Immunoelectrophoresis of blood proteins

Paraproteins are normally absent in blood serum.

Immunoglobulinopathies, or gammopathies, comprise a large group of pathological conditions characterized by polyclonal or monoclonal hypergammaglobulinemia. Immunoglobulins consist of two heavy (H) chains (molecular weight 50,000) and two light (L) chains (molecular weight 25,000). The chains are linked by disulfide bridges and consist of structures called domains (H - 4, L - 2 domains). Under the action of proteolytic enzymes, Ig is divided into fragments: Fc fragment and Fab fragment. Human Ig heavy chains are represented by five structural variants, which are designated by letters of the Greek alphabet: γ, α, μ, δ, ε. They correspond to 5 Ig classes - G, A, M, D, E. Light chains are represented by two structurally different variants: κ (kappa) and λ (lambda), which correspond to two Ig types of each class. In each Ig molecule, both heavy and both 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 κ and λ molecules within different Ig classes is also not the same. Detection of disturbances in the ratios of Ig or their fragments plays a key role in the diagnosis of monoclonal immunoglobulinopathies.

Monoclonal immunoglobulinopathy (paraproteinemia) is a syndrome manifested in the accumulation of Ig or their fragments homogeneous in all physicochemical and biological parameters in the blood serum and/or urine of patients. Monoclonal Ig (paraproteins, M-proteins) are a product of secretion of one clone of B-lymphocytes (plasma cells), therefore they represent a pool of structurally homogeneous molecules with heavy chains of one class (subclass), light chains of one type and variable regions of the same structure. Monoclonal immunoglobulinopathies are usually divided into benign and malignant. In benign forms of monoclonal gammopathies, the 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.

Classification of monoclonal immunoglobulinopathies

Category of onoclonal gammopathies	Nature of pathology	Concentration of pathological Ig in blood serum, g/l
B-cell malignancies	Multiple myeloma, Waldenstrom's macroglobulinemia	More than 25
	Plasmacytoma (solitary - bone and extramedullary), lymphoma, chronic lymphocytic leukemia, heavy chain disease	Well below 25
B-cell benign	Monoclonal gammopathies of unknown genesis	Below 25
Immunodeficiency states with imbalance of T and B links of the immune system	Primary (Wiskott-Aldrich, DiGeorge, Neselef, severe combined immunodeficiency syndromes)	Below 25
	Secondary (age-related, caused by the use of immunosuppressants, associated with non-lymphoid oncological diseases (for example, colon cancer, breast cancer, prostate cancer, etc.)	Below 2.5
Immunodeficiency states with imbalance of T and B links of the immune system	Restructuring of the immune system after red bone marrow transplantation	Below 25
	Antigenic stimulation in early ontogenesis (intrauterine infection)	Below 25
Homogeneous immune response	Bacterial infections	Below 25
	Autoimmune diseases such as cryoglobulinemia, SLE, rheumatoid arthritis, etc.	Below 25

Immunoelectrophoresis of blood serum proteins allows to detect monoclonal (pathological) IgA, IgM, IgG, H and L chains, paraproteins. During conventional electrophoresis, normal Ig, heterogeneous in properties, are located in the γ zone, forming a plateau or a wide band. Monoclonal Ig, due to their homogeneity, migrate mainly to the γ zone, occasionally to the β zone and even to the α region, where they form a high peak or a clearly delimited band (M-gradient).

Multiple myeloma (Rustitsky-Kahler disease) is the most common paraproteinemic hemoblastosis; it is detected no less frequently 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 generally correlates with the ratio of classes and types of normal Ig in healthy people.

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

To make a diagnosis of multiple myeloma, the following criteria must be met.

Big onescriteria

1. Plasmacytoma based on biopsy results.
2. Plasmacytosis in the red bone marrow (more than 30% of cells).
3. Monoclonal (pathological) Ig peaks on serum protein electrophoresis: more than 35 g/L for IgG peak or more than 20 g/L for IgA peak. Excretion of κ and λ chains in amounts of 1 g/day or more, detected by urine electrophoresis in a patient without amyloidosis.

Smallcriteria

1. Plasmacytosis in red bone marrow 10-30% of cells.
2. The peak PIg in serum is less than the amount indicated above.
3. Lytic bone lesions.
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.

To diagnose multiple myeloma, at least 1 major and 1 minor criterion or 3 minor criteria with the mandatory presence of the criteria listed in points 1 and 2 are required.

To determine the stage of myeloma, the Durie-Salmon standardization system is used, which reflects the volume of tumor damage.

All myeloma groups are divided into subclasses depending on the state of renal function: A - serum creatinine concentration below 2 mg% (176.8 μmol/l), B - more than 2 mg%. In myeloma disease, a high concentration of β ₂ -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 set at 30 °C), anemia, renal failure, hypercalcemia, hypoalbuminemia and a large tumor volume.

Light chain diseases (Bence Jones myeloma) account for approximately 20% of myeloma cases. In Bence Jones myeloma, only free light chains are formed, which are detected in the urine (Bence Jones protein), in the absence of serum pathological Ig (M-gradient).

Stages of Multiple Myeloma

Stage	Criteria	Tumor mass (number of cells), x10 12 /m 2
I	Small myeloma if the following criteria are met: The concentration of hemoglobin in the blood is higher than 100 g/l; The concentration of total calcium in the blood serum is normal (<3 mmol/l); No bone changes on radiography or solitary plasmacytoma of bone; Low concentration of paraproteins in the blood serum (IgG below 50 g/l, IgA below 30 g/l); L-chains (Bence Jones protein) in urine less than 4 g/24 h	<0.6
II	Intermediate myeloma (criteria are between stages I and III)	0.6-1.2
III	Major myeloma with one or more of the following criteria: The concentration of hemoglobin in the blood is below 85 g/l; Total serum calcium concentration is above 12 mg% (3 mmol/L); Extensive skeletal damage or major fractures; High concentration of paraproteins in the blood serum (IgG more than 70 g/l, IgA more than 50 g/l); L-chains (Bence Jones protein) in urine more than 12 g/24 h.	>1,2

Rare immunochemical variants of myeloma disease include non-secretory myeloma, in which paraproteins can be detected only in the cytoplasm of myeloma cells, as well as diclonal myelomas and M-myeloma.

Waldenström's macroglobulinemia is a chronic subleukemic leukemia of B-cell origin, morphologically represented by lymphocytes, plasma cells and all transitional forms of cells 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. An electrophoregram of blood serum proteins reveals an M-gradient in the β- or γ-globulin zone, less often the paraprotein does not migrate in an electric field, remaining in place. Immunochemically, it is PIgM with one type of light chains. The concentration of PIgM in the blood serum in Waldenström's macroglobulinemia ranges from 30 to 79 g / l. Bence Jones protein is found in urine in 55-80% of patients. The concentration of normal Ig in the blood decreases. Renal failure develops infrequently.

Lymphomas. IgM-secreting lymphomas are the most frequently recorded, second place is occupied by paraproteinemic lymphomas secreting IgG, lymphomas with IgA paraproteinemia are detected extremely rarely. A decrease in the concentration of normal Ig (usually to a small extent) in lymphomas is recorded in most patients.

Heavy chain diseases are B-cell lymphomas that produce monoclonal fragments of Ig heavy chains. Heavy chain diseases are very rare. There are 4 types of heavy chain disease: α, γ, μ, δ. Heavy chain disease γ usually occurs in men under 40 years of age and is characterized by enlargement of the liver, spleen, lymph nodes, swelling of the soft palate and tongue, erythema, and fever. Bone destruction usually does not develop. The concentration of pathological globulin in the blood serum is low, and ESR is normal. Lymphoid cells and plasma cells of varying degrees of maturity are found in the bone marrow. The disease progresses rapidly and ends in death within a few months. Heavy chain disease is detected mainly in the elderly and is most often manifested by hepatosplenomegaly. The tumor substrate is lymphoid elements of varying maturity. Isolated cases of δ heavy chain disease have been described, it occurs as myeloma. α heavy chain disease is the most common form, developing mainly in children and people under 30, 85% of cases are registered in the Mediterranean. Immunoelectrophoresis of blood serum and urine is the only method for diagnosing the disease, since the classic M-gradient on the serum protein electropherogram is often absent.

Reactive paraproteinemias occur in the presence of a genetic predisposition in response to bacterial and viral infections (hepatitis, CMV infection) or parasitic invasions (leishmaniasis, toxoplasmosis, schistosomiasis). This form of monoclonal immunoglobulinopathy has been registered in organ transplantation, treatment with cytostatics, hereditary or acquired immunodeficiencies. Transient paraproteinemias are characterized by low serum PIg concentrations, absence or trace amounts of Bence Jones protein in the urine.

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

Idiopathic paraproteinemias occur in elderly people and may represent premyeloma conditions. In such cases, a thorough examination is necessary to identify the initial stage of the disease and long-term dynamic observation.

Signs of benign paraproteinemia include: absence of Bence Jones protein, changes in the concentration of normal Ig, the number of plasma cells in the bone marrow aspirate less than 15%, lymphocytes less than 20%, the concentration of serum paraprotein below 30 g/L.

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