Immunophenotyping of hemoblastoses

Significant progress in hematological research in recent years is associated with the use of modern immunological methods and automated means of analysis and sorting of peripheral blood and bone marrow cells - flow cytometers. Traditional morphological and cytochemical studies of disease substrate cells (blood, red bone marrow, lymph nodes, spleen, etc.) in many cases, especially in lymphoproliferative diseases, do not allow us to identify the full variety of variants among morphologically similar forms and establish the source of origin of the pathological clone. These problems can be solved only by studying the immunological characteristics of the cells. Each stage of differentiation of hematopoietic cells corresponds to its own set of antigens, which according to the international classification are called differentiation and are divided into differentiation clusters, designated CD.

In neoplastic changes, a differentiation block can occur at any stage of normal cell development, resulting in the formation of a clone of pathological cells that determine the disease substrate and have the same immunological (or phenotypic) characteristics. By conducting studies of these markers on cells, it is possible to determine what form and variant of the disease they correspond to, that is, based on the immunological phenotype of cells, to conduct differential diagnostics, which is most difficult in lymphoproliferative diseases, because the main cell of the pathological substrate of the disease are morphologically almost identical cells.

Phenotyping allows using monoclonal antibodies to type blast and mature blood cells of the myelo-, mono-, lymphocytic series by the presence of differentiation antigens (receptors) in the cell wall. The section "Assessment of the immune status of the body" partially describes the characteristics and diagnostic value of the study of cellular markers; below is a brief description of the antigen markers of cells in relation to the diagnosis of hemoblastoses. The following antigens (markers) can be detected on the membranes of blood cells and red bone marrow.

• CD2 is a monomeric transmembrane glycoprotein. It is present on the surface of all T-lymphocytes circulating in the blood and on some NK-lymphocytes. CD2 is involved in the process of alternative activation of T-lymphocytes. Detection of CD2 using monoclonal antibodies in clinical practice is used for phenotyping acute T-cell leukemia, lymphomas, chronic inflammatory and immunodeficiency conditions.
• CD3 is a protein complex associated with the antigen-specific T-cell receptor, it is the main functional marker of T-lymphocytes. It facilitates the transfer of the activation signal from the membrane to the cell cytoplasm. Determination of CD3 is indicated for the diagnosis of acute T-cell leukemia, lymphomas (CD3 is not expressed in non-T-cell lymphoid neoplasms) and immunodeficiency diseases.
• CD4 is a transmembrane glycoprotein expressed by a subpopulation of T-helpers (inducers), which constitute 45% of peripheral blood lymphocytes. In the early stages of lymphocyte development in the thymus, CD4 antigens, as well as CD8, are expressed by all cortical lymphocytes. Medullary thymocytes, the phenotype of which is similar to mature CD4+ T-cells of peripheral blood (T-helpers), already express either CD4 or CD8 receptors. In peripheral blood, up to 5% of cells carry both CD4 and CD8 markers. Minor expression of CD4 is possible on some cells of the monocytic series. CD4 is expressed in most cases of T-cell lymphomas, including mycosis fungoides, as well as in HTLV-associated T-cell leukemia (HTLV - human T-lymphotropic virus).
• CD5 is a single-chain glycoprotein present on all mature T lymphocytes and most thymocytes, and is weakly expressed by B lymphocytes. CD5 is detected on neoplastic cells of B-cell chronic lymphocytic leukemia and centrocytic lymphoma. In other types of malignant lymphoid diseases - follicular lymphoma, hairy cell leukemia, large cell lymphoma - CD5 is not expressed.
• CD7 is a single-chain protein, the earliest marker of T-cell differentiation. It is expressed by pro-T-lymphocytes even before their migration to the thymus. CD7 is detected on most NK cells, weak expression is noted on monocytes. B-lymphocytes and granulocytes do not contain this antigen. CD7 determination is used to diagnose lymphomas, childhood T-cell lymphoblastic leukemia.
• CD8 is a protein consisting of two polypeptide chains linked by disulfide bridges. It is expressed by a subpopulation of cytotoxic and suppressor T lymphocytes, which make up 20-35% of peripheral blood lymphocytes. This antigen is also expressed by NK lymphocytes, cortical thymocytes, 30% of medullary thymocytes, and a subpopulation of red bone marrow cells. CD8 is studied to quantify the content of T suppressors (see the section “Suppressor T lymphocytes in the blood” above).

• CD10 is a cell membrane-associated endopeptidase. CD10 is expressed by young forms of B lymphocytes and a subpopulation of cortical lymphocytes. CD10 is expressed by all ALL cells.
• CD11c is expressed on the cell membrane by macrophages, monocytes, granulocytes, NK cells and hairy cell leukemia cells.
• CD13 is a glycoprotein expressed by cells of the myelomonocytic lineage (progenitor cells, neutrophils, basophils, eosinophils, monocytes, and myeloid leukemia cells). It is absent from T and B lymphocytes, erythrocytes, and platelets.
• CD14 is a surface membrane glycoprotein. It is expressed mainly by monocytes and macrophages. CD14 is detected on more than 95% of monocytes in peripheral blood and bone marrow. Strong expression of CD14 is observed in acute myeloblastic leukemia. This antigen is not expressed in acute and chronic lymphoblastic leukemia.
• CD15 is an oligosaccharide. It is involved in phagocytosis and chemotaxis. This antigen is present on the surface of mature granulocytes and Berezovsky-Sternberg cells. Expression of the CD15 antigen is detected in Hodgkin's disease. In non-Hodgkin's lymphomas, CD15 is not detected in most cases.
• CD16 is expressed on the surface of granulocytes, monocytes, macrophages and NK cells. All lymphocytes expressing this antigen have the capacity for antibody-dependent cellular cytotoxicity. CD16 is determined during typing of chronic myelocytic leukemias, to characterize NK cells.
• CD19 is a glycoprotein present on all peripheral B lymphocytes and all B-cell precursors. It is absent from plasma cells. It is the earliest marker of B cells and plays an important role in regulating B-cell activation and proliferation. CD19 is expressed on all neoplastic cells of acute leukemia of B-cell origin and is also present in some forms of acute monoblastic leukemia.
• CD20 is a nonglycosylated protein. In the ontogenesis of B-lymphocytes, the CD20 antigen appears after CD19 at the stage of pre-B-cell differentiation of lymphocytes. It is absent from the plasma membrane of plasma cells. It is expressed in ALL, B-cell chronic lymphocytic leukemia, hairy cell leukemia, Burkitt's lymphoma and very rarely in acute monoblastic leukemia.
• CD21 is a glycoprotein present in significant amounts on B-lymphocytes in lymphoid organs and in small amounts on B-cells in peripheral blood. CD21 is a receptor for the Epstein-Barr virus.
• CD22 is a protein consisting of two polypeptide chains. It is expressed on the membrane of most B lymphocytes, including precursor cells (prolymphocytes). The antigen is not expressed on B lymphocytes (plasma cells) after their activation. The most pronounced expression of CD22 is detected on cells in hairy cell leukemia, weak - in myeloid leukemia and non-T-cell ALL.
• CD23 is a glycoprotein expressed to a much higher extent by activated peripheral blood B lymphocytes. CD23 mediates IgE-dependent cytotoxicity and phagocytosis by macrophages and eosinophils.
• CD25 is a single-chain glycoprotein identified as a low-affinity receptor for IL-2. This receptor is expressed on activated T lymphocytes and, to a lesser density, on activated B cells. In the peripheral blood of healthy individuals, the antigen is present on more than 5% of lymphoid cells.
• CD29 is a fibronectin receptor. It is widely distributed in tissues and is expressed by leukocytes. CD29 detection on peripheral blood cells is used to type a subpopulation of T cells with the CD4+CD29+ phenotype, which are called type 2 helpers (Th2). These cells participate in the humoral immune response by producing lymphokines.
• CD33 is a transmembrane glycoprotein. It is present on the surface of cells of the myeloid and monocytic series. It is found on the surface of monocytes and, to a lesser extent, granulocytes in peripheral blood. Approximately 30% of red bone marrow cells express CD33, including myeloblasts, promyelocytes, and myelocytes. The antigen is absent from the membranes of pluripotent stem cells. CD33 determination is used to characterize cells in leukemias of myeloid origin. Leukemia cells of lymphoid and erythroid origin do not express CD33.
• CD34 is a phosphoglycoprotein expressed by hematopoietic progenitor cells, including monopotent stem cells. The most pronounced expression of Ag is observed in early progenitors; as cells mature, marker expression decreases. CD34 is also found on endothelial cells. CD34 determination is used to characterize cells in acute myeloblastic and lymphoblastic leukemias. In chronic lymphocytic leukemias and lymphomas, CD34 antigen expression is not detected.

• CD41a is expressed by platelets and megakaryocytes. Monoclonal antibodies to detect CD41a are used to diagnose megakaryoblastic leukemia. In Glanzmann's thrombasthenia, expression of this antigen is absent or significantly suppressed.
• CD42b is a membrane glycoprotein consisting of two polypeptide chains. The marker is detected on the surface of platelets and megakaryocytes. In clinical practice, detection of CD42b is used to diagnose thrombocytopathy - Bernard-Soulier syndrome.
• CD45RA belongs to the class of transmembrane glycoproteins. It is a common leukocyte antigen. It is expressed on the cell membrane of B lymphocytes, to a lesser extent T lymphocytes and on mature medullary thymocytes. The marker is not expressed by granulocytes.
• CD45RO is a low-molecular isoform of CD45RA, a common leukocyte antigen. It is detected on T cells (memory T lymphocytes), a subpopulation of B lymphocytes, monocytes, and macrophages. Monoclonal antibodies to CD45RO interact with most thymocytes, a subpopulation of resting CD4+ and CD8+ T lymphocytes, and mature activated T cells. Cells of myelomonocytic origin, granulocytes, and monocytes also carry this antigen. It is detected in centroblastic and immunoblastic lymphomas.
• CD46 is an O-glycosylated dimer. It is widely distributed in tissues and is expressed by T and B lymphocytes, monocytes, granulocytes, NK cells, platelets, endothelial cells, fibroblasts, but is absent from the surface of red blood cells. CD46 provides tissue protection from complement.
• CD61 is a platelet antigen. It is expressed on platelets of peripheral blood and red bone marrow, as well as on megakaryocytes and megakaryoblasts. Its determination is used as a marker in acute megakaryoblastic leukemia. Antigen expression is absent or suppressed in patients with Glanzmann's thrombasthenia.
• CD95, also called Fas or APO-1, is a transmembrane glycoprotein, a member of the tumor necrosis factor receptor family. It is expressed in significant amounts on T lymphocytes (CD4+ and CD8+) in peripheral blood and, to a lesser extent, on B lymphocytes and NK cells. This antigen is also expressed on granulocytes, monocytes, tissue cells, and neoplastic cells. Binding of CD95 to Fas ligand (CD95L) induces apoptosis in cells.
• CD95L, or Fas ligand, is a membrane protein belonging to the tumor necrosis factor receptor family. This antigen is expressed by cytotoxic T lymphocytes, NK cells, and very often tumor cells; it is the main inducer of apoptosis in cells.
• HLA-DR is a monomorphic determinant of class II molecules of the human major histocompatibility complex (HLA). The marker is expressed on Langerhans cells, dendritic cells of lymphoid organs, certain types of macrophages, B lymphocytes, activated T cells and thymic epithelial cells. The study of this marker is used for the quantitative determination of activated T lymphocytes with the CD3+ HLA-DR+ phenotype.

Using a different selection of monoclonal antibodies to markers, it is possible to create a phenotypic portrait of cells characteristic of a given form of leukemia.

In addition to the use of immunophenotyping methods for diagnostics and differential diagnostics of hemoblastoses, their use in the treatment process to assess the state of remission and the residual population of leukemic cells has proven to be especially important. Knowing the phenotypic "portrait" of blast cells during the period of diagnosis, these markers make it possible to detect cells of the leukemic clone during the period of remission, and by the increase in their number - to predict the development of a relapse long before (1-4 months) the appearance of its clinical and morphological signs.

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