OC 77.1 - Unravelling APTT discrepancies in the presence of in vitro haemolysis

Background: In vitro haemolysis interferes with the activated partial thromboplastin time (APTT) and manifests as shortened/prolonged clotting times. Possible causes include sample activation, and tissue factor (TF) / platelet factor 4 (PF4) release.

Aims: This study evaluated possible mechanisms underlying discrepant APTT results in paired haemolysed samples (HLS) versus non-haemolysed replacement samples (NHLS).

Methods: Citrated HLS and NHLS from the same patient were identified on the Sysmex CN-6000 via the haemolysis index. Paired-matched samples (PMS) from the same patient were frozen (- 80°C) within 4 hours of sample collection. PMS were analysed on the CN-6000 for APTT (5 different reagents) and APTT-based one-stage clotting assays (OSCA). Factor VIIa (FVIIa) was quantified on the Sysmex CS-5100 and PF4 via ELISA methodology (HYPHEN Biomed ZYMUTEST). Statistical significance was assessed via a Wilcoxon signed rank test (p < 0.05) and correlation between the difference in APTT versus the difference in factor concentration between PMS via Spearman’s rank correlation coefficient. Clinical significance was assessed against a local reference range (RR) using 30 normal samples for APTT/FVIIa and the manufacturer’s RR for OSCA.

Results: Clinically significant differences in APTT and OSCA assays were observed in the presence of haemolysis in some samples (Table 1). A statistically/clinically significant marked increase in PF4 in HLS was observed by an average of 348.2 IU/mL (RR < 10 IU/mL). FVIIa showed no statistically significant difference. In HLS with shorter APTTs, OSCA was marginally increased but marginally decreased in HLS with prolonged APTTs. The correlation between the difference in APTT versus the difference in factor concentrations was weak for XII, moderate for VIII/XI, but strong for IX (Figure 1).

Conclusion(s): HLS should be rejected for APTT/OSCA/PF4/Heparin assays to maintain result integrity. In vitro platelet activation was confirmed by significantly elevated PF4 in HLS. The change in factor concentrations between PMS was inconclusive.