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Abbreviations-. LMWH, low molecular weight heparin; HIPA, heparin-induced platelet activation; UFH, unfractionated heparin.

with danaparoid

Abbreviations-. LMWH, low molecular weight heparin; HIPA, heparin-induced platelet activation; UFH, unfractionated heparin.

for 5 min, and 50 mL of supernatant fluid is transferred to tubes containing scintillation fluid for detection of [14C]serotonin released during platelet activation.

Carbon-14 is a radioisotope with a long half-life (5730 yr) that emits b-particles (electrons). Laboratories require special licenses to handle radioisotopes, thus limiting widespread use of this platelet-activation marker. However, it is also possible to quantitate serotonin by nonradioactive analysis (Gobbi et al., 2003; Fouassier et al., 2006). Results are expressed as percentage of serotonin released. This is calculated based on comparison with maximal possible release (determined following detergent-induced platelet lysis), and adjusted for background release (determined by quantitating serotonin release from a sample incubated with buffer alone). Acceptable experiments should have less than 5% background release, with both buffer and negative control serum testing being negative for HIT antibodies.

Aggregation of Washed Platelets. A convenient and useful activation endpoint— platelet aggregation—was reported in the HIPA assay (Greinacher et al., 1991; Eichler et al., 1999). Test serum and heparin buffer are placed in U-bottomed polystyrene microtiter wells containing two stainless steel spheres, and the platelets are stirred at approximately 500 rpm, using a magnetic stirrer. At 5-min intervals, the wells are examined against an indirect light source: a change in appearance of the reaction mixture from turbidity (nonaggregated platelets) to transparency (aggregated platelets) is a positive result. Although the activation endpoint is evaluated subjectively, interobserver agreement is good. A further advantage of this technique is its repeated evaluation of platelet activation over time. Thus, strong HIT sera that cause the typical activation profile of HIT (i.e., activation at low, but not high, heparin concentrations) within 15-30 min are readily identified. In contrast, such a strong HIT serum might eventually cause platelet activation even at the high heparin concentration and thus cause an "indeterminate" reaction pattern (activation at both low and high heparin concentrations) if activation is assessed at a later time point only. Occasionally there is interference with visual interpretation (e.g., a lipemic serum).

Luminography. Stewart et al. (1995) reported luminography to detect platelet activation, using a commercially available lumiaggregometer. Adenosine tripho-sphate (ATP) is released from platelet-dense granules during platelet activation. In the presence of luciferin-luciferase reagent, a light flash is generated in the presence of ATP, which is detected and quantitated. Another group reported similar results using a standard scintillation counter (Teitel et al., 1996). It is uncertain how the sensitivity and specificity of these assays compare with other markers of platelet activation.

Platelet-Derived Microparticle Generation. Generation of platelet-derived micro-particles occurs when washed platelets are activated by HIT sera (Warkentin et al., 1994). With the use of a fluorescein-labeled anti-GPIa murine monoclonal antibody, a method for quantitating microparticles using flow cytometry was reported by Lee et al. (1996). Although both platelets and microparticles bind fluorescein-labeled anti-GPIa monoclonal antibodies, they can be distinguished by their size and scatter parameters using flow cytometry, with microparticles quantitated in relation to platelet numbers (Lee et al., 1996).

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