Immunologic studies in urology

Prescribing an immunogram to a urological patient means that the attending physician suspects the presence of disorders in the immune system. Recurring bacterial, viral, fungal infections, allergic manifestations, systemic diseases may 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 manifest itself as a predisposition to infectious and oncological diseases (oncological syndrome). Predisposition to infections may occur against the background of secondary immunodeficiency, which developed as a result of a lymphoproliferative disease, such as leukemia. There are three main groups of pathological changes in the immune system:

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

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

B-cell immunity

Screening methods

• Determination of the relative and absolute number of B-lymphocytes using immunofluorescence or flow cytofluorometry with monoclonal antibodies to B-cell antigens (CD19, CD20, where CD is clusters of differentiation). The normal content of B-lymphocytes in adults is 8-19% of the total number of leukocytes or 190-380 cells/μl. An increase in the content of B-lymphocytes occurs in acute and chronic bacterial and fungal infections, chronic liver diseases, systemic connective tissue diseases, chronic lymphocytic leukemia, and 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 in the same pathological conditions in which an increase in the content of B-lymphocytes occurs. A decrease in the concentration of immunoglobulins occurs in congenital hypogammaglobulinemia, neoplasms of the immune system, removal of the spleen, protein loss, kidney or intestinal diseases, treatment with cytostatics and immunosuppressants.

Clarifying methods

• Determination of circulating immune complexes in the blood by selective precipitation in polyethylene glycol followed by spectrophotometric density testing (normal 80-20 U). An increase in circulating immune complexes is typical for acute bacterial, fungal, viral infections, autoimmune, immune complex diseases, serum sickness, allergic reactions of type 3;
• Determination of specific immunoglobulins in the blood in relation to bacterial and viral antigens, deoxyribonucleic acid (DNA) in autoimmune diseases, detection of antisperm (autoimmune infertility) and antirenal antibodies (pyelonephritis and glomerulonephritis) by the radial immunodiffusion or ELISA method.
• Determination of antisperm antibodies in sperm [MAR test (mixed antiglobulin reaction)], normal - negative result.
• Determination of the concentration of immunoglobulins in urine for the purpose of differential diagnosis between pyelonephritis and glomerulonephritis (selectivity of proteinuria).
• Determination of IgE content in prostate juice for the purpose of diagnosing allergic prostatitis using the radial immunodiffusion method or ELISA.
• Study of the response in the reaction of B-lymphocyte blast transformation to B-cell mitogen (pokeweed mitogen for stimulation of the reaction of B-lymphocyte blast transformation 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 immunofluorescence reaction or flow cytofluorometry using monoclonal anti-CD3 antibodies. The norm for adults is 58-76% or 1100-1700 cells/μl. A decrease in the number of T-lymphocytes is an indicator of 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, injuries, stress, aging, malnutrition, treatment with cytostatics, exposure to ionizing radiation). An increase in the number of T-lymphocytes occurs against the background of immune hyperactivity or in lymphoproliferative diseases. With inflammation, the number of T-lymphocytes first increases and then decreases. The absence of a decrease in T-lymphocytes indicates a chronic inflammatory process.
• Evaluation of lymphocyte subpopulations.
  • Determination of the number of T-helpers (anti-CD4 antibodies). Normally 36-55% or 400-1100 cells/mcl. An increase in the number of these cells occurs in autoimmune diseases, Waldenstrom's disease, activation of antitransplant immunity; a decrease in the number of T-helpers occurs in chronic bacterial, viral, protozoan infections, tuberculosis, acquired immunodeficiency syndrome, malignant tumors, burns, injuries, malnutrition, aging, treatment with cytostatics, exposure to ionizing radiation.
  • Determination of the number of T-suppressors (anti-CD4 antibodies). Normally 17-37% or 300-700 cells/μl. An increase in the number of T-suppressors occurs in the same conditions in which the number of T-helpers decreases, and their decrease occurs in the same conditions in which the content of T-helpers increases.
  • Immunoregulatory index CD4/CD8, normally 1.5-2.5. Hyperactivity with values over 2.5 (allergic and autoimmune diseases); hypoactivity - less than 1.0 (predisposition to chronic infections). At the beginning of the inflammatory process, the immunoregulatory index increases, and when it subsides, it normalizes.

Clarifying 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 during transplant rejection, a decrease - with viral infections, cancer, primary and secondary immunodeficiencies, burns, injuries and stress, treatment with cytostatics and exposure to ionizing radiation.
• Determination of the number of T-lymphocytes with a receptor to 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, a decrease - in the same diseases in which there is a decrease in the number of NK cells.
• Study of expression of activation marker - class II histocompatibility molecule HLA-DR. Increased expression occurs in inflammatory processes, in patients with hepatitis C, celiac disease, syphilis, acute respiratory diseases.
• Evaluation of lymphocyte apoptosis. A rough idea of the readiness of lymphocytes for apoptosis can be determined by the expression of the Fas receptor (CD95) on their surface and the bd-2 proto-oncogene in the mitochondria. Lymphocyte apoptosis is assessed by treating them with two fluorescent dyes: propidium iodide, which binds to DNA fragments, and annexin Y, which binds to phosphatidylserine, which appears on the cell membrane at the onset of apoptosis. The results are evaluated using a flow cytofluorometer. The results are calculated based on the ratio of cells stained with different dyes. Unstained cells are viable, cells bound only to annexin Y are early manifestations of apoptosis, with propidium iodide and annexin Y are late manifestations of apoptosis, staining with only propidium iodide indicates necrosis.
• Evaluation of T-lymphocyte proliferation in vitro.
  • Changes in cell blastogenesis - lymphocyte blast transformation reaction. Leukocytes are incubated with any mitogen of plant origin (lectins). Phytohemagglutinin is most often used for 72 hours, then a smear is taken, stained and the number of blasts is counted! 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 lymphocyte blast transformation reaction can be assessed by the inclusion of a radioactive label (ZN-thymndinum) in the cultured cells, since DNA synthesis increases during cell division. Disturbances in the proliferative response occur in both primary and secondary immunodeficiencies associated with infections, cancer, renal failure, and surgical interventions.
  • In these studies, the expression of activation markers (CD25, transferrin receptor - CD71) and the molecule of the major histocompatibility complex class II HLA-DR, which are practically absent on resting T-lymphocytes, are assessed. T-lymphocytes are stimulated with phytohemagglutinin, after 3 days the expression of activation markers is analyzed by the method of direct or indirect immunofluorescence reaction, flow cytofluorometry, using monoclonal antibodies to the isolated receptors.
  • Measurement of the amount of mediators synthesized by activated T-lymphocytes [interleukin (IL) 2, IL-4, IL-5, IL-6, γ-interferon, etc.] using radioimmunoassay or ELISA. Of particular importance is the assessment of the concentration of γ-interferon and IL-4 as markers of Th1 and Th2 in the supernatant of activated cultures and inside the cell. If possible, it is useful to determine the gene expression for the corresponding cytokine by the level of matrix ribonucleic acid in the producer cell and the intensity of expression of receptors for the corresponding cytokines.
    
• Lymphocyte migration inhibition reaction. Sensitized T-lymphocytes in reaction with antigen secrete lymphokines, including factors inhibiting lymphocyte migration. The inhibition phenomenon is observed when mitogens are introduced into the cell culture. Evaluation of the inhibition degree allows us to judge the ability of lymphocytes to secrete cytokines. Normally, the migration frequency, depending on the specific mitogen, is 20-80%.
• Assessment of NK cell cytotoxicity. The ability of natural killer cells to kill target cells of the K-562 erythromyeloid line is determined. If antibody-dependent cytotoxicity is assessed, 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 assessed by the release of the radioactive label into the solution. A decrease in cytotoxicity occurs in malignant neoplasms. In some cases, when it is necessary to predict the effectiveness of treatment with interleukins, the cytotoxicity of NK cells is assessed during incubation with certain cytokines.

Study of phagocyte function

Screening methods

Study of the intensity of absorption of microbial cells by phagocytes (phagocytosis of latex particles, test culture of staphylococcus, E. coli or microorganisms isolated from the patient). By centrifuging 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 minutes, taking samples for analysis 30.90.120 minutes after the start of incubation. Smears are made from the collected leukocyte suspension. The following phagocytosis indicators are determined:

• phagocytic index - the percentage of cells that entered phagocytosis within 30 min and 120 min of incubation; the standard value of the phagocytic index (30) is 94%, the phagocytic index (120) is 92%;
• phagocytic number - the average number of bacteria located intracellularly; the standard value of the phagocytic number (30) is 11%, the phagocytic number (120) is 9.8%;
• phagocytic number coefficient - the ratio of the phagocytic number (30) to the phagocytic number (120); normally 1.16;
• Neutrophil bactericidal index - the ratio of the number of microbes killed inside phagocytes to the total number of microbes absorbed; normally 66%.

Clarifying methods

• Study of bactericidal ability of phagocytes in the test with nitroblue tetrazolium (NBT) - NBT test. Yellow nitroblue tetrazolium dye is added to leukocytes. When a neutrophil absorbs the dye, a reduction process occurs under the influence of free oxygen radicals, resulting in blue coloration. The reaction is carried out in a 96-well flat-bottomed plate. Hanks' solution (spontaneous NBT) is added to the first three wells with a mixture of NBT and leukocytes, and latex particles are added to the second; the mixture is incubated at 37 C for 25 min. The results are read at 540 nm on a reader and expressed in arbitrary units. The stimulation coefficient (K _st ) is calculated, equal to the ratio of the optical density in the stimulated wells to the average optical density in the wells without stimulation. In healthy people, NBT _spont = 90 ± 45 CU, NBT _stim = 140 ± 60 CU. K _st = 1.78±0.36.
• Adhesion molecule study. Flow cytofluorometry is used to determine the expression of surface antigens CD11a/CD18, CD11b/CD18, CD11c/CD18. Immunodeficiencies with impaired adhesion are manifested by recurrent infections, slow wound healing, and the absence of pus in the foci of infection.

Study of the complement system

Screening methods

Determination of hemolytic activity of complement is a study of the classical pathway of complement activation. Different dilutions of serum from a sick and healthy person are added to ram erythrocytes coated with antibodies. The unit of hemolytic activity is the reciprocal of the serum dilution at which 50% of erythrocytes are destroyed. The degree of hemolysis is estimated photometrically by the release of hemoglobin into the solution. A decrease in hemolytic activity of complement is observed in systemic lupus erythematosus with kidney damage, acute glomerulonephritis, combined immunodeficiencies, myasthenia, viral hepatitis, lymphomas, an increase - in obstructive jaundice, Hashimoto's thyroiditis, rheumatism, rheumatoid arthritis, nodular periarteritis. dermatomyositis, myocardial infarction, ulcerative colitis, Reiter's syndrome, gout.

Clarifying methods

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

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