Technical Specialist of Coagulation Health New Zealand, Te Whatu Ora- Waikato Hamilton, Waikato, New Zealand
Background: BIOPHENTM FVIII variant kit is a chromogenic assay for quantifiable determination of Factor VIII activity. (1) Utilising this kit for setup on the STAR Max 2® proved challenging as there wasn’t a known protocol available. A significant issue in the calibration curve were ‘error’ results given at the high end of the curve.
Aims: To determine if further diluting SXa-11 chromogenic substrate using water can rectify optical density errors in high calibrator points on the calibration curve on a STAR Max 2®.
Methods: SXa-11 chromogenic substrate was reconstituted using 2.5 ml of ‘water for irrigation’ by Baxter, as per the manufacturer’s instructions. (2) A further diluted substrate solution was also prepared by mixing 1ml of reconstituted substrate with 2 ml of water. Calibration curves were run using each respective substrate, along with the other reagents provided in the kit. Siemens Standard Human plasma (SHP) was double diluted with STA®-ImmunoDef VIII to form a series of dilutions. The dilutions of SHP were run on both protocols as “known values.” Siemens normal (N) and pathological (P) quality control were run after each calibration. To test FVIII values >100% a patient with a FVIII level of 150% was used and run on both calibration curves. This level was determined by a 1-stage FVIII assay using TriniCLOT aPTT HS and STA®-ImmunoDef VIII. A low curve protocol was created for FVIII results < 10%. This was achieved by diluting SHP, N and P controls to a ¼ dilution using STA®-ImmunoDef VIII. Known values from the diluted SHP that were < 10% were run using both protocols.
Results: Diluted chromogenic substrate allowed higher calibrator points to have quantitative optical density values. A low curve was required for both protocols.
Conclusion(s): Diluting the chromogenic substrate is a worthwhile option to allow higher ODs to be expressed on a STAR Max 2®.
