Blood group O may add to the effect of VWF variants including p.Y1584C and to non-VWF factors to reduce VWF plasma levels. In summary, ‘type 1 VWD’ includes a heterogeneous patient group with VWF levels from just detectable into the normal range, some with minor multimer abnormalities, a wide range of

bleeding severities and variable desmopressin responses. Phenotype-genotype relationships are being identified. Two-stage chromogenic substrate (CS) methods, which can be considered as variants of the two-stage (TS) clotting method, for the determination of FVIII activity in plasma and concentrates have been commercially available as kits for up to 25 years [13,14]. All kit methods measure the ability of FVIII to potentiate activation of FX by FIXa in the presence learn more of calcium ions and phospholipids. Similar

to the TS clotting method, the first step comprises activation of FVIII and FX and the generated FXa is measured in a second step through hydrolysis of a chromogenic FXa substrate. Thrombin required for activation of FVIII is generated during the assay [13] or present in a reagent. Assays are designed such that the amount of FXa formed should be directly proportional to FVIII activity in the sample. Chromogenic methods typically provide two measuring ranges, indicating levels of 0.2–2.0 IU mL−1 in one range and down to 0.005–0.01 IU mL−1 in a low range, the latter being used

for e.g. diagnosis and classification of haemophilia A. All CS methods are easy to automate and therewith offer cost-efficient use, e.g. when applied on microplates. RG7204 clinical trial In contrast to one-stage clotting (OS) methods, CS methods are not sensitive to preactivation of FVIII due to fast and complete FVIII activation during the assay. The sensitivity of the OS method for preactivated selleck screening library FVIII results in overassignment of FVIII potency, noticed for intermediate purity plasma-derived FVIII concentrates in the 1980s and again observed in the calibration of the plasma-derived standard Mega 2/BRP 3, where partial activation/structural modifications during manufacturing resulted in ∼30% over-assignment of FVIII potency [15]. For reasons of use of a relatively high plasma dilution and involving only the tenase complex, CS methods are minimally influenced by variable levels of plasma components. This also holds for lupus anticoagulants, which may result in a pronounced underestimation of FVIII activity in OS methods [16]. Robustness combined with high assay precision and accuracy led to adoption of the chromogenic method as the reference method in the European Pharmacopoeia in 1994 [17]. Importantly, this method requires predilution of FVIII concentrates in FVIII deficient plasma to 1 IU mL−1 followed by further dilution in buffer containing 1% albumin, the quality of which should always be carefully checked.