title: Paraformaldehyde fixation induces a systematic activation of platelets.
abstract: In whole blood flow cytometric platelet assays sample fixation using paraformaldehyde (PFA) is considered very advantageous to prevent spontaneous activation of platelets in vitro. However, fixation is an important variable in activation assays and its influence on platelets is poorly understood. Using a direct immunofluorescence labelling technique and whole blood flow cytometry, the effect of PFA fixation was investigated for 4 different epitopes on platelet surface each of which mirrors a different aspect of platelet activation, namely P-selectin (CD62P), GP IIbIIa complex (CD41), the fibrinogen binding site of the activated GP IIaIIIb complex (PAC-1) and GP Ib-V-IX complex (CD42b). Platelets fixed with PFA (0.5%) before antibody labelling showed significant (P<0.01) increases in mean fluorescence intensity (MFI) of CD62P (1.10 +/- 0.14 vs. 0.94 +/- 0.12 arbitrary units of fluorescence), CD41 (27.3 +/- 6.3 vs. 15.6 +/- 2.1) and PAC-1 (6.21 +/- 1.25 vs. 0.55 +/- 0.12) when compared to unfixed samples. At the same time, MFI of CD42b was reduced from 28.2 +/- 1.6 to 22.6 +/- 2.3 (P<0.01). When fixation was initiated after antibody labelling, we observed less prominent increases in MFI of CD41 (P<0.05) and PAC-1 (P<0.05) while there was no significant difference for CD62P and rather a moderate rise in CD42b than a decrease (P<0.05). Because these alterations cannot be explained by unspecific effects only, it must be concluded that PFA induces a systematic stimulation of platelets. The lowest in vitro platelet activation was found when antibody labelling was started immediately after blood sampling and when samples were analysed within 10 minutes after being stored without fixation of 4 degrees C in the dark.
authors.affiliation: Department of Sports Medicine, Friedrich-Schiller-Universität Jena, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Schmidt
authors.forename: Thomas
authors.initials: T
authors.lastname: Hilberg
authors.forename: Gabriele
authors.initials: G
authors.lastname: Franke
authors.forename: Doreen
authors.initials: D
authors.lastname: Gläser
authors.forename: Holger H W
authors.initials: HH
authors.lastname: Gabriel
mesh_terms: D015415:Biomarkers / Q000032:analysis; D001792:Blood Platelets / Q000737:chemistry; D054641:Dual Specificity Phosphatase 2; D005434:Flow Cytometry; D005557:Formaldehyde / Q000494:pharmacology*; D006801:Humans; D019007:P-Selectin / Q000032:analysis; D015539:Platelet Activation / Q000187:drug effects*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000032:analysis; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000032:analysis; D011108:Polymers / Q000494:pharmacology*; D054648:Protein Phosphatase 2; D017027:Protein Tyrosine Phosphatases / Q000032:analysis; D016707:Tissue Fixation
chemical_list: D015415:Biomarkers; D019007:P-Selectin; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex; D011108:Polymers; D005557:Formaldehyde; D054648:Protein Phosphatase 2; C517032:DUSP2 protein, human; D054641:Dual Specificity Phosphatase 2; D017027:Protein Tyrosine Phosphatases; C003043:paraform