Influence of source of phospholipids for APTT‐based factor IX assays and potential consequences for the diagnosis of mild haemophilia B

Abstract

Several reagents are available to perform activated partial thromboplastin time (APTT) that is a key test in routine haemostasis laboratories. However, the ability of APTT reagents to detect specific coagulation factor deficiencies may be affected by several variables, including the origin (human, animal or vegetable), composition, configuration of phospholipids and the type of surface activator. These variations in sensitivity explain why specialized haemostasis laboratories often use several different APTT reagents for specific and variable applications, i.e. control of heparin therapy, screening for lupus anticoagulant, evaluation of coagulation status before surgery and one-stage APTT-based assays for measurement of factor VIII (FVIII), factor IX (FIX), factor XI (FXI) and factor XII (FXII) plasma levels.We have recently tested the Actin FS Ò reagent (Dade Behring, Marburg, Germany) in our laboratory and recorded higher FIX activity levels (FIX:C) in several haemophilia B (HB) patients than those previously assayed with our usual reagent. We therefore initiated a multicentre study in a larger sample of patients to confirm these results and to identify the reasons explaining such discrepancies in the results obtained.Three hospital haematology laboratories participated in this study: Tours (central laboratory), Caen and Nantes. Blood samples were obtained from 40 patients previously diagnosed as having HB and not regularly treated with plasma-derived or recombinant factor IX (rFIX) concentrates. The basal FIX:C level in these patients was £1 IU dL )1 (n = 6), between 1 and 5 IU dL )1 (n = 10), or between 5 and 40 IU dL )1 (n = 24). FIX antigen levels (FIX:Ag) were also measured in these 40 untreated HB patients using Asserachrom Ò IX:Ag (Stago, Asnières, France), and the values obtained were compared with FIX:C levels. In 13 patients FIX:Ag levels were comparable with those of procoagulant activity (FIX:C/FIX:Ag >0.7), and these patients were defined as having a Ôtype 1Õ FIX deficiency. In contrast, the amount of FIX measured by immunological assay in the plasma from 27 HB patients was markedly higher than functional level. These patients therefore exhibited a FIX:C/FIX:Ag ratio lower than 0.7 and were defined as having a Ôtype 2Õ FIX deficiency.Factor IX:C level was measured by one-stage assay carried out on a STAR Analyzer (Stago) which is used in the majority of reference laboratories in France. All analyses were performed in duplicate on plasma aliquots which had been stored at )80°C after double centrifugation to ensure efficient platelet depletion and the same reagent batches were used in all three centres.Three APTT reagents were used: CKPrest Ò from Stago (rabbit brain thromboplastin + Kaolin), PTTA Ò from Stago (rabbit brain thromboplastin + silica) and Actin FS Ò from Dade Behring (soy phospholipids + ellagic acid). For each APTT reagent, calibration curves from 100 to 12.5 IU dL )1 were obtained by diluting standard human plasma (RKL) from 1/5 to 1/80 in FIX-deficient plasma (both reagents from Dade...

Effect of Ca2+ and Mg2+ concentration in culture medium on the activation of recombinant factor IX produced in Chinese hamster ovary cells

“…A total activity of rFIX including both aFIX and FIXa was determined using an activated partial thromboplastin time (APTT) assay (Parsi et al, 2007;Pouplard et al, 2009). Briefly, 50 L of sample, diluted in CA system buffer (Seimens, Marburg, Germany), was mixed with 50 L of FIX-deficient plasma (Seimens) and 50 L of Dade ® Actin ® (Seimens).…”

Hemophilia B: Molecular Basis

“…The terms cross-reacting material (CRM) negative and CRM positive (or CRM reduced) approximate to types I and II [9,17]. Type I or quantitative mutations result in reduced levels of an otherwise normal protein.…”

Recombinant FIX Fc fusion protein activity assessment with the one‐stage clotting assay: A multicenter, assessor‐blinded, prospective study in Japan (J‐Field Study)

“…This method is based on the activated partial thromboplastin time (aPTT), for which a variety of activators, phospholipid components, calibrator plasmas, and instruments are commercially available. Although the calibrator plasmas are traceable to an international standard for FIX activity (commissioned by the World Health Organization), the diversity of available reagents and instruments has resulted in significant assay variability, particularly reported for recombinant FIX (rFIX) products . Furthermore, there is greater variability for some of the newer extended half‐life FIX products compared with the conventional rFIX products.…”