Caron C, Dautzenberg M-D, Delahousse B, Droulle C, Pouzol P, Dubanchet A, Rothschild C
Laboratoire d'Hématologie, Hôpital Cardiologique, Lille, France.
Haemophilia. 2002 Sep;8(5):639-43. doi: 10.1046/j.1365-2516.2002.00666.x.
Assay of factor VIII (FVIII) in patient samples is routinely carried out using the one-stage assay rather than the chromogenic substrate assay. The introduction of new FVIII preparations for the treatment of haemophilia A, including immunopurified FVIII and particularly, recombinant FVIII (rFVIII) concentrates, has led to discrepancies between the results obtained with the two assays. In patients treated with rFVIII concentrates, FVIII levels measured with the one-stage assay can be 20-50% lower than those measured with the chromogenic assay. In this study, the one-stage assay was performed with cephalin dilutions higher than those recommended by the manufacturer. B-domain-deleted recombinant FVIII, Refacto, was diluted to eight different concentrations, ranging from 1-100 IU dL(-1), in FVIII-deficient plasma and the FVIII activity of the eight solutions was determined by the chromogenic method in a central laboratory. Aliquots were then assayed by the one-stage method in the four participating laboratories, using different dilutions of CK-Prest. When CK-Prest was reconstituted according to the manufacturer's recommendations (dilution 1 : 1), the difference between the one-stage and chromogenic methods was close to 30%. CK-Prest cephalin dilutions of 1 : 5 and 1 : 8 gave very similar results with the two methods, without increasing the interlaboratory coefficient of variation. These findings confirm the influence of phospholipids on the one-stage assay, particularly the importance of using a phospholipid concentration close to the physiological value in platelets. This modified one-stage method may therefore offer an alternative to the use of a concentrate-specific standard.
在患者样本中，VIII因子（FVIII）的检测通常采用一期法而非发色底物法。用于治疗甲型血友病的新型FVIII制剂的出现，包括免疫纯化的FVIII，尤其是重组FVIII（重组FVIII）浓缩物，导致两种检测方法所得结果出现差异。在用重组FVIII浓缩物治疗的患者中，一期法测得的FVIII水平可能比发色法测得的低20%-50%。在本研究中，一期法采用了高于制造商推荐的脑磷脂稀释度。将B结构域缺失的重组FVIII（Refacto）在FVIII缺乏血浆中稀释至八种不同浓度，范围为1-100 IU dL(-1)，并在中心实验室通过发色法测定这八种溶液的FVIII活性。然后，在四个参与实验室中，使用不同稀释度的CK-Prest通过一期法对等分试样进行检测。当按照制造商的建议（1:1稀释）重构CK-Prest时，一期法和发色法之间的差异接近30%。CK-Prest脑磷脂1:5和1:8的稀释度在两种方法中得到了非常相似的结果，且未增加实验室间变异系数。这些发现证实了磷脂对一期法的影响，特别是使用接近血小板生理值的磷脂浓度的重要性。因此，这种改良的一期法可能为使用浓缩物特异性标准提供一种替代方法。
