RU2138811C1 - Method for diagnosing myasthenia - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves examining healthy human blood treated with acetyl choline solution and counting the number of rosette-forming cells in a smear before and after its being incubating with inactivated whole blood serum of ill patient at 37-37.5 C during 30-40 min and adding it to acetyl choline-loaded sheep erythrocytes suspension. The number of rosette-forming lymphocytes in smears is counted. The number being 1.5 or more times so large as the background value, myasthenia diagnosis is to be set. EFFECT: simplified method. 3 tbl

Description

 The invention relates to the field of clinical medicine, namely to laboratory diagnosis of myasthenia gravis.

Known in the literature is a method for detecting antibodies to cholinergic receptors (HR) in the blood plasma of patients with myasthenia gravis, based on the ability of antibodies passively transferred to the body of an animal (C ₅₇ mice) to block H - XP and reduce the animal’s mobility in the tail-raising test "(Smirnov V.A., Saykova L.A., Lobzin BC. On two-phase action on mice of immunoglobulins isolated from the blood of a patient with myasthenia. Bulletin of Experimental Biology and Medicine, 1988, No. 11, pp. 549-551).

 The disadvantage of this method is its unsuitability for use in the clinic due to the need for animals, personnel trained in working with animals, vivarium.

 The invention is aimed at solving the problem: simplification of the method, increasing specificity, efficiency, safety.

This task is achieved by studying the interaction of blood serum of a patient with myasthenia gravis with cholinergic receptors of a healthy donor’s blood lymphocyte membrane, previously activated in vitro with acetylcholine (AX), in the reaction of rosette formation specific to acetylcholine. Among blood donor blood lymphocytes, preactivated with AX at a dose of 100 μg in vitro at a temperature of + 37 - 37.5 ^o C for 30-40 minutes, the number of rosette-forming cells specific for AX is studied before and after the patient’s blood serum is inactivated. at a temperature of + 56-57 ^o C for 30-35 minutes, followed by incubation at a temperature of + 37-37.5 ^o C for 30-40 minutes, and after adding sheep erythrocytes sensitized with acetylcholine, the number of rosette-forming lymphocytes is counted in a smear ; with a decrease in their number by 1.5 or more times compared with the background, myasthenia gravis is diagnosed.

The method is as follows: from the vein of the subject, blood is collected in an amount of 1.5-2 ml in a dry clean centrifuge tube, after settling, serum is isolated by centrifugation for 5 minutes at a speed of 1500 rpm, then the serum is inactivated for 30-40 minutes incubating at a temperature of +56 - 57 ^o C.

2.5-3 ml of blood are taken from a donor vein into a centrifuge tube containing 0.8-1.0 ml of a 5% sodium citrate solution, the mixture is stirred. After sedimentation, 0.4 ml of this suspension is added to 2 clean centrifuge tubes (one to determine the initial content of rosettes in the donor blood lymphocyte suspension activated by acetylcholine [healthy person, animal], the second to study the effect on the activated blood cells of the blood serum of the subject ) 3.0 ml of distilled water to add red blood cell lysis is added to each tube, followed by 3.0 ml of a 1.8% sodium chloride solution to restore a pH of 7.2. Centrifuged for 5 minutes at a speed of 1.5 thousand revolutions per minute. Having drawn off the supernatant from all the tubes, leaving 0.2 ml in them, add 1 ml of 199 medium to each tube, shake and leave in the thermostat for 15 minutes at a temperature of + 37 - 37.5 ^o C. Then the suspension of lymphocytes is centrifuged for 5 minutes at a speed of 1.5 thousand revolutions per minute, the supernatant is aspirated with a pipette. Precipitation of the tubes is activated with acetylcholine, for which 0.1 ml of a solution containing 100 μg of acetylcholine is added to the tubes (1 mg of acetylcholine chloride is dissolved in 1 ml of 199 medium). The mixture is shaken and incubated for 30 - 35 minutes at a temperature of + 37 - 37.5 ^o C. After incubation, the cell suspension is centrifuged for 5 minutes at a speed of 1.5 thousand revolutions per minute, the supernatant is aspirated with a pipette. Then washed twice with isotonic sodium chloride solution (pH 7.2) by centrifugation at a speed of 1500 rpm. The first tube is left temporarily unaffected, and the pellet of the second tube is combined with 0.4 ml of the patient’s whole blood test. The mixture is gently shaken and incubated for 30 - 35 minutes at a temperature of +37 - 37.5 ^o C. After incubation with serum, the lymphocytes are washed twice with isotonic sodium chloride solution (pH 7.2) by centrifugation at a speed of 1500 rpm for 5 minutes, the supernatant is aspirated with a pipette. Then, 1 ml of 199 medium is added to both tubes and incubated at a temperature of + 37 - 37.5 ^o C. 15 minutes, then centrifuged for 5 minutes at a speed of 1500 rpm, the supernatant is removed. The precipitate was resuspended in all test tubes in 0.1 ml of 199 medium. 0.1 ml of 0.5% suspension of erythrocyte diagnosticum was added to the obtained leukocyte suspensions, the mixture was incubated at a temperature of + 37-37.5 ^o C for 15 min, centrifuged 5 min at 1500 rpm, incubated for 1 hour in a refrigerator (at a temperature of +4 - +6 ^o C). After incubation in all tubes, pulling off the supernatant and carefully pipetting the precipitate, add 1 drop of 0.5% glutaraldehyde solution, kept at room temperature for 20 minutes. Then, 0.75 ml of distilled water is washed, and the supernatant is drained with a pipette. 0.5 ml of a 50% solution of bovine serum is added to all tubes, the mixture is centrifuged for 5 minutes at 1500 rpm, the supernatant is drained, and smears are made from the precipitate, which are dried in air, fixed with ethyl alcohol for 30 minutes and stained with Romanovsky-Giemsa. They are examined under a microscope at a 900-fold increase in 100 lymphocytes, noting among them the number (in percent) of lymphocytes that have fixed on themselves 3 or more red blood cells (ROC). With a decrease in the number of ROCKs by 1.5 or more times in the second smear, in comparison with the first (background), myasthenia gravis is diagnosed.

To prepare an acetylcholine diagnosticum, sheep erythrocytes treated with glutaraldehyde are used. Fresh sheep erythrocytes are washed three times with isotonic sodium chloride solution (pH 7.2) by centrifuging the suspension for 5 minutes at 1500 rpm. A 5% suspension is prepared from the washed red blood cells, combining 0.5 ml of the erythrocyte sediment and 9.5 ml of a buffered isotonic sodium chloride solution of pH 7.2. During glutarization, a 5% suspension of red blood cells is mixed with a 0.25% freshly prepared solution of glutaraldehyde in a 1: 1 ratio. The mixture is kept at a temperature of + 37 - 37.5 ^o C for 15 minutes, after which the red blood cells are thoroughly washed from glutaraldehyde with a buffered isotonic sodium chloride solution and the volume is adjusted to the original solution with the same solution (5% suspension). Thus prepared red blood cells are stored for 6 months at a temperature of +4 ^o C.

Upon sensitization of erythrocytes with acetylcholine, the glutarized erythrocytes are washed twice with isotonic sodium chloride pH 6.4 and adjusted to the initial volume of suspension with the same suspension (1 ml). Then combined with 10 mg of acetylcholine, previously dissolved in 0.1 ml of an isotonic solution of sodium chloride, pH 6.4. The mixture is gently shaken, then kept in an thermostat for 30-40 minutes at a temperature of +37 - 37.5 ^o C, periodically shaking, and then washed twice with a buffered solution of sodium chloride pH 7.2. Drain the supernatant, bring the volume back to the original (5% suspension). The reaction uses a 0.5% suspension of sensitized red blood cells, which is prepared from the original (5%) suspension by combining 1 ml of it with 9 ml of 199 medium.

 The inventive method investigated the blood serum of 16 patients with a reliable diagnosis of myasthenia gravis, 15 patients with diseases of the peripheral nervous system (comparison group) and 15 healthy individuals (control group). The results of the study are shown in tables 1 to 3.

 Studies have shown that whole blood serum of patients with myasthenia gravis causes a decrease in the ability of donor lymphocytes previously activated with an acetylcholine solution to develop rosette with an acetylcholine diagnosticum. The treatment of the same lymphocytes with the blood serum of healthy individuals (table 2) and patients with diseases of the peripheral nervous system (table 3) did not cause a similar effect and the number of acetylcholine rosettes remained high. The effect of inhibition of rosette formation is associated with the action of antibodies to cholinergic receptors found in the blood serum of patients with myasthenia gravis.

 Examples of specific performance.

 Example 1. Patient B-va A. S., 39 years old, was admitted to the neurology clinic on December 16, 1997 with complaints of increased fatigue after moderate physical exertion, general weakness, choking when swallowing solid food, drooping eyelids in the evening. From the anamnesis of the disease it is known that she’s been sick since December 1989, when doubling, general weakness, fatigue, and trouble swallowing appeared against the background of full health. Symptoms appeared suddenly, within 2-3 days, progressed rapidly and after a week the patient was unable to swallow on her own. The patient was hospitalized in the neurological department where thymoma was detected. On January 18, 1990, a thimectomy was performed; from 1990 to 1992, the patient was in group II disability. Constantly takes Kalimin in a dose of 1-1.5 tab. per day. In an objective study, small bilateral ptosis, swallowing with choking, sometimes liquid food enters the nose, the phonation is satisfactory, the pharyngeal reflex is absent. There are no sensitive disorders, diffuse muscle hypotension and hypotrophy, muscle strength of the extremities 4 points, tendon and periosteal reflexes are reduced, symmetrical. The coordination of movements and the function of the pelvic organs are not impaired. Clinical diagnosis: Myasthenia gravis, generalized form.

The patient was immunologically tested by the claimed method. For this purpose, 2.5 ml of blood was taken from the ulnar vein of the patient. By centrifugation for 5 minutes at a speed of 1500 rpm, serum was isolated, which was incubated at +56 ^° C for 35 minutes in order to inactivate.

3 ml blood was taken from the donor vein into a centrifuge tube containing 0.8-1.0 ml of a 5% sodium citrate solution. The mixture was mixed. After settling, 0.4 ml of this suspension was added to 2 clean centrifuge tubes. Red blood cells were lysed, adding 3.0 ml of distilled water, then 3.0 ml of 1.8% sodium chloride solution, centrifuged for 5 min at a speed of 1.5 thousand rpm. Having drawn off the supernatant from all the tubes, 1 ml of medium 199 was added to each tube, shaken and left in the thermostat for 15 minutes at a temperature of +37 ^o C. Then the suspension of lymphocytes was centrifuged for 5 minutes at a speed of 1.5 thousand revolutions per minute, the supernatant the liquid was removed with a pipette. Then, 0.1 ml of a solution containing 100 μg of acetylcholine was added. The mixture was shaken and kept in a thermostat for 30 minutes at a temperature of +37 ^o C, centrifuged for 5 minutes at 1.5 thousand rpm, the supernatant was removed. Then it was washed twice with isotonic sodium chloride solution (pH 7.2) by centrifugation at a speed of 1.5 thousand rpm. The first tube was left temporarily unaffected, and the sediment of the second tube was combined with 0.4 ml of the patient's blood serum. The mixture was gently shaken and incubated for 30 minutes at a temperature of 37 ^o C. After incubation with serum, the lymphocytes were washed twice with isotonic sodium chloride solution (pH 7.2), the supernatant was removed. Then, 1 ml of 199 medium was added to both tubes and incubated at +37 ^° C. for 15 minutes, then centrifuged for 5 minutes at 1.5 thousand rpm, and the supernatant was removed. The precipitate was resuspended in all three tubes in 0.1 ml of medium 199. 0.1 ml of 0.5 suspension of erythrocyte diagnosticum was added to the obtained suspensions of leukocytes, the mixtures were incubated at a temperature of +37 ^o C for 15 min, centrifuged for 5 min at 1500 rpm min, kept for 1 hour in a refrigerator (at a temperature of +5 ^o C). After incubation in all test tubes, pulling off the supernatant and carefully pipetting the precipitate, 1 drop of 0.5% glutaraldehyde solution was added, kept at room temperature for 20 minutes. Then, 0.75 ml of distilled water was washed, and the supernatant was pipetted off. 0.5 ml of a 50% solution of bovine serum was added to all tubes, the mixture was centrifuged for 5 min at 1500 rpm, the supernatant was drained, and smears were made from the precipitate, which were dried in air, fixed with ethanol for 30 minutes and stained with Romanovsky-Giemsa. Smears were examined under a microscope at 900x magnification.

 The results of the study showed that the blood serum of a patient with myasthenia gravis causes a decrease in the ability of donor lymphocytes, previously activated with an acetylcholine solution, to develop rosette with an acetylcholine diagnosticum. If the initial number of rosette-forming cells in a suspension of donor blood cells was 30%, then after adding blood serum to the patient, only 9%. Thus, the number of rosette-forming cells decreased by more than 1.5 times (3.3 times) compared to the background. Immunological analysis confirmed the clinical diagnosis: Myasthenia gravis.

 Example 2. Patient K-on O. Yu., 34 years old, was admitted to the neurology clinic on 10/14/1997 with complaints of nasal voice growing during the conversation, fatigue of the masticatory muscles when eating, choking with liquid food, fatigue of the muscles of the neck with little physical exertion , general weakness, increasing when walking. From the history of the disease it is known that the first signs of the disease appeared in April 1993 in the form of episodes of a nasal voice when talking and difficulty swallowing solid food, in June 1993 these symptoms became permanent. With these complaints I turned to an ENT doctor, for some time I was treated with a diagnosis of Chr. pharyngitis. A neurologist examined in May 1995, a course of treatment that included corticosteroids (prednisone 50 mg) and AChE drugs gave a good positive effect: an almost complete regression of symptoms. In December 1995, a nasal voice appeared again during conversation and after eating. Symptoms of the disease gradually increased.

 An objective study draws attention to mild hypotrophy of the masticatory muscles, nasal voice, increasing during a conversation. It is difficult to swallow solid food, soft palate hangs, more to the right. Palatine and pharyngeal reflexes are reduced. There are no sensitive disorders, sufficient muscle strength. Tendon and periosteal reflexes are lively, symmetrical. The coordination of movements is not broken. Proserine test is positive. When computed tomography of the thymus gland, thymomas were not detected. Clinical diagnosis: Myasthenia gravis, local cranial form.

The patient conducted a study of blood serum of the claimed method. For this, 2.0 ml of blood was taken from the ulnar vein of the patient. By centrifugation for 5 minutes at 1500 rpm, serum was isolated, which was incubated at +56 ^° C for 30 minutes for inactivation purposes.

2.5 ml blood was taken from the donor vein into a centrifuge tube containing 1.0 ml of 5% sodium citrate solution, the mixture was mixed. After settling, 0.4 ml of this suspension was added to 2 clean centrifuge tubes. 3.0 ml of distilled water was added to each tube for lysing red blood cells, followed by 3.0 ml of a 1.8% sodium chloride solution to restore a pH of 7.2. It was centrifuged for 5 minutes at a speed of 1.5 thousand revolutions per minute. Having drawn off the supernatant from all the tubes, 1 ml of 199 medium was added to each tube, shaken and left in the thermostat for 15 minutes at a temperature of +37.5 ^o C. Then the suspension of lymphocytes was centrifuged for 5 minutes at 1.5 thousand rpm, the supernatant was removed and 0.1 ml of a solution containing 100 μg of acetylcholine was added. The mixture was shaken and kept in a thermostat for 30 minutes at a temperature of +37.5 ^o C. Then the cell suspension was centrifuged for 5 minutes at 1.5 thousand rpm, the supernatant was removed, washed twice with isotonic sodium chloride solution (pH 7.2) by centrifugation at a speed of 1500 rpm. The first tube was left temporarily unaffected, and the pellet of the second tube was combined with 0.4 ml of the patient's blood serum. The mixture was gently shaken and incubated for 35 minutes at a temperature of +37.5 ^o C, then the lymphocytes were washed twice with isotonic sodium chloride solution (pH 7.2) by centrifugation at 1500 rpm for 5 minutes, the supernatant was removed. Then, 1 ml of 199 medium was added to both tubes and incubated at +37.5 ^° C. for 15 minutes, centrifuged for 5 minutes at 1500 rpm, and the supernatant was removed. To the obtained leukocyte suspensions, 0.1 ml of a 0.5% suspension of erythrocyte diagnosticum was added, the mixture was incubated at a temperature of +37.5 ^o C for 15 minutes, centrifuged for 5 minutes at 1500 rpm, kept for 1 hour in a refrigerator (at temperature +5 ^o C). After incubation in all test tubes, pulling off the supernatant and carefully whipping the precipitate, 1 drop of 0.5% glutaraldehyde solution was added, kept at room temperature for 20 minutes. Then, 0.75 ml of distilled water was washed, the supernatant was drained, 0.5 ml of a 50% bovine serum solution was added, the mixture was centrifuged for 5 min at 1500 rpm, the supernatant was drained. Smears were made from the precipitate, which were dried in air, fixed with ethyl alcohol for 30 minutes and stained according to Romanovsky-Giemsa.

 When viewed under a microscope at 900x magnification, 27% of rosette-forming cells were found in the first smear and 8% in the second. Thus, the number of rosette-forming cells in the second smear decreased by more than 1.5 times compared to the background, and an immunological test confirms the clinical diagnosis: Myasthenia gravis.

 Example 3. Patient D., aged 43, was admitted to a neurology clinic with complaints of pain in the lumbar spine radiating along the back-side surface of the right foot to the foot, which intensified in the sitting position, numbness of the side surface of the right foot. Sick for 4 years, exacerbations with a frequency of 1-2 times a year, associated with weight lifting, hypothermia. Last exacerbation 2 weeks before admission to the clinic. An objective examination determines the vertebral syndrome in the form of smoothed lumbar lordosis, lumbar muscle deflection, movement restrictions in the lumbar spine, positive tension symptoms (Lasega on both sides, more on the right), hypotension and hopotrophy of the muscles of the right lower leg, hypesthesia in dermatomas L5-S1 on the right , Achilles reflex decrease on the right. An X-ray examination of the lumbar spine in two projections determined a decrease in the height of the intervertebral discs L4-L5, L5-S1, ladder spondylolisthesis. In computed tomography, posterolateral prolapse of the L5-S intervertebral disc.

 Clinical diagnosis: Osteochondrosis of the lumbar spine, herniation of the intervertebral disc L5-S1, radicular syndrome.

 The patient was immunologically tested by the claimed method. For this purpose, 2.5 ml of blood was taken from the patient's ulnar vein, serum was isolated, followed by its inactivation.

2.0 ml blood was taken from the donor vein into a centrifuge tube containing 0.8 ml of 5% sodium citrate solution, the mixture was mixed. After sedimentation, 0.4 ml of this suspension was added to 2 clean centrifuge tubes, red blood cells were lysed, 1 ml of medium 199 was added to each tube, left in an incubator for 20 min at a temperature of +37 ^o C. Then, the supernatant was removed by centrifugation for 5 min at 1,5 thousand rpm. 0.1 ml of a solution containing 100 μg of acetylcholine was added and kept in an incubator for 35 minutes at a temperature of +37 ^o C. The cell suspension was washed twice with isotonic sodium chloride solution (pH 7.2). The first tube was left temporarily without exposure. The precipitate of the second test tube was combined with 0.4 ml of the patient’s test blood serum, incubated for 30 minutes at a temperature of +37 ^o C, then washed twice with isotonic sodium chloride solution (pH 7.2). 1 ml of 199 medium was added to both tubes and incubated at +37 ^° C. for 20 minutes, centrifuged for 5 minutes at 1500 rpm, the supernatant was removed and 0.1 ml of a 0.5% suspension of erythrocyte diagnosticum was added. The mixture was kept for 20 minutes at a temperature of +37 ^o C and 1 hour in a refrigerator (at a temperature of +4 ^o C). Pulling off the supernatant and carefully whipping the precipitate, 1 drop of a 0.5% solution of glutaraldehyde was added, kept at room temperature for 20 minutes, washing 0.75 ml of distilled water, 0.5 ml of a 50% solution of bovine serum was added. , the mixture was centrifuged for 5 min at 1500 rpm, the supernatant was drained. Smears were made from the sediment, which were dried in air, fixed with ethyl alcohol for 30 minutes and stained according to Romanovsky-Giemsa.

 When immunological blood tests by the claimed method, the number of RACs specific for acetylcholine in the donor's initial blood was 28%, with the addition of patient serum - 24%. An immune response is not characteristic of myasthenia gravis.

 Example 4. V-va I.V., 27 years old, is practically healthy. The inventive method, the study of blood serum. Results: the number of donor-forming donor lymphocytes activated by acetylcholine was 34%, after treatment with the patient's blood serum - 28%. Conclusion: antibodies to cholinergic receptors in the blood not found.

 The advantages of the method are its high specificity and sensitivity, technical simplicity of execution, the need for minimum quantities of available reagents and simple equipment, which allows the method to be carried out in a clinic.

 The method is of great practical importance, as it enables early diagnosis of the disease and its debuts, erased, atypical forms and differential diagnosis.

Claims (1)

A method for diagnosing myasthenia gravis, including blood sampling of a patient with myasthenia gravis and serum excretion, characterized in that they examine the blood of a healthy person treated with a solution of acetylcholine followed by counting rosette-forming cells in a smear before and after incubation with inactivated whole blood serum of the patient at a temperature of 37 - 37.5 ^o C for 30 - 40 min and adding to the suspension of sheep erythrocytes loaded with acetylcholine, the number of rosette-forming lymphocytes is counted in smears and with a decrease in their number by 1.5 and more e compared with background determined gravis.

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