Rapid Assay Particle Gel Immunoassay IDHPF4 Test

Figure 4 illustrates a rapid assay for anti-PF4-H antibodies, the particle gel immunoassay [(PaGIA], H/PF4-PaGIA) (DiaMed, Cressier sur Morat, Switzerland). This assay utilizes PF4-H complexes bound to red, high-density polystyrene particles; after addition of patient serum or plasma, the anti-PF4-H antibodies bind to the antigen-coated beads (Meyer et al., 1999; Eichler et al., 2001). However, IgG class antibodies do not agglutinate the polystyrene beads well, and therefore a secondary antihuman immunoglobulin antibody is added into the sephacryl gel. The principle of this (and other gel centrifugation assays) is that upon centrifugation, the agglutinated beads (indicating the presence of anti-PF4-H antibodies) do not migrate through the sephacryl gel (strong positive result), whereas nonagglutinated beads (indicating absence of antibodies) pass through the gel (negative result), thus forming a red band at the bottom. A weak positive result is indicated by dispersal of the particles throughout the gel. The assay is technically easy, can be performed rapidly, and is readily automated. Results are read visually. The method is available to blood banks that utilize a gel centrifugation technology system. Currently, the PaGIA is available in Europe and Canada, and is under investigation in the United States.

Eichler and colleagues (2001) compared this new assay with two functional assays (HIPA test; SRA) and both commercially available solid-phase PF4-depen-dent EIAs. In preselected samples, the H/PF4-PaGIA had a sensitivity intermediate between that of the functional and commercial antigen assays. The specificity appeared to resemble that of the functional assays.

In contrast, Risch and coworkers (2003) found many more sera to test positive using the H/PF4-PaGIA, compared to a commercial EIA (Asserochrom®), among 42 patients sampled 10-18 days following cardiac surgery (69% vs. 26%). Since none of the patients had clinical evidence of HIT, this suggested that the diagnostic specificity of the H/PF4-PaGIA was far less than the solid-phase EIA. These authors did not test sera from patients with HIT, and therefore were unable to assess test sensitivity (Warkentin, 2003b).

The manufacturer's instructions indicate that the assay is to be read as "positive" (any agglutination within the gel), "negative" (no agglutination) using neat (undiluted) serum, or "borderline." However, when a positive or borderline test result was obtained, Alberio et al. (2003) repeated the assay with undiluted and serially diluted plasma (up to one in 1024) until the result was negative. The reported titer was the last positive result followed by either borderline or negative results. Patients judged clinically to have had "probable" or "highly probable/ definite" HIT had antibody titers of four or more in 39 of 54 (72%) cases, compared with only two of 85 (2%) judged "unlikely" to have had HIT. Further, all 19 of the patient samples that tested positive in a c-PRP aggregation assay tested positive in the PaGIA (generally, in a titer of eight or higher). Among all patients studied, the percentage with associated thrombotic complications increased from 8% (negative or low titer) to 55% (positive titer 4-16) to 74% (positive titer 32-256). This study suggests that reporting quantitatively the results of the H/PF4-PaGIA—with a titer of four or more being clinically significant— may increase diagnostic usefulness.

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