*Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies DOI: http://dx.doi.org/10.5772/intechopen.89743*

before adding the detection antibody. This fixation/permeabilization treatment allows the antibody to pass through the plasma membrane into the cell while maintaining morphological characteristics. While this technique is informative and very useful to identify total defects of a particular protein, detecting expression does not rule out the defect, as there may be mutations that do not alter the expression but the function. Some examples of this type of application to PID diagnosis are the evaluation of the following proteins:

For intracellular staining, there are several commercial options, including laboratory permeability and fixation reagents. It is important to take into account that not all commercial kits work the same way for different cell types or for different techniques. It is therefore necessary to adjust the conditions in each particular case.

Intracellular staining protocol:

*Innovations in Cell Research and Therapy*

group of congenital defects of the number or function of phagocytes in which the following leukocyte populations are affected: CD56bright NK cell, B lymphocytes, monocytes and peripheral dendritic cells populations are affected. Patients with this PID have susceptibility to infections by mycobacteria and human papillomavirus (HPV), histoplasmosis, alveolar proteinosis, myelodysplastic syndromes such

*NK/monocytes/DC dendritic cells (DC) gating strategy. NK cells and monocyte populations were analyzed in the CD3−CD19− gate. NK subpopulations (NKdim and NKbright) were studied using CD56 and CD16 expression markers. The markers CD16 and CD14 were used to identify classical monocytes (CD14+CD16−) and non-classical monocytes (CD16+CD14−). DCs were studied selecting the population negative for the following markers: CD3, CD14, CD16, CD19, CD20, and CD56. High expression of HLA-DR and CD11c and CD123 was used to identify plasmacytoid DCs (HLA-DR+CD123+) and myeloid DCs (HLA-DR+CD11c+).*

**3. Evaluation of intracellular proteins and activation markers involved** 

Intracellular protein and specific cell surface antigen detection is a screening test for PIDs. Depending on the specific clinical presentation of the patient, the diagnostic suspicion can be assessed by evaluating the expression of the affected protein by flow cytometry. The most widely used procedure for staining intracellular molecules requires the fixation of cells in suspension and subsequent permeabilization

acute myelogenous leukemia and lymphedema [24]. See the immunophenotype protocol in Section 2.

**3.1 Evaluation of intracellular proteins**

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**in immunity**

**Figure 5.**


(This protocol may change slightly depending on the commercial kit used).
