The Utility of Detection Populations of T-Lymphocytes, T-Killer Cells and Natural Killer Cells by CD2 Expression In White Blood Cell Differential Using Flow Cytometry

The differential leukocyte count using Cytodiff™ reagent from Beckman Coulter (BEC) allows to detect a wide spectrum of normal and pathological cells in peripheral blood and is useful as a screening test. The CytoDiff™ tube is a 5-color/6-marker panel that provides an extended cytometric differential for whole blood specimens and comprises of: CD36-FITC, (CD2+CD294)-PE, CD19-ECD, CD16-PC5, and CD45-PC7. This provides clinicians with a diagnostic tool to determine appropriate therapies for a variety of blood-related diseases rapidly. CytoDiff™ allows to detect T&NK lymphocytes in the mutual gate using the gating strategy. The aim of this study was to analyze the CD2 expression on T-lymphocytes, T killer cells and natural killer cells in order to separate T&NK lymphocytes, as it was published by Catovsky (Catovsky et al., 1996) and to investigate if CytoDiff™ reagent can be useful to differentiate these types of lymphocytes.

18 normal peripheral blood (PB) samples in K²EDTA were enrolled into this study. Each sample was stained with the CytoDiff™ reagent and with the reference mixture of monoclonals to detect T-killer, NK-cells and T-Lymphocytes (CD3-FITC, CD(16+56)-PE, CD45-ECD, CD2-PC7, all BEC). The PB was lyzed using VersaLyse (BEC) to prevent selective loss of cells and to conserve light scatter characteristics. PB leukocytes were analyzed according to their immunofluorescence reactivity. At least 20,000 CD45+ events per tube were acquired on FC500 Cytomics flow cytometer (BEC) and analyzed using CXP software (BEC). In the reference tube we identified three populations using the CD45^brightSS^low lymphocyte gate in the CD3-FITC/CD(16+56)-PE plot: CD3⁺CD(16+56)^- T lymphocytes, CD3⁺CD(16+56)⁺ T killer cells and CD3^-CD(16+56)⁺ natural killer cells. Each population after detecting was applied to CD2/SS plot and X-mean value of CD2 population was calculated. For the CytoDiff tube we analyzed the expression of CD2 and CD16 on the different sub-populations of the T&NK lymphocytes, The X-mean values of CD2 expression and percentages of sub-populations of T&NK lymphocytes in the reference tube and in the CytoDiff™ tube were calculated. For statistic analysis we used Statistica 6.0. The study was approved by The Pavlov State Medical University's Institutional Review Board.

The data, obtained with reference tube, shows that the populations of T-lymphocytes, T killer cells and natural killer cells are different by X-mean of CD2 expression (p<0.001). The natural killer cells have the lower intensity the CD2 antigen compared with CD3⁺CD(16+56)^- T lymphocytes (p<0.001), while the highest CD2 expression was found in the CD3⁺CD(16+56)⁺ T killer cells (p<0.001). According to Mann-Whithey test the percentages and X-mean value of CD2 of the described populations are not different in the reference and in the CytoDiff™ tubes.

These findings show that different populations of T&NK cells can be differentiated by CD2 expression and flow cytometric WBC differential can provide more wide possibilities for screening of pathology, especially in cases of viral infection and T-cell leukemia.

Sukhacheva: Beckman Coulter: Employment. Simon-Lopez: Beckman Coulter: Employment.