Volume 53, Issue 2

Laboratory and radiographic tests are often ordered unnecessarily. This excess testing has financial costs and is a burden on patients. We performed a systematic review to determine the effectiveness interventions to reduce test utilization by physicians. The MEDLINE and EMBASE databases were searched for the years 1946 through to September 2013 for English articles that had themes of test utilization and cost containment or optimization. Bibliographies of included papers were scanned to identify other potentially relevant studies. Our search resulted in 3236 articles of which 109 met the inclusion criteria of having an intervention aimed at reducing test utilization with results that could be expressed as a percent reduction in test use relative to the comparator. Each intervention was categorized into one or more non-exclusive category of education, audit and feedback, system based, or incentive or penalty. A rating of study quality was also performed. The percent reductions in test use ranged from a 99.7% reduction to a 27.7% increase in test use. Each category of intervention was effective in reducing test utilization. Heterogeneity between interventions, poor study quality, and limited time horizons makes generalizations difficult and calls into question the validity of results. Very few studies measure any patient safety or quality of care outcomes affected by reduced test use. There are numerous studies that use low investment strategies to reduce test utilization with one time changes in the ordering system. These low investment strategies are the most promising for achievable and durable reductions in inappropriate test use.

A new generation of antithrombotic agents, which are conventionally known as direct oral anticoagulants (DOACs), have recently emerged and are continuing to be developed. These provide direct inhibition of either thrombin (factor IIa; FIIa) or activated factor X (FXa) and currently include dabigatran (FIIa inhibitor) and rivaroxaban, apixaban, and edoxaban (FXa inhibitors). The dogma that DOACs do not require laboratory monitoring is countered by ongoing recognition that laboratory testing for drug effects is needed in many situations. In this review, we summarize the background to establishment of DOACs, assess which tests were found to be useful to screen for or quantitate drug effects/levels, and then review published guidelines/recommendations to assess concordance. In brief, (a) for the anti-FIIa agent dabigatran, the recommended screening assays are activated partial thromboplastin time (APTT) and/or thrombin time (TT), and the quantitative assays (using a dabigatran standard) are dilute TT/direct thrombin inhibitor assay (Hemoclot thrombin inhibitor) or an ecarin-based assay such as the ecarin clot time (ECT); (b) for the anti-FXa agent rivaroxaban, the recommended screening assay is the prothrombin time (PT), but this was not endorsed by all guidelines, and the quantitative assay (using a specific rivaroxaban standard) is an anti-FXa assay; (c) for the anti-FXa agent apixaban, the general insensitivity of PT and APTT prevented most groups from providing recommendation, and instead there was generalized support for direct quantitative assessment using anti-FXa assays and specific apixaban standard; (d) there is insufficient data for other direct anti-FXa agents and limited guidance in the literature.

The VII European Symposium of the Clinical Laboratory and In Vitro Diagnostic Industry, co-organized between the Catalan Association for Clinical Laboratory Sciences (ACCLC) and the Catalan Society of Biology, was held on May 28th–29th, 2013 in Barcelona (Catalonia, Spain) under the IFCC auspices and the IUPAC sponsorship. The subject of the present Symposium was “Molecular Genetics in the Clinical Laboratory” and began with an opening conference that was a stroll through the history of molecular genetics in the context of the clinical laboratory. The scientific program was structured in several 2-h length roundtables that dealt with the following topics: recent advances in molecular genetics for clinical microbiology, latest evidences and real applicability of pharmacogenetics in the clinical practice, quality assurance of a molecular genetics laboratory, and latest trends in prenatal genetic diagnosis. The aim of the Symposium was the discussion of the transformation that molecular genetics has generated on clinical laboratories in terms of organization, specialization, interpretation of results and fast technical and knowledge evolution. High-qualified professionals from several countries together with in-country experts formed the roundtables. Attendants participated actively in the debates, increasing the overall interest.

Background: Analysis of circulating cell-free fetal DNA (cffDNA) in maternal plasma is very promising for early diagnosis of monogenic diseases. However, this approach is not yet available for routine use and remains technically challenging because of the low concentration of cffDNA, which is swamped by the overwhelming maternal DNA. Methods: To make clinical applications more readily accessible, we propose a new approach based on mutant enrichment with 3′-modified oligonucleotides (MEMO) PCR along with real-time PCR to selectively amplify from the maternal blood the paternally inherited fetal allele that is not present in the maternal genome. Results: The first proof of concept of this strategy was displayed for cystic fibrosis by the accuracy of our detection of the p.Gly542* mutation used as the initial developmental model. Subsequently, a retrospective study of plasmas originating from two pregnant women carrying a fetus with private mutation confirmed the effectiveness of our method. We confirmed the presence of cffDNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. Conclusions: This new non-invasive prenatal diagnosis test offers numerous advantages over current methods: it is simple, cost effective, time efficient and does not require complex equipment or bioinformatics settings. Moreover, our assays for different private mutations demonstrate the viability of this approach in clinical settings for monogenic disorders.

Background: A number of studies have indicated that the conversion of clopidogrel to its active metabolite is reduced in patients who carry the CYP2C19 *2, *3, *4 or *5 loss-of-function allele, resulting in decreased response of platelet to clopidogrel treatment and worse cardiovascular outcome. The aim of this study was to develop a novel biosensor-based microarray to visually detect CYP2C19 polymorphisms. Methods: The target DNA was amplified from regions flanking the respective alleles using 5′-biotinylated reverse primer, and plasmids were prepared for the respective alleles. High stringency reversed hybridization, horseradish peroxidase-labeled streptavidin reaction, and color development, with multiple washes in different steps, were carried out and the results were recorded with an optical camera. The gene chips were tested for specificity, detection limit, intra- and inter-batch variations using the constructed plasmids. Finally, 88 clinical samples were assayed with this microarray as well as direct sequencing. Results: The results could be seen with the naked eye. Concordance tests indicated that for alleles *2, *3, *4, and *5, the κ values between this assay and plasmids all reached 1.000. The detection limit was 5×10 2 cells/mL. Concordance test between direct sequencing and the microarray assay using 88 clinical samples gave rise to the κ value of 0.983, and p<0.01, indicating very high concordance. Conclusions: This novel biosensor-based microarray assay can amplify the signal in situ so that it can be detected by simple instruments or even the naked eyes. It is promising for clinical application in hospital laboratories.

Background: The presence of errors in the preanalytical phase is a widely studied topic. However, information regarding the perspective of those professionals involved is rather scant. Methods: Two focus groups of professionals from Primary Care involved in the preanalytical phase (general practitioners [GP], community nurses [CN], and other auxiliary health workers, including administrative personnel [AHW]) were convened. A qualitative analysis with a phenomenological approach was performed by using the structure of SWOT (Strengths, Weaknesses, Opportunities and Threats) analysis as a guide, and results were categorized by grouping the resultant dimensions according to this structure. Results: Overall, 12 professionals (3 GP, 6 CN, and 3 AHW) were distributed in two groups. Age and gender distribution were similar between groups. The most commented strengths were organizational capability and teamwork. The main weakness was the workload increase (compared to the short time spent on sample collection). Opportunities were related to workload optimization through on-line analytical requests. Threats were related to the long time elapsed between sample drawing at Primary Care and delivery to the Central Laboratory. Conclusions: The phenomenological approach allows revealing those aspects that cannot be entirely elucidated by objective data measurement. Attitudes considered as positive can be exploited by the institution, whereas those considered as negative alert us to possible future problems. Primary Care professionals offered a different point of view to laboratory staff, but both recognized high workload as the main threat and on-line analytical request as the best opportunity. These perspectives may help to improve detection and decrease the number of errors.

Background: Validation of blood collection tubes are important to determine the role of different collection tubes which influence the assurance of laboratory results. We compared two different tubes (Improvacuter ™ and Becton Dickinson [BD] Vacutainer ™ ) and investigated the effect of gel and storage time in comparison with each other. Methods: We compared the results of nine immunoassays performed on UniCel ® DxI 800 using blood samples collected in BD Vacutainer SST II Advance tubes, Improvacuter Gel and Clot Activator tubes, BD Vacutainer Clot Activator tubes and Improvacuter tubes. Analytes were measured in all tubes on 3 consecutive days to study the effect of long-term storage. Evaluation of clinical significance was performed based on total allowable error. Results: Estradiol and testosterone concentrations obtained from Improvacuter Gel and Clot Activator tubes and BD Vacutainer SST II Advance tubes remained below the lower limits of analytical range for the same analytes while they were within the limits in BD Vacutainer Clot Activator tubes and Improvacuter tubes. Statistical significance of stability was not clinically significant for the hormone parameters we tested in all four tubes. Conclusions: Gel containing tubes (both BD and Improve) gave comparable results with the tubes which do not contain gel except for estradiol and testosterone. The use of gel containing tubes for estradiol and testosterone are not recommended on UniCel ® DxI 800 according to our results. The change in the analyte concentrations over 48 h remained within the TE A limits for the studied analytes. Improve tubes gave similar results to BD tubes.

Background: Point-of-care testing (POCT), like other laboratory tests, can be affected by errors throughout the total testing process. To evaluate quality error rates, the use of quality indicators (QIs) is recommended; however, little information is available on the quality error rate associated with POCT. The objective of this study was to investigate quality error rates related to POCT and compare them with central laboratory (CL) testing. Methods: We studied standardized QIs for POCT in comparison to CL testing. We compared error rates related to requests, collection, and handling of samples and results from external quality assessment program (EQAP) and internal quality control (IQC). Results: The highest difference between POCT and CL testing was observed for QI related to patient identification, 45.3% vs. 0.02% (p<0.001). Regarding specimen collection and handling, the QI related to samples without results was also higher in POCT than in CL testing, 15.8% vs. 3.3% (p<0.001). For the QI related to insufficient sample volume, we obtained 2.9% vs. 0.9% (p=0.27). Unlike QIs for the preanalytical phase, QIs for the analytical phase had better results in POCT than CL testing. We obtained 8.3% vs. 16.6% (p=0.13) for QI related to unacceptable results in EQAP and 0.8% vs. 22.5% (p<0.001) for QI related to unacceptable results in IQC. Conclusions: Our results show that the preanalytical phase remains the main problem in POCT like in CL testing and that monitoring of quality indicators is a very valuable tool in reducing errors in POCT.

Background: A significant proportion of patients undergoing cardiopulmonary bypass develop anti-protamine antibodies, with or without the association of thromboembolic events. Methods: We extensively investigated the serological features of protamine antibodies, which developed in six patients who were clinically suspected to have heparin-induced thrombocytopenia (HIT). Three patients had thrombotic events. Sera were tested by four different commercially available immunoassays, a heparin-platelet aggregation test, and for their binding properties to heparin, platelet factor 4 (PF4), complex heparin-PF4, protamine, and protamine complex with heparin. Sera from four patients were also tested for the capability to induce platelet activation and the formation of platelet-monocyte heterotypic aggregates. Results: The ELISA assay Zymutest HIA was strongly positive in all cases, the HPIA Asserachrome was borderline, and the gel centrifugation test PaDGIA was positive in two tested patients. Platelet aggregation tests were negative. Using a variation of the Zymutest HIA we demonstrate that IgG antibodies bound only to protamine or protamine complex with heparin, but not to heparin or PF4 only. Sera-induced platelet P-selectin expression and the formation of platelet-monocyte aggregates. Blood samples from one patient proofed positive concomitantly with the thromboembolic event. However, serological characteristics did not differ between antibodies associated with thromboembolic events from those without. Conclusions: These data show that protamine-induced antibodies are specific and may induce platelet activation, which explains their association with thromboembolic events.

Background: Heparin-induced thrombocytopenia (HIT) is caused by platelet activating antibodies that recognize platelet factor 4/heparin (PF4/H) complexes. Laboratory testing plays a key role in the diagnosis of HIT. As functional assays are unfeasible for most clinical laboratories, antigen binding assays are commonly used in routine testing. However, their low specificity leads to overdiagnosis of HIT. Therefore, it is advisable to improve screening tests in this setting. Methods: Blood samples from 114 patients in whom HIT was suspected were investigated using a chemiluminescence test (HemosIL ® AcuStar HIT-IgG), a PF4/H IgG enzyme immunoassay (Lifecodes PF4 IgG), an IgG-specific lateral flow immunoassay heparin-induced thrombocytopenia (LFI-HIT, STic Expert ® HIT) and the heparin-induced platelet aggregation (HIPA) test. Results: Twenty-nine (25.4%) out of 114 subjects with suspected HIT had a positive HIPA test. None of patients with a 4Ts score <4 were positive at HIPA. HemosIL ® AcuStar HIT-IgG showed the best performance in term of sensitivity and specificity when used as single test. Receiver operating characteristic (ROC) analysis showed optimization of sensitivity and specificity using a cut-off of 1.13 U/mL (0.95 and 0.98, respectively). As an alternative approach, a strategy based on screening samples by STic Expert ® HIT and then retesting positive results by Lifecodes PF4 IgG (cut-off 1 OD) or HemosIL ® AcuStar HIT-IgG (cut-off 1.3 U/mL) showed a performance compared to a single test approach by HemosIL ® AcuStar HIT-IgG. Conclusions: The HemosIL ® AcuStar HIT or a combinatorial approach with the STic Expert ® HIT and the PF4/H IgG enzyme immunoassay provide an accurate diagnosis of immune HIT.

Background: Apixaban is a newly developed direct oral anticoagulant targeting activated factor X (FXa). The degree of interference of apixaban with coagulation parameters has not been thoroughly investigated. Methods: Increasing amounts of apixaban were added to aliquots of a pooled normal plasma (PNP) to mimic a large range of concentrations (n=8) that are observed in treated patients. Upon preparation, samples were stored frozen and tested for a vast array of coagulation parameters (including procoagulant and anticoagulant factors) in three laboratories, using three widely used coagulation platforms (reagent/coagulometer combinations). Results: Results for each parameter were expressed as ratios of the value corresponding to each apixaban concentration to the value corresponding to the PNP without apixaban. By definition, ratios higher or lower than the unity define overestimation or underestimation, respectively. Prothrombin and activated partial thromboplastin times were barely prolonged by apixaban 200 ng/mL (ratios <1.29 or <1.19, respectively). Conversely, antithrombin was considerably overestimated when the measurement was made with FXa as target enzyme (ratios up to 1.43). Protein C and protein S were overestimated when measured as anticoagulant activities (ratios up to 1.20 or 1.95, respectively), and most measurements for individual coagulation factors (except fibrinogen) were considerably underestimated (ratios from 0.62 to 1.01). Conclusions: This large multicenter multiplatform study investigating a common set of test plasmas shows that apixaban interferes with the measurement of most coagulation parameters requested for investigation of hemostasis and highlights the need for a careful interpretation of results obtained in patients under treatment.

Background: The rate of auto-validation is dependent on the ability of the laboratory information system (LIS) to integrate historical data, on the frequency and methods for identifying analyzer errors, and on the criteria for reflex testing, including the need for peripheral smear review. The rate of auto-validation in outpatient laboratories, however, is unclear. Methods: We examined 45,925 consecutive complete blood count (CBC) test results (1 January, 2014–31 January, 2014) from patients aged 50±24 years. The LIS auto-validates all samples according to set criteria. Technicians validated test results when previous CBC test results were required to determine: 1) the need for peripheral slide review and/or sample rerun or 2) the need for reflex testing to detect autoimmune hemolytic anemia or β-thalassemia minor. Results: The auto-validation rates were 97.6% after rejecting results requiring validation to determine the need for a peripheral smear review and/or sample rerun. This decreased to 92.9% after including reflex testing to determine the reasons for normocytic and microcytic anemia. We estimated that auto-validation decreased the workload by 7.7–11.6 h per 3000 test results. Conclusions: We conclude that very high auto-validation rates are possible in outpatient general laboratories, leading to conformity in the validation process and a considerable estimated savings in technician time. Further studies are needed in other settings.

Background: The XN-Series (Sysmex, Kobe, Japan) have been equipped with the automated digital cell imaging analyzer DI-60, which provides complete automation of the sample processing with automated complete blood counts (CBC), slide making/staining, and digital scanning with cell pre-classification. The aim of this study was to evaluate the efficacy of the XN-Series as an integrated blood cell analysis system. Methods: White blood cell (WBC) morphological analysis by the DI-60 was evaluated using 232 blood samples from patients. Routine analysis of a total of 2000 blood samples has been performed to evaluate the processing ability of the XN-Series connected to the DI-60. Results: The overall analysis accuracy of pre-classification of WBC by the DI-60 was 88.4%. Good correlation was observed between final results of the DI-60 analysis and manual differentiation with high sensitivity and specificity for blasts and immature granulocytes. The sample processing time of the XN-Series, from automated CBC to cell pre-classification, was 38±1 min/single run and 165±12 min/500 CBC samples run (slide preparation rate 15.6%) with no sample hold-up at the DI-60. Conclusions: The automated morphological analysis capability of the DI-60 has potential usefulness in the integrated automated hematology analysis system of XN-Series.

Background: The aim of this study was to evaluate the plasma keratan sulfate (KS) level as a potential marker of joint damage in children with juvenile idiopathic arthritis (JIA). The influence of growth factors as well as proteolytic and prooxidative agents on aggrecan alterations were evaluated in this study. Methods: Plasma levels of KS, transforming growth factor β1 (TGF-β1), platelet-derived growth factor BB (PDGF-BB), a disintegrin and metalloproteinase with thrombospondin motifs 4 and 5 (ADAMTS-4 and ADAMTS-5), and thiol groups (TG) were quantified in samples obtained from 30 healthy subjects and 30 patients with JIA before and after treatment. Results: Increased (p<0.01) plasma KS was observed in JIA patients before treatment. Therapy resulted in a decrease in KS level. However, plasma KS level remained higher (p<0.05) than in controls. Increased levels of TGF-β1 (p<0.01) and PDGF-BB (p<0.05) in untreated JIA patients were recorded. Clinical improvement was accompanied by significant decrease in TGF-β1 and PDGF-BB, compared with a pretreatment condition and a control group. The concentrations of proteinases were characterized by different trends of alterations. When the ADAMTS-4 level increased (p<0.01) in the blood of untreated patients, the concentration of ADAMTS-5 was found to be reduced (p<0.0001), compared with controls. JIA treatment resulted in the normalization of ADAMTS-4 level. Plasma TG concentration was decreased only in untreated patients (p<0.05). We have revealed a significant correlation between plasma KS level and ADAMTS-4, TGF-β1, TG, C-reactive protein, and erythrocyte sedimentation rate levels. Conclusions: Plasma KS level in JIA patients, reflecting the aggrecan structure, indicates that treatment that modifies inflammation simultaneously does not contribute to total regeneration of articular matrix components and signalizes the need for further treatment.

Background: Numerical data on the components of biological variation (BV) have many uses in laboratory medicine, including in the setting of analytical quality specifications, generation of reference change values and assessment of the utility of conventional reference values. Methods: Generation of a series of up-to-date comprehensive database of components of BV was initiated in 1997, integrating the more relevant information found in publications concerning BV. A scoring system was designed to evaluate the robustness of the data included. The database has been updated every 2 years, made available on the Internet and derived analytical quality specifications for imprecision, bias and total allowable error included in the tabulation of data. Results and conclusions: Our aim here is to document, in detail, the methodology we used to evaluate the reliability of the included data compiled from the published literature. To date, our approach has not been explicitly documented, although the principles have been presented at many symposia, courses and conferences.

Background: Inflammation plays an imperative role in the etiology of cardiovascular diseases (CVD). The role of cytokines in the development and progression of idiopathic dilated cardiomyopathy (IDCM) is still uncertain. The current study was conducted to evaluate the association of tumor necrosis factor-α ( TNF-α ) –238G/A and IL-6 –572G/C gene polymorphism with IDCM in a Pakistani population. Methods: IDCM cases (n=250) and healthy controls (n=300) were genotyped using PCR-RFLP. Results: The variant genotypes of both the loci showed significant differences between patients and controls (p<0.05). However, –238G/A polymorphism did not show association with the disease in the presence of covariates. We also conducted a meta-analysis of both the loci with regards to CVD in accordance with the Prisma checklist. No significant relation of the TNF-α –238G/A polymorphism with CVD was found; however, this single nucleotide polymorphism (SNP) showed an association with the disease in the Asian population after subgroup analysis (p=0.01). Whereas, the IL-6 –572G/C polymorphism showed that the variant genotype (GC+CC) was associated with higher risk of CVD in contrast to the GG genotype. Furthermore, subgroup meta-analysis demonstrated a significant association of the –572 polymorphism with CVD in Asians, but no association was observed among Western populations with this SNP. Conclusions: Our findings support an association between the IL-6 –572G/C polymorphism and IDCM risk. The role of the TNF-α –238G/A polymorphism in IDCM is still unclear. Further studies are warranted to determine the serum cytokine levels in relation to the cytokines’ SNP in diverse ethnic groups to ascertain the molecular basis of the disease pathology.

Background: The accurate and precise quantification of HbA 1c is essential for the diagnosis and routine monitoring of patients with diabetes. We report an evaluation of the Trinity Biotech Premier Hb9210 analyser (Bray, Ireland/Kansas City, MO, USA), a boronate affinity chromatography-based high performance liquid chromatography (HPLC) system for the measurement of glycated haemoglobin. Methods: We evaluated the analytical performance of the Hb9210 as part of a multicentre evaluation. The effect of haemoglobin variants, other potential interferences and the performance in comparison to both the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and National Glycohemoglobin Standardization Program (NGSP) reference systems, was assessed. Most of the centres participating also act as reference laboratories for both the IFCC standardisation network for HbA 1c and the NGSP. Results: The combined data from all centres showed total coefficients of variation (CV) of 2.71%, 2.32% and 2.14% at low, medium and high values, respectively, for mmol/mol (SI units) and 1.62%, 1.59% and 1.68% for % (NGSP units), which are well below the recommended upper limits of 3% CV for mmol/mol (SI units) and 2% CV for % (NGSP). The analyser showed a good correlation to HbA 1c methods currently used in clinical practice and the IFCC reference method procedure. Haemoglobin variants AC, AS, AE and AD do not affect the measurement of HbA 1c . Overall the Hb9210 performs well across the whole analytical range. Conclusions: The Hb9210 performs well and is suitable for clinical application in the analysis of HbA 1c .

Background: An effective quality control (QC) program requires the establishment of control limits within which the results of the QC sample is expected to fall. Traditionally, the mean plus/minus two standard deviations calculated for a set of QC sample results is used to establish control limits. Allowable total error (TEa) and Westgard rules aid in interpreting QC sample results. Westgard rules assume QC sample results are normally distributed and TEa assumes commutability between the QC sample and patient results. None of these paradigms apply to infectious disease testing. Methods: Results from the NRL’s QC program were extracted and sorted into assay/QC lot number-specific data. Control limits for selected QC samples used to monitor 64 commonly used serological assays were calculated and validated using the within- and between-QC lot variance of data from each of the assay/QC combinations. Results: No assay/QC combination had more than 10% of results less than the lower control limit or greater than the upper control limit. Of the 423 assay/QC lot combinations, 14 (3.3%) had more than 5% of results less than the lower limit and 48 (11.3%) had more than 5% of results greater than the upper limit calculated for that assay/QC combination. Conclusions: The control limits, established by this novel method, are based on more than a decade of QC test results from >300 laboratories from 30 countries and provides users of the NRL QC program evidence-based control limits that can be applied in isolation or in conjunction with more traditional methods for establishing control limits.