Quantitative Flow Cytometric Assessment of CD36 (Platelet Glycoprotein IV) Expression on Monocytes and Platelets: Assay Development, Role in Plasmodium falciparum Risk Assessment, and Relationship to Sickle Hemoglobin.
Authors: Cserti Christine M. , Xu Songyi , Sutherland Robert , Nayar Rakesh , Reis Marciano , Dorn Michelle E. , Quagliaroli Robert O. , Sullivan John D. , Preffer Frederic I. , Dzik Walter H. 
Published: April 2020

Background: CD36 (platelet glycoprotein IV) is a known binding site for red cells infected with Plasmodium falciparum (iRBC). The pathologic adhesion of iRBC to CD36 accounts for much of the endothelial sequestration and platelet rosetting observed. Deficient expression of CD36 is hypothesized to be another adaptation to P. falciparum malaria, because a 10-fold higher prevalence of deficiency (3–12%) exists in malaria-endemic areas.

Purpose: A technique to quantitatively assess CD36 expression was developed for a future study on factors affecting P. falciparum malaria severity. In this pilot, CD36 levels were assessed in African-Americans (AA), non-African-Americans (non-AA), and a cohort of patients screened for sickle hemoglobin (HbS), in order to determine the prevalence and types of CD36 deficiency, and deficiency co-inheritance with HbS, a known malaria-adaptive mutation.

Method: EDTA-anticoagulated blood was retrieved from clinical specimens. Antibody-conjugated fluorochromes (CD36-PE, CD14-FITC/− APC, CD61-PerCP, and CD54-FITC) were used. Effects of different assay conditions were investigated on samples from AA (N=57), non-AA (N=36), and those screened for HbS (N=27). FacsLyse lysis was used in the AA and non-AA subjects, but NH4Cl was selected in HbSS samples because of its unique ability to lyse otherwise lysisresistant sickle cells. After washing, samples were analysed by flow cytometry (FACSCalibur). Monocytes and platelets were identified by light-scatter and CD14- and CD61-expression respectively, and analyzed for CD36 at different storage times. Viability was assessed in 2 non-AA controls with 7-AAD. The median fluorescence intensity (MFI) of CD36 expression was log transformed.

Results: Monocyte CD36 MFI follows a log-normal distribution:

number tested mean SD *5% of AA were type I CD36-deficient (logMFI &lt;1.5; MFI&lt;32.) AA* 57 2.42 .45 non-AA 36 2.58 .22 HbS-pos 15 2.51 .35 HbS-neg 12 2.73 .28

Monocyte CD36 MFI at day 4 of room temperature storage was not significantly different from day 1 [N=28; mean MFI 96% of fresh, mean Δ = 65±241, p=NS, paired t-test], despite reduced monocyte viability and CD14 expression after 3 days. Results of platelet CD36 paralleled monocyte CD36. Platelet-monocyte aggregates gave spuriously high CD36, occurring commonly in citrated, heparinized, and ACD anticoagulants, but were rare in EDTA. Sample size was insufficient to detect an association between reduced CD36 and HbS+ (p=0.23, Fisher’s exact test).

Conclusions: Flow cytometry using EDTA blood can quantify CD36 expression on monocytes and platelets. NH4Cl lysis is preferred for Hb SS specimens. Monocyte CD36 follows a log-normal distribution with 5% of AA deficient in CD36. Monocyte CD36 appears stable during room temperature storage for 4 days. This assay should be useful in studies of malaria and CD36-expression. Further work is required to assess if the reduced expression of CD36 is associated with adaptations to malaria.