Both CD62 and CD162 antibodies prevent formation of CD36‐dependent platelets, rosettes, and artefactual pseudoexpression of platelet markers on white blood cells: A study with ImageStream^®

Abstract

Fluorescent labeled monoclonal antibodies (mAbs) against CD36 are routinely used as monocyte, erythroid, or platelet markers in clinical cytometry. CD36 has recently been proposed by various authors as a valuable marker helping to enumerate leukocyte's subpopulations by flow cytometry. However, it is known that binding of CD36 may induce platelet activation and formation of platelet's rosettes on leukocytes, resulting in false expression of platelet markers on white blood cells. To study this phenomenon, we have combined classical flow cytometry and a new quantitative flow imaging technique with the ImageStream 1 analyzer. We show that CD36 ligation induces activation of platelets with CD62 expression and their adhesion on leukocytes due to CD62 and CD162 interactions. Preincubation of whole blood samples with either anti-CD62 or anti-CD162 antibodies could prevent formation of these rosettes. Our approach also emphasizes the fact that immunomorphological analysis of cell events with ImageStream technology is a useful tool to validate the specificity of marker's labeling or to elucidate incoherent results obtained with classical flow cytometry. We thus propose to prevent false platelet labeling on leukocytes by preincubation with either anti-CD62 or anti-CD162 antibodies when using CD36 mAbs. ; platelet-leukocyte rosettes EXPRESSION of platelet markers on the surface of leukocytes, such as blast cells of acute leukemia is very often difficult to demonstrate by flow cytometry. Indeed, formation of platelet's rosettes on the surface of white blood cells (WBCs) can never be excluded, mainly when labeling is performed on whole blood samples and when using a combination of antibodies for multicolor flow cytometry. These labeling artifacts are very likely to be due to in vitro platelet activation during the labeling procedure. Indeed, among antibodies able to activate platelets are those against CD36, such as NL07, OKM5, OKM8, or FA6-152 (1-4).CD36, also called GPIV, is one of the numerous receptors expressed at the surface of platelets. CD36 belongs to the class B scavenger receptor family, which includes the receptor for selective cholesteryl ester uptake, scavenger receptor class B type I (SR-BI), and lysosomal integral membrane protein II (LIMP-II) (5). CD36 has a molecular weight of 53 kDa (6) and is expressed on an extensive range of cells and tissues, including microvascular endothelial cells, monocytes and macrophages, dendritic cells, adipocytes, keratinocytes, cardiac and skeletal muscle, retinal pigment epithelium, microglia, cells of the erythroid lineage (7), breast, gut, renal epithelium, and platelets (8). The protein is heavily N-linked glycosylated, a modification that

Cell Interaction Analysis by Imaging Flow Cytometry

“…The percentage of neutrophils with adherent platelets decreased from 64 to 3% in the presence of the anti-CD162 mAb, ranging from 1 to 3 particles per FITC-fluorescent cells [22]. These observations suggest that MIFC can be a useful tool to study in depth the formation of cell aggregates, and their constituents.…”

“…MIFC technology has also been used to study the cellular composition of cell aggregates, as is the case during the formation of rosettes by the adhesion of platelets and leukocytes [22]. CD36 is frequently used as a marker of monocytes, erythrocytes, or platelets in clinical cytometry, while antibodies binding to CD36 may induce platelet activation and formation of platelet's rosettes on leukocytes.…”

“…This frequently results in the false identification of expression of platelet markers on white blood cells. MIFC analysis has been used to visualize, quantify, and further confirm platelet rosetting on leukocytes after CD36 activation, and the application of anti-CD162 and anti-CD62 antibodies to prevent the formation of these complexes [22]. Using the ''Spot Count'' function of the IDEAS software applied to multispectral images acquired using MIFC, a direct relation between the number of adherent platelets and CD36-FITC intensity for neutrophils, monocytes and lymphocytes was discovered.…”

Aggregation of monocytes and platelets interferes in measurement of monocyte viability with phosphatidylserine expression but not with mitochondrial membrane potential in whole blood

“…It has previously been shown that leukocyte-platelet aggregates or coincidence of platelets can interfere with measurement of expression on leukocytes in whole blood (5,6). To our knowledge, the interfering effect of leukocyte-platelet aggregates on leukocyte viability measurement has not yet been studied.…”

Imaging Flow Cytometry

“…IFC combines the statistical advantage of FC with the ability to identify each event based on a real image, which allows it to analyze protein expression in single cells in heterogeneous cell populations, where the level of expression of one of the proteins is low and could be described by Poisson distribution (rare cell subpopulations with <0.01% of expression). In recent years, IFC was also employed for the evaluation of asymmetric cell division (Filby et al 2011), internalization of CypHer5E-conjugated antibodies and PKH-labeled exosomes (Xu et al 2010;Vallhov et al 2011), intercellular communication by exchange of cytoplasmic material (Domhan et al 2011), analysis of cell interactions and immune synapse (Ahmed et al 2009;Ouk et al 2011), and some other experimental applications (Ponomarev et al 2011). In recent years, IFC was also employed for the evaluation of asymmetric cell division (Filby et al 2011), internalization of CypHer5E-conjugated antibodies and PKH-labeled exosomes (Xu et al 2010;Vallhov et al 2011), intercellular communication by exchange of cytoplasmic material (Domhan et al 2011), analysis of cell interactions and immune synapse (Ahmed et al 2009;Ouk et al 2011), and some other experimental applications (Ponomarev et al 2011).…”