**2. Materials and methods**

We studied the subpopulation composition of B lymphocytes of peripheral blood in patients with gastric cancer. The possible influence of splenectomy on the subpopulation composition of B lymphocytes was investigated. The B cell link of peripheral blood lymphocytes in patients with gastric cancer was studied in dynamics (3 months after the operation).

To assess the effect of the splenectomy on the B cell link, the subpopulation composition of B cells of peripheral blood after surgery was assessed both in patients after gastrectomy with standard D2 lymphodissection (splenectomy) (14 people) and in patients after gastrectomy with spleen-protective D2 lymphodissection (12 people).

The reaction was taken into account on flow cytometers (FACScan, Lysys II and FACSCanto II, FACSDiva program). Data processing: WinMDI 2.8 and FCS 3.0 applications. Evaluation of the expression of membrane antigens was carried out in the gate of CD19+ B cells. Cells were stained at the same time with three monoclonal antibodies labeled with different fluorochromes. In 42 peripheral blood samples before the operation and 23 samples after the operation on the hematological analyzer Micros 60, the hemogram was calculated, which allowed to estimate the absolute number of B cells.

The expression of the following antigens is analyzed: CD20, CD21, CD23, CD38, HLA-DR, CD71, CD10, CD95, CD25, CD5, CD56, IgG-λ, and IgG-κ light chain immunoglobulins (**Table 1**). Direct conjugates of monoclonal antibodies with fluorochromes were used: FITC, fluorescein; PE, phycoerythrin; PerCP, peridinin


**137**

**Figure 1.**

*Splenectomy in Gastric Cancer: Influence of B Lymphocytes*

antibodies directly labeled with various fluorochromes.

the absolute content of B cells fit within the limits of the norm.

chlorophyll protein; and PE-Cy5, double (tandem) dye-combining phycoerythrin with cyanine 5. The expression of the above antigens was detected by monoclonal

An analysis of the B cell subpopulation was performed in the gate of CD19+ cells. At the first stage of the analysis, the number of CD19+ B cells within the lymphocytes was cytometrically estimated (**Figure 1a**). Further, within the limits of only B cells [gate CD19+, **Figure 1b**], the expression of two antigens associated with B cells was analyzed. In these samples, there is an estimate of CD56 antigens

The main indices of peripheral blood samples before and after surgery are given in **Table 2**. In general, in the group before the surgical intervention in comparison with the physiological norm, the average indices of both the relative quantity and

In 33% of patients with gastric cancer at the preoperative period, a decrease in the relative number of B cells (less than 5%) were detected. Moreover in 38% of patients decrease in the absolute number were revealed. Three months after the operation, in 52% of cases, the relative number of B cells was reduced, in 31% of the

The average relative number of B cells, as well as their mean absolute content in 1 μl of peripheral blood, was slightly elevated 3 months after surgery in comparison with these parameters in preoperative assessment. The average percentage of B lymphocytes after the operation was slightly lower than before surgery. However, these differences were statistically unreliable (T-test when comparing two independent variables). This can be explained by a significant spread of the analyzed indicators

Investigation of subpopulations of peripheral blood B lymphocytes in patients with gastric cancer at the preoperative stage established that all B cells had immunophenotype of naïve mature B2 cells: CD19+ CD20+ HLA-DR+ CD10-CD21low/+. The data of subpopulation composition of B lymphocytes of peripheral blood of gastric cancer patients are given in **Table 3**. B2 cells have a long life, localized mainly

*(a) Estimating the total number of CD19+ B cells within the lymphocytes. The abscissa is the expression of pan-B cell antigen CD19+; along the ordinate axis—the parameter of lateral light scattering of the laser beam, reflecting the working cytometric concept—the granularity of the cells. (b) Cells that fall only in the region of CD19+, i.e., B cells accumulated during sample collection. (c) Evaluation of the expression of two antigens* 

**3. The effect of the splenectomy on humoral immunity in patients** 

*DOI: http://dx.doi.org/10.5772/intechopen.80075*

**with gastric cancer**

absolute content of B cells.

**3.1 B2 cells**

concurrently with CD21 (**Figure 1c**).

both in patients before and after surgery.

*associated with B cells in the CD19+CD56 and CD21.*

#### **Table 1.**

*Monoclonal antibodies and antigens.*

chlorophyll protein; and PE-Cy5, double (tandem) dye-combining phycoerythrin with cyanine 5. The expression of the above antigens was detected by monoclonal antibodies directly labeled with various fluorochromes.
