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Immunoelectrophoresis of serum proteins

  • Immunoelectrophoresis of serum proteins

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    Paraproteins in the blood serum are normally absent.

    Immunoglobulinopathies, or gammopathies, combine a large group of pathological conditions characterized by polyclonal or monoclonal hypergammaglobulinemia. Ig consist of two heavy( H) chains( molecular weight 50,000) and two light( L) chains( molecular weight 25,000).Chains are connected by disulfide bridges and consist of structures called domains( H-out of 4, L-of 2 domains).Under the action of proteolytic enzymes, Ig is separated into

    fragments: the Fc fragment and the Fab fragment. Heavy chains of human Ig are represented by five structural variants, which are denoted by the letters of the Greek alphabet. They correspond to 5 classes Ig - G, A, M, D, E. Light chains are represented by two structurally different variants: k( kappa) and X( lambda), which correspond to two Ig types of each class. In each Ig molecule, both heavy and light chains are identical. All people normally have Ig of all classes and both types, but their relative content is not the same. The ratio of molecules to and X within different classes of Ig is also not the same. The detection of a violation of Ig or their fragments plays a crucial role in the diagnosis of monoclonal immunoglobulinopathies.

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    Monoclonal immunoglobulinopathy( paraproteinemia) is a syndrome expressed in the accumulation in the blood serum and / or urine of patients that are homogeneous in terms of all physicochemical and biological Ig parameters or fragments thereof. Monoclonal Ig( paraproteins, M-proteins) is a secretion product of a single clone of B-lymphocytes( plasma cells), therefore they represent a pool of structurally homogeneous molecules having heavy chains of the same class( subclass), light chains of the same type and variable regions of the same structure. Monoclonal immuno-globulinopathies are usually divided into benign and malignant. In benign forms of monoclonal gammapathies, proliferation of plasma cells is controlled( possibly by the immune system) in such a way that clinical symptoms are absent. In malignant forms, uncontrolled proliferation of lymphoid or plasma cells occurs, which determines the clinical picture of the disease. The classification of monoclonal immunoglobulopathy is given in the table.

    Table Classification of monoclonal immunoglobulinopathies

    Table Classification of monoclonal immunoglobulinopathies



    Immunoelectrophoresis of serum proteins allows detection of monoclonal( pathological) IgA, IgM, IgG, chains H and L, paraproteins. With conventional electrophoresis, normal Ig, heterogeneous in properties, are located in the y region, forming a plateau or a wide band. Because of their homogeneity, monoclonal Ig migrate mainly to the zone Y, occasionally to the p region and even to the region a, where they form a high peak or a clearly delineated band( M gradient).

    Multiple myeloma( Rustitzky-Kahler's disease) is the most frequent paraproteinemic hemoblastosis;it is detected no less often than chronic myelo- and lymphocytic leukemia, lymphogranulomatosis and acute leukemia. The class and type of pathological Ig secreted by myeloma determines the immunochemical variant of the disease. The frequency of classes and types of pathological Ig in myeloma as a whole correlates with the ratio of classes and types of normal Ig in healthy people( Table).

    Along with an increase in the content of pathological Ig in the serum of patients with multiple myeloma, normal Ig in a reduced concentration is determined. The content of total protein is sharply increased - up to 100 g / l. The activity of the process with G-myeloma is estimated by the number of plasmocytes in the sternal point, the concentration of creatinine and calcium in the serum( their increase in calcium indicates a progression of the disease).The concentration of M-protein( in the urine it is called the Bens-Jones protein) serves as a criterion for evaluating the progression of the disease in A-myeloma. The concentration of paraproteins in the serum and urine varies during the course of the disease under the influence of therapy.

    For the diagnosis of multiple myeloma, the following criteria are necessary [DeVita V. T. et al., 1989].

    Large criteria

    1. Plasmacytoma based on biopsy results.

    2. Plasmocytosis in the red bone marrow( more than 30% of the cells).

    3. Peaks of monoclonal( pathological) Ig in the electrophoresis of whey protein: more than 35 g / l for the peak of IgG or more than 20 g / l for the peak of IgA.Excretion of k- and lambda chains in the amount of 1 g / day or more, revealed by urine electrophoresis in a patient without amyloidosis.

    Small criteria

    1. Plasmocytosis in the red bone marrow of 10-30% of cells.

    2. Peak PIg in serum in an amount less than that indicated above.

    3. Lethal lesions of bones.

    4. The concentration of normal IgM is below 0.5 g / l, IgA is below 1 g / l or IgG is below 0.6 g / l.

    Table Basic immunochemical variants of multiple myeloma and their characteristics

    Table Basic immunochemical variants of multiple myeloma and their characteristics


    For the diagnosis of multiple myeloma, at least 1 large and 1 small criterion or 3 small ones are required, with the mandatory criteria given in points 1 and 2.

    To determine the stage of myeloma, a standardizing Dury-Salmon system is used, which reflects the volume of tumor damage( Table) [Munker R. et al., 2000].

    All myeloma groups are divided into subclasses depending on the state of kidney function: A - serum creatinine concentration below 2 mg%( 176.8 μmol / l), B - more than 2 mg%.In myeloma, a high concentration of b2-microglobulin in the blood serum( more than 6000 ng / ml) suggests an unfavorable prognosis, as well as high LDH activity( above 300 IU / L, reaction at 30 ° C), anemia, kidney failure, hypercalcemia, hypoalbuminemia and a large tumor volume.

    Diseases of the lung chains( Bence-Jones myeloma) account for approximately 20% of myeloma cases. With Bence-Jones myeloma, exclusively free light chains are formed that are detected in the urine( Bens-John-sa protein), in the absence of serum pathological Ig( M-gradient).

    Table of the Stage of Multiple Myeloma

    Table of the Stage of Multiple Myeloma


    The rare immunochemical variants of myeloma include non-secretory myeloma, in which paraproteins can be found only in the cytoplasm of myeloma cells, as well as diclone myeloma and M-myeloma.

    Macroglobulinemia Waldenström is a chronic subleukemic leukemia of B-cell nature, morphologically represented by lymphocytes, plasmocytes and all transitional cell forms synthesizing PIgM( macroglobulin).The tumor has a low degree of malignancy. In the red bone marrow, proliferation of small basophilic lymphocytes( plasmacytoid lymphocytes) is detected, the number of mast cells is increased. On the electrophoregram of serum proteins, an M gradient is detected in the b- or y-globulin zone, less often the paraprotein does not migrate in the electric field, remaining in place. Immunochemically, he represents PIgM with one type of light chain. The concentration of PIgM in the blood serum with Waldenstrom's macroglobulinemia ranges from 30 to 79 g / l. In 55-80% of patients, Bens-Jones protein is detected in the urine. The concentration of normal Ig in the blood decreases. Renal insufficiency develops infrequently.

    Lymphomas. IgM secreting lymphomas are most often recorded, the second place is occupied by paraproteinemic lymphomas secreting IgG, lymphomas with IgA paraproteinemia are extremely rare. Reduction in the concentration of normal Ig( usually to a small extent) with lim-phom is recorded in most patients.

    Diseases of heavy chains - B-cell lymphatic tumors, accompanied by the production of monoclonal fragments of heavy chains Ig. Diseases of heavy chains are very rare. There are 4 types of heavy chain disease. The disease of heavy chains in men usually occurs in men younger than 40 years, characterized by an increase in the liver, spleen, lymph nodes, edema of the soft palate and tongue, erythema, fever. The destruction of bones, as a rule, does not develop. The concentration of pathological globulin in the blood serum is low, the ESR is normal. Lymphoid cells and plasma cells of different degrees of maturity are found in the bone marrow. The disease proceeds quickly and ends with death within a few months. Disease of heavy chains is detected mainly in the elderly, it is more often manifested by hepatosplenomegaly. Substrate tumor - lymphoid elements of varying degrees of maturity. Single cases of heavy chain disease 8 are described, it proceeds as a myeloma. The disease of heavy chains is the most frequent form, developing mainly in children and persons under 30 years of age, 85% of cases are registered in the Mediterranean. Immunoelectrophoresis of blood serum and urine is the only method of diagnosing the disease, since the classical M-gradient on the electrophoregram of serum proteins is often absent.

    Reactive paraproteinemia occurs when there is a genetic predisposition in response to bacterial and viral infections( hepatitis, CMV infection) or parasitic infestations( leishmaniasis, toxoplasmosis, schistosomiasis).This form of monoclonal immunoglobulinopathy was registered during organ transplantation, treatment with cytostatics, hereditary or acquired immunodeficiencies. Transient paraproteinemias are characterized by low serum PIg concentrations, the absence or trace amounts of the Bence-Jones protein in the urine.

    Associated paraproteinemia accompanies a number of diseases in the pathogenesis of which the immune mechanisms play a role: autoimmune diseases, tumors, chronic infections. These diseases include AL-amyloidosis and cryoglobulinemia.

    Idiopathic paraproteinemia occurs in the elderly and may be premilye. In such cases, a thorough examination is necessary to identify the initial stage of the disease and prolonged dynamic observation.

    Symptoms of benign paraproteinemia include: absence of Bens-Jones protein, changes in normal Ig concentration, plasma cell count in the red bone marrow puncture less than 15%, lymphocytes less than 20%, serum paraprotein concentration below 30 g / l.