Differential Expression of CD46 in Leukocytes by Flow Cytometry: Methodological Aspects

SABRINA ROTONDO; CÉLIA DOS SANTOS; MARÍA FABIANA ALBERTO; ANALÍA SÁNCHEZ-LUCEROS

Congreso; ISTH 2020 Virtual Congress; 2020

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Background: Ubiquitously expressed CD46 (MCP) is a transmembrane protein that acts as negative regulator preventing host cells injury from complement activation. CD46 alterations are associated to atypical Hemolytic Uremic Syndrome (aHUS). Literature data about CD46 screening for diagnosis by flow cytometry are scarce.Aims: To establish a protocol of CD46 expression on lymphocytes (L) and neutrophils (N) for clinical test.Methods: Whole blood (EDTA) from healthy donors (control group, CG) was collected and processed on the same day (T0, n=23) and at 24h (T24, n=17) with CD46-PE, CD45-PerCP, isotype control-PE markers. Staining was measured through median fluorescence intensity (MFI); reference intervals (RI) were calculated using ratio CD46/isotype. Lymphocyte subpopulations were identified using CD3-PerCp/Cy5.5, CD4-FITC, CD8-APC/H7, CD19APC/H7 markers.Results: Neutrophils presented more CD46 homogeneous staining and greater cell death after 24h, compared with lymphocytes. Median of ratio and 5th-95th percentiles were calculated in neutrophils (T0: 42; 32-54/T24: 37; 24-62) and lymphocytes (T0: 124; 60-168/T24: 99; 47-175). No significant difference was observed between T0 and T24 (pL=0,49/pN=0,29). Lymphocytes from two CG samples (C1, C2) showed ratio values in RI?s lower limit (table 1) and presented distinct cell populations with differential CD46 expression (table 2). In C1, isotype unspecific staining was observed on lymphocytes only (table 1) compared to CG (median T0:66; median T24:70). Lymphocyte subpopulations analysis in C1, C2 and C3 (representative sample from CG), demonstrated that CD46 expression was higher in T cells compared to B and NK cells. Higher percentages of B and NK cells in C1 and C2 could explain the dual expression. Conclusions: Our results highlighted that CD46 staining on lymphocytes from CG samples showed higher variability, which might depend on the subject?s lymphocyte phenotype. Hence neutrophils would be the population of choice despite showing higher cell death rate in vitro. In both populations, screening can be performed 24h following the venipuncture.explain the dual expression.