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

### **3.1 Evaluation of intracellular proteins**

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

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*(HLH)*

*Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies*

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

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.

1.Fix 50 μL of EDTA whole blood with the appropriate volume of fixative

*DOI: http://dx.doi.org/10.5772/intechopen.89743*

evaluation of the following proteins:

Intracellular staining protocol:

2.Incubate for 15 min in the dark at RT.

4.Add the extracellular antibodies mix.

5.Add the appropriate intracellular antibody.

6.Incubate for 20 min at RT and in the dark.

7.Add the appropriate volume of wash buffer.

*3.1.1 BTK expression in X-linked agammaglobulinemia*

3.Add the appropriate volume of permeabilizing reagent.

8.Discard the wash buffer after 5 min of centrifugation at 1500 rpm.

that intracellular BTK protein has to be studied by gating monocytes [25].

*3.1.2 SAP, XIAP, and perforin expression in Hemophagocytic lymphohistiocytosis* 

Measurement of intracellular signaling lymphocyte activation moleculeassociated protein and X-linked inhibitor of apoptosis are used as screening tools to evaluate X-linked lymphoproliferative syndrome disorder type 1 and 2, respectively. This approach has been shown to provide good sensitivity and specificity compared

See the intracellular staining protocol in Section 3.1.

9.Add 100 μL of PBS and the sample is ready to be acquired by flow cytometry.

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

X-linked agammaglobulinemia or Bruton tyrosine kinase (BTK) deficiency is a disorder caused for mutations in the gene encoding BTK. It is a tyrosine-protein kinase localized intracellularly next to the B-cell receptor (BCR), and it is crucial for the B cell development. The expression of BTK protein is evaluated in monocytes or platelets as patients with suspected X-linked agammaglobulinemia have an absent or significantly reduced number of circulating B lymphocytes. This leads to the fact

reagent.
