Immunological research in urology

Assignment of an immunogram to a urological patient means that the attending physician assumes the presence of disturbances in the immune system. Repeated bacterial, viral, fungal infections, allergic manifestations, systemic diseases can be signs of these disorders, which are characterized by a number of syndromes (infectious, oncological, allergic, autoimmune, lymphoproliferative). One patient may have several syndromes. For example, chronic infectious diseases (infectious syndrome) can cause immunodeficiency, and immunodeficiency can be manifested by a predisposition to infectious and oncological diseases (oncological syndrome). Predisposition to infections can occur against a background of secondary immunodeficiency, which has developed as a result of a lymphoproliferative disease, for example, leukemia. There are three main groups of pathological changes in the immune system:

• the quantitative or functional insufficiency of one or another link of immunity, which leads to the development of an immunodeficiency state;
• a violation in the recognition of the antigen by the immune system, leading to the development of autoimmune processes;
• hyperreactive or "perverted" immune response, manifested by the development of allergic diseases.

There are screening (tests of level 1) and qualifying (tests of the 2 level) methods of immunodiagnostics. The former exist to fix violations in the immune system, the latter - to establish the mechanisms involved in their implementation for the purpose of further immunocorrection.

B-Cellular link of immunity

Screening methods

• Determination of the relative and absolute number of B lymphocytes by means of an immunofluorescence reaction or flow cytofluorimetry using monoclonal antibodies to B cell antigens (CD19, CD20, where CD clusters are differentiated). The B-lymphocyte content is normal in adults: 8-19% of the total number of leukocytes or 190-380 cells / μl. An increase in B-lymphocyte levels occurs with acute and chronic bacterial and fungal infections, chronic liver diseases, systemic connective tissue diseases, chronic lymphocytic leukemia, myeloma.
• Determination of the concentration of non-specific immunoglobulins (F, M, G, E) by simple radial immunodiffusion, nephelometry or turbometry, radioimmunoassay or enzyme immunoassay (ELISA). Norms for adults: immunoglobulin (Ig) A 0.9-4.5 g / l. IgM 03-3.7 g / l. IgG 8.0-17 g / l. An increase in the concentration of immunoglobulins occurs with the same pathological conditions in which an increase in the content of B lymphocytes occurs. Reducing the concentration of immunoglobulins occurs with congenital hypogammaglobulinemia, neoplasms of the immune system, removal of the spleen, loss of protein, with kidney or intestinal diseases, treatment with cytostatics and immunodepressionants.

Specifying methods

• Determination of circulating immune complexes in the blood by selective precipitation in polysilene glycol followed by spectrophotometric density testing (normal 80-20 UE). Increase of circulating immune complexes is characteristic for acute bacterial, fungal, viral infections, autoimmune, immunocomplex diseases, serum sickness, allergic reactions of type 3;
• Determination of specific immunoglobulins in blood with respect to bacterial and viral antigens, deoxyribonucleic acid (DNA) in autoimmune diseases, detection of antispermal (autoimmune infertility) and contraceptive antibodies (pyelonephritis and glomerulonephritis) by radial immunodiffusion or ELISA.
• Determination of antisperm antibodies in the sperm [MAR-test (mixed antiglobulin reaction)], the norm is negative.
• Determination of the concentration of immunoglobulins in urine for the purpose of differential diagnosis between pyelonephritis and glomerulonephritis (selectivity of proteinuria).
• Determination of IgE in the prostate juice for the diagnosis of allergic prostatitis by radial immunodiffusion or ELISA. 
• Study of the response in the reaction of B-lymphocyte blast transformation to B-cell mitogen (mitogen of the lakonos for stimulating the reaction of the blast-transformation of B-lymphocytes in the presence of T-lymphocytes), the normative value of which is 95-100%.

T-cell link of immunity

Screening methods

• Determination of the relative and absolute number of mature CD3 T-lymphocytes by the method of the immunofluorescence reaction or flow cytofluorometry using monoclonal anti-CDS antibodies. The norm for adults is 58-76% or 1100-1700 cells / μl. Decrease in the number of T-lymphocytes is an indicator of the insufficiency of the cellular link of immunity. This is typical for some secondary and primary immunodeficiencies (chronic bacterial and viral infections: tuberculosis, acquired immunodeficiency syndrome, malignant tumors, chronic renal failure, trauma, stress, aging, malnutrition, cytostatics, ionizing radiation). An increase in the number of T-lymphocytes occurs against a background of hyperactivity of immunity or in lymphoproliferative diseases. With inflammation, the number of T-lymphocytes first increases, and then decreases. Absence of a decrease in T-lymphocytes suggests chronicization of the inflammatory process.
• Assessment of the subpopulations of lymphocytes.
  • Determination of the number of T-helpers (anti-CD4 antibody). Normally, 36-55% or 400-1100 cells / μl. An increase in the number of these cells occurs in autoimmune diseases, Waldenstrom's disease, activation of antigraft implantation; a decrease in the number of T-helpers occurs with chronic bacterial, viral, protozoal infections, tuberculosis, acquired immunodeficiency syndrome, malignant tumors, burns, trauma, malnutrition, aging, treatment with cytostatics, and the effects of ionizing radiation.
  • Determination of the number of T-suppressors (anti-CD4 antibody). Normally, 17-37% or 300-700 cells / μl. An increase in the number of T-suppressors occurs with the same conditions in which the number of T-helpers decreases, and their decrease at the same conditions, when the content of T-helpers rises.
  • Immunoregulatory index CD4 / CD8, in the norm 1,5-2,5. Hyperactivity at rates more than 2.5 (allergic and autoimmune diseases); hypoactivity - less than 1.0 (predisposition to chronic infections). At the onset of the inflammatory process, the immunoregulatory index rises, and when it subsides, it normalizes.

Specifying methods

• Determination of the number of natural killers (NK cells) - anti-CD16 and anti-CD56-antibodies. The norm for CD 16-lymphocytes is 6-26%, CD56 - 9-19%. An increase in the number of NK cells occurs with rejection of the graft, a decrease in viral infections, oncological diseases, primary and secondary immunodeficiencies, burns, trauma and stress, treatment with cytostatics and exposure to ionizing radiation. 
• Determination of the number of T-lymphocytes with the receptor for interleukin-2 (activation marker) - anti-CD25 antibodies. The norm is 10-15%. An increase in their number is observed in allergic diseases, transplant rejection, response to thymus-dependent antigens in the acute period of primary infection, decrease in the same diseases in which the number of NK cells decreases.
• Examination of the expression of the activation marker - the histocompatibility molecule of class II HLA-DR. Increased expression occurs in inflammatory processes, in patients with hepatitis C, celiac disease, syphilis, acute respiratory infections.
• Evaluation of apoptosis of lymphocytes. An approximate idea of the readiness of lymphocytes for apoptosis can be determined by the expression on their surface of the Fas receptor (CD95) and in mitochondria of the proto-oncogene bd-2. Lymphocyte apoptosis is assessed by treatment with two fluorescent dyes: iodide propidium, which binds to DNA fragments, and annexin Y, binding to phosphatidylserine appearing on the cell membrane at the onset of apoptosis. Evaluation of the results is carried out on a flow cytofluorimeter. The calculation of results is based on the ratio of cells stained with various dyes. Unpainted cells are viable, cells associated only with annexin Y - early manifestations of apoptosis, with propidium iodide and annexin Y - late manifestations of apoptosis, staining only propidium iodide indicates necrosis.
• Evaluation of the proliferation of T-lymphocytes in vitro.
  • Change in cell blastogenesis is a reaction of the blast-transformation of lymphocytes. Leukocytes are incubated with any mitogen of plant origin (lectins). More commonly used phytohemagglutinin for 72 hours, then make a smear, stain it and count the number of blasts! The stimulation index is the ratio of the percentage of transformed cells in the experiment (culture with phytohemagglutinin) to the percentage of transformed cells in the control (culture without phytohemagglutinin). The reaction of the blast-transformation of lymphocytes can be assessed by the inclusion of a radioactive label (ZN-thymdin) in cultured cells, as DNA synthesis increases when dividing cells. Disturbances in the proliferative response occur both in primary and secondary immunodeficiencies associated with infections, oncological diseases, renal insufficiency, and surgical interventions.
  • Evaluation of the expression of activation markers (CD25, receptor for transferrin - CD71) and molecules of the main histocompatibility complex of class II HLA-DR, which are practically absent on resting T lymphocytes, in these studies. T-lymphocytes are stimulated with phytohemagglutinin; after 3 days, the expression of activation markers is analyzed by direct or indirect immunofluorescence reaction, flow cytometry, using monoclonal antibodies to secreted receptors.
  • Measurement of the number of mediators synthesized by activated T-lymphocytes [interleukin (IL) 2, IL-4, IL-5, IL-6, γ-interferon, etc.], by radioimmunoassay or ELISA. Especially important is the evaluation of the concentration of y-interferon and IL-4 as markers of TH and Th2 in the supernatant of activated cultures and within the cell. If possible, it is useful to determine the expression of the gene for the corresponding cytokine by the level of the matrix ribonucleic acid in the producing cell and the intensity of expression of the receptors for the corresponding cytokines.
    
• The reaction of inhibition of migration of lymphocytes. Sensitized T-lymphocytes in the reaction with the antigen release lymphokines, including factors. Inhibiting the migration of lymphocytes. The phenomenon of inhibition is observed when mitogens are introduced into the culture of cells. Evaluation of the degree of inhibition makes it possible to judge the ability of lymphocytes to release cytokines. Normally, the frequency of migration, depending on the specific mitogen, is 20-80%.
• Evaluation of cytotoxicity of NK cells. Determine the ability of natural killer cells to kill target cells of the erythromyeloid line K-562. If antibody-dependent cytotoxicity is evaluated, target cells coated with IgG antibodies are used. Target cells are labeled with 3H-uridine and incubated with effector cells. The death of target cells is estimated from the release of the radioactive label into the solution. Reduction of cytotoxicity occurs with malignant neoplasms. In some cases, when a prognosis of the effectiveness of treatment with interleukins is required, the cytotoxicity of NK cells is evaluated when incubated with certain cytokines.

Investigation of the function of phagocytes

Screening methods

Investigation of the absorption of microbial cells by phagocytes (phagocytosis of latex particles, test culture of staphylococcus, Escherichia coli, or microorganisms isolated from the patient). By centrifuging the heparinized blood, a suspension of leukocytes is isolated, serum of the IV blood group is added for opsonization (opsonins are proteins that enhance phagocytosis). The microbial suspension is diluted, mixed with leukocytes and incubated for 120 min, sampling for analysis after 30.90.120 min after the start of incubation. Of the selected leukocyte suspensions make smears. Determine the following indicators of phagocytosis:

• phagocytic index - the percentage of cells that entered phagocytosis in 30 min and 120 min incubation; normative value of the phagocytic index (30) 94% of the phagocytic index (120) - 92%;
• phagocytic number - the average number of bacteria that are intracellular; the normative value of phagocytic number (30) 11%, phagocytic number (120) - 9.8%;  
• coefficient of phagocytic number - ratio of phagocytic number (30) to phagocytic number (120); normal 1.16;
• the bactericidal index of neutrophils is the ratio of the number of microbes killed inside the phagocytes to the total number of microbes taken up; in the norm of 66%.

Specifying methods

• Investigation of the bactericidal activity of phagocytes in the test with nitrosine tetrazolium (NST) is the NST test. To the leukocytes, dye of nitrous tetrazolium yellow is added. When the dye is absorbed by the neutrophil, the process of reduction takes place under the action of free radicals of oxygen, resulting in a blue staining. The reaction is carried out in a 96-well flat-bottomed plate. In the first three wells with a mixture of HCT and leukocytes, Hanks solution (spontaneous HCT) is added, in the latter - latex particles; incubate at 37 ° C for 25 minutes. The results are read at 540 nm on the reader and expressed in conventional units. Calculate the stimulation factor (K _st ), equal to the ratio of optical density in the stimulated wells to the average optical density in the wells without stimulation. In healthy people, _{HCT spont} = 90 ± 45 UE, NST _Stim = 140 ± 60 UE. K _st = 1.78 ± 0.36.
• Investigation of adhesion molecules. With the help of flow cytofluorimetry, the expression of surface antigens CD11a / CD18, CD11b / CD18, CD11c / CD18 is determined. Immunodeficiencies with violation of adhesion are manifested by relapsing infections, slow healing of wounds and absence of pus in the foci of infection.

Investigation of the complement system

Screening methods

Determination of hemolytic activity of complement - a study of the classical pathway of complement activation. Different dilutions of the serum of the patient and a healthy person are added to the erythrocytes of a ram covered with antibodies. A unit of hemolytic activity is taken as the inverse of the dilution of serum, in which 50% of the erythrocytes are destroyed. The degree of hemolysis is evaluated photometrically by the yield of hemoglobin in the solution. Reduction of hemolytic activity of complement is observed in systemic lupus erythematosus with renal involvement, acute glomerulonephritis. Combined immunodeficiencies, myasthenia gravis, viral hepatitis, lymphomas, increase - with obstructive jaundice, thyroiditis Hashimoto. Rheumatism, rheumatoid arthritis, nodular periarteritis. Dermatomyositis, myocardial infarction, ulcerative colitis, Reiter's syndrome, gout.

Specifying methods

• Determination of complement components. The quantitative determination is carried out by the method of radial immunodiffusion and nephelometry.
  The study is not informative, unless the antigenic properties of complement components are changed.
• It was found that the Clq-component of complement enhances phagocytosis and mediates cellular cytotoxicity. Its decrease occurs in diseases of immune complexes, systemic lupus erythematosus, purulent infections and tumors.
• The C3-component participates in the activation of the classical and alternative complement pathway. Reduction of its concentration is associated with chronic bacterial and fungal infections, the presence of circulating or tissue immune complexes.
• The C4 component participates in the activation of the classical pathway. Reduction of its concentration is associated with a prolonged activation of complement with immune complexes and a decrease in the concentration of the C1 inhibitor, which controls the activation of the classical complement pathway. C4 deficiency occurs with systemic lupus erythematosus, an increase in C4 occurs with kidney disease, transplant rejection, acute inflammation, and diseases of the gastrointestinal tract.
• C5a is a small fragment of the C5 molecule, split off from it as a result of activation of the complement system. Increase in its concentration occurs with inflammation, sepsis, atopic and allergic diseases.
• Cl-inhibitor is a multifunctional factor. It controls the activation of the C1 component of complement, inhibits the activity of kallikrein, plasmin and activated factor Hageman, proteases Cls and Or. Deficiency of C1-inhibitor leads to angioedema.
• complementary studies. To the standard serum lacking any complement component, the test serum is added and the hemolytic activity of the complement is determined. If the hemolytic activity is not restored to normal, it is believed that the activity of this complement component in the test serum is reduced.

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