Christopher Reilly‐Stitt

Introduction: Improvements in treatment of patients with haemophilia A have meant that their quality of life has majorly improved. However, a disadvantage to these developments has come at a cost to the laboratory when monitoring patient Factor VIII (FVIII) levels and there is the potential for under‐ or overtreatment leading to clinical risk towards the patient. Methods: A global study performed in 2023 to investigate the differences in FVIII assay results in a large cohort of laboratories has enabled users to understand the challenges that new products can cause. Five FVIII modified/extended half‐life (EHL) products were studied by distributing spiked samples via 3 external quality assessment (EQA) schemes (ECAT, NEQAS and RCPAQAP). Results: Participating laboratories used either one‐stage assays (OSA), chromogenic assays (CA), or both methods when performing the assays. Most centres running the OSA used IL Hemosil Synthasil, Siemens Actin FS, Siemens Pathromtin SL, or Stago Cephalin/Kaolin/C.K. Prest reagents, and for the CA, the Chromogenix Coamatic FVIII kit and the Siemens chromogenic FVIII kit were utilised. Conclusion: The FVIII results submitted by participants showed that currently available OSA and CA do not provide consistent results in some products with both an under‐ and over‐estimation of the expected recovery based on potency at either concentration level. Results for Afstyla Lonoctocog alfa suggest that centres were not clear on whether OSA results were before or after application of the correction factor (multiplication of initial result by 2).

Direct oral anticoagulants (DOACs), also referred to as non-vitamin K oral anticoagulants (NOACs), have revolutionised anticoagulant therapy since their introduction in the late 2000s. These agents – dabigatran (a direct thrombin inhibitor) and the factor Xa inhibitors apixaban, edoxaban, and rivaroxaban – offer more predictable pharmacokinetics, fewer food and drug interactions, and do not typically require routine laboratory monitoring, unlike traditional anticoagulants such as warfarin and heparin. Despite these advantages, laboratory assessment of DOAC activity remains clinically important in specific circumstances, including bleeding, thrombosis during therapy, emergency surgery, renal or hepatic impairment, and suspected non-compliance. This chapter reviews the pharmacology, clinical applications, and laboratory evaluation of DOACs, outlining available assays and their principles. The gold standard for quantification is liquid chromatography-tandem mass spectrometry (LC-MS/MS), though chromogenic anti-Xa and dilute thrombin time (DTT) or ecarin-based assays are more commonly used in clinical laboratories. The impact of DOACs on routine coagulation tests such as PT, APTT, and TT is variable and reagent dependent, underscoring the need for drug-specific calibrators and assay validation. Quality assurance through both internal quality control (IQC) and external quality assessment (EQA) is essential to maintain assay accuracy and reproducibility, in accordance with ISO 15189:2022 standards. Furthermore, DOACs can interfere with other haemostasis assays – including factor assays, antithrombin, and lupus anticoagulant testing – leading to potential diagnostic errors. Recent studies demonstrate that activated charcoal-based reagents, such as DOAC Remove™ and DOAC Stop™, can effectively neutralise DOAC interference in such assays. In conclusion, while DOACs have largely supplanted warfarin in the management of non-valvular atrial fibrillation and venous thromboembolism due to their safety and convenience, laboratory testing retains a critical role in ensuring safe and effective patient management under specific clinical scenarios.

Background: Efanesoctocog alfa (EFA) is an ultra‐long half‐life FVIII product. However, measuring the level of EFA using standard one stage (OSA) and Chromogenic assays (CA) is affected by both underestimation and overestimation when using some commonly used APTT reagents for OSA and overestimation when using CA assays. Objectives: ECAT and NEQAS BC distributed samples containing EFA to centres for measurement of EFA using assays available locally. The aim was to provide method specific data and recommendations for additional OSA or CA assays. Methods: In Autumn 2024, four samples with levels of EFA between 5 and 100 IU/dL (based on potency label) were buffered and lyophilized for distribution to 300 hospital and university laboratories. Results: Two‐hundred participants returned 174 OSA and 148 CA results for samples EFA 24:01‐24:04. Twelve different APTT reagents, six were used by ( n ≥ 10) and 10 different chromogenic assays were employed, of which four were used by ( n ≥ 10). Conclusion: The NEQAS BC and ECAT exercise confirmed the field study: over‐estimation for Actin FS OSA and all CA assays at all EFA levels plus an under‐estimation for SynthasIL OSA at EFA levels of: 20, 50 and 100 IU/dL. Centres using Actin FSL on a Siemens/Sysmex or Stago analyser plus centres using Synthafax on a Werfen analysers had median recoveries within ±25% compared to the assigned potency for samples at EFA levels of: 20; 50 and 100 IU/dL. Centres using CK Prest and Pathromtin had median recoveries within ±25% compared to the assigned potency for samples at an EFA levels of 100 IU/dL.

Background: To achieve accreditation for ISO 15189, laboratories are required to either participate in EQA exercises or intra‐laboratory comparisons to meet the standard. Light transmission aggregometry performed by laboratory scientists for the clinical investigation of possible platelet function defects is time dependent. Current EQA available audits the interpretation of platelet aggregation traces.Objectives: Could NEQAS BC provide external quality assessment (EQA) material to centres employing Light Transmission Aggregometry LTA for clinical investigation of platelet function? The use of fresh donor platelets could audit more of the analytical and post analytical aspects of light transmission aggregation than currently available.Methods: A pool of donor platelets was split into aliquots that were distributed to testing centres across England, Scotland and Wales for testing within a 72 h window. Participating centres employed their locally validated testing methods for LTA assays, for agonists including ADP; Arachidonic acid; Collagen; Epinephrine; Ristocetin; TRAP and U46619.Results: Five different aggregation platforms were used including: Chronolog 700; Helena Aggram; PAP‐8; Stago TA‐8V and Sysmex CN‐series. Sample packs were tested through the 72 h window with most sites performing LTA on the EQA material on day one of the three.Conclusion: The % consensus of interpretations provided by participants for agonists including: ADP; Arachidonic acid; Collagen; Epinephrine and Ristocetin ranged from 94% to 100% indicating that the material is stable plus centres using different aggregometers returned the same clinical interpretations.

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.