simultaneously using microtiter plates

Note: See text for further details on differences between washed platelet and citrated plasma assays (pp. 234-235).

Note: See text for further details on differences between washed platelet and citrated plasma assays (pp. 234-235).

2. Apyrase is used to prevent accumulation of ADP during platelet washing. This prevents platelets from becoming refractory to subsequent ADP-mediated platelet activation (Ardlie et al., 1970). Empirically, apyrase grade III (Sigma) is acceptable for use: grades I and II are too impure, and grades IV and higher are expensive.

3. Physiological calcium concentrations are present when washed platelets are used. Under these conditions, ADP produces only primary platelet aggregation. However, as observed by Packham et al. (1971), traces of immunoglobu-lin complexes in amounts too low to cause aggregation themselves will cause secondary aggregation to occur following the addition of ADP. The importance of ADP in mediating HIT antibody-induced platelet activation has been reported by Polgar and colleagues (1998). Thus, the reaction conditions that exist when washed platelets are used appear to maximize HIT antibody-induced platelet activation because the platelets retain sensitivity to ADP-mediated platelet activation.

4. Low concentrations of IgG are present in the final washed platelet reaction mixture: there is a fivefold reduction in IgG compared with citrated platelet-rich plasma (c-PRP) assays, because only IgG from the test serum is present in the final reaction mixture. Chong and colleagues (1993a) showed that high plasma IgG levels in one platelet donor's blood seemed to explain the discrepancy between studies using donor c-PRP (poor reactivity) and donor washed platelets (good reactivity). These and other investigators (Greinacher et al., 1994c) also observed that addition of IgG inhibits HIT serum-induced activation of washed platelets in a dose-dependent fashion.

5. Low concentrations of fibrinogen and other plasma proteins could reduce the potential for nonidiosyncratic heparin-induced platelet aggregation (Salzman et al., 1980; Chong et al., 1993a). In contrast, low concentrations of heparin rarely cause significant activation of washed platelets. It is possible that acute-phase reactant proteins such as fibrinogen could lead to false-positive activation assays for HIT using c-PRP.

6. Room temperature conditions are used for washed platelet assays; in contrast, c-PRP studies are performed at 37°C. Although this is a major difference between the assays, it is unknown whether there are advantages or disadvantages of performing washed platelet assays at room temperature. In Greifswald, all buffers are warmed to 37°C, and all incubation steps are performed at this temperature; only the final incubation on the microtiter plates is performed at room temperature.

7. Multiple serum-platelet reactions in microtiter plates can be performed, and even several hundred reactions studied in parallel. Quality control is thereby enhanced by the large number of control and test reaction conditions that can be analyzed, and the long incubation period employed (up to 60 min). The incubation period in HIT assays should be at least 20-30 min (Stewart et al., 1995).

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