ROSETTING TESTS — the laboratory methods of identification and quantitative definition of T-or B-lymphocytes, subpopulations of these lymphocytes and nek-ry other cells based on their ability to fix erythrocytes on the surface. A rosetting lymphocyte, or a rosette-forming cell (FATE), call a lymphocyte (a granulocyte, the macrophage, etc.), to-ry fixes three and more erythrocytes (see. Immune sticking ). The arrangement of erythrocytes on a surface FATE can be various: in one cases they form a nimbus, in others — an incomplete nimbus, sometimes FATE is completely surrounded with erythrocytes — such socket received the name «morula» (fig). Sometimes erythrocytes are located in several layers. When to FATE more than 36 erythrocytes are attached, sockets call huge. The phenomenon of rosetting is most often used for studying of superficial receptors on a membrane lymphocytes (see).
River of t. were entered into practice immunol. and immunochemical laboratories in the late sixties — the beginning of the 70th of 20 century for assessment of an immune response of experimental animals on immunization by xenogenic erythrocytes. Then R. of t. were applied to a research of lymphocytes of blood of the person.
Depending on a way of statement of R. of t. they are divided into two groups: tests of spontaneous rosetting and tests of indirect rosetting. Below they are considered on the example of lymphocytic rosetting.
Tests of spontaneous rosetting
Spontaneous rosetting happens at accession to a surface of lymphocytes of blood of the person of heterological erythrocytes, most often erythrocytes of a ram or mouse. T lymphocytes have on the surface receptors for erythrocytes of a ram (see. Immunocompetent cells ). The formed sockets received the name E-POR. This type of sockets is most investigated. V-lymphocytes have the receptors specific to erythrocytes of mice on the surface. The formed sockets designate I Eat - ROK. This test is less studied and is less applied in a lab. to practice, than test for formation of E-POK.
Test for formation of E-POK. Determine by this way the general population of T lymphocytes. The technique of statement of the test consists in the following: mix a suspension of lymphocytes with 30 — 100-fold quantity of erythrocytes; mix incubate at 37 ° within 5 min., then centrifuge and maintain at 4 °; carefully stir up and count percent of the lymphocytes forming sockets in Goryaev's camera; at the same time sockets can be fixed glutaraldegidy, to do smears on glass, to paint them and to count abundance of E-POK. At the same time count absolute quantity of E-POK in unit volume of the studied sample of blood.
According to many researchers, E-POK can be formed to 70% of all lymphocytes of blood of the person. Decrease in number of E-POK at a number of allergic, autoallergichesky and infectious and allergic diseases is noted that demonstrates oppression of the mechanism cellular immunity (see). However the number of E-POK is not always adequate to a condition of cellular immunity therefore additional methods of a research are required.
Little changes in a technique of statement of R. of t. allow to define not all but only a part of population of T lymphocytes. On the nature of formation of E-POK distinguish the following types of subpopulations of T lymphocytes.
Subpopulation of T lymphocytes with the high connecting activity in relation to erythrocytes of a ram forms so-called active E-POK. This population makes more apprx. 22 — 32% of all T lymphocytes of pi total quantity of the T-cells attaching erythrocytes of a ram to the surface correlates with expressiveness of cellular immunity, than. It is established that such subpopulation of T lymphocytes has no on the surface of a Fc-receptor for IgG it. the lake, obviously, has no suppressor activity. For identification of subpopulation of the T lymphocytes forming active E-POK on test take smaller quantity of erythrocytes and after centrifuging do not expose test to cold.
If after centrifuging a suspension incubate within 30 min. at 37 °, then there is a disintegration of sockets. Usually them there are about 3%. At activation of T lymphocytes at active hepatitis this figure increases, at treatment by glucocorticoid hormones — decreases. Consider that ability to form stable sockets is more characteristic of less differentiated T lymphocytes.
T lymphocytes of healthy people form the insignificant number of huge sockets. Their number increases at R.'s statement t. with the lymphocytes cultivated by in vitro and stimulated phytohemagglutinin.
If to replace erythrocytes of a ram auto-or allogenic erythrocytes of the person, then ability of T lymphocytes to form sockets and with such erythrocytes comes to light. Consider that special subpopulation of T lymphocytes has this ability. The number of these sockets depends on a technique of statement of R. of t. Change of their number in comparison with norm at nek-ry tumors and autoallergichesky diseases is noted.
I Eat the test for education - ROK. The general principle of statement of this test same, as well as at statement of the test about E-POK, distinction consists only that erythrocytes of a mouse are used. Decrease or total absence of formation of sockets at an agammaglobulinemia is noted and increase in their education at hron. lymphoid leukosis. At healthy people about 17% come to light on average I Eat - ROK.
Tests of indirect rosetting
by these tests for surfaces of lymphocytes of the person reveal various receptors not specific in relation to erythrocytes. Therefore attach so-called inserted components to erythrocytes, to-rye can connect to this or that receptor on a surface of a lymphocyte. Erythrocytes in these cases are fixed on a lymphocyte through this inserted component. Therefore such R. of t. called tests of indirect rosetting. Distinguish the following types of indirect rosetting.
Test for formation of Fc-sockets (EA-sockets). On a surface of lymphocytes, granules of otsit, macrophages and nek-ry other cells of the person there are receptors specific to a Fc-fragment of different classes immunoglobulins (see). On a surface both V-lymphocytes, and T lymphocytes they are revealed for IgG, IgM, IgA and IgE, and various subpopulations of T - or V-lymphocytes have Fc-receptors only for certain immunogobulin. There are data that T lymphocytes with a Fc-receptor for IgM — a Tji-cell can perform the T-helperov function (English helper the assistant), promoting proliferation and differentiation of V-lymphocytes in plasmocytes (see), and T lymphocytes with a Fc-receptor for IgG — Tu cell can be the T-suppressors which are slowing down education antibodies (see) against own antigens (see). For identification of the cells bearing Fc-retseitor, erythrocytes of cattle sensibilize rabbit anti-erythrocyte IgG-or IgM — antibodies. The complex an erythrocyte — an antibody, to-ry is usually designated by EA (E — an erythrocyte, And — an antibody), add to lymphocytes and incubate. Live lymphocytes form Fc-sockets (EA-socket). At healthy people according to different researchers, education EA-ro-zetok considerably varies that is explained by lack of the standard test. At nek-ry pathologies, napr, at hron. a lymphoid leukosis (see. Leukoses ), considerable deviations in formation of EA-sockets in comparison with average data on norm come to light.
Test for formation of complementary sockets (EAS-sockets). Lymphocytes of people have on the surface two types of receptors to a complement (C): the immunoconnecting receptors — SZ (a fragment of the third component C) and the S3s1-retsep-tory (a fragment of a receptor of SZ) having specificity to different parts of molecules SZ and S4. Most often both receptors are present at the lymphocytes having the receptor specific to Page. The general principle of definition of this type of lymphocytes is the sensitization of erythrocytes of a ram rabbit antieritro-cytic IgM-antibodies and addition of a complement of the person. The formed EAS complex (E — erythrocytes, And — antibodies, With — a complement) is connected to a suspension of lymphocytes. Sensibilized erythrocytes are fixed on lymphocytes through a complement. Sockets are formed, to-rye received the name «EAC-sockets». There are various modifications of this test with use of erythrocytes of other animal species and the person, and also complement (see) (e.g., a complement of a mouse) that allows to reveal this or that complementary receptor. Consider that EAS-cells are most likely V-limfotsita-mi, however the receptor specific to SZ is available also on T lymphocytes, «zero cells» and monocytes. The quantity of lymphocytes determined by method of EAS-sockets in blood of the person depends on a technique and fluctuates from 15% to 25%.
Test for formation of antigensvyazyvayushchy sockets. By this test reveal the sensibilized lymphocytes connecting antigens of own fabrics. Sockets around sensibilized lymphocytes are formed by the erythrocytes of the person loaded with these or those fabric antigens. At nek-ry collagenoses (see. Collagenic diseases ) increase in number of such sockets is established.
Test for formation of histamine sockets. Apprx. 30% of V-lymphocytes and 10% of T lymphocytes of blood of the person have a receptor, specific concerning a histamine, on the surface. It comes to light on the differentiated T - and V-cells. This receptor can serve in population of T lymphocytes as a marker of the mature cells having suppressor or cytotoxic activity. For identification of the lymphocytes bearing a histamine receptor on erythrocytes fix a histamine, and then these erythrocytes mix with lymphocytes. Sockets are formed, in to-rykh erythrocytes are fixed on a surface of a lymphocyte by means of a histamine.
Test for formation of adenosine sockets. Apprx. 11% of lymphocytes of blood of the person contain the receptors specific to adenosine. For their identification on erythrocytes of cattle fix a complex adenosine — a seralbumin of the person and mix it with lymphocytes. Due to linkng of adenosine with the corresponding receptors on a surface of lymphocytes sockets are formed. This group of lymphocytes belongs hl. obr. to T lymphocytes.
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M. V. Egorova.