Clin Chem Lab Med Research Articles

Biological variation of free plasma amino acids in healthy individuals

Biological variation data for free plasma amino acids are lacking from the more comprehensive databases. Therefore, we determined the intra- and inter-individual components of variation in healthy subjects. These data were used to define desirable goals for imprecision, bias and total error, indices of individuality and reference change values. Plasma samples were collected from 11 volunteers at weekly intervals over 5 weeks. Free plasma amino acids were analyzed by reversed-phase HPLC and analytical and biological variation data were derived using ANOVA. Intra-individual coefficients of variation ranged from 9.5% to 46.4%, with lower values among the essential amino acids. The mean inter-individual coefficient of variation was 46.6% and was higher than intra-individual values for all amino acids. Thus, indices of individuality were below 0.8. Reference change values ranged from 30.9% to 128.4% and total error values ranged from 15.2% to 61.0%. Plasma amino acids exhibit relatively low intra-individual coefficients of variation, with essential amino acids showing tighter homeostatic control. Analytical quality goals can be reasonably achieved with current methods. Reference intervals are of limited value in the detection of unusual results in an individual. Therefore, comparison of serial results by means of the reference change values is recommended.

Inverse relationship between serum high density lipoprotein and negative syndrome in antipsychotic-naive schizophrenia

Recent literature suggests a role for apolipoprotein L (apoL) aberrations in the pathogenesis of schizophrenia. ApoL is almost exclusively associated with apolipoprotein A-I in high-density lipoproteins (HDLs). The objective of this study was to examine the correlation between symptom scores and serum HDL in antipsychotic-naive schizophrenia patients. In this cross-sectional study, 60 antipsychotic-naive schizophrenia patients were systematically examined for their symptom scores, with good inter-rater reliability. Concurrently, an overnight fasting serum lipid profile from these patients was assessed. Serum HDL had a significant inverse correlation with a total negative syndrome score (rho=-0.43; p=0.001). The study observation supports the potential role for HDL abnormalities in the genesis of negative symptoms in schizophrenia.

Differences in serum potassium concentrations between Chinese, Indians and Malays

It has been suggested that potassium concentrations may vary between different geographical regions, possibly reflecting ethnic differences in potassium status. This study compared the serum potassium concentrations of three Asian ethnicities in a single geographical location. Details of simultaneous serum potassium, creatinine, cholesterol, triglyceride and serum index measurements for samples from polyclinics and health screening were extracted for multivariable linear regression. Haemolysed and duplicate patient samples were excluded. Separate analysis was performed based on measurement platform (Roche or Beckman-Coulter) and patient location. Eighty-five thousand nine hundred and ninety-seven records met the inclusion criteria. When controlled for age, gender, serum creatinine, cholesterol and triglyceride, the average serum potassium concentration in Indians was 0.13-0.16 mmol/L higher than in Malays, who in turn had average serum potassium concentrations 0.05-0.06 mmol/L higher than Chinese when controlled for age, gender, serum creatinine, cholesterol and triglyceride concentrations. For patients undergoing health screening, the average serum potassium concentration in Indians and Malays was 0.12 mmol/L higher than in Chinese. Chinese individuals have lower average serum potassium concentrations than Indians and Malays. This may have clinical implications in relation to the high occurrence of thyrotoxic hypokalaemic paralysis and the aetiology of sudden unexplained death syndrome (SUDS) in Asians.

Managing quality vs. measuring uncertainty in the medical laboratory

ISO 15189's particular requirements for quality management in medical laboratories provide guidance for (a) relating performance specifications to the intended use of a test or examination procedure, (b) designing internal quality control (IQC) procedures to verify the attainment of the intended quality of test results, as well as (c) determining the uncertainty or results, where relevant and possible. This guidance has particular implications for analytical quality management, specifically for validating method performance relative to quality goals or requirements (intended use), designing statistical quality control procedures on the basis of the quality required for a test and the precision and bias observed for a method, and characterizing the quality achieved in practice by calculating measurement uncertainty. There already exists an error framework that provides practical tools and guidance for managing analytical quality, along with an existing concept of total error that can be used to characterize the quality of laboratory tests, thus there is considerable concern and debate on the merits and usefulness of measurement uncertainty. This paper argues that total error provides a practical top-down estimate of measurement uncertainty in the laboratory, and that the ISO/GUM model should be primarily directed to and applied by manufacturers.

The role of anti-core antibody response in the detection of occult hepatitis B virus infection

Occult hepatitis B virus (HBV) infection is characterized by the presence of HBV DNA in serum and/or in the liver of patients negative for hepatitis B surface antigen (HBsAg). Occult infection may impact in several different clinical contexts including the risk of HBV transmission with transfusion or transplantation, and endogenous viral reactivation. The gold standard test for detection of occult infection is the amplification of HBV DNA. However, the serological assay for the long-lasting antibody response to the highly immunogenic HBV core antigen (anti-HBc) represents a qualified candidate as a surrogate for DNA amplification, or for increasing overall sensitivity when assessing the risk of occult hepatitis in peripheral blood. The risk of occult hepatitis associated with anti-HBc seropositivity has been demonstrated extensively, and the presence of antibody response to HBc can be considered a sentinel marker of occult HBV infection.

Achieving continuous improvement in laboratory organization through performance measurements: a seven-year experience

Laboratory performance can be measured using a set of model key performance indicators (KPIs). The design and implementation of KPIs are important issues. KPI results from 7 years are reported and their implementation, monitoring, objectives, interventions, result reporting and delivery are analyzed. The KPIs of the entire laboratory process were obtained using Laboratory Information System (LIS) registers. These were collected automatically using a data warehouse application, spreadsheets and external quality program reports. Customer satisfaction was assessed using surveys. Nine model laboratory KPIs were proposed and measured. The results of some examples of KPIs used in our laboratory are reported. Their corrective measurements or the implementation of objectives led to improvement in the associated KPIs results. Measurement of laboratory performance using KPIs and a data warehouse application that continuously collects registers and calculates KPIs confirmed the reliability of indicators, indicator acceptability and usability for users, and continuous process improvement.

Impact of calibration fitting models on the clinical value of chromogranin A

The clinical relevance of chromogranin A (CgA) concentrations depends on the analytical performance of the assay. The goal of the present study was to define the clinical involvements in CgA calibration models by evaluating the confidence intervals (CIs) for values from patients who were undergoing monitoring for disease. Thirty calibration curves for the CgA assay [immunoradiometric assay (IRMA), (CIS-BIO)] were built using linear regression (LR), and four-parameter logistic models were used to estimate CIs for patient concentrations. We reported the inadequacy of the LR curve estimation procedure. We showed: 1) no evidence that the straight calibration line could fit the average responses, 2) non-constant and non-uniform variance of the replicated calibration responses. All tests performed in the analysis of variance and CI calculation for the calibration curve should be invalidated. The four-parameter logistic function yielded results for 16 curves only; this result could be due to the low number and inappropriate concentration of calibrators. This suggests that some aspects of the assay design should be reviewed. However, using the variance function estimated in this model, we could assess the CI for calibration curves and patient samples. We showed that the four-parameter logistic calibration model with estimated variance function should better support clinical interpretation of marker concentration changes in patients serially tested.

Serum thyrotropin and free thyroxine reference ranges as defined in a disease-free sample of French middle-aged adults

The goal of this study was to provide reference ranges for thyrotropin (TSH) and free thyroxine (fT(4)) based on data collected from a disease-free sample of French middle-aged adults. A total of 3218 subjects participating in the Supplémentation en Vitamines et Minéraux Antioxydants (SU.VI.MAX) study had measurements for TSH, fT(4) and urinary iodine concentrations. Thyroid volume and structure were evaluated using standardized ultrasonography. We selected a disease-free sample which included 2338 subjects (1313 females aged 35-60 years and 1025 males aged 45-60 years) with normal thyroid imaging, no previously reported thyroid disease or use of thyroid medication, and no risk factors for thyroid dysfunction. Distribution of TSH and fT(4) was estimated in males and females. The median (central 95% range) TSH serum concentrations for females were 1.79 mU/L (0.29-5.21 mU/L) for ages 35-44 years and 1.98 mU/L (0.27-6.94 mU/L) for ages 45-60 years (p<0.0001, for age). The median (central 95% range) for males 45-60-year-old was 1.63 mU/L (0.28-4.54 mU/L) (p<0.0001, for sex). Sex- and age-specific mean fT(4) concentrations did not differ significantly (p=0.06) between males and females and (p=0.08) between female age groups. However, median fT(4) concentrations between 45-60-year-old males and females differed (p<0.001). In middle-aged adults, the TSH distribution was associated with gender and, among females, with age. Stratification according to gender and age should be considered when TSH ranges are used in the diagnosis, treatment and monitoring of thyroid disease. Clin Chem Lab Med 2009;47:1497-505.

Multiple gene interaction and modulation of hemostatic balance

Recent progress in the understanding of the pathophysiology of hemostasis has established that blood coagulation disorders causing bleeding or thrombosis often indicate a multi-casual nature in which the interaction between genetic and acquired factors plays a major mechanistic role. The interaction of two or more factors may attenuate clinical symptoms. However, a synergic worsening may also occur, resulting in a higher risk than expected from the combination of the separate effects. The role of genetic factors on the modulation of the phenotypic expression of coagulation disorders will be addressed in this review, with particular emphasis on the underlying pathogenic mechanisms and clinical implications.

Therapeutic drug monitoring and drugs of abuse testing on the cobas® 6000 analyzer series: analytical performance under routine-like conditions

The analytical performance of the clinical chemistry module c 501 (cobas 6000 analyzer series) was evaluated for therapeutic drug monitoring and drugs of abuse testing using a spectrum of representative assays. Particular attention was paid to potential interactions between reagents using a simulated routine workload. Within-run and total imprecision were assessed using a selection of representative reagents. Deviation from a consensus mean was tested using samples from a proficiency testing scheme. Method comparison using routine samples was carried out against the MODULAR ANALYTICS SWA and COBAS INTEGRA 800 analysis systems. Total coefficients of variation (CV) ranged from 1.9% to 7.8% for individual drugs, and from 3.2% to 8.6% for drugs of abuse testing. Results from proficiency test samples were between 81% and 125% of the consensus mean for therapeutic drugs. Method comparisons (Passing-Bablok regression) showed overall good comparability to MODULAR ANALYTICS SWA and COBAS INTEGRA 800 systems, with slopes from 0.93 to 1.17 and correlation coefficients r > 0.98. Imprecision in a simulated routine run was tested using a total of 42 methods (10 therapeutic drug monitoring, 9 drugs of abuse testing, 3 enzymes, 12 substrates, 8 specific protein assays). Imprecision in the reference batch run ranged from 0.7% to 5.0% CV for therapeutic drug monitoring assays, except for digoxin (DIG) (7.3%), and from 0.9% to 7.7% for drugs of abuse testing. The CVs of general clinical chemistry and specific protein tests were within the expected limits of 2% and 4%. CV changes in the simulated routine run were within the expected limits for most assays. Negative DeltaCVs (> or = 2%) for DIG, digitoxin (DIGIT), cannabinoids (THC), and phencyclidine (PCP) may indicate improved performance when running these assays in a simulated routine operation. A positive DeltaCV (> or = 3%) was found for amphetamines (AMPHs). In conclusion, the cobas c 501 module seems to be well-suited for routine use as consolidated workstation. Except for a potential interaction with AMPH, as indicated by the positive DeltaCV, no significant interferences from different reagents could be observed during this study.

Potential utility of the new Sysmex XE 5000 red blood cell extended parameters in the study of disorders of iron metabolism

New erythrocyte parameters are reported by the Sysmex XE 5000 analyzer. This instrument measures the hemoglobin (Hb) content of individual red cells, calculates the percentage of hypochromic red cells (%Hypo He) and the percentage of hyperchromic red cells (%Hyper He) and quantifies the proportion of marginally sized erythrocytes (%Micro R and %Macro R). The goals of the study were to establish the reference range for erythrocyte extended parameters, their value in different types of anemia and to investigate their reliability in the study of disorders of iron metabolism. Three hundred and ninety samples were analyzed. The Kolmogorov-Smirnoff test, independent samples t-test and Pearson correlation were calculated; receiver operating characteristic (ROC) curve analysis was used to determine their diagnostic performance. The values of the four parameters studied were normally distributed and statistically different (p<0.0001) in the different groups of patients; the only exception was %Hypo He in cases of iron deficiency and thalassemia (p=0.3758). Results of ROC curve analysis for %Hypo He in the diagnosis of restricted erythropoiesis reticulocyte Hb content (reticulocyte hemoglobin content, Ret He <29 pg) were: area under the curve 0.963; cut-off 1.8%; sensitivity 98.3%; specificity 91.1%. The new parameters appear to be sensitive for detecting small changes in the number of red cells with inadequate hemoglobinization and volume.

New European guidelines for cardiovascular disease prevention in clinical practice

In 2007, the Fourth Joint European Societies' Task Force on cardiovascular disease (CVD) prevention in clinical practice issued an update of the guidelines on how to prevent CVD. These guidelines are endorsed by 10 international societies interested in the subject. Particular attention is given to lifestyle changes, risk factor management, and the use of cardioprotective drugs. In this paper, the content of the guidelines is summarized with emphasis on what is new compared to the previous version and on what could be of particular interest to clinical chemists. For those who would like to read more on this, please refer to the full document (112 pages including 1105 references).

Detection of myelin autoantibodies: evaluation of an assay system for diagnosis of multiple sclerosis in differentiation from other central nervous system diseases

Multiple sclerosis (MS) is a frequent and often severe autoimmune disease of the central nervous system. We describe a newly developed enzyme-linked immunosorbent assay (ELISA)-based test system for the assessment of neuronal autoantibodies in serum and cerebrospinal fluid (CSF). This tool could help define autoimmune status and thus be a potential means of therapeutic surveillance. We used an assay system (ELISA, E100, Mediagnost) based on purified bovine antigens [myelin-oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), myelin-associated glycoprotein (MAG), proteolipid protein (PLP) and alpha-B-crystalline (CRY)] antibodies for the measurement of specific immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies. Assay characteristics and preliminary validation were conducted by measurement of serum and CSF samples from 41 MS patients and 128 patients with other neurological diseases (OND). We measured the inter- (17.8/10.1%) and intra-assay variability (5.5/6.7%); linearity (1:250- 1:16,000), and specificity of IgG and IgM. We demonstrate that by the results of this test system MS patients can be differentiated from patients with OND. The ELISA kit we evaluated is suitable for the measurement of neuronal autoantibodies. The initial validation demonstrates its potential use in the differential diagnosis of central neuronal system diseases.

Clinical relevance of circulating midkine in ulcerative colitis

Non-invasive biochemical markers are needed to support the diagnosis of ulcerative colitis (UC), an incurable disease of unknown pathology. Midkine is an angiogenic cytokine, chemotactic towards neutrophils and macrophages, and a T-regulatory cell suppressor. Serum midkine was measured immunoenzymatically in 93 UC patients and 108 healthy subjects, and evaluated with respect to disease status, endoscopic, inflammatory and angiogenic activity. The diagnostic value of midkine was compared to C-reactive protein (CRP) using receiver operating characteristics (ROC) analysis. Midkine was higher (p<0.0001) in inactive (199 ng/L) and active UC (351 ng/L) compared with controls (93 ng/L), and reflected disease activity (r=0.427, p<0.001). Midkine was correlated with CRP, erythrocyte sedimentation rate (ESR), leukocytes, platelets, interleukin-6, paraoxonase-1, albumin, transferrin, iron, hemoglobin, and hematocrit. Midkine correlated with angiogenic factors: vascular endothelial growth factor-A and platelet-derived growth factor-BB. As a marker of UC, midkine showed a diagnostic accuracy of 85%, sensitivity of 72%, specificity of 82%, whereas CRP showed 83%, 65% and 91%, respectively. As a marker of active UC, midkine showed a diagnostic accuracy of 87%, sensitivity of 84%, specificity of 75%, whereas CRP showed 75%, 63% and 83%, respectively. Combined assessment of midkine and CRP improved sensitivity but substantially decreased specificity. UC is associated with increased circulating midkine, which corresponds with clinical, endoscopic, inflammatory and angiogenic activity, and anemia. Performance of midkine as a marker of UC or active UC was comparable to that of CRP.

Expression of lineage markers using real-time quantitative polymerase chain reaction (RT-qPCR) in normal and in leukemia bone marrow

The study of lineage markers by real-time quantitative polymerase chain reaction (RT-qPCR) at diagnosis enables differentiation between acute myeloblastic leukemia, B- or T-lineage acute lymphoblastic leukemia, without cell sorting. Our objective was to assess the relationship between protein expression and the amount of lineage marker mRNA in acute leukemia samples and to determine whether four lineage markers could be used to differentiate between normal and acute leukemia bone marrow (BM) without cell sorting. Quantification of the mRNA of CD19, CD79a, CD3e, and myeloperoxidase was performed by RT-qPCR on 130 acute leukemia BM samples at diagnosis and on 20 BM samples from healthy donors, without cell sorting. Immunophenotyping of leukemia samples was performed after manual gating around the blastic population. Reference values for the four lineage markers were established by RT-qPCR for normal BM. The mRNA expression levels of these four lineage markers allowed the distinction between normal samples and 100% of acute leukemia samples. With 92% congruence for protein expression and amount of mRNA in acute leukemias, these four lineage markers, essential for diagnosis and subclassification of acute leukemias by flow cytometry, also represent excellent candidate genes when using RT-qPCR technology as a diagnostic tool for molecular cancer class prediction.

Pancreatic cancer biomarkers discovery by surface-enhanced laser desorption and ionization time-of-flight mass spectrometry

Surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF/MS), a laboratory-friendly technique, is used to identify biomarkers for cancer. The aim of the present study was to explore the application of SELDI proteomic patterns in serum for distinguishing between cases of pancreatic cancer, chronic pancreatitis, type 2 diabetes mellitus and healthy controls. Sera from 12 healthy controls, 24 patients with type 2 diabetes mellitus, 126 with pancreatic cancer, including 84 with diabetes, and 61 with chronic pancreatitis, 32 of which were diabetics, were analyzed using SELDI-TOF/MS. Spectra (IMAC-30) were clustered and classified using Biomarker Wizard and Biomarker Pattern software. Two decision tree classification algorithms, one with and one without CA 19-9, were constructed. In the absence of CA 19-9, the splitting protein peaks were: m/z 1526, 1211, and 3519; when CA 19-9 was used in the analysis, it replaced the m/z 3519 splitter. The two algorithms performed equally for classifying patients. A classification tree that considered diabetic patients only was constructed; the main splitters were: 1211, CA 19-9, 7903, 3359, 1802. With this algorithm, 100% of patients with type 2 diabetes mellitus, 97% with chronic pancreatitis and 77% of patients with pancreatic cancer were correctly classified. SELDI-TOF/MS features improved the diagnostic accuracy of CA 19-9 (AUC = 0.883 for CA 19-9; AUC = 0.935 for CA 19-9 and SELDI-TOF/MS features combined). SELDI-TOF/MS allows identification of new peptides which, in addition to CA 19-9, enable the correct classification of the vast majority of patients with pancreatic cancer, which can be distinguished from patients with chronic pancreatitis or type 2 diabetes mellitus.

Contributions in Biochemistry and Molecular Biology from China and other top-ranking countries: a 10-year survey of the literature

Over the past decade, research in Biochemistry and Molecular Biology has developed rapidly worldwide. The present study was designed to study the characteristics of publications in Biochemistry and Molecular Biology journals between 1999 and 2008 from the six top-ranking countries - the United States (USA), Japan, Germany, United Kingdom (UK), China and France. We also examined the research output from three different regions - Mainland China (ML), Hong Kong (HK) and Taiwan (TW). The USA contributed 34.1% of the world's total output and ranked first, but its percentage of research articles in this field went down. In total, 26,867 articles were published in journals with an impact factor (IF) <10.000, and 46.5% of these were from the USA. China contributed 4.2% of the total in 268 journals, 0.5% of which were journals with the top 10 IF. Our analysis describes the output from each country and region, and reveals the positive trend in China during the period of 1999-2008. In contrast to other countries, our results suggest that China is behind in conducting high-quality research.

The future of hospital laboratories. Position statement from the Royal Belgian Society of Clinical Chemistry (RBSCC)

To face the economic pressures arising from the current socio-economic conjuncture, hospital laboratories are endangered by an increasing trend towards the outsourcing of clinical laboratory tests to external (mega-) laboratories. This should allow hospitals to meet their economic requirements, but with an increased risk of loss of medical quality and, mid- to long-term, loss of cost effectiveness of healthcare at the national level. To anticipate current developments (economical and technological) that inevitably will affect the future of laboratory medicine, hospital laboratories should be proactive and enhance efficiency, reduce costs by consolidation, integrate into regional networks, and form alliances or partnerships. To create additional value, the core competency of laboratory professionals must be refocused to provide medical knowledge services (consultative support to clinicians) related to in vitro diagnostic testing. To integrate cost-efficiency with medical quality, implementation of a matricial organization - operational vs. biomedical level - could be an interesting approach. This integrated structure should create total quality of laboratory testing, managing the entire medical diagnostic cycle from the pre-preanalytical to post-postanalytical phase.

Novel rare alleles of ABCA1 are exclusively associated with extreme high-density lipoprotein-cholesterol levels among the Han Chinese

High-density lipoprotein (HDL) is a major plasma lipoprotein directly associated with cholesterol metabolism. The ATP binding cassette transporter 1 gene (ABCA1) is one of the major genes modulating plasma levels of HDL-cholesterol (HDL-C). Rare alleles of ABCA1 associated with extreme HDL-C concentrations have not been previously investigated in the Chinese. Blood samples were collected from 470 subjects whose HDL-C concentrations were within the top 5% of the distribution, 335 subjects in the lowest 5%, and 220 within the range 5%-95%. First, we sequenced all exons of the ABCA1 gene from 50 subjects from the group with extremely high HDL-C, and 50 from the group with extremely low HDL-C concentrations. Next, in the remaining subjects, we genotyped the non-synonymous variants identified exclusively with either extreme group. Four novel non-synonymous alleles were identified; all were rare. Alleles c.3029C>T (p.Ala1010Val) and c.5399A>G (p.Asn1800Ser) were found exclusively in the low group, c.2031C>A (p.Asp677Glu) and c.2660G>T (p.Cys887Phe) exclusively in the high group. Our results show that some rare alleles of ABCA1 are associated with marked phenotypes, supporting the "rare-variant common-disease" hypothesis. Certain alleles also provide tools for identifying individuals at high risk of dyslipidaemia, allowing for early therapeutic intervention.

Potential of the Sebia Capillarys® neonat fast automated system for neonatal screening of sickle cell disease

Most screening programs for sickle cell disease (SCD) utilize isoelectric focusing (IEF) or high performance liquid chromatography (HPLC) to detect haemoglobin (Hb) variants. The first method is not automated and becomes too tedious when many samples have to be investigated. The aim of this work is to explore the capacity of an automated capillary electrophoresis (CE) system, with full traceability, as a tool for newborn screening of SCD. The Capillarys neonat fast automated system has been developed by Sebia for newborn screening. We performed separate studies using different types of samples to evaluate the utility of the Capillarys for (i) separating Hb S and other variants, and (ii) for performing the routine activity of our laboratory for 20 working days. A throughput of 48 samples per hour with a loading capacity of 192 samples was achieved. Migration times of the major Hb variants were distinct. There were few variants showing similar migration times to Hb S and Hb C and thalassaemia could be detected. In addition, late screening, screening of premature or transfused babies and screening performed using poor quality Guthrie's cards did not interfere with reporting of accurate phenotypes. Sebia Capillarys neonat fast automated system is a reliable tool for haemoglobinopathy neonatal screening.