Flow Cytometric Diagnosis of Cellular Defects of Hemostasis

Prof.Dr.D. Tschöpe

Herz- und Diabeteszentrum Nordrhein-Westfalen
Universitätsklinik der Ruhr-Universität Bochum
Georgstr.11, D-32545 Bad Oeynhausen, Germany
Tel: +49/5731/972292, Fax: +49/5731/971967

- Bleeding and thrombosis are clinical manifestations of defects in the coagulation system. Hemorrhagic diathesis and prethrombotic state represent prodromi. They are frequently only rationalized when a stigmatized patient has suffered from a typical exposure situation and consecutively developed severe clinical manifestations. The components of the cellular hemostasis system exert an important regulatory function between offer and demand in the total coagulatory system.

- Endothelial cells and vessel wall matrix as well as luminally streaming corpuscular blood elements like thrombo- and leukocytes balance the different pro- and anticoagulatory regulation circuits of plasmatic coagulation and reparative fibrinolysis.

- Diagnostic efforts aim at the recognition of such regulatory changes in view of a prospective risk assessment in individual patients. The in vitro investigation of thrombocyte aggregation as an example for conventional investigation methods shows substantial disadvantages in this respect: Different, functionally relevant thrombocyte populations in the same sample may influence the total result in an uncontrollable way. Furthermore, ultrastructural factors leading to changes of functional thrombocyte properties cannot be adequately described by global cellular assays. The sensitivity of global assays is furthermore insufficient to detect small variations of functional properties of a few barely activated thrombocytes.

- A representative characterization of the cellular hemostasis system in individual patients is, however, well possible by the flow cytometric single cell analysis of coagulation competent cells like peripheral blood thrombocytes.

- Constitutive parameters like thrombocyte concentration and size as well as presence of specific surface receptors provide information on the thrombosis potential. Bleeding anomalies like Bernard-Soulier syndrome or Glanzmann's thrombasthenia can be clearly demonstrated by isolated expression defects of the GPIb or the GPIIbIIIa cell membrane receptor. The increased thrombotic potency of the peripheral blood thrombocyte pool in diabetes mellitus patients has been attributed to an increased expression of these receptors.

- The analysis of constitutive thrombocyte parameters is less suitable for the thrombotic risk assessment in individual patients because they do not indicate the actual activity of the cellular coagulation system. The analysis of the expression of activation dependent neo-antigens like e.g. CD62, CD63 and thrombospondin as well as the fibrinogen receptor (GPIIb/IIIa) activation on thrombocytes, in contrast, shows quite promising aspects.

- With the Düsseldorf III assay system it was shown in platelet rich plasma (PRP) of diabetic patients that large, activated thrombocytes can be observed in an early disease phase similarly as in patients following acute myocardial infarction. The predictive value of this cellular technique was furthermore demonstrated in the Düsseldorf PTCA-thrombocyte study for ischemic coronary events following catheter angioplasty.

- A further approximation to the in-vivo complexity of all participating cellular systems consists in the measurement of activated thrombocytes in whole blood. Using the Düsseldorf IV test system it was possible by this direct two colour flow cytometric immunofluorescence assay to determine the amount of CD62-positive thrombocytes in M.Crohn and in patients with highly developed cerebral sklerosis exhbiting sonographically demonstrably embolisation events. The essential advantage of this assay consists in the dynami investigation of the interaction of activated thrombocytes and leukocytes in the same assay.

- Flow cytometry, in summary, permits rapid discrimination of clonal thrombopathies in the differential diagnosis of bleeding events. It furthermore permits the characterization of intravascular thrombocyte activation in vascular risk patients. The two colour whole blood methodology permits furthermore the determination of thrombocyte-leukocyte interactions. This may lead to a reassessment of the significance of activated cells in the coagulation system and to an improvement of the therapeutic pharmacological intervention by monitoring the coagulation cell activation status.

Last Update: Jan 02,2012