HEPARIN COFACTOR II ASSAY : IMPORTANCE AND CONTROL OF THE NON-CONTAMINATION BY HEPARIN OF DERMATAN SULFATE

A chromogenic substrate method for the determination of heparin cofactor II (HC II) in plasma, based on its anti Fc Ila activity, has been developped in our laboratory, using dermatan sulfate (D.S.) as activator. We observed that D.S. from porcine skin, which was claimed not to be contaminated by heparin, was sometimes contaminated, depending upon the batch used. Moreover, a classical treatment of D.S. by nitrous acid, that specifically degrades heparin-like glycosaminoglycans, is not always sufficient and in some cases, needs to be repeated. It is thus important to check the absence of heparin contamination for the validity of the HC II assay. Indeed, we have shown that for an amount of contaminating heparin as small as 5×10⁻⁵ iu per μg of D.S., the HC II level of a normal plasma was increased by 50%. Such a low contamination is undetected by routine tests (thrombin time, APTT, heparin activity assay). Thus, we propose two useful methods for this purpose : (i) an anti-Fc Xa heparin cofactor activity assay on normal plasma, using D.S. as activator ; HC II being devoid of any anti-Fc Xa activity, if a Fc Xa inactivation is observed, it is due to AT III potentiation by the contaminating heparin, (ii) an HC II assay using a normal plasma and an AT III depleted plasma ; in the case of D.S. contamination, a higher level of HC II is obtained with normal plasma, reflecting the AT III potentiation by heparin. For example, with 2.5×10⁻⁵ iu of heparin per μg of D.S. : (i) an anti-Fc Xa activity of 50% was found, decreasing to 0% after decontamination, (ii) the HC II. level which was 125% in normal plasma, compared to 100% in AT III depleted plasma was identical (100%) in both plasmas after decontamination. These results demonstrate the validity of the two methods and the usefulness of a control of each batch of D.S. used in HC II assay, even after nitrous acid treatment of the glycosaminoglycan.