Pre-analytical practices for routine coagulation tests in European laboratories. A collaborative study from the European Organisation for External Quality Assurance Providers in Laboratory Medicine (EQALM).

Background Correct handling and storage of blood samples for coagulation tests are important to assure correct diagnosis and monitoring. The aim of this study was to assess the pre-analytical practices for routine coagulation testing in European laboratories. Methods In 2013-2014, European laboratories were invited to fill in a questionnaire addressing pre-analytical requirements regarding tube fill volume, citrate concentration, sample stability, centrifugation and storage conditions for routine coagulation testing (activated partial thromboplastin time [APTT], prothrombin time in seconds [PT-sec] and as international normalised ratio [PT-INR] and fibrinogen). Results A total of 662 laboratories from 28 different countries responded. The recommended 3.2% (105-109 mmol/L) citrate tubes are used by 74% of the laboratories. Tube fill volumes ≥90% were required by 73%-76% of the laboratories, depending upon the coagulation test and tube size. The variation in centrifugation force and duration was large (median 2500 g [10- and 90-percentiles 1500 and 4000] and 10 min [5 and 15], respectively). Large variations were also seen in the accepted storage time for different tests and sample materials, for example, for citrated blood at room temperature the accepted storage time ranged from 0.5-72 h and 0.5-189 h for PT-INR and fibrinogen, respectively. If the storage time or the tube fill requirements are not fulfilled, 72% and 84% of the respondents, respectively, would reject the samples. Conclusions There was a large variation in pre-analytical practices for routine coagulation testing in European laboratories, especially for centrifugation conditions and storage time requirements.

Alternative Sample-Homogeneity Test for Quantitative and Qualitative Proficiency Testing Schemes.

Proficiency Testing (PT) External Quality Assessment (EQA) schemes are designed to ascertain the ability of individual laboratories to perform satisfactorily with respect to their peer laboratories or to limits imposed by external sources. Observed deviation of a laboratory result for a PT sample must be entirely attributed to the laboratory and not to the PT provider. To minimize the probability that deviations could be attributed to the PT provider, sample homogeneity should be assured. It is generally required that for quantitative parameters, the standard deviation among PT units should be calculated on the basis of duplicate measurements of at least 10 samples chosen at random, and the standard deviation among PT units should not exceed 0.3 times the standard deviation used to evaluate laboratories. Because this approach has important drawbacks, an alternative procedure is proposed by applying the theory of acceptance sampling to the assessment of sample heterogeneity for both quantitative and qualitative data and deriving acceptance limits on the basis of minimizing the probability of falsely evaluating laboratories. For obtaining acceptance limits for quantitative parameters, a distinction is made between laboratory evaluation using fixed limits on the one hand and laboratory evaluation using limits that are based on the variability of the reported results on the other hand. Sequential tests are proposed to evaluate sample heterogeneity by means of a comparison with the χ2 distribution. For qualitative parameters, acceptance-sampling plans are proposed that are based on minimizing the joint probability of rejecting batches that have a satisfactory amount of defective samples and accepting batches unnecessarily. The approach for quantitative parameters is applied on samples for a PT scheme of ethanol quantification and for qualitative parameters on the presence of monoblasts in a blood smear. It was found that five samples could already be enough to prove that the batch was homogeneous for quantitative parameters, although more than 20 samples were needed to prove homogeneity for qualitative parameters. This study describes a direct relation among the objective of an PT round, the criteria for evaluating the results, and the sample heterogeneity. When samples are effectively homogeneous, less measurements are needed than current practices require. A drawback of the proposed approach is that the number of samples to be tested is not known beforehand, and good knowledge of the analytical variability is crucial. The formulas to be applied are relatively simple. Despite the drawbacks, the proposed approach is generally applicable for both quantitative and qualitative data.

ANA IIF Automation: Moving towards Harmonization? Results of a Multicenter Study.

Background. Our study aimed to investigate whether the introduction of automated anti-nuclear antibody (ANA) indirect immunofluorescence (IIF) analysis decreases the interlaboratory variability of ANA titer results. Method. Three serum samples were sent to 10 laboratories using the QUANTA-Lyser® in combination with the NOVA View®. Each laboratory performed the ANA IIF analysis 10x in 1 run and 1x in 10 different runs and determined the endpoint titer by dilution. One of the three samples had been sent in 2012, before the era of ANA IIF automation, by the Belgian National External Quality Assessment (EQA) Scheme. Harmonization was evaluated in terms of variability in fluorescence intensity (LIU) and ANA IIF titer. Results. The evaluation of the intra- and interrun LIU variability revealed a larger variability for 2 laboratories, due to preanalytical and analytical problems. Reanalysis of the EQA sample resulted in a lower titer variability. Diluted endpoint titers were similar to the estimated single well titer and the overall median titer as reported by the EQA in 2012. Conclusion. The introduction of automated microscopic analysis allows more harmonized ANA IIF reporting, provided that this totally automated process is controlled by a thorough quality assurance program, covering the total ANA IIF process.

A new approach to define acceptance limits for hematology in external quality assessment schemes.

BACKGROUND: A study performed in 2007 comparing the evaluation procedures used in European external quality assessment schemes (EQAS) for hemoglobin and leukocyte concentrations showed that acceptance criteria vary widely. For this reason, the Hematology working group from the European Organisation for External Quality Assurance Providers in Laboratory Medicine (EQALM) decided to perform a statistical study with the aim of establishing appropriate acceptance limits (AL) allowing harmonization between the evaluation procedures of European EQAS organizers. METHODS: Eight EQAS organizers from seven European countries provided their hematology survey results from 2010 to 2012 for red blood cells (RBC), hemoglobin, hematocrit, mean corpuscular volume (MCV), white blood cells (WBC), platelets and reticulocytes. More than 440,000 data were collected. The relation between the absolute value of the relative differences between reported EQA results and their corresponding assigned value (U-scores) was modeled by means of an adaptation of Thompson's "characteristic function". Quantile regression was used to investigate the percentiles of the U-scores for each target concentration range. For deriving AL, focus was mainly on the upper percentiles (90th, 95th and 99th). RESULTS: For RBC, hemoglobin, hematocrit and MCV, no relation was found between the U-scores and the target concentrations for any of the percentiles. For WBC, platelets and reticulocytes, a relation with the target concentrations was found and concentration-dependent ALs were determined. CONCLUSIONS: The approach enabled to determine state of the art-based ALs, that were concentration-dependent when necessary and usable by various EQA providers. It could also easily be applied to other domains.

Influence of dabigatran and rivaroxaban on routine coagulation assays. A nationwide Belgian survey.

The Belgian national External Quality Assessment Scheme performed a nationwide survey using lyophilised plasma samples spiked with dabigatran or rivaroxaban to demonstrate to the Belgian clinical laboratories how these drugs affect their routine coagulation assays prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and antithrombin. Virtually all Belgian laboratories performing routine coagulation testing (189/192) participated in the survey. Both, dabigatran and rivaroxaban significantly prolonged the PT and aPTT in a concentration- and reagent-dependent manner. PT reagents were more influenced by rivaroxaban than by dabigatran and aPTT reagents more influenced by dabigatran than by rivaroxaban. Among PT reagents, Neoplastin R® was the most sensitive to rivaroxaban and Innovin® and Thromborel S® the least sensitive. Converting PT results to INR only increased the variability between reagents. Among aPTT reagents, Actin FSL® was the least sensitive to dabigatran while the other aPTT reagents showed slightly higher sensitivities. The presence of dabigatran led to falsely reduced fibrinogen concentrations when measured with a low thrombin concentration reagent. The presence of dabigatran caused an overestimation of the antithrombin level when measured with a thrombin-based activity assay and the presence of rivaroxaban an overestimation of the antithrombin level when measured with a FXa-based assay. Instrument-related differences were found for all tested parameters. In conclusion, this paper provides detailed information on the effect of dabigatran and rivaroxaban on routine coagulation assays as performed with a large number of reagent/instrument combinations.

From ANA-screening to antigen-specificity: an EASI-survey on the daily practice in European countries.

OBJECTIVES: One of the main goals of the European Autoimmunity Standardisation Initiative (EASI) is the harmonisation of test-algorithms for autoantibodies related to systemic autoimmune rheumatic diseases (SARD). METHODS: A questionnaire was used to gather information on methodology, interpretation, and the algorithm for detection of anti-nuclear antibodies (ANA) in relation to their antigen-specificity. The questionnaire was sent to 1200 laboratories in 12 European countries. RESULTS: The response rate was 47.2%. The results reveal not only apparent differences between countries, but also within countries. CONCLUSIONS: Awareness of these differences may as such already stimulate harmonisation, but the observed differences may also direct recommendations that may further contribute to achieving the EASI goal of harmonisation of autoimmune diagnostics for SARD.

Comparison of different approaches to evaluate External Quality Assessment Data.

In EQA programs, Z-scores are used to evaluate laboratory performance. They should indicate poorly performing laboratories, regardless of the presence of outliers. For this, two different types of approaches exist. The first type are "outlier-based" approaches, which first exclude outlying values, calculate the average and standard deviation on the remaining data and obtain Z-scores for all values (e.g., Grubbs and Dixon). The second type includes the "robust" approaches (e.g., Tukey and Qn or the algorithm recommended by ISO). The different approaches were assessed by randomly generated samples from the Normal and Student t distributions. Part of the sample data were contaminated with outliers. The number of false and true outliers was recorded and subsequently, Positive and Negative Predictive Values were derived. Also, the sampling mean and variability were calculated for location and scale estimators. The various approaches performed similarly for sample sizes above 10 and when outliers were at good distance from the centre. For smaller sample sizes and closer outliers, however, the approaches performed quite differently. Tukey's method was characterised by a high true and a high false outlier rate, while the ISO and Qn approaches demonstrated weak performance. Grubbs test yielded overall the best results.

A new statistical method for evaluating long-term analytical performance of laboratories applied to an external quality assessment scheme for flow cytometry.

BACKGROUND: The Belgian External Quality Assessment Scheme for Flow Cytometry evaluates the long-term analytical performance of participating laboratories by calculating a regression line between the target and reported values of each parameter for each laboratory during the past 3 years. This study aims to develop a method to find laboratories with aberrant variability or bias using robust techniques and to obtain robust estimates of the variability. METHODS: A method is proposed to find outliers with respect to the individual regression line, followed by a step to find regression lines with excessive variability and finally a step to find regression lines with high bias. RESULTS: The model was applied to the results obtained by 52 laboratories for CD4%. From the 1340 data points, 35 were determined to be regression outliers. The second step revealed one regression line with excessive variability; the third step detected three regression lines with exceeding bias. CONCLUSIONS: The methodology allows assessment of the long-term performance of laboratories, taking into account samples with different target values. Outliers in the first step indicate accidental mistakes, outliers in the second and third step point to high analytical variability or bias.

Current practices in antinuclear antibody testing: results from the Belgian External Quality Assessment Scheme.

BACKGROUND: This study aimed to assess the state-of-the-art of antinuclear antibody (ANA) testing as practiced in the Belgian and Luxembourg laboratories, using the results obtained in the Belgian National External Quality Assessment Scheme from 2000 to 2005. METHODS: During this period, nine samples with different specificities were sent for analysis. Participants were surveyed for methodology used and were asked to report staining pattern and titer of ANAs. In 2002, an attempt was made to improve the comparability of quantitative ANA results by the provision of a commercial reference material and to relate observed differences to methodology. RESULTS: With one exception, all participants employed a microscope-based indirect immunofluorescence assay with human epithelial cell line 2 cells. Most laboratories were accurate in describing the pattern. The percentage of unacceptable answers was greater for samples with borderline levels of antibody and for samples showing a cytoplasmic pattern. An improvement in the detection of anticentromere antibodies was observed. For all samples, a wide range of titers was reported. The provision of the secondary reference preparation led to improved inter-laboratory concordance. Comparison of methodology variables revealed a correlation between unstandardized titers and the power of the lamp of the microscope and the use of a dark room. CONCLUSIONS: The EQAS results presented in this work provide valuable insights into the state of the art of ANA testing as practiced in the Belgian and Luxembourg Laboratories and illustrate the important value of a national EQAS for ANA testing as a tool to improve performance and interlaboratory comparability of laboratory results.

Guidelines for setting up an external quality assessment scheme for blood smear interpretation. Part II: survey preparation, statistical evaluation and reporting.

Blood smear analysis is a well-known technique in medical laboratories. The clinical relevance of this analysis and its interpretation are very important. Consequently, monitoring of laboratory performance by an external quality assessment scheme is strongly recommended. This article represents a very practical guidance document for External Quality Assessment Scheme (EQAS) organizers for setting up blood smear schemes. In the first part of the guidelines, the Hematology Working Group of the European External Committee for External Quality Assurance Programmes in Laboratory Medicine (EQALM) published practical information for the preparation of blood smears for use in an EQAS. Part II focuses on aspects such as survey preparation, statistical evaluation and reporting, and describes particular details for organizing blood morphology EQA surveys by means of virtual microscopy.

Guidelines for blood smear preparation and staining procedure for setting up an external quality assessment scheme for blood smear interpretation. Part I: Control material.

Blood smear analysis is a well known technique in medical laboratories. Clinical relevance of this analysis and its interpretation are very important. Consequently, monitoring of laboratory performance by an external quality assessment scheme is strongly recommended. Most starting external quality organizers set up a scheme for clinical chemistry. Due to a lack of guidance documents, many organizers are reluctant to offer a hematology scheme. This article aims to be a very practical guidance document for external quality assessment organizers for the set up of blood smear schemes.