Serum samples and platelet donors are ranked from strongest to weakest (S1-S10 and p1-p10, respectively), according to the mean percentage of [l4C]serotonin release when considering all 100 serum-platelet donor pairs (10 pairs corresponding to each HIT serum and each normal platelet donor). For each serum-platelet donor pair, the individual amount of serotonin release is summarized as follows: 80-100% release, + + + +; 60-79% release, + + +; 40-59% release, + +; 20-39% release, +; <20% release, - . Overall, there is a graded pattern of reactivity among the individual reaction pairs that is hierarchical (i.e., there are no unexpected weak or strong reactions among the pairs). All negative reactions (<20% release) were found in the lower right portion of the table. Conversely, the strongest reactions (>80% release) were found in the upper left portion of the table.

Source: Warkentin et al., 1992.

Hierarchical Versus Idiosyncratic Platelet Activation by HIT Sera. The results of a systematic investigation, summarized in Table 5, showed that both HIT sera and platelet donors exhibit variable reactivity in a hierarchical, rather than an idiosyncratic, manner. The strongest reactions were produced by strong HIT sera against strongly reactive platelet donors. All of the negative reactions occurred when the weakest sera were mixed with the weakest platelets. Importantly, no unexpected negative reactions occurred elsewhere in the 10 X 10 serum-platelet grid (Table 5). Furthermore, the relative ranking of platelet donors appeared to be stable over time, an observation also reported by Chong and colleagues (1993a) in their study of platelet donor variability using c-PRP.

The finding of a hierarchical pattern of reactivity has important implications for quality control in diagnostic testing for HIT using activation assays. First, it indicates that platelets from certain donors who tend to respond well to HIT sera should be chosen. Second, relatively weak HIT sera should be included as positive controls (~20-50% serotonin release, or 25 min lag time in the HIPA).

Heparin-Independent Platelet Activation: Indeterminate Results. About 5% of test sera or plasma give an indeterminate result in an activation assay. This is defined as platelet activation that occurs at both therapeutic (0.1-0.3 U/mL) and suprather-apeutic (10-100 U/mL) heparin concentrations. Often an interpretable result is obtained when the assay is repeated using another heat-inactivated aliquot. This suggests that the first result may have been an artifact caused by heat-aggregated IgG generated ex vivo. However, some serum and plasma samples repeatedly demonstrate heparin-independent platelet activation. Biological explanations include circulating immune complexes (e.g., systemic lupus erythematosus), high-titer HLA class I alloantibodies, and, possibly, other platelet-activating factors (e.g., thrombotic thrombocytopenic purpura). An antigen assay is required for further investigation when an indeterminate result is consistently obtained.

Inhibition by High Heparin Concentrations. Sheridan and colleagues (1986) first emphasized that there was a relatively specific activation profile triggered by HIT sera and plasmas: activation at therapeutic heparin concentrations (maximal at 0.10.3 U/mL) that progressively diminished with increasing heparin concentrations, typically falling to background activation at very high (100 U/mL) heparin concentrations. Classically, a positive test was deemed as greater than 20% serotonin release at 0.1 U/mL heparin and less than 20% serotonin release at 100 U/mL heparin. These criteria should not be applied indiscriminately, however. For example, a very strong HIT serum could produce more than a 90% release at 0.1 U/mL heparin and 25% release at 100 U/mL heparin. Alternatively, a serum or plasma sample that was not adequately heat-inactivated could produce a similar reaction profile (i.e., residual thrombin is inhibited by the high, but not low, heparin concentration). The strength of reactivity caused by patient serum can be helpful: clinically significant HIT antibodies almost always cause more than 50% serotonin release using optimally reactive platelets (Warkentin et al., 2000; Warkentin and Heddle, 2003). In the HIPA test, differences in the lag time to platelet aggregation provide useful information.

Inhibition by Fc Receptor Blockade. Platelet activation by HIT antibodies is inhibited in the presence of a murine IgG2b monoclonal antibody (IV.3) that recognizes the platelet FcgIIa receptor (Kelton et al., 1988; Chong et al., 1989) and can be used to enhance test specificity.

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