RU2349917C1 - Method of patients condition diagnostics for hypertension cases complicated by ischemic stroke - Google Patents

Abstract

FIELD: medicine; cardiology and neurology.

SUBSTANCE: patient's peripheral venous blood is centrifuged in ficoll-verografin density gradient ρ=1.077, and, count of 100 lymphocytes with identification by external cytoplasm membrane vesiculation is performed in 10 visual fields, using phase-contrast microscopy. Blebbing distinction degree of lymphocytes corresponding to patient's condition is defined.

EFFECT: enhanced reliability of patient's condition diagnostics, possibility to perform adequate therapy timely.

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Description

The invention relates to medicine, namely to cardiology and neurology, and can be used to assess the severity of the condition of patients with hypertension complicated by ischemic stroke.

Stroke is a leading cause of adult disability and one of the leading causes of death. In developed countries, the incidence of stroke is 2400 cases per 1 million people per year.

The main risk factors for ischemic stroke include arterial hypertension. In patients with confirmed arterial hypertension, the stroke rate is 3 times higher than in patients with normotension. More than 80% of patients hospitalized in the neurological department for ischemic stroke are patients with arterial hypertension (Gusev E.I. Possibilities of secondary prevention of stroke: the importance of antihypertensive and antiplatelet therapy / E.I. Gusev, A.B.Gecht // Journal neurology and psychiatry named after S. S. Korsakov (supplement to the journal "Stroke") - 2002. - No. 5. - P.43-49). Patients with stroke need to provide emergency and effective medical care, which is the solution to the problem of not only reducing mortality, but also effective rehabilitation. Progressive damage to the vascular wall - endothelial dysfunction, which is the basis of arterial hypertension, and the focus of ischemia and cerebral infarction that form during a stroke, necessitate a comprehensive assessment of the severity of a patient with ischemic stroke developed against the background of arterial hypertension. Existing scales for assessing the severity of a patient with a stroke, as a rule, include the determination of only neurological status, while the functional state of the endothelium, which has numerous functions, is not taken into account. In particular, the endothelium regulates the rheological properties of blood, the balance of vasoconstriction and vasodilation, the diapedesis of cells of the immune system into the focus of aseptic inflammation (Gusev E.I. Ischemia of the brain / E.I. Gusev, V.I. Skvortsova. - M .: Medicine, 2001. - 328 p .; Gitkina L.S. Quantitative assessment of motor impairment and disability in patients after cerebral stroke, traumatic brain injury.Instruction for use / L.S. Gitkina, V. B. Smychek, etc. // Research Institute of Medical and Social Expertise and Rehabilitation. - Minsk, 2003. - 18 p .; Min-Lai, S. Evaluation of the Am erican Heart Association Stroke Outcome Classification / S. Min-Lai, P.W. Duncan // Stroke. - 1999. - Vol.30. - P.40-43).

Available markers of the severity of the condition of patients with ischemic stroke, which constitute a comprehensive assessment of oxidative stress and the functional state of the vascular wall as key predictors of the outcome of the disease, can be considered peripheral blood cells, in particular peripheral blood lymphocytes, which meet the following necessary parameters: they are highly sensitive to the action of apoptogenic and activation factors, are a substrate of the immune response (aseptic inflammation and form stimulation of immune synapses); the lymphocyte population is dynamic due to the relatively short stay in the systemic circulation of lymphocytes; as well as the participation of lymphocytes in the initiation of apoptosis of target cells and the formation of endothelial dysfunction (Moiseeva, O.M. Transforming factor - beta 1 and leukocyte activation markers in hypertension / O.M. Moiseeva, E.A. Lyasnikova, etc. // Arterial hypertension. - 2003. - No. 1. - P.20-23; Martinez, MC Shed membrane microparticles from circulating and vascular cells in regulating vascular function / MC Martinez, A. Tesse, F. Zobair // Am. J. Physiol Heart Circ. Physiol. - 2005. - Vol. 288. - P.1004-1009).

There are several well-known approaches to assessing the severity of stroke patients based on the study of cellular and humoral parameters of peripheral blood.

A known method for assessing the severity of ischemic stroke in people under the age of 50 years (RF Patent No. 2312356; G01N 33/68, 10.12.2007), which consists in the fact that in the serum of venous blood of patients with ischemic stroke determine the content of homocysteine, and the concentration of homocysteine assess the severity of ischemic stroke. The disadvantage of this method is that the question of the correlation of homocysteinemia and inflammation, endothelial dysfunction, hypercoagulation in patients with stroke (Dusitanond P.) is still debatable. A drug-induced decrease in homocysteine levels is not accompanied by a decrease in the concentration of inflammatory biomarkers, endothelial dysfunction, hypercoagulation in patients with stroke / R. Dusitanond et al. // Stroke 2005 - Cardiosayt.ru; Ray, JGHomocysteine-Lowering Therapy and Risk for Venous Thromboembolism / Ray JG, Kearon C., Yi Q. et al. // A Randomized Trial. Arm Intern Med. - 2007 - 146 - P.761-767).

There is a method for predicting the functional outcome of ischemic stroke in people under the age of 50 (RF Patent No. 2312362; G01N 33/68, 12/10/2007) and consists in determining the concentration of homocysteine (Hz) and fibrinogen (Fg) in the patient’s venous blood , which calculate the level of Y independence from outside assistance: Y = 132.3-8.5 × Fg-2.5 × Hz. With a Y value of more than 85, the patient is predicted to be completely independent of outside help; with a Y value of more than 75 to 85, a slight dependence of the patient on outside assistance is predicted; with a Y value of more than 55 to 75, a moderate dependence of the patient on outside assistance is predicted; with a Y value of 35 to 55, a pronounced dependence of the patient on outside help is predicted; with a Y value of less than 35, the patient is predicted to be completely dependent on outside help.

The authors of the method carried out an original approach to assessing the state of damage to brain tissue cells and the degree of reperfusion of the ischemic lesion, which can be indirectly reflected by the concentration of homocysteine and, in the hemostasis parameter, the content of fibrinogen. However, homocysteine and fibrinogen do not reliably indicate the functional state of the vascular wall and do not reflect the level of oxidative stress, the state of the immune system.

Another method for predicting the clinical course of ischemic stroke (RF Patent No. 2217754; G01N 33/49, G01N 33/68, 11/27/2003) is that the patient’s venous blood is examined for hemostatic activation parameters such as degradation products of D- fibrinogen dimer, soluble complexes of fibrin monomer (RKFM), as well as aggregation activity of platelets (AT). Measurements are taken throughout the acute and acute periods of ischemic stroke. In the case of even a single increase in the concentration of the D-dimer over 1 μg / ml and / or the concentration of RKFM more than 50 mg /% in combination with a significant decrease in AT to 10-15% compared with the values of AT at the beginning of the acute period, the probability of a lethal outcome of the course of ischemic a stroke. The disadvantage of this method is that despite the listed factors of hemostatic activation, which can indirectly reflect the degree of damage to the vascular wall, the degree of oxidative stress and activation of the body's immune system are not taken into account.

A known method for predicting the outcome of cerebral stroke (RF Patent No. 2079287; А61В 10/00, 05/20/1997), which consists in determining the aggregation of peripheral blood platelets caused by a collagen solution, and in the presence of decreases in the maximum amplitude and angle of inclination of the aggregogram, the development of retrombosis with cerebral stroke. The disadvantage of this method is the inability to give a complete assessment of the severity of the patient's condition and, therefore, to select the necessary pathogenetic therapy in addition to the correction of hemostasis parameters.

There is a method for predicting the course of ischemic stroke (RF Patent No. 2236685; G01N 33/577, 09/20/2004), which consists in the fact that on the 1st, 10th, 21st day from the moment of the development of ischemic stroke, the average level of CD95 lymphocytes is determined,%, when the level of CD95 lymphocytes on the 1st day is more than 7.2 ± 2.8% and return to normal on the 21st day, a favorable outcome is predicted, when the level of CD95 lymphocytes is above 7.2 ± 2.8% and maintaining an average the level of the indicator on the 21st day - they predict a favorable prognosis for life and unfavorable for the restoration of neurological deficit, and when the average value of CD95 lymphocytes is higher than 29.12 ± 3.1%, an unfavorable prognosis for life is predicted. The disadvantage of this method is the incomplete assessment of the processes aggravating the course of ischemic stroke and the rehabilitation period, in particular endothelial dysfunction and oxidative stress.

Closest to the claimed method from the sources of information described in the prior art is a method for diagnosing the severity of ischemic stroke (RF Patent No. 2238566; G01N 33/577, 10/20/2004), which consists in determining the level of antigen-positive cells in venous blood by immunophenotyping ( CB95 lymphocytes) as the percentage of fluorescent cells when looking at 200 lymphocytes minus the percentage of fluorescent cells observed in the negative control preparation. 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%, an ischemic stroke is severe.

The CD95 molecule (Fas / APO-1) belongs to the family of tumor necrosis factor alpha, which regulates the process of programmed cell death (apoptosis). Therefore, lymphocytes expressing the Fas receptor of apoptotic death are CD95 +. Thus, the disadvantage of this method is the determination of lymphocytes in a state of apoptosis without taking into account the fact that oxidative stress or endothelial dysfunction during ischemic stroke can trigger a cascade of changes not related to the launch of programmed cell death (Huot, J. SAPK2 / p38 - dependent F-actin reorganization regulates early membrane blebbing during stress-induced apoptosis / J. Huot, F. Houle, S. Rousseau // J. Cell. Biol. - 1998. - Vol.143, No. 5. - P.1361-1373), and, therefore, an assessment of the content of CD95 + lymphocytes cannot give a complete picture of the severity of the condition of patients with ischemic insu tom and hypertension.

Recently, an actively studied marker of oxidative stress, the functional state of the vascular wall and the immune system is bleeding (protrusion) of the outer plasma membrane of lymphocytes, which is a consequence of changes in membrane-cytoskeletal interactions due to the influence of stimuli of various origins (apoptosis, activation, oxidative stress) on the cell ( Egorova A.B. Damage to the cytoskeleton and cell membranes during apoptosis / A.B. Egorova, Yu.A. Uspenskaya // Advances in modern biology. - 2001. - T. 121 - No. 5. - S. 502-510).

The objective of the invention: to reduce disability and mortality by increasing the reliability of determining the severity of the condition of patients with ischemic stroke and arterial hypertension and providing the possibility of timely adequate therapy.

The task is carried out due to the fact that after centrifugation of the patient’s peripheral blood on a density gradient of ficoll-verographin ρ = 1.077 using phase contrast microscopy, 10 lymphocytes are counted in 10 fields of view with identification by vesiculation of the outer cytoplasmic membrane; the degree of severity of peripheral blood lymphocyte bleeding is determined by the ratio of lymphocytes with an intact membrane and in the state of terminal blebing: when the values of lymphocytes with an intact plasma membrane are more than 60% and in the state of terminal blebing from 10% to 30%, the severity of blebing is regarded as grade I; with values of lymphocytes with an intact membrane from 41% to 60% and in a state of terminal blebbing from 31% to 50% - II degree; with the values of lymphocytes with an intact membrane from 21% to 40% and in the state of terminal blebbing from 51% to 60% - III degree; when the values of lymphocytes with an intact membrane are from 10% to 20% and in the state of terminal blebing more than 60% - IV degree; the severity of blebing corresponds to the severity of the condition of a patient with ischemic stroke and arterial hypertension.

The method is as follows. A peripheral blood sampling of 5 ml is carried out with a thick needle from the patient's elbow vein into a plastic test tube with risks containing 0.15 ml of heparin; then thoroughly mix the blood with heparin in vitro. It is acceptable to store blood tubes in the refrigerator at + 5 ° C for 3-4 hours. In laboratory conditions, repeated resuspension of blood is carried out in vitro.

To isolate the lymphocytic fraction of peripheral blood, a density gradient of ficoll-verographin is used. To do this, add 2 ml of ficoll-verographin (Pharmacia hemical, Uppsala, Sweden) with a density of ρ = 1.077 into a separate plastic tube, after which 4 ml of resuspended blood is carefully layered on the wall of the tube carefully, slowly with a dispenser-verographin, and centrifuged at room temperature for 15 minutes at a speed of 3000 rpm.

After centrifugation at the interface, a cell ring is collected using a Pasteur pipette into a separate plastic tube. A tube with an isolated cell ring is centrifuged at room temperature for 5 minutes at a speed of 3000 rpm. After centrifugation, the supernatant is removed, 500 μl of PBS (phosphate buffer) or Hanks solution is added to the precipitate, and the suspension with a large tip is carefully resuspended.

Before microscopy, 50 μl of a cell suspension is applied to a previously defatted glass slide, and a small amount of emersion oil is applied to the coverslip.

Microscope in 10 fields of view using a phase-contrast nozzle; magnification × 900. In the field of view of the microscope, lymphocytes are visible (round cells of 9-10 microns in size).

According to the morphology of the cytoplasmic membrane, the following types of lymphocytes are differentiated: 1) intact cells (with a visually unchanged plasma membrane; round, flat surface); 2) cells in the state of initial blebing (small vesicles on the membrane - up to 1/3 of the radius of the cell); 3) cells in a state of terminal blebing (large membrane bubbles - more than 1/3 of the cell radius); 4) necrotic lymphocytes.

In figure 1. a photograph of a lymphocyte with protrusions of the cytoplasmic membrane in the lower pole of the cell (blebbing), phase contrast microscopy, magnification x900.

A study of the plasma membrane of peripheral blood lymphocytes revealed a predominance of lymphocytes in the state of blebing on the 1st day of hospitalization in the blood of patients with hypertension complicated by ischemic stroke, by 70% compared with the lymphocyte content in the state of bleeding in the same patients on the 20th day hospitalization (35%). The peripheral blood of patients with hypertension, not complicated by stroke, also contained lymphocytes in the state of blebing, which amounted to 20.9% of all identified lymphocytes by a change in the outer cytoplasmic membrane. In figure 2. a diagram of the content of lymphocytes with an intact membrane is presented (30% of all identified categories of lymphocytes on the 1st day of hospitalization and 65% on the 20th day of hospitalization), lymphocytes in the state of terminal blebbing (on the 1st day - 48%; by 20 day (15%), lymphocytes in the state of initial blebing (day 1 - 25%; day 20 - 20%).

In figure 3. a diagram of the content of lymphocytes in the state of bleeding in patients with hypertension without vascular catastrophes (control group) is presented: cells with an intact membrane - 83.7%; in the state of initial blebbing - 9.7%; in state of terminal blebing - 6.6%; necrotic lymphocytes - 1%.

Taking into account the obtained differences in the ratio of the lymphocyte content of the identified categories in patients with hypertensive disease complicated by stroke, on the 1st and 20th days of hospitalization and patients with hypertension without stroke, a scale for assessing the severity of peripheral blood lymphocyte bleeding was developed, which indirectly reflects the severity of the patient's condition (Table. one).

Table 1
Lymphocyte bleeding severity scale Identified cell category The number of lymphocytes,% IV degree III degree II degree I degree Intact plasma membrane 10-20 21-40 41-60 > 60 Terminal blebbing > 60 51-60 31-50 10-30

The severity of blebing in the peripheral blood lymphocyte population was significantly (p <0.05) higher in patients on the 1st day of hospitalization compared to patients in the hospital for 20 days and the control group. At the same time, on the 1st day of hospitalization, the severity of bleeding, as a rule, was III or IV, while in the future there was a tendency to a decrease in the severity of blebing by 7 days of hospitalization to grade II and by 20 days of hospitalization to grade I. The control group was dominated by patients with I degree of lymphocyte bleeding.

High correlation ratios (0.87) were found between the content of bleeding peripheral blood lymphocytes and the highly sensitive and specific humoral marker of sPECAM-1 endothelial dysfunction (Table 2). To calculate the correlation coefficient, Pearson's parametric correlation analysis was used. Correlation relations of medium strength were revealed between the content of lymphocytes in the state of blebing and a violation of the hemostatic system, blood lipid spectrum (0.53), as well as with the severity of the patient's condition (see clinical examples). It follows that lymphocyte bleeding reflects a wide range of pathological conditions of patients associated with endothelial dysfunction and oxidative stress due to ischemia. Therefore, the severity of lymphocyte bleeding can be considered as a fairly reliable and affordable way to determine the severity of the condition of patients with ischemic stroke and arterial hypertension.

table 2
Correlation analysis between lymphocyte bleeding and a humoral marker of endothelial dysfunction Studied parameter Correlation indicator Lymphocyte Bleeding Degree Lymphocyte Bleeding Degree one SPECAM-1 concentration 0.871

We give examples - extracts from the medical history.

Example 1

Patient M., 57 years old, and / No. No. 3990-2005. ONMK by ischemic type in the pool of the right midbrain artery with left-side hemiparesis. Background: Hypertension III, risk 4.

From the anamnesis: about 15 years suffering from hypertension, the maximum numbers of blood pressure 180/100 mm Hg, "average" blood pressure 150/95 mm Hg I did not receive regular antihypertensive therapy. He took medications (capoten, corinfar) as needed (severe headache, nausea, dizziness, "flies before the eyes").

Received in serious condition 1 hour after the onset of neurological symptoms.

At the time of admission: blood pressure 210/100 mm Hg, sinus rhythm with a heart rate of 80 per min, left-side hemiparesis, left facial hypesthesia.

In the biochemical analysis of blood: hypercholesterolemia (8.3 mmol / liter), hyperfibrinogenemia (6.22), severe thrombinemia, inhibition of fibrinolysis.

According to the content of blebbing lymphocytes, a 4 degree of severity of blebbing is diagnosed, which corresponds to 4 severity of the condition.

On the 7th day of hospitalization: against the background of standard neurotropic and antihypertensive therapy (perindopril 4 mg 1 tablet × 2 times / day, hypothiazide 12.5 mg / day), moderate severity, blood pressure 130/80 mm Hg, heart rate 75 per minute, the strength in the left hand is restored (5 points), in the left leg - 3 points.

According to laboratory tests: 3 severity of the condition according to the severity of lymphocyte bleeding.

20 days of hospitalization: satisfactory condition, neurological symptoms regressed, blood pressure 130/70 mm Hg, heart rate 70 per min.

Laboratory indicators: 2 severity of the condition (according to the severity of peripheral blood lymphocyte bleeding).

These results indicate the reversibility of endothelial dysfunction, a decrease in the level of oxidative stress during pathogenetic antihypertensive therapy, and also that the repair period after the action of oxidative stress is not limited to 20 days of hospitalization.

Example 2

Patient S., 52 years old, and / b No. 30482-2005. ONMK by ischemic type in the pool of the right midbrain artery with left-side hemiparesis. Background: Hypertension III, risk 4.

From the anamnesis: hypertension for more than 10 years, maximum blood pressure 160/100 mm Hg, blood pressure "average" 130/80 mm Hg I regularly took enalapril at a dose of 10 mg 1 tab. × 2 times / day.

Upon admission to the hospital: blood pressure 140/90 mm Hg, sinus rhythm with a heart rate of 76 per minute, headache, left-side hemiparesis, left hypesthesia, the left nasolabial fold is smoothed.

Biochemical analysis of blood: dyslipidemia due to an increase in the concentration of β-lipoproteins; fibrinogenemia (4.66 mg / l), impaired fibrinolysis.

Laboratory indicators: 4 degree of severity of blebbing (= severity of the patient's condition).

Conducted standard neurotropic therapy, hypotensive - perindopril 4 mg 1 table. per day, hypothiazide 12.5 mg / day.

7 days of hospitalization: a state of moderate severity. There is a decrease in strength in the left lower limb to 4 points, blood pressure 120/80 mm Hg, heart rate 70 per min.

Laboratory: 2 severity of the condition according to the severity of blebbing.

20 days of hospitalization: satisfactory condition, neurological symptoms completely regressed, blood pressure 120/70 mm Hg, heart rate 70 per min.

According to laboratory indicators: 1 severity of the condition according to the severity of blebbing.

Thus, after the recovery of the patient, neurological symptoms regressed.

Example 3

Patient B., 60 years old, and / b No. 31687-2004. Repeated ONMK by ischemic type in the pool of the right midbrain artery with left-side hemiparesis. Background: hypertension III, risk 4. Dyscirculatory encephalopathy III (mixed genesis: hypertensive, dysmetabolic).

Received in extremely serious condition: stunning I-II art., Contact difficult, dysarthria, gaze to the right, left-side hemiparesis; HELL 180/100 mm Hg, sinus rhythm with a heart rate of 90 per min.

According to relatives, he suffers from hypertension for about 20 years, blood pressure maximum 220/120 mm Hg, blood pressure "average" 160/100 mm Hg The previous ONMK in 2003 against the background of HELL 220/120 mm Hg I did not monitor BP, antihypertensive drugs (Adelfan) were rarely taken.

Blood biochemical analysis: hypercholesterolemia (8.7 mmol / liter), hyperfibrinogenemia (7.2 mg / liter), hypertrombinemia, severe inhibition of fibrinolysis.

Laboratory: 4 degree of severity of blebbing.

Intensive neurotropic therapy was carried out, infusion cardiovascular (polarizing mixture with magnesia), enalapril 20 mg 1 tablet was established through an established gastric tube. × 2 times / day.

7 days of hospitalization: negative dynamics of the patient’s condition, stunning III stage, an increase in cerebral symptoms, severe endotoxemia, blood pressure 140/100 mm Hg, heart rate 100 per minute, deep left-side hemiparesis.

Laboratory: 4 degree of severity of blebbing.

20 days of hospitalization: HELL 140/90 mm Hg, heart rate 66 per minute, stunning III art., The eye does not fix, the previous focal and cerebral symptoms, no meningeal signs.

Laboratory: 4 degree of severity of blebbing.

On the 23rd day of hospitalization, the patient died. Pathological and anatomical diagnosis: Repeated ONMK by ischemic type. A heart attack up to 2/3 of the volume of the right hemisphere in the temporal-occipital region. Cerebral edema, dislocation of the brain stem. Generalized atherosclerosis of blood vessels.

In the patient’s case, initially and during inpatient treatment, a degree of severity of peripheral blood lymphocyte bleeding was diagnosed, which corresponded to the patient’s serious condition.

Using the proposed method makes it possible to efficiently and at low cost to assess the severity of the condition of patients with hypertension complicated by ischemic stroke, which makes it possible to start early pathogenetic hypotensive therapy aimed at correcting blood pressure and endothelial dysfunction, oxidative stress, as well as to evaluate the dynamics of patient recovery and the effectiveness of the therapy in terms of the severity of peripheral blood lymphocyte bleeding is ultimately It reduces the risk of disability and mortality by improving the reliability of determining the degree of severity of the patient with ischemic stroke and hypertension, and to be able to conduct timely adequate therapy.

Claims (1)

A method for diagnosing the severity of the condition of patients with hypertension complicated by ischemic stroke, including the collection of peripheral blood from the patient's ulnar vein in a volume of 5 ml, characterized in that after centrifugation of the patient's peripheral blood on a density gradient ficoll-verographin ρ = 1,077, using phase-contrast microscopy counts in 10 fields of view 100 lymphocytes with identification by vesiculation of the outer cytoplasmic membrane; the degree of severity of peripheral blood lymphocyte bleeding is determined by the ratio of lymphocytes with an intact membrane and in the state of terminal blebing: when the values of lymphocytes with an intact plasma membrane are more than 60% and in the state of terminal blebing from 10 to 30%, the severity of blebing is regarded as grade I; at values of lymphocytes with an intact membrane from 41 to 60% and in a state of terminal blebing from 31 to 50% - II degree; when the values of lymphocytes with an intact membrane are from 21 to 40% and in the state of terminal blebbing from 51 to 60% - III degree; with values of lymphocytes with an intact membrane from 10 to 20% and in a state of terminal blebing greater than 60% - IV degree; the severity of blebbing corresponds to the severity of the patient's condition.

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