IMEX   05356
INSTITUTO DE MEDICINA EXPERIMENTAL
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Differential expression of CD46 on leukocytes by flow cytometry: methodological aspects
Autor/es:
SABRINA ROTONDO; ANALÍA SÁNCHEZ-LUCEROS; CELIA DOS SANTOS; FABIANA ALBERTO
Lugar:
VIRTUAL
Reunión:
Congreso; XXVIII Congress of the International Society on Thrombosis and Haemostasis (ISTH); 2020
Institución organizadora:
International Society on Thrombosis and Haemostasis (ISTH)
Resumen:
Background: Ubiquitously expressed CD46 (MCP) is a transmembrane protein that acts as negativeregulator 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 (T , n=23) and at 24h (T , 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 5 -95 percentiles were calculated in neutrophils (T : 42; 32-54/T : 37; 24-62) and lymphocytes (T : 124; 60-168/T : 99; 47-175). No significant difference was observed between T and T (p =0,49/p =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 T :66; median T :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 showedhigher variability, which might depend on the subject´s lymphocyte phenotype. Hence neutrophilswould be the population of choice despite showing higher cell death rate in vitro. In both populations, screening can be performed 24h following the venipuncture.