Immunophenotyping of hemoblastoses

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

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

Phenotyping allows the typing of blast and mature blood cells of the myelo-, mono-, and lymphocytic series using monoclonal antibodies by the presence of differentiation antigens (receptors) in the cell wall. In the section "Evaluation of the immune status of the organism," the characteristics and diagnostic significance of cell markers are partially described; Below is a brief description of antigenic cell markers applied to the diagnosis of hemoblastosis. On the membranes of blood cells and red bone marrow, the following antigens (markers) can be identified.

• CD2 is a monomeric transmembrane glycoprotein. It is present on the surface of all circulating T-lymphocytes and some NK-lymphocytes. CD2 takes part in the process of alternative activation of T-lymphocytes. Detection of CD2 using monoclonal antibodies in clinical practice is used for phenotyping of acute T-cell leukemias, lymphomas, chronic inflammatory and immunodeficient conditions.
• CD3 - a protein complex associated with an antigen-specific T-cell receptor, is the main functional marker of T-lymphocytes. It facilitates the transfer of activation signal from the membrane to the cytoplasm of the cell. CD3 detection is indicated for the diagnosis of acute T-cell leukemia, lymphoma (CD3 is not expressed in non-T-cell lymphoid tumors) and immunodeficiency diseases.
• CD4 is a transmembrane glycoprotein expressed by a subpopulation of T-helpers (inducers) constituting 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, whose phenotype 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 the cells are simultaneously labeled CD4 and CD8. Slight expression of CD4 is possible on some monocyte cells. CD4 is expressed in most cases by T-cell lymphomas, including mushroom mycosis, as well as HTLV-associated T-cell leukemia (HTLV-human T-lymphotropic virus).
• CD5 - a single-chain glycoprotein, present on all mature T-lymphocytes and most thymocytes, is weakly expressed by B-lymphocytes. CD5 is detected on neoplastic cells of B-cell chronic lymphocytic leukemia and centro- cytic lymphoma. In other types of malignant lymphoid diseases - follicular lymphoma, hairy cell leukemia, large cell lymphoma - CD5 is not expressed.
• CD7 is a single-stranded protein, the earliest marker of T-cell differentiation. It is expressed by pro-T-lymphocytes even before they migrate 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. The definition of CD7 is used for the diagnosis of lymphomas, children's 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 comprise 20-35% of peripheral blood lymphocytes. This antigen also has NK lymphocytes, cortical thymocytes, 30% of medullary thymocytes, and a subpopulation of red bone marrow cells. CD8 is examined for quantitative evaluation of T-suppressor content (see section "T-lymphocytes-suppressors in the blood" above).

• CD10 is the endopeptidase associated with the cell membrane. CD10 expresses young forms of B lymphocytes and a subpopulation of cortical lymphocytes. CD10 express all cells of ALL.
• CD11c express macrophages, monocytes, granulocytes, NK cells and hairy cell leukemia cells on the cell membrane.
• CD13 is a glycoprotein expressed by myelomonocytic cells (progenitor cells, neutrophils, basophils, eosinophils, monocytes and myeloid leukemia cells). It is absent in T and B lymphocytes, erythrocytes and platelets.
• CD14 is a surface membrane glycoprotein. It is expressed mainly by monocytes and macrophages. CD14 is detected in more than 95% monocytes of peripheral blood and bone marrow. Strong expression of CD14 is observed in acute myeloblastic leukemia. In acute and chronic lymphoblastic leukemia, this antigen is not expressed.
• CD15 is an oligosaccharide. He takes part in the processes of phagocytosis and chemotaxis. This antigen is present on the surface of mature granulocytes and Berezovsky-Sternberg cells. CD15 antigen expression 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 ability for antibody-dependent cellular cytotoxicity. CD16 is determined when typing chronic myelocytic leukemias, to characterize NK cells.
• CD19 is a glycoprotein present on all peripheral B lymphocytes, as well as on all B cell precursors. It is absent on plasma cells. This is the earliest marker of B-cells, plays an important role in regulating the activation and proliferation of B-lymphocytes. CD19 is expressed on all neoplastic cells of acute leukemia of B-cell origin, and is also present in some forms of acute monoblast leukemia.
• CD20 is a non-glycosylated 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 on 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 monoblast leukemia.
• CD21 is a glycoprotein, in a significant amount is present on B-lymphocytes in lymphoid organs and in a small amount on B-cells of 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 progenitor 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 with hairy cell leukemia, weak - in myeloid leukemias and non-T-cell ALL.
• CD23 is a glycoprotein expressed by activated B-lymphocytes of peripheral blood to a much greater extent. 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, at a lower density, on activated B-cells. In the peripheral blood of healthy people, the antigen is present in more than 5% of the lymphoid cells.
• CD29 is a fibronectin receptor. It is widely distributed in tissues, it is expressed by leukocytes. CD29 detection on peripheral blood cells is used to typify a subpopulation of T cells having the CD4 + CD29 + phenotype, which are called Type 2 helper (Th2). These cells, through the production of lymphokines, participate in the realization of the humoral immune response.
• 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 of peripheral blood. Approximately 30% of red bone marrow cells express CD33, including myeloblasts, promyelocytes and myelocytes. Antigen is absent on membranes of pluripotent stem cells. CD33 is used to characterize cells in myeloid leukemias. 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 Ar is observed in early progenitors; when the cells mature, the expression of the marker falls. CD34 is also found on endothelial cells. CD34 is used to characterize cells in acute myelogenous and lymphoblastic leukemia. With chronic lymphocytic leukemia and lymphomas, CD34 antigen expression is not detected.

• CD41a is expressed by platelets and megakaryocytes. Monoclonal antibodies for CD41a detection are used to diagnose megakaryoblastic leukemia. With Glązmann thrombasthenia, the expression of this antigen is absent or significantly suppressed.
• CD42b is a membrane glycoprotein consisting of two polypeptide chains. The marker is found 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. This 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 weight isoform of CD45RA - a common leukocyte Ag. They are found on T-cells (memory T-lymphocytes), subpopulations 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 - 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 absent on the surface of red blood cells. CD46 provides tissue protection from complement action.
• CD61 is a platelet antigen. It is expressed on platelets of peripheral blood and red bone marrow, as well as on megakaryocytes and megacaryoblasts. Its definition is used as a marker for acute megakaryoblastic leukemia. Expression of antigen is absent or suppressed in patients with Glanzmann thrombastenia.
• CD95, also called Fas or APO-1, is a transmembrane glycoprotein, a member of the family of receptors of tumor necrosis factor. It is expressed in significant amounts on peripheral blood T-lymphocytes (CD4 + and CD8 +) 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 the Fas ligand (CD95L) induces apoptosis in the cells.
• CD95L, or Fas ligand, a membrane protein belonging to the family of receptors of tumor necrosis factor. This antigen is expressed by cytotoxic T lymphocytes, NK cells and very often by tumor cells; the main inducer of apoptosis in cells.
• HLA-DR is a monomorphic determinant of class II molecules of the main human 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 epithelial cells of the thymus. The test for this marker is used to quantify activated T-lymphocytes with the phenotype CD3 + HLA-DR +.

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

In addition to the use of immunophenotyping for the diagnosis and differential diagnosis of hemoblastoses, it was especially important to use them in the treatment to assess the state of remission and the residual population of leukemia cells. Knowing the phenotypic "portrait" of blast cells at the time of diagnosis, these markers manage to detect cells of the leukemic clone during the period of remission, and as their number increases, it is predicted the development of relapse long before the appearance of its clinical and morphological signs (1-4 months).

[1], [2], [3], [4], [5], [6]