Characterization of Shear Stress Mediated Platelet Dysfunction: Data from an Ex Vivo Model for Extracorporeal Circulation and a Prospective Clinical Study.

Extracorporeal circulation (ECC) is frequently used in intensive care patients with impaired lung or cardiac function. Despite being a life-saving therapeutic option, ECC is associated with increased risk for both bleeding and thrombosis. The management of bleeding and thromboembolic events in ECC patients is still challenging partly due to the lack of information on the pathophysiological changes in hemostasis and platelet function during the procedure. Using a combination of an ex vivo model for shear stress and a sensitive and easy-to-use laboratory method, we analyzed platelet responsiveness during ECC. After shear stress simulation in an ex vivo closed-loop ECC model, we found a significantly decreased response of α-granules after activation with adenosine diphosphate and thrombin receptor activating peptide (TRAP-6) and CD63 expression after activation with TRAP-6. Mepacrine uptake was also significantly reduced in the ex vivo shear stress model.In the same line, platelets from patients under ECC with venovenous systems and venoarterial systems showed impaired CD62P degranulation after stimulation with ADP and TRAP-6 compared with healthy control on day 1, 6, and 10 after implantation of ECC. However, no correlation between platelet degranulation and the occurrence of bleeding or thromboembolic events was observed.The used whole blood flow cytometry with immediate fixation after drawing introduces a sensitive and easy-to-use method to determine platelet activation status and our data confirm that increased shear stress conditions under ECC can cause impaired degranulation of platelet.

[Acquired immune thrombocytopenia: An update on aspects of diagnosis and management relevant for intensive care medicine]. / Erworbene immunvermittelte Thrombozytopenie in der Intensivmedizin: Ein Update über relevante Aspekte der Diagnostik und Therapie.

Acquired thrombocytopenias represent a group of bleeding diseases, which can be mediated by immune or non-immune factors. Acquired immune thrombocytopenia (AITP) leads to an accelerated decrease in platelet count by platelet reactive antibodies arising from several mechanisms. In AITP, autoantibodies, alloantibodies or drug-dependent antibodies are usually targeting platelet surface glycoproteins. The consequence of this is a significant decrease in the number of circulating platelets, leading to clinic pathological disorders including immune thrombocytopenia, heparin-induced thrombocytopenia or drug-induced thrombocytopenia, respectively. The aforementioned disorders are characterized by a severe reduction in platelet count (< 20 × 109/l), which is, with the exception of HIT, associated with high bleeding risk. In this review we provide current insight into recent achievements regarding diagnosis and management of AITP.

Heparin-induced thrombocytopenia: Diagnostic challenges in intensive care patients especially with extracorporeal circulation.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a serious drug induced reaction that may be associated with life threatening complications. Platelet-activating antibodies directed against platelet factor 4 (PF4)/heparin complexes cause the disease. The diagnosis of HIT is challenging, as thrombocytopenia is a frequent finding in intensive care (ICU) patient population, especially during extracorporeal membrane oxygenation. OBJECTIVE: To investigate the performance of a diagnostic algorithm for HIT in ICU patients. METHODS: ICU patients who developed thrombocytopenia or thrombosis under heparin treatment were included in this study. The pretest probability for HIT was estimated using the 4Ts-score and patient's sera were tested using two rapid immunoassays (RA) LFI-HIT and PaGIA (from Milenia Biotec and DiaMed), and within 72 h using the IgG enzyme immunoassay (EIA) from Hyphen and the heparin induced platelet activation assay (HIPA). RESULTS: 392 consecutive ICU patients with suspected HIT were enrolled in this study, of whom 83/392 (21.2%) patients had extracorporeal circulation. Sera from 120/392 (30.6%) and 98/392 (25.0%) patients revealed positive results in RA and IgG EIA, respectively. The HIPA test revealed heparin-dependent platelet activation in a total of 15/392 (3.8%) ICU patients (3 medical and 12 surgical patients). In addition, sera from 7 patients revealed indeterminate HIPA results, of whom 2 patients had a clinical course compatible with HIT. CONCLUSIONS: Data from our study confirm the high frequency of IgG PF4/heparin antibodies in ICU patients under unfractionated heparin and shows that the combination of 4Ts-score and RA does not reduce the laboratory overinvestigation for HIT in these patients.

Evaluation of a flow cytometer-based functional assay using platelet-rich plasma in the diagnosis of heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4/heparin (PF4/hep)-complexes. The in vitro demonstration of PF4/hep antibodies using functional assays is essential for an optimal management of patients suspected to have HIT. However, conventional functional assays are technically challenging and limited to specialized laboratories. In contrast, flow cytometers are commonly used in routine laboratories. The aim of this study is to investigate the performance characteristics of a commercially available, flow cytometer based assay in the diagnosis of HIT. STUDY DESIGN: Sera of consecutive patients with suspected HIT were investigated using the Emo-test HIT Confirm® assay and compared to the standard method consisting of an IgG-specific enzyme immunoassay (EIA) for anti-PF4/hep antibodies and the heparin induced platelet aggregation (HIPA) test. RESULTS: 390 sera were included in the study, 164 sera tested IgG EIA-positive, of which 33 also tested HIPA-positive. No HIPA-positive samples were EIA-negative. In the Emo-test HIT Confirm® assay, 112 sera revealed positive results (%Hepla > 13); however, 51 (45.5%) were EIA-negative. Of the 33 HIPA-positive/EIA-positive HIT sera, 23 tested positive in the Emo-test HIT Confirm® assay, 2 gave ambiguous results, and 8 sera yielded false-negative results. Accordingly, the HIT Confirm® assay showed a sensitivity of 69.7% with a slightly better specificity of 75.4% compared to the EIA (sensitivity 100%, specificity 63.3%). An increase in diagnostic specificity for HIT to 85% was found when positive results were obtained in both the Emo-test HIT Confirm® assay and EIA. CONCLUSION: The Emo-Test HIT Confirm® assay may improve the specificity of laboratory investigations of HIT. However, the assay can only be recommended in combination with an immunoassay due to the high rate of false negativity. Our observation indicates a need to establish external quality assessment for functional assays to avoid such clinically relevant pitfalls.