IVBT-documented platelet function correlates with flow cytometric data.

Thrombocytopenic patients with identical platelet counts often show different bleeding tendencies owing to significant differences in the platelet function. This could be demonstrated by the in vitro bleeding test (IVBT). Using flow cytometry, we tried to find characteristics of platelet antigen expression in order to explain these differences in function. Thirty patients with bone marrow hypoplasia receiving 65 platelet transfusions (mainly from a cell separator) were observed for 3 to 29 days. Size, granulation and fluorescence of platelet-rich plasma (n = 522 samples) were evaluated using monoclonal antibodies against GP IIIb (collagen receptor), GP IIb/IIIa (fibrinogen receptor) and GP Ib (thrombin receptor). We defined separate gates for each antibody using the results from 50 normals and by laying an orthograde cross over the gate to divide the gate into four equal quadrants. The platelet populations were divided into four different groups according to the occlusion time (OT) of the IVBT and the Simplate time (ST). The thrombocytes with the most impaired function (OT > or = 485 s/ST > 30 min) had significantly less platelet fluorescence when marked with antibodies against GP IIIb and GP Ib than those with short OT and ST (OT < 100 s/ST < 15 min). Similar results were obtained when evaluating the data relative to the bone marrow status: patients with < 1000 WBC/microliters showed significantly less platelet fluorescence when marked with anti-GP IIIb and anti-GP Ib than thrombocytopenic patients, who had a spontaneous platelet rise beyond 30,000 platelets/microliters a few days later. One day after platelet transfusion, significantly more platelets with high GP IIIb and Ib expression could be found. We were also able to document better transfusion efficacy of platelet concentrates with high GP IIIb and Ib expression. Finally, patients with high bleeding scores showed less GP Ib expression on the platelets than patients with low bleeding scores. In summary, the IVBT-documented platelet function clearly corresponded to an increased expression of the collagen receptor and the thrombin receptor of platelets.

Thrombocytopenia-adapted in vitro bleeding test assesses platelet function in thrombocytopenic patients.

Platelet counts do not always reflect the true bleeding risk in chronically thrombocytopenic patients, and the posttransfusion platelet increments do not necessarily demonstrate that therapeutic efficacy. There are no easy and reliable tests yet permitting the determination of platelet function in thrombocytopenic patients. The in-vitro bleeding test (IVBT) with the Thrombostat 4000 proved to be a very sensitive and specific test for the detection of platelet disorders. In order to become suitable for the investigation of thrombocytopenic blood with platelet count between 5 x 10(9)/L and 50 x 10(9)/L, special modifications were necessary. We report on the evaluation of two thrombocytopenia-adapted modifications (TP-IVBT 150/120), first with blood of healthy donors made thrombocytopenic (three experiments with six blood samples each of different platelet concentrations and identical hematocrit) and then in a clinical study on 77 thrombocytopenic patients (69 with bone marrow hypoplasia, eight with autoimmune thrombocytopenia) receiving 267 platelet transfusions. The patients were followed over 15 days on average (1-67 days) by daily examinations (total 1,285 observation days). Most TP-IVBT measurements were carried out in triplicate, using the modification with the 120-microns filter (TP-IVBT 120) because it proved to be superior to the other modification. Additionally, cell counts, hematocrit, body temperature, platelet volume, platelet distribution width, expression of CD 36, 41a, 42b on platelets, Simplate bleeding time, and detailed analysis of bleeding signs were performed for the calculation of a bleeding score. There was a close correlation between TP-IVBT and platelet counts with thrombocytopenic normal blood (r2 = 0.81-0.94). This indicated the suitability of this test modification to examine platelet function in thrombocytopenic patients. The clinical study showed that the TP-IVBT helped at least to determine the platelet-related bleeding risk in thrombocytopenic patients. It allowed differentiation between hypoplastic and autoimmune thrombocytopenia in most cases. In addition, significant differences in platelet function of various diseases and of different bone marrow regeneration could be demonstrated. The TP-IVBT is well-suited for the control of platelet transfusion efficacy and may replace the in-vivo bleeding time in most cases. On the other hand, the test still shows too much of a variation and involves too much labor and cost for routine application.