International Council for Standardization in Haematology Guidance for New Lot Verification of Coagulation Reagents, Calibrators, and Controls.

The clinical laboratory uses commercial products with limited shelf life or certain expiry dates requiring frequent lot changes. Prior to implementation for clinical use, laboratories should determine the performance of the new reagent lot to ensure that there is no significant shift in reagent performance or reporting of patient data. This guideline has been written on behalf of the International Council for Standardization in Haematology (ICSH) to provide the framework and provisional guidance for clinical laboratories for evaluating and verifying the performance of new lot reagents used for coagulation testing. These ICSH Working Party consensus recommendations are based on good laboratory practice, regulatory recommendations, evidence emerged from scientific publications, and expert opinion and are meant to supplement regional standards, regulations, or requirements.

A next generation FVIII mimetic bispecific antibody, Mim8, the impact on non-factor VIII related haemostasis assays.

INTRODUCTION AND AIMS: Mim8 is a next generation bispecific antibody developed for the treatment of haemophilia A (HA). Mim8 has an increased potency compared to first generation molecules. The impact on Mim8 on non-FVIII measuring haemostasis assays was assessed in plasma containing Mim8. METHODS: Congenital severe HA plasma was spiked with increasing concentrations of Mim8 (0-20 µg/mL). 28 routine and specialist haemostasis assays were used to measure activities. These included tests for prothrombin time (PT), fibrinogen, thrombin, D-dimer, anti-Xa, heparin induced thrombocytopenia (HIT), clotting factors II-XII, factor XIII, von Willebrand factor (VWF), thrombophilia and DRVVT. RESULTS AND CONCLUSIONS: Less than 10 % difference was calculated between plasma without Mim8 and plasma spiked to 15 µg/mL Mim8 in all assays except thrombin time (-10.5%), APTT-based factor IX, XI and XII, Werfen VWF:RCo (10.6%) and Siemens LA1 (-26.4%) and LA2 (-16.9%). At the expected therapeutic steady state levels of Mim8 (5-8 µg/mL), less than 10% difference was calculated for thrombin time and Werfen VWF:RCo. APTT-based assays of FIX, XI and XII are significantly elevated in the presence of Mim8 and should not be performed. A chromogenic FIX assay could be used to accurately measure FIX activity in the presence of Mim8. There was some interference in the DRVVT method we used so local assessment of other DRVVT methods is advised. Differences in all other tests would not be predicted to affect patient management.

Recommendations for clinical laboratory testing for protein C deficiency, for the subcommittee on plasma coagulation inhibitors of the ISTH.

Inherited protein C (PC) deficiency increases risk of venous thromboembolism (VTE) by 5 to 10-fold in thrombosis-prone families; however, heterozygous PC deficiency alone does not determine that a subject has thrombophilia. Protein C deficient subjects, who lack additional inherited risk factors such as factor V Leiden or have no major acquired risk factors, may not suffer from VTE. In addition, PC deficiency may be acquired, often due to vitamin K antagonist treatment or liver disease. In contrast, homozygous or compound heterozygous PC deficiencies are rare and serious disorders, and affected infants are often in families with no history of PC deficiency or thrombosis. Laboratories commonly use the chromogenic PC assay to diagnose deficiency. Chromogenic assay is recommended due to its good specificity, but this assay fails to detect the rare type 2b deficiency where the defect is due to poor interaction with calcium ions, phospholipid, protein S, and factor Va and factor VIIIa. The clotting-based assay of PC is capable of detecting type 2b deficiency but it has reduced specificity. Importantly, PC level varies with age, adult reference ranges cannot be applied to babies or children and levels may not reach those of adults even in adolescence. Pre-analytical variables in the specimen affect measurement of PC, and can be assay-dependent; for example, a partially clotted sample will have falsely raised PC level by chromogenic assay but reduced level by clotting-based assay. Direct oral anticoagulants falsely raise PC level in the clotting-based assay but the standard chromogenic assay is unaffected.

Biomarkers for the diagnosis of venous thromboembolism: D-dimer, thrombin generation, procoagulant phospholipid and soluble P-selectin.

BACKGROUND: The diagnostic algorithm for venous thromboembolism (VTE) currently involves a composite of pre-test probability, D-dimer and imaging. Other laboratory tests, however, may assist in the identification of patients with VTE. AIM: To assess the accuracy of different coagulation tests (D-dimer, thrombin generation, phospholipid-dependent (PPL) clotting time, soluble P-selectin (sP-selectin)) as biomarkers of acute VTE. METHODS: Random samples arriving at the Coagulation Laboratory at Mater Dei Hospital (Msida, Malta) from the Accident and Emergency Department with a request for D-dimer measurement were collected between August 2015 and February 2016. The following tests were performed: Innovance D-dimer (Siemens Healthcare Diagnostics), HemosIL D-dimer HS (Instrumentation Laboratory), thrombin generation (using the calibrated automated thrombogram), STA Procoag PPL (Diagnostica Stago) and sP-selectin (Affymetrix; eBioscience). VTE was objectively confirmed by compression ultrasonography, CT pulmonary angiography or ventilation/perfusion lung scan. RESULTS: 100 samples were collected (33 with VTE). A strong positive linear correlation was found between the two D-dimer tests (r=0.97, p<0.001). Patients with VTE showed significantly higher sP-selectin concentrations compared with patients without VTE (75.7 ng/mL vs 53.0 ng/mL, p<0.001). In the random forest plot, the two D-dimer assays showed the highest variable importance, followed by sP-selectin. A sP-selectin cut-off of 74.8 ng/mL was associated with 72.7% sensitivity and 78.2% specificity for acute VTE in our cohort. CONCLUSION: Our results confirmed D-dimer as the main biomarker of VTE and speculated a role for sP-selectin. The impact of thrombin generation was limited and no role emerged for the PPL clotting time. These observations need to be confirmed in large management studies.