Band 55, Heft 3

Elevated serum uric acid (SUA) levels may increase the risk of prehypertension. However, the findings from these studies remain conflicting. The objective of this study was to determine the relationship between SUA levels and risk of prehypertension by conducting a meta-analysis. We conducted a comprehensive literature search of PubMed, Embase, China National Knowledge Infrastructure, VIP, and the Wangfang database without language restrictions through May 2015. Observational studies assessing the relationship between SUA levels and prevalence of prehypertension were included. Pooled adjust odds ratio (OR) and corresponding 95% confidence intervals (CI) of prehypertension were calculated for the highest vs. lowest SUA levels. Prehypertension was defined as systolic blood pressure (BP) ranging from 120 to 139 mmHg or diastolic BP ranging from 80 to 89 mmHg. Eight cross-sectional studies with a total of 21,832 prehypertensive individuals were included. Meta-analysis showed that elevated SUA levels were associated with increased risk of prehypertension (OR: 1.84; 95% CI: 1.42–2.38) comparing the highest vs. lowest level of SUA levels. Subgroup analyses showed that elevated SUA levels significantly increased the risk of prehypertension among men (OR: 1.60; 95% CI: 1.12–2.21) and women (OR: 1.59; 95% CI: 1.17–2.16). Elevated SUA levels are positively associated with the risk of prehypertension in the general population. However, more well-designed longitudinal studies are needed before a definitive conclusion can be drawn due to the cross-sectional studies included are susceptible to bias.

Lactate is one of the most crucial intermediates in carbohydrate and nonessential amino acid metabolism. The complexity of cellular interactions and metabolism means that lactate can be considered a waste product for one cell but a useful substrate for another. The presence of elevated lactate levels in critically ill patients has important implications for morbidity and mortality. In this review, we provide a brief outline of the metabolism of lactate, the pathophysiology of lactic acidosis, the clinical significance of D-lactate, the role of lactate measurement in acutely ill patients, the methods used to measure lactate in blood or plasma and some of the methodological issues related to interferences in these assays, especially in the case of ethylene glycol poisoning.

International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has established reference measurement procedures (RMPs) for the most popular enzymes. Manufacturers should assign values to commercial calibrators traceable to these RMPs to achieve equivalent results in clinical samples, independent of reagent kits, instruments, and laboratory where the measurement is carried out. The situation is, however, far from acceptable. Some manufacturers continue to market assays giving results that are not traceable to internationally accepted RMPs. Meanwhile, end-users often do not abandon assays with demonstrated insufficient quality. Of the enzyme measurements, creatine kinase (CK) is satisfactorily standardized and a substantial improvement in performance of marketed γ-glutamyltranspeptidase (GGT) assays has been demonstrated. Conversely, aminotransferase measurements often exceed the desirable analytical performance because of the lack of pyridoxal-5-phosphate addition in the commercial reagents. Measurements of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and α-amylase (AMY) still show major disagreement, suggesting the need for improvement in implementing traceability to higher-order references. This is mainly the result of using assays with different analytical selectivities for these enzymes. The definition by laboratory professionals of the clinically acceptable measurement uncertainty for each enzyme together with the adoption by EQAS of commutable materials and use of an evaluation approach based on trueness represent the way forward for reaching standardization in clinical enzymology.

In a recent EFLM recommendation on reference intervals by Henny et al., the direct approach for determining reference intervals was proposed as the only presently accepted “gold” standard. Some essential drawbacks of the direct approach were not sufficiently emphasized, such as unacceptably wide confidence limits due to the limited number of observations claimed and the practical usability for only a limited age range. Indirect procedures avoid these disadvantages of the direct approach. Furthermore, indirect approaches are well suited for reference limits with large variations during lifetime and for common reference limits.

The knowledge of error rates is essential in all clinical laboratories as it enables them to accurately identify their risk level, and compare it with those of other laboratories in order to evaluate their performance in relation to the State-of-the-Art (i.e. benchmarking) and define priorities for improvement actions. Although no activity is risk free, it is widely accepted that the risk of error is minimized by the use of Quality Indicators (QIs) managed as a part of laboratory improvement strategy and proven to be suitable monitoring and improvement tools. The purpose of QIs is to keep the error risk at a level that minimizes the likelihood of patients. However, identifying a suitable State-of-the-Art is challenging, because it calls for the knowledge of error rates measured in a variety of laboratories throughout world that differ in their organization and management, context, and the population they serve. Moreover, it also depends on the choice of the events to keep under control and the individual procedure for measurement. Although many laboratory professionals believe that the systemic use of QIs in Laboratory Medicine may be effective in decreasing errors occurring throughout the total testing process (TTP), to improve patient safety as well as to satisfy the requirements of International Standard ISO 15189, they find it difficult to maintain standardized and systematic data collection, and to promote continued high level of interest, commitment and dedication in the entire staff. Although many laboratories worldwide express a willingness to participate to the Model of QIs (MQI) project of IFCC Working Group “Laboratory Errors and Patient Safety”, few systematically enter/record their own results and/or use a number of QIs designed to cover all phases of the TTP. Many laboratories justify their inadequate participation in data collection of QIs by claiming that the number of QIs included in the MQI is excessive. However, an analysis of results suggests that QIs need to be split into further measurements. As the International Standard on Laboratory Accreditation and approved guidelines do not specify the appropriate number of QIs to be used in the laboratory, and the MQI project does not compel laboratories to use all the QIs proposed, it appears appropriate to include in the MQI all the indicators of apparent utility in monitoring critical activities. The individual laboratory should also be able to decide how many and which QIs can be adopted. In conclusion, the MQI project is proving to be an important tool that, besides providing the TTP error rate and spreading the importance of the use of QIs in enhancing patient safety, highlights critical aspects compromising the widespread and appropriate use of QIs.

Background: Spinal muscular atrophy (SMA) is mainly caused by deletions in SMA-related genes. The objective of this study was to develop gene-dosage assays for diagnosing SMA. Methods: A multiplex, quantitative PCR assay and a CNVplex assay were developed for determining the copy number of SMN1 , SMN2 , and NAIP . Reproducibility and specificity of the two assays were compared to a multiple ligation-dependent probe amplification (MLPA) assay. To evaluate reproducibility, 30 samples were analyzed three times using the three assays. A total of 317 samples were used to assess the specificity of the two assays. Results: The multiplex quantitative PCR (qPCR) assay had higher reproducibility. Intra-assay CVs were 3.01%–8.52% and inter-assay CVs were 4.12%–6.24%. The CNVplex assay had ratios that were closer to expected (0.49–0.5 for one copy, 1.03–1.0 for two copies, and 1.50–1.50 for three copies). Diagnostic accuracy rates for the two assays were 100%. Conclusions: The multiplex qPCR assay was a simple, rapid, and cost-effective method for routine SMA diagnosis and carrier screening. The CNVplex assay could be used to detect SMAs with complicated gene structures. The assays were reliable and could be used as alternative methods for clinical diagnosis of SMA.

Background: Improving quality and patient safety in the medical biochemistry laboratory accredited according to the International Standard Organization (ISO 15189:2012) requires the patient-centered evaluation of errors based on the implementation of quality indicators (QIs) across the total testing process. Our main goal was to achieve quality improvement of the preanalytical process in an emergency laboratory which had the highest error rate using risk management principles. Methods: Failure mode and effects analysis (FMEA) was applied to analyze predefined preanalytical QIs and score laboratory failures for the failure demerit value (FDV), probability of failure (PF) and probability of failure remedy (PFR). Based on obtained scores (on a 10-point scale) risk priority numbers (RPNs) were calculated. Results: A total of five failure modes were identified in the preanalytic process. The calculated risks were “sample hemolysis” (RPN, 168),“misidentified samples” (RPN, 108),“samples clotted” (RPN, 90),“sample volume error” (RPN, 72) and “samples transported at inappropriate temperature” (RPN, 24). The activation of corrective risk-reducing measures for failure modes with RPN≥30 resulted in quality improvement with the significant decrease in reevaluated RPNs. Conclusions: The implementation of a preanalytical quality monitoring system based on observation of evidence-based QIs and patient-centered evaluation of errors through risk analysis with regular tailored education as well as implementing process improvements can effectively reduce preanalytical errors in the emergency laboratory and improve patient safety.

Background: We provide a clinical and analytical evaluation of the reformulated version of the Abbott Architect 25-hydroxyvitamin D assay. We compared this assay with three commercial automated immunoassays and against a VDSP-traceable liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in six different populations. We also supplemented 40 healthy volunteers with either 600,000 IU of vitamin D2 or 100,000 of vitamin D3 to evaluate the performance of the immunoassays vs. the LC-MS/MS. Methods: Precision and limit of quantification were assessed, 25(OH)D2 and C3-epimer recovery were calculated. Two hundred and forty samples obtained in healthy Caucasians and Africans, osteoporotic, hemodialyzed and intensive care patients and 3rd trimester pregnant women were analyzed by all methods. Correlation was studied using Passing-Bablok and Bland-Altman analysis. Concordance correlation coefficient (CCC) was calculated to evaluate agreement between immunoassays and LC-MS/MS. We verified if patients were homogeneously classified with the immunoassays when they took vitamin D2 or vitamin D3 after 1, 7 and 28 days. Results: We observed excellent analytical features and showed a very good correlation to the LC-MS/MS results in the overall population. Compared to the other immunoassays, concordance of the new Abbott assay with the LC-MS/MS was at least similar, and often better in diseased populations. Althought the cross-reactivity with 25(OH)D2 was not of 100%, there was no significant difference in the classifications of the patients, either supplemented with D2 or D3 or after 7 or 28 days. Conclusions: This modified version of the Abbott Architect assay is clearly improved compared to the previous one and presents a better agreement with the LC-MS/MS.

Background: The systematic evaluation of the clinical concordance of various 25-hydroxyvitamin D (25OHD) testing methods is presented. The need for this approach is raised by the discrepancies in the analytical performance of the available assays. Methods: The analytical and clinical performance of six automated 25OHD assays and an in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was investigated. Leftover serum samples (n=162, SA: n=114) were analyzed and all 21 assay combinations were evaluated. The utility of Cohen’s κ values was assessed by transforming them into minimum percentage agreement (MPA). McNemar’s hypothesis test was employed for testing the symmetry of the disagreeing classification outcomes within each method pair. Results: Depending on the assay method, the ratio of results classified as positive (<20 ng/mL) was 13.5%–40.0%. The percentage agreement (PA) was 74.1%–92.6%. Compared to other methods, significantly more hypovitaminosis cases were delivered by DiaSorin Liaison® 25 OH vitamin D Total (DL) and significantly fewer by IDS-iSYS 25-Hydroxy Vitamin DS (II). The strongest clinical concordance was exerted by II vs. LC-MS/MS. The κ-derived MPA showed close similarity to the PA scores. McNemar’s tests confirmed the asymmetry of the disagreement in the classification in 14 method combinations. Conclusions: The presented approach allows the prediction of the clinical consequences of a 25OHD method transfer. Differences in the clinical classification of assay results are likely encountered when transferring to a new method, even between assays standardized according to the Vitamin D Standardization Program (VDSP) Reference Method Procedure (RMP).

Background: Osmotic gradient ektacytometry is part of the laboratory diagnosis process of hereditary spherocytosis (HS) and other red blood cell (RBC) membrane disorders. We here present the experience of two independent institutions with a next-generation ektacytometer, the LoRRca MaxSis analyzer, in HS diagnostic settings. Methods: Inter- and intra-assay variability and sample stability were analyzed. Samples from patients with HS (n=40), probable HS (n=21), auto-immune hemolytic anemia (n=7), and other pathologies (n=37) were studied. Daily controls were run in parallel with patient samples. Results were expressed as percent of change compared to mean of controls. Results: Analytical performances showed an inter-assay variability between 0.2% and 3%. Samples were stable for 48–72 h depending of temperature storage and anticoagulant used. The following diagnostic cut-offs were established for HS: an increase of more than 21.5% for the osmolality point at the minimal elongation index (O min), a decrease of more than 8.5% for the maximal elongation index (EI max), and a decreased area under the curve (AUC) of more than 18.5% compared to the mean of controls. Conclusions: As the previous instrument, the next-generation ektacytometer is an efficient tool for laboratory diagnosis of HS. Sample stability and standardized reporting of results allow inter-laboratory exchange and comparison. The most useful parameters for HS diagnosis were AUC, EI max, and O min; unfortunately, this method does not differentiate between HS and auto-immune hemolytic anemia (AIHA), but it distinguishes HS from other hereditary membrane pathologies. It can thus be considered as an intermediate step between screening and diagnostic tests.

Background: Lysosphingolipids (LysoSLs) are derivatives of sphingolipids which have lost the amide-linked acyl chain. More recently, LysoSLs have been identified as storage compounds in several sphingolipidoses, including Gaucher, Fabry and Niemann-Pick diseases. To date, different methods have been developed to measure each individual lysosphingolipid in plasma. This report describes a rapid liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of several LysoSLs in plasma. Methods: We analyzed the following compounds: hexosylsphingosine (HexSph), globotriaosylsphingosine (LysoGb3), lysosphingomyelin (LysoSM) and lysosphingomyelin-509 (LysoSM-509). The sample preparation requires only 100 μL of plasma and consists of an extraction with a mixture of MeOH/acetone/H 2 O (45:45:10, v/v). Results: The method validation showed high sensitivity, an excellent accuracy and precision. Reference ranges were determined in healthy adult and pediatric population. The results demonstrate that the LC-MS/MS method can quantify different LysoSLs and can be used to identify patients with Fabry (LysoGb3), Gaucher and Krabbe (HexSph) diseases, prosaposine deficiency (LysoGb3 and HexSph), and Niemann-Pick disease types A/B and C (LysoSM and LysoSM-509). Conclusions: This LC-MS/MS method allows a rapid and simultaneous quantification of LysoSLs and is useful as a biochemical diagnostic tool for sphingolipidoses.

Background: Electronic cigarettes (e-cig) known as electronic nicotine devices recently gained popularity among smokers. Despite many studies investigating their safety and toxicity, few examined the delivery of e-cig-derived nicotine and its metabolites in alternative biological fluids. Methods: We performed a randomized, crossover, and controlled clinical trial in nine healthy smokers. Nicotine (NIC), cotinine (COT), and trans-3′-hydroxycotinine (3-HCOT) were measured in plasma and oral fluid by liquid chromatography-tandem mass spectrometry after consumption of two consecutive e-cig administrations or two consecutive tobacco cigarettes. Results: NIC and its metabolites were detected both in oral fluid and plasma following both administration conditions. Concentrations in oral fluid resulted various orders of magnitude higher than those observed in plasma. Oral fluid concentration of tobacco cigarette and e-cig-derived NIC peaked at 15 min after each administration and ranged between 1.0 and 1396 μg/L and from 0.3 to 860 μg/L; those of COT between 52.8 and 110 μg/L and from 33.8 to 94.7 μg/L; and those of 3-HCOT between 12.4 and 23.5 μg/L and from 8.5 to 24.4 μg/L. The oral fluid to plasma concentration ratio of both e-cig- and tobacco cigarette-derived NIC peaked at 15 min after both administrations and correlated with oral fluid NIC concentration. Conclusions: The obtained results support the measurement of NIC and metabolites in oral fluid in the assessment of intake after e-cig use and appear to be a suitable alternative to plasma when monitoring nicotine delivery from e-cig for clinical and toxicological studies.

Background: Serum free light chains (FLC) are sensitive biomarkers used for the diagnosis and management of plasma cell dyscrasias, such as multiple myeloma (MM), and are central to clinical screening algorithms and therapy response criteria. We have developed a portable, near-patient, lateral-flow test (Seralite ® ) that quantitates serum FLC in 10 min, and is designed to eliminate sample processing delays and accelerate decision-making in the clinic. Methods: Assay interference, imprecision, lot-to-lot variability, linearity, and the utility of a competitive-inhibition design for the elimination of antigen-excess (‘hook effect’) were assessed. Reference ranges were calculated from 91 healthy donor sera. Preliminary clinical validation was conducted by retrospective analysis of sera from 329 patients. Quantitative and diagnostic results were compared to Freelite ® . Results: Seralite ® gave a broad competitive-inhibition calibration curve from below 2.5 mg/L to above 200 mg/L, provided good assay linearity (between 1.6 and 208.7 mg/L for κ FLC and between 3.5 and 249.7 mg/L for λ FLC) and sensitivity (1.4 mg/L for κ FLC and 1.7 mg/L for λ FLC), and eliminated anomalous results from antigen-excess. Seralite ® gave good diagnostic concordance with Freelite ® (Roche Hitachi Cobas C501) identifying an abnormal FLC ratio and FLC difference in 209 patients with newly diagnosed MM and differentiating these patients from normal healthy donors with polyclonal FLC. Conclusions: Seralite ® sensitively quantitates FLC and rapidly identifies clinical conditions where FLC are abnormal, including MM.

Background: Pressure to cut health-care costs has involved clinical laboratories underpinning the need to reduce cost per test through programs designed to consolidate activities and increase volumes. Currently, however, there is little evidence of the effectiveness of these measures. The aim of the present study was to verify whether a rational, evidence-based decision-making process might be achieved based on an activity-based cost analysis performed by collecting the data of all variables affecting cost per test. Methods: An activity-based costing analysis was performed using a program that provides collected data on performance indicators, benchmark between different laboratories based on performance indicators, and information on reorganization initiatives. Results: The data provided were used in two different settings to (1) verify the results of the internal re-organization of specific protein assay and (2) simulate some scenarios for the reorganization of autoimmune testing in the network of clinical laboratories in a large territory. Conclusions: The data produced by the e-Valuate project enabled the quantification of variation in costs, the utilization of human and technological resources and efficiency, both as final result of a reorganization project (proteins) and as a simulation of a possible future organization (autoimmune tests).

Background: Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor caused by a malignant transformation in the parafollicular C-cells of the thyroid, where calcitonin (CT) is released. Nowadays the main tumor markers (TM) used in the diagnosis and follow-up of MTC patients are CT and carcinoembryonic antigen (CEA). Nonetheless, progastrin releasing peptide (proGRP) has been recently proposed as a TM useful in the MTC. Our aims were to investigate the release of proGRP in thyroid tumors, its role in the assessment of advanced MTC and its utility in the differential diagnosis between MTC and non-MTC thyroid tumors. Methods: Serum samples from 22 patients with MTC and 16 with non-MTC were collected. Patients were classified into advanced cancer or no evidence of disease (NED). ProGRP was performed by Architect (Abbot Diagnostics), CT by Liaison (Diasorin) and CEA by Cobas E601(Roche Diagnostics). Results: ProGRP median concentration in advanced MTC was significantly higher (1398.4 pg/mL) when compared with non-MTC, either in advanced disease (24.9 pg/mL) or NED (14.6 pg/mL). In non-MTC patients, proGRP median concentration was below its cutoff level (50 pg/mL). Similar to CT, proGRP was able to detect 88.9% of MTC patients, but with a slightly lower specificity of 76.9%. Using proGRP together with CT the sensitivity increased to 100%. Conclusions: The low prevalence of this malignancy strongly recommends further collaborative studies, mainly focused on monitoring proGRP during tyrosine kinase inhibitors treatment for early detection of resistance and assessing its usefulness to avoid the observed false positive fluctuations that occur with CT and CEA.

Background: Cholesterol homeostasis disorders may cause dyslipidemia, atherosclerosis progression and coronary artery disease (CAD) development. Evaluation of non-cholesterol sterols (NCSs) as synthesis and absorption markers, and lipoprotein particles quality may indicate the dyslipidemia early development. This study investigates associations of different cholesterol homeostasis patterns with low-density (LDL) and high-density lipoproteins (HDL) subclasses distribution in statin-treated and statin-untreated CAD patients, and potential use of aforementioned markers for CAD treatment optimization. Methods: The study included 78 CAD patients (47 statin-untreated and 31 statin-treated) and 31 controls (CG). NCSs concentrations were quantified using gas chromatography- flame ionization detection (GC-FID). Lipoprotein subclasses were separated by gradient gel electrophoresis. Results: In patients, cholesterol-synthesis markers were significantly higher comparing to CG. Cholesterol-synthesis markers were inversely associated with LDL size in all groups. For cholesterol homeostasis estimation, each group was divided to good and/or poor synthetizers and/or absorbers according to desmosterol and β-sitosterol median values. In CG, participants with reduced cholesterol absorption, the relative proportion of small, dense LDL was higher in those with increased cholesterol synthesis compared to those with reduced synthesis (p<0.01). LDL I fraction was significantly higher in poor synthetizers/poor absorbers subgroup compared to poor synthetizers/good absorbers (p<0.01), and good synthetizers/poor absorbers (p<0.01). Statin-treated patients with increased cholesterol absorption had increased proportion of LDL IVB (p<0.05). Conclusions: The results suggest the existence of different lipoprotein abnormalities according to various patterns of cholesterol homeostasis. Desmosterol/β-sitosterol ratio could be used for estimating individual propensity toward dyslipidemia development and direct the future treatment.

Background: Consolidation of laboratories has left many hospitals and satellite laboratories with minimal microbiologic testing. In many hospitals and satellite laboratories, Gram stains on primary specimens are still performed despite difficultly in maintaining proficiency. Methods: To maintain Gram stain proficiency at a community 450-bed hospital with an active emergency room we designed bimonthly challenges that require reporting Gram staining and morphology of different organisms. The challenges consist of five specimens prepared by the reference microbiology laboratory from cultures and primary specimens. Twenty to 23 medical laboratory scientists participate reading the challenges. Results from the challenges are discussed with each medical laboratory scientists. In addition, printed images from the challenges are presented at huddle to add microbiology knowledge. Results: On the first three challenges, Gram staining was read correctly in 71%–77% of the time while morphology 53%–66%. In the last six challenges correct answers for Gram stain were 77%–99% while morphology 73%–96%. Conclusions: We observed statistically significant improvement when reading Gram stains by providing frequent challenges to medical laboratory scientists. The clinical importance of Gram stain results is emphasized during huddle presentations increasing knowledge and motivation to perform the test for patients.

Background: Microsampling techniques have several advantages over traditional blood collection. Dried blood spot (DBS) sampling and blood collection with heparinized capillaries are the standard techniques. Volumetric absorptive microsampling (VAMS) is a novel technique that collects a fixed volume of blood by applying an absorbent tip to a blood drop. In the present study we explored the feasibility of HbA 1c monitoring with VAMS sampling at home and analysis in the laboratory. Methods: Diabetic patients were enrolled in this study during consultation with the endocrinologist. A venous (adults) or capillary (children) sample was taken for immediate HbA 1c analysis. DBS (n=1) and dried VAMS (n=2) were collected at home and sent to the laboratory. For 25 pediatric patients one VAMS was collected during consultation for immediate analysis (without drying), referred to as “wet VAMS”. HbA 1c analyses were performed on a Tosoh HLC-723 G8 high-performance liquid chromatography (HPLC) analyzer. Results: The median time between sampling at home and analysis was 3 days. Results of HbA 1c in dried VAMS showed a poor agreement with venous/capillary blood collected in hospital (concordance correlation coefficient CCC=0.72). Similar observations were found with standard DBS. An excellent agreement was obtained between HbA 1c results on wet VAMS (CCC=0.996) and standard blood samples. Patients experienced VAMS and DBS as easy and convenient to use. Conclusions: Utilizing equipment standard available in the clinical laboratory, the use of home-sampled dried VAMS and DBS is not a reliable tool for the monitoring of HbA 1c . However, perfect agreement between HbA 1c measured on wet VAMS and capillary microsamples was obtained.