D-dimer is a soluble fibrin degradation product deriving from the plasmin-mediated degradation of cross-linked fibrin. D-dimer can hence be considered a biomarker of activation of coagulation and fibrinolysis, and it is routinely used for ruling out venous thromboembolism (VTE). D-dimer is increasingly used to assess the risk of VTE recurrence and to help define the optimal duration of anticoagulation treatment in patients with VTE, for diagnosing disseminated intravascular coagulation, and for screening medical patients at increased risk of VTE. This review is aimed at (1) revising the definition of D-dimer; (2) discussing preanalytical variables affecting the measurement of D-dimer; (3) reviewing and comparing assay performance and some postanalytical variables (e.g. different units and age-adjusted cutoffs); and (4) discussing the use of D-dimer measurement across different clinical settings.

Despite the ongoing development of automated hematology analyzers to optimize complete blood count results, platelet count still suffers from pre-analytical or analytical pitfalls, including EDTA-induced pseudothrombocytopenia. Although most of these interferences are widely known, laboratory practices remain highly heterogeneous. In order to harmonize and standardize cellular hematology practices, the French-speaking Cellular Hematology Group (GFHC) wants to focus on interferences that could affect the platelet count and to detail the verification steps with minimal recommendations, taking into account the different technologies employed nowadays. The conclusions of the GFHC presented here met with a "strong professional agreement" and are explained with their rationale to define the course of actions, in case thrombocytopenia or thrombocytosis is detected. They are proposed as minimum recommendations to be used by each specialist in laboratory medicine who remains free to use more restrictive guidelines based on the patient’s condition.

The impact of direct oral anticoagulants (DOACs) on laboratory assays used for thrombophilia testing (e.g., antithrombin, protein S, protein C, lupus anticoagulant and activated protein-C resistance) is a well-known issue and may cause false-positive and -negative results. Therefore, the correct interpretation of tests that are performed in patients taking DOACs is mandatory to prevent misclassification and the subsequent clinical consequences. We aimed at evaluating the efficiency of a new and simple procedure (DOAC-Stop®; Haematex Research, Hornsby, Australia) to overcome the effect of all DOACs in real-life settings and to assess the percentage of erroneous results due to the presence of DOACs on thrombophilia screening tests. For this purpose, 135 DOAC-treated patients (38 apixaban, 40 dabigatran, 15 edoxaban, and 42 rivaroxaban) and 20 control patients were enrolled. A significant drop in apixaban, dabigatran, edoxaban, and rivaroxaban plasma concentrations following the DOAC-Stop® treatment was observed (74.8–8.2 ng/mL [p < 0.0001], 95.9–4.7 ng/mL [p < 0.0001], 102.1–8.8 ng/mL [p = 0.001], and 111.3–7.0 ng/mL [p < 0.0001], respectively). The DOAC-Stop® treatment was mostly effective to overcome the effect of DOACs on PTT-LA, dilute Russell's viper venom time (dRVVT) screen, and dRVVT confirm tests. Using our procedures, false-positive results due to DOACs were observed only with lupus anticoagulant tests (up to 75%) and fell to zero after the DOAC-Stop® procedure, regardless of the DOAC considered. In conclusion, the DOAC-Stop® adsorbent procedure appeared to be an effective and simple way to overcome the interference of DOAC on coagulation tests and should facilitate the interpretation of thrombophilia screening tests in patients taking DOACs.

ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 repeats, member 13) is responsible for the proteolysis of ultra-large von Willebrand factor (VWF) multimers into smaller multimers.[1] Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy (TMA) caused by severe ADAMTS13 deficiency (activity < 10%) leading to the formation of platelet-rich microthrombi within small arterioles responsible for the destruction of platelets and erythrocytes.[2] [3] TTP is a life-threatening condition with a mortality rate of 10 to 20% despite correct management.[1] The assessment of ADAMTS13 is of clinical importance as it is the only marker to be specific and sensitive toward TTP.[3] The rapid differential diagnosis between TTP and other TMA (e.g., haemolytic uraemia syndrome [HUS, no ADAMTS13 deficiency]) is essential to initiate the appropriate treatment as it can be difficult to differentiate TMA based on clinical presentation.[1] [4] Furthermore, monitoring of ADAMTS13 activity is useful to ensure biological remission (ADAMTS13 > 10%) as well as predicting relapse (usually preceding clinical signs).[5] [6] It has effectively been demonstrated that 40% of patients diagnosed with autoimmune TTP will experience at least one relapse.[1] It has been shown that any delay in ADAMTS13 results will have a negative impact on the diagnosis, treatment and prognosis of the patient.[3] [7] [8] Today, in-house methods using full-length VWF and the fluorescence resonance energy transfer (FRET)-VWF73 are considered reference methods.[5] [9] [10] However, these methods require considerable skill, are time-consuming and not widely available.[1] In our laboratory (Université catholique de Louvain, CHU UCL Namur, Hematology Laboratory, Yvoir, Belgium), the activity of ADAMTS13 is determined using the Technozym chromogenic enzyme-linked immunosorbent assay (ELISA) assay (Technozym, Technoclone, Vienna, Austria) based on the direct detection of cleavage products from a VWF peptide substrate (recombinant GST-VWF73 peptide [glutathione S-transferase-von Willebrand factor of 73 amino acids]) containing the cleavage site of the ADAMTS13 (Tyr1605-Met1606) (Technozym insert kit). This assay is calibrated against the first World Health Organization international standard ADAMTS13 plasma.[11] Unfortunately, the Technozym chromogenic ELISA assay is time-consuming, highly manual (risk of error) and requires a new calibration at each run. Therefore, we aimed to compare this method with the new and first fully automated HemosIL AcuStar ADAMTS13 activity assay (Instrumentation Laboratory, Bedford, Massachusetts, United States). This latter assay is a two-step immunoassay also using the GST-VWF73 substrate but with magnetic particles and chemiluminescence technology with a release of first results within 33 minutes (HemosIL AcuStar preliminary insert kit). For this method comparison, citrated plasma from 38 patients were selected from our sample collection preserved at –20°C for 1 to 11 months. These samples included 8 TTP patients, 2 patients with moderate ADAMTS13 activity (> 10–40%) and 28 patients with normal ADAMTS13 activity level (> 40%) based on our routine assay (Technozym). Assays were performed according to the manufacturers' instructions during October 2017. MedCalc software was used for statistical analysis (version 12.7.0). The mean bias between methods was determined with a Bland–Altman plot. Inherent characteristics and the pros and cons of each method were also compared ([Table 1]). The results of our external controls for the Technozym assay obtained from the ECAT Foundation (External quality Control for Assays and Tests with a focus on Thrombosis and Haemostasis) between July 2015 and August 2017 were also analysed (n = 16). The absolute mean bias on Bland–Altman plot was 4.8%, but was not constant (95% confidence interval [CI], –0.27 to 9.83%) and the limits of agreement (95% CI) were –25.3% and +34.9%, reflecting a high variability between both assays ([Fig. 1]). This variability has already been reported for several commercial ADAMTS13 assays.[12] Based on these results, the same method should be used for the monitoring of ADAMTS13 and each laboratory must determine their own reference values. We should note that for long-term monitoring purposes, biological and analytical variation should be further investigated. More important to notice is that based on the well acceptable TTP diagnosis cut-off (< 10%), no misclassification was pointed out. If considering only values < 10%, the bias was near to zero. Consequently, both methods appeared to be effective to identify acute TTP. Based on our external quality control program, the calculated Z-score always fell into the –2 to +2 interval except in one sample (August 2016, sample 16.58) (data not shown) but no misclassification was observed based on the < 10% cut-off value. In this method comparison, we used the Technozym assay as reference which showed a non-significant mean bias of –0.02% (95% CI, –1.75 to 1.71%) with 95% CI limits of agreement of –16.83 to 16.80% in 97 patients compared with the well-established FRET reference method developed by Kokame et al in 2005.[9] [10] Another study evaluating the Technozym assay showed that 88% (83/94) of TTP patients were correctly correlated to an in-house FRETS-VWF73 assay considered being the reference.[5] In this same study, 2.6% (4/154) of non-TTP patients were classified as falsely positive with the Technozym assay.[5] Taken together, this study calculated the sensitivity, specificity, predictive positive value and predictive negative value of the Technozym assay for the diagnosis of TTP at 88, 97, 95 and 93%, respectively, which supported its use in our daily routine practice. Recently, Langley et al also claimed that the Technozym assay was suitable for use in clinical settings.[8] In...

Flow cytometry is broadly used for the identification, characterization, and monitoring of hematological malignancies. However, the use of clinical flow cytometry is restricted by its lack of reproducibility across multiple centers. Since 2006, the EuroFlow consortium has been developing a standardized procedure detailing the whole process from instrument settings to data analysis. The FranceFlow group was created in 2010 with the intention to educate participating centers in France about the standardized instrument setting protocol (SOP) developed by the EuroFlow consortium and to organise several rounds of quality controls (QCs) in order to evaluate the feasibility of its application and its results. Here, we report the 5 year experience of the FranceFlow group and the results of the seven QCs of 23 instruments, involving up to 19 centers, in France and in Belgium. The FranceFlow group demonstrates that both the distribution and applicability of the SOP have been successful. Intercenter reproducibility was evaluated using both normal and pathological blood samples. Coefficients of variation (CVs) across the centers were <7% for the percentages of cell subsets and <30% for the median fluorescence intensities (MFIs) of the markers tested. Intracenter reproducibility provided similar results with CVs of <3% for the percentages of the majority of cell subsets, and CVs of <20% for the MFI values for the majority of markers. Altogether, the FranceFlow group show that the 19 participating labs might be considered as one unique laboratory with 23 identical flow cytometers able to reproduce identical results. Therefore, SOP significantly improves reproducibility of clinical flow in hematology and opens new avenues by providing a robust companion diagnostic tool for clinical trials in hematology. © 2019 International Society for Advancement of Cytometry

Continuous intravenous unfractionated heparin (UFH) is administered routinely in the intensive care unit (ICU) for the anticoagulation of patients, and monitoring is performed by the activated partial thromboplastin time (APTT) or anti-Xa activity. However, these strategies are associated with potentially large time intervals before dose adjustments, which could be detrimental to the patient. The aim of the study was to compare a point-of-care (POCT) version of the APTT to (i) laboratory-based APTT and (ii) measurements of anti-Xa activity in terms of correlation, agreement and turnaround time (TAT). Thirty-five ICU patients requiring UFH therapy were prospectively included and followed longitudinally for a maximum duration of 15 days. UFH was administered according to a local adaptation of Raschke and Amanzadeh’s aPTT nomograms. Simultaneous measurements of POCT-APTT (CoaguCheck^® aPTT Test, Roche Diagnostics) on a drop of fresh whole blood, laboratory-based APTT (C.K. Prest^®, Stago) and anti-Xa activity (STA^®Liquid anti-Xa, Stago) were systematically performed two to six times a day. Antithrombin, C-reactive protein, fibrinogen, factor VIII and lupus anticoagulant were measured. The time tracking of sampling and analysis was recorded. The overall correlation between POCT-APTT and laboratory APTT (n = 795 pairs) was strongly positive (rs = 0.77, p < 0.0001), and between POCT-APTT and anti-Xa activity (n = 729 pairs) was weakly positive (rs = 0.46, p < 0.0001). Inter-method agreement (Cohen’s kappa (k)) between POCT and laboratory APTT was 0.27, and between POCT and anti-Xa activity was 0.30. The median TATs from sample collection to the lab delivery of results for lab-APTT and anti-Xa were 50.9 min (interquartile range (IQR), 38.4–69.1) and 66.3 min (IQR, 49.0–91.8), respectively, while the POCT delivered results in less than 5 min (p < 0.0001). Although the use of the POCT-APTT device significantly reduced the time to results, the results obtained were poorly consistent with those obtained by lab-APTT or anti-Xa activity, and therefore it should not be used with the nomograms developed for lab-APTT.