Comparison of clot-based and chromogenic assay for the determination of protein c activity

Abstract

Activated protein C inactivates factor Va and VIIIa. Deficiency of this natural anticoagulant may result in recurrent venous thrombosis. Performance characteristics of clot-based and chromogenic protein C activity assays are different. The clot-based assay has limitations because of interference with coagulation inhibitors resulting in spuriously increased protein C levels or underestimation because of elevated levels of factor VIII and Factor V-Leiden mutation. The chromogenic assay is not influenced by such interferences but only detects functional defects of protein C that involve the active site rendering it insensitive to rare mutations. To compare two methods, we conducted a retrospective study from January 2015 to June 2017. Our results showed a good correlation between clot-based and chromogenic assay ( R = 0.94 and r 2 = 0.88). The study of agreement between the two methods by the Bland–Altman method showed that chromogenic method on an average measures 7.8% more protein C than that of clot-based. The results also showed that the bias between the two methods is significant. The positive trend noted was contributed by the values of less than 20% of protein C. Both clot-based and chromogenic assays had high sensitivity; however, the chromogenic assay showed better specificity (97%) as compared with the clot-based assay (93%). In conclusion, we recommend the chromogenic method as the assay of choice, which is also recommended by the College of American Pathologist Consensus Study over activated partial thromboplastin time-based assay. We have shown here that despite a good correlation between the two techniques, there is a difference as highlighted by the difference plots.

PC Deficiency Testing: Thrombin-Thrombomodulin as PC Activator and Aptamer-Based Enzyme Capturing Increase Diagnostic Accuracy

“…Comparison of chromogenic and clot-based assay results showed a good agreement, but the chromogenic assay measures approximately 10% more PC activity than the clot-based assay. This is supported by previously published data ( 23 ). The most likely explanation for this finding is that APC, that is complexed to its inhibitor alpha-2-macroglobulin, retains the ability to cleave small peptide substrates, while the macromolecular substrates FVa and FVIIIa have no access to alpha-2-macroglobulin-complexed APC.…”

“…This advantage of clot-based PC activity tests is outweighed by limitations caused through interferences that result in underestimation of PC levels. Another disadvantage of clot-based PC activity testing is its sensitivity to coagulation inhibitors such as heparins, direct acting oral anticoagulants and phospholipid antibodies, that result in overestimation of PC levels ( 23 ). Attempting to overcome these limitations, we used the APC-OECA for quantification of generated APC.…”

Monocyte activation and acquired autoimmune protein S deficiency promote disseminated intravascular coagulation in a patient with primary antiphospholipid syndrome

“…While PC activity in clotting-based assays depends on PS cofactor function, PS is dispensable in chromogenic PC activity assays. 12 Since patient IgG also interfered with chromogenic PC activity (Figure S3D), we cannot exclude the possibility that the patient additionally had an IgG PC inhibitor, contributing to the procoagulant state.…”

Laboratory Limitations of Excluding Hereditary Protein C Deficiency by Chromogenic Assay: Discrepancies of Phenotype and Genotype

“…The comparably lower NPV for the chromogenic versus clotting time–based PC assay (88% vs 97%) indicates that the lower reference range of the manufacturer’s reagent does not meet the requirements of a suitable cutoff for PC activity, which is illustrated in Figure 2. A recent comparison between the chromogenic and clotting time–based PC assay showed a good correlation ( R = 0.94 and r 2 = 0.88) but also a significant bias, measuring on average 7.8% more PC by chromogenic than by clotting time–based PC assay, 39 which might be more relevant for patients with borderline results than with extreme alterations of PC activity. A recent study in a Spanish cohort found 4 individuals carrying a PC variation with PC activity ranging from 72% to 77% and 10 relatives ranging from 75% to 94%.…”