Volume 43, Issue 2

Circulating cell detection using reverse transcriptase-polymerase chain reaction (RT-PCR) techniques has been studied as a new prognostic factor in colorectal cancer patients. With the view of enhancing detection sensitivity, we developed a new multiplex RT-PCR assay for circulating cell detection based on the expression of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5; formerly CEA) and CEACAM7 (formerly CGM2). Between November 2002 and December 2003, 45 stage III-IV, 39 stage I-II colorectal cancer patients, 32 non-colorectal cancer patients and 41 healthy individuals were included. Positive selection using HEA-125 immunobeads was applied to blood samples before mRNA extraction, cDNA synthesis and a multiplex CEACAM5/CEACAM7 RT-PCR assay. For both CEACAM5 and CEACAM7, the limit of detection was found to be as low as 1 expressing cell in 10 6 nucleated blood cells. The multiplex RT-PCR assay was negative for the 41 healthy individuals and the 32 non-colorectal cancer patients. The test was positive in 53/84 (63%) of the colorectal cancer patients for CEACAM5 and/or CEACAM7, whereas 32/84 (38%) were positive for both markers. Colorectal cancer patients were positive for one of the two markers in 80% of cases (36/45) for stage III-IV patients (CEACAM5 73%, CEACAM7 51%) and in 44% of cases (17/39) for stage I-II patients. This multiplex RT-PCR assay with two markers proved to be more sensitive than use of a single marker in detecting circulating tumour cells. The discrepant expression of CEACAM5 and CEACAM7 may label circulating tumour cells that have different levels of differentiation and subsequent aggressive behaviour.

Surface-enhanced laser desorption/ionization (SELDI) time-of-flight mass spectrometry with protein arrays has facilitated the discovery of disease-specific protein profiles in serum. Such results raise hopes that protein profiles may become a powerful diagnostic tool. To this end, reliable and reproducible protein profiles need to be generated from many samples, accurate mass peak heights are necessary, and the experimental variation of the profiles must be known. We adapted the entire processing of protein arrays to a robotics system, thus improving the intra-assay coefficients of variation (CVs) from 45.1% to 27.8% (p<0.001). In addition, we assessed up to 16 technical replicates, and demonstrated that analysis of 2–4 replicates significantly increases the reliability of the protein profiles. A recent report on limited long-term reproducibility seemed to concord with our initial inter-assay CVs, which varied widely and reached up to 56.7%. However, we discovered that the inter-assay CV is strongly dependent on the drying time before application of the matrix molecule. Therefore, we devised a standardized drying process and demonstrated that our optimized SELDI procedure generates reliable and long-term reproducible protein profiles with CVs ranging from 25.7% to 32.6%, depending on the signal-to-noise ratio threshold used.

HIV in particular, as well as hepatitis B and C, present a burden on healthcare systems worldwide. Early detection of these diseases may prevent further infections and improve the outcome for patients. In particular, transmission of HIV from mother to child can be significantly reduced when preventive measures are taken before birth. We have developed and optimized a method for the simultaneous detection of HIV 1 and hepatitis B and C from dried blood specimensusing the Luminex multi-analyte profiling technology (LabMap). Dried blood spots provide a convenient method for mailing, analysis and storage of samples. Specimens from known HIV-positive children (n=46) as well as hepatitis B- (n=8) and hepatitis C-positive patients (n=7) tested positive in our assay. Storage for up to 10years did not interfere with the test in the case of HIV-positive patients. Results for five different antibodies and one antigen were obtained in approximately 80seconds. Furthermore, antibody levels in infants of HIV-positive mothers were monitored over a period of 1year. Antibodies were no longer detectable after 260–360days, which compared well with results independently obtained by ELISA and Western blot analysis. We demonstrated the feasibility of the simultaneous detection of infectious diseases from dried blood. Our novel method also provides a convenient tool for monitoring children from HIV-positive mothers and for possible screening efforts.

Hematopoietic cytokines (HCs) can affect the growth and spread of cancer. Therefore, in the present study, we investigated in pancreatic cancer patients the serum levels of selected HCs, such as stem cell factor (SCF), interleukin 3 (IL-3), granulocyte-macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF) and macrophage-colony stimulating factor (M-CSF) in relation to a control group and to a group of patients with chronic pancreatitis. Classical tumor markers such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9) were also tested. We compared the serum level of cytokines with the tumor stage. The diagnostic sensitivity, specificity, positive and negative predictive values and receiver-operating characteristics (ROC) curve for cytokines and classical tumor markers were defined. The cytokines were measured in 48 patients with pancreatic cancer, in 23 patients with chronic pancreatitis and in 40 healthy subjects. HCs were determined using ELISA. CEA and CA 19-9 were measured by microparticle enzyme immunoassay. There were significant differences in the levels of circulating SCF, IL-3, GM-CSF, M-CSF, CEA and CA 19-9 in the pancreatic cancer patients compared to the control group. The serum levels of M-CSF and tumor markers were significantly higher in pancreatic cancer patients compared to the pancreatitis group. The levels of SCF, M-CSF and tumor markers were higher in patients with a more advanced tumor stage. The M-CSF serum levels in the pancreatitis group correlated positively with the tumor markers tested – CEA and CA 19-9. The diagnostic sensitivity of SCF and specificity of M-CSF and tumor markers were the highest. The SCF and M-CSF areas under the ROC curve were greater than the areas for other cytokines. These results suggest the potential usefulness of HCs in pancreatic cancer detection; however, further investigations of early-stage pancreatic cancer patients and confirmation by a prospective study are necessary.

The histone deacetylase inhibitors sodium butyrate (NaBu) and trichostatin A (TSA) exhibit anti-proliferative activity by causing cell cycle arrest and apoptosis. The mechanisms by which NaBu and TSA cause apoptosis and cell cycle arrest are not yet completely clarified, although these agents are known to modulate the expression of several genes including cell-cycle- and apoptosis-related genes. The enzymes involved in the process of translation have important roles in controlling cell growth and apoptosis, and several of these translation factors have been described as having a causal role in the development of cancer. The expression patterns of the translation mechanism, namely of the elongation factors eEF1A1 and eEF1A2, and of the termination factors eRF1 and eRF3, were studied in the breast cancer cell line MCF-7 by real-time quantitative reverse transcription-polymerase chain reaction after a 24-h treatment with NaBu and TSA. NaBu induced inhibition of translation factors' transcription, whereas TSA caused an increase in mRNA levels. Thus, these two agents may modulate the expression of translation factors through different pathways. We propose that the inhibition caused by NaBu may, in part, be responsible for the cell cycle arrest and apoptosis induced by this agent in MCF-7 cells.

While buccal cells provide an easily accessible source of genomic DNA, the amount extracted may be insufficient for many studies. Whole genome amplification (WGA) using multiple displacement amplification (MDA) may optimize buccal cell genomic DNA yield. We compared the usefulness, in epidemiological surveys, of DNA derived from buccal cells collected by alcohol mouthwash and amplified by WGA protocol and standard protocols. Buccal cell collection kits were mailed to 300 randomly selected members of a large cohort study, and 189 subjects returned buccal cell samples. We determined: (i) which QIAamp ® DNA Blood Mini Kit extraction protocol (tissue or blood) produced more DNA; and (ii) whether it is feasible to use MDA to prepare DNA for single nucleotide polymorphism (SNP) genotyping of markers such as the methylenetetrahydrofolate reductase ( MTHFR ) and vitamin D receptor ( VDR ) genes. The two DNA extraction protocols were tested on 20 different patient samples each. The tissue protocol yielded more DNA than the blood protocol (15.4±8.6 vs. 7.6±7.1μg, p<0.0001). The 20 DNA samples extracted using the tissue protocol were then subjected to pre- and post-MDA genotyping using amplicons for the MTHFR SNP at C677T and the intron 8 VDR SNP. No genotyping discrepancies were detected in pair-wise comparisons of pre- and post-MDA. Genotyping DNA from MDA-based WGA is indistinguishable from routine polymerase chain reaction and offers a stable DNA source for genomic research and clinical diagnosis.

Given the lack of information about Y haplotypes for Campania (southern Italy), we analyzed eight Y short tandem repeats in a sample of males from this region with the aim of establishing a Y-haplotype database that can be used for forensic purposes. The eight Y short tandem repeats were amplified by two PCR multiplex reactions: multiplex A for loci DYS19 , DYS385 , DYS392 and DYS393 , and multiplex B for DYS389 I and II , DYS390 , DYS391 and DYS385 . The proportion of unique haplotypes was 100% (108 Y-chromosome haplotypes in 108 unrelated males) and the haplotype diversity discrimination index was 0.99. These data reflect the high heterogeneity of male lineage in our population and are similar to those obtained in other regions of southern Italy.

Multiple single-nucleotide polymorphisms in the BAT1-NFKBIL1-LTA region on the short arm of chromosome 6 have been found to be associated with susceptibility to myocardial infarction in a recent case-control study including individuals from Japan. Specifically, the association relates to homozygosity for the minor alleles of five individual polymorphisms (one each in BAT1 and NFKBIL1 and three in LTA ) and a haplotype defined by a particular allele combination of 10 polymorphisms (three each in BAT1 and NFKBIL1 and four in LTA ). In the Japanese study, genotype determinations were carried out using a multiplex PCR-Invader assay or DNA sequencing. As an alternative to these methods, we have established TaqMan assays for genotyping of nine of the 10 polymorphisms. Accuracy of the genotyping results obtained with the TaqMan reactions was demonstrated in experiments involving restriction enzyme analysis or DNA sequencing. Using the new TaqMan assays, we have genotyped the polymorphisms in a group of 1211 Caucasians who presented without symptoms or signs of acute or previous myocardial infarction. Genotype distributions of each of the five myocardial infarction-associated polymorphisms were significantly different between the present study group and two Japanese control groups (p≤0.01). High linkage disequilibrium measures between polymorphisms suggested that the nine polymorphisms reside in a single haplotype block. Based on the nine polymorphisms, eight different haplotypes with allele frequencies above 1% were identified, which together represented 98.4% of the total number of chromosome 6 in the 1211 individuals. This Caucasian study group and the Japanese sample showed strong linkage disequilibrium in the myocardial infarction-associated BAT1-NFKBIL1-LTA genomic region and related haplotype content, but significantly different genotype frequencies. The new TaqMan systems provide relatively simple and fast assays to test the clinical relevance of these polymorphisms.

Neuroblastoma is the most common extracranial solid tumor in children. Abnormal secretion of catechol-amines in tissues and body fluids allows for the differential diagnosis of neuroblastoma from other neoplasms and its distinction from non-neoplastic inflammatory diseases. This is achieved by assaying homovanillic acid and 4-hydroxy-3-methoxymandelic acid, the catabolites of catecholamine metabolism. In the course of an evaluation of children with suspected neuroblastoma, homovanillic acid and 4-hydroxy-3-methoxymandelic acid were analyzed in urine samples by capillary gas chromatography with flame ionization detection after extraction and derivatization of these compounds as trimethylsilyl derivatives. In three urine samples a significant increase in biogenic amines was observed, but these results were not confirmed by thin-layer chromatography. Patient history revealed that these children had been treated with ibuprofen, an analgesic and anti-inflammatory drug. To verify how ibuprofen or its metabolites may have interfered with capillary gas chromatography with flame ionization detection, we analyzed the same samples by capillary gas chromatography-mass spectrometry. In urine samples from patients on the drug, the presence of a peak identified as the trimethylsilyl ester of hydroxyibuprofen, which had the same retention time as 4-hydroxy-3-methoxymandelic acid, was found to interfere with the capillary gas chromatography with flame ionization detection analysis of the metabolite. This interference must be taken into account during the laboratory diagnosis of neuroblastoma.

Quantification of formaldehyde (HCHO) in urine was recently shown to be a promising tool in the investigation of cancer, particularly bladder cancer. Development of a low-maintenance, inexpensive and rapid analyzer for HCHO in urine would greatly facilitate future research and the potential diagnosis of bladder cancer. We examine here the application of an off-the-shelf system, originally designed for gas-phase atmospheric monitoring of HCHO, for the quantification of HCHO in urine. Under strict dietary protocols, e.g., avoidance of foods rich in free or chemically bound HCHO, an increase in HCHO in urine is an indirect indicator of cancer in the urogenital system. The concentration of HCHO in urine samples from an individual over a several-month period was determined, with a range from 39 to 1400μM and a mean of 600μM. The limit of detection for the present method was 0.1μM. The proposed technique provides a direct, low-cost and greatly simplified analytical method for the quantification of HCHO in urine compared to other available techniques.

An increased concentration of erythrocyte protoporphyrin-IX in whole blood or erythrocytes is a valuable diagnostic indicator for acquired porphyrias, e.g., iron deficiency anemia and lead poisoning, and for inherited porphyrias. We developed a spectrophotometric micromethod for determining erythrocyte protoporphyrin-IX. In this method, exhaustive release of erythrocyte porphyrins is achieved using hydroquinone and formic acid. The clean-up procedure for 50μL of whole blood or erythrocytes covers three steps of liquid/liquid solvent partition: two partitions using diethyl and diisopropyl ether and HCl 2.5mol/L, and one buffered step using ammonium formate. Determinations of erythrocyte protoporphyrin-IX are possible by: (a) absorption using three wavelengths, Rimington's constant and a millimolar absorptivity coefficient mɛ 408.8 =294.3L·mmol -1 ·cm -1 according to With; and (b) 2nd derivative, which is linked to mɛ 408.8 . Determination of erythrocyte protoporphyrin-IX using a 2nd derivative algorithm showed better spectral resolution and higher sensitivity at a five-fold lower detection limit compared to absorption. Within-run precision of medium and high levels was found for absorption and for 2nd derivative with a coefficient of variation (CV) of 1.4–1.9% (n=10). Total precision evaluated was CV=2.5–8.3% (n=20). Levels of reference intervals could only be measured using the 2nd derivative (CV 2.9%). Linearity was proved to E=1.0. Recoveries of protoporphyrin-IX ranged from 95.3% to 103.0%. Method comparison was carried out using a fluorimetric reference method (Piomelli). Reference intervals for gender groups are discussed.

P-Glycoprotein (PGP) encoded by the multi-drug-resistance 1 ( MDR1 ) gene is a member of theATP-binding cassette (ABC) transporter family, drug-transporting proteins involved in the bioavailability and pharmacokinetics of various drugs. Several single nucleotide polymorphisms (SNPs) in the MDR1 gene have been identified so far that may influence PGP expression levels and function. Thus, genotyping for MDR1 polymorphisms and determining specific haplotypes may become an important tool in predicting individual susceptibility to developing drug resistance. We developed a new multiplexed allele-specific PCR method based on the principle of mutagenically separated PCR (MS-PCR) for rapid and reliable simultaneous genoptyping of the C3435T polymorphism in exon 26 of the MDR1 gene and two additional SNPs (G2677T/A in exon 21 and C1236T in exon 12), which are in linkage disequilibrium with MDR1 C3435T. The accuracy and reliability of this method was confirmed by sequencing the respective regions in the MDR1 gene. This newly developed MDR1 MS-PCR will facilitate fast, accurate and economic analysis of MDR1 genotypes and will provide important information in optimizing individual therapeutic approaches.

New parameters correlated with the hemoglobin content in reticulocytes (RET-Y) and in red blood cells (RBC-Y) have been suggested as helpful in diagnosing iron deficiency anemia. We have studied RET-Y and RBC-Y indices in two groups of patients with microcytosis to verify if these parameters could be used to differentiate iron deficiency anemia from β-thalassemia minor. Blood samples from 33 iron-deficient patients, 25 β-thalassemic minor patients and 50 normal individuals were analyzed on a Sysmex XE-2100 instrument. A significant difference was observed in reticulocyte counting and immature reticulocyte fraction between iron deficiency anemia and β-thalassemia minor groups, but not in RBC-X and RET-Y parameters. Reticulocyte counting was higher in β-thalassemia minor and the immature reticulocyte fraction was higher in severe iron deficiency anemia. The ratio RET-Y/mean cell volume was tested and was significantly different when β-thalassemia minor was compared with mild and severe iron deficiency anemia, and showed better performance than the Mentzer ratio and the Green and King function. A great overlap of RET-Y and RBC-Y individual values was observed in both groups of microcytic anemias; we conclude that these new indices may be used with caution as indicative of iron deficiency, mainly in populations where β-thalassemia minor is frequent.

Chitotriosidase (ChT), a protein produced by activated macrophages, belongs to the chitinases, a group of enzymes able to hydrolyze chitin, a structural component of fungi and nematodes. A codominant inherited deficiency in ChT activity is frequently reported in plasma of Caucasian subjects, whereas in the African population this deficiency is rare. This study compares ChT activity in colostrum of 53 African women and 50 Caucasian women. Samples were collected at 24–48 and 72h after delivery. We found elevated ChT in colostrum of African women on the first day after delivery (1230±662nmol/mL/h) which decreased to 275±235nmol/mL/h on the third day. The ChT activity on the first day after delivery in the colostrum of Caucasian women, however, was significantly lower (293±74nmol/mL/h) and decreased to 25±20 and 22±19nmol/mL/h on the 2nd and 3rd day, respectively. The ChT activity in plasma of African women was also higher (101±80nmol/mL/h) than that of Caucasian women (46±16nmol/mL/h), but no correlation was found between plasma and colostrum ChT activity. The elevated ChT activity in colostrum of African women suggests the presence of activated macrophages in human milk, consistent with the genetic characteristics of the African population.

To achieve rapid assessment of chest pain in emergency/cardiology departments, a short turnaround time for cardiac marker testing is necessary. Nevertheless, Total Quality Management principles must be incorporated into the management of point-of-care testing (POCT); in this setting we implemented the Stratus CS ® assay as POCT for cardiac markers in our emergency/cardiology department. The analytical performance of the troponin I method was evaluated; information connectivity between the Stratus CS ® data management system and the laboratory information system was implemented and practical training of testing personnel was carried out at the POCT site. A total of 41 non-ST-segment elevation patients admitted to the hospital were followed to evaluate the appropriateness of hospital admission, formulated on the basis of the cardiac troponin-I level measured at the POCT site by clinical staff. Our preliminary clinical data suggest that the high sensitivity of the Stratus CS ® troponin method could play an important role in the early identification of patients with acute myocardial infarction in a low to intermediate-risk population for acute coronary syndrome. Our POCT model suggests that the central laboratory could ensure that the POCT program remains in compliance with quality requirements. Nevertheless, our comparison studies suggest that the implementation of POCT requires a high level of integration between cardiologists and pathologists to guarantee appropriate interpretation of the monitoring results for suspected ACS patients.

Blood specimens from primary care centres are normally transported to central laboratories by mail. This necessitates centrifugation and separation, especially since the potassium ion concentration in whole blood changes during storage at ambient temperature. Thus, because of the growing awareness of and concern for pre-analytical contributions to the uncertainty of measurements, we investigated 27 components and their stability under controlled temperature conditions from 17 to 23°C. We found that storage of whole blood can be prolonged by up to 8–12h for all components examined, including potassium ions, when stored at 20±0.2°C. We conclude that this opens the possibility for establishing a pick-up service, by which whole blood specimens stored at 20–21°C can be collected at the doctor's office, making centrifugation, separation and mailing superfluous. In addition, the turn-around time from sample drawing to reporting the analytical result would be shortened. After investments in thermostatted boxes and logistics, the system could reduce costs for transporting blood samples from general practice centres to central laboratories.

A questionnaire to assess knowledge of the expected elevation in serum K measurement with different grades of hemolysis was administered to medical technologists working in biochemistry laboratories, hospital physicians and nurses. The questions involved different grades of hemolysis (mild, 1.0, moderate, 2.5 and severe, 5.0g/L) and different final K measurements (2.9, 4.0, 5.2 and 8.2mmol/L). Subjects estimated the K concentration in a non-hemolyzed sample for each scenario. Adjustment values (difference between final hemolyzed K concentration and subject's response) were calculated. For the 132 respondees, the mean correct score was 1.7/12. Mean adjustment values were: mild, 0.43mmol/L (K 2.9), 0.55 (4.0), 0.88 (5.2) and 1.53 (8.2); moderate, 0.85 (2.9), 0.92 (4.0), 1.33 (5.2) and 2.50 (8.2); and severe, 0.93 (2.9), 1.48 (4.0), 1.96 (5.2), 2.96 (8.2). Correct adjustments were: mild, 0.28; moderate, 0.70; and severe, 1.40mmol/L. Healthcare staff overestimated the effect of hemolysis on potassium measurement and used an incorrect proportional adjustment approach to the problem. Such poor knowledge and faulty thinking could lead to diagnostic delays or misdiagnoses. There is potential for such faulty thinking in all areas of laboratory medicine, and laboratories should review their educational responsibilities and reporting practices in light of this.

Twenty-one healthy male volunteers aged 20–70years were given transdermally 25mg of 7-oxo-dehydroepiandrosterone daily in the form of an emulgel for 8 consecutive days. Morning blood was collected as follows: before application, and after the first, fourth and eighth doses (days 0, 2, 5 and 9), and then at different time intervals after termination of the treatment (days 16, 23, 37, 51, 72 and 100). Cortisol, testosterone, epitestosterone, estradiol, dehydroepiandrosterone and its sulfate, 7α- and 7β-hydroxy-dehydroepiandrosterone, luteinizing hormone, follicle-stimulating hormone and sex hormone-binding globulin were measured in blood sera. In the course of treatment 7β-hydroxy-dehydroepiandrosterone was significantly increased; testosterone and gonadotropins were lowered, but only after the first dose. All other significant changes were observed duringthe period after termination of the application:7β-hydroxy-dehydroepiandrosterone remained increased for 28days, 7α-hydroxy-dehydroepiandrosterone, testosterone, estradiol and sex hormone-binding globulin were decreased as late as day 63 and 91, respectively. On the other hand, epitestosterone was significantly increased between days 23 and 100. The levels of all other parameters studied were not significantly changed. The study points to an immediate as well as delayed effect of the short-term transdermal application of 7-oxo-dehydroepiandrosterone on relevant hormonal parameters.

The problem in the measurement of cyclosporin (CyA) is that the widely used immuno-based assays suffer from interference by metabolites present in unpredictable excess. To resolve this, the consensus view has been to develop more specific and robust procedures for the measurement of CyA alone in order to give values similar to those obtained by HPLC. We developed an alternative strategy based on Abbott poly- and monoclonal assays to derive an adjusted monoclonal value as an equivalent measurement to HPLC. We have now evaluated a recently developed semi-automated HPLC procedure and used it to test the validity of the adjusted monoclonal value. The automated HPLC procedure with online clean-up was optimised for the separation of CyA and internal standard CyD. The assay was simple to use, precise and gave good recovery of cyclosporin from whole blood. Comparisons with the more specific immunoassays Abbott AxSym and EMIT showed close agreement, whereas Abbott monoclonal values indicated up to 20% positive bias. In contrast, the adjusted monoclonal values gave good agreement with HPLC. Data obtained from HPLC linked to tandem mass spectrometry (MS) indicated closer agreement with Abbott monoclonal values than expected, suggesting some positive bias with MS. The benefit of using an adjusted monoclonal value is that a result equivalent to HPLC is obtained, as well as an indication of the concentration of metabolites from the Abbott polyclonal measurement.

In this work data obtained on the VARIANT™ II hemoglobin analyzer using the Dual Kit elution system were compared to those obtained with the β-Thalassemia Short Program. Since many laboratories still use an earlier model of the hemoglobin analyzer, the Variant™ 1, these data were also compared to those obtained with the latter instrument. Our study is divided into two parts. The first is an evaluation of the precision of the VARIANT™ II for determining the levels of hemoglobin (Hb)A 2 and Hb F. This was carried out for normal subjects, thalassemic patients and patients with HbS. The second part concerns the potential of this instrument in the presumptive identification of Hb variants.

The Dade Behring BCS is a coagulation analyser with optical reaction detection (standard 405nm). The present study was conducted to evaluate measurement at 570nm for analyses in interfering plasma samples. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and D-dimer in normal (n=50), lipaemic (n=60), icteric (n=113), and haemolytic (n=58) samples were measured at 405 and 570nm. As they are unaffected by the optical properties of the sample, the mechanical STAcompact analyser (Roche Diagnostics) and an ELISA technique were defined as the “comparison” methods. The percentage of valid PT results using the 570-nm method varied from 54% (lipaemic samples) to 97% (haemolytic samples). Valid aPTT measurements were found in 67% (lipaemic samples) up to 93% (icteric samples) of samples. Fibrinogen measurement revealed valid results in 58% (lipaemic samples) to 100% (haemolytic samples) of samples. The number of valid D-dimer results varied from 28% (lipaemic material) up to 100% (haemolytic material). Significant inter-method differences were found: aPTT in lipaemic (BCS 405 vs. 570nm) and icteric samples (STAcompact vs. BCS 405 and 570nm); fibrinogen in lipaemic (BCS 405 vs. 570nm), icteric (BCS 405 vs. 570nm; STAcompact vs. BCS 570nm) and haemolytic samples (STAcompact vs. BCS 405 and 570nm). Differences between the BCS 570-nm and the STAcompact methods were in most cases low and less pronounced than between the BCS 570- and 405-nm methods, making the BCS 570-nm method an alternative to measurement at 405nm. Limitations have to be taken into account regarding lipaemic plasma.