Platelet Analysis Using Flowcytometric Procedures

Abstract

'Ex vivo' testing of functional platelet properties using conventional techniques reflects the overall behaviour of the whole platelet population in the sample under investigation. Since many functional aspects depend on ultrastructural constituents which may vary from one cell to another, multiparameter single cell analysis of platelets may be advantageous in providing direct insight into deviations at the cellular level relevant to the pathophysiology of disease states such as bleeding disorders or thrombophilia. Immunolabelling with monoclonal antibodies against membrane antigens has been combined with flowcytometry to provide a standardized, highly specific and sensitive analytical tool. The assay has been optimized for simultaneous two colour fluorescence staining, and this allows the testing of whole blood to provide a quick monitoring method for the differential diagnosis of thrombasthenic diseases like Bernard Soulier's syndrome or Glanzmann's thrombasthenia in which typical staining patterns lack the specific fluorescence for glycoproteins Ib and IIb/IIIa respectively. Also changes in the antigenicity of the outer membrane of activated platelets are detectable with monoclonal antibodies against specific antigenic epitopes such as thrombospondin (a secretion marker) or α-granule and lysosomal proteins (extrusion markers). However, for detection of activated platelets in diseases associated with a prethrombotic state, the procedures for immunolabelling platelets with monoclonal antibodies and instrumental detection sensitivity remain to be optimized. After further development, flowcytometric assays of the functional status of individual platelets may be superior to the measurement of the indirect plasma markers such as platelet factor 4 or β-thromboglobulin for routine diagnosis of the prethrombotic state.

State of the Art in Platelet Function Testing

“…Flow cytometry allows the analysis of individual platelet functional capability and the measurement of the expression of platelet activation markers on individual platelets as well as the quantitation of associates between platelets and other blood cells [145,146]. Overviews on the methodology of platelet flow cytometry are given by Michelson [147,148] and Knight [149].…”

Standardization of the Flow Cytometric Determination of HLA Class I Antigens, 'Platelet-Specific' Glycoproteins and Activation Markers

“…Platelets were activated after isolation, then fixed and immunolabelled with 1 NIH-U thrombin/1 PBS, pH 7.2 (108 platelets in 100 pi thrombin/PBS), for 10 min at room temperature, as recommended by Tschöpe et al [16]. The FC parameters of the glycoproteins on activa ted and unfixed platelets, on platelets from the immediately fixed whole blood and on platelets fixed after isolation were compared.…”

“…Protocol B was performed according to the procedure described by Tschöpe et al [16], using differential centrifugation.…”

“…Among a group of several different sub-| stances such as collagen or ADP, thrombin has been recom mended by other authors [4,16] for in vitro activation exper-1 iments. In platelets, the receptors for thrombin are GPIb and !…”

“…Efforts in FC to calculate the number of binding sites of glycoproteins on the surface of cells by means of beads coated with a definite number of fluorochrome molecules in combination with beads carrying a definite number of MAB binding a-ms IgG have been described [17,25]. This method has also been adapted for the determination of platelet gly coproteins [16]. In our study the 'platelet-specific' glyco proteins CD41a and CD41b, the HLA-class I molecules (both in the dimeric form and as a native single heavy chain), ß2-m and the activation marker CD36 could be enu merated on activated and nonactivated platelets by FC.…”