Non-Specific Parameters of Immune Status
Lymphoid system characteristics
- The total quantity of blood lymphocytes, granulocytes and monocytes is determined by total leukocyte count and leukocyte differential count.
- Lymphocytes make 18-37% of total leukocyte count, monocytes – 3-11%, neutrophils – 47-72%, basophils – 0-1%, eosinophils – 1-5%.
- The total quantity of leukocytes is 4-9*109/l, lymphocytes – about 2*109/l.
- Evaluation of different lymphocyte subsets is performed by determination of their specific cellular markers – membrane CD molecules.
- The major method for identification of subpopulations of immune cells is the indirect fluorescent assay with anti-CD mouse monoclonal antibodies It is carried out as follows: patient’s white blood cell suspension is fixed on the slide.
- Mouse monoclonal antibodies to appropriate CD-Ag are added to the cells. They should interact directly with CD antigens upon the membranes of particular cell subset.
- After incubation and subsequent wash, the slide is treated with a fluorescent-labelled anti-mouse immunoglobulin, which binds to the immune complex Ab-CD Ag.
- Finally, the slide is examined by luminescent microscopy. Cells, bearing specific CD-Ags show bright luminous halo. The part of specific cell subset among all leukocytes is evaluated.
- Likewise, the immune fluorescent technique is applied to automatic cell subpopulation count that also allows automatic cell sorting.
- This technology is named flow cytometry. Fluorescence-activated cell sorter (FACS) is used for automatic cell sorting.
- It analyzes a single-cell suspension flowing along with the capillary unit of sorter crossing several laser beams.
- It measures the light scattering of cells, thereby estimating total cell count, and the relative fluorescence of cell subpopulations, tagged with monoclonal fluorescent antibodies. Cells, bearing specific CD antigens, are detected by luminescence sensors.
- By means of the electrostatic field, the cells passing through the capillary unit and bearing fluorescent label can be separated from the total cell population. This technology is widely used in clinical medicine and biomedical research.
- Using the above-mentioned methods different lymphoid subpopulation can be readily estimated.
- Basic markers of total T cells are CD2 and CD3, T helpers – СD4 (35-50% of total lymphocytes), cytotoxic T cells – СD8 (18-25%).
Th/Tc ratio – immunoregulatory index – is in the range 1.4-2.0. It declines in patients with immunodeficiency (e.g., in AIDS patients it may be less than 0.04) and arises in autoimmune diseases.
B cells markers are CD19-22, CD40, CD72.
The quantity of interleukins and other cytokines produced by different cell types is evaluated by ELISA or radioimmunoassay.
Evaluation of functions of T- and B cell
- Different methods can be used to determine T-cell or B cells functional activities.
- One of the basic methods is the test of lymphocyte blast transformation. It estimates the proliferative response of T- and B cells to mitogens (mitogen-activated blast transformation) and antigens (specific blast transformation).
- For these purposes, lymphocyte culture taken from the examined patient is cultivated in the presence of mitogens (cell mitotic activity stimulators) or antigens. After incubation, the responding cells differentiate into their blast forms, which can be identified by microscopy. Blast cells have a large basophilic nucleus surrounded with a narrow ring of cytoplasm.
- Also, the radiometric count is used, where DNA synthesis in proliferating cells is determined by detection of 3H-thymidine incorporation into replicating cell DNA.
- The example of T cell mitogen is phytohemagglutinin (PHA). Almost 40-70% of lymphocytes respond to PHA. Bacterial lipopolysaccharide (LPS) is the strong mitogen for B cells; 15-25% of B cells respond to it.
Specific antigens, involved in blast transformation reaction, activate populations of antigen-specific T- and B cells that can be detected in similar ways.
Investigation of the complement system
- Serum concentration of complement proteins is determined by ELISA. Serum content of major complement fractions varies significantly (C1s – 0.12 g/l; C4 – 0.43 g/l; C3 – 1.30 g/l; C9 – 0.16 g/l).
- Functional activity of the complement system is estimated by hemolysis reaction. It is expressed in 50% hemolysis units (CH50).
- During infection after the initial increase, complement serum concentration is diminished substantially due to immune complex formation that results in complement consumption.
Determination of immunoglobulin concentration
- Quantitation of major serum and secretory immunoglobulins of G, M and A classes is estimated by ELISA or Mancini single radial immune diffusion test.
- Serum IgE concentration is evaluated by ELISA or RIA taking into account negligible serum IgE content.
Macrophage and granulocyte system assessment
- The total quantity of blood granulocytes and monocytes is determined by leukocyte differential count.
- Different aspects of phagocyte activity could be examined. Bacterial engulfment and phagocyte digestive capacity are evaluated by incubation of leukocyte culture with model microorganisms with subsequent Giemsa stain.
- Phagocytic index and phagocytic number are determined here.
The phagocytic number means an average quantity of ingested bacteria within the single phagocyte (normally 3-8). The phagocytic index is the percentage of phagocytes taking part in phagocytosis (about 70-80% in immune-competent persons).
- Digestion is estimated by inoculation of phagocyte lysates into nutrient media after the processing of native bacteria by leukocytes. If the abundant growth of bacteria has appeared, the phagocytosis is regarded as incomplete and the digestion is impaired.
Respiratory burst in phagocytes is measured by nitroblue tetrazolium reduction test (NBT-test).
- Metabolic activity of phagocytes (e.g., neutrophils) that reflects respiratory burst activation is determined by cytoplasmic oxidative conversion of nitroblue tetrazolium (NBT) dye into insoluble formazan. In normalcy about 15-18% of neutrophils are positive. In case of serious infections this index rises above 40%.
- Indirect immune fluorescent test with monoclonal antibodies is used for phagocyte cell typing. Phagocytes express the vast number of membrane markers; among them are СD14, СD11/СD18, CD16, CD32, CD64, CD35, etc.
- Cytokines, produced by phagocytic cells, are determined by the ELISA test.