IMMUNOLOGY TEXTBOOK - Mercury Podillia 2013

CONGENITAL IMMUNE DEFICIENCY

Evaluation of Cellular Immunity

Analysis of T-lymphocyte surface Antigens. Determining T-lymphocyte surface antigens via flow cytometry allows for the assessment of their maturation, differentiation, and activation.

In vitro T-lymphocyte stimulation. Impairments in T-lymphocyte maturation and differentiation in immunodeficiencies with cellular immune defects occur at various levels. For instance, in severe combined immunodeficiency, thymic maturation of T-lymphocyte is disrupted, manifested by the absence of the CD2 antigen on the T-lymphocyte surface. This condition may also involve the absence of CD3 and CD4, along with an inability of T-lymphocytes to synthesize cytokines. In bare lymphocyte syndrome, MHC Class II antigens are absent from the membrane of activated T-lymphocytes. Wiskott-Aldrich syndrome exhibits decreased expression of the CD43 antigen, which is involved in T-lymphocyte activation. Severe immunodeficiencies with cellular immune defects are accompanied by pronounced impairment of T-lymphocyte function, even though the absolute and relative numbers of these Cells may remain normal.

Lymphocyte blastogenesis assay (lymphocyte transformation test). Under METABOLISM/18.html">The Influence of mitogens, antigens, and allogeneic cells, resting B-lymphocytes become activated, transform into blast cells, and begin to divide. Spontaneous lymphocyte proliferation (blastogenesis) can be elevated in patients who have received multiple Blood transfusions, individuals with allergic and autoimmune disorders, during bacterial and viral infections, and in newborns.

Mixed lymphocyte reaction is used to assess the capacity of T-lymphocytes to recognize HLA antigens on allogeneic B-lymphocytes and monocytes. Stimulating cells (allogeneic B-lymphocytes) are inactivated by irradiation or mitomycin C. The patient's lymphocyte response is evaluated by measuring the incorporation of radiolabeled thymidine into DNA.

Biochemical testing. When combined humoral and cellular immunodeficiency is suspected, the activities of adenosine deaminase and purine nucleoside phosphorylase (involved in nucleoside metabolism) are measured. In ataxia-telangiectasia, serum alpha-fetoprotein levels are almost always elevated, which helps differentiate this condition from other neurological disorders. Rare Metabolic Disorders accompanied by cellular immune deficiency include orotic aciduria and biotin-dependent carboxylase deficiency (manifested by alopecia and neurological symptoms). Transcobalamin II deficiency (involved in Vitamin B12 transport) damages rapidly proliferating Tissues, making this condition characteristic for humoral immune deficiency, impaired hematopoiesis (anemia, thrombocytopenia), diarrhea, and developmental delay.

Genetic testing. Patients with severe cellular immune deficiencies may exhibit chimerism (the presence of cells with different genotypes within a single Organism). This occurs when maternal Blood Cells cross into the Fetal Circulation, following blood component transfusions, or after Bone Marrow transplantation. If a patient's blood contains Cells of the opposite sex, chimerism is readily detected by identifying cells with female and male karyotypes. In other cases, the patient's blood cells undergo HLA typing. This analysis also helps detect the absence of HLA class II antigens on activated T-lymphocytes in bare lymphocyte syndrome.

Scanning Electron Cell/15.html">Microscopy reveals T-lymphocytes lacking microvilli on their surface, a feature characteristic of Wiskott-Aldrich syndrome.

Thymic biopsy is performed in certain cases to confirm the Diagnosis of severe combined immunodeficiency. In cellular immune deficiency, the Thymus shows clusters of reticuloepithelial cells, an absence of Hassall's corpuscles, a loss of distinct corticomedullary demarcation, and a dramatic reduction in thymocyte count.

Lymph node biopsy. In cellular immune deficiency, lymph node biopsy specimens reveal depletion of the paracortical zone. Due to the risk of wound infection and anesthesia-related complications, lymph node biopsies are performed only when other Laboratory tests fail to confirm the diagnosis.

Evaluation of phagocytosis is indicated in chronic and recurrent bacterial infections when humoral and cellular Immunity tests show no abnormalities. Phagocytic deficiency may result from impaired migration, chemotaxis, or adhesion of phagocytes, as well as defects in the phagocytic process itself. Furthermore, phagocyte dysfunction can be caused by opsonin deficiency (Antibodies and Complement) and disturbed phagocytic metabolism.

Nitroblue tetrazolium reduction test is used in the diagnosis of chronic granulomatous disease. THE PRINCIPLE OF the assay is as follows: the yellow dye nitroblue tetrazolium is added to phagocytes; normally, upon its engulfment, phagocytic metabolic activity increases, reducing the nitroblue tetrazolium into blue-colored reaction products. Phagocytic metabolic impairment is indicated by a reduction in blue color intensity. When abnormalities are detected, the levels of cytochrome b558 and other phagocyte Proteins are determined.

Chemiluminescence also allows for the assessment of phagocytic functional activity. Normally, phagocytosis generates a large quantity of free oxygen radicals that oxidize a substrate, such as Introduction/37.html">Bacterial Cell wall components. This oxidation is accompanied by the emission of visible or ultraviolet light. The emission intensity reflects the functional activity of the phagocytes.

Assessment of phagocytic activity is the most informative method for studying opsonins and the functional state of phagocytes. Normally, approximately 95% of Bacteria are engulfed and destroyed by phagocytes within 2 hours. In chronic granulomatous disease, the proportion of destroyed bacteria does not exceed 10%, and viable bacteria are detected within leukocytes. The presence of live bacteria inside leukocytes during incubation with healthy human serum indicates impaired bacterial Digestion without a decrease in the capacity for bacterial engulfment. An elevated content of viable bacteria in the supernatant following incubation with patient serum points to an opsonin deficiency.

Leukocyte chemotaxis. Impaired chemotaxis may be caused by phagocytic defects, the presence of chemotaxis inhibitors, or a deficiency of serum or tissue chemotactic factors.

To investigate leukocyte chemotaxis, the Skin window technique is used. In vitro chemotaxis assays rely on stimulating isolated blood phagocytes with chemotactic factors. The capacity of phagocytes for directed migration can be assessed by placing them in a Boyden chamber or an agarose Petri dish.

Leukocyte adhesion. Impaired leukocyte adhesion is caused by decreased expression or absence of adhesion molecules, such as CD11/CD18, on their surface. Flow cytometry is used to identify adhesion molecules. The absence of CD11/CD18 on neutrophils and monocytes manifests clinically as delayed umbilical cord sloughing, recurrent bacterial infections, and periodontitis.

Diagnosis of asplenia. The Spleen plays a crucial role in defense against infection because it contains a massive number of macrophages and plasma cells. Patients with asplenia frequently experience Sepsis, and blood smears reveal deformed erythrocytes and Howell-Jolly bodies. Asplenia is diagnosed using Ultrasonography.

Other investigations. Assays for myeloperoxidase, Glutathione peroxidase, Lysozyme, glucose-6-phosphate dehydrogenase, and Pyruvate kinase activities, along with electron microscopy, are performed to detect minor phagocytic dysfunctions and for research purposes. In neutropenia, indicated Procedures include repeat leukocyte counts, leukocyte counts following administration of corticosteroids, adrenaline, and endotoxin, testing for antileukocyte antibodies, and bone marrow examination.

Bone marrow examination is performed in cases of persistent leukopenia or leukocytosis, altered leukocyte Morphology, or the appearance of blast cells in the blood.

Prenatal diagnosis and Genetic Counseling. Today, it is established that many immunodeficiencies are hereditary disorders: their inheritance patterns are known, the localization of the defective Gene has been identified, and the gene product has been characterized (Table 49). Carrier detection for defective genes has now become feasible. For instance, heterozygous carriage of a defective gene encoding a specific enzyme can be identified by reduced activity of that enzyme; Examples include decreased adenosine deaminase activity in autosomal recessive severe combined immunodeficiency, Respiratory Chain enzyme deficiencies in chronic granulomatous disease, and Tyrosine kinase deficiency in B-lymphocytes in X-linked agammaglobulinemia. A number of non-enzyme defects have also been identified, such as impaired Synthesis of the interleukin-2 receptor gamma chain in X-linked severe combined immunodeficiency, and abnormal synthesis of The Cell membrane glycoprotein gp39 (the Ligand for the B-lymphocyte CD40 receptor) in hyper-IgM syndrome. In female carriers of X-linked immunodeficiencies characterized by impaired lymphocyte differentiation (X-linked agammaglobulinemia, X-linked severe combined immunodeficiency, Wiskott-Aldrich syndrome), both differentiated and undifferentiated lymphocytes are found in the blood. This occurs because the X chromosome carrying the defective gene is inactivated in only a fraction of the cells. The presence of undifferentiated lymphocytes in the absence of clinical manifestations of these immunodeficiencies indicates carrier status for the defective gene. Restriction fragment length polymorphism analysis also enables the identification of defective gene carriers within a family.

Table 49. Hereditary immunodeficiencies with identified Gene Mutations

Laboratory Methods of prenatal diagnosis are based on the analysis of umbilical cord blood, Amniotic Fluid, and chorionic villi. Specifically, in all forms of severe combined immunodeficiency, T lymphocytes are absent in the cord blood, whereas Wiskott-Aldrich syndrome is characterized by thrombocytopenia and T lymphocytes lacking microvilli. Table 49 shows the inheritance pattern and the applicability of Genetic Methods for diagnosing certain Primary immunodeficiencies.