlo Hegedüs

Page range: 1058-1064

Abstract

Background : It has been shown that the level of serum thyroid antibodies affects serum thyrotropin (TSH) concentrations in men and women, and that these autoantibodies in combination with serum TSH are predictive of future thyroid disease. As the biological variation of these autoantibodies is unknown, we investigated this in fertile women during one complete regular menstrual cycle. Methods : A total of 24 healthy women (23–46 years) were investigated twice a week between 07:30 and 11:00 h. Antibodies against thyroid peroxidase (TPOAb), thyroglobulin (TgAb), and thyrotropin receptor (TRAb) were measured in serum, as well as thyroglobulin (Tg). TPOAb, TgAb and Tg were determined on an AutoDELFIA system (Perkin Elmer/Wallac) and TRAb by a radioreceptor assay from Brahms Diagnostica. Results: All 24 women had measurable levels of TPOAb and TgAb in all samples, and nine women had antibodies above the upper reference limit of the laboratory (6 had TPOAb >10 kIU/L, 6 had TgAb >20 kIU/L and 1 had TRAb >0.75 IU/L). Eight women had Tg below the lower reference limit, five of whom had elevated TgAb. Variations in the thyroid antibodies were random and not related to the menstrual cycle. For TPOAb (2.5–258 kIU/L), the CV biological was 11.3%, while the CV analytical was 10.6%. For TgAb (5.6 to 148 kIU/L) CV biological was 8.5% and CV analytical was 9.0%. The woman with TRAb had a CV biological of 4.8%, while the analytical variation in duplicates was 3.9% at a level of 2.8 IU/L. Conclusions : It is possible to measure TPOAb and TgAb in all samples with the AutoDELFIA. There is no systematic variation in autoantibodies during the menstrual cycle. The biological coefficient of variation for TPOAb and TgAb was 11.3% and 8.5%, respectively. Clin Chem Lab Med 2007;45:1058–64.

August 9, 2007

Monitoring glycaemic control: is there evidence for appropriate use of routine measurement of glycated haemoglobin?

Gian Luca Salvagno, Giuseppe Lippi, Giovanni Targher, Martina Montagnana, Gian Cesare Guidi

Page range: 1065-1067

Abstract

Background : Regardless of the available recommendations to perform glycated haemoglobin testing at a 2- to 3-month frequency, there is increasing evidence of an inappropriate laboratory use of this test in clinical practice. Methods : Data from our Laboratory Information System were analysed for glycated haemoglobin test orders over a 3-year period using Microsoft ® Excel to calculate the order intervals and the test frequency for each patient. To assess the appropriateness of repeat testing, only data for patients who had at least two separate glycated haemoglobin test results were included in the analysis. Inappropriate test orders were defined as any order for a given patient taking place within a 29- or 89-day period following the previous order. Results : The results of our investigation demonstrate that inappropriate laboratory utilisation of this test is commonplace (26% of total repeat orders within 90 days), especially for inpatients (63.7% of inpatient repeat orders in less than 90 days). When stratifying glycated haemoglobin test results according to the >7% threshold, the frequency of inappropriate laboratory use (>90 days) was surprisingly greater among inpatients with a previous value of <7% than among those with a previous value of >7% (57.6% vs. 42.4%). The frequency of inappropriate glycated haemoglobin repeat test orders was lower among outpatients with a previous value of <7% than in outpatients with a previous value of >7% (64.8% vs. 35.2%). Conclusions : We conclude that more accurate application of the current recommendations would be advisable to decrease unnecessary testing and prevent avoidable health expenditure. Clin Chem Lab Med 2007;45:1065–7.

August 9, 2007

Performance and utility of a cost-effective collagen-binding assay for the laboratory diagnosis of Von Willebrand disease

Muriel Meiring, Philip N. Badenhorst, Mareli Kelderman

Page range: 1068-1072

Abstract

Background : The collagen-binding assay, a functional assay of Von Willebrand factor (VWF), discriminates the subtypes of Von Willebrand disease (VWD). Commercial collagen binding assays have the advantage of immediate use for fast results, but are expensive. Methods : In this study we evaluated an in-house collagen-binding assay using type III collagen. We included it in the diagnostic work-up of 44 patients with VWD and 40 normal subjects. Other assays included VWF antigen, ristocetin cofactor activity, ristocetin-induced platelet agglutination and VWF multimeric analysis. Results : The cost of this collagen-binding assay is 10-fold lower than that of commercial kits. The intra- and inter-assay coefficients of variation were <8% and <9% for normal values, respectively, and the normal reference range varies between 51% and 143%. This assay is sensitive to large VWF multimer representation, with a mean collagen-binding activity/antigen level (CB/Ag) ratio of 0.18 and 0.45 for type 2A and type 2B VWD, respectively, indicating functional discordance. It correlates with the antigen levels in type 2M and type 1 VWD, with mean CB/Ag ratios of 1.1 and 1, respectively. Conclusions : Our cost-effective in-house collagen-binding assay produced reliable results. We recommend the use of this test together with the ristocetin cofactor test in the diagnostic work-up of VWD. Clin Chem Lab Med 2007;45:1068–72.

August 1, 2007

Hook effect in calcitonin immunoradiometric assay

Mariasilvia Tommasi, Silvia Raspanti

Page range: 1073-1074

August 1, 2007

Rapid genotyping of the Ser447Stop variant of the lipoprotein lipase gene using real-time fluorescent PCR

Arizumi Kikuchi, Yuko Kuramoto, Nobuyasu Noritake, Hiroshi Murase, Osami Daimaru, Takeo Nakakita, Shinichi Itoh

Page range: 1075-1076

August 9, 2007

Global standardization of glycated hemoglobin measurement: the position of the IFCC Working Group

International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) IFCC Scientific Division, Working Group on Standardization of HbA1c (WG-HbA1c): Andrea Mosca, Ian Goodall, Tadao Hoshino, Jan O. Jeppsson, W. Garry John, Randie R. Little, Kor Miedema, Gary L. Myers, Hans Reinauer, David B. Sacks, Cas W. Weykamp

Page range: 1077-1080

Abstract

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 βN1-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. Clin Chem Lab Med 2007;45:1077–80.

August 9, 2007

Recommendation for term and measurement unit for “HbA1c”

International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) IFCC Scientific Division, Committee on Nomenclature, Properties and Units (C-NPU): Gunnar Nordin, René Dybkær

Page range: 1081-1082

Abstract

Clin Chem Lab Med 2007;45:1081–2.

August 9, 2007

Desirable performance standards for HbA_1c analysis – precision, accuracy and standardisation Consensus statement of the Australasian Association of Clinical Biochemists (AACB), the Australian Diabetes Society (ADS), the Royal College of Pathologists of Australasia (RCPA), Endocrine Society of Australia (ESA), and the Australian Diabetes Educators Association (ADEA)

Ian Goodall, Peter G. Colman, Hans G. Schneider, Mark McLean, George Barker

Page range: 1083-1097

Abstract

Background : HbA 1c (glycohaemoglobin) is universally used in the ongoing monitoring of all patients with diabetes. There are many % HbA 1c target control rating recommendations by national, regional and international expert bodies for diabetes patients and these are variable around the world. General patient target control ratings are currently most often recommended as either <6.5% or <7.0% HbA 1c , with <6.0% HbA 1c stated for individual patients where clinically possible. This necessitates very precise HbA 1c assays and the same patient values, irrespective of HbA 1c method or area of the world. Methods : HbA 1c targets recommended by major expert groups and published HbA 1c assay precision (coefficient of variation, %CV) levels have been detailed. These have been compared with published biological variation levels and with calculated HbA 1c error ranges at various HbA 1c levels and %CV levels. In addition, these have been compared with the analytical precision necessary to differentiate between the upper limit of the normal range for HbA 1c and targets recommended by expert groups for diabetes control. Results : Intralaboratory analytical CVs of <2% are necessary and are achievable on automated HPLC analysers, and are supported on grounds of both clinical need and biological variation, as well as the need to differentiate the national, regional and international target recommendations from the upper limit of the normal range (<6.0% HbA 1c level). Conclusions : Routine methods with tight long-term imprecision with CVs of <2% are recommended. International HbA 1c targets essentially require that all HbA 1c methods be precise, and have minimal standardisation bias and minimal methodological interferences in individual patients. Clin Chem Lab Med 2007;45:1083–97.

August 9, 2007

IFCC Position Paper: Report of the IFCC Taskforce on Ethics: Introduction and framework

Leslie Burnett, Matthew J. McQueen, Jon Johannes Jonsson, Francesca Torricelli

Page range: 1098-1104

Abstract

Laboratory Medicine organizations and their professional members have a goal and responsibility to benefit the health and wellbeing of the patients and communities they serve. Newer genetics and biochemical techniques raise significant issues of community concern, impacting on privacy, informed consent, access to and retention of samples and information. Balance may be required to ensure protection of individual rights against potential benefits to the broader community. While many national organizations may already have appropriate policies addressing various ethics issues, there is a need for an international framework to assist those nations that have not yet developed such policies, as well as to enable alignment of existing national policies. We have proposed a generic ethics framework, incorporating a hierarchy of four fundamental guiding principles: autonomy, justice, non-maleficence and beneficence. Proposals or issues requiring policy development can be considered and tested against this hierarchy, resulting in the development of policy and positions consistent with the above framework, acceptable to all participating stakeholders. Clin Chem Lab Med 2007;45:1098–104.

August 1, 2007

IFCC Awards: Call for Nominations

Page range: 1105-1105

Publicly Available August 1, 2007

Call for papers, ideas and initiatives

Page range: 1106-1106

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Clinical Chemistry and Laboratory Medicine (CCLM) publishes articles on novel teaching and training methods applicable to laboratory medicine. It is focused on basic and applied research and cutting-edge clinical laboratory medicine. CCLM is one of the leading journals in the field, with an impact factor over 8. All contributions submitted for publication in CCLM are single-blind peer reviewed by at least two experts in the field. CCLM is led by a multi-institutional editorial board. It is issued monthly, both in print and electronically. Letters to the Editor and Congress Abstracts are published online only.

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