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Role of Flow Cytometry in the Diagnosis of Chronic Granulomatous Disease: the Egyptian Experience

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Abstract

Introduction

Chronic granulomatous disease (CGD) is an inherited mutational defect in any of the NADPH oxidase complex, CYBB (gp91-phox), NCF1 (p47-phox), CYBA (p22-phox), NCF2 (p67-phox), or NCF4 (p40-phox) leading to inability of phagocytes to perform effective respiratory burst and thus diminished killing of bacteria and fungi. The identification of defective proteins aids in establishing a diagnosis prior to genetic analysis, which is rather labor-intensive, expensive, and time-consuming.

Aim

The present study aims at assessing the NADPH proteins by performing the intracellular staining with specific monoclonal antibodies and their assessment on flow cytometry. The use of flow cytometry is less laborious and faster to perform than western blot. It also confirms the diagnosis of CGD and detects the affected components allowing proper management of patients.

Materials and Methods

Twenty-eight patients from 25 different kindred, clinically suspected as CGD were recruited in Egypt. Dihydrorhodamine test was performed to confirm the diagnosis of the patients. Intracellular staining of NADPH components using specific monoclonal antibodies was performed followed by flow cytometric analysis.

Results

The present study revealed that the most common defective protein in our cohort is p22-phox, found in 13 patients (46.4 % of cases) followed by p47-phox in 8 patients (28.6 %), gp91-phox in 5 patients (17.9 %), and finally p67-phox in 2 patients (7.1 %).

Conclusion

In countries with limited resources and yet large number of CGD patients, the analysis of the defective proteins by flow cytometry is an optimum solution for confirming the diagnosis and is a step for targeted sequencing in families seeking prenatal diagnosis.

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Acknowledgments

This work is funded by Cairo University. We would like to thank the Molecular Genetics Laboratory, Centre for Cardiovascular Surgery and Transplantation, Brno, Czech Republic, for performing Sanger sequencing to detect the molecular mutations and The Laboratory of Human Genetics of Infectious Diseases which is supported by institutional grants from INSERM, University Paris Descartes, The Rockefeller University, and the St. Giles Foundation and grants from the French National Research Agency (ANR) under the “Investments for the Future” program (grant no. ANR-10-IAHU-01) and grant IFNGPHOX (no. ANR13-ISV3-0001-01).

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    Authors and Affiliations

    1. Clinical Pathology Department, Faculty of Medicine, Cairo University, Cairo, 11562, Egypt

      Rabab El Hawary, Safa Meshaal, Mahitab Abdelkawy & Taghrid Gaafar

    2. Laboratory of Human Genetics of Infectious Diseases, Necker Branch, Institut National de la Santé et de la Recherche Médicale, INSERM UMR1163, Paris, France

      Caroline Deswarte & Jacinta Bustamante

    3. Imagine Institute, Paris Descartes University, Paris, France

      Caroline Deswarte & Jacinta Bustamante

    4. Pediatrics Department, Faculty of Medicine, Cairo University, Cairo, Egypt

      Nermeen Galal, Radwa Alkady, Dalia Abd Elaziz, Jeannette Boutros & Aisha Elmarsafy

    5. Molecular Genetics Laboratory, Centre for Cardiovascular Surgery and Transplantation, Brno, Czech Republic

      Tomas Freiberger & Barbora Ravcukova

    6. Department of Clinical Immunology and Allergology, Medical Faculty, Masaryk University, Brno, Czech Republic

      Tomas Freiberger & Jiri Litzman

    7. Center for the Study of Primary Immunodeficiencies, Assistance Publique-Hôpitaux de Paris AP-HP, Necker Hospital for Sick Children, Paris, France

      Jacinta Bustamante

    8. St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY, USA

      Jacinta Bustamante

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    1. Rabab El Hawary
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    Correspondence to Rabab El Hawary.

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    Additional information

    Rabab El Hawary and Safa Meshaal equally contributed to this work, Taghrid Gaafar and Aisha Elmarsafy share senior coauthorship.

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    El Hawary, R., Meshaal, S., Deswarte, C. et al. Role of Flow Cytometry in the Diagnosis of Chronic Granulomatous Disease: the Egyptian Experience. J Clin Immunol 36, 610–618 (2016). https://doi.org/10.1007/s10875-016-0297-y

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