**4. Evaluation of cellular function**

The assessment of lymphocyte function typically refers to the study of T cell function through cell proliferation in response to polyclonal nonspecific and specific stimuli; however, this concept is more extensive and refers to any method that evaluates any of the various effectors or regulatory aspects of subset of lymphocytes [33]. As we have seen previously, these studies are of particular importance when the functional impact of a mutation is unknown; in such cases, flow cytometry provides a number of different immune function assessment techniques, including evaluation of specific immune cell line functions, cell activation, proliferation, and cytokine production.

### **4.1 Lymphocyte proliferation assay**

Lymphocyte phenotype studies should be accompanied by laboratory tests assessing the functional immunity of T cells when a primary immunodeficiency is suspected [34]. Lymphocyte proliferation assay is used to determine lymphocyte activation and their ability of carry out cell-mediated immune response. Ligand binding and transmembrane signal transduction result in cell activation, proliferation of B and T cells which ultimately leads to clonal expansion. Flow cytometry techniques are used to evaluate *in vitro* different cell types proliferation, including the use of fluorescent follow-up cell tracking intracellular dyes [e.g., succinimidyl ester of carboxyfluorescein diacetate (CFSE)] which are capable of covalent binding to cytoplasmic structures of the cell and remain there for a long period of time, being redistributed into daughter cells by each cell division resulting in a 50% decrease in fluorescence intensity after activation for each cell division cycle [35]. Almost all lymphocytes can be stimulated to proliferate non-specifically by stimulating them *in vitro* with polyclonal or mitogenic stimulants such as phytohemagglutinin (PHA), concanavaline A, mitogen pokeweed (PWM), or anti-CD3 antibodies alone or in combination with anti-CD28 or IL-2. Generally, these trials involve at least 72-h or 6–7 days of culture depending on the stimulation (**Figure 7**). The use of flow cytometry has gradually replaced "gold standard" methods such as lymphocyte proliferation by incorporating [3H] thymidine into the DNA of dividing cells and measured using a liquid scintillation counter, which is proportional to the amount of cell proliferation. In terms of complexity, methods based on flow cytometry are considerably simpler to perform, and the undoubted advantage of this method is that the cells grow without any negative influence during the entire culture period, compared to thymidine. However, the interpretation is less objective and time-consuming, thus requiring experienced staff [36].

Cell proliferation induced by an activation signal is a critical parameter in the diagnosis of patients with a possible T-cell defect, including infants with abnormal T-cell receptor excision circles (TRECs) during newborn screening test. In these cases, the T-cell response to polyclonal stimuli is usually less than 10% of the lower limit of the reference value. However, there are patients with hypomorphic mutations that allow some degree of T cell response, often <30% of the lower limit of the reference value. Some laboratories use more than one dose of mitogen or mitogens, although this is not done routinely and is generally not necessary to detect significant defects in T cell immunity. There have been rare cases where mitogen-induced

### **Figure 7.**

*Histograms of lymphocyte proliferation analysis after stimulation with PMA plus ionomicin. Lymphocytes staining with CFSE lose fluorescence after cell division. The histogram on the right shows the result of a patient, with no cell division shown compared to the result of a healthy donor on the histogram on the left.*

**39**

*Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies*

Lymphocyte proliferation assay protocol (CFSE):

2.Manually gently stir the sample during 5 min.

gate at 1500 rpm (324 g) during 5 min.

has been correctly stained with the CFSE.

8.Incubate during 6 days at 37°C and in the dark.

10.Stain with appropriate mix of antibodies of interest.

12.Wash with 2 mL of phosphate buffered saline (PBS).

**4.2 Functional analysis studying STAT phosphorylation**

13.Discard the PBS after 5 min of centrifugation at 1500 rpm.

6.Dispense 100 μL of the PBMCs at 106

proliferation was found to be abnormal, but further evaluation with a combination of agents that directly activate T cells [forbol 12-miristate 13-acetate (PMA) and ionomycin] showed that the proliferative capacity of T cells was normal, but the signaling apparatus (i.e., T[TCR]-CD3 cell antigen receptor-complex) was dysfunctional [7]. Obviously, one of the advantages of using cytometry to evaluate lymphocyte proliferation is the elimination of radioactive reagents. It also allows distinguishing proliferating cell subpopulations, but evaluation of the results is

1.Add 1 μL of carboxyfluorescein succinimidyl ester (CFSE) in 1 mL of PBMCs

3.Add 10 mL of Roswell Park Memorial Institute (RPMI) medium and centrifu-

5.Acquire 50 μL of the sample by flow cytometry just to make sure your sample

7.Leave one well unstimulated (with RPMI) and add 100 μL of mitogens of interest in each different well [phorbol myristate acetate (PMA), ionomycin, phytohaemagglutinin (PHA), muromonab-CD3 (OKT3), interleukin-2 (IL-2),

9.Resuspend cell button and transfer it to fluorescence-activated cell sorter

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

Signal transducer and activator of transcription (STAT) control cellular activation processes after cell activation through cytokines. All STAT molecules are phosphorylated by receptor-associated kinases, that causes activation, by forming homo- or heterodimers and finally translocate to nucleus to function as transcription factors [37]. The study of the different STATs by flow cytometry is of great interest not only of STAT deficiencies (STAT1, STAT3 or STAT5 deficiencies) but also to study the proper

pokeweed mitogen (PWM), and concanavalin A (ConA)].

11.Incubate for 20 min at room temperature (RT) and in the dark.

cells/mL in a 96 well plate.

cells/mL.

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

(all the PBMCs of your sample).

4.Count and adjust cells at 106

(FACS) tubes.

more subjective.
