Diagnostic Usage of Intracellular Protein Staining by Flow Cytometer in Primary Immune Deficiencies; Marmara Experience

İsmail Öğülür
Safa Barış
Ahmet Özen
Ercan Nain
Ayça Kıykım
Elif Karakoç Aydıner

Abstract

Objective: The aim of our study was the optimization and standardization of intracellular dedicator of cytokinesis 8 (DOCK8), LPS-responsive beige-like anchor protein (LRBA), SH2D1A/SLAMassociated protein (SAP) and X-linked inhibitor of apoptosis protein (XIAP) protein expressions in healthy controls with a single flow cytometer protocol and to concomitantly evaluate the possible use of this method for diagnosis.

Materials and Methods: Peripheral blood mononuclear cells were isolated from heparinized blood samples. Protein expressions were analyzed as mean fluorescein intensity difference (∆MFI) according to the isotype.

Results: ∆MFI values obtained by DOCK8 antibody staining were 21.3±4 in CD3+ T cells and 25±3.3 in CD20+ T cells in healthy controls. These values in patients with DOCK8 deficiency were either very low or completely absent. ∆MFI values obtained by LRBA protein antibody staining were 36±7.7 in healthy controls, while they were at the very low levels of 5.9±1.8 in the LRBA protein deficiency patients. The values obtained by SAP and XIAP antibody staining were 30.2±3 in CD8+ T cells for SAP, 13.9±3.2 in CD3+ T and 14.6±3.5 in CD20+ B cells for XIAP. Since the SAP and XIAP results were not confirmed by gene sequencing, the results were not compared to healthy controls.

Conclusion: Due to its rapid and reliable results in clinically relevant cases for DOCK8 and LRBA deficiencies, analysis of protein expression is primarily suitable to evaluate intracellular staining protocol by flow cytometer. In addition, this particular method could be suitable for patients considered to be SAP and XIAP deficient.

Keywords

Flow cytometer, intracellular protein expression, mean fluorescence intensity, primary immunodeficiency
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