Hemolysis and Heparin Anti-Xa

A message from “Danagah:” Hi, George, How would you handle a sample for a chromogenic heparin anti-Xa assay if a patient is experiencing in vivo hemolysis and you cannot obtain a sample that is not hemolyzed? Would it make a difference as to the degree of hemolysis as to even accepting the sample? We reject all markedly hemolyzed samples for any hemostasis testing. Would you reject a sample with any visible hemolysis? Thank you.

Hello, Danagah, and thanks for your question. Chromogenic anti-Xa assays, in which the plasma is diluted and the test absorbance is generated as a ratio to a plasma blank, claim no interference up to 2 mg/mL plasma hemoglobin. Plasma hemolysis first becomes visible at 0.5 mg/mL, so the method provides some leeway, but read on.

The key reference about hemolysis is CLSI H21-A5; Collection, transport, and processing of blood specimens for testing plasma-based coagulation assays and molecular hemostasis assays; approved guideline–5th edition, 2007. CLSI, PA. H21-A5 recommends rejecting “severely hemolyzed” specimens, so you are correct to reject markedly hemolyzed specimens.

The trouble is, what do “severely,” or “markedly” mean? In fact, we assume any visible hemolysis implies platelet activation, and upon activation, platelets release platelet factor 4 (PF4), which neutralizes heparin. Consequently, while the heparin anti-Xa assay istechnically accurate despite visible hemolysis up to 2 mg/mL, it is likely to generate an unpredictable and falsely reduced plasma heparin concentration because of the preanalytical variable. Consequently, I recommend rejecting all coagulation specimens with visible hemolysis, especially when monitoring heparin, be it by chromogenic substrate or clot-based assay.

Most blood specimen hemolysis is caused by collection variations, especially collection through a vascular access device. See Ernst DJ. Blood Specimen Collection FAQs, Center for Phlebotomy Education, 2008. In the case of in vivo hemolysis, you have no choice. I recommend you perform the assay and append a comment describing the hemolysis and explaining it could affect all test results and could falsely reduce the reported heparin concentration. The information provided may meet the clinician’s needs despite its being equivocal. I hope this has helped!