Blood platelet activation evaluated by flow cytometry: optimised methods for clinical studies.
A variety of flow cytometry techniques are in use to evaluate in vivo blood platelet activation. We have in this study further developed and optimised these methods to be suitable for use in clinical studies. By preloading the Monovette EDTA vacuum blood sampling tubes with 1/8 vol 4% (w/v) paraformaldehyde (PFA), we were able to assess platelet CD62P (P-selectin) expression in whole blood with less than 0.2% activated platelets. No washing or neutralising steps were required to remove excess fixative. Both basal and agonist-stimulated CD62P expression were stable for at least 48 h after sampling. The standard curve was linear from 1.9 (basal) to 8.1 x 10(3) (TRAP-stimulated) molecules of equivalent soluble fluorochrome units (MESF) in phycoerythrein-conjugated anti-CD62P labelled whole blood samples. These assay conditions were also well suited for assessment of platelet expression of CD41, CD42a, CD61 and CD63. The preanalytic storage period was extended from 10 min to at least 2 h for platelet PAC-1 and fibrinogen binding analysis by preloading Monovette citrate tubes with 8/10 vol buffer. With PFA preloading, blood sampled into citrated tubes could be analysed for fractions of microparticles and platelet-platelet aggregates as well as for aggregate size.
Ullevaal University Hospital, Oslo, Norway. ingeranne.hagberg@ulleval.no
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D000328:Adult; D000911:Antibodies, Monoclonal; D000925:Anticoagulants / Q000494:pharmacology; D015703:Antigens, CD / Q000378:metabolism; D001793:Blood Preservation; D019343:Citric Acid / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005189:False Positive Reactions; D005340:Fibrinogen / Q000276:immunology / Q000378:metabolism; D005404:Fixatives / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D005455:Fluorescent Antibody Technique; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D039661:Integrin beta3; D008875:Middle Aged; D019007:P-Selectin / Q000097:blood / Q000187:drug effects / Q000276:immunology; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology*; D010974:Platelet Aggregation / Q000187:drug effects; D010979:Platelet Function Tests / Q000295:instrumentation / Q000379:methods / Q000592:standards; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011108:Polymers / Q000494:pharmacology; D011506:Proteins / Q000494:pharmacology; D018179:Receptors, Thrombin; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D013696:Temperature; D060149:Tetraspanin 30; D013997:Time Factors
D000911:Antibodies, Monoclonal; D000925:Anticoagulants; D015703:Antigens, CD; C554586:CD63 protein, human; D005404:Fixatives; D039661:Integrin beta3; D019007:P-Selectin; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011108:Polymers; D011506:Proteins; D018179:Receptors, Thrombin; D060149:Tetraspanin 30; D005557:Formaldehyde; D019343:Citric Acid; D005340:Fibrinogen; D004492:Edetic Acid; C003043:paraform