When monocytes and platelets compete: The effect of platelet count on the flow cytometric measurement of monocyte CD36

Abstract

Background: Flow cytometric measurement of monocyte surface CD36 is relevant to several conditions including diabetes, cardiovascular disease, lipid disorders, platelet isoimmunization, and susceptibility to P falciparum malaria. CD36 is also strongly expressed on platelets where it is also known as platelet glycoprotein IV.Methods: Whole blood samples, containing identical monocyte concentrations, were adjusted to contain platelets ranging from 20,000/uL to 600,000/uL, were stained with fluorescent-labeled anti-CD36, and analyzed by flow cytometry.Results: CD36 median fluorescent intensity (MFI) observed on monocytes decreased as the platelet concentration in the sample increased with more than a 50% decline in monocyte MFI over the normal range of platelet values. The effect was not abolished by using larger volumes of monoclonal antibody and was observed with different clones of reagent anti-CD36. The findings were most consistent with competition by platelets for the CD36 reagent. Similar findings were observed with antibody to class I HLA. Under defined assay conditions, monocyte CD36 MFI declined with rising platelet concentration in a predictable fashion following an inverse linear relationship.Conclusions: Measurement of CD36 expression on monocytes by flow cytometry in whole blood samples is affected by the sample platelet count. When comparing the monocyte CD36 expression among different individuals, our approach can be used to adjust measured monocyte CD36 expression for the effect of the platelet concentration in the sample. Competition by platelets for monoclonal reagents may occur in other settings when whole blood assays are used and when the target antigen is strongly expressed on both platelets and leukocytes.

Combined measurement of soluble and cellular ICAM-1 among children with Plasmodium falciparum malaria in Uganda

“…Peripheral blood, collected in EDTA, was tested within 48 hours of collection. Previous work demonstrated that monocyte ICAM-1 cell-surface levels were stable for 72 hours after sample collection and were unaffected by the platelet concentration in the sample[26]. Prior to beginning the study, serial testing of samples stored as anticoagulated whole blood in EDTA without continuous mixing at local temperatures demonstrated no statistical difference between monocyte ICAM-1 measurements at day 0 (day of phlebotomy) and day 3 of storage.…”

Frequency of CD36 deficiency in Thais analyzed by quantification of CD36 on cell surfaces and in plasma

“…This approach is limited by the requirement for fresh blood cells, especially for the analysis of CD36 on monocytes. In addition, measurement of CD36 expression on monocytes in whole blood samples is affected by the platelet count . Accordingly, this quantification approach is not easy to standardize.…”

“…This variability may be caused by factors including the origin (plasma or serum), handling (preparation, storage) of the blood samples, and by other specific factors such as different mutations. Several studies have demonstrated up regulation of sCD36 in certain diseases …”

Cytoadherence in paediatric malaria:ABOblood group,CD36, andICAM1 expression and severePlasmodium falciparuminfection

“…Values for monocyte CD36 MFI were corrected for the effect of platelet count prior to analysis (Dzik et al , 2010). Complete blood counts were determined by Coulter AC*T8 (Coulter Corp, Miami, FL, USA).…”