RU2273026C1 - Method for diagnosing the cases of hyperparaproteinemia - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves studying blood serum with wedge dehydration. Blood serum drop is first dried on object-plate and the prepared facia is examined with microscope. Anomalous reticular radial structures being observed in central and/or intermediate zones of the facia, moderate hyperparaproteinemia degree is diagnosed. Isolated lumps and/or lumps located on crack periphery in central zones of the facia being observed, highly marked hyperparaproteinemia degree is diagnosed.

EFFECT: simplified accelerated method.

3 dwg, 1 tbl

Description

The invention relates to medicine, namely to laboratory diagnostics, and can be used to diagnose hyperparaproteinemia and its severity in patients with suspected multiple myeloma.

Paraprotein, myeloma proteins, M-gradient are structurally abnormal, functionally inert proteins that do not exist in plasma normally. The appearance of abnormal proteins in the plasma that are absent under normal conditions is called paraproteinemia. M-gradient (paraproteins) are detected with plasmacytoma (myeloma) and Waldenstrom macroglobulinemia, lymphoreticular neoplasias and some malignant epithelial tumors. In addition, in small amounts, these proteins are found in healthy people. The appearance of the M-gradient is due to the production of monoclonal proteins by malignant plasmacytoma cells and atypical lymphocyte cells in Waldenstrom macroglobulinemia. All molecules of this protein have the same characteristics (charge, size, configuration) and appear on the electrophoregram as a narrow clear strip of the marker protein, which is represented by a peak with a narrow base on the densitogram. In 90% of cases, the M-gradient is found in the zone of γ-globulins, in 6-7% in the region of β-globulins, and extremely rarely in the α2-fraction of globulins [4, 5].

Paraproteinemic hemoblastoses (multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease) are tumors of the B-lymphocyte system (cells that perform the functions of humoral immunity). The main feature of hemoblastosis is the preservation of the ability to differentiate to the stage of immunoglobulin-secreting cells. However, secreted immunoglobulins are characterized by uniformity of structure (monoclonal paraprotein PIg), which is explained by their origin from a single tumor cell clone [3].

Multiple myeloma is characterized by tumor proliferation of plasma cells. The disease usually manifests itself in the elderly, cases of the disease under the age of 40 years are rare. The frequency of multiple myeloma is 3 per 100,000 of the population per year, men are sick more often [1, 2, 3].

In the diagnosis of multiple myeloma, three criteria are leading [1, 3]:

1. the presence of more than 10% of plasma cells in the myelogram and / or plasma cell infiltration in the biopsy of the affected tissues;

2. monoclonal paraprotein for immunoelectrophoresis in blood serum, containing either immunoglobulin G more than 35 g / l or immunoglobulin A more than 20 g / l; in urine κ or λ - light chains more than 0.05 g / day; hyperpaproteinemia more than 15%.

3. the presence of osteolytic lesions of the skeleton and / or diffuse osteoporosis.

The diagnosis of multiple myeloma is made with a combination of at least 2 out of 3 of the above symptoms, the presence of the first is mandatory.

Hyperparaproteinemia is associated with increased production of paraprotein, but additional immunological studies are needed to identify monoclonal paraprotein. When electrophoresis of serum proteins in 80% of patients with myeloma is detected M-paraprotein. 10% have hypogammaglobulinemia, and 10% have no changes in the protein spectrum. Immunoelectrophoresis and immunofixation are the most informative methods to clarify the presence of paraproteinemia in more than 90% of cases. Immunoelectrophoresis and immunofixation of urine reveals paraproteinuria in 80% of patients with a ratio of κ / λ = 2/1. Typical symptoms of myeloma are a decrease in the level of normal serum immunoglobulin [5].

Closest to the proposed invention is the study of the protein spectrum of blood serum by electrophoresis and immunoelectrophoresis. Electrophoresis allows you to detect the presence of the M-gradient (paraprotein - a band of monoclonal protein in the migration zone of globulins and a decrease in the fraction outside this zone) and to establish the degree of hyperparaproteinemia. Immunoelectrophoresis allows you to establish the absolute number of immunoglobulins of different classes, including PIg.

For the prototype of the present invention, a method for the diagnosis of hyperparaproteinemia using electrophoresis of serum proteins was selected. For research, take fresh, non-hemolyzed fasting serum. Preliminary determine the content of total protein biuret method. Necessary equipment:

1) power supply

2) electrophoretic chamber with plastic electrodes,

3) foregram analyzer (densitometer),

4) an applicator for applying samples,

5) cellulose acetate membranes (ACM),

6) containers for staining and bleaching phonoplates.

Definition process: ACMs are placed on the surface of the electrode buffer so that they suck in the liquid only from below using capillary forces. After removing from the buffer, the ACM get wet between the sheets of filter paper, preventing drying. The ACM soaked in the buffer is placed in a frame and then in an electrophoresis chamber. Samples using an applicator are applied to the cathode edge. After applying the samples, a current is connected. After 20 minutes, the current is turned off and the ACM is transferred to a coloring solution, then washed until the background is bleached in a solution of ethyl alcohol with acetic acid. Enlightenment of the ACM is carried out on the apparatus FOMK using ethyl alcohol and onion (glacial acetic acid). The dried ACM is scanned on a densitometer at a wavelength of 600 nm. Data on the content of protein fractions in blood serum are presented in relative units (%). Hyperparaproteinemia is judged by a relative amount of paraprotein of more than 10%. The disadvantages of the prototype method are:

1) the need for special expensive equipment,

2) the need for a large number of expensive reagents,

3) the complexity of the analysis,

4) the need for a specially equipped laboratory,

5) long duration of work of qualified personnel (at least 3 hours).

An interesting method is the wedge-shaped dehydration, which makes it possible to make the molecular organization of biological fluids visible by transferring it to the macro level. After the droplet dries under standard conditions on a solid substrate, the amount of salts increases from the periphery to the center, and the amount of organic substances increases from the center to the periphery [8].

Among pathological structures in the blood serum facies, V.N.Shabalin and S.N.Shatokhin [6] distinguished systemic and systemic anomalies. The main systemic disorders in the serum facies are manifested in the form of a loss of symmetry in the location of its main elements, a change in their shape or complete absence. These changes indicate pathological processes in the body, they are not specific to a particular disease, but reflect only the severity of its course. Violations of the subsystem structural engineering can manifest themselves in the form of the following formations:

- structures such as the languages of Arnold and the Sierpinski fields that appear during inflammatory processes of any nature;

- “wrinkles” resulting from increased levels of protein-peptide and protein-amino acid aggregates in the blood;

- leaf type structures that appear with atherosclerotic vascular lesions;

- plaque-like structures indicating an increased content of exo- and endotoxins in the body;

- “silver cracks”, indicating a violation of the elasticity of blood vessels;

 - “tourniquets” in the central zone of the facies (cracks in the form of rings, half rings and waves), indicating hypoxia, primarily of the brain.

A systematic analysis of the structure of the serum facies can be used to assess the state of homeostasis: normal or pathological, stable or not [7]. The authors do not indicate a relationship between specific structures and biochemical parameters of blood serum.

The objective of the invention is to reduce the cost and simplify the diagnosis of hyperparaproteinemia.

The task in the method for the diagnosis of hyperparaproteinemia according to the results of a study of blood serum is achieved by the fact that the blood serum is examined by the method of wedge-shaped dehydration, while a drop of serum is preliminarily dried on a glass slide and then the structure of the obtained facies is analyzed under a microscope, and in the presence of central and / or intermediate zones facies of abnormal mesh radial structures diagnosed with a moderate severity of paraproteinemia, and in the presence of separately lying "clumps" and / or "g ybok "disposed on the edges of cracks in the central zone of the facies diagnosed paraproteinemia high severity.

Necessary equipment:

1) glass slides,

2) pipette microdoser,

3) a light microscope.

The proposed method has the following advantages in comparison with the prototype:

1) simplification (does not require special expensive equipment and reagents),

2) cheaper (the average cost of the study of blood proteins by electrophoresis is 160 rubles, and the method we offer is 50 rubles),

3) acceleration (while working with the sample is on average about 15 minutes).

We examined 15 patients with paraproteinemia, the severity of which is indicated in Table. 1. All patients underwent a comprehensive clinical and laboratory examination. The blood serum of all patients was examined by wedge-shaped dehydration. Using a pipette microdoser, a drop of serum with a volume of 0.01 ml was applied to a clean slide. This drop was dried at room temperature in a closed cabinet. After 18-24 hours, the resulting facies was examined under a microscope in transmitted light.

Table 1 The severity of paraproteinemia Number of observations Low (less than 15%) four Medium (15-30%) 6 High (over 30%) 5

In patients with a low severity of paraproteinemia (less than 15%), multiple sclerosis (1 person) and cancer (3 people) were diagnosed. In patients with moderate and high severity of paraproteinemia, multiple myeloma was diagnosed.

When comparing the morphological picture of blood serum and protein spectrum, the following data were obtained: with a low severity of paraproteinemia (less than 15%), abnormal structures were not detected in the serum facies. Figure 1 presents micrographs (B - increase × 25, C - increase × 100) of the blood serum facies of patient K. with suspected paraneoplastic syndrome (13% paraprotein is a diagnostically insignificant level for the diagnosis of multiple myeloma, A is a phoregram, the arrow shows a strip corresponding to the M-gradient), in which there are no abnormal structures. With an average degree of severity of paraproteinemia (from 15 to 30%) in all cases in the intermediate and / or central zones of the facies, abnormal net radial structures were detected, a description of which is not available in the available literature. Figure 2 presents microphotographs (B - increase × 25, C - increase × 100) of the blood serum facies of patient K. with multiple myeloma (25.6% paraprotein is a diagnostically significant level for multiple myeloma, A is a phoreogram, the arrow shows a strip, corresponding to the M-gradient), abnormal mesh radial structures in the central zone are indicated by arrows. With a high degree of severity of paraproteinemia (more than 30%), in all cases, separately lying “clumps” and / or “clumps” were identified located along the edges of cracks in the central zone of the facies, a description of which is not available in the available literature. Figure 3 presents microphotographs (B - increase × 25, C - increase × 100) of the blood serum facies of patient X. with multiple myeloma (46% paraprotein is a high level, A is a phoregram, the arrow shows a strip corresponding to the M-gradient), abnormal separately lying “clumps” and “clumps” located along the edges of cracks in the central zone of the facies are indicated by arrows.

When examining the morphological picture of the blood serum of 600 patients with various diseases (coronary heart disease, calculous cholecystitis, autoimmune diseases, neurological diseases, tumors of different locations, chronic renal failure), the above abnormal signs were not found.

The proposed method is as follows. Patients take blood from the ulnar vein on an empty stomach, centrifuge it for 10 minutes at 1500 rpm, serum in an amount of 0.01 ml using a pipette microdoser is applied to a clean glass slide and dried at room temperature in a closed cabinet. After 18-24 hours, the resulting facies is examined under a microscope in transmitted light at low magnification. In the presence of abnormal net radial structures in the intermediate and / or central zone of the facies, a conclusion is drawn on the average severity of paraproteinemia (from 15 to 30%). In the presence of abnormal separate “clumps” and / or “clumps” along the edges of cracks in the central zone of the facies, a conclusion is made about the high degree of severity of paraproteinemia (more than 30%).

Necessary equipment: slides, pipette microdoser, microscope with transmitted light. No reagents are required. The method is cheap and simple to execute, does not require special expensive equipment and a specially equipped laboratory.

As an illustration of the claimed diagnostic technology, we give examples confirming the practical application of this technique.

Patient X., 44 years old, has been under the supervision of hematologists since May 2002, when she was first diagnosed with multiple myeloma, diffuse focal form, secreting IgG type λ, paraproteinemic kidney CRF _0-I , steroid diabetes mellitus. The diagnosis was confirmed by histological examination of trepanobioptates of the ilium and x-ray of the skeleton bones (foci of destruction were detected mainly in the flat bones, pathological fractures of the skeleton - compression fractures of the Th 9 and Th 10 vertebral bodies). In the myelogram, the number of plasma cells was 20.8%. Polychemotherapy was repeatedly performed. Arrives January 30, 04 in the hematological department of the Nokol. N.A.Semashko in order to conduct the next course of polychemotherapy (and / b No. 0402701).

According to laboratory tests: a complete blood count - Hb-113 g / l, Er - 3.1 · 10 ¹² / l, CP-1.0, Ret 10%, Tr - 240 · 10 ³ / l, ESR - 56 mm / hour, Le - 3.9 · 10 ⁹ / l, P - 1%, C - 70%, EO - 1%, lymphocytes - 25%, monocytes - 3%; immunogram - IgA less than 0.6 g / l, IgM 0.29 g / l, IgG more than 25.7 g / l; β2 - serum microglobulin - 4.9 mg / ml; fractions of blood proteins - albumin 37%, alpha1 2%, alpha2 6%, beta 8%, paraprotein 46%, gamma 1%, total protein 93 g / l; circulating immune complexes of 30 PIECES / ml; total protein in daily urine of 0.42 g / day. Other biochemical blood counts were within normal limits.

In the study of the serum facies, both separate “clumps” and “clumps” located along the edges of cracks in the central zone of the facies were revealed (Fig. 3).

Patient U., 46 years old, is being monitored at the neurological clinic of the NKB them. N.A.Semashko about multiple sclerosis, cerebrospinal form, remitting type of course. The patient considers himself for 10 years, when after physical exertion appeared weakness in the lower extremities, more on the right. The diagnosis was made in 2000, taking into account the clinical and neurological examination, medical history and results of magnetic resonance imaging of the brain (the presence of foci of demyelination in the periventricular region). Received December 10, 03 in the 2nd neurological department of the Nokol. N.A.Semashko in order to conduct the next course of pulse therapy with glucocorticosteroid drugs in connection with exacerbation (and / b No. 0330703).

According to a laboratory study: a complete blood count - Hb - 132 g / l, Er - 4.42 · 10 ¹² / l, CP - 0.9, ESR - 13 mm / h, Le - 5.4 · 10 ⁹ / l , segmented - 53.1%, lymphocytes - 40.7%, monocytes - 6.2%; immunogram - IgA 2.9 g / l, IgM - 1.75 g / l, IgG - 17.0 g / l; blood protein fractions - albumin 59%, alpha1 2%, alpha2 16%, beta 19%, paraprotein 11%, gamma 10%, total protein 70 g / l; circulating immune complexes of 120 PIECES / ml. Other biochemical blood counts were within normal limits. Plasma cells were not detected during sternal puncture. Immunoelectrophoresis and immunofixation of blood and urine showed no pathological changes. There is no data for multiple myeloma.

When studying the serum facies by the method of wedge-shaped dehydration, the above pathological structures were not detected.

Information sources

1. Alekseev G.A., Andreeva N.E. Myeloma - M.: Medicine, 1966 .-- 246 p.

2. Diagnostic and prognostic value of determination of ceruloplasmin, acetylcholinesterase and total proteolytic activity in the blood serum of patients with multiple myeloma / Bessmeltsev SS, Rybakova LP, Gritskevich NL, Golota GZ .. Blinova MN , Abdulkadyrov K.M. // Questions of oncology. - 1999. - T.45 - No. 4. - S. 405-410.

3. Clinical oncohematology / Under. ed. M.A. Volkova. - M.: Medicine, 2001 .-- 572 p.

4. Clinical use of tumor markers. Critical Assessment / Under. ed. Academician of the Academy of Medical Sciences of the SSR N.N. Trapeznikov. - M.: NPO Soyuzmedinform., 1989. - 76 p.

5. Titov V.N., Amelyushkina V.A. Electrophoresis of serum proteins. M .: OPTIMUM PRESS, 1994 .-- 63 p.

6. Shabalin V.N., Shatokhina S.N. Morphology of human biological fluids. - M .: Chrysostom, 2001 .-- 304 p.

7. Shabalin V.N., Shatokhina S.N., Shabalin V.V. A method for assessing the state of homeostasis. - RF patent No. 2147124, 2000.

8. Shatokhina S.N., Shabalin V.N. Early diagnosis of urolithiasis, determining the degree of its activity and the composition of the stone-forming salts of urine (Cast system) // Urology and Nephrology. - 1998, No. 1. - S. 19-23.

Claims (1)

A method for the diagnosis of hyperparaproteinemia, including the study of blood serum, characterized in that the blood serum is examined by the method of wedge-shaped dehydration, while a drop of serum is preliminarily dried on a glass slide and then the structure of the obtained facies is analyzed under a microscope and, in the presence of abnormal mesh in the central and / or intermediate zones of the facies radial structures diagnose a moderate severity of paraproteinemia, and in the presence of separately lying “blocks” and / or “blocks” located along wells of cracks in the central zone of the facies, diagnose high severity paraproteinemia.

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