RU2128840C1 - Method for diagnosing the cases of multiple sclerosis - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves combining patient blood lymphocytes taken before and 30 min later after making contact with serotonin solution, sheep erythrocytes treated with serotonin. Patient blood lymphocytes are mixed with serotonin solution in vitro selecting 1:10 mcg/ml, 1: 100 mcg/ml, 1: 1000 mcg/ml dilution proportions and then sheep erythrocytes treated with serotonin are added. The suspension is hold at 37 C - 37.5 C during 15-20 min and at +2 C - +4 C during 30-40 min. Then the number of serotonin-specific rosette-forming complexes is counted. No changes in number being detected before and after adding serotonin, the disease is diagnosed. EFFECT: enhanced accuracy of diagnosis. 2 tbl

Description

 The invention relates to medicine, namely to neurology and can be used for laboratory diagnosis of multiple sclerosis.

 A known method for the diagnosis of multiple sclerosis / patent N 2007714, published 02.15.94. Bull.N3 /. The method consists in studying the response of a patient's blood lymphocytes to a 0.1% solution of serotonin administered subcutaneously in a plaque formation reaction.

 Disadvantages: the method is complicated in formulation, it requires expensive reagents / Difco agar, ficol-verographin, dry complement, chromium chloride /. In addition, subcutaneous administration of a solution of serotonin is associated with the risk of an allergic reaction and infection in the patient.

 The invention is aimed at solving the problem: safety and simplification of the method with high accuracy and specificity.

 The essence of the proposed method lies in the fact that in vitro the response of the patient’s blood lymphocytes to serotonin introduced into the suspension of his blood cells is studied, followed by determination of the number of rosette-forming / ROCK / cells specific to serotonin. And in the absence of changes in their number before and after the introduction of serotonin, multiple sclerosis is diagnosed (see Tables 1 and 2).

 The method is as follows: 3 ml of the patient’s blood is taken into a 5% solution of sodium citrate, then it is left to stand at room temperature for 3 hours. After settling, 0.4 ml of the leukocyte suspension is collected in 4 tubes with an automatic pipette. For the purpose of hemolysis of erythrocytes, add 3 ml of distilled water to the suspension of leukocytes, shake for 30 s and add 3 ml of 1.8% sodium chloride solution. The mixture is centrifuged for 5 minutes at 1,500 rpm. The supernatant is removed with a pipette.

 In one tube with a leukocyte suspension add 1 ml pit. environment 199 to determine the initial number of ROCK.

Dilutions of serotonin 1: 10, 1: 100 and 1: 1000 μg / ml in a volume of 0.2 ml are successively added to 2, 3, 4 tubes. The mixture is incubated in an incubator at 37-37.5 ^o C for 30 minutes After incubation, add 1 ml of a pH 7.2 buffer solution, shake for 30 s, centrifuge for 5 min at 1,500 rpm to remove excess serotonin. Delay the supernatant, add 1 ml pit. 199 medium, shaken for 30 s and kept at room temperature for 20 min. Centrifuged at 1,500 rpm.

Delay the supernatant, add 0.1 ml pit. medium 199, shaken for 30 s and add 0.1 ml of serotonin diagnosticum to the original volume of 0.2 ml of suspension. After adding diagnosticum, the mixture is incubated for 15-20 minutes at 37-37.5 ^o C and 30-40 minutes at 2-4 ^o C.

 After incubation in the refrigerator, the supernatant is drained, the precipitate is gently whipped, 0.01 ml of 0.5% glutaraldehyde solution is added. Stand for 20 minutes at room temperature. Then 0.75 ml of distilled water is added, centrifuged at 1.500 rpm for 5 minutes. Strain the supernatant, add 0.5 ml of bovine serum. Centrifuge at 1,500 rpm for 5 minutes. The supernatant is pipetted, the precipitate is carefully beaten, pipetting it. Smears are prepared from it, dried in air, and fixed in ethanol for 30 minutes. Stain smears according to Romanovsky-Giemsa. Count under a microscope with a 900-fold increase in the number of ROCK among 100 medium lymphocytes.

 A lymphocyte is considered rosette-forming, fixing on its membrane 3 or more mutton erythrocytes sensitized with serotonin. Multiple sclerosis is diagnosed by the absence of changes in the number of ROC specific for serotonin before and 30 minutes after the introduction of serotonin.

Preparation of serotonin diagnosticum:
On a torsion balance, 0.1 mg of dry serotonin is weighed. To the dry matter add 5 ml of a buffer solution of pH 6.4. Take 0.1 ml of a suspension of glutar erythrocytes, centrifuged for 5 min at 1,500 rpm. The supernatant is drawn off, 0.5 ml of pH 6.4 buffer solution is added again, centrifuged for 5 minutes at 1,500 rpm. The supernatant is drawn off, 0.1 ml of serotonin solution is added to the precipitate. The mixture is incubated in an incubator at 3 ^° C for 30 minutes, periodically shaking at intervals of 10 minutes. Centrifuge for 5 minutes at 1,500 rpm. Strain the supernatant, add 0.5 ml of pH 7.2 buffer solution to the precipitate, centrifuge for 5 minutes at 1,500 rpm. Strain the supernatant, add 0.1 ml of a buffer solution of pH 7.2 and 0.9 ml of pit. Wednesday 199.

 Method of glutarization of sheep erythrocytes.

1 ml of sheep erythrocytes is diluted with 9 ml of 0.9% physical. solution, shake and centrifuged for 10 min at 1,500 rpm The supernatant is removed with a syringe with a long needle, 0.9% nat. 9 ml solution and centrifuged for 10 minutes at 1,500 rpm. Thus, washed sheep red blood cells 3 times. A 5% suspension is prepared: 0.5 ml of a erythrocyte sediment with a solution of 0.25% (glutaraldehyde 1: 1 and kept at 3 ^° C in an thermostat for 3 hours and a day in a refrigerator. Store for up to 6 months.

 Example 1. Patient R., 24, was admitted to the neurology clinic 8.01.96, with complaints of instability when walking, weakness in the arms and legs, dizziness, imperative urination, trembling head, fuzzy speech. In neurological status: spastic tetraparesis with dysfunction of the pelvic organs of the central type and cerebellar syndrome, represented by dysarthria, horizontal nystagmus, static and dynamic ataxia, tremulous hyperkinesis. From the anamnesis it is known that he is sick for a year, the course of the disease is steadily progressing. The clinical diagnosis was established: Multiple sclerosis, cerebrospinal form, hyperkinetic variant, progressive course.

 The patient underwent immunological blood tests. For this purpose, a blood sample was taken, 0.4 ml of a suspension of leukocytes was taken, erythrocyte hemolysis was performed, then 1 ml of pit was added to 1 tube. Wednesday 199.

To determine the initial number of ROCK, in 2, 3, 4, dilutions of serotonin 1:10, 1: 100 and 1: 1000 μg / ml were added sequentially. After incubation at 37 ^° C, a suspension of leukocytes was washed to remove excess serotonin. Then, 0.1 ml of serotonin diagnosticum was added to a suspension of leukocytes, incubated at 37 ^° C for 15 minutes and at 2 ^° C for 30 minutes. The rosettes were fixed with 0.01 ml of 0.5% glutaraldehyde solution for 20 minutes, then the suspension was washed with distilled water, smears were prepared and rosette-forming lymphocytes were recorded under the microscope, fixing 3 or more ram red blood cells sensitized with serotonin on their surface,
During the study, the patient found: the content of serotonin rosettes in the leukocyte suspension was 1%, after introducing the serotonin into the cell suspension, the number of ROS did not change and amounted to 2% at a dose of 10 μg / ml per 10 UCS, 4% at a dose of 100 μg / ml, 4% at a dose of 1000 mcg / ml.

 Conclusion: immunological analysis confirmed the clinical diagnosis of multiple sclerosis.

 Example 2. Patient K., 42 years old, was admitted to the clinic of neurology 06/18/96. with complaints of periodic dizziness, headaches, weakness in the legs. From the anamnesis it is known that she is sick for about 10 years, the course of the disease with remissions.

 In neurological status: cranial innervation without pathology, tendon reflexes from the legs without pathological signs are revived, there are no abdominal reflexes, slight instability in the Romberg position. Clinical diagnosis: suspected multiple sclerosis.

Blood was examined by incubating a suspension of leukocytes at 37.5 ^° C for 20 minutes and at 4 ^° C for 40 minutes. The examined revealed among 10 ⁶ YSC of blood 3% ROCK specific for serotonin. After incubation of blood cells with serotonin at a dose of 10 μg / ml, the number of ROS increased to 28%, during incubation with serotonin at a dose of 100 μg / ml increased to 25%, while incubation with serotonin at a dose of 1000 μg / ml increased to 20%.

 Conclusion: the patient’s blood lymphocytes respond to in vitro serotonin. The reaction is not typical for a patient with multiple sclerosis, the diagnosis of multiple sclerosis is rejected. With further monitoring of the patient, the diagnosis of multiple sclerosis is completely rejected.

Example 3. Surveyed A., 18 years old, almost healthy. A blood test revealed among 10 ⁶ YSC - 2% ROCK specific for serotonin. After incubation of blood cells with serotonin at a dose of 10 μg / ml, the number of ROS increased to 16%, at a dose of 100 μg / ml, the number of ROS increased to 25%, and at a dose of 1000 μg / ml it increased to 18%.

 Conclusion: the reaction is not typical for a patient with multiple sclerosis.

 The advantages of the method are its accuracy, specificity, sensitivity, technical simplicity, the possibility of using it to carry out early diagnosis of the disease even before clinical manifestations, the possibility of diagnosing the debuts of multiple sclerosis, its erased and atypical forms.

Claims (1)

A method for diagnosing multiple sclerosis by combining a patient’s blood lymphocytes taken before and 30 minutes after contact with a solution of serotonin, with sheep erythrocytes treated with serotonin, characterized in that the patient’s blood lymphocytes are combined with a solution of serotonin in dilutions of 10, 100 and 1000 μg / ml , then with sheep erythrocytes treated with serotonin slurry was maintained at 37 - 37,5 ^o C for 30 - 40 minutes, then count the number of ROCK specific serotonin in the absence of changes in their numbers before and after the serotonin Diagna tiruyut disease.

Images