A collaborative study by the Working Group on Hemostasis and Thrombosis of the Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC) on the interference of haemolysis on five routine blood coagulation tests by evaluation of 269 paired haemolysed/non-haemolysed samples.

Introduction: Haemolysis is the leading cause of sample rejection in laboratory haemostasis. Most studies focused on artificially haemolysed samples. The aim of this study was a prospective assessment of spontaneous haemolysis on haemostasis tests, by comparing results of haemolysed (H) new, non-haemolysed (NH) specimens, collected within 4hrs. As new coagulometers can identify interfering substances, visual assessment of haemolysis was also compared with instrumental haemolysis index and stratified in subclasses.

Materials And Methods: Two hundred and sixty nine paired samples were collected and analysed using ACL TOP750-CTS (Instrumentation Laboratory, Bedford, USA), for prothrombin time (PT), activated partial thromboplastin time (aPTT), D-Dimer (DD), fibrinogen (Fib) and antithrombin (AT). Bias between H and NH was calculated and compared with the respective critical difference (CD).

Results: Mean bias was - 0.1 s for PT (P = 0.057), - 1.1 s for aPTT (P < 0.001), 1025 ng/mL for DD (P < 0.001), - 0.04 g/L for Fib (P = 0.258) and 1.4% for AT (P = 0.013). Bias exceeding the CD varied according to the method, with larger differences for aPTT (36.1%) and DD (17.1%) and < 8% for PT, Fib and AT. No correlation emerged between free haemoglobin values and difference in haemostasis tests in H and NH samples for any tests. Moderate/severe haemolysis involved > 95% of samples. The agreement between visual assessment and instrumental evaluation of haemolysis was 0.62.

Conclusion: Spurious haemolysis deeply influences aPTT and DD, and to a lesser extent AT and Fib. Prothrombin time seems only slightly influenced, suggesting that PT can be accepted also in haemolysed samples. Although a good inter-observer correlation of haemolysis evaluation was found, the instrumental assessment of haemolysis seems recommendable.