A flow cytometric assay using mepacrine for study of uptake and release of platelet dense granule contents

Abstract

Diagnosis of platelet dense granule storage pool disease and release defects at present requires a combination of studies including lumiaggregometry, conventional platelet aggregation, radioactive serotonin uptake and release, and electron microscopy. Flow cytometric methods have been developed to study platelet activation, aggregation, and alpha-granule protein release. Here, we have investigated the use of flow cytometry for analysis of platelet dense granule content uptake and release using mepacrine as a fluorescent marker. Mepacrine (quinacrine) is rapidly taken up and localized in dense granules of platelets. For the assay, as little as 20 microliters of blood from a fingerstick collected without anticoagulant or venous blood collected in 3.8% sodium citrate were diluted 1:40 with 2 ml Hanks balanced salt solution (BSS). 300 microliters of this cell suspension were incubated with mepacrine alone, or simultaneously with a mouse monoclonal antibody to human platelet glycoprotein IIb (Tab), used as a platelet-specific marker. The bound monoclonal antibody was then indirectly labelled with the fluorochrome, RED670. 100 microliters of the sample were further diluted with Hanks BSS for one- or two-colour flow cytometric analysis. To verify that mepacrine uptake was related to platelet dense granule content, platelets of beige mice, a strain with dense granule deficiency, were examined. Their mepacrine uptake was substantially decreased compared to that of normal mice. Decreased mepacrine uptake also was demonstrated in platelets of a patient with Hermansky-Pudlak syndrome in which a deficiency of platelet dense granules is characteristic. In both human and mouse platelets, mepacrine uptake was proportional to platelet size. Thrombin induced mepacrine release in a dose-dependent manner from 0.003 to 0.4 U/ml. Therefore both platelet uptake and release of mepacrine can be readily detected by flow cytometry. Flow cytometry provides an attractive alternative to aggregation and radioactive serotonin as methods to study defects in platelet dense granule function.

Platelet activation and hypercoagulability following treatment with porcine factor VIII (HYATE:C)

“…After incubation with antibody, the samples were analyzed within 4 hr. Platelet dense granule release was assessed by mepacrine staining [10,11].…”

A flow cytometric assay for the study of dense granule storage and release in human platelets

“…1 and 2), who had normal staining without stimulation but who did not release a normal amount of granule contents upon stimulation. This patient would have been classified as normal in analyses performed as previously described 2,10 , since defects in the release reaction were not detected there.…”

“…The development of the flow cytometer opened a new possibility of studying platelet dense granules with mepacrine in a simple and rapid way. This has been described both using whole blood 10 and platelet rich plasma (PRP) 2 . Both studies have reported reduced staining of platelets from patients with -storage pool disease compared with the staining of platelets from normal controls.…”

A flow cytometric assay for the study of dense granule storage and release in human platelets

“…1 and 2), who had normal staining without stimulation but who did not release a normal amount of granule contents upon stimulation. This patient would have been classified as normal in analyses performed as previously described 2,10 , since defects in the release reaction were not detected there.…”

“…The development of the flow cytometer opened a new possibility of studying platelet dense granules with mepacrine in a simple and rapid way. This has been described both using whole blood 10 and platelet rich plasma (PRP) 2 . Both studies have reported reduced staining of platelets from patients with -storage pool disease compared with the staining of platelets from normal controls.…”