Adequate laboratorial diagnostics of hemophilia is essential and could be done by chromogenic and one-stage assays based on APTT methodologies. Adverse effects of medications became less severe recently, but it remains important to be aware of to ensure that treatment is effective and ensure the best possible conditions for affected individuals. Classification of hemophilia according to FVIII activity: mild 25% - 5%; moderate, 5% - 1% and severe< 1%. For new therapies such as emicizumab and some drugs with an extended half-life, the World Federation of Hemophilia recommends the use of a chromogenic assay of FVIII containing bovine FX for monitoring factor FVIII activity.

evaluate the applicability of a low calibration curve (LCC) in the chromogenic factor VIII (FVIII-Chr) assay by standardizing the dilution of the standard (calibrator) and thus ensuring the accuracy of the analyses in the lower values like severe hemophilia range.

The samples were obtained from H&H LAB SAS, which guarantees all requirements. Sysmex® CS 2100i Siemens Healthineers. Calibration was performed with Standard Human Plasma (SHP), together with FVIII-Chr. Quality Control (QC); the accuracy of the LCC was checked.

Preparation of the LCC: SHP reconstitution, followed by 1:10 dilution between the SHP and the plasma deficient in FVIII. The curve was named FVIII-ChrLow. QC was performed through pathological control (PC) with 2 dilutions: 1) identical to the formation of the low curve, i.e., dilution 1:10 (2.3 – 3.9%). 2) dilution1:30 (0.2 - 2%) to accommodate the range of activity comprising severe hemophilia. The values found confirms the curve's quality. Discussion: This study sought to develop a way to detect FVIII activities < 1.5% in a reproductive manner, even with a reduced “N” number of samples for severe state. The chromogenic test presents a limit of detection for the optical density of D.O. 0.0080 reporting < 0,3% FVIII-Chr activity, which is very close to the most diluted point of the LCC.

QC performance with PC 1/10 and 1/30 showed excellent reproducibility. With the protocol FVIII Low and LCC (SHP+PDF) it is possible to obtain results of FVIII-Chr < 1.0% compatible patients with severe hemophilia clinical condition.