**Conflict of interest**

*Innovations in Cell Research and Therapy*

5.Wash with 2 mL of PBS.

4.Aliquot 200 μL of sample into different FACS tubes.

7.Fix with the appropriate volume of fixative reagent.

9.Add the appropriate volume of permeabilizing reagent.

8.Incubate for 15 min at RT and in the dark.

10.Add the extracellular antibodies mix.

11.Add the intracellular antibodies mix.

**5. Conclusions**

diated diseases.

immune abnormalities.

**Acknowledgements**

d'Hebron Institut de Recerca (VHIR).

for the study of cell function (3

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

13.Add the appropriate volume of wash buffer.

6.Discard the PBS after 5 min of centrifugation at 1500 rpm (324 g).

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

15.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).

Flow cytometry is an accessible technique in clinical laboratories; the implementation of this technology has progressed together with the significant improvements in instrumentation and the availability of a wide variety of monoclonal reagents. While the main clinical indications for immunophenotyping typically have been the quantification of CD4 T lymphocytes in HIV infection, the study of the extended immunophenotype and functional studies by flow cytometry offers nowadays advanced diagnostic approaches. Previously used techniques

etc.) using radioactivity have been gradually replaced by flow cytometry. Flow cytometry can provide rapid and accurate identification of expanded lymphocyte subpopulations, which play an important role in the evaluation and understanding of complex disorders such as primary immunodeficiencies and other immunome-

The application of flow cytometry techniques in the evaluation of PID assesses lymphocyte proliferation, intracellular changes associated with activation, cytokine production, and biological effects associated with immune defects and functional

We are grateful for the support of Immunology Division and Diagnostic Immunology Research Group from Hospital Universitario Vall d'Hebron and Vall

H-thymidine incorporation, 51Cr-release assay,

**46**

The authors declare no conflict of interest.
