RU2104543C1 - Method of diagnosing disseminated sclerosis - Google Patents

Abstract

FIELD: immunoneurology. SUBSTANCE: blood of patient (0.1-0.2 ml) is examined by adding 0.1-0.2 ml of 0.5% suspension of morphine-loaded ram erythrocytes to lymphocytes in sample. Mixture is incubated at 37-37.5 C, after which rosette-forming lymphocytes in the smear are calculated. When percentage of rosette-forming cells exceeds 10%, disseminated sclerosis is diagnosed. EFFECT: simplified and made less expensive diagnostics; increased its accuracy and specificity. 5 tbl

Description

 The invention relates to medicine, namely to immunoneurology.

A known method for the diagnosis of multiple sclerosis by subcutaneous injection of a patient with a solution of rocedil, followed by studying the effect of its effect, namely, to diagnose the disease, the blood is examined before and after the introduction of a solution of rocedil, isolating lymphocytes, combining them with 0.1 - 0.2 ml A 0.5% suspension of ram erythrocytes loaded with serotonin, incubating the mixture at 37 - 37.5 ^o C for 10 - 15 minutes, followed by counting the number of rosette-forming lymphocytes in the smear and in the absence of a significant increase in the inter both tests diagnose multiple sclerosis [1].

 The disadvantages of the method are the technical complexity of its formulation, the need for parenteral administration of the drug and the associated risk of infection, the need for a large number of disposable syringes, the possibility of an allergic reaction of the patient to the administration of the drug, the absence of ampouled racedil, which is not currently available in Russia or in European states (England, France, Germany, Hungary, Yugoslavia, Czechoslovakia), the need for double blood sampling from a vein.

 The invention is aimed at solving the problem - the simplification and cheapening of the method for the diagnosis of multiple sclerosis with high accuracy and specificity.

The task is achieved by determining the number of rosette-forming lymphocytes specific to the hapten in the patient’s blood. New in the method is that the patient’s original blood is examined by adding to the leukocyte blood sample 0.1 - 0.2 ml of a 0.5% suspension of sheep erythrocytes loaded with morphine, the mixture is incubated at 37 - 37.5 ^o C, s subsequent counting in a smear of rosette-forming lymphocytes. If there are more rosette-forming cells (ROC) in it, more than 10% are diagnosed with multiple sclerosis.

The method is as follows: from the finger, the blood is taken by micromethod in an amount of 0.1 - 0.2 ml into a centrifuge tube containing the same amount of a 5% sodium citrate solution. The tube is shaken. For lysis of red blood cells, 0.8 - 0.9 ml of distilled water is added, then the mixture is stirred by rolling the tube between the palms of the hands. After 20-30 seconds, 0.8-0.9 ml of a 1.8% sodium chloride solution is added, after which the mixture is centrifuged for 5 minutes at 1500 rpm. The supernatant is decanted, 1 ml of medium 199 is added to the precipitate and incubated at 37-37.5 ^° C for 15 minutes. After incubation, the mixture is centrifuged for 5 minutes. Then the supernatant is removed and 0.1 ml of medium 199 is added, the precipitate is resuspended. Then the suspension is kept at room temperature for 30 minutes, after which 0.1 - 0.2 ml of a 0.5% solution of erythrocyte morphine diagnosticum is added to it. The mixture is thoroughly mixed and incubated for 15 minutes at 37 - 37.5 ^o C, and then for 18 hours in a refrigerator. After incubation, the suspension is centrifuged for 5 min at 1500 rpm, the supernatant is drained. To the precipitate was added 1 drop of a 0.5% solution of glutaraldehyde, after which the suspension was incubated for 20 minutes at room temperature, then 0.75 ml of distilled water was added and centrifuged for 5 minutes at 1500 rpm. The supernatant is drained, 0.5 ml of 5% bovine serum solution is added to the precipitate, and then centrifuged again. A smear is prepared from the precipitate, dried in air, fixed with ethyl alcohol for 30 minutes and stained according to Romanovsky-Giemsa. Count under a microscope with a 900-fold increase in 100 lymphocytes, among them the number (in percent) of cells that have fixed 3 or more sheep erythrocytes (ROCs) on their surface, and with a number of ROCs of more than 10%, multiple sclerosis is determined.

To prepare a morphine diagnosticum, sheep erythrocytes treated with glutaraldehyde are used. Fresh sheep erythrocytes are washed three times with an isotonic sodium chloride solution of pH 7.2, and the suspension is centrifuged for 5 minutes at 1500 rpm. A 5% suspension is prepared from the washed red blood cells by combining 0.5 ml of a red blood cell 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 37-37.5 ^o C for 15 minutes, after which the red blood cells are thoroughly washed from glutaraldehyde with a buffered isotonic solution of sodium chloride, pH 7.2, and the volume is adjusted to the original solution with the same solution (5% suspension). The red blood cells thus prepared are stored at 4 ^° C. for 6 months.

To sensitize red blood cells with morphine, the glutarized red blood cells are washed twice with isotonic sodium chloride pH 6.4. Then combined with a solution of morphine in a ratio of 1: 1 (0.05 ml of a 1% solution of morphine is added to 0.45 ml of a phosphate buffer solution, pH 6.4; 0.1 ml of this suspension contains 0.1 mg of morphine). The mixture was incubated for 30 min at 37 ^° C, shaking periodically, after which it was washed twice with a buffered solution of sodium chloride, pH 7.2. After draining the supernatant, the volume is adjusted to the original volume with the same buffer (5% suspension). The reaction uses a 0.5% suspension of sensitized red blood cells, which is prepared from the original (5%) suspension by connecting it to medium 199.

 The results of the studies are shown in table. 15.

The results of the proposed method with incubation of the indicator system for 30 and 60 minutes, at 37 and 37.5 ^o C are given in table. 5.

 Examples of practical application of the method.

 Example 1. Patient H., 47 years old, complains of bouts of systemic dizziness, unsteady gait, decreased visual acuity in both eyes to 0.05, fatigue.

 The patient considers himself from the age of 35, when retrobulbar neuritis was first diagnosed. Almost complete restoration of vision under the influence of hormonal therapy occurred after 2 to 3 weeks. After 12 months after respiratory disease, retrobulbar neuritis again developed with a deep and persistent decrease in visual acuity. A third exacerbation of the disease occurred 6 years later while taking a hot bath: retrobulbar neuritis developed again, which was soon joined by cerebellar syndrome and pyramidal disorders in the form of right-sided spastic hemiparesis. After a course of hormone therapy, the symptoms of the disease completely regressed.

 Neurologically determined is the symmetrical revitalization of tendon reflexes on both sides, the absence of lower abdominal reflexes and asymmetry of the upper and middle, mild ataxia.

 Clinical diagnosis: multiple sclerosis, cerebrospinal form, remitting course, remission phase.

The inventive method was carried out an immunological study of the patient’s blood, for which 0.1 ml of a 0.5% suspension of sheep erythrocytes loaded with morphine was added to the leukocytes of the initial blood, after which the mixture was incubated at 37 ^o C. When counting rosette-forming lymphocytes in the smear, their number amounted to 11%. Thus, using the proposed method immunologically confirmed the clinical diagnosis of multiple sclerosis.

 Example 2. Patient B., 39 years old, fell ill 8 years ago, when shortly after a respiratory illness, visual acuity suddenly decreased in his left and then in his right eye. He was treated in the clinic of eye diseases with a diagnosis of bilateral retrobulbar neuritis, but visual acuity was not fully restored. A year later, a weakness appeared in the right leg, which gradually increased. Then weakness in the left leg joined. With an interval of one year in the summer, the following symptoms appeared consecutively: weakness in the upper extremities, impaired function of the pelvic organs as an imperative urge, diplopia, tinnitus and unsteady gait.

 The neurological status reveals: pronounced bilateral horizontal nystagmus, a decrease in visual acuity to 0.04 - 0.05 in both eyes; spastic tetraparesis with bilateral pathological symptoms, the absence of abdominal reflexes; lack of deep sensitivity in the lower and its sharp decrease in the upper limbs, lack of surface sensitivity below the C3 segment; dysfunction of the pelvic organs as imperative incontinence. The patient is euphoric, uncritical of his condition.

 Diagnosis: multiple sclerosis, cerebrospinal form, progressive course.

An immunological study of the patient’s blood was carried out: the leukocytes of the initial blood were combined with 0.2 ml of a 0.5% suspension of sheep erythrocytes loaded with morphine, the mixture was incubated at 37.5 ^o C, and then the number of morphine-specific ROS was calculated in a smear, which amounted to 29%. The claimed method immunologically confirmed the clinical diagnosis of multiple sclerosis.

 Example 3. Patient Ch., 32 years old, was admitted to the clinic of nervous diseases with complaints of weakness and crawling in the left extremities, staggering when walking, recurring headaches of an oppressive nature in the form of a "hoop", fatigue, and low mood.

 Sick for 1 year, when after a traumatic situation a feeling of awkwardness appeared, and then weakness in the left arm, to which weakness in the left leg gradually joined. Notes a deterioration in the form of increased headaches and increased weakness in the left limbs after thermal procedures and in the warm season.

 Neurological status upon admission determined: central paresis of the facial and hyoid nerves on the right, symmetrical revitalization of tendon and periosteal reflexes, a decrease in muscle strength in the left limbs to three points, a decrease in deep sensitivity in the left limbs and astereognosis in the fingers of the left hand, staggering in the Romberg and when walking, overshooting and intentional trembling when performing finger-bearing and heel-to-knee tests on the left.

 Diagnosed with multiple sclerosis, cerebrospinal form, progressive course.

 An immunological blood test by the claimed method established the presence of 7% ROCK specific for morphine. In the immunological reaction, the diagnosis of multiple sclerosis was not confirmed.

 With further clinical observation, the diagnosis of multiple sclerosis was rejected, the diagnosis was hysterical neurosis.

 Example 4. Patient K., 38 years old, complains of lower back pain radiating to the outer surface of the right thigh and lower leg, numbness of the outer edge of the right foot. The pain intensifies with movement. Sick 5 years, exacerbations are 1 - 2 times a year after physical exertion.

 During the examination, vertebral syndrome was noted in the form of limiting the range of movements in the lumbar spine, sharp smoothing of lumbar lordosis, right-sided scoliosis, and lower back muscle defenses; a positive symptom of Laseg and Neri on the right, hypesthesia on the outer surface of the right lower leg. Knee reflexes are living, equal. The left Achilles reflex is reduced, the right is not caused.

 On the radiograph of the lumbosacral spine, indirect signs of a hernia of the L4 disc are determined.

 Clinical diagnosis: osteochondrosis of the lumbar spine, disc herniation L4, radicular compression syndrome.

 When immunological blood tests by the claimed method, the number of ROC specific for morphine in the patient's initial blood was 5%. The immune response for multiple sclerosis is not characteristic.

 Example 5. Donor V. (male), 28 years old, practically chlorine. When immunological blood tests by the claimed method, the number of ROC specific for morphine was 7%. The immune response for multiple sclerosis is not characteristic.

 The advantages of the proposed method are its technical simplicity, the absence of the need to administer medications to patients associated with the danger of infection or the occurrence of an allergic side effect in a patient, the absence of the need for disposable syringes.

 The method provides a sensitive and specific immunodiagnosis of multiple sclerosis. The method allows you to early diagnose the debut of the disease; its erased and atypical forms; enables differential diagnosis in unclear, doubtful cases.

Claims (1)

A method for diagnosing multiple sclerosis by determining the number of rosette-forming cells specific for the hapten in the patient’s blood, characterized in that the patient’s original blood is examined by adding 0.1-0.2 ml of a 0.5% suspension of sheep erythrocytes loaded with morphine to blood leukocytes, the mixture is incubated at a temperature of 37.37.5 ^° C, followed by counting of rosette-forming lymphocytes in a smear and in the presence of a number of rosette-forming cells in it more than 10%, multiple sclerosis is diagnosed.

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