**4.1 Single protein evaluation in related cell population by flow cytometry**

The following protocol is applied to the patients who have suggestive clinical history related to LRBA, STK4, DOCK8 and BTK deficiencies before and after sequencing to evaluate the alteration of designated protein expressions.


*Flow Cytometric Approach in the Diagnosis of Primary Immunodeficiencies DOI: http://dx.doi.org/10.5772/intechopen.96004*


#### *4.1.1 LRBA deficiency*

(Lipopolysaccharide responsive beige-like anchor protein) LRBA plays important roles in vesicle trafficking and receptor recycling. LRBA is responsible for CTLA4 trafficking from vesicular compartments to the cell membrane. In patients with LRBA mutations, an autosomal recessive form of combined immunodeficiency arises and this deficiency is associated with hypogammaglobulinemia, recurrent respiratory infections, multiple autoimmune manifestations and frequently susceptibility to inflammatory bowel disease and malignity in some cases [4, 6, 7, 43–45]. See the Section 4.1. for the staining protocol. **Figure 7** shows a representative image of LRBA expression in LRBA deficient patient and a healthy control.

#### *4.1.2 STK4 (MST1) deficiency*

STK4 (serine–threonine protein kinase 4), also known as MST1 (Macrophage Stimulating 1), was first found in *Drosophila* as a member of the Hippo pathway, which regulates proliferation and cell survival. Human STK4 is principally discovered as a constitutively expressed kinase, structurally homologous to the *Drosophila* Hippo, and plays roles in vital biologic processes such as morphogenesis, proliferation, apoptosis, and stress response [46–49]. STK4 deficiency was first defined in 2012 by 3 separate groups as causing a novel autosomal recessive CID, which is characterized by a profoundly decreased level of CD4+ T cells with the concomitant tendency to recurrent viral and bacterial infections and mucocutaneous candidiasis [46, 49]. Mutations in STK4 gene cause the lack of protein expression or severely reduced level of protein expression [50] (**Figure 8**). See the Section 4.1. for the staining protocol.

#### **Figure 7.**

*A representative image of LRBA expression in a negative control (NC 0r isotype control), positive or healthy control (PC) and a patient (P). Decreased LRBA expression was observed in the patient compared the PC.*

#### *4.1.3 DOCK8 deficiency*

DOCK8 is a member of DOCK-C family and is responsible for activation of GTPases such as CDC42 and RAC. Therefore it transmit the signals from the membrane to intracellular compartment of cells and involves the cytoskeletal rearrangement of the cells. Decreased expression or total loss of DOCK8 protein due to bi-allelic mutations of DOCK8 gene cause Autosomal-Recessive Hyper-IgE Syndrome (AR-HIES) which is associated with eosinophilia and elevated IgE levels in the effected patients [51–53] (**Figure 9**). See the Section 4.1. for the staining protocol.

#### *4.1.4 BTK deficiency in XLA*

BTK is a member of Tec family of non-receptor tyrosine kinases and plays a role in the transmission of the signals from the membrane into the cell. BTK localizes

#### **Figure 8.**

*A representative image of STK4 expression in isotype control (blue), healthy control (green) and the patient (red). Decreased STK4 expression was observed in the patient compared to the healthy control [50].*

#### **Figure 9.**

*A representative image of DOCK8 expression in healthy control (top) and the patient (below). Decreased DOCK8 expression was observed in the patient compared to the healthy control.*

*Flow Cytometric Approach in the Diagnosis of Primary Immunodeficiencies DOI: http://dx.doi.org/10.5772/intechopen.96004*

next to BCR in B cells, therefore it is important for B cell development. In mutations of BTK which is present on X-chromosome cause X-linked agammaglobulinemia in patients who suffered from recurrent bacterial infections due to low or nearly undetectable immunoglobulins and B lymphocytes [54]. Lymphocyte phenotyping is frequently used to diagnose the diseases in patients with suspicious clinical findings and BTK expression is analyzed for molecular diagnosis underlying the XLA. **Figure 10** demonstrates the BTK expression in a patients' and a healthy controls' samples. See the Section 4.1. for the staining protocol.

## **4.2 Pathway characterization in PIDs**

## *4.2.1 PI3K pathway characterization*

Activated phosphoinositide-3 kinase-δ syndrome (APDS) also known as p110δactivating mutation causing senescent T cells, lymphadenopathy and immunodeficiency (PASLI) occurs in patients with combined immunodeficiency due to gain of function mutations of phosphoinositide 3-kinase (PI3K) genes PIK3CD and

#### **Figure 10.**

*BTK expression in isotype control (top) healthy control (middle) and the patient (below). BTK expression was lower in the patient than the healthy control.*

**Figure 11.**

*Ratio of cells expressing p-Akt and p-mTOR in a patient with PIK3*δ *GOF deficiency and a healthy control following pathway stimulation as described in section 4.2.1.1.*

PIK3R1 [14, 16–18]. Although clinical manifestations are heterogenous among the patients, recurrent and persistent infections with herpes family viruses, lymphoproliferation, immune cytopenia are observed in the majority of the patients. Investigating the pathway in patients with suggestive to APDS or PASLI, PI3K pathway analysis, downstream kinase phosphorylations with or without stimulation with specific receptors such as TCR or BCR are investigated by flow cytometry [16]. In the latter section, staining protocol of the PIK3δ, p-Akt and p-mTOR are summarized. **Figure 11** shows a representative image of p-Akt and p-mTOR expression in a patient with PIK3δ GOF deficiency and a healthy control sample.

*4.2.1.1 PIK3*δ *and downstream pathway activation and staining protocol*


*Flow Cytometric Approach in the Diagnosis of Primary Immunodeficiencies DOI: http://dx.doi.org/10.5772/intechopen.96004*

