title: Formaldehyde-fixation of platelets for flow cytometric measurement of phosphatidylserine exposure is feasible.
abstract: Strong platelet activation results in a redistribution of negatively charged phospholipids from the cytosolic to the outer leaflet of the cellular membrane. Annexin V has a high affinity to negatively charged phospholipids and can be used to identify procoagulant platelets. Formaldehyde fixation can cause factitious Annexin V binding. Our aim was to evaluate a method for fixing platelets avoiding additional Annexin V binding. We induced expression of negatively charged phospholipids on the surface of a fraction of platelets by combined activation with convulxin and thrombin in the presence of Annexin V-fluorescein isothiocyanate and calcium. Aliquots of resting and activated platelets were fixed with a low concentration, calcium-free formaldehyde solution. Both native platelets and fixed platelets were analyzed by flow cytometry immediately and after a 24-h storage at 4°C. We observed that the percentage of Annexin V positive resting platelets ranged from 1.5 to 9.3% for the native samples and from 0.4 to 12.8% for the fixed samples (P=0.706, paired t-test). The amount of Annexin V positive convulxin/thrombin activated platelets varied from 12.9 to 35.4% without fixation and from 15.3 to 36.3% after formalin fixation (P=0.450). After a 24-h storage at 4°C, Annexin V positive platelets significantly increased both in the resting and in the convulxin/thrombin activated samples of native platelets (both P<0.001), while results for formalin fixed platelets did not differ from baseline values (P=0.318 for resting fixed platelets; P=0.673 for activated fixed platelets). We conclude that platelet fixation with a low concentration, calcium-free formaldehyde solution does not alter the proportion of Annexin V positive platelets. This method can be used to investigate properties of procoagulant platelets by multicolor flow-cytometric analysis requiring fixation steps.
authors.affiliation: University Department of Haematology and Central Haematology Laboratory, Inselspital, Bern University Hospital and University of Bern, Bern, Switzerland.
authors.forename: Sophie
authors.initials: S
authors.lastname: Rochat
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Alberio
mesh_terms: D017304:Annexin A5 / Q000737:chemistry; D001792:Blood Platelets / Q000737:chemistry / Q000166:cytology / Q000187:drug effects*; D002118:Calcium / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D003435:Crotalid Venoms / Q000494:pharmacology; D005404:Fixatives / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry*; D006801:Humans; D037181:Lectins, C-Type; D010718:Phosphatidylserines / Q000032:analysis* / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects*; D013917:Thrombin / Q000494:pharmacology; D016707:Tissue Fixation / Q000379:methods
chemical_list: D017304:Annexin A5; D003435:Crotalid Venoms; C492760:FITC-annexin A5; D005404:Fixatives; D037181:Lectins, C-Type; D010718:Phosphatidylserines; D005557:Formaldehyde; C028890:convulxin; D013917:Thrombin; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium
keywords: Annexin V; fixation; flow cytometry; formaldehyde; phosphatidylserine; platelets