Functional Assays for Hit Confirmation

Diagnosis of HIT, in patients with a significant HIT clinical probability (4Ts` score or HIT Expert Probability score), and positive for heparin dependent antibodies, must be confirmed with functional assays. These assays are performed at a low (0.10 to 1.00 lU/ml) and a high (10 to 100 lU/ml) heparin concentration, and possibly without heparin, as this helps identification of aspecific reactions. Positive criteria for assayed samples are a positive platelet activation at a low, but not at a high, heparin concentration. Many different assays are available and have been recently reviewed elegantly by F. Mullier`s group. Assays are designed with the use of platelet rich citrated plasma, or with washed platelets. Platelet donors need to be characterized and selected, as there is an important donor`s variability for platelets` activation by heparin dependent antibodies. FcyRIIA (CD32) platelet surface density and polymorphism (His or Arg at position 131) could be one of the parameters explaining the platelet donor variability. However, sensitivity is higher when washed platelets are used, and platelet donors` variability is lower, probably as the result of a slight platelet activation and PF4 release and binding onto the platelet surface  during washing  process.

Among functional assays, the C14-Serotonin Release Assay (SRA) is considered as the gold standard for the diagnosis of HIT. Usually, four donors are used, and the assay is positive if SRA is > 20 % at low heparin concentration and < 20% at high concentration, or without heparin. Some inconclusive results can be faced, especially when SRA is borderline or slightly positive, and differences without heparin or at a high concentration are weak. These inconclusive cases account for most of discrepancies when comparing assays` performances. SRA is considered to offer up to date the highest specificity and sensitivity for diagnosis of HIT. Recently, an assay variant performed with PF4 was proposed (PF4-SRA) and reported as offering a higher sensitivity but probably a lower specificity.

The Platelet Aggregation Test (PAT) is frequently used in laboratories where SRA is not available and it is performed with platelet rich plasmas (PRP) from 4 selected donors tested separately, each PRP being mixed with the tested patient plasma at a low and a high heparin concentration. As for SRA, the test is positive if at least platelets from 2 out of the 4 donors are activated at a low heparin concentration, but not at a high one.