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Flow cytometry represents a modern analytical method useful for an assessment of selected cellular subpopulations in suspension (peripheral blood, aspirate of bone marrow, liquid fluid etc. and also in suspensions prepared from non-fixed solid tumors) in clinical and research laboratories. The method provides information on numerous surface or intracellular markers of the analyzed elements at the same time. The usage of fluorescent monoclonal antibodies against surface or intracellular antigens associated with specific type of cells (T-lymphocytes, B-lymphocytes etc.), with their developmental stage, or with monoclonality (for example antibodies against light chains of immunoglobulins), are the most valuable for the clinical practice. The most important application of flow cytometry in pathology has remained in hematologic malignancies, where this fast and exact method provides, practically in a real time, crucial information for correct diagnosis as well as for the choice of optimal therapy, and subsequently for assessing the effect of the therapy.

Objective: CD154 (also called CD40L) is a transmembrane glycoprotein predominantly expressed on the surface membrane of activated CD4+ T cells. Its receptor CD40 is present on all B cells, but also on other cells. The interaction CD154-CD40 is necessary for the optimal development of the adaptive immune response and also has consequences for the modulation of the inflammatory response. A defect in the expression of CD154 is pathognomonic of congenital immunodeficiency called X-linked Hyper-IgM syndrome (XHIGM). To detect the abnormality of CD154 is essential for making the diagnosis of XHIGM., Material and Methods: We worked out a microtest for the detection of CD154 expression on in vitro activated CD4+ T cells in whole blood and compared it with that on isolated cells from peripheral blood. Heparinized peripheral blood was activated with phorbol 12-myristate 13-acetate and ionomycin for 4 hours, labeled with monoclonal antibodies and analyzed by flow cytometry. Considering that the CD4 marker on the plasma membrane surface decreases during the activation, CD4+ T cells are mostly recognized as CD5+/CD8- cells. Their activation is monitored based on the expression of CD69. Three-colour immunofluorescence staining was used for simultaneous detection of CD154., Results: Ten blood donors were tested. As little as 0.5 ml of heparinized whole blood is needed to complete the test. Optimal time for activation and detection of CD154 on T lymphocytes is 4 hours. We found that the number of CD4 molecules on the surface of T cells decreases during the activation. The expression of CD154 in our whole blood microtest is fully comparable with that in the test on isolated leukocytes., Conclusion: The presented microtest for the detection of CD154 on activated lymphocytes in whole blood is fast and blood saving, since as little as 0.5 ml of blood is needed to complete it. It can be recommended as the initial test for suspected hyper-IgM syndrome in children. We demonstrate that this screening method can help to detect also carriers of XHIGM.