Clinical Chemistry and Laboratory Medicine (CCLM)

Published by De Gruyter

Volume 39 Issue 5

Issue of Clinical Chemistry and Laboratory Medicine (CCLM)

Contents
Journal Overview

October 7, 2005

SIBioC 2001 33rd National Congress of the Italian Society of Clinical Biochemistry and Clinical Molecular Biology

Page range: A1-A24

June 1, 2005

CCLM: The Near Future

Marek H. Dominiczak

Page range: 373-373

June 1, 2005

Granulocyte-Colony Stimulating Factor and Macrophage-Colony Stimulating Factor in Patients with Non-Small-Cell Lung Cancer

Barbara Mroczko, Maciej Szmitkowski, Jacek Niklinski

Page range: 374-379

Abstract

We have investigated the serum level of granulocyte-colony stimulating factor (G-CSF) and macrophagecolony stimulating factor (M-CSF) in non-small-cell lung cancer (NSCLC), in relation to the control group and commonly accepted tumor markers, such as carcinoembryonic antigen (CEA) and cytokeratin fragment 19 (CYFRA 21-1). Additionally, we have defined the diagnostic sensitivity, specificity, positive predictive value, negative predictive value and receiver-operating characteristics (ROC) curve of G-CSF and M-CSF. Serum levels of cytokines were measured in 61 patients with NSCLC and in 20 healthy subjects. G-CSF and M-CSF were determined using ELISA. CYFRA 21-1 was measured by radioimmunoassay and CEA by microparticle enzyme immunoassay. There were significant increases in the level of circulating G-CSF in the lung cancer patients compared to the control group. Moreover, the diagnostic sensitivity of G-CSF was higher (56%) than the sensitivity of CYFRA 21-1 (51%), but lower than the CEA sensitivity (62%). The diagnostic specificity of G-CSF was higher (70%) than the M-CSF specificity (40%) and the G-CSF predictive values were higher in relation to the predictive values of M-CSF. These results suggest a potential role of G-CSF as a tumor marker for NSCLC.

June 1, 2005

Circulating Matrix Metalloproteinases and Their Inhibitors in Premature Coronary Atherosclerosis

Yoshihiro Noji, Kouji Kajinami, Masa-aki Kawashiri, Yasuhiro Todo, Takahiro Horita, Atsushi Nohara, Toshinori Higashikata, Akihiro Inazu, Junji Koizumi, Tadayoshi Takegoshi, Hiroshi Mabuchi

Page range: 380-384

Abstract

To investigate the clinical significance of circulating matrix metalloproteinases (MMPs) and their tissue inhibitos (TIMPs) in patients with premature coronary atheroscrelosis, we studied 53 consecutive male patients with angiographically defined premature (<65 years) and stable coronary artery disease. Plasma levels of MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 were determined in peripheral blood by a sandwich enzyme immunoassay, and the results were compared with those from 133 age-matched control males. There were significant differences in all the MMPs and TIMPs (p<0.001) between patients and controls. In the patient group, the levels of MMP-9 (mean±SD (ng/ml) 27.2±15.2/21.8±15.2) and TIMP-1 (130.4±55.7/94.5±26.3) were significantly higher, and the levels of MMP-2 (632.5±191.6/727.6±171.4), MMP-3 (53.1±31.2/79.6± 29.9), and TIMP-2 (24.7±15.2/35.4±16.4) were significantly lower than those of controls. We found significant positive correlation between plasma MMP-9 levels and low-density lipoprotein (LDL)-cholesterol levels (Rs=0.168, p=0.022), and significant negative correlation between plasma MMP-9 levels and high-density lipoprotein (HDL)-cholesterol levels (Rs=−0.164, p=0.026) by Spearman rank correlation test. In contrast, plasma MMP-2 (Rs=0.181, p=0.014) and MMP-3 (Rs=0.260, p=0.0004) levels were positively correlated with HDL-cholesterol levels. TIMP-2 levels were negatively correlated with total cholesterol (Rs=−0.197, p=0.007) and LDL-cholesterol (Rs=−0.168, p=0.022) levels. These results suggest that the circulating levels of MMPs and TIMPs are altered in patients with premature coronary atherosclerosis and that plasma lipoprotein cholesterol levels correlate with these, possibly as a result of the lipoprotein-vessel wall interactions.

June 1, 2005

Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) for the Measurement of Prostate-Specific Antigen mRNA in the Peripheral Blood of Patients with Prostate Carcinoma Using the TaqManTM Detection System

Stefania Gelmini, Carmela Tricarico, Giovanna Vona, Lorenzo Livi, Alessandro Della Melina, Sergio Serni, Enrico Cellai, Stefano Magrini, Donata Villari, Marco Carini, Mario Serio, Gianni Forti, Mario Pazzagli, Claudio Orlando

Page range: 385-391

Abstract

Circulating prostate cells can be detected in peripheral blood of patients with clinically localized or advanced prostate carcinoma. Traditionally, nested reverse transcriptase-polymerase chain reaction (RT-PCR) is used for this as a sensitive, but qualitative only, detection system. We developed a quantitative real-time RT-PCR method for measuring prostate-specific antigen (PSA) mRNA in peripheral blood of prostate cancer patients. A quantitative assay was developed using an external standard reference curve generated with RNA from the human prostate cell line LNCaP. Basal blood samples were collected from 44 patients without evidence of distant metastases and from 30 healthy controls. In 29 patients surgically treated with radical prostatectomy, the measurement of PSA mRNA was performed in blood samples collected before, at the end and 6 days after surgery. In 14 patients treated with radiotherapy, the measurements were repeated at 3-month intervals to evaluate time-related changes during therapy. The measurements were also performed for one year at 3-month intervals in one patient treated with anti-androgen therapy. We found detectable PSA mRNA in 14/44 (32%) basal blood samples. A wide range of values were observed in these patients, ranging from 0.5 to 1724 pg of total LNCaP RNA/ml blood. In patients undergoing radical prostatectomy, circulating PSA mRNA was detectable in eight patients in basal samples, and in seven of them also in blood specimens collected at the end of surgery, showing an increase in only two patients. In blood samples collected 6 days later, PSA mRNA was dramatically reduced in all patients, but still present in seven of them. In four patients, whose basal samples were negative, PSA mRNA was detectable in samples collected at the end of surgery and three of them were negative after 6 days. In patients who did not receive surgical treatment, a rapid decrease in PSA mRNA was demonstrated in five patients treated with radiotherapy and in one patient undergoing androgen deprivation. No detectable PSA mRNA was found in healthy controls. The levels of PSA mRNA in peripheral blood from patients with prostate carcinoma can be easily measured by this sensitive, quantitative and reliable procedure. This assay is a promising tool for the detection and follow-up of these patients.

June 1, 2005

Serum Cardiac Troponin I after Conventional and Minimal Invasive Coronary Artery Bypass Surgery

Giampaolo Cattozzo, Sergio Finazzi, Sandro Ferrarese, Andrea Sala, Gian Vico Melzi D'Eril

Page range: 392-395

Abstract

We evaluated myocardial release of cardiac troponin I (cTnI) in patients treated with conventional coronary artery bypass grafting (CABG), which employs extra-corporeal circulation, and different kinds of minimal invasive coronary artery bypass grafting (MICABG), a surgical technique where the operation is performed without extra-corporeal circulation. Furthermore, we evaluated the usefulness of serum cTnI measurement to detect perioperative myocardial infarction (PMI) after coronary artery bypass surgery. Thirty-one patients were included: sixteen underwent CABG, fifteen underwent different MICABG and five patients had PMI. Blood specimens for cTnI measurements were collected up to 72 hours after opening the graft. Aortic cross-clamping time was a minor determinant of myocardial damage; on the other side, the trauma during surgery correlated with the number of involved arteries and with the manoeuvre employed to obtain heart dislocation, and appeared a more important determinant of myocardial damage. In patients with PMI, the cumulative release of cTnI was higher than in patients free from PMI; however, only after 24–72 hours we observed significant differences in serum cTnI values, because the increased perioperative values of cTnI complicated the interpretation of the myocardial status and a single cut-off could not be used to exclude PMI.

June 1, 2005

Improvement of the Quantitative Method for Glucose Determination Using Hexokinase and Glucose 6-Phosphate Dehydrogenase

Zensuke Ogawa, Motohito Kanashima, Hiroshi Nishioka

Page range: 396-400

Abstract

This paper has two aims. The first one is to point out the shortcomings of Food and Drug Administration's (FDA's) reference method for the measurement of glucose. We found that the quantity of enzyme used in the method recommended by the FDA was more than the exact quantity needed for accurate measurement. The use of exact quantity of enzyme is important to minimize the negative effects due to impurity and side reactions of enzymes. The second aim is to simulate the coupling enzyme reaction using computer. We have successfully established the exact quantity of enzyme needed in the assay through the computer simulation. The quantity of the enzyme was lesser than the that recommended by FDA, but the reaction ended at the same time as in the FDA method. In addition, optimum conditions and inhibitory effects of various reagents have also been successfully analyzed using computer. In conclusion, we suggest a reference method using computer simulation to determine the exact quantity of the coupling enzyme needed in the assay.

June 1, 2005

Conditions for Single-Strand Conformation Polymorphism (SSCP) Analysis of BRCA1 Gene Using an Automated Electrophoresis Unit

Berta Campos, Orland Díez, Joan Cortés, Montserrat Domènech, Carles Pericay, Carmen Alonso, Montserrat Baiget

Page range: 401-404

Abstract

The single-strand conformation polymorphism procedure has been applied in routine testing for hereditary diseases and cancer. However, temperature, running time, gel composition, fragment length, etc . can influence its sensitivity. Mutation detection in the clinical setting depends on the development of automated technology, especially for large genes such as the breast cancer gene BRCA1 . We analysed DNA samples with BRCA1 mutations in an automated system (GenePhor System; Amersham-Pharmacia Biotech, Uppsala, Sweden). The concentrations of DNA template and PCR primers, the effect of chilling after denaturation, and the temperature and time of the electrophoresis were investigated. All band-shifts were detected by electrophoresis at 5 °C for 2 h 15 min. Concentrations of DNA and samples used in the PCR did not affect the SSCP pattern, but chilling the PCR product in ice after denaturation was required. The type and position of mutation in the fragments did not influence the probability of a mobility shift, although SSCP analysis was more sensitive for fragments shorter than 350 bp. This automated SSCP method meets the requirements of fast turnaround and sensitivity and can be readily adapted to the screening of large genes such as BRCA1 .

June 1, 2005

Discrepancies between Apolipoprotein E Phenotyping and Genotyping in the Elderly

Anne Marie Dupuy, Stéphanie Badiou, Karen Ritchie, Emilie Mas, Bernard Descomps, Jean Paul Cristol, Jacques Touchon

Page range: 405-413

Abstract

We estimated the frequencies of phenotype (isoelectric focusing; IEF) vs. genotype (PCR/HhaI) discordance in a sample of an aged population (> 65 years). Both phenotype and genotype techniques have been used in the study of apolipoprotein E (apoE) polymorphism in 125 elderly subjects. The discordance between phenotype and genotype was unresolved in 11 (8.8%) of the 125 unrelated subjects studied. We observed a significant association between the presence of the E4 allele and both Alzheimer's disease (χ 2 =13 p<0.001) and increased cholesterol concentration (Mann Whitney, p<0.03). These relationships were not affected by the techniques used. Our results indicate that transcriptional modulation and post-transductional modifications in normal ageing and in aged-related diseases may explain in part discrepancies between gene analysis and protein characterisation.

June 1, 2005

Markers of Bone Turnover in Postmenopausal Women Receiving Hormone Replacement Therapy

Ahmad Hamwi, Abdel-Halim Ganem, Christina Grebe, Katharina Kerschan-Schindl, Elisabeth Preisinger, Ewald Boschitsch, Christian Bieglmayer

Page range: 414-417

Abstract

We investigated the effects of hormone replacement therapy (n = 27) on biochemical markers of bone turnover in a cross-sectional study of 127 postmenopausal women (according to WHO guidelines 18 patients had normal bone mineral density and 109 suffered from bone loss). Urinary excretion of free deoxypyridinoline and C- or N-telopeptide fragments of type I collagen served as bone resorption markers, serum osteocalcin as a bone formation marker. In women with no hormone replacement therapy, only C-and N-telopeptides correlated significantly with the lumbal T-score as an index for bone mineral density. Patients with bone loss receiving hormone replacement therapy exhibited significantly lower C-telopeptide, N-telopeptide and osteocalcin levels than those with no therapy (mean −45%, −43% and −26%, respectively), while deoxypyridinoline showed no significant differences. Among the markers investigated, C- and N-telopeptides seemed to be more reliable to detect therapeutic effects on bone metabolism. We present a preliminary model to evaluate bone turnover and resorption/formation rate.

June 1, 2005

Reference Intervals for the Markers of Proteinuria with a Standardised Bed-Rest Collection of Urine

Timo Kouri, Aimo Harmoinen, Kaija Laurila, Ilpo Ala-Houhala, Timo Koivula, Amos Pasternack

Page range: 418-425

Abstract

Reference intervals for markers of proteinuria or glomerular charge selectivity were measured in 61 healthy female and 61 healthy male individuals. Timed bed-rest and daytime collections were used to assess significance of preanalytical variability of results. Bed-rest collections are advisable for research on renal damage, whereas in routine care, robust protein/creatinine ratios work as practical estimates of protein excretion rates, the correlations to excretion rates improving with increasing proteinuria. For glomerular charge selectivity, pancreatic/salivary isoamylase clearance ratio showed lower within-subject biological variation than IgG/IgG4 clearance ratio, allowing more accurate classification into normal and reduced charge selectivity. With our method, the lower 2.5% reference intervals for isoamylase clearance ratio were 1.1 in men and 1.9 in women.

June 1, 2005

The Friedewald Formula Underestimates LDL Cholesterol at low Concentrations

Hubert Scharnagl, Matthias Nauck, Heinrich Wieland, Winfried März

Page range: 426-431

Abstract

Due to recent advances in the treatment of hypercholesterolemia, low density lipoprotein (LDL) cholesterol concentrations below 2.6 mmol/l have become attainable. In general, LDL cholesterol is determined indirectly according to Friedewald. We examined the performance of the Friedewald formula at low concentrations of LDL cholesterol in comparison with a β-quantification method. We analyzed 176 samples from individuals treated by LDL apheresis with a mean LDL cholesterol concentration of 3.07 mmol/l and found that the Friedewald formula underestimated LDL cholesterol with a bias of −18.5%, −14.5%, −7.3%, and −3.8% at mean LDL cholesterol levels of 1.58, 2.4, 3.49, and 4.67 mmol/l, respectively. Thus, the lower the LDL cholesterol concentration was, the greater the negative bias. We conclude that the Friedewald formula may not be reliable at low LDL cholesterol concentrations produced by LDL apheresis. This finding may also be of relevance to the monitoring of patients being treated with lipid lowering drugs.

June 1, 2005

The White Blood Cell Differential: Three Methods Compared

Rüdiger Siekmeier, Alexa Bierlich, Werner Jaroß

Page range: 432-445

Abstract

The analysis of the automated blood cell count is an essential tool in haematological diagnostics. However, in the case of the white blood cell differential the microscopy method, although tedious, often serves as reference. We evaluated the ABX Pentra 120 Retic haematology analyser in comparison to the Coulter STKS haematology system and the microscopy method with respect to accuracy, precision and reliability. We compared 308 samples (239 samples from adults and 69 from children) including patients with oncological diseases. The comparison of the white blood cell differential revealed strong correlations between the results obtained with the ABX Pentra 120 Retic and the microscopy method, the Coulter STKS and the microscopy method and both automated methods (values of paediatric samples in parentheses; neutrophils: r S ≥0.933 (r S ≥0.951), lymphocytes: r S ≥0.907 (r S ≥0.945), monocytes: r S ≥0.584 (r S ≥0.459) and eosinophils: r S ≥0.963 (r S ≥0.966)). The analytical performance of automatic analysers for the detection of the morphological “left shift” was determined for all samples in comparison to the microscopical white blood cell differential. The sensitivity, specificity and efficiency depended strongly on the chosen threshold levels and were different for both analysers. The sensitivity for flagging a left shift increased with an increasing proportion of neutrophil bands, metamyelocytes, myelocytes and promyelocytes. Our study suggests that the ABX Pentra 120 Retic haematology analyser, as well as the Coulter STKS haematology system are useful tools for routine analysis in haematology.

June 1, 2005

Plasma Brain Natriuretic Peptide Measured by Fully-Automated Immunoassay and by Immunoradiometric Assay Compared

Silvia Del Ry, Daniela Giannessi, Aldo Clerico

Page range: 446-450

Abstract

The clinical relevance of measuring plasma brain natriuretic peptide (BNP) is well-known, especially in patients with heart failure. Recently, a new method for measuring BNP, called TRIAGE ® , has been developed which can be used for point-of-care testing of patients with congestive heart failure. The aim of the present study is to compare the analytical performance of this fully-automated method to that of an immunoradiometric assay (IRMA), routinely used to measure BNP. The TRIAGE ® method is a non-competitive immunofluorometric assay which uses two different binding phases, specific for two different epitopes of the BNP amino acid chain, to form a sandwich with the specific ligand ( i.e. , BNP). A polyclonal antibody is included in the fluorescent immunoassay reagents which are contained in the assay devices and a monoclonal antibody is immobilized in the detection lane of the assay device. The imprecision of the TRIAGE ® method was approximately 12% for BNP concentrations in the normal range and about 18% for BNP concentrations above the normal range. The mean reading time of the TRIAGE ® method was 14.5±8.6 min. A close linear relationship was found between the BNP values measured with the two methods (TRIAGE=24.6+0.933 IRMA; r=0.932, n=83). The TRIAGE ® method is indicated for BNP assay in ambulatory and coronary or emergency units, where usually only a few samples (preferentially whole blood samples) must be measured in a short time. The IRMA method should be preferred for pathophysiological studies, requiring the highest degree of precision and sensitivity for simultaneous measurement of several stored plasma samples or tissue extracts.

June 1, 2005

Length of Sedimentation Reaction in Undiluted Blood (Erythrocyte Sedimentation Rate): Variations with Sex and Age and Reference Limits

Elisa Piva, Maria Colomba Sanzari, Giuseppe Servidio, Mario Plebani

Page range: 451-454

Abstract

Although the length of sedimentation reaction in blood (LSRB) (commonly, but improperly called erythrocyte sedimentation rate, ESR) has long been used in clinical laboratories because it is simple and low-cost, its sensitivity and specificity are unsatisfactory. Usually, the values are reported using the Westergren method with sodium citrate-anticoagulated specimens. We used a new procedure, the Test1™, which measures the length of sedimentation reaction in undiluted K 3 EDTA anticoagulated blood samples following ICSH (International Committee for Standardization in Haematology) recommendations. Samples obtained from 840 reference individuals (430 females and 410 males, mean age 44 and 46.5 years respectively, range 1 to 90 years) were utilised to estimate the reference limits. The subjects, classified by sex, were subdivided into four statistically different age groups to determine the reference limits (2.5th and 97.5th percentiles). Sex difference was statistically significant in two age groups, from 14 to 50 (p<0.0001) and from 50 to 70 years (p<0.01). We did not observe significant sex difference within the age bracket from 1 to 14 years and from 70 to 90 years. In both sexes LSRB values increased with age, in significant correlation with fibrinogen concentration (p<0.0001), and became significantly higher in subjects older than 70 compared to all the younger subjects (p<0.01 in females and p<0.02 in males). Thus, we defined adequate reference ranges in elderly.

June 1, 2005

Biochemical Analysis of the Sperm and Infertility

Owona Dieudonné, Pierre-Arnaud Godin, Anne Van-Langendonckt, Jacques Jamart, Laurence Galanti

Page range: 455-457

Abstract

The clinical value of biochemical analysis of sperm is still unclear. In this study, we evaluated the potential of several biochemical markers in the seminal plasma (zinc, citrate, acid phosphatase, fructose and neutral α-glucosidase) as a screening method in male infertility investigation.

June 1, 2005

Evaluation of a Rapid Bedside Immunochromatographic Assay for Detection of Cardiac Troponin I in Whole Blood

Franca Pagani, Cristina Serena, Carla Bosio, Claudio Cuccia, Mauro Panteghini

Page range: 458-459

June 1, 2005

Correct Graphic Presentation of Method Comparison Data

Carlo Franzini

Page range: 460-461

July 27, 2005

European Symposium on Clinical Laboratory and In Vitro Diagnostics Industry

Rene Dybkaer

Page range: 462-463

About this journal

Objective
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 3.5. 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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Topics

clinical biochemistry
clinical genomics and molecular biology
clinical haematology and coagulation
clinical immunology and autoimmunity
clinical microbiology
drug monitoring and analysis
evaluation of diagnostic biomarkers
disease-oriented topics (cardiovascular disease, cancer diagnostics, diabetes)
new reagents, instrumentation and technologies
new methodologies
reference materials and methods
reference values and decision limits
quality and safety in laboratory medicine
translational laboratory medicine
clinical metrology

Article formats
Research Articles, Reviews, Mini Reviews and Opinion Papers on all aspects of clinical chemistry and laboratory medicine, Guidelines and Recommendations, Point/Counterpoint Articles, Letters to the Editor, Editorials