RU2712105C1 - Method for diagnosing severity of ischemic stroke - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, namely to neurology, and can be used for diagnosing severity of ischemic stroke. That is ensured by examining a patient's venous blood on the first day of admission. RNA is recovered, amount of micro-RNA-20a is determined, then, the number of microRNAs is normalized to endogenous control of GAPDH, and with a multiplicity of microRNA-20a expression relative to GAPDH expression from -25 to -5.1, a minor degree of stroke is predicted, from -5.0 to 0 – moderate severity, from 0.1 to 25 predict severe severity of ischemic stroke.EFFECT: use of the invention provides more accurate diagnosis of the severity of ischemic stroke at the earliest stages of the disease, thereby reducing the incidence of disability and mortality and providing a timely adequate therapy.1 cl, 9 ex

Description

The invention relates to medicine, namely to neurology, can be used to predict the severity of ischemic stroke.

A stroke is an acute disturbance of cerebral circulation (ONMK), which is characterized by a sudden (within minutes, less often - hours) occurrence of focal neurological symptoms (motor, speech, sensory, coordinating, visual and other disorders) and / or cerebral disorders (changes in consciousness, headache, vomiting, etc.) that persist for more than 24 hours or lead to the death of the patient in a short period of time due to the cause of cerebrovascular origin [Clinical recommendations now the IV All-Russian Congress of General Practitioners (Family Doctors) of the Russian Federation on November 15, 2013, Kazan].

In turn, according to the most commonly used TOAST classification (TrialofOrg 10172 inAcuteStrokeTreatment) [Adams H.P., Bendixen B.H., KappelleL.J. etal 1993] distinguish five subtypes of ischemic stroke:

1. atherosclerosis of the large arteries of the brain;

2. cardioembolic stroke;

3. lacunar stroke (blockage of small vessels);

4. stroke due to other reasons;

5. stroke of unknown etiology.

Different subtypes of ischemic stroke are determined by the difference in the mechanisms of ischemia formation. The cause of a stroke may be atherosclerosis of the cerebral arteries or clogging of the embolarterimi brain. Ischemic stroke is a classic example of the multifactorial nature of pathology, in which the implementation of the pathological process is carried out by the interaction of genetic factors with environmental factors.

Depending on the severity of the stroke, the outcome of the disease varies greatly. It is imperative to identify more accurate markers of the severity of ischemic stroke in order to select adequate therapy in a timely manner.

A known method for predicting the course of ischemic stroke, characterized in that on the first day of acute cerebrovascular accident analysis of blood serum and cerebrospinal fluid for uric acid content. When the content of uric acid in the blood serum is higher than 390 μmol / L, in the cerebrospinal fluid above 140 μmol / L, an unfavorable course (lethal outcome) of ischemic stroke is predicted [patent RU 2324941 from 05.20.2008]. However, this method allows you to predict only the development of a fatal outcome, without determining the severity of the disease.

A known method for diagnosing the severity of ischemic stroke, which consists in the fact that on the 1st day in the venous blood the method of immunophenotyping determines the level of antigen-positive cells (CD95 lymphocytes) as a percentage of fluorescent cells when viewing 200 lymphocytes minus the percentage of fluorescent cells observed in the preparation negative control. With an increase in the value of CD95 lymphocytes to 9.6 ± 3.65%, a mild ischemic stroke is diagnosed. With an increase in the value of CD95 lymphocytes to 16.8 ± 2.7%, an ischemic stroke of moderate severity. With an increase in the value of CD95 lymphocytes to 28.1 ± 3.2%, a severe ischemic stroke [patent RU 2238566 from 10/20/2004]. The disadvantage of this method is the need for expensive specific equipment (fluorescence microscope) and highly qualified personnel with experience in phenotyping of lymphocytes labeled with FITCem.

A method for diagnosing an atherothrombotic subtype of ischemic stroke has been registered, based on the fact that a cell suspension enriched in white blood cells at a concentration of 50,000 in 1 μl is isolated from the venous blood of a patient after an ischemic stroke, it is cultured in sterile vials for 62 hours, and the supernatant is collected, determine in it the level of total cholesterol, and with its value exceeding 0.150 mmol / l, an atherothrombotic subtype of ischemic stroke is diagnosed [patent RU 2266545 of 12.20.2005]. A significant disadvantage of this method is the long lead time of the analysis, while there is a need to carry out treatment as early as possible.

The prototype of the invention is a method for assessing the severity of ischemic stroke in people under the age of 50 years, which consists in determining the homocysteine content (Hz) in the serum of venous blood of patients with ischemic stroke, and ischemic stroke is assessed at a homocysteine concentration of less than 10.4 μmol / L as mild severity, at a concentration of homocysteine in the range of 10.4-15.1 μmol / L, ischemic stroke is assessed as moderate in severity, at a concentration of homocysteine over 15.1 μmol / L, ischemic stroke is assessed severe degrees of severity [patent RU 2312356 of 10.12.2007]. The main disadvantage of this method is the following: the severity of ischemic stroke is determined only before the age of 50, while most patients are much older.

The result of recent scientific studies has been the understanding of the important role of determining the level of expression of CD 46 and its miRNAs inhibiting it in the development of ischemic stroke.

CD 46 is a membrane protein inhibiting the complement receptor. It is known that CD46 gene mRNA can be used as a potential marker for the differential diagnosis of different subtypes of ischemic stroke, since CD46 is part of the complement cascade that regulates inflammation, a critical pathological process in the pathophysiology of the atherothrombotic subtype of ischemic stroke [JicklingGC, XuH, StamovaB, Ander ZhanX, TianY, etal. Signaturesofcardioembolicandlarge-vesselischemicstroke.Ann Neurol. 2010; 68 (5): 681-92. doi: 10.1002 / ana.22187, Jickling GC, Stamova B, Ander BP, Zhan X, Liu D, Sison SM, et al. Prediction of cardioembolic, arterial, and lacunar causes of cryptogenic stroke by gene expression and infarct location. Stroke. 2012; 43 (8): 2036-41. doi: 10.1161 / STROKEAHA.111.648725].

The objective of the invention is to develop a method for diagnosing the severity of ischemic stroke in different age categories.

The technical result when using the invention is to increase the accuracy of diagnosis by determining the severity of ischemic stroke in patients older than 50 years.

The proposed method for diagnosing the severity of ischemic stroke is as follows. On the first day of admission of a patient with ischemic stroke, venous blood of the patient is taken.

RNA samples are isolated from peripheral venous blood lymphocytes. RNA integrity is assessed by electrophoresis on a 1% agarose gel by the ratio of the intensities of the bands in the gel corresponding to 28S and 18S rRNA.

The synthesis of the first cDNA strand is carried out using a reverse transcription reaction. To study the amount of miRNA-20a, PCR is carried out with miRNA-specific primers, then the miRNA amount is normalized to the endogenous control of GAPDH. GAPDH, the “household” gene, is necessary to normalize quantitative expression data. Expression of this gene is stable in almost all cells of the body. A quantitative analysis of microRNA is then carried out using specific TaqMan probes. Then carry out a statistical analysis of the expression of miRNA-20A.

With a multiplicity of expression of miRNA-20a relative to GAPDH expression from -25 to -5.1, a moderate degree of severity of a stroke is predicted, with a multiplicity of expression from -5.0 to 0, a moderate degree of severity is expected, with a multiplicity of expression from 0.1 to 25 a severe severity is predicted.

RNA samples of 89 patients with a diagnosis of ischemic stroke in the acute period at the age of 36 to 80 years were used. The average age was 64.87 ± 11.61 years. 49 people were patients with mild severity of the disease, 9 patients with moderate severity, 13 patients with severe severity of ischemic stroke. The diagnosis of stroke is established on the basis of generally accepted criteria in accordance with the international classification of diseases and health problems [ICD 10, WHO, 1992].

The control group consisted of 18 practically healthy individuals, residents of the Republic of Bashkortostan, comparable in age, gender and ethnicity, without chronic diseases, including a history of cardiovascular system pathology. The study included the collection of complaints and medical history, the study of ambulatory patient records. Neurological status was studied according to the generally accepted scheme. An objective assessment of the severity of neurological deficits and the severity of patients was assessed according to the National Institutes of Health StrokeScale (NIHSS) stroke scale at the time of admission and at the time of discharge.

The NIHSS scale is based on an assessment of the level of consciousness, visual functions, anosognosia, motor, coordinating, speech and sensory disturbances [BrottT., AdamsH.P., OlingerC.P. etal. Measu rement sofacut ecerebralin farction: a clinical examination scale // Stroke. -1989. - Vol. 20 (7). - p. 864-70]. The minimum score on the NIHSS stroke scale is 0 points, the maximum is 34 points. The absence of a neurological deficit on this scale of stroke was rated as 0 points; from 1 to 8 points - a neurological deficit is defined as a mild severity; from 9 to 12 points - moderate severity; 13-15 points - severe neurological disorders, from 16 to 33 points neurological disorders of extreme severity, 34 points corresponds to a coma.

The multiplicity of miRNA-20a expression in the group with a mild disease severity was low and ranged from -25 to -5.1, in patients with moderate severity the expression ratio was from -5.0 to 0, in patients with severe severity the expression rate was in the range from 0.1 to 25 (F = 4.05, p = 0.023).

The invention is illustrated by the following clinical examples:

Example 1

Patient K., born in 1978, was admitted to the emergency department of the ambulance hospital with a diagnosis of ischemic stroke, an acute period. In order to determine further treatment tactics, it is necessary to determine the subtype of stroke and make a prognosis of the severity of the disease. On examination: the consciousness is clear, oriented in self, time, place completely, correctly. FMN: Equal palpebral fissures. The photoreaction is live. Corneal reflexes saved. Convergence is complete, vision is not broken. Pupils D = C, the movement of the eyeballs in full, nystagmus, diplopia is not. The nasolabial folds are smoothed to the left, the tongue centered. The phonation is preserved, swallowing is not disturbed, the pharyngeal reflex is alive. Hearing saved, sensitivity preserved. There are no speech disorders. CXP live D = C. The strength in the left limbs is 4 points, in the right - 5 points. KP performs with intention on both sides. In the Romberg position is unstable. NIHSS National Institutes of Health Stroke Score on admission 5 points. Ultrasound tests of MAG, echocardiography, CT of the brain of the patient were performed. For molecular genetic testing, 8 ml of venous blood was taken from the patient, followed by determination of miRNA-20a by reverse transcription followed by PCR with miRNA-specific primers, normalization of the miRNA amount to endogenous GAPDH control and quantitative analysis of miRNA using specific TaqMan probes. The multiplicity of the relative expression of miRNA-20a was -17.2, the patient was diagnosed with ischemic stroke of mild severity. The prognosis of the disease is favorable. The patient was discharged on the 10th day with a regression of neurological deficit. NIHSS at discharge 0 points.

Example 2

Patient S., born in 1947. He was admitted to the hospital in the acute period of ischemic stroke. On examination: the consciousness is clear, oriented in self, time, place completely, correctly. FMN: Equal palpebral fissures. The photoreaction is live. Corneal reflexes saved. Convergence is complete, vision is not broken. Pupils D = C, full movement of the eyeballs, horizontal nystagmus, more to the left, no diplopia. The nasolabial folds are smoothed to the left, the tongue deviates to the left. Speech: dysarthria. The phonation is preserved, swallowing is not disturbed, the pharyngeal reflex is alive. Hearing saved. Sensitivity - not broken. CXP live D = C. Strength in the left limbs 3 points, in the right - 5 points. KP does not perform on the left. In the pose of Romberg NIHSS upon admission 7 points. The patient underwent ultrasound testing of the MAG, echocardiography, CT of the brain. During molecular genetic testing, 8 ml of venous blood was taken from the patient, followed by determination of miRNA-20a by reverse transcription followed by PCR with miRNA-specific primers, normalization of the miRNA amount to endogenous GAPDH control and quantitative analysis of miRNA using specific TaqMan probes. The multiplicity of relative expression of miRNA-20a was -25, the patient was diagnosed with ischemic stroke of mild severity. The prognosis of the disease is favorable. The patient was discharged on day 12 with minimal neurological deficit. NIHSS at discharge: 2 points.

Example 3

Patient O., born in 1955. He was admitted to the hospital in the acute period of ischemic stroke. On examination: the consciousness is clear, oriented in self, time, place completely, correctly. FMN: Equal palpebral fissures. The photoreaction is live. Corneal reflexes saved. Convergence is complete, vision is not broken. Pupils D = C, the movement of the eyeballs in full, nystagmus, diplopia is not. The nasolabial folds are smoothed to the right, the tongue deviates to the right. Speech: motor aphasia. The phonation is preserved, swallowing is not disturbed, the pharyngeal reflex is alive. Hearing is preserved, sensitivity is reduced on the right. CXP live D = C. Strength in the right limbs 3 points, in the left - 5 points. KP does not perform right. In the Romberg position is unstable. NIHSS at admission 9 points. During molecular genetic testing, 8 ml of venous blood was taken from the patient with subsequent determination of miRNA-20a by reverse transcription followed by PCR with miRNA-specific primers, normalization of the miRNA amount to endogenous control of GAPDH and quantitative analysis of miRNA using specific TaqMan probes. The multiplicity of the relative expression of miRNA-20a was -5.1, the patient was diagnosed with ischemic stroke of mild severity. The prognosis of the disease is favorable. The patient was discharged on day 13 with minimal neurological deficit. NIHSS at discharge: 2 points.

Example 4

Patient K., born in 1974. He was admitted to the hospital in the acute period of ischemic stroke. On examination: consciousness is stunned 1, disoriented in self, time, place. FMN: Equal palpebral fissures. The photoreaction is live. Corneal reflexes saved. Convergence failure on both sides, vision is not impaired. Pupils D = C, eyeball movement in full, horizontal bilateral nystagmus, no diplopia. The nasolabial folds are smoothed to the right, the tongue deviates to the right. Speech: moderate motor aphasia. Swallowing is disturbed (choking). Hearing is preserved, sensitivity is reduced on the right. CXP live D = C. Strength in the right limbs 2 points, in the left - 5 points. KP does not perform right. In the Romberg position is not worth it. NIHSS at admission 11 points. The patient was diagnosed with a cardioembolic subtype of AI using ultrasound MAG, echocardiography, ECG, CT of the brain. During molecular genetic testing, 8 ml of venous blood was taken from the patient with subsequent determination of miRNA-20a by reverse transcription followed by PCR with miRNA-specific primers, normalization of the miRNA amount to endogenous control of GAPDH and quantitative analysis of miRNA using specific TaqMan probes. The multiplicity of the relative expression of miRNA-20a was -2.2, the patient was diagnosed with moderate ischemic stroke. The prognosis of the disease is dubious. The patient was discharged on day 17 with a mild neurological deficit: strength in the right extremities recovered to 4 points, speech recovered to a mild degree of motor aphasia. NIHSS at discharge: 4 points.

Example 5

Patient D., born in 1963, was admitted to the emergency department of the emergency hospital. At a neurological examination: the consciousness is clear, disoriented by the individual personality, time, place. FMN: Equal palpebral fissures. The photoreaction is live. Corneal reflexes saved. Convergence failure on both sides, vision is not impaired. Pupils D = C, the movement of the eyeballs in full, nystagmus, diplopia is not. The nasolabial folds are smoothed to the left, the tongue centered. Speech: dysarthria. Swallowing is not broken. Hearing saved. Sensitivity is reduced on the left. CXP live D = C. Strength in the right limbs 5 points, in the left limbs 3 points. KP performs with a crude intention on the left. In the Romberg position is not worth it. NIHSS at admission 9 points. The patient underwent ultrasound testing of the MAG, MAG, ECG, CT of the brain. Diagnosed with ischemic stroke. For molecular genetic testing, 8 ml of venous blood was taken from the patient with subsequent determination of miRNA-20a by reverse transcription followed by PCR with miRNA-specific primers, normalization of the miRNA amount to endogenous control of GAPDH and quantitative analysis of miRNA using specific TaqMan probes. The multiplicity of the relative expression of miRNA-20a was-5.0, the patient was diagnosed with moderate ischemic stroke. The prognosis of the disease is dubious. The patient was discharged on day 12 with a slight neurological deficit (strength recovered to 4 points). NIHSS at discharge: 3 points.

Example 6

Patient T., born in 1963. Was admitted to the hospital in the acute period of ischemic stroke. The diagnosis was made on the basis of clinical, neurological and neurophysiological studies, data of ultrasound duplex scanning of cerebral vessels and computed tomography of the brain. On examination: consciousness stunned 1-2, the patient is disoriented in his own personality, time, place. FMN: Equal palpebral fissures. The photoreaction is live. Corneal reflexes saved. Pupils D = C, eyeball movement in full, horizontal bilateral nystagmus, no diplopia. The nasolabial folds are smoothed to the right, the tongue deviates to the right. Speech: failed to verify, the patient does not speak (sensory-motor aphasia). Swallowing is not broken. Hearing is preserved, sensitivity is reduced on the right. CXP live D = C. Strength in the right limbs 2 points, in the left - 5 points. KP does not perform on the right. In the Romberg position is not worth it. NIHSS upon admission 13 points. During molecular genetic testing, 8 ml of venous blood was taken from the patient, followed by determination of miRNA-20a by reverse transcription followed by PCR with miRNA-specific primers, normalization of the miRNA amount to endogenous GAPDH control and quantitative analysis of miRNA using specific TaqMan probes. The multiplicity of the relative expression of miRNA-20a was 0, the patient was diagnosed with moderate ischemic stroke. The prognosis of the disease is dubious. The patient was discharged on the 18th day, with moderate neurological deficit (strength in the right limbs 3 points, moderate motor aphasia). NIHSS at discharge: 9 points.

Example 7

Patient F., born in 1959. Was admitted to the hospital in the acute period of ischemic stroke in the right carotid basin. The diagnosis was made on the basis of clinical, neurological and neurophysiological studies, data of ultrasound duplex scanning of cerebral vessels, echocardiography and computed tomography of the brain. Multiplicity of consciousness - stunning 2. FMN: Eye slots equal. The photoreaction is live. Corneal reflexes saved. Pupils D = C, the movement of the eyeballs in full, nystagmus, diplopia is not. The nasolabial folds are smoothed to the left. Voice contact is difficult. CXP live D = C. Strength in the left limbs 0-1 point, in the right limbs 5 points. KP does not comply. In the Romberg position is not worth it. NIHSS at receipt of 20 points. FMN: Pupils D = C, palpebral fissures equal. Nystagmus, no diplopia. Weak convergence on both sides. Asymmetry of the face, smoothed right nasolabial fold, tongue in the center. Speech is not intelligible (sensory-motor aphasia). Strength in limbs on the right 2 points, left 5 points. CXR are brisk on the right. KP on the right does not perform, in the Romberg position is not worth it. During molecular genetic testing, 8 ml of venous blood was taken from the patient with subsequent determination of miRNA-20a by reverse transcription followed by PCR with miRNA-specific primers, normalization of the miRNA amount to endogenous control of GAPDH and quantitative analysis of miRNA using specific TaqMan probes. The multiplicity of the relative expression of miRNA-20a was 7.7, the patient was diagnosed with severe ischemic stroke. The prognosis of the disease is unfavorable. The patient was discharged on day 21 with a deep neurological deficit (strength in the extremities 2 points, moderate sensory-motor aphasia remains) NIHSS at discharge 14 points.

Example 8

Patient P., born in 1957. He was admitted to the hospital in the acute period of ischemic stroke. The diagnosis was made on the basis of clinical, neurological and neurophysiological studies, data of ultrasound duplex scanning of cerebral vessels, echocardiography and computed tomography of the brain. On examination: Level of consciousness - stupor. Voice contact is difficult. Paresis of the gaze, asymmetry of the facial muscles are noted. Deep hemiparesis on the right - 2 points. NIHSS upon admission 16 points. During molecular genetic testing, 8 ml of venous blood was taken from the patient, followed by determination of miRNA-20a by reverse transcription followed by PCR with miRNA-specific primers, normalization of the miRNA amount to endogenous GAPDH control and quantitative analysis of miRNA using specific TaqMan probes. The multiplicity of the relative expression of miRNA-20a was 0.1, the patient was diagnosed with severe ischemic stroke. The prognosis of the disease is unfavorable. The patient was discharged on the 17th day with a moderate neurological deficit (strength in the limbs on the right 3 points, pseudobulbar dysarthria). NIHSS at discharge: 11 points.

Example 9

Patient A., born in 1947. Was admitted to the hospital in the acute period of ischemic stroke. The diagnosis was made on the basis of clinical, neurological and neurophysiological studies, data of ultrasound duplex scanning of cerebral vessels and computed tomography of the brain. The level of consciousness is coma 1-2. FMN: Pupils D = C, palpebral fissures equal. Asymmetry of the face, smoothed left nasolabial fold. Voice contact is difficult. The force could not be verified, the command does not execute. Forced lying position. NIHSS upon admission 25 points. During molecular genetic testing, 8 ml of venous blood was taken from the patient, followed by determination of miRNA-20a by reverse transcription followed by PCR with miRNA-specific primers, normalization of the miRNA amount to endogenous GAPDH control and quantitative analysis of miRNA using specific TaqMan probes. The multiplicity of the relative expression of miRNA-20a was 25, the patient was diagnosed with severe ischemic stroke. The prognosis of the disease is unfavorable. The patient is dead.

Claims (1)

A method for diagnosing the severity of ischemic stroke, including examining the patient's venous blood on the first day of admission, characterized in that RNA is isolated, the amount of miRNA-20a is determined, then the amount of miRNA is normalized to the endogenous control of GAPDH, and when the expression of miRNA-20a is expressed relative to expression GAPDH from -25 to -5.1 predict a mild stroke severity, from -5.0 to 0 a moderate severity, from 0.1 to 25 predict a severe severity of ischemic stroke.