Variability in the analysis of 25-hydroxyvitamin D by liquid chromatography–tandem mass spectrometry: The devil is in the detail

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Lewis Couchman, Christopher M. Benton, Cajetan F. Moniz

Background Liquid chromatography–tandem mass spectrometry (LC–MS/MS) is increasingly used in clinical laboratories for the analysis of 25-hydroxyvitamin D (25OHD), but measurement is not straightforward. Importantly, LC–MS/MS is not a single technique: variables in sample preparation, chromatography and ionisation/fragmentation should each be considered. Methods We analysed results from a survey organised by the international Vitamin D External Quality Assessment Scheme (DEQAS), to determine the influence of such variables on the results for two DEQAS distributions. Results 65 laboratories returned questionnaires. 346 (57%) individual results were from laboratories using electrospray ionisation (ESI), and 259 (43%) from laboratories using atmospheric pressure chemical ionisation (APCI). Although the mean ratio of results was not significantly different between ESI and APCI (P=0.5828), there was greater variation (P<0.0001) in results obtained by laboratories using ESI. Greater variation (P<0.05) was also observed between results from laboratories monitoring non-specific water-loss transitions. Only 3 laboratories (5%) could resolve the isobaric metabolite 3-epi-25OHD3 from 25OHD3. Conclusions There are many variables to consider when using LC–MS/MS, including assay standardisation/calibration, chromatography and MS conditions. MS/MS alone cannot distinguish isobaric metabolites such as 3-epi-25OHD3. Interference can also occur if non-specific transitions are used. Laboratories should always subscribe to an EQA scheme for 25OHD analysis.

Highlights

Posted on 27 May 2012 | 1:12 am

Multi-center analytical performance evaluation of the Access Hybritech® p2PSA immunoassay

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Lori J. Sokoll, Daniel W. Chan, George G. Klee, William L. Roberts, Ron H.N. van Schaik, Dorothy A. Arockiasamy, Dennis L. Broyles, Corey M. Carlson, Isaac A. Mizrahi, Tina B. Pierson, Jeffrey E. Tam

Background Total PSA assays measure both complexed and non-complexed forms of PSA while free PSA assays only measure non-complexed forms. Free PSA is a mixture of isoforms including immature PSA (proPSA) with retained portions of the leader sequence (e.g. [?7], [?4], and [?2]proPSA) and nicked forms (BPSA). ProPSA isoforms in male sera have been associated with prostate cancer. This study characterized the analytical performance of a chemiluminescent immunoassay for [?2]proPSA. Methods The Access Hybritech p2PSA assay is a sandwich immunoassay using an anti-[?2]proPSA monoclonal antibody attached to paramagnetic beads and an anti-PSA monoclonal antibody conjugated to alkaline phosphatase calibrated with recombinant [?2]proPSA. Analytical studies including sensitivity (CLSI EP17-A) and imprecision (CLSI EP5-A2) were performed. Results The Access Hybritech p2PSA assay for [?2]proPSA had a dynamic range of 0.5 to 5000pg/ml. The total CV of the assay was <7% for [?2]proPSA concentrations between 20 and 1000pg/ml. The LOB was 0.50pg/ml, LOD 0.69pg/ml, and LOQ 3.23pg/ml (20% CV). There was no hook effect up to 15,000pg/ml. There was a <5% difference between calibrator and reagent lots and no interference from normal serum constituents. Conclusions The Access Hybritech p2PSA assay is a robust immunoassay for the measurement of serum [?2]proPSA.

Highlights

Posted on 27 May 2012 | 1:12 am

Discrimination of clinical stages in non-small cell lung cancer patients by serum HSP27 and HSP70: A multi-institutional case–control study

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Matthias Zimmermann, Stefanie Nickl, Christopher Lambers, Stefan Hacker, Andreas Mitterbauer, Konrad Hoetzenecker, Anita Rozsas, Gyula Ostoros, Viktoria Laszlo, Helmut Hofbauer, Ferenc Renyi-Vamos, Walter Klepetko, Balazs Dome, Hendrik Jan Ankersmit

Introduction Lung cancer represents a major healthcare problem. Accordingly, there is an urgent need to identify serum biomarkers for early diagnosis of lung pathology. We have recently described that patients with manifest COPD evidence elevated levels of heat shock proteins (HSPs). Based on these data, we speculated whether HSPs are also increased in patients with diagnosed lung cancer. Methods Serum levels of HSP27, phospho-HSP27 (pHSP27) and HSP70 in patients with non-small cell lung cancer (NSCLC) diagnosed at an early (stages I–II, n =37) or advanced (stages IIIA–IV, n =72) stage were determined by using ELISA. Healthy smokers (n =24), healthy never-smoker volunteers (n =33) and COPD patients (n =34) according to GOLD classification served as control population. Results Serum levels of HSP27 were elevated in patients with NSCLC diagnosed at an early or advanced stage when compared with both healthy control groups (P <0.005 and P <0.0001 respectively). Statistically significant differences were furthermore found between the groups of patients with early vs. advanced stage NSCLC (P =0.0021). Serum levels of HSP70 were also significantly elevated in patients with NSCLC diagnosed at an early or at an advanced stage when compared with either healthy control groups (P =0.0028 and P <0.0001 respectively). In univariate logistic regression models including healthy subjects and patients with NSCLC, HSP70 had an area under the curve (AUC) of 0.779 (P <0.0001) and HSP27 showed an AUC of 0.870 (P <0.0001). Conclusion Our data suggest that serum HSP27 levels might serve as a possible tool to discriminate between early and advanced stages NSCLC.

Highlights

Posted on 27 May 2012 | 1:12 am

Evaluation of a novel, commercially available mass spectrometry kit for newborn screening including succinylacetone without hydrazine

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Thomas F. Metz, Thomas P. Mechtler, Michael Merk, Anne Gottschalk, Richard Luka?in, Kurt R. Herkner, David C. Kasper

Newborn screening for tyrosinemia type I (Tyr-I) is mandatory to identify infants at risk before life-threatening symptoms occur. The analysis of tyrosine alone is limited, and might lead to false-negative results. Consequently, the analysis of succinylacetone (SUAC) is needed. Current protocols are time-consuming, and above all, include hazardous reagents such hydrazine. We evaluated a novel, commercial kit to analyze amino acids, acylcarnitines and SUAC with a significantly less harmful hydrazine derivative in a newborn screening laboratory. Dried blood spot specimens from 4683 newborns and samples from known patients with inborn errors of metabolism (IEM) were analyzed by a novel protocol and compared to an in-house screening assay. All samples were derivatized with butanol–HCl after extraction from 1/8-inch DBS punches. For the novel protocol, the residual blood spots were extracted separately for SUAC, converted into hydrazone, combined with amino acids and acylcarnitines, and subsequently analyzed by mass spectrometry using internal isotope-labeled standards. All newborns were successfully tested, and 74 patients with IEMs including three with Tyr-I (SUAC 1.50, 4.80 and 6.49; tyrosine levels 93.10, 172.40 and 317.73, respectively) were detected accurately. The mean SUAC level in non-affected newborns was 0.68?mol/l (cut-off 1.29?mol/l). The novel assay was demonstrated to be accurate in the detection of newborns with IEM, robust, and above all, without the risk of the exposure to highly toxic reagents and requirement of additional equipment for toxic fume evacuation.

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Posted on 27 May 2012 | 1:12 am

Interleukin-1 (IL-1) family of cytokines: Role in Type 2 Diabetes

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Monisha Banerjee, Madhukar Saxena

Cytokines are small cell signaling protein molecules which encompass a large and diverse family. They consist of immunomodulating agents such as interleukins and inteferons. Virtually all nucleated cells, especially endo/epithelial cells and macrophages are potent producers of IL-1, IL-6 and TNF-?. IL-1 family is a group of cytokines which play a central role in the regulation of immune and inflammatory responses. Type 2 diabetes (T2D) has been recognized as an immune mediated disease leading to impaired insulin signaling and selective destruction of insulin producing ?-cells in which cytokines play an important role. Disturbance of anti-inflammatory response could be a critical component of the chronic inflammation resulting in T2D. IL-1 family of cytokines has important roles in endocrinology and in the regulation of responses associated with inflammatory stress. The IL-1 family consists of two pro-inflammatory cytokines, IL-1? and IL-1?, and a naturally occurring anti-inflammatory agent, the IL-1 receptor antagonist (IL-1Ra or IL-1RN). This review is an insight into the different types of cytokines belonging to IL-1 family, their modes of action and association with Type 2 diabetes.

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Posted on 27 May 2012 | 1:12 am

XAGE-1a and XAGE-1d are potential biomarkers of lung squamous cell carcinoma

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Jong Seong Ha, Hye Yeong Sung, Seon-Young Kim, Heon M. Lim, Hark Kyun Kim, Sung Sup Park

Background Lung cancer is the leading cause of cancer deaths worldwide. We evaluated the diagnostic potential of sera XAGE-1a and XAGE-1d in lung cancer, both of which are variants of the X antigen family, member 1. Methods The expression levels of XAGE-1a and XAGE-1d in cell lines were determined using western blot analysis. Competitive ELISA was used to analyze XAGE-1a and XAGE-1d levels in culture supernatants and sera from 194 lung cancer patients and 194 healthy sex- and age-group-matched controls. To evaluate the diagnostic performance of these proteins, we also analyzed carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA 21–1) in culture supernatants and 388 sera using commercial ELISA kits. Results XAGE-1a and XAGE-1d proteins were expressed in both breast cancer and lung cancer cell lines, but they were only secreted by the latter. The areas under the curves (AUCs) for XAGE-1a and XAGE-1d were 0.787 and 0.806, respectively. The cutoff values (sensitivity, specificity) for XAGE-1a and XAGE-1d were 1.62ng/ml (0.866, 0.572) and 2.51ng/ml (0.871, 0.613), respectively. The diagnostic performance was improved for patients with squamous cell carcinoma. The AUC values for XAGE-1a and XAGE-1d for patients with squamous cell carcinoma versus a group containing all healthy participants and patients with any illness other than squamous cell carcinoma were similar to those for CEA and CYFRA 21–1. Better performance (AUC: 0.914) for all patients was obtained when using a combination of four markers (Random Forest). Conclusions Sera XAGE-1a and XAGE-1d are potential biomarkers for lung cancer; they display a diagnostic performance comparable to that of CEA or CYFRA 21–1. Further studies are needed to evaluate the diagnostic and prognostic potential of XAGE-1a and XAGE-1d in lung cancer.

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Posted on 27 May 2012 | 1:12 am

Analysis of serum genome-wide microRNAs for breast cancer detection

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Qian Wu, Chao Wang, Zuhong Lu, Li Guo, Qinyu Ge

Objective Among methods for profiling levels of miRNAs, next-generation sequencing (NGS) has an effective one for genome-wide profiles, which not only can accurately quantify known miRNAs expression, but also discovery novel miRNAs. In this paper, we investigated that whether specific miRNAs were co-expressed in the serum and tissue of breast cancer (BC) patients as novel biomarkers by SOLiD sequencing. Methods Different miRNA expression profiles of serum and tissue in breast cancer patients and control subjects were obtained by NGS -SOLiD sequencing. Real-time PCR was used to selected and validated candidate miRNA-biomarkers. Novel miRNAs were predicted by computational pipeline, and validated by Northern blot analysis. Results Of genome-wide miRNA analysis using SOLiD sequencing, 7 miRNAs were found to be co-upregulated (i.e., miR-103, miR-23a, miR-29a, miR-222, miR-23b, miR-24 and miR-25). miR-222 was significantly increased in the serum of BC patients by further validation(P<0.05), which may be a useful biomarker for differentiating BC patients from controls with receiver operating characteristic (ROC) curve area 0.67 of (95% CI=0.5649 to 0.7775). A novel miRNA, named miR-BS1 was preliminarily identified and validated. Pre-miR-BS1 has a characteristic secondary structure. Mature miR-BS1 expression was detected in MCF-7 and MDA-MB-231 cells. Through gene ontology analysis, predicted target genes of miR-BS1, such as FOXO3 and KRAS, were involved in cancer-related signaling pathway. Conclusions This study presented a connection between serum- and tissue- based miRNA of breast cancer which suggested that serum-miRNAs may be potential biomarkers for BC detection. And next-generation sequencing will provide a robust platform for miRNA profilings.

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Posted on 27 May 2012 | 1:12 am

Evaluation of commutability of several materials for harmonization alkaline phosphatase catalytic concentration measurements

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Laura Rami, Montserrat Roura, Francesca Canalias

Background The International Standard ISO 18153 establish that one of the requirements to assure the metrological traceability of values for catalytic concentration of enzymes is the commutability of calibrator and control materials used in the reference measurement systems. This approach was applied to verify the commutability of several commercial stabilized materials using the recently published alkaline phosphatase IFCC primary reference procedure and two routine procedures. Methods ALP catalytic activity was measured in 50 serum samples and 16 commercial materials, including control materials from EQAS programs, using primary reference measurement procedure and two routine measurement procedures with AMP and DEA as buffers. Calibration materials with a value assigned by reference procedure which were proved to be commutable were used to recalculate the serum values obtained by routine procedures. Results All commercial materials showed a similar behaviour to the patient specimens when AMP vs IFCC procedures were compared. For DEA vs IFCC comparison only one calibration material and two quality control materials were commutable. Recalculation of serum results with a commutable common calibrator improves the agreement between methods changing the ratio AMP vs IFCC from 1.44 to 1.04 and DEA vs IFCC from 3.02 to 1.05. Conclusions The use of a common commutable calibration material allows harmonizing ALP measurements and made traceable patient results to reference procedure.

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Posted on 27 May 2012 | 1:12 am

Human biomarkers in breath by photoacoustic spectroscopy

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

M.J. Navas, A.M. Jiménez, A.G. Asuero

Photoacoustic spectrometry provides a novel approach to the analysis of human breath biomarkers. This unique methodology has specific applications for determination of ethylene oxide, nitrous oxide, ammonia as well as other compounds of interest including sevoflurane, halothane, isoflurane, methane, ethane and propofol. The advantages and disadvantages of photoacoustic spectrometry for this purpose are evaluated.

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Posted on 27 May 2012 | 1:12 am

Lysosomal storage disorder 4+1 multiplex assay for newborn screening using tandem mass spectrometry: Application to a small-scale population study for five lysosomal storage disorders

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Joseph J. Orsini, Monica M. Martin, Amanda L. Showers, Olaf A. Bodamer, X. Kate Zhang, Michael H. Gelb, Michele Caggana

Background We sought to modify a previously published tandem mass spectrometry method of screening for 5 lysosomal storage disorders (LSDs) in order to make it better suited for high-throughput newborn screening. Methods Two 3-mm dried blood spot (DBS) punches were incubated, each with a different assay solution. The quadruplex solution was used for screening for Gaucher, Pompe, Krabbe and Fabry diseases, while a separate solution was used for Niemann–Pick A/B disease. Results The mean activities of acid-?-glucocerebrosidase (ABG), acid sphingomyelinase (ASM), acid glucosidase (GAA), galactocerebroside-?-galactosidase (GALC) and acid-galactosidase A (GLA) were measured on 5055 unidentified newborns. The mean activities (compared with their disease controls) were, 15.1 (0.35), 22.2 (1.34), 16.8 (0.51), 3.61 (0.23), and 20.7 (1.43) (?mol/L/h), respectively. The number of specimens that fell below our retest level cutoff of <20% daily mean activity (DMA) for each analyte is: ABG (6), ASM (0), GAA (5), GALC (17), and GLA (2). Conclusions This method provides a simplified and reliable assay for screening for five LSDs with clear distinction between activities from normal and disease samples. Advantages of this new method include significant decreases in processing time and the number of required assay solutions and overall decreased complexity.

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Posted on 27 May 2012 | 1:12 am

Hypokalemic paralysis in a young obese female

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Wen-Fang Chiang, Yu-Juei Hsu, Chin-Chun Chang, Shih-Hua Lin

Background Profound hypokalemia with paralysis usually poses a diagnostic and therapeutic challenge. Methods We report on a 28-y-old obese Chinese female presenting with sudden onset of flaccid quadriparesis upon awaking in the morning. There is no family history of hyperthyroidism. She experienced body weight loss of 7kg in 2months. Results The most conspicuous blood biochemistry is marked hypokalemia (1.8mmol/l) and hypophosphatemia (0.5mmol/l) associated with low urine K⁺ and phosphate excretion. Surreptitious laxatives and/or diuretics abuse-related hypokalemic paralysis were tentatively made. However, her relatively normal blood acid–base status and the absence of low urine Na⁺ and/or Cl^? excretion made these diagnoses unlikely. Furthermore, she developed rebound hyperkalemia (5.7mmol/l) after only 80mmol K⁺ supplementation. Thyroid function test confirmed hyperthyroidism due to Graves' disease. Control of the hyperthyroidism completely abolished her periodic paralysis. Conclusions Thyrotoxic periodic paralysis (TPP) should be kept in mind as a cause of paralysis in female, even with obesity, despite its predominance in adult males.

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Posted on 27 May 2012 | 1:12 am

Cortisol assay in dried blood spots to reduce false positive rate in congenital adrenal hyperplasia screening

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Julie Brossaud, Pascal Barat, Laurence Fagour, Jean-Benoît Corcuff

Posted on 27 May 2012 | 1:12 am

Biologic variability of C-reactive protein: Is the available information reliable?

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Federica Braga, Mauro Panteghini

Background C-reactive protein (CRP) is recognized as a marker of cardiovascular risk. The biologic variability of CRP is crucial to understanding its significance in estimation of individual risk and subsequent changes in serial analyses. Methods We systematically reviewed publications on biologic variation of CRP to evaluate the consistency of available data. Data was evaluated with attention to number and type of enrolled subjects, duration of study, frequency of sample collection, sample type, sample storage, analytical methodology, assay sensitivity and statistical analysis. Results A total of eleven studies on CRP biologic variability were recruited from literature. The majority of studies were limited by choice of analytic methodology, population selection, protocol application, and statistical analysis. Unfortunately, the only study that fulfilled all major pre-analytical, analytical and post-analytical requirements derived biologic variability from logarithmically transformed data, thus making application to clinical practice difficult. Conclusions There is a paucity of robust data on biologic CRP variability in serum. It is obvious that additional well defined studies are needed to define reliable values of reference change values and of number of samples required to estimate the individual's cardiovascular risk by CRP.

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Posted on 27 May 2012 | 1:12 am

Comparison of Sebia Capillarys Flex capillary electrophoresis with the BioRad Variant II high pressure liquid chromatography in the evaluation of hemoglobinopathies

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Dina N. Greene, Amy L. Pyle, Judy S. Chang, Carolyn Hoke, Thomas Lorey

Background There are multiple biochemical screening techniques for assessing hemoglobinopathies. Here we compare a new instrument, the Sebia Capillarys Flex (capillary zone electrophoresis (CE)), with the BioRad Variant II (high pressure liquid chromatography (HPLC) in the evaluation of hemoglobinopathies. Methods This was a retrospective study using 174 whole blood samples encompassing the 5 most common (Hb A, A2?, S, C, and E) and 10 rare (Hb GPhilidelphia, D, H, Bart's, OArab, S/GPhilidelphia, Hasharoon, QIndia, NBaltimore, and Malmo) hemoglobin variants. An additional 126 samples were used to establish a CE reference interval for Hb A2. Results Hb A measurements agreed well between the 2 methods (bias=?0.06;r=0.999). The agreement of Hb F was also very good (bias=?0.17; r=0.994). When samples with the highest Hb F concentration were excluded, agreement was less precise (bias=?0.44; r=0.811). When no variant was present, the Hb A2 concentrations showed excellent agreement (bias=0.00; r=0.994). Positive bias for Hb A2 is seen when Hb C is present using CE. The Hb A2 reference interval using CE was <3.2%. Conclusion The Capillarys Flex is capable of identifying and quantifying hemoglobin species, consistent with existing HPLC methods.

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Posted on 27 May 2012 | 1:12 am

The clinical performance of the EGV1 self-monitoring blood glucose system

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Chien-Chih Chen, Jui-Jane Lin, Sheng-tien Hung, Peng-Ting Chun, Yiu-Kay Lai

Background The novel technique of blood volume detection can improve the reliability and accuracy of a self-monitoring blood glucose system. Self-management of diabetes can be improved, and the glycemic range can be efficiently controlled. Methods A total of 153 patients with diabetes mellitus participated in the clinical study. The accuracy, blood volume detection, interference, and altitude effect of the EGV1 self-monitoring blood glucose system were evaluated and compared among the fingerstick, alternative site testing, and venous blood. Results The EGV1 self-monitoring blood glucose system with fingertip demonstrated an excellent correlation with venous blood (linear regression analysis: slope=1.01, intercept=?0.8972mg/dl, r² =0.96), and with other brands of glucose systems (linear regression analysis: slope=0.99, intercept=+3.5632mg/dl, r² =0.94). The Clarke error grid analysis indicated that the results of fingertip and alternative sites were in the acceptable zones, A and B. The system required 0.6 ul of a blood sample to obtain an accurate reading, and was unaffected by several interferents and altitude. Conclusions The EGV1 self-monitoring blood glucose system using various blood samples demonstrated acceptable accuracy and reliability compared to the laboratory reference and other self-monitoring blood glucose systems.

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Posted on 27 May 2012 | 1:12 am

Validation and comparison of tumor-associated trypsin inhibitor (TATI) immunoassays

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Elisabete Janeiro, Joana Guimarães, Ulf-Håkan Stenman, Manuela Catarino, Outi Itkonen

Background Tumor-associated trypsin inhibitor (TATI) is mainly proposed as a tumor marker for ovarian cancer. However, recent discoveries of TATI in cancer and inflammatory diseases show that assay of TATI in biological samples is of increasing interest. Methods We validated a previously described TR-IFMA and a newly developed dual-monoclonal sandwich ELISA for TATI. These methods were compared with a commercial radioimmunoassay (RIA). We studied preanalytical factors affecting serum TATI concentrations and established age- and gender specific reference intervals using serum samples from 195 healthy volunteers. Results The assay range and precision were 0.8–45?g/L and <9.1% for the ELISA, and 0.13?g/L–150?g/L and <12.5% for the TR-IFMA, respectively. Both assays correlated well with a RIA. Type of blood sample and nutritional status were not critical and TATI was stable in serum samples when stored at +4°C and ?20°C for 4weeks and at ?80°C for 8weeks. The 95% reference limits for serum TATI in adults were 5.2–15.3?g/L in the age group 18–70years and 7.5–21.3?g/L in the age group >70years. Conclusions Significantly higher concentrations of serum TATI were observed in elderly women and in men. Both ELISA and TR-IFMA technologies can be employed to develop sensitive and robust immunoassays for TATI using monoclonal antibodies.

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Posted on 27 May 2012 | 1:12 am

Diagnostic value of transferrin

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Dominika Sz?ke, Mauro Panteghini

Despite the growing interest in hepcidin and other relatively new biomarkers, guidelines and clinical pathways continue to recommend traditional markers, such as serum transferrin (Tf) and ferritin, as laboratory tests for the diagnostic evaluation of iron-related disorders. In this study, we aimed to critically evaluate the diagnostic role of Tf relying on the highest level of available evidence by a comprehensive literature search. The role of Tf in iron deficiency (ID) and iron overload (IO) syndrome as well as a risk marker was evaluated. The low accuracy of Tf and Tf saturation (TS) in the diagnosis and management of ID conditions does not permit definitively recommending their use, even if recently published guidelines still consider the TS investigation as a complementary test for ferritin. If a tissue IO is suspected, TS is often used, even if it may not be the best test for detecting this condition. Nevertheless, clinical guidelines strongly recommend the use of TS as a first-level test for performing genetic diagnosis of hereditary hemochromatosis. Recently reported data indicating elevated TS as a risk factor for diabetes mellitus, cancer, and total mortality, may provide useful additions to the debate over whether or not to screen for IO using TS.

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Posted on 27 May 2012 | 1:12 am

Multiplex primer extension reaction and capillary electrophoresis to study the frequency of thrombophilia-related mutations in a spanish population

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Mercedes Zafra-Ceres, Tomás de Haro, José Antonio Gómez-Capilla, Esther Farez-Vidal, Antonio Poyatos, Carolina Gómez-Llorente

Background Thrombophilia is defined as an inherited or acquired abnormality of hemostasis predisposing to thrombosis. While the most common thrombophilia has a genetic origin and is manifested by elevated circulating antiphospholipid antibodies, about 40% of cases presenting with thrombosis are acquired. Factor V Leiden G1691A, prothrombin G20210A, MTHFR C677T, and Factor XII C46T mutations are associated with the risk of developing thrombophilia. Methods In this study, a method using single base extension assay coupled with fluorescent detection and capillary electrophoresis was applied to simultaneously detect G1691A, G20210A, C677T and C46T mutations in 1499 patients from Spain with suspicion of thrombotic disease. Results Out of these individuals, 5.4% were heterozygous for G20210A mutation, 9.21% were heterozygous and 0.20% homozygous for G1691A mutation, 46.36% were heterozygous and 20.71% homozygous for MTHFR mutation, and 30.41% were heterozygous and 3.4% homozygous for C46T mutation. Conclusion We applied an accurate, simple, semi-automatic, and cost-effective method to simultaneously detect the main thrombophilia-related mutations, allowing us to determine the frequency of these mutations in a Spanish population.

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Posted on 27 May 2012 | 1:12 am

A new screening method for proteinuria using Erythrosin B and an automated analyzer—Rapid, sensitive and inexpensive determination

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Satoshi Horikoshi, Asami Higurashi, Emiko Kaneko, Hajime Yoshimura, Isao Ohsawa, Yusuke Suzuki, Chieko Hamada, Yasuhiko Tomino

Background In spite of the urgent necessity for a screening test of urinary protein for the early diagnosis of kidney diseases, a rapid, accurate and cost-effective method for their detection has yet to be developed. Methods A solution containing a buffer agent (pH 2.3) and surfactants and a solution of Erythrosin B are added to a urine sample. After letting the mixture stand for 5min at 37°C, the dye-bound protein is measured by a spectrophotometer at 546nm using a Hitachi 7170S automated analyzer. Results The calibration curve was linear with human serum albumin concentration in the range of 2.4–200mg/l. The detection limit, 2.4mg/l was superior to conventional dye-binding methods by one order of magnitude and comparable to the turbidimetric immunoassay (TIA). Spot urine samples from 70 patients who showed (?) or (±) in the dip-stick screening test for proteinuria and 79 healthy volunteers were analyzed. There was an excellent correlation (r=0.978, n=149) between the results given by the proposed method and those by the TIA. Conclusions This method provides a viable alternative to the conventional immunoassay-based methods for urinary protein measurement, and will be useful in the diagnosis of early stage kidney disease.

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Posted on 27 May 2012 | 1:12 am

Evaluation of factors influencing accuracy in the analysis of succinylacetone in dried blood spots

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Minzhi Peng, Li Liu, Lijun Peng

Background Dried blood spots offer specific advantages over conventional blood collection methods, but with certain limitations. This article aims to evaluate factors which affect succinylacetone test in dried blood spots. Methods Whole blood with defined hematocrit and blood volume spiked with succinylacetone was spotted on filter paper, and analyzed by liquid chromatography?tandem mass spectrometry (LC-MS/MS). Four hematocrit levels (30%, 40%, 50%, and 60%) and five blood volume levels (10, 30, 50, 70, and 100?l) were tested. Results Succinylacetone concentration increased with increasing hematocrit, large bias from added concentration was found to be - 45% when hematocrit was 30%, as the difference of hematocrit level between the calibrator and QC sample increased, the bias from nominal value was increased. Blood volume also has effect on succinylacetone concentration level, but the accuracy was <15% when blood volume was 10 to 50?l, and >20% as the blood volume went to ?70?l. Conclusions Both hematocrit and blood volume have effect on analysis of succinylacetone in dried blood spots, the effect of hematocrit is more significant, due to hematocrit level of majority Type I tyrosinemia patients is low, diagnoses may be missed by using dried blood spots to analysis.

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Posted on 27 May 2012 | 1:12 am

Serum proteomics for biomarker discovery in nonalcoholic fatty liver disease

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Yusuf Yilmaz

Proteomic platforms have gained increasing attention in the clinical spectrum of nonalcoholic fatty liver disease (NAFLD). This approach allows for the unbiased discovery of circulating biochemical markers, i.e., it is not limited to known molecules of presumed importance. This manuscript provides an overview of proteomic serum biomarker discovery in NAFLD. Hemoglobin is currently the most widely replicated proteomic circulating biomarker of NAFLD; it was identified as a biomarker of fatty liver in two distinct proteomic studies and subsequently validated using distinct analytical methods by independent research groups in large replication cohorts. Given the increasing availability of numerous serum samples and the refinement of the technological platforms available to scrutinize the blood proteome, large collaborative studies between academia and industry are warmly encouraged to identify novel, unbiased circulating biomarkers of NAFLD.

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Posted on 27 May 2012 | 1:12 am

Predicting prostate biopsy outcome: prostate health index (phi) and prostate cancer antigen 3 (PCA3) are useful biomarkers

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Matteo Ferro, Dario Bruzzese, Sisto Perdonà, Claudia Mazzarella, Ada Marino, Alessandra Sorrentino, Angelina Di Carlo, Riccardo Autorino, Giuseppe Di Lorenzo, Carlo Buonerba, Vincenzo Altieri, Angela Mariano, Vincenzo Macchia, Daniela Terracciano

Indication for prostate biopsy is presently mainly based on prostate-specific antigen (PSA) serum levels and digital-rectal examination (DRE). In view of the unsatisfactory accuracy of these two diagnostic exams, research has focused on novel markers to improve pre-biopsy prostate cancer detection, such as phi and PCA3. The purpose of this prospective study was to assess the diagnostic accuracy of phi and PCA3 for prostate cancer using biopsy as gold standard. Phi index (Beckman coulter immunoassay), PCA3 score (Progensa PCA3 assay) and other established biomarkers (tPSA, fPSA and %fPSA) were assessed before a 18-core prostate biopsy in a group of 251 subjects at their first biopsy. Values of %p2PSA and phi were significantly higher in patients with PCa compared with PCa-negative group (p<0.001) and also compared with high grade prostatic intraepithelial neoplasia (HGPIN) (p<0.001). PCA3 score values were significantly higher in PCa compared with PCa-negative subjects (p<0.001) and in HGPIN vs PCa-negative patients (p<0.001). ROC curve analysis showed that %p2PSA, phi and PCA3 are predictive of malignancy. In conclusion, %p2PSA, phi and PCA3 may predict a diagnosis of PCa in men undergoing their first prostate biopsy. PCA3 score is more useful in discriminating between HGPIN and non-cancer.

Highlights

Posted on 27 May 2012 | 1:12 am

Editorial Board

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Posted on 27 May 2012 | 1:12 am

High incidence of severe neutropenia after gemcitabine-based chemotherapy in Chinese cancer patients with CDA 79A>C mutation

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Jialin Xu, Yuhong Zhou, Jing Zhang, Yuancheng Chen, Rongyuan Zhuang, Tianshu Liu, Weimin Cai

Cytidine deaminase (CDA) is a crucial enzyme in gemcitabine inactivation. Mutations in CDA gene have been found to influence the activity of CDA enzyme, which might lead to altered pharmacokinetics profile and clinical outcome of gemcitabine. In this study, the screening for the presence of CDA 79A>C and CDA 208G>A was performed by allele-specific PCR and restriction fragment length polymorphism, respectively. No difference in CDA allele frequencies was found between Chinese cancer patients and healthy volunteers. The frequencies for CDA 79A>C and 208G>A in the studied population were 12.2% and 1.0%, respectively. While a high incidence of grade 3–4 neutropenia was noted as 5 out of 8 (62.5%) patients heterozygous or homozygous for CDA 79A>C mutation developed it, in patients homozygous for the wild-type allele, the incidence was only 17.2% (5 out of 29) (p =0.021). Our study suggests that CDA 79A>C mutation might be a potential risk factor of gemcitabine-induced neutropenia toxicity.

Highlights

Posted on 27 May 2012 | 1:12 am

Effects of cytokine and cytokine receptor gene variation on high anti-HB titers: Following up on Taiwan's neonatal hepatitis B immunization program

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Ying-Ju Lin, Yu-Ching Lan, Yu-Chuen Huang, Ting-Hsu Lin, Shao-Mei Huang, Chien-Chen Lai, Chiu-Shong Liu, Cheng-Wen Lin, Shih-Yin Chen, Fuu-Jen Tsai

Background A significant percentage of Taiwanese neonatal HB immunization recipients have subsequently exhibited low anti-HB titers at non-protective or undetectable levels. Several mechanisms have been proposed to explain this phenomenon, including low vaccination responsiveness, deficient lymphocyte function, inappropriate antigen processing and presentation, and abnormal cytokine secretion. Methods To determine genetic influences resulting in high anti-HB titers, we divided a study cohort of 183 individuals into an anti-HBs?1000mIU/mL group and a 10–1000mIU/mL anti-HBs titer group. Chi-square tests were used to compare genotype and allelic frequencies between the two groups. Results Data from univariate and multivariate regression analyses of cytokine and cytokine receptor gene variants indicate (a) increased potential of high anti-HB titers in the presence of the TT genotype of the IL-4 rs2243250 SNP (OR=3.19; p =0.012) and the AA genotype of the IL-4R rs1805010 SNP (OR=2.25; p =0.048), and (b) individuals carrying the TT genotype of the IL-4 rs2243250 SNP had anti-HB titers at levels that were almost twice as high as those in individuals carrying the CC genotype (478.8mIU/mL and 290.3mIU/mL, respectively; p =0.033). Conclusion Genetic determinants, especially IL-4 and IL-4R, may contribute to high anti-HB titers in immune responses to HB vaccinations.

Highlights

Posted on 27 May 2012 | 1:12 am

Plasma myostatin measured by a competitive ELISA using a highly specific antiserum

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Karl Florian Wintgens, Thomas Dschietzig, Stanka Stoeva, Mats Paulsson, Franz Paul Armbruster

Background Understanding the (patho)physiology of the negative muscle regulator myostatin (Myo) is important for patients with skeletal muscle disorders or cardiac disease. However, a reliable tool for measuring plasma Myo immunoreactivity is still lacking. Methods Human full-length proMyo was used to raise a polyclonal rabbit antiserum for a competitive Myo ELISA that was validated in patients with decompensated congestive heart failure (CHF) and in control patients (n=20 each). Results The Myo antiserum detected all subunits of human proMyo. The calibration curve showed an optimal range between 0.3 and 83.3ng/ml (7.5–2100pmol/l), with no cross-reactivity to growth differentiation factor-11, follistatin and follistatin-related gene protein. The inter-assay and intra-assay variances in human serum were ?15% and ?10%, respectively; the detection limit was 270pg/ml (6.75pmol/l). The assay showed excellent linearity in human plasma. Plasma NT-proBNP and Myo were significantly elevated in decompensated CHF compared with control patients and decreased significantly upon recompensating therapy. Conclusion We describe the development of the first ELISA for myostatin immunoreactivity and its validation during recompensating therapy for CHF. This assay will be valuable for investigating neurological and cardiac diseases and states of cachexia, insulin resistance, and obesity.

Highlights

Posted on 27 May 2012 | 1:12 am

Characterization of urinary bile acids in a pediatric BRIC-1 patient: Effect of rifampicin treatment

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Tatsuki Mizuochi, Akihiko Kimura, Atsushi Tanaka, Akina Muto, Hiroshi Nittono, Yoshitaka Seki, Tomoyuki Takahashi, Takao Kurosawa, Masayoshi Kage, Hajime Takikawa, Toyojiro Matsuishi

Background Benign recurrent intrahepatic cholestasis type 1 (BRIC-1), a rare autosomal recessive disorder characterized by recurrent episodes of jaundice and pruritus, is caused by mutations in the ATP8B1 gene. Rifampicin has been reported to be an effective treatment of jaundice and pruritus in patients with BRIC. Proposed mechanisms of effect for rifampicin include enhancement of multidrug-resistance protein 2 expression, activation of the enzymes of uridine diphosphate glucuronosyltransferase 1A1 and cytochrome P450 3A4, and stimulation of 6?-hydroxylation of bile acids. Methods To confirm the diagnosis of BRIC-1 and demonstrate the effect of rifampicin treatment on bile acid metabolism, we analyzed the patient's ATP8B1 gene and bile acids in urine. Results We detected 2 heterozygous mutations in the ATP8B1 gene, and increasing amounts of unusual bile acids such as 1?-hydroxylated cholic acid, 2?-hydroxylated cholic acid, 4?-hydroxylated cholic acid, 6?-hydroxylated cholic acid, and hyocholic acid in urine during rifampicin treatment. Conclusions We diagnosed a jaundiced pediatric patient with BRIC-1 caused by 2 novel mutations (1226delA/2210delA) in the ATP8B1 gene. Rifampicin was effective in treating cholestasis. Results of urinary bile acid analyses during rifampicin treatment in this patient, suggested that rifampicin might stimulate 1?-, 2?-, and 4?-hydroxylation of bile acids in addition to 6?-hydroxylation.

Posted on 27 May 2012 | 1:12 am

Plasma poltergeists: A negative cortisol interference leading to a false diagnosis of adrenal insufficiency

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Damon A. Bell, Chris M. Florkowski, John G. Lewis, Michael J. Crooke

Introduction Adrenal insufficiency is a rare life threatening condition with non-specific clinical signs and symptoms. This necessitates a high level of reliance on laboratory results for making the diagnosis. Methods We report a case involving an interference in cortisol measurement leading to spuriously low-cortisol measurements and the incorrect diagnosis and treatment of adrenal insufficiency. We also discuss the testing used to confirm the presence and nature of the interferent. Results Interference with an IgG antibody was demonstrated, and the patient was found to have a normally functioning adrenal axis. Conclusion Interference in immunoassay is well described but often difficult to detect in clinical practice. Negative interference in cortisol measurement can occur and may lead to inappropriate diagnosis and treatment.

Highlights

Posted on 27 May 2012 | 1:12 am

Therapeutic monitoring of benzodiazepines in the management of pain: Current limitations of point of care immunoassays suggest testing by mass spectrometry to assure accuracy and improve patient safety

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Charles Mikel, Amadeo J. Pesce, Murray Rosenthal, Cameron West

Background Concomitant use of opioids and benzodiazepines can result in significant untoward effects. Point of care (POC) urine testing devices are commonly used tools to monitor patient use of medications. These useful devices are relatively inexpensive and yield immediate results that can be acted upon at the time of the appointment, although numerous limitations have been identified for specific medications or medication classes. We established the diagnostic accuracy of a commonly used POC testing method for benzodiazepines. Methods One thousand patients, from a single interventional pain practice receiving opioid therapy provided urine specimens as part of the usual practice of monitoring consistency with prescribed medications. These de-identified urine specimens were tested using LC-MS/MS and the results were compared using the standard calculations for sensitivity, specificity, and predicted value. Five specimens were excluded from the study because the prescribed flurazepam could not be confirmed by LC-MS/MS (the LC-MS/MS instrumentation was not set to identify flurazepam), resulting in 995 specimens. Results Point of care assays yielded false negative results for patients prescribed benzodiazepines nearly 20% of the time (98 out of 498 patients). The point of care cup often failed to produce positive results for persons who were shown by LC-MS/MS to be taking lorazepam or clonazepam. Although only 26 out of 498 patients (5%) were prescribed ?2 benzodiazepines, 73 out of 498 patients (15%) were found to be positive for that drug class. Conclusions POC immunoassay for benzodiazepines could fail to provide accurate information regarding patient specific medication use. The false positive and false negative rates of the immunoassay were particularly high for clonazepam and lorazepam. Further testing of patient specimens using more accurate methods such as LC-MS/MS is necessary to provide definitive data that can assist in clinical decision making, and potentially protect these patients from untoward effects, morbidity and mortality.

Highlights

Posted on 27 May 2012 | 1:12 am

Biospecimen reporting for improved study quality (BRISQ)

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Helen M. Moore, Andrea Kelly, Lisa M. McShane, Jim Vaught

Posted on 27 May 2012 | 1:12 am

Integrative role of neuropeptides and cytokines in cancer anorexia–cachexia syndrome

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Surajeet K. Patra, Sarika Arora

Background The cachexia anorexia syndrome is a complex metabolic syndrome associated with cancer and some other palliative conditions characterized by involuntary weight loss involving fat and muscle, weight loss, anorexia, early satiety, fatigue, weakness due to shifts in metabolism caused by tumour by-products and cytokines. Various neuropeptides like Leptin, neuropeptide Y, melanocortin, agouti-related peptides have been known to regulate appetite and body weight. Method A comprehensive literature search was carried out on the websites of Pubmed Central (http://www.pubmedcentral.nih.gov/), National Library of Medicine (http://www.ncbl.nlm.nih.gov) and various other net resources. Result Data from observational studies shows that various cytokines (TNF-?, IL-6 and IL-1) are associated with metabolic changes resulting in cachexia in cancer patients. These cytokines may mimic the action of various neuropeptides resulting in anorexia, various metabolic effects resulting from enhanced catabolic state and weight loss. Conclusion There is a need to understand and explore the role of various neuropeptides and cytokines in the pathophysiology of cancer-anorexia syndrome so that therapeutic measures may be designed for effective palliative care.

Highlights

Posted on 27 May 2012 | 1:12 am

Editorial Board

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Posted on 27 May 2012 | 1:12 am

Analysis of laboratory sample rejections in the pre-analytical stage at an oncology center

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Vivek Bhat, Manikchandra Tiwari, Preeti Chavan, Rohini Kelkar

Background Effective patient management depends on the accuracy of laboratory results. Sample collection errors constitute an important reason for repeat collections. This study was conducted at the laboratory diagnostic services of a tertiary care oncology center with a hematopoietic stem cell transplant unit to determine the common causes of sample rejections and see the effects of corrective action. Methods A retrospective, intervention and prospective analysis of the samples rejected from the total samples received in our laboratories, during a nine month period from January to September 2011 was undertaken. Causes of sample rejections were determined and intervention in the form of training relevant staff was instituted. Results Out of 32,548 samples received during Jan–Sep 2011, 177 samples (0.54%) were rejected. The most common reasons for rejection in hematology and biochemistry areas were clotted blood specimen (51.2%), improperly labeled specimen containers (14.46%) and hemolyzed blood samples (11.45%). For microbiology these included labeling errors, collection of specimen in wrong containers and specimen collection date and time not being entered, unacceptable specimen source and delayed transit time (18.2% each). Conclusions Directed interventions may help reduce the incidence of sample rejections.

Highlights

Posted on 27 May 2012 | 1:12 am

Gene transcripts in spermatozoa: Markers of male infertility

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Chunjin Li, Xu Zhou

The presence of a complex population of gene transcripts in mature human sperm is well established, and numerous mRNAs and non-coding mRNAs have been identified in sperm of men and other mammalian species using microarray and RT-PCR. The traditional concept that RNAs in mature sperm are only remnants from spermatogenesis and have no biological functions is in doubt. The findings that reverse transcriptases in sperm are active and that sperm can independently activate translation of stored mRNAs suggest that sperm RNAs may have significant effects on male fertility. The differences in expression profiles among RNAs in mature sperm from fertile and infertile men, and the regulation of sperm RNAs in embryonic development make them appealing markers for therapeutic and diagnostic tools in male infertility. In this review, methods for the detection and description of the diversity of gene transcript in sperm are discussed along with their putative roles in functional aspects of sperm and in embryogenesis.

Highlights

Posted on 27 May 2012 | 1:12 am

A novel non-synonymous mutation in the homeodomain of HOXD13 causes synpolydactyly in a Chinese family

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Binbin Wang, Baoqiang Xu, Zhi Cheng, Xueya Zhou, Jing Wang, Guang Yang, Longfei Cheng, Jun Yang, Xu Ma

Purpose The 5? HoxD genes and their paralogs in the HoxD cluster are crucial for normal vertebrate limb development. Mutations in HOXD13 and HOXD13 have been found to cause human limb malformation. Here we describe a two-generation Chinese family with a variant form of mild synpolydactyly. Methods Sequence analysis of HOXD13 gene in a two-generation Chinese family with six individuals. Results Gene scan and linkage analysis suggested that HOXD13 might be responsible for the disease of this family. An LOD around 1.8 was observed at three markers (P =2E^?3). We identified a novel c.893G>A (p.Arg298Gln) mutation in the HOXD13 homeodomain. And the mutation affected the transcriptional activation ability of HOXD13. Conclusion This finding expands the phenotypic spectrum associated with HOXD13 mutations and advances our understanding of human limb development.

Highlights

Posted on 27 May 2012 | 1:12 am

Case series: CSF LDH, proteins and electrolyte levels in patients of acute lymphocytic leukemia

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Deepa Rao, Veena S. Ghalaut, P.S. Ghalaut, Shashi Rao

Background Central nervous system (CNS) involvement is common in hemoncologic diseases especially in patients with acute lymphocytic leukemia (ALL). Currently available modalities have limitations in diagnosing CNS involvement in early stages of disease and have a limited prognostic value. Raised cerebrospinal fluid (CSF) total lactate dehydrogenase (LDH) levels can predict CNS involvement in patients with various neurological disorders including CNS leukemia. Methods This study was conducted in 23 consecutive freshly diagnosed patients of ALL without any previous CNS disease. Analysis of CSF was done for total LDH, proteins and electrolytes in all the patients before the start of chemotherapy and when the patients were in remission or 6weeks after chemotherapy whichever was earlier. Twenty-three age and sex matched controls were also studied to set the normal reference range. The results were analyzed statistically by Student's t test and coefficient of co-relation between CSF LDH and protein in patients with raised CSF LDH at the time of presentation was also calculated. Results CSF LDH was increased in 4 out of 6 patients with signs and symptoms of CNS involvement (Group A) and 3 of these patients also had increased CSF protein levels. 2 out of 17 patients without signs and symptoms of CNS involvement (Group B) had both elevated CSF LDH and protein levels. The increased levels came down to normal reference values after chemotherapy except in one Group A patient in whom CSF LDH remained high. However, no significant change in CSF electrolytes was noted in these patients. Conclusion Raised CSF LDH and CSF protein levels may indicate CNS involvement in patients with ALL.

Highlights

Posted on 27 May 2012 | 1:12 am

A new method for the measurement of lysosomal acid lipase in dried blood spots using the inhibitor Lalistat 2

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

John Hamilton, Iain Jones, Rajeev Srivastava, Peter Galloway

Background Cholesterol ester storage disease (CESD) and Wolman Disease (WD) are due to deficiency of lysosomal acid lipase (LAL). A new method is described for the measurement of LAL in dried blood spots (DBS) using Lalistat 2 an inhibitor of LAL. Methods LAL activity in DBS extracts was measured using the substrate 4-methylumbelliferyl palmitate. LAL activity was determined by measuring total lipase activity and lipase activity in the presence of Lalistat 2. The specificity of Lalistat 2 was investigated using human recombinant LAL (hrLAL) and human pancreatic lipase (hPL). Results Lalistat 2 inhibited hrLAL with 1% residual activity at 1?M inhibitor but had no effect on hPL up to 10?M. LAL activity in DBS samples obtained from normal controls (n =140) was 0.50–2.30nmol/punch/h and in patients with CESD was <0.03nmol/punch/h (n =11). Activity in carriers showed intermediate activity: 0.15–0.40nmol/punch/h (n =15). Conclusions Measurement of LAL using DBS is made difficult by the presence of other lipases in whole blood. Lalistat 2 is a specific inhibitor of LAL which allows the determination of LAL in DBS. Results show the method differentiates clearly between normal controls, carriers and affected cases.

Highlights

Posted on 27 May 2012 | 1:12 am

Associations between microRNA (miR-21, 126, 155 and 221), albuminuria and heavy metals in Hong Kong Chinese adolescents

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Alice P.S. Kong, Kang Xiao, Kai Chow Choi, Gang Wang, Michael H.M. Chan, Chung Shun Ho, Iris Chan, Chun Kwok Wong, Juliana C.N. Chan, Cheuk Chun Szeto

Background and aim Pathogenetic mechanisms underlying albuminuria are not completely understood. Heavy metals might lead to atherosclerosis and kidney damage. miR-21, 126, 155 and 221 regulated endothelial function and might contribute to the development of albuminuria. To date, no clinical trial has explored the relationship between miRNAs, microalbuminuria and heavy metals in human. In this study, we aimed to examine the association between microalbuminuria, miRNAs and heavy metals in adolescents. Materials and methods From a cross-sectional, population-recruited study, we identified 60 school children aged 12–19years with microalbuminuria (defined as spot urine albumin–creatinine ratio >3.5mg/mmol). We compared the urine heavy metals (arsenic, mercury, cadmium and lead) and miRNAs levels (miR-21,126, 155 and 221) with another 60 age-and sex-matched normoalbuminuric adolescents as control. Results Mean age of the study cohort was 15.5±2.1years. 43% were boys. Among the four miRNAs tested, only miR-21 was associated with microalbuminuria (p=0.02). Urinary arsenic and lead levels had a negative association with both miR-21 and miR-221. No significant association was found between heavy metals examined and microalbuminuria. Conclusion The results of our study suggest an association between microalbuminuria, miR-21 and heavy metals (arsenic and lead). This might imply that miR-21 is involved in the pathogenetic mechanisms linking heavy metals exposure and albuminuria.

Highlights

Posted on 27 May 2012 | 1:12 am

A novel and precise method for simultaneous measurement of serum HDL and LDL subfractions and lipoprotein (a) cholesterol by ultracentrifugation and high-performance liquid chromatography

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Jun Dong, Hanbang Guo, Ruiyue Yang, Hongxia Li, Shu Wang, Jiangtao Zhang, Weiyan Zhou, Wenxiang Chen

Background We developed an ultracentrifugation and high-performance liquid chromatography (HPLC) method for simultaneous measurement of cholesterol in serum high density lipoprotein (HDL) and low density lipoprotein (LDL) subfractions and lipoprotein (a) [Lp(a)]. Methods Serum aliquots of 0.05ml were centrifuged at background densities of 1.006, 1.044kg/l, and in the presence of ?-mercaptoethanol (ME) at background densities of 1.044, 1.063 and 1.125kg/l for the separation of lipoprotein subfractions and Lp(a). Cholesterol levels in the ultracentrifugal bottom fractions were analyzed by HPLC. Results ME effectively dissociated Lp(a) into apolipoprotein (a) and Lp(a) remnant [Lp(a-)]. Lp(a-) showed a distinctive density distribution from that of the native Lp(a). Based on these data, a method was developed to separate lipoprotein into subfractions and Lp(a) by ultracentrifugation. The separated HDL and LDL subfractions were not contaminated with Lp(a). This method is highly precise with the total CVs for the measurement of HDL2-C, HDL3-C, LDLa-C, LDLb-C and Lp(a)-C 0.85%–2.66%, 0.87%–3.21%, 0.86%–1.11%, 2.59%–6.35% and 4.42%–12.29%, respectively. Conclusion A new method for the separation of HDL and LDL subfractions and Lp(a) and simultaneous measurement of cholesterol by ultracentrifugation and HPLC have been established. It is precise and sensitive and can be used in research or clinical laboratories for lipoprotein profiling.

Highlights

Posted on 27 May 2012 | 1:12 am

Evaluation of saliva collection devices for the analysis of proteins

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Eleni Topkas, Patricia Keith, Goce Dimeski, Justin Cooper-White, Chamindie Punyadeera

Background Human saliva mirrors the body's health and can be collected non-invasively, does not require specialized skills and is suitable for large population based screening programs. The aims were twofold: to evaluate the suitability of commercially available saliva collection devices for quantifying proteins present in saliva and to provide levels for C-reactive protein (CRP), myoglobin, and immunoglobin E (IgE) in saliva of healthy individuals as a baseline for future studies. Methods Saliva was collected from healthy volunteers (n =17, ages 18–33years). The following collection methods were evaluated: drool; Salimetrics® Oral Swab (SOS); Salivette® Cotton and Synthetic (Sarstedt) and Greiner Bio-One Saliva Collection System (GBO SCS®). We used AlphaLISA® assays to measure CRP, IgE and myoglobin levels in human saliva. Results Significant (p <0.05) differences in the salivary flow rates were observed based on the method of collection, i.e. salivary flow rates were significantly lower (p <0.05) in unstimulated saliva (i.e. drool and SOS), when compared with mechanically stimulated methods (p <0.05) (Salivette® Cotton and Synthetic) and acid stimulated method (p <0.05) (SCS®). Saliva collected using SOS yielded significantly (p <0.05) lower concentrations of myoglobin and CRP, whilst, saliva collected using the Salivette® Cotton and Synthetic swab yielded significantly (p <0.05) lower myoglobin and IgE concentrations respectively. Conclusions The results demonstrated significantly relevant differences in analyte levels based on the collection method. Significant differences in the salivary flow rates were also observed depending on the saliva collection method. The data provide preliminary baseline values for salivary CRP, myoglobin, and IgE levels in healthy participants and based on the collection method.

Highlights

Posted on 27 May 2012 | 1:12 am

Diagnostic and clinical utility of antibodies against the nuclear body promyelocytic leukaemia and Sp100 antigens in patients with primary biliary cirrhosis

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Maria G. Mytilinaiou, Wolfgang Meyer, Thomas Scheper, Eirini I. Rigopoulou, Christian Probst, Andreas L. Koutsoumpas, Daniel Abeles, Andrew K. Burroughs, Lars Komorowski, Diego Vergani, Dimitrios P. Bogdanos

Background The lack of an immunoassay that detects antibodies to promyelocytic leukaemia (PML) protein, the primary biliary cirrhosis (PBC)-specific multiple nuclear dot (MND) antigen, has prompted us to develop a line immunoassay (LIA) for the simultaneous detection of PML and Sp100 MND-specific autoantibodies. Methods PML and Sp100 were expressed in Escherichia coli, and analysed by SDS-PAGE and immunoblotting using a monoclonal antibody and MALDI-ToF fingerprinting. A quantitative PML and Sp100 LIA were developed and testing was performed in 150 anti-mitochondrial antibody (AMA) positive, 20 AMA-PBCs and 130 controls. Results Thirty-five (23%) of 150 AMA+ PBCs (18 anti-MND+) were anti-PML+ (12%) or anti-Sp100+ (20%), 10 being anti-PML+/Sp100+, 5 single anti-PML+ and 20 single anti-Sp100+. Six (30%, 5 anti-MND+) AMA-PBCs were anti-PML+ or Sp100+. Only 2 (1.7%) pathological controls were anti-PML+ and/or anti-Sp100+. Levels of anti-PML correlated with those of anti-Sp100 (R=0.64, p <0.0001). The autoantibody profile largely remained unchanged over a 10year-follow up (52 patients, 352 samples). Anti-PML, Sp100 or MND-reactive PBCs were younger and had longer disease duration than the seronegative (p=0.06, for both). Anti-Sp100 levels correlated with the Mayo risk score (r=0.63, p =0.01). Anti-PML+/Sp100+ patients had more advanced disease compared to patients negative for anti-PML/Sp100 (p =0.04). Conclusion The new line immunoassay offers a robust and accurate method for the detection of clinically-relevant PBC-specific anti-MND antibodies.

Highlights

Posted on 27 May 2012 | 1:12 am

The characteristics of remnant lipoproteins in the fasting and postprandial plasma

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Katsuyuki Nakajima, Takamitsu Nakano, Yoshiharu Tokita, Takeaki Nagamine, Shin-ichi Yatsuzuka, Younosuke Shimomura, Akira Tanaka, Hiroyuki Sumino, Makoto Nara, Tetsuo Machida, Masami Murakami

Background Remnant-like lipoprotein particles (RLP) have been measured by cholesterol as RLP-C for CHD risk assessment in the fasting plasma. However, RLP-triglyceride (TG) is a better marker of the characteristics of remnant lipoproteins in the postprandial plasma, especially in plasma with TG concentrations <150mg/dl. Method The RLP-TG and RLP-C concentrations in subjects undergoing a health check-up and in volunteers receiving an oral fat load were determined in the fasting and postprandial plasma. TC, TG, HDL-C, LDL-C, apoB 100, apoB48, RLP apoB-100 and RLP apoB48 were also determined. Results When fasting TG concentrations were <150mg/dl, the 95th percentile of RLP-TG was 20mg/dl and the RLP-C 7.5mg/dl in healthy subjects. The prevalence of RLP-TG and RLP-C above the cut-off values with a TG concentration <150mg/dl was significantly higher in the metabolic syndrome cases than in the controls. RLP-TG increased significantly in plasma to >20mg/dl after an oral fat load in cases with TG concentrations >80mg/dl. Further, RLP apoB100, but not RLP apoB48 was highly correlated with the increase of TG in the postprandial plasma. Conclusion RLP-TG and RLP-C were increased significantly above the cut-off values in the postprandial plasma in healthy volunteers from a TG concentration >80mg/dl. RLP apoB100, but not RLP apoB48, increased significantly when the plasma TG increased after an oral fat load despite the increase of plasma apoB48. The results show that the major lipoproteins which were increased in postprandial plasma were VLDL remnants, not CM remnants.

Highlights

Posted on 27 May 2012 | 1:12 am

Proteomic identification of serum biomarkers for gastric cancer using multi-dimensional liquid chromatography and 2D differential gel electrophoresis

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Wentao Liu, Bingya Liu, Qu Cai, Jianfang Li, Xuehua Chen, Zhenggang Zhu

Background Early diagnosis and treatment of gastric cancer patients is essential for improving prognosis. However, no available serum-based test provides sufficient sensitivity or specificity for widespread use. Therefore, in this study we aimed to identify cancer biomarkers in human sera using 2-dimensional difference gel electrophoresis (2D-DIGE), and to characterize protein biomarkers with tandem mass spectrometry. Methods We compared the serum proteomic profiles of 20 gastric cancer patients and 10 healthy volunteers. Serum samples were first chromatographed using an immunoaffinity high-performance liquid chromatography (HPLC) column to selectively remove albumin, immunoglobulins, transferrin, haptoglobin, and antitrypsin. Differential protein analysis was then performed using DIGE. Significantly increased and decreased protein spot features were excised, trypsin digested, and analyzed by tandem matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF)/TOF and a linear trap quadrupole (LTQ) mass spectrometer. Results Seventeen protein spot features were significantly increased and 7 were significantly decreased in cancer serum samples compared to healthy controls. We identified 7 unique proteins that were upregulated, including plasminogen, apolipoprotein A-IV, Kininogen-1, complex-forming glycoprotein HC, complement component C4A, apolipoprotein J, and clusterin, and 5 that were decreased. Conclusions These results suggest that the combination of multi-dimensional HPLC and 2D-DIGE provides a valuable tool for serum proteomics in gastric cancer.

Highlights

Posted on 27 May 2012 | 1:12 am

High-resolution melting (HRM) analysis for the detection of single nucleotide polymorphisms in microRNA target sites

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Pei-Chin Lin, Ta-Chih Liu, Chun-Chi Chang, Yung-Hsiu Chen, Jan-Gowth Chang

Background The function of microRNAs (miRNAs) depends on the binding of miRNAs to their target sequences in the 3'UTR of messenger RNAs (mRNAs), which enhances the degradation of mRNAs and consequently, represses their expression. Single nucleotide polymorphisms (SNPs) in the miRNA target sequences may affect or impair the binding of miRNAs. Studies have shown that SNPs in miRNA target sites (miR-TS-SNPs) have a great influence on diverse biological functions, including pharmacogenomics and disease susceptibilities in human. Methods High-resolution melting (HRM) analysis was applied for investigating the allele frequencies of 3 miR-TS-SNPs (PLA2G2A, IL-16, and NOD2) in acute leukemia. We also compared the genotypes of acute lymphoblastic leukemia patients at initial diagnosis and complete remission. Results HRM analysis revealed 3 genotypes (both homozygous and heterozygous) in the 3 miR-TS-SNPs. The allele frequencies of all 3 miR-TS-SNPs were similar in normal individuals and patients with acute myelogenous leukemia. Most patients with acute lymphoblastic leukemia had the same genotypes at initial diagnosis and complete remission. Conclusions Large scale scanning of case–control studies for miR-TS-SNPs may contribute to the investigation of their roles and pathogenesis mechanisms in human diseases. Our study showed that HRM analysis can be an efficient tool for studies of miR-TS-SNPs.

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Posted on 27 May 2012 | 1:12 am

Improved analysis of C26:0-lysophosphatidylcholine in dried-blood spots via negative ion mode HPLC-ESI-MS/MS for X-linked adrenoleukodystrophy newborn screening

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

Christopher A. Haynes, Víctor R. De Jesús

Background X-linked adrenoleukodystrophy (X-ALD) is the most common human peroxisomal disorder, and is caused by mutations in the peroxisomal transmembrane ALD protein (ALDP, ABCD1). The biochemical defect associated with X-ALD is an accumulation of very long-chain fatty acids (VLCFA, e.g. C24:0 and C26:0), which has been shown to result in the accumulation of C26:0-lysophosphatidylcholine (C26:0-LPC). Methods We describe the analysis of C26:0-LPC in dried-blood spots (DBS) using a rapid (30min) and simple extraction procedure, isocratic HPLC resolution of LPC, and structure-specific analysis via negative ion mode tandem mass spectrometry. Results In putative normal DBS specimens from newborns (N=223) C26:0-LPC was 0.09±0.03?mol/l whole blood, while in peroxisomal biogenesis disorder (including X-ALD) patients (N=28) C26:0-LPC was 1.13±0.67?mol/l whole blood. Both multiple reaction monitoring and a neutral loss scan (225.1Da) analysis of DBS were used to analyze LPC. Conclusions Compared to a previous report of C26:0-LPC analysis in DBS, the method described here is simpler, faster, and more structure-specific for LPC with C26:0 acyl chains.

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Posted on 27 May 2012 | 1:12 am

Reference interval computation: which method (not) to choose?

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Igor Y. Pavlov, Andrew R. Wilson, Julio C. Delgado

Background When different methods are applied to reference interval (RI) calculation the results can sometimes be substantially different, especially for small reference groups. If there are no reliable RI data available, there is no way to confirm which method generates results closest to the true RI. Methods We randomly drawn samples obtained from a public database for 33 markers. For each sample, RIs were calculated by bootstrapping, parametric, and Box-Cox transformed parametric methods. Results were compared to the values of the population RI. Results For approximately half of the 33 markers, results of all 3 methods were within 3% of the true reference value. For other markers, parametric results were either unavailable or deviated considerably from the true values. The transformed parametric method was more accurate than bootstrapping for sample size of 60, very close to bootstrapping for sample size 120, but in some cases unavailable. Conclusions We recommend against using parametric calculations to determine RIs. The transformed parametric method utilizing Box-Cox transformation would be preferable way of RI calculation, if it satisfies normality test. If not, the bootstrapping is always available, and is almost as accurate and precise as the transformed parametric method.

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Posted on 27 May 2012 | 1:12 am

Current 25-hydroxyvitamin D assays: Do they pass the test?

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Lizhen Ong, Sharon Saw, Noorulhijjah Bte Sahabdeen, Kiat Teng Tey, Chung Shun Ho, Sunil Kumar Sethi

Background Vitamin D testing is becoming increasingly important with recent research demonstrating a correlation between vitamin D insufficiency and metabolic diseases, immunodeficiencies and other diseases. However, existing 25-hydroxyvitamin D (25OHD) assays lack comparability to the candidate reference method, causing difficulties in diagnosis and monitoring of vitamin D deficiency. Methods We looked at the accuracy of 3 automated assays (Roche Diagnostics Elecsys® Total 25OHD assay, Abbott Architect® Total vitamin D assay, Advia Centaur® vitamin D Total assay) and Diasorin® Radioimmunoassay (RIA) compared to a routine laboratory Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS). Results The correlation based on Passing Bablok regression was good with the slopes between 0.95 and 1.31 and the intercepts between ?3.24 and 3.68. However, a significant positive bias was observed using the Abbott Architect and the Diasorin RIA. Using published analytical goals of coefficient of variation (CV) <10% and bias <5%, most methods did not meet these criteria. Using measurement of uncertainty of 9%, most methods were able to meet criteria using quality control materials but not patient samples. Conclusion Inadequacies of these assay performances are contributed by differences in method of extraction of vitamin D from vitamin D binding protein, cross-reactivities to 25OHD2, 25OHD3 and other vitamin D metabolites, matrix interferences and a lack of standardization.

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Posted on 27 May 2012 | 1:12 am

Prognostic value of matrix metalloproteinase 9 expression in patients with non-small cell lung cancer

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Wen-Jia Peng, Jun-Qing Zhang, Bing-Xiang Wang, Hai-Feng Pan, Man-Man Lu, Jing Wang

Background The role of matrix metalloproteinase 9 (MMP-9) expression in non-small cell lung cancer (NSCLC) remains controversial. We performed a systematic review of the literature with meta-analysis. Methods Electronic databases were used to identify published studies before December 1, 2011. Pooled hazard ratio (HR) with 95% confidence interval (95% CI) was used to estimate the strength of the association between MMP-9 expression survival of NSCLC patients. Heterogeneity and publication bias were also assessed. Results The final analysis of 2029 NSCLC cases from 17 studies is presented. The combined HR of 1.84 (95% CI: 1.62–2.09) suggested that MMP-9 over-expression had a poor prognosis in patients with NSCLC. Subgroup analyses also detected significant association. Heterogeneity and publication bias was absent in current meta-analysis. Sensitivity analyses suggested that the summary statistics obtained should approximate the actual average. Conclusion High MMP-9 expression is associated with a poor prognosis in patients with NSCLC.

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Posted on 27 May 2012 | 1:12 am

Association between FTO 1st intron tagging variant and telomere length in middle aged females. 3PMFs study

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 15–16

D. Dlouha, J. Pitha, V. Lanska, J.A. Hubacek

The FTO gene plays an important role in the determination of body weight and BMI and it has been suspected of being associated with all-cause mortality, cardiovascular disease, cancer and end stage renal disease, but the causal mechanism of these effects is still unknown. One of the possibilities is the potential association with telomere length. Telomeres are repetitive DNA-sequences located at the ends of eukaryotic chromosomes' length of which is reduced in all somatic cells during ageing. Out of the 908 females (3PMFs study), in 783 females both FTO 1st intron tagging polymorphism (G>T, rs17817449) and the relative telomere length were successfully analysed. The relative telomere length was calculated as the ratio of telomere repeats to single-copy gene copies. The frequencies of the FTO genotypes were similar to other populations (GG=18.3%, GT=49.1% and TT=32.6%). We have detected, that the relative telomere length was significantly shorter (P<0.02, P<0.01 after adjustment for age, BMI, waist and subcutaneous fat), in carriers of at least one FTO risky (G) allele (0.85±0.39) in comparison to the carriers of the protective TT genotype (0.93±0.48). We have demonstrated that the FTO variant could be associated with the relative telomere length. Whether this represents a causality of association between the FTO variant and the non-communicable diseases needs to be further analysed.

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Posted on 27 May 2012 | 1:12 am

High-sensitive cardiac troponin T outperforms novel diagnostic biomarkers in patients with acute chest pain

Publication year: 2012
Source:Clinica Chimica Acta, Volume 413, Issues 13–14

Kai M. Eggers, Per Venge, Bertil Lindahl

Background Measurement of high-sensitive cardiac troponin (hs-cTn) has facilitated the early diagnostic assessment of chest pain patients. However, the information obtained from hs-cTnT levels might be improved when combined with results of other biomarkers of myocardial injury. Methods We measured admission levels of hs-cTnT (Roche Diagnostics), heart-type fatty-acid binding protein (H-FABP; Randox Laboratories) and copeptin using a novel ultra-sensitive (us) assay (Thermo Fisher Scientific) in 360 chest pain patients with a non-diagnostic ECG. Non-STEMI was defined according to the Universal Definition using cardiac troponin I (Stratus CS; Siemens Healthcare Diagnostics) as biochemical gold standard. Results Non-STEMI was diagnosed in 128 (36%) patients. Hs-cTnT had a greater diagnostic accuracy regarding non-STEMI (C-statistics 0.84) compared to H-FABP (C-statistics 0.80; p=0.04) and us-copeptin C-statistics(0.62; p<0.001). Compared to hs-cTnT alone, no increase in the C-statistics was noted for the combination of hs-cTnT with H-FABP (0.85; p=0.43) or with us-copeptin (0.84; p=0.88). Due to suboptimal sensitivities and/or specificities, neither H-FABP nor us-copeptin dichotomized at commonly applied diagnostic thresholds added information to hs-cTnT that would have facilitated early diagnostic assessment. Conclusions Hs-cTnT provides an excellent early diagnostic accuracy regarding non-STEMI already on admission. Neither H-FABP nor us-copeptin perform better or provide diagnostic increment to hs-cTnT levels.

Highlights

Posted on 27 May 2012 | 1:12 am