Advantages, disadvantages and optimization of one‐stage and chromogenic factor activity assays in haemophilia A and B

Abstract

Haemophilia A and B diagnosis and disease severity classification are determined on the basis of results from factor VIII (FVIII) and factor FIX (FIX) activity assays, respectively. These assays are also used for potency labelling, postinfusion monitoring of factor replacement products and testing for FVIII/FIX inhibitors. This review focuses on activated partial thromboplastin time (APTT)-based one-stage assays (OSAs) and two-stage chromogenic substrate assays (CSAs). Currently, there is considerable inter-laboratory variability in the results obtained using OSAs, which can be intensified in a reagent-specific manner by the presence of the new modified recombinant factor replacement products that are entering the market. Furthermore, the use of CSAs, which tend to show less variability, especially with the new modified products, is recommended in a number of clinical scenarios. Clinical laboratories may, therefore, need to establish CSAs for routine use. In this review, we aim to improve understanding and help establish best practices by describing the methodology behind OSAs and CSAs and highlighting assay advantages and limitations. We argue that there can be value in offering both assay methodologies in clinical laboratories that contribute to the care of patients with haemophilia A or B. Educating both laboratory scientists and clinicians about the strengths and weaknesses of each type of assay will help to establish the necessary dialogue that is key to ensuring not only that the appropriate assays are used in the right clinical situations, but also that the results are interpreted correctly.

Performance evaluation of Revohem™ FVIII chromogenic and Revohem™ FIX chromogenic in the CS‐5100 autoanalyser

“…It is crucial to ensure that mild and/or moderate HA are not misclassified due to discrepancies in results between OSA and CSA . Adcock et al mentioned that sufficient data are available to recommend CSA in combination with OSA . Also, as haemophilia treatment has developed, CSAs have recently entered the spotlight.…”

“…Haemophilia A (HA) and haemophilia B (HB) are X‐linked bleeding disorders that result from deficiencies in coagulation factor VIII (FVIII) and factor IX (FIX), respectively. The diagnosis and disease severity classifications of haemophilia are determined based on results from FVIII and FIX activity assays . Factor activity assays are also used for potency labelling and postinfusion monitoring of factor replacement products, as well as FVIII/FIX inhibitor testing .…”

“…CSAs use activated factor X (FXa)‐specific chromophores. OSAs are the most widely used tests in diagnostic laboratories, but several factors can limit their performance . Many different aPTT reagents and factor‐deficient plasmas are available, and vast numbers of combinations exist.…”

Clinical utility and impact of the use of the chromogenic vs one‐stage factor activity assays in haemophilia A and B

“…A burst of thrombin formation occurs after sufficient levels of activated FVIII (FVIIIa) have been generated through feedback activation by thrombin, leading to the formation of a clot . Adapted with permission from Adcock et al…”

A cost‐effective approach to factor assay calibration using a truncated live calibration curve

“…They further suggest recalibration should be performed every 6 months or following major maintenance on the analyser. More recently following the increased use of CSA, Adcock et al have stated that in their experience, stored calibration curves can also be utilized for FVIII:C CSA. UK NEQAS reported that in 2007, around 50% of centres (n = 39) used stored calibration curves for up to 6 months without recalibration.…”