Volume 50, Issue 2

In order to meet the challenges of effective healthcare, the clinical laboratory is constantly striving to improve testing sensitivity while reducing the required time and cost. Gold nanoparticles (AuNPs) are proposed as one of the most promising tools to meet such goals. They have unique optophysical properties which enable sensitive detection of biomarkers, and are easily amenable to modification for use in different assay formats including immunoassays and molecular assays. Additionally, their preparation is relatively simple and their detection methods are quite versatile. AuNPs are showing substantial promise for effective practical applications and commercial utilization is already underway. This article covers the principles of preparation of AuNPs and their use for development of different diagnostic platforms.

The human kallikrein-related peptidase 6 ( KLK6 ) gene belongs to the 15-member kallikrein ( KLK ) gene family mapping to chromosome 19q13.3–13.4. Encoding for an enzyme with trypsin-like properties, KLK6 can degrade components of the extracellular matrix. The successful utilisation of another KLK member (KLK3/PSA) for prostate cancer diagnosis has led many to evaluate KLK6 as a potential biomarker for other cancer and diseased states. The observed dysregulated expression in cancers, neurodegenerative diseases and skin conditions has led to the discovery that KLK6 participates in other cellular pathways including inflammation, receptor activation and regulation of apoptosis. Moreover, the improvements in high-throughput genomics have not only enabled the identification of sequence polymorphisms, but of transcript variants, whose functional significances have yet to be realised. This comprehensive review will summarise the current findings of KLK6 pathophysiology and discuss its potential as a viable biomarker.

Clinical Proteomics biomarker discovery programs lead to the selection of putative new biomarkers of human pathologies. Following an initial discovery phase, validation of these candidates in larger populations is a major task that recently started relying upon the use of mass spectrometry approaches, especially in cases where classical immune-detection methods were lacking. Thanks to highly sensitive spectrometers, adapted measurement methods like selective reaction monitoring (SRM) and various pre-fractionation methods, the quantitative detection of protein/peptide biomarkers in low concentrations is now feasible from complex biological fluids. This possibility leads to the use of similar methodologies in clinical biology laboratories, within a new proteomic field that we shall name “Clinical Chemistry Proteomics” (CCP). Such evolution of Clinical Proteomics adds important constraints with regards to the in vitro diagnostic (IVD) application. As measured values of analytes will be used to diagnose, follow-up and adapt patient treatment on a routine basis; medical utility, robustness, reference materials and clinical feasibility are among the new issues of CCP to consider.

The evaluation of patients admitted at the emergency department (ED) for chest pain is challenging and involves many different clinical specialists including emergency physicians, laboratory professionals and cardiologists. The preferable approach to deal with this issue is to develop joint protocols that will assist the clinical decision-making to quickly and accurately rule-out patients with non life-threatening conditions that can be considered for early and safe discharge or further outpatient follow-up, rule-in patients with acute coronary syndrome and raise the degree of alert of the emergency physicians on non-cardiac life-threatening emergencies. The introduction of novel biomarkers alongside the well-established troponins might support this process and also provide prognostic information about acute short-term or chronic long-term risk and severity. Among the various biomarkers, copeptin measurement holds appealing perspectives. The utility of combining troponin with copeptin might be cost-effective due to the high negative predictive value of the latter biomarker in the rule-out of an acute coronary syndrome. Moreover, in the presence of a remarkably increased concentration (e.g., more than 10 times the upper limit of the reference range), to reveal the presence of acute life-threatening conditions that may not necessarily be identified with the use of troponin alone. The aim of this article is to review current evidence about the clinical significance of copeptin testing in the ED as well as its appropriate placing within diagnostic protocols.

The success of genome-wide association studies (GWAS) in identifying replicating associations has greatly contributed to understanding of the genetic aetiology of complex diseases. This review discusses and provides examples of the potential of GWAS findings to be translated into clinical practice, i.e., diagnosis, prediction, prognosis, novel treatments and response to treatment of common diseases. The biological insights afforded by newly-identified robust associations represent the largest, albeit indirect, translational contribution of GWAS.

Background: Circulating cell-free (ccf) DNA in blood has been suggested as a potential biomarker in many conditions regarding early diagnosis and prognosis. However, misdiagnosis can result due to the limited DNA resources in Biobank’s plasma samples or insufficient DNA targets from a predominant DNA background in genetic tests. This study explored several strategies for an efficient DNA extraction to increase DNA amount from limited plasma input. Methods: Ccf plasma DNA was extracted with three different methods, a phenol-chloroform-isoamylalcohol (PCI) method, a High Pure PCR Template Preparation Kit method and a method used for single cell PCR in this group. Subsequently, the total DNA was measured by Nanodrop and the genome equivalents (GE) of the GAPDH housekeeping gene and MTATP 8 gene were measured using a multiplex real-time quantitative PCR for the quantitative assessment of nDNA and mtDNA. Results: Instead of 400–800 μL (routine input in the laboratory), 50 μLof plasma input enabled the extraction of ccf DNA sufficient for quantitative analysis. Using the PCI method and the kit method, both nDNA and mtDNA could be successfully detected in plasma samples, but nDNA extracted using protocol for single cell PCR was not detectable in 25% of plasma samples. In comparison to the other two methods, the PCI method showed lower DNA purity, but higher concentrations and more GE of nDNA and mtDNA. Conclusions: The PCI method was more efficient than the other two methods in the extraction of ccf DNA in plasma. Limited plasma is available for ccf DNA analysis.

Background: The aim of the study was to assess the performance of Promega, Maxwell ® 16 for the extraction of genomic DNA from whole blood and FFPE tissue. Methods: DNA was extracted from 10 whole blood and 10 FFPE specimens using six different commercial kits. Results: For whole blood, the mean DNA concentration obtained by Maxwell ® 16 was significantly greater than either easyMAG ® (p<0.0001) or QIAamp ® Blood DNA kit (p<0.001). For FFPE, the mean DNA concentration obtained by the AllPrep ® FFPE specific DNA/RNA kit was significantly greater than either the Maxwell ® 16 (p<0.0001) or the general AllPrep ® DNA/RNA kit (p<0.0001). Conclusions: Comparative evaluation of the six DNA extraction kits indicated that the semi-automated Maxwell ® 16 was superior for whole blood extraction while the manual AllPrep ® FFPE DNA/RNA kit (Qiagen) performed better for FFPE DNA extraction in terms of quantity of DNA obtained. All six extraction methods (blood and FFPE) performed well in terms of purity. Although there were variances in the quantity of DNA obtained, there were no significant differences in the efficiency of these methods in yielding amplifiable DNA extracts, as demonstrated by β-actin for whole blood specimens. In evaluation of FFPE DNA extraction methods, the Qiagen AllPrep ® FFPE DNA/RNA Mini Kit was the best for applications requiring larger amplicons, but for smaller amplicons the Maxwell was most consistent.

Background: α-Thalassemia is one of the most commonly inherited single-gene disorders in southern China. It is important to identify non-deletional α-thalassemia in areas where α-thalassemia is prevalent, since non-deletional HbH disease ( --/α T α or --/αα T ) is caused by the interaction of a non-deletional α-thalassemia with α-thalassemia-1 trait ( --/αα ). In this study, we developed an optimized molecular protocol for screening for α-globin gene mutations and validated the feasibility of using it as a rapid detection method. Methods: An approach based on high-resolution melting (HRM) analysis was used. A total of 74 samples, including 54 abnormal α-chain samples and 20 control samples, were tested. Results: All of the 54 samples with point mutations at the exons 1, 2 or 3 of the α-globin genes, including 33 non-deletional α-thalassemia, were successfully detected. Conclusions: HRM has the potential to become an efficient, rapid screening method for non-deletional α-thalassemia.

Background: Several studies report difficulties in genotyping HNF1β rs757210 using TaqMan probes. This is possibly due to the tri-allelic nature of this single nucleotide polymorphism (SNP). The aim of the present research was to develop alternative methods for genotyping rs757210. Methods: Pyrosequencing and high resolution melting analysis of small amplicons (HRM) were developed and tested in panels of type 2 diabetes mellitus patients (n=258) and healthy blood donors (n=183). Results were confirmed by Sanger sequencing. Results: With pyrosequencing, allele frequencies for the A, G and C allele of 0.42, 0.56, 0.02 and 0.37, 0.62, 0.01 were established in the panel of type 2 diabetes mellitus patients and healthy blood donors, respectively. Similar results were found using the more routinely available HRM method. Results for pyrosequencing and HRM were in 99.6% concordance. Conclusions: Pyrosequencing and HRM can be used to genotype the tri-allelic SNP rs757210 in the HNF1β gene and have the advantage over the commercially available TaqMan analysis that they can determine the rare C-allele variant.

Background: Apolipoprotein C3 ( APOC3 ) gene polymorphisms are associated with cardiometabolic risk factors, varying in ethnicities. This study aimed to investigate such association between the APOC3 –482C>T polymorphism and cardiometabolic risk factors in the turkish adult risk factor (TARF) study cohort, stratifying by gender and obesity. Methods: Randomly selected 1548 individuals (757 male and 791 female, mean age 49.9±11.8 years) were genotyped for –482C>T polymorphism using hybridization probes in a Real-Time PCR LC480 device. Results: The –482TT genotype prevailed 9.9% in men and 11.5% in women. Association between 482C>T polymorphism and dyslipidemia (p=0.036, OR=1.42, 95%Cl=1.02–1.97) was found only in men. Analysis of variance showed that anthropometric and metabolic variables were not differently distributed in APOC3 –482C>T genotypes in the study population. In relation to dyslipidemia and obesity, the –482C>T polymorphism showed significant gender-by-genotype interactions (p<0.01). When the study population was stratified according to gender and obesity, homozygotes for the T allele were associated strongly with (by 45%) elevated fasting triglyceride concentrations in obese men (p=0.009) and homeostatic model assessment (HOMA) index in non-obese women (p=0.013). Furthermore, in the same subgroups, the associations of the fasting triglyceride concentrations and HOMA index with the TT genotype remained after adjustment for risk factors (p<0.05). Conclusions: APOC3 –482TT genotype is independently associated with elevated fasting triglyceride concentrations in obese men. Presence of obesity seems to be required for this genotype to induce markedly elevated triglycerides. Furthermore, it is associated with the dyslipidemia in men, without requirement of obesity.

Background: In critically ill patients independent studies have shown contradictory findings regarding the prognostic significance of the D/D genotype of the I/D angiotensin converting enzyme (ACE) polymorphism. The study aim was to evaluate the effect of both ACE I/D polymorphism and ACE serum levels on the clinical outcomes of critically ill septic patients. Methods: This study recruited 186 Caucasian patients with sepsis, severe sepsis or septic shock. Epidemiological, clinical data, co-morbidities and severity scores were recorded. Measurements of serum ACE activity and genotyping for ACE I/D polymorphism were carried out. Primary outcomes were the 28- and the 90-day mortality; secondary outcomes included the number of days without renal or cardiovascular failure and ventilation-free days over the 28-day period following study enrolment. Results: Neither 28- nor 90-day mortality were associated with ACE I/D polymorphism (p=0.59 and 0.34, respectively) or circulating ACE levels (p=0.17 and 0.25, respectively). Similarly, ACE polymorphism and levels were not related to ventilation-free days (p=0.14 and 0.25, respectively), days without cardiovascular failure (p=0.14 and 0.81, respectively) and days without renal failure (p=0.64 and 0.27, respectively). Conclusions: Neither ACE I/D polymorphism nor serum ACE levels seem to be significant prognostic factors of clinical outcomes in septic, critically ill patients.

Background: In acute myeloid leukemia (AML), the internal tandem duplication (ITD) in the juxtamembrane domain of the FLT3 (Fms-like tyrosine kinase 3) gene is one of the most frequent genetic alterations associated with poor prognosis. Methods: A complex evaluation of the analytical properties of the three most frequently used detection methods – PCR followed by agarose (AGE), polyacrylamide (PAGE) or capillary electrophoresis (CE) – was performed on 95 DNA samples obtained from 73 AML patients. Results: All the three methods verified the presence of a mutant allele in 20 samples from 18 patients. AGE and PAGE could detect the presence of 1%–2% mutant allele, while the detection limit of CE was 0.28%. However, acceptable reproducibility (inter-assay CV <25%) of the mutant allele rate determination was only achievable above 1.5% mutant/total allele rate. The reproducibility of the ITD size determination by CE was much better, but the ITD size calculated by PeakScanner or GeneScan analysis was 7% lower as compared to values obtained by DNA sequencing. The presence of multiple ITD was over-estimated by PAGE and AGE due to the formation of heteroduplexes. Conclusions: This study suggests the use of PCR+CE in the diagnostics and the follow-up of AML patients. The data further supports the importance of proper analytical evaluation of home-made molecular biological diagnostic tests.

Background: Asthma is an airway disorder characterized by bronchial inflammation. An imbalance between the oxidative forces and the antioxidant defense systems has been implicated in the pathogenesis of asthma. Glutathione S-transferases (GSTs) play an important role in cellular protection against inflammation. Several studies have investigated the genetic variability of GSTM1, GSTP1 and GSTT1 enzymes in asthma development with conflicting results. Moreover, in our previous independent case-control study on GSTs and asthma, we have found that GSTA1 and GSTO2 gene polymorphisms are associated with asthma. The aim of the present study is to analyze if some functional polymorphisms of GSTA1 , GSTM1 , GSTP1 , GSTO2 and GSTT1 are associated with asthma in pediatric patients from Chieti (Italy). Methods: In this study, we performed an association study on 127 asthmatic children and 126 controls. We screened single nucleotide polymorphisms at GSTA1 , GSTO2 and GSTP1 loci. The effects of GSTM1 and GSTT1 null genotype were also investigated. Results: The GSTA1*-69T and GSTO2*D142 variants are associated with the significant increased risk of asthma development in our study population, while GSTM1 , GSTP1 and GSTT1 genotype distributions were nearly equal between the control group and asthmatics. Conclusions: Confirming our previous study, these findings suggest that the GSTA1 and the GSTO2 are asthma susceptible genes involved in increasing the risk of asthma development in the Italian population.

Background: Manganese (Mn) toxicity has been reported in patients receiving total parenteral nutrition. To avoid unnecessary exposure it is recommended by NICE (National Institute for Clinical Excellence) that blood Mn concentrations are monitored. The aim of the study was to develop a method using inductively coupled plasma mass spectrometry (ICP-MS) for the reliable determination of Mn in plasma and whole blood, as indices of acute and chronic exposure. Methods: Whole blood and plasma samples were prepared by appropriate dilution (diluent containing 0.005% Triton X-100, 0.2% propan-2-ol, 0.2% butan-1-ol and 1% nitric acid) addition of an internal standard gallium, followed by centrifugation to remove cell debris. Thermo Fisher Scientific ExCell and X Series ICP-MS instruments were used to define and correct for polyatomic interference on Mn assay. Results: Mn was quantified at mass 55 using aqueous calibration and the polyatomic interference from FeH was successfully eliminated by modified (Xt) skimmer cones but not with the collision cell (collision gas 7% H 2 in He, flow rate 4–7 mL/min). The assay was validated showing good precision, limit of detection and percentage recovery. Good agreement was observed with the All Laboratory Trimmed Mean of External Quality Assurance samples y (in house)=1.1 (ALTM)–45.0 between values of 250 and 750 nmol/L. Conclusions: A method has been developed using ICP-MS for the analysis of whole blood and plasma Mn incorporating a novel method of eliminating interference by utilizing the different geometries of the Xt interface cones. The procedure is simple and robust with good precision and recovery over a wide dynamic range.

Background: Antibody microarrays (Ab-array) represent a new, innovative proteomics platform for high-throughput protein expression profiling in body fluids. Because they allow for multiplexed measurements in small sample volumes, Ab-arrays are an interesting alternative to conventional indirect sandwich immunoassay (ELISA or DELFIA) tests in clinical or population screening if sets of markers are to be analyzed simultaneously. However, to allow implementation of Ab-arrays in clinical or population screening programs, it is of vital importance to establish that this method is both sensitive and quantitative. Methods: This study developed and optimized a duplex Ab-array for pregnancy-associated plasma protein A (PAPP-A) and human chorion gonadotropin (fβ-hCG), two serum biomarkers currently analyzed by conventional biochemical techniques in prenatal screening. Serum samples from pregnant women, representing the dynamic range of both markers, were analyzed on Ab-arrays, and validated to the, in prenatal screening routinely applied, AutoDelfia system. Results: Two different array hybridization conditions were tested, i.e., direct and indirect labeling, of which the indirect method displayed a sensitive and quantitative performance and a low intra- and inter-assay variation. Conclusions: Taken together, these findings indicate that Ab-array technology is a promising alternative for ELISA or DELFIA in population screening programs, allowing future quantitative analysis of multiple biomarkers simultaneously in small volumes of serum.

Background: To investigate the mechanism by which strontium ranelate (SrR) inhibits the bone resorption, this study compared the effects of SrR and calcium on parathyroid hormone (PTH) and the biochemical marker of bone resorption (serum type 1 collagen cross-linked C-telopeptide, βCTX). Methods: In 10 healthy young subjects, after overnight fasting, 1000 mg of elemental calcium and 2000 mg of SrR containing 600 mg Sr 2+ were administered consecutively with a 1 week washout period. During the control period no drug was given. Fasting blood samples were drawn at baseline and throughout the next 5-h period. Results: After the ingestion of either calcium or SrR, there was a significant increase in serum calcium and strontium concentrations, and a decrease in serum βCTX and intact PTH concentrations as compared to the baseline values (p<0.05). In the fasting subjects, no significant differences in the variable were found as compared to the baseline values. Conclusions: The decrease in PTH and the marker of bone resorption observed after the SrR administration is comparable to the decrease observed after the calcium administration in young adults.

Background: This study investigated whether a dot immunoassay (DIA) can provide simultaneous detection of anti-tissue transglutaminase (tTG), anti-deamidated gliadin (DG) and total IgA antibodies, as required in the work-up of celiac disease (CD) patients. Methods: Celiac disease patients (n=111) consecutively diagnosed from 2001 to 2011 at the Children’s Hospital and Institute of Immunology (Technical University Dresden) were tested for anti-tTG, anti-DG and total IgA by enzyme-linked immunosorbent assay (ELISA) and DIA retrospectively. Blood donors (n=45) and non-CD individuals with low IgA serum levels (n=8) were included as controls. Antibodies to endomysial antigens (EmA) were assessed by indirect immunofluorescence (IIF). Results: Four (3.6%) of 111 CD patients demonstrated an IgA deficiency with total IgA below 50 mg/L by ELISA. Total IgA of the 107 IgA-non-deficient CD patients varied from 70 to 6000 mg/L. All four IgA-deficient CD patients were detected by a reduced reaction control of DIA and demonstrated positive anti-tTG or anti-DG IgG by DIA or ELISA. Detection of anti-tTG and anti-DG by DIA and ELISA showed a very good agreement (IgA: κ=0.972, 0.856, respectively; IgG: 0.921, 0.895, respectively). Conclusions: Immunodot assay is a reliable and easy-to-use technique for the detection of IgA-deficient CD patients. Simultaneous assessment of anti-tTG and anti-DG IgA antibodies, and IgA deficiency by DIA can improve the efficacy of CD serology.

Background: Patients with primary Sjögren’s syndrome (pSS) mostly have high levels of polyclonal immunoglobulin (IgG) and selective increase of IgG subclasses. As IgG subclasses profiles represent specific immunological processes and may be indicative of the underlying pathological pattern in pSS, it would be of great clinical importance to investigate the distribution of IgG subclasses in pSS patients. Methods: In archived sera from 155 patients with pSS, 50 patients with systemic lupus erythematosus (SLE) and 50 normal controls were analyzed by immunonephelometric assay. Results: The median levels of IgG1, IgG2, IgG3 and IgG4 in pSS patients were 11300 mg/L, 6130 mg/L, 961 mg/L and 142 mg/L, respectively. When compared with the normal controls, significant increases of IgG1 (p<0.001), IgG2 (p<0.001) and IgG3 (p<0.001) was observed in pSS patients, while the IgG4 level was significantly decreased (p=0.002). The ratio of IgG1/IgG2 in pSS patients was 2.3±1.4, which was significantly higher than that in the normal controls (p<0.001). When the pSS patients were divided into different groups, a higher level of IgG3 was observed in pSS with systematic disorders (p=0.008). In addition, the level of IgG1 was statistically higher in the anti-Ro or anti-La positive group than that in the negative group (p<0.001), but the IgG4 concentration was lower than that in the negative group (p=0.046). A significant positive correlation between the concentration of IgG3 and the disease duration (p<0.001) was also identified. Conclusions: The data supported the pathogenic role of IgG1 and IgG3 subclasses in the process of pSS. Further investigations to elucidate the reasons for the increase of IgG2 and whether IgG2 has any contribution to the pathogenesis of pSS are needed.

Background: There is no certified control material for hemoglobin analysis which has the hemoglobin (Hb)A 2 /E level as high as found in β-thalassemia trait, HbE trait, β-thalassemia/HbE disease and homozygote of HbE, the thalassemia types found frequently in the Southeast Asian population. The aim of this study was to prepare the lyophilized hemoglobin control materials for hemoglobin analysis. Methods: Washed and dialysed erythrocytes of normal individuals and patients with β-thalassemia trait, HbE trait, β-thalassemia/HbE disease, homozygous HbE were lysed in 5% sucrose solution. The lyophilized hemoglobin control materials were prepared by using a freeze-drying (lyophilization) method. The high performance liquid chromatography (HPLC) analysis of lyophilized hemoglobin was performed after storing at –20°C for 1, 15 and 30 days and for 3 months. Results: The chromatograms of lyophilized hemoglobin control materials showed similar patterns and similar levels of HbA, HbA 2 /E and HbF when compared with equivalent fresh whole blood. Moreover, the lyophilized hemoglobin presented a good correlation coefficient (r>0.990) of relationships between HPLC, low pressure liquid chromatography (LPLC) and capillary electrophoresis (CE) methods. Conclusions: The lyophilized hemoglobin could be developed and used as control materials for hemoglobin analysis.

Background: Nucleated red blood cells (NRBCs) are present in the peripheral blood of several hematological and non-hematological conditions, usually associated with bad prognosis. The lack of an easy, rapid and reliable NRBCs count method did no’t allow one to know the incidence of NRBCs and to quantify them: the count was usually done during the microscopic revision of a blood smear; this is the reason we found few studies on NRBCs automated count in the literature. The aim of this study was the evaluation of the presence and the quantification of NRBCs in some onco-hematological disorders. Methods: This study analyzed 478 patients with the automated hematology analyzer Sysmex XE2100. The range of NRBCs were calculated in the peripheral blood at diagnosis, at hematological remission and during therapy. Results: NRBCs are present in the peripheral blood of a high number of hematological diseases and are related to ineffective erythropoiesis or stress erythropoiesis or primary alterations of hematopoiesis. NRBCs were found in nearly all onco-hematological diseases at diagnosis, but not in all patients. NRBCs were frequently found during chemotherapy and absent at remission. Conclusions: To the authors’ knowledge, this is the first study that gives a range for NRBCs count in the peripheral blood of these diseases.

Background: The aim of the study was to evaluate the effect of hematocrit (HCT) on glucose meter assays based on different measurement techniques. Methods: This paper studied glucose meters utilizing the glucose dehydrogenase reaction and four measurement techniques: colorimetry (HemoCue), reflectometry (Accu-Chek Active), amperometry (Optium Xido) and coulometry (Optium Omega). The EDTA venous blood samples HCT were modified by adding or removing defined aliquots of plasma. Glucose concentration was measured using each meter in 27 batches of blood samples, with HCT ranging from 20% to 60% in 10% increments. The data were analyzed using repeated measures models and a linear random effects model. Results: A significant relationship between HCT and glucose reading in all meters was found and, for all meters except Optium Xido, there was a significant modification of this relationship by glucose level. The relative decrease in glucose concentration per 1% increase of the HCT value varied from 0.30% for Optium Omega in samples with glucose concentrations <5.55 mmol/L to 1.37% for Optium Xido in the same stratum (p<0.0001). The 5% glucose meter error (the ADA recommendation) was reached in the <5.55 mmol/L stratum after HCT change by 3.9%–16.7%. Conclusions: There is a significant continuous effect of HCT on measurement accuracy of glucose meters across its wide range of values and glucose concentrations. The most sensitive to the HCT interference was the system utilizing amperometric technique (Optium Xido) followed by the one with reflectometric technique (Accu-Chek Active), while the systems with the coulometric technique (Optium Omega) or colorimetric measurements in whole blood haemolysate (HemoCue) were less sensitive.

Background: For automation it is important to know the effect of analyzing fresh samples after a single rather than after two centrifugations, and to determine test stability. Methods: This study compared silica clotting times (SCT) and dilute Russel’s viper venom tests (dRVVT) after one and two centrifugations in 50 fresh plasma samples. Then it compared test results within 4 h to those after 6–8 h of blood drawing in 40 samples. Means, minimums, maximums and quartiles of the paired screen were compared, test ratios were confirmed and correlations, linear regressions and Bland-Altman statistics were calculated. Results: The distributions of test results were nearly identical, regardless of the number of centrifugations or timing of the analysis. The first centrifugation explained 97.7% and 94.8% of the variance of tests results after the second centrifugation for the SCT and dRVVT, respectively. The test results after 6–8 h explained 98.3% and 96.3% of the variance of the SCT-ratios and dRVVT-ratios, respectively, tested within 4 h. Inter-day coefficients of correlation of ratio comparisons were similar to those of the controls values. Conclusions: Testing of fresh samples after a single centrifugation might replace batch testing of frozen samples after double centrifugation, providing timelier reporting of results and resulting in savings of technician time.

Background: Automated leukocyte differential counts of synovial fluid (SF) can be influenced by laboratory artefacts. Pseudoeosinophilia of SF has recently been first described in association with monosodium urate and calcium pyrophosphate crystals. This study compared automated measurements of the percentages of SF leukocyte fractions by two haematology analysers in order to elucidate the underlying mechanism of pseudoeosinophilia. Methods: The percentages of the leukocyte fractions of 17 crystal-containing and 28 crystal-free specimens were compared using the Wilcoxon test. Measurements were performed using the Cell-Dyn 3200 and the ADVIA 2120i, which are based on different techniques. Results: The percentages of eosinophils of the crystal-positive samples determined by the Cell-Dyn 3200 were significantly higher than those assessed by the ADVIA (p<0.0001), whereas the percentages of eosinophils of the controls did not differ significantly between the two devices (p=0.95). The Cell-Dyn 3200 clearly showed the phenomenon of crystal-associated pseudoeosinophilia (p<0.0001), which did not occur in the ADVIA measurements (p=0.28). The percentage of neutrophils was to a lower degree elevated in the crystal group (p=0.015). Conclusions: It was confirmed that SF crystals interfere with the typical light scattering fractions of leukocyte granules and may thus lead to spuriously elevated percentages of eosinophils and neutrophils in SF specimens.

Background: An increase in urine calcium compared to the consensus value was observed in some urine samples of the Dutch External Quality Assessment Scheme (EQAS). It appeared that the increase was due to the addition of oxalate by the EQAS organizers and preanalytical acidification of the samples by some of the participants. Because of this observation, the effect of urine acidification on urine calcium level in EQAS and patient samples with added oxalate was investigated. Methods: Twenty-four EQAS urine samples and 20 patient urine samples were subject to recovery measurements of urine calcium before and after addition of sodium oxalate and acidification. Results: Differences in urine calcium between acidified and non-acidified samples up to 30.9% have been observed in EQAS samples with added oxalate. Patient samples show differences up to 80%. Differences between acidified and non-acidified samples are minimal for low calcium oxalate levels but increase with higher levels. Samples without added oxalate show equal urine calcium results between acidified and non-acidified samples. Conclusions: Urine calcium results are decreased in non-acidified samples with an excess of oxalate. In case of hyperoxaluria, acidification of patient urine collections and EQAS samples is recommended for correct urine calcium values.

Background: Recently, several polymorphisms located on human chromosome 8q24 were found to be associated with prostate cancer risk with different frequency and incidence among the investigated populations. The authors conducted a prostate cancer case-control study in the Greek population to evaluate the association of the single nucleotide polymorphism (SNP) rs6983267, located at region 3 of chromosome 8q24, with this type of cancer. Methods: Samples of total blood from 86 patients with histologically confirmed prostate cancer and 99 healthy individuals were genotyped using real time polymerase chain reaction (PCR). Tumor-node-metastasis (TNM) stage, Gleason score and levels of prostate-specific antigen (PSA) at diagnosis were included in the analysis. Results: A highly significant association (odds ratio=2.84 and p-value=0.002) was found between rs6983267 and prostate cancer in the Greek population. The sensitivity, specificity, negative and positive predictive values of the presence of G allele for the discrimination between patients and controls were 81.40%, 39.4%, 53.9% and 70.9%, respectively. A lower proportion of homozygotes was found in patients with PSA level <4 ng/mL compared to those with PSA level more than 4 ng/mL (p=0.019). None of the other clinical factors nor the aggressiveness of the disease were found to be significantly associated with rs6983267 genotype. Conclusions: The SNP rs6983267 is an established marker for a range of cancers. In prostate cancer, it indicates an enhanced risk for carriers to develop the disease in general. In our study it showed no association with aggressive forms or familial and early-onset prostate cancer families.

Background: Heart-type fatty acid-binding protein (H-FABP) is a marker of myocardial necrosis, but whether it increases during myocardial ischemia is not known. This study investigated whether serum levels of H-FABP change during adenosine stress testing and nuclear imaging in patients with stable coronary artery disease. Methods: Thirty stable patients with established coronary artery disease on their medications were studied. Sampling was performed before the stress test, at the end of adenosine infusion, as well as 1, 2 and 3 h after the completion of the infusion. Results: No difference in H-FABP serum levels were found at the five pre-specified time points in the overall group (p=0.99); furthermore, there was no significant difference regardless of the test result – positive (p=1) or negative (p=0.98). Conclusions: It is concluded that H-FABP does not change significantly during pharmacologic stress testing in patients with known coronary artery disease and there is no difference whether there is inducible ischemia or not.

Background: The effects of fibrates on C-reactive protein (CRP) are controversial. This meta-analysis was conducted to synthesize the available clinical trial evidence and summarize the effects of fibrates on CRP concentrations. In addition, this study assessed the relationship between changes in CRP and lipid measures. Methods: A systematic search was conducted of randomized controlled trials on the effects of fibrates on CRP concentrations in the PubMed, Embase and Cochrane Library Database up to January 2011. A meta-analysis was performed using a random effect model. Meta-regression analysis was employed to assess the relationships between average change in CRP and lipid profiles. Results: Sixteen randomized controlled trials were included in the meta-analysis. Compared with placebo, treatment with fibrates significantly decreased CRP concentrations (weighted mean difference –0.47 mg/L, 95% confidence interval –0.93 to –0.01 mg/L, p=0.046). Fibrates significantly reduced CRP concentrations in trials with a higher baseline CRP concentrations (≥3 mg/L). There was a significant correlation between change in CRP and change in high-density lipoprotein cholesterol (regression coefficient or slope=–2.03, 95% CI –3.20 to –0.87, p=0.001). Conclusions: Fibrates can reduce CRP concentrations and change in CRP was correlated with change in high-density lipoprotein cholesterol but not with triglyceride. These findings suggest that patients with dyslipidemia could benefit from fibrates treatment by CRP lowering and this benefit is associated with lipid profile improving.