Tests for Detection of CMI

Tests for Detection of CMI

The CMI can be detected by in vivo and in vitro tests as follows:

◗ CMI in vivo tests

Skin tests are useful to detect delayed hypersensitivity reac-tion to common antigens that come in contact with the body. Purified protein derivative in tuberculin test, dinitrochloro-benzene, or dinitrofluorobenzene are the antigens used for the skin testing.

Most of the normal people respond with delayed type reac-tions to these skin antigens. Absence of reactions to these skin tests suggests impairment of the CMI.

◗ CMI in vitro tests

Many in vitro tests are available for detection of CMI. These are:

a)  Migration inhibition factor (MIF) test: This test isperformed to determine the CMI by making a semiquantita-tive assessment of the migration inhibition of leukocytes. This test depends on the principle that cultured T cells pro-duce macrophage migration inhibition factor on exposure to the antigen to which they are sensitized. In this test, human peripheral leukocytes are incubated in capillary tubes in cul-ture chamber containing culture fluid. The leukocytes, in the absence of antigen, migrate out to the open end of the tube to form a fan-like pattern. In the presence of antigen, the leuko-cytes are prevented from migrating.

b) Lymphocyte blast transformation: A large number ofT cells undergo blast transformation when exposed to certain mitogens, such as the phytohemagglutinin and concavalin. Sensitized T lymphocytes are transformed into large blast cells with great increase in the synthesis of DNA, on exposure to the specific antigen. An increase in DNA synthesis is then measured by incorporation of tritiated thymidine.

c)  Enumeration of T cells, B cells, and subpopulation: 

Fluorescence-activated cell sorter is used to count the number of each type of cells, whether T or B cells. In this method, cells are labeled with monoclonal antibody tagged with a fluores-cent dye, such as fluorescent or rhodamine. The number of cells that fluoresce is registered by passing those single cells through a laser light beam.

The total number of B cells can be counted by using fluorescein-labeled antibodies against all immunoglobulin classes. Specific monoclonal antibodies directed against T-cell marker allows the counting of T cells, CD4 helper cells, CD8 suppressor cells, and other cells. The ratio of CD4 to CD8 in normal person is 1.5 or more but becomes less than one in persons with AIDS.

d)       Rosette formation: Rosette is a lymphocyte to which threeor more sheep erythrocytes are attached. Most T cells form rosettes when mixed with sheep erythrocytes. T-cell rosette is referred to as E-rosette. T cells can be determined by counting E-rosettes. Rosette formation is useful to detect T cells, hence the CMI of the host.