Principal Biomedical Scientist Viapath Analytics London, England, United Kingdom
Background: The glycoprotein Pre-Kallikrein (PK) plays a role in the in vitro intrinsic coagulation pathway. The role of PK in vivo remains unclear and cases of deficiency are rare and largely asymptomatic. Diagnosis of PK deficiency is required to avoid the administration of unnecessary prohaemostatic therapy or delay to surgical procedures. Additionally there are occasional case reports of bleeding and cardiovascular co-morbidities associated with PK deficiency which further necessitate the availability of a reliable and reproducible assay.
Aims: In response to frequent assay underperformance it was decided to investigate an amended Pre-kallekrein protocol to improve assay functionality on the Sysmex CS and CN series analysers.
Methods: PK assay employs a one-stage activated partial thromboplastin time (APTT) using PK (Fletcher trait) deficient plasma. Reference plasma or sample is diluted 1:10, mixed with PK deficient plasma and incubated with APTT reagent. Calcium Chloride is added to trigger the coagulation cascade and time to clot recorded by light transmission. The assay was reprogrammed to perform a broader range of calibrator values using initial dilutions of 1:10, 1:20 and 1:40. QC material and reference plasma was assayed at a range of dilutions to establish which protocol showed the best linearity and parallel recovery of PK concentrations.
Results: The extended protocol using 1:20 pre-dilution gave the broadest range of calibrator curve values maintaining linearity (R2 >0.99). Figure 1 shows comparison of the existing and proposed calibration curves. Measurement of a range of quality control and reference plasma material showed much improved recoveries using the dilute protocol. PK recoveries from serially diluted reference plasma displayed non-parallelism in the original assay. This was improved using the proposed protocol using 1:20 pre-dilution (Table 1).
Conclusion(s): The revised protocol has been demonstrated to display improved PK assay parameters will be employed as the assay migrates to the next generation Sysmex CN analyser platform.
