Diagnosis and management of heparin-induced thrombocytopenia: third edition

This guideline updates and widens the scope of the previ-ous British Society for Haematology (BSH) Clinical guide-lines for Diagnosis and Management of Heparin-Induced Thrombocytopenia: Second Edition1 to include functional assays in the diagnosis of heparin-induced thrombocytope-nia (HIT), when to use direct-acting oral anti-coagulants, and the role of intravenous (IV) immunoglobulins and plasma exchange in the management of HIT and spontane-ous HIT.HIT is an immune-mediated, highly pro-thrombotic dis-order of platelet activation caused by pathogenic antibodies against a platelet factor 4 (PF4)­heparin complex. It is the most frequent drug-induced immune thrombocytopenia and may lead to life-threatening thrombosis. There are two distinct forms of HIT: type I, also known as heparin-asso-ciated thrombocytopenia, which is a non-immunological response to heparin treatment, mediated by a direct interac-tion between heparin and circulating platelets causing plate-let clumping or sequestration, and type II, which is immune mediated.

A global comparative field study to evaluate the factor VIII activity of efanesoctocog alfa by one-stage clotting and chromogenic substrate assays at clinical haemostasis laboratories.

INTRODUCTION: Structural and chemical modifications of factor VIII (FVIII) products may influence their behaviour in FVIII activity assays. Hence, it is important to assess the performance of FVIII products in these assays. Efanesoctocog alfa is a new class of FVIII replacement therapy designed to provide both high sustained factor activity levels and prolonged plasma half-life. AIM: Evaluate the accuracy of measuring efanesoctocog alfa FVIII activity in one-stage clotting assays (OSAs) and chromogenic substrate assays (CSAs). METHODS: Human plasma with no detectable FVIII activity was spiked with efanesoctocog alfa or a full-length recombinant FVIII product comparator, octocog alfa, at nominal concentrations of 0.80 IU/mL, 0.20 IU/mL, or 0.05 IU/mL, based on labelled potency. Clinical haemostasis laboratories (N = 35) tested blinded samples using in-house assays. Data from 51 OSAs (14 activated partial thromboplastin time [aPTT] reagents) and 42 CSAs (eight kits) were analyzed. RESULTS: Efanesoctocog alfa activity was reliably (±25% of nominal activity) measured across all concentrations using OSAs with Actin FSL and multiple other aPTT reagents. Under- and overestimation of FVIII activity occurred with some reagents. No specific trend was observed for any class of aPTT activators. A two- to three-fold overestimation was consistently observed using CSAs and the OSA with Actin FS as the aPTT reagent across evaluated concentrations. CONCLUSION: Under- or overestimation occurred with some specific OSAs and most CSAs, which has been previously observed with other modified FVIII replacement products. Efanesoctocog alfa FVIII activity was measured with acceptable accuracy and reliability using several OSA methods and commercial plasma standards.

Agreement between one stage and chromogenic assays in samples from patients receiving recombinant porcine FVIII (Obizur, Susoctocog-alfa).

INTRODUCTION: Recombinant porcine FVIII (rpFVIII) (Obizur, Susoctcog-alfa, Takeda, Japan) is licensed for the treatment of bleeding in acquired Haemophilia A (AHA). The summary of product characteristics state that monitoring should be by one stage assay (OSA) rather than chromogenic assay (CSA). CSA have been shown to underestimate activity when rpFVIII is added to plasma in vitro. METHODS: Samples from three AHA patients (n = 21) (pre- and post rpFVIII) were assessed using FVIII:C assays; OSA methods: Actin, Actin FS, Actin FSL and Pathromtin SL performed on CS5100i (Sysmex, Kobe, Japan); APTT-SP, SynthASil and SynthAFax performed on ACL TOP (Werfen, Barcelona, Spain). CSA methods on CS5100i: Siemens Chromogenic Assay, Biophen FVIII:C, Technochrom FVIII:C; on ACL TOP: Rox Factor VIII, Coamatic Factor VIII and CRYOcheck Factor VIII. RESULTS: OSA and CSA varied according to reagent used median OSA 61 IU/dL (range 41.5-81 IU/dL (ANOVA p < 0.0001)) median CSA 46.5 IU/dL (range of method specific medians 36.5-84 IU/dL (ANOVA p < 0.0001)). Amongst OSA, Actin FS was associated with the highest FVIII:C, APTT-SP was associated with the lowest. Variation in CSA results by different methods was also seen with highest FVIII:C levels obtained using the Technochrom FVIII:C and the lowest levels obtained with Siemens Assay. CONCLUSION: The relationship between OSA and CSA was not consistent between method or patient. Previously there has been reports of underestimation by CSA in in vitro spiked samples. Investigation into concentration of phospholipids in the APTT reagents may explain some of these variations.

Defining a metrologically traceable and sustainable calibration hierarchy of international normalized ratio for monitoring of vitamin K antagonist treatment in accordance with International Organization for Standardization (ISO) 17511:2020 standard: communication from the International Federation of Clinical Chemistry and Laboratory Medicine-SSC/ISTH working group on prothrombin time/international normalized ratio standardization.

Calibration of prothrombin time (PT) in terms of international normalized ratio (INR) has been outlined in "Guidelines for thromboplastins and plasmas used to control oral anticoagulant therapy" (World Health Organization, 2013). The international standard ISO 17511:2020 presents requirements for manufacturers of in vitro diagnostic (IVD) medical devices (MDs) for documenting the calibration hierarchy for a measured quantity in human samples using a specified IVD MD. The objective of this article is to define an unequivocal, metrologically traceable calibration hierarchy for the INR measured in plasma as well as in whole blood samples. Calibration of PT and INR for IVD MDs according to World Health Organization guidelines is similar to that in cases where there is a reference measurement procedure that defines the measurand for value assignment as described in ISO 17511:2020. We conclude that, for PT/INR standardization, the optimal calibration hierarchy includes a primary process to prepare an international reference reagent and measurement procedure that defines the measurand by a value assignment protocol conforming to clause 5.3 of ISO 17511:2020. A panel of freshly prepared human plasma samples from healthy adult individuals and patients on vitamin K antagonists is used as a commutable secondary calibrator as described in ISO 17511:2020. A sustainable metrologically traceable calibration hierarchy for INR should be based on an international protocol for value assignment with a single primary reference thromboplastin and the harmonized manual tilt tube technique for clotting time determination. The primary international reference thromboplastin reagent should be used only for calibration of successive batches of the secondary reference thromboplastin reagent.

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.

Internal Quality Control in Hemostasis Assays.

Internal quality control (IQC) for routine and specialist hemostasis testing represents a mandatory requirement for assays offered by clinical laboratories under International Organization for Standardization, Code of Federal Regulations, and Clinical and Laboratory Standards Institute standards. The underlying principle is that regular IQC audits the analytical performance of automated, semiautomated, and manual methods. This review investigates IQC practices, including benefits, limitations, frequency per time period or batch, sources of material used, primary supplier, third party or in-house, plus troubleshooting when IQC falls outside acceptance criteria. To assess IQC practice, the UK National External Quality Assessment Scheme (NEQAS) Blood Coagulation distributed a questionnaire to 1,200 participants enrolled in our scheme that collected details of the local practices for IQC testing. We received returns from 127 centers that described their local practices for the frequency of IQC, the type of IQC material employed, acceptance criteria for IQC data, and troubleshooting protocols for IQC failures. The data collected as part of an NEQAS BC questionnaire confirmed that all the participants returning answers to the questionnaire meet the standards for regular IQC testing for the hemostasis assays they perform.

International Council for Standardization in Haematology recommendations for laboratory measurement of factor VIII and FIX type I inhibitors.

This guidance document has been prepared on behalf of the International Council for Standardisation in Hematology. The aim of the document is to provide guidance and recommendations on the measurement of factor VIII (FVIII) and factor IX (FIX) inhibitors. After an introduction on the clinical background and relevance of factor VIII and factor IX inhibitor testing, the following aspects of laboratory testing are included: screening for inhibitors, assay principle, sample requirements, testing requirements and interpretation, quality assurance, interferences and recent developments. This guidance document focusses on recommendations for a standardised procedure for the laboratory measurement of FVIII and FIX type I inhibitors. The recommendations are based on published data in peer-reviewed literature and expert opinion.

ICSH guidance for INR and D-dimer testing using point of care testing in primary care.

This guideline has been written on behalf of the International Council for Standardisation in Haematology (ICSH) and focuses on two point of care haematology tests used within primary care, namely International Normalised Ratio (INR) and D-dimer. Primary care covers out of hospital settings and can include General Practice (GP), Pharmacy and other non-hospital settings (although these guidelines would also be applicable to hospital out-patient settings). The recommendations are based on published data in peer reviewed literature and expert opinion; they should supplement regional requirements, regulations or standards.

The combination of emicizumab and recombinant factor VIII in plasma: Which assays can we use for accurate measurement?

INTRODUCTION: The bispecific antibody, emicizumab, is a prophylactic therapy used for the treatment of haemophilia A (HA). Patients may require additional replacement factor VIII (FVIII) to ensure adequate haemostasis. This study investigated the laboratory measurement of severe HA (SHA) plasma spiked with 36 combinations of emicizumab plus recombinant (r) FVIII concentrates. METHOD: FVIII assays were performed by one stage assay (OSA) using eight APTT reagents from three manufacturers and chromogenic assays (CSA) using seven kits. CSA kits comprised a range of bovine FX/FIXa, bovine FX/human FIXa or human FX/FIXa. Thrombin generation (TG) was assessed by CAT and ST-Genesia. RESULTS: Emicizumab-calibrated modified OSA and human FX CSA both overestimated rFVIII in the presence of emicizumab; median FVIII:C of up to 89% higher was observed in plasma spiked with both drugs compared to just rFVIII. In bovine FX CSA assays, there was a FVIII:C increase of up to 11% in plasmas spiked with both drugs compared to rFVIII alone. TG parameters were not all normalized by the presence of emicizumab however addition of rFVIII increased TG. ETP and peak thrombin were normalized at 50 µg/ml emicizumab using ST-Genesia but were still reduced at 75 µg/ml with CAT. Addition of rFVIII further normalized results. CONCLUSION: Modified OSA and human FX CSA could not distinguish between rFVIII or emicizumab. The presence of both emicizumab and rFVIII increased thrombin generation to normal levels compared to each drug alone. Bovine FX CSA can be used to accurately determine FVIII activity of rFVIII in plasma which also contains emicizumab.

The effect of a next generation factor VIII mimetic bispecific antibody (Mim8) on assays of factor VIII activity and thrombin generation.

BACKGROUND: Mim8 is a next generation bispecific antibody developed for the prophylactic treatment of hemophilia A. The sensitivity of activated plasma thromboplastin time (APTT), assays measuring factor VIII activity (FVIII:C) and thrombin generation to plasma containing Mim8 was assessed. METHODS: Congenital severe hemophilia A plasma was spiked with Mim8 at 0 µg/mL to 20 µg/mL. APTT was measured with 4 reagents, one-stage FVIII with 9 reagents, and chromogenic FVIII with 6 assays. Thrombin generation was assessed in a low tissue factor system. RESULTS: At 1-µg/mL Mim8, the APTT was shortened to within normal limits and one-stage FVIII:C was >1.00 IU/mL with all APTT reagents. Modified one-stage assays calibrated with Mim8 reference material calibrated recovered within 20% of the target Mim8 concentration with most APTT reagents. Factor VIII:C of bovine only chromogenic assays was <0.04 IU/mL at all Mim8 concentrations. Factor VIII:C of human only chromogenic assay was >4.00 IU/mL at 20-µg/mL Mim8. Factor VIII:C of hybrid bovine FX, human FIXa chromogenic assays ranged from 0.076 to >3.00 IU/mL at 20-µg/mL Mim8. Normal thrombin generation was restored at 5-µg/mL Mim8. CONCLUSIONS: APTT-based assays are sensitive to Mim8 and should not be performed in the presence of the drug. Chromogenic assays containing human proteins or hybrid human/bovine proteins demonstrated variable sensitivity to Mim8. Bovine only chromogenic assays were largely insensitive to the presence of Mim8. Thrombin generation normalized at increased Mim8 concentrations. Modified one-stage and chromogenic assays could be used to quantify the Mim8 concentration in plasma.

How to assess parallelism in factor assays: coefficient of variation of results with different dilutions or slope ratio?

INTRODUCTION: Non-parallelism in factor assays can lead to incorrect factor activities. Parallelism can be assessed by calculating the coefficient of variation (CV) of results obtained on 3 dilutions of the same sample. Some authors have proposed that if there is <15% then the average activity is reportable. Some analysers use a slope ratio (SR) to calculate parallelism, with an acceptance range of approximately 0.9-1.1. METHODS: We evaluated CV and SR in one stage FII-FXII assays on Sysmex CS5100i using Innovin or Actin FS. Frozen normal and pathological plasmas, plasmas containing Direct Oral Anticoagulants, Direct Thrombin Inhibitors or Lupus Anticoagulant were analysed to assess possible non-parallelism. RESULTS: In plasmas with factor levels >25 IU/dl (plus no interfering substances) all CVs were < 15%. One sample (low factor activities 10-15 IU/dl), had CVs > 15% in FII, FVII and FXII assays only. SR outside of 0.9-1.1 were seen in FII and FXII assays at different levels of clotting factor including some within the normal range. Non-parallelism was detected more frequently with SR than CV for those with interfering substances. CONCLUSIONS: SR outside of 0.9-1.1 were seen in different levels of clotting factors, including samples which did not contain interfering substances. The target of 15% CV was a better discriminator than a SR for acceptance. When factor levels were reduced to around 10-15 IU/dl, a target 20 %CV was more appropriate than 15%. It might be appropriate for laboratories to assess locally whether their acceptance criteria need to be wider at low levels of clotting factors.

Differences in wild-type- and R338L-tenase complex formation are at the root of R338L-factor IX assay discrepancies.

Adeno-associated virus (AAV) gene therapy has the potential to functionally cure hemophilia B by restoring factor (F)IX concentrations into the normal range. Next-generation AAV therapies express a naturally occurring gain-of-function FIX variant, FIX-Padua (R338L-FIX), that increases FIX activity (FIX:C) by approximately eightfold compared with wild-type FIX (FIX-WT). Previous studies have shown that R338L-FIX activity varies dramatically across different clinical FIX:C assays, which complicates the monitoring and management of patients. To better understand mechanisms that contribute to R338L-FIX assay discrepancies, we characterized the performance of R338L-FIX in 13 1-stage clotting assays (OSAs) and 2 chromogenic substrate assays (CSAs) in a global field study. This study produced the largest R338L-FIX assay dataset to date and confirmed that clinical FIX:C assay results vary over threefold. Both phospholipid and activating reagents play a role in OSA discrepancies. CSA generated the most divergent FIX:C results. Manipulation of FIX:C CSA kits demonstrated that specific activity gains for R338L-FIX were most profound at lower FIX:C concentrations and that these effects were enhanced during the early phases of FXa generation. Supplementing FX into CSA had the effect of dampening FIX-WT activity relative to R338L-FIX activity, suggesting that FX impairs WT tenase formation to a greater extent than R338L-FIX tenase. Our data describe the scale of R338L-FIX assay discrepancies and provide insights into the causative mechanisms that will help establish best practices for the measurement of R338L-FIX activity in patients after gene therapy.

External Quality Assessment Data for Investigation of von Willebrand Disease: Focus on Relative Utility of Contemporary Functional von Willebrand Factor Assays. The United Kingdom National External Quality Assessment Scheme (UK NEQAS) Experience.

Von Willebrand disease (VWD) is one of the most common hereditary bleeding disorders. Effective management of patients and their families depends on accurate diagnosis and subtype classification, and quality assurance including participation in proficiency testing programs is essential to ensure the accuracy of the panel of assays required to achieve this diagnosis. We report here findings from recent external quality assessment (EQA) exercises, as well as from a questionnaire about diagnostic practices employed by centers in the United Kingdom National Quality Assessment Scheme (UK NEQAS) performing von Willebrand factor (VWF) assays. Plasma samples from donors with VWD, "normal" donors, the International Society for Thrombosis and Haemostasis Scientific Subcommittee (ISTH SSC) plasma standard, and whole blood samples were sent to participants in the UK NEQAS BC program for VWF investigation. Calibration of lot#5 of the ISTH SSC plasma standard was shown to give improved comparability between the recovered value from an EQA exercise and the assigned potency for VWF activity assays. Diagnostic accuracy and precision amongst UK NEQAS participants was good, with an average 99% of centers reporting the correct interpretation for normal, type 1 and type 2 VWD samples, 100% diagnostic accuracy for centers performing FVIII binding assays, and good agreement amongst centers performing multimeric analysis. Genetic analysis of the VWF gene by specialist centers demonstrated errors in the genotyping process in one center, but also demonstrated failings in the interpretation of results in other centers. Despite evidence of good laboratory accuracy and precision in their assays, a questionnaire identified marked variation in diagnostic criteria employed, underlining the importance of guidelines to support the diagnosis of VWD.

Anti-PF4 testing for vaccine-induced immune thrombocytopenia and thrombosis (VITT): Results from a NEQAS, ECAT and SSC collaborative exercise in 385 centers worldwide.

BACKGROUND: Vaccine-induced immune thrombocytopenia and thrombosis (VITT) following the administration of the AstraZeneca (AZ) ChAdOx1 nCOV-19 vaccine is a well recognized clinical phenomenon. The associated clinical and laboratory features have included thrombosis at unusual sites, thrombocytopenia, raised D-dimer levels and positivity for immunoglobulin G (IgG) anti-platelet factor 4 (PF4) antibodies. OBJECTIVES: A collaborative external quality assessment (EQA) exercise was carried out by distributing five lyophilized samples from subjects with VITT and one from a healthy subject to 500 centers performing heparin-induced thrombocytopenia (HIT) testing. METHODS: Participating centers employed their locally validated testing methods for HIT assays, with some participants additionally reporting results for VITT modified assays. RESULTS: A total of 385 centers returned results for anti-PF4 immunoassay and functional assays. The ELISA assays used in the detection of anti-PF4 antibodies for the samples distributed had superior sensitivities compared with both the functional assays and the non-ELISA methods. CONCLUSION: ELISA-based methods to detect anti PF4 antibodies have a greater sensitivity in confirmation of VITT compared with functional assays regardless of whether such functional assays were modified to be specific for VITT. Rapid immunoassays should not be employed to detect VITT antibodies.

Advantages of external quality assessment-EQA programs.

INTRODUCTION: The first external quality assessment (EQA) in Thrombosis and Haemostasis was elaborated over 20 years ago, and since then, several national and international EQA institutions have been established. AIM: Display the benefits of EQA programs. METHODS: The spectrum of EQA action was evaluated ranges from improving the performance of the local laboratory to highlighting inadequate diagnostic tests that need to be replaced by new technologies. RESULTS: The first result approach is related to a national management of quality in laboratories. In recent years, Brazil has invested in an EQA program to aid public policy in the laboratory area. During this period, a group of haemostasis laboratory specialists were invited to manage the results and help the Ministry of Health with applying these results as a strategy to improve laboratories. Thus, in collaboration with NEQAS-BC, the University of Campinas - UNICAMP, established a Brazilian EQA program for Blood Coagulation. The second result approach is related to FVIII inhibitor assay performance evaluation, which is another type of EQA program benefit. Despite the assay being considered the gold standard to measure neutralised immunoglobulins for FVIII since 1975, over 40 years ago, the test still has a high coefficient of variation. Results from NEQAS-BC and WFH IEQAS program demonstrate the inter-laboratory variation across the United Kingdom over the last years and among emergent countries. CONCLUSION: The EQA programs have an important educational role in helping countries manage their public policy and in the international inquiry regarding the necessity of new technologies in haemostasis.