RU2324939C1 - Method of evaluation of degree of endothelial dysfunction - Google Patents

Abstract

FIELD: medicine; cardiology; neurology.

SUBSTANCE: blood sample is centrifuged at density gradient ficoll-verografin ρ=1.077 and the number of cast-off endotheliocytes per 100 lymphocytes is calculated in phase-contrast microscopy, with simultaneous identification of 100 lymphocytes by vesiculation of the external cytoplasmic membrane. The ratio of membrane-released microparticles is determined and the summing titer of antibodies to sPECAM-1 and phospholipids is calculated. The degree of endothelial dysfunction is diagnosed, depending on total titer of sPECAM-1 and antibodies to phospholipids.

EFFECT: enables determination of humoral markers by registration of cell markers, evaluation of degree of endothelial dysfunction, which provides early pathogenic therapy.

3 dwg, 8 tbl, 3 ex

Description

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

The pathogenesis of ischemic stroke is based on a persistent change in the normal functioning of the vascular wall, which leads to an increase in its resistance, a decrease in elasticity, and an increase in smooth muscle cells. All this not only persistently narrows the lumen of the arteries, but also leads to aggravation of damage to the endothelium.

In physiological conditions, the vascular endothelium provides adequate vasodilation, controls the growth of smooth muscle cells, inhibits platelet activation and adhesion, inhibits blood coagulation, and inhibits inflammatory processes. Endothelium plays an active role in cell-mediated immunity, which is caused, in particular, by the interaction of endotheliocytes with T-lymphocytes. Such interactions are determined by the presentation of antigens by endotheliocytes, the diapedesis of lymphocytes through the endothelium to the inflammation site, their binding to endotheliocyte receptors (Cines, DB Endothelial cells in physiology and in the pathophysiology of vascular disorders. / DB Cines, ES Pollak et al. // Blood - 1998 - - Vol. 91 - N. 10 - P. 3527-3561).

An increase in vasoconstriction, a decrease in the elasticity of the vascular wall, and the progression of atherothrombosis form chronic ischemia of organs and tissues, which is an inducer of apoptosis of cells not only of organs and tissues, in which conditions are created for the formation of an imbalance between the need for oxygen and its delivery, but also endotheliocytes themselves. Under the conditions of acute vascular catastrophe, when aggravation and progression of endothelial dysfunction and the formation of acute ischemic stress occur, along with necrotic changes in the affected organs, the processes of massive apoptosis of cells of the affected organ and endotheliocytes (Flotats, A. Non-invasive in vivo imaging of myocardial apoptosis and necrosis. / A.Flotats, I. Carrio // Eur J Nucl Med Mol Imaging - 2003 - V.30 - N.4 - P. 615-630).

Potential humoral markers of endothelial dysfunction are nitric oxide deficiency, adhesion molecules, insulin-like growth factor, and angiotensin II. At the present stage, there are some difficulties associated with determining the concentration of markers of endothelial dysfunction, such as nitric oxide. This is due to the extreme instability of the NO molecule. According to biochemical criteria, NO-producing activity can only be estimated indirectly - by recording NO metabolites, which approximately reflects reality. In this regard, the majority of researchers studying endothelial dysfunction in various cardiovascular diseases propose to evaluate the concentration of adhesion molecules as reliable markers of endothelial dysfunction. According to modern concepts, endothelial dysfunction directly correlates with a sharp increase in the expression of sPECAM-1 adhesion molecules (Kurbanov, R.D. Humoral markers of endothelial dysfunction in essential hypertension. / R.D. Kurbanov, M.R. Eliseeva, PPTursunov, etc. // Cardiology - 2003 - No. 7 - P.61-64).

The role of RESAM is transmembrane signal transmission of intercellular interaction, which, in particular, leads to thrombosis, leukocyte diapedesis through the vascular wall to the site of inflammation. A 2001 article by Neta Ilan et al. Reports that RESAM-1 is capable of enhancing apoptotic processes in the endothelium (Ilan, N. RESAM-1 shedding during apoptosis generates a membrane-anchored truncated molecule with unique signaling characteristics. / N Ilan, A. Mohsenin, L. Cheung et al // The FASEB journal - 2001 - V.15 - P.362-370). Antibodies to RESAM show high sensitivity and specificity to endothelial cells in normal and pathological conditions (endothelial dysfunction). To determine the titer of sPECAM-1 in blood plasma, the ELISA method (human sPECAM-1 ELISA, Bender Med Systems GmbH, Vienna, Austria) is used.

Another marker of endothelial dysfunction, proposed and patented by Krasnova L.G. et al. (RF Patent No. 2234094; G01N 33/49, 08/10/2004), is the determination of the amount of desquamated endotheliocytes in peripheral blood by the method of Hladovec J. (Hladovec, J. Circulating endothelial cells in acute myocardial infarction and angina pectoris. / J. Hladovec, I. Prerovsky, V. Stanek, J. Fabian // Klin Wochenschr - 1978 - No. 56 - P.1033-1036). According to the method proposed by the authors, endothelial dysfunction is diagnosed with an indicator value exceeding 2.77 · 10 ⁵ in 1 liter of plasma. The disadvantage of this method is the inaccuracy of the Hladovec J. method already indicated by the author, which consists in determining the normal amount of endotheliocytes circulating in the peripheral blood, while endothelial dysfunction is not confirmed instrumentally. The authors improved the method by proposing that prior to blood sampling, a positive pressure in the shoulder region should be created for 4 minutes, exceeding systolic blood pressure by 40-50 mm Hg. When comparing with the initial number of endotheliocytes, according to the authors' interpretation, latent endothelial dysfunction can be detected. However, given the technically complex procedure, which in most clinics should be performed without the participation of a doctor, the time costs and the duration of the Hladovec J. method itself, the proposed method seems difficult to apply in the conditions of clinical and laboratory studies.

Dignat-George F. et al. in 2003 published the results of a study of circulating endotheliocytes in the blood in patients with various diseases, including acute coronary syndrome and in patients after angioplasty. Using immunomagnetocytochemical binding of CD 146 (monoclonal antibodies responsible for intercellular interaction) with desquamated endotheliocyte receptors, the authors found that in acute coronary syndrome in 1 ml of blood an average of 7.5 circulating endotheliocytes is detected, while after angioplasty - from 6 up to 10 desquamated endotheliocytes. In the control group (healthy people), according to the authors, the average number of circulating endotheliocytes was less than 3 cells per 1 ml of blood (Dignat-George, F. Circulating endothelial cells: realities and promises in vascular disorders. / F.Dignat-George, J. Sampol, G. Lip, AD Blann // Pathophysiol Haemost Thromb - 2004 - No. 33 - P.495-499). The method is expensive, which limits its use in clinics and clinics.

Recently, an actively studied marker of oxidative stress is the plasma cell membrane blebbing. It should be especially noted that peripheral blood lymphocytes can be considered as an accessible object for studying the severity of endothelial dysfunction and the effect of oxidative stress, which meet the following necessary conditions: they are highly sensitive to the action of apoptogenic and activation factors, they are a substrate of the immune response (aseptic inflammation and the formation of immune synapses); the lymphocyte population is dynamic due to the relatively short stay in the systemic circulation of lymphocytes; participate in the initiation of apoptosis of target cells and the formation of endothelial dysfunction.

The bleeding of the plasma membrane of lymphocytes, especially at its initial, reversible stages, may result in the release of membrane microparticles formed by desquamation of vesicles - blebs. However, the peripheral blood also contains membranes released active microparticles of leukocyte, platelet, endothelial origin. Recently, membranes released microparticles play a significant role in the development of typical pathological conditions such as inflammation, hypercoagulation, as well as the presentation of antigens. The higher the degree of endothelial dysfunction, the higher the membrane content of released microparticles in the peripheral blood (Preston, RA Effects of severe hypertension on endothelial and platelet Microparticles. / RAPreston, W.Jy, JRAPreston et all // Hypertension. - 2003 - No. 41 - P.211-213).

Endothelial dysfunction is accompanied by an increase in the titer of antibodies to phospholipids. Antiphospholipid antibodies have the ability to cross-react with components of the membranes of endothelial cells and extracellular matrix, including phosphatidylserine, inhibit prostacyclin synthesis, stimulate procoagulant activity. The binding of antiphospholipid antibodies and endothelial cells leads to overexpression of cell adhesion molecules and apoptosis of endothelial cells. To determine the total number of antibodies to phosphatidylserine, phosphatidyl-inositol, cardiolipid and phosphatidyl acid, the Anti-phospholipid Screen IgG / IgM method, ORGENTEC, Germany, is used.

The Anti-phospholipid Screen method does not allow to diagnose antiphospholipid syndrome, but antibodies to phospholipids are an indirect marker of endothelial dysfunction, a violation of the rheological properties of the blood. However, this method is also expensive and does not allow blood testing in one patient (the kit is designed for simultaneous study of blood plasma in 96 patients). However, an increase in the content of antibodies to phospholipids and sPECAM-1 is the most accurate modern marker of endothelial dysfunction.

Object of the invention: increasing the sensitivity of evaluating the effectiveness of antihypertensive therapy.

The task is carried out due to the fact that after centrifugation of the patient’s blood on a density gradient ficoll-verographin ρ = 1.077 using phase contrast microscopy, the number of desquamated endotheliocytes per 100 lymphocytes is counted in 10 fields of view and the 100 lymphocytes are simultaneously identified by vesiculation of the outer cytoplasmic membrane, and also calculate the coefficient of membrane content of released microparticles using the Adobe Photoshope CS program, then calculate the total titer of sPECAM-1 and antibodies to phosphorus olipids according to the formula sPECAM + a / TFL = 6.9913 + 0.1043 × c × (b + m), where sPECAM is the titer of sPECAM-1 intercellular adhesion molecules (ng / ml), and / TFL is the titer of antibodies to phospholipids ( U / ml), s is the number of desquamated endotheliocytes (pcs.), B is the degree of lymphocyte bleeding (estimated value), m is the membrane content coefficient of released microparticles (estimated value), 6.9913 and 0.1043 are calculated correction factors; with estimated values of the total titer of sPECAM-1 and antibodies to phospholipids 7-8, 1 degree of endothelial dysfunction is diagnosed; 8.15-10.0 - 2 degree; 10.5-17.0 - 3 degree; 17.5 and above - 4 degree; intermediate values are evaluated as a transitional degree of endothelial dysfunction; an extremely severe endothelial lesion corresponds to an estimated value of 26.5 or higher.

The method is as follows: the venous blood is taken in a volume of 5 ml by a thick needle from the patient's elbow vein into a plastic tube with rhiza 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. For this, 2 ml of ficoll-verographin (Pharmacia hemical, Uppsala, Sweden) with a density of ρ = 1.077 is added to a separate plastic tube; then, carefully, slowly, 4 ml of resuspended blood are layered onto the ficoll-verographin on the wall of the tube 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; x900 magnification. Endothelial cells (polygonal cells with a size of 20-50 microns), lymphocytes (round cells with a size of 9-10 microns), membranes released microparticles up to 0.3 microns (a hallmark of platelets) are visible in the field of view of the microscope.

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.

The calculation is carried out per 100 lymphocytes.

By the content of lymphocytes with an intact membrane and in the state of terminal blebing, the severity of blebing is determined (Table 1)

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

Using the Adobe Photoshope CS program, quantitatively assess the content of images of membranes of released microparticles in the field of view in pixels. Then calculate the percentage image of the microparticles in relation to the entire field of view and translate the values into the coefficient of the membrane content of the released microparticles (table 2).

table 2 Scale of interpretation of the content of membranes of released microparticles in the lymphocytic fraction of peripheral blood The coefficient of the membrane content of released microparticles Membrane content of microparticles released in percent relative to the field of view 0 <0.1% 1 (low content) 0.1-3% 2 (average content) 4-9% 3 (high content) 10-19% 4 (very high content) ≥20%

The relationship between individual indicators and their compositions can be assessed using a correlation analysis between the studied cellular (lymphocyte bleeding, membranes released microparticles, desquamated endotheliocytes) and humoral (sPECAM-1, antibodies to phospholipids) indicators of endothelial dysfunction (Table 3).

Table 3 Correlation analysis between cellular and humoral markers of endothelial dysfunction Studied parameter Correlation indicator (b) (m) (from) sPECAM a / TFL sPECAM x a / TFL sPECAM + a / TFL b + m b + m + s (b + m) × c bl one mvm 0.771 one se 0.853 0.918 one sPECAM 0.871 0.897 0.967 one a / TFL 0.817 0.879 0.954 0.981 one sPECAM × a / TFL 0.803 0.833 0.927 0.967 0,994 one sPECAM + a / TFL 0.847 0.892 0.964 0,995 0,995 0.986 one bl + mvm 0.901 0.945 0.917 0.921 0.871 0.832 0,900 one bl + mvm + se 0.878 0.941 0,996 0.970 0.951 0.922 0.965 0.945 one (b + m) × s 0.897 0.932 0.984 0.971 0.963 0.942 0.972 0.938 0,991 one bl (b) - the severity of blebbing (estimated value)
mvm (m) - coefficient of membrane content of released microparticles (estimated value)
se (s) - number of desquamated endotheliocytes (pcs.)
a / tFL - antibodies to phospholipids (U / ml)
sPECAM - cell-cell adhesion molecule sPECAM-1 (ng / ml)

Close correlation is observed between the individual analyzed indicators. The most tight ties are complex, formed by adding and / or product of certain indicators known and proposed by us. Cellular markers of the pathology of homeostasis and endothelial dysfunction are closely correlated with humoral markers.

The highest correlation coefficient (0.97) is observed between the sum of the known indicators sPECAM + a / tFL and our generalized indicator in the form of (b + m) × s.

In figure 1. a graph of the functional relationship between the sum of sPECAM-1 and antibodies to phospholipids and the generalized indicator [(degree of blebbing + coefficient of membrane content of released microparticles) x number of desquamated endotheliocytes] is presented. According to the revealed linear relationship between these parameters, the total titer of sPECAM-1 and antibodies to phospholipids can be calculated by the formula: sPECAM + a / tFL = 6.9913 + 0.1043 × c × (b + m), where c is the number of desquamated endotheliocytes , b is the degree of severity of blebbing, m is the coefficient of the content of membranes of released microparticles, and / TFL - antibodies to phospholipids; calculated correction factors are 6.9913 and 0.1043.

In table 4. The calculation of the adequacy of the method according to the Fisher criterion (F) is presented.

Table 4 Calculation of the adequacy of the method Indicators Observed Values Estimated Values Average 17.33 17.34 Dispersion 19.55 18.47 F calculated 1.06 F tabular 1,59

Fisher’s calculated criterion is less than tabular for the accepted significance level, that is, the proposed method adequately describes the sum of sPECAM-1 and antibodies to phospholipids.

The method for calculating the total titer of sPECAM-1 and antibodies to phospholipids allows rapid diagnostics of the degree of endothelial dysfunction according to the scale developed by us (Table 5).

Table 5 The scale for translating calculated values of the sum of humoral markers into the degree of endothelial dysfunction Degree of endothelial dysfunction Estimated total titer (estimated value) sPECAM + a / TFL = 6.9913 + 0.1043 × c × (b + m) 1 degree 7-8 2 degree 8.15-10.0 3 degree 10.50-17.0 4 degree ≥17.5

In this case, an extremely severe endothelial lesion corresponds to a calculated index of ≥26.5.

The humoral markers sPECAM-1 and antibodies to phospholipids display endothelial dysfunction. However, each indicator individually shows the degree of increase in the content of biologically active substances associated with endothelial dysfunction. So, the sPECAM-1 titer is the concentration of cell adhesion molecules responsible for the connection with endothelium and diapedes through it. Antibodies to phospholipids - dysfunction of cell membranes, hypercoagulation (correlation coefficient 0.46) and violation of the lipid spectrum of the blood (correlation coefficient 0.59).

In figure 2. presents a graph of the ratio of the content of the total titer of sPECAM-1 and antibodies to phospholipids (Z axis) with the number of desquamated endotheliocytes (Y axis) and the degree of severity of peripheral blood lymphocytes bleeding (X axis), the highest total titer of sPECAM-1 and antibodies to phospholipids corresponds to the largest the content of desquamated endotheliocytes and a high degree of severity of lymphocyte bleeding.

In figure 3. The graph of the ratio of the calculated titer of sPECAM-1 humoral markers and antibodies to phospholipids (Y axis) with the degree of endothelial dysfunction (X axis) is presented, the higher the total titer of sPECAM-1 and antibodies to phospholipids, the higher the degree of endothelial dysfunction.

Thus, the most reliable way to diagnose endothelial dysfunction is to determine the titer of humoral markers (sPECAM-1 and antibodies to phospholipids) in the plasma of peripheral blood, which reflect the pathology of the functioning of the endothelium in the aggregate form, including hypercoagulation, dyslipidemic, immune shifts; increased adhesive properties of the endothelium. In our study, we found high correlation ratios (0.97) between the total titer of humoral markers of endothelial dysfunction (sPECAM-1 and antibodies to phospholipids) and the combination of cellular markers of endothelial dysfunction [(severity of blebbing + membrane content score of released microparticles) × number of desquamated endocytes ]. According to our proposed method, the total content of humoral markers is calculated by the totality of cell markers:

sPECAM + a / TFL = 6.9913 + 0.1043 × c × (b + m)

The cost of determining markers of endothelial dysfunction (hereinafter calculations in 2006 prices) in the plasma of peripheral blood for one patient is 2000 rubles (taking into account the cost of the reagent, consumables, depreciation of equipment). A set of reagents for determining sPECAM-1 and antibodies to phospholipids, costing 947 euros, is designed for simultaneous analysis of blood plasma in 96 people. The cost of calculating the total concentration of these humoral markers of endothelial dysfunction by cell markers is 30 rubles per patient (taking into account the reagent, consumables, depreciation of equipment). Our proposed method allows you to save time and money, as well as to determine the presence of endothelial dysfunction individually in one patient.

We give examples - extracts from the medical history.

1. Patient 3., 56 years old, was admitted to the neurological department with a diagnosis of stroke of ischemic type in the pool of the right midbrain artery with left-side hemiparesis. Background: hypertension III, risk 4.

On admission: a serious condition, blood pressure 220/140 mm Hg, sinus rhythm with a heart rate of 80 per minute.

According to computed tomography of the brain (CT), the focus of ischemic stroke is determined near the body of the right lateral ventricle and in the parietal lobe of 53 × 30 mm in size.

Biochemical analysis of blood: dyslipidemia due to an increase in the content of low density lipoproteins, hypercoagulation.

Conducted standard neurotropic therapy. Antihypertensive therapy included perindopril 4 mg, 1 tab. × 2 times a day, hypothiazide 12.5 mg in the morning.

After 7 days: moderate severity, blood pressure 130/70 mm Hg, heart rate 70 per min, strength in the left hand restored - 5 points, in the left leg - 3 points, tendon reflexes on the left more than on the right.

After 20 days: satisfactory condition, blood pressure 130/80 mm Hg, heart rate 68 min, nerve functions restored.

The parameters we analyze are presented in Table 6.

Table 6 Indicators of cellular and humoral markers of endothelial dysfunction and patient severity b 1 day b 7 days b 20 days m 1 day m 20 days from 1 day from 20 days sP 1 day sP 20 days a / tFL 1 day a / tFL 20 days four 3 2 four one 17 one 15.78 5.68 5.40 1,62 b - severity of blebbing (estimated value)
m - coefficient of membrane content of released microparticles (estimated value)
C is the number of desquamated endotheliocytes (pcs.)
sP - sPECAM-1 (titer, ng / ml)
a / tFL - antibodies to phospholipids (titer, U / ml)

We calculate the total indicator of humoral markers based on the values of cell markers of endothelial dysfunction:

sPECAM + a / TFL = 6.9913 + 0.1043 × c × (b + m) = 6.9913 + 0.1043 × 17 (4 + 4) = 21.18

The obtained calculated value corresponds to the experimental one and belongs to 4 degrees of endothelial dysfunction on the 1st day of hospitalization of the patient.

On the 20th day of hospitalization in a patient, a degree of endothelial dysfunction is diagnosed according to the calculated indicator of humoral markers. The results indicate the effectiveness of the antihypertensive drug on the vascular wall and rheological properties of blood, while the restoration of metabolic processes at the level of the vascular wall and brain tissue continues for several months after inpatient treatment.

2. Patient K., 53 years old, was admitted with a diagnosis of stroke of ischemic type in the basin of the left midbrain artery with right-sided hemiparesis, motor aphasia. Background: hypertension III, risk 4.

On admission: a serious condition, blood pressure 190/100 mm Hg, sinus rhythm with a heart rate of 84 per minute.

According to the CT scan of the brain, the focus of ischemic stroke is determined in the left hemisphere in the temporoparietal region measuring 25 × 46 mm.

Biochemical analysis of blood: hypercholesterolemia (8.4 mmol / L), hyperfibrinogenemia (6.8 mg / liter), inhibition of fibrinolysis.

Conducted standard neurotropic therapy (see above). Antihypertensive therapy included enalapril 20 mg, 1 tab. × 2 times a day, hypothiazide 12.5 mg in the morning.

After 7 days: moderate severity, blood pressure 130/80 mm Hg, heart rate 75 min, elements of motor aphasia, strength in the right hand - 4 points, in the right leg - 3 points, tendon reflexes on the right more than the left .

After 20 days: the condition is closer to satisfactory, blood pressure 130/80 mm Hg, heart rate 78 per min, residual effects of motor aphasia, strength in the right leg - 4 points, in the right hand - 5 points, impaired cognitive function.

The parameters we analyze are presented in Table 7.

Table 7 Indicators of cellular and humoral markers of endothelial dysfunction and patient severity b 1 day b 7 days b 20 days m1 day m 20 days from 1 day from 20 days sP 1 day sP 20 days a / tFL 1 day a / tFL 20 days four 3 2 3 one 23 10 17.54 6.56 6.24 3.56 b - severity of blebbing (estimated value)
m - coefficient of membrane content of released microparticles (estimated value)
c - number of desquamated endotheliocytes (pcs.)
sP - sPECAM-1 (titer, ng / ml)
a / tFL - antibodies to phospholipids (titer, U / ml)

The obtained calculated value (sPECAM + a / TFL = 6.9913 + 0.1043 × c × (b + m) = 23.78) corresponds to the experimental one and belongs to 4 degrees of endothelial dysfunction on the 1st day of hospitalization of the patient. On the 20th day of hospitalization in a patient, a transitional value between the 2nd and 3rd degree of endothelial dysfunction is determined by the calculated indicator of humoral markers. On the 20th day of hospitalization, the patient is diagnosed with grade 3 endothelial dysfunction. This can be interpreted as an insufficient effect of the applied antihypertensive agent on the restoration of endothelial function, which retains the risk of a new complication of hypertension, while the cardiovascular and neurotropic therapy had a sufficient reparative effect to restore blood supply to the affected brain focus, i.e. the risk of disability is low.

3. Patient A., 59 years old, was admitted with a diagnosis of stroke in ischemic type in the pool of the right midbrain artery with gross left-side hemiparesis. Background: hypertension III, risk 4.

On admission: a serious condition, blood pressure 160/100 mm Hg, sinus rhythm with a heart rate of 70 per minute; dysarthria, gaze to the right, deviation of the tongue to the left, paresis of the face to the left, gross left-side hemiparesis.

Due to the severity of the state of the CT scan of the brain was not performed.

Blood biochemical analysis: hypercholesterolemia (9.2 mmol / L), dyslipidemia, hyperfibrinogenemia (7.4 mg / liter), hypertrombinemia, inhibition of fibrinolysis.

Intensive neurotropic therapy was prescribed, through a gastric tube - enalapril 10 mg, 1 tablet × 2 times a day, hypothiazide 12.5 mg in the morning.

After 7 days: the condition is extremely serious, stunning III degree, blood pressure 150/90 mm Hg, heart rate 95 per min.

The patient died on the 13th day of hospitalization. Pathological and anatomical diagnosis: cerebral infarction in the right hemisphere, edema and dislocation of the brain stem. Generalized atherosclerosis of the arteries.

The parameters we analyze are presented in Table 8.

Table 8 Indicators of cellular and humoral markers of endothelial dysfunction and patient severity b 1 day b 7 days m 1 day m 7 days from 1 day from 7 days sP 1 day sP 7 days a / tFL 1 day a / tFL 7 days four four 3 four 25 26 16.09 19.49 9.15 9.53 b - severity of blebbing (estimated value)
m - coefficient of membrane content of released microparticles (estimated value)
C is the number of desquamated endotheliocytes (pcs.)
sP - sPECAM-1 (titer, ng / ml)
a / tFL - antibodies to phospholipids (titer, U / ml)

In the patient, on the 1st day of hospitalization, the calculated value (sPECAM + a / TFL = 6.9913 + 0.1043 × c × (b + m) = 25.24) corresponds to the experimental one and belongs to 4 degrees of endothelial dysfunction.

On the 7th day of hospitalization in a patient, an extremely severe endothelial lesion is diagnosed by the calculated indicator of humoral markers (28.69).

The results obtained correspond to the clinical picture: initially, the patient was admitted with a severe degree of endothelial dysfunction, severe damage to the nervous tissue. In the dynamics of the disease, endothelial dysfunction progressed, adequate perfusion of brain tissue was not restored, resulting in swelling and dislocation of the brain stem, followed by death.

Using the proposed method allows you to determine the content of humoral markers by registering cell, the method is specific, sensitive, low cost and makes it possible to start early pathogenetic therapy of endothelial dysfunction and associated pathological conditions, to evaluate the effectiveness of antihypertensive therapy in terms of the effect on restoration of endothelial function, to reduce the risk of developing complications of hypertension and disability against the background of developed ischemic insu ta.

Claims (1)

A method for assessing the degree of endothelial dysfunction, including the collection of peripheral venous blood from a patient in a volume of 5 ml, characterized in that after centrifugation of the patient’s blood on a density gradient of ficoll-verographin ρ = 1,077 using phase contrast microscopy, the number of desquamated endotheliocytes is counted in 10 fields of view per 100 lymphocytes and the simultaneous identification of 100 lymphocytes by vesiculation of the outer cytoplasmic membrane, and the coefficient of the content of membrane-released microparticles is also calculated c using the Adobe Photoshope CS program, then calculate the total titer of antibodies to phospholipids and sPECAM-1 according to the formula sPECAM + a / TFL = 6.9913 + 0.1043 · s · (b + m), where sPECAM is the titer of intercellular adhesion molecules sPECAM-1, ng / ml; a / tFL - titer of antibodies to phospholipids, U / ml; C is the number of desquamated endotheliocytes, pcs; b - severity of lymphocyte bleeding, estimated value; m is the coefficient of the content of membrane-released microparticles, the estimated value; 6.9913 and 0.1043 are calculated correction factors; with estimated values of the total titer of sPECAM-1 and antibodies to phospholipids 7-8, 1 degree of endothelial dysfunction is diagnosed; 8.15-10.0 - 2 degree, 10.5-17.0 - 3 degree; 17.5 and above - 4 degree; intermediate values are evaluated as a transitional degree of endothelial dysfunction; an extremely severe endothelial lesion corresponds to an estimated value of 26.5 or higher.

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