Method for studying thrombocytic aggregation in patients with arterial hypertension at metabolic syndrome

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: the suggested studying should be carried out on the glass simultaneously with several inductors by applying minimal inter-taking antilogarithms concentrations of aggregation inductors which correspond at double combination of inductors: ADP 5.0 x 10-8 M, adrenaline 3.0 x 10-9, collagen - dissolving the main suspension 1:8, thrombin 0.075 U/ml; at triple combination of inductors: ADP 10-9 M, adrenaline 10-9, collagen - dissolving the main suspension 1:9, thrombin 0.060 U/ml. The development of aggregation means thrombocytic activation in patients with arterial hypertension at metabolic syndrome. The method enables to evaluate the changes of thrombocytic functional state with combination of inductors more probably present in area of vascular lesion by applying minimal necessary concentrations that develops real conditions at hemostatic initiation in human vessels.

EFFECT: higher efficiency of studying.

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The invention relates to biology and medicine in the field of Hematology and can be used to assess platelet aggregation in conditions close to intravascular, in order to detect platelet activation in hypertensive patients with metabolic syndrome.

The closest to the invention is a method of determining platelet aggregation on a glass slide using a single inductor (Shitikova AS. Visual micromethod studies of platelet aggregation. In the book: Hemostasis. Physiological mechanisms, principles of diagnostics of the main forms of hemorrhagic diseases. Edited Yea, Lpparam, SPb.: 1999. 49 - 52. The described method does not allow to simulate the real conditions of platelet aggregation in the human body and its implementation does not provide the minimum vzaimootmenyaemy concentrations of inducers aggregation.

There is a method of study of platelet aggregation in aggregometry "Python" (USA) with a combination of inductors (Akopov SE, Martirosyan BORN About using combinations of inductors aggregation in estimating changes in platelet function in patients with disorders of cerebral circulation. // Circulation. 1988. - T, No. 4. P.47 - 49). The method allows to evaluate platelet aggregation with a combination of inductors on aggregometry. However,it can be applied not every laboratory because of the high cost of aggregometer, as well as the research costs of the plasma, significantly exceeding its expense when evaluating aggregation on the glass. In addition, the authors of the method was not intended to use the minimum vzaimootmenyaemy doses of aggregation inducers.

The purpose of the invention: using technically simple, affordable and quickly implementable techniques to assess changes in the functional status of platelets in a small volume of plasma with a combination of inductors, most likely present in the damage zone of the vessel.

The essence of the proposed method lies in the fact that after blood collection, its stabilization by sodium citrate 3.8 percent, the separation of blood into plasma and erythrocytes by centrifugation to obtain platelet-rich plasma (BTP) and is on a slide test for platelet aggregation with multiple inductors. Visual assessment of platelet aggregation under these conditions allows a small volume of 0.02 ml) of plasma and low volume coils (total volume of 0.02 ml) to simulate the real conditions of blood flow in humans and to assess changes in the functional status of platelets at a minimum vzaimootmenyaemye concentrations of inducers.

The inventive method is carried out as follows. After taking blood in sodium citrate 3.8% in the ratio of 9:1, it is centrifuged for 5 min at 100 rpm, to obtain the BTP. Part of the plasma is taken, and the remainder centrifuged at 3000 rpm for 20 min, receiving platelet-poor plasma (Betp). BTP standardise the number of platelets by dilution of the original BTP autologous sample Betp (up to 200·109/l).

The standardized volume of plasma is withdrawn from the calculation of 0.02 ml of plasma for each of the studied combination of inductors. The remaining volume of plasma can be used for other hematological and biochemical investigations.

Selected standardized volume of plasma on a glass slide cause of 0.02 ml of plasma and different pipette solutions multiple inductors in minimum vzaimootmenyaemy concentrations (figure 1 and 2) with a total volume equal to the volume of plasma. Volumes agonists are defined by the formula:

where n is the number of used simultaneously inductors aggregation (figure 1 and 2).

As agonists may use adenosine diphosphate (ADP), collagen, thrombin, epinephrine, and other inductors. Minimum vzaimootmenyaemye dose inductors always below standard (table 1 and 2). Glass wand mix plasma with inductors so that the liquid held a circle with a diameter of about 2 cm (figure 3). Shaking the glass in a circular motion in transmitted light illuminator on a black background is watched through a magnifying glass for the occurrence of aggregates. With the appearance of distinct units, the enlightenment of the solution and the adhesion of some units fixed platelet aggregation.

It should be noted that the study of the aggregation ability of blood platelets with multiple inductors simulates events that occur in blood flow in the damaged area of the vessel and the initiation of the process of hemostasis, because really in these conditions, platelets are activated by several agonists. For example, in hypertensive patients with metabolic syndrome often found regularities expressed more than in healthy people due to platelet activation functions.

Table 1

The standard dose of inductors, causing platelet aggregation when they are a stand-alone application
InductorsADP, MAdrenaline, MCollagen, the main breeding suspensionThrombin, IU/ml
Dose0,5·10-45·10-61:20,125

Table 2

Minimum vzaimootmenyaemye concentrations of inducers aggregation in healthy individuals and patients with arterial hypertension with metabolic syndrome Ave is double and triple combination
Category surveyedCombinations of inducers of platelet aggregation
ADP, MAdrenaline, MCollagen, the main breeding suspensionThrombin, IU/ml
with the same agonistwith two agonistswith the same agonistwith two agonistswith the same agonistwith two agonistswith the same agonistwith two agonists
Healthy people, n=2110-65·10-72,5·10710-71:41:50,1000,090
Sick people, n=1255,0·10-810-93,0·10-910-91:81:90,075to 0.060

From tables 1 and 2 it follows that the minimum causing aggregation dose inductors when their combined use is significantly lower than in standard conditions when using effects on platelets of the same agonist. At simultaneous presence in BTP three inductors, the dose required for aggregation, even lower than in the case of two inducto the RC. Moreover, in hypertensive patients with metabolic syndrome, these values are even lower, which indicates they expressed the activation of platelets. Simultaneous use of multiple inductors brings our understanding of the required minimum concentration of inducers in real conditions at the initiation of hemostasis in blood vessels of a person.

Example 1. Citizen N., 31 years old, was admitted to the sanatorium-resort treatment in the sanatorium. Ideonexus.com, Kursk. Upon examination and objective examination of pathological changes were observed. In the morning on an empty stomach was blood sampling for biochemical and hematological studies, does not produce deviations from accepted norms. Platelet aggregation with ADP, collagen, thrombin, ristomycin, H2O2and the adrenaline was within normal limits. Evaluation of the aggregation of blood platelets in a standardized number of platelets plasma (200·1009Tr/l) with a combination of inductors ADP + epinephrine, ADP + collagen, epinephrine

+ collagen, ADP + collagen + adrenaline and ADP + collagen + thrombin recorded its development. Minimum vzaimootmenyaemye concentration when using two inductors accounted for ADF - 10-6M, collagen - 1:4 dilution of the primary suspension, adrenaline - 2,5·10-7M, for thrombin - 0,100 u/m is. In case of simultaneous use of the three inductors minimum vzaimootmenyaemy concentration amounted to ADP - 5,0·10-7M, collagen 1:5 dilution of the primary suspension, adrenaline - 10-7M, thrombin - 0,090 u/ml lower concentrations of inducers never had examined platelet aggregation. Thus, the development of platelet aggregation in response only to vzaimootmenyaemye dose inductors typical of healthy humans suggest that the normal function of platelets in patients.

Example 2. Patient B., 53, in the dispensary of hypertension in the clinic MUSES GB NSR, Kursk and having as comorbidities of the metabolic syndrome were examined in a planned manner.

The survey revealed complaints of episodic headaches in the occipital region, flashing "flies" before the eyes associated with a rise in blood pressure. The patient also complained of overweight localized fat primarily in the belly.

At objective examination revealed Android obesity type II and Art. and the offset of the left border of the heart to the left by 1.5 cm Electrocardiographically revealed the presence of the patient hypertrophy of the left ventricle. Ophthalmologist diagnostical the patient the hypertensive angiopathy of the retina. Laboratory on the following revealed her impaired glucose tolerance, hyperlipidemia type IIB with mild hypercholesterolemia (5.8 mmol/l).

Platelet aggregation was initiated, the patient with a combination of inductors: ADP + epinephrine, ADP + collagen, adrenaline + collagen, ADP + collagen + epinephrine, ADP + collagen + thrombin. Minimum vzaimootmenyaemy aggregation concentration in the case of two inductors amounted to ADP - 5·10-8M, collagen - 1:8 dilution of the primary suspension of collagen, adrenaline - 3,0·10-9M, thrombin - of 0.075 u/ml In the case of the simultaneous use of three inductors their minimum concentration was lower. For ADF - 10-9M, for collagen - 1:9 dilution of the primary suspension, adrenaline - 10-9M, thrombin - to 0.060 u/ml

Development of platelet aggregation with minimal vzaimopodderzhivayuschimi doses of inductors indicates a violation (activation) aggregation of platelets.

The patient was assigned adequate hypotensive therapy Diroton 10 mg 1 time per day, individually chosen a low-calorie diet and exercise training. This allowed after 3 months of treatment to cause minimal vzaimootmenyaemye dose of inductors to the level of healthy people, thereby normalizing platelet aggregation.

Example 3. Patient C., 60 years in the dispensary of hypertension in the clinic who is 7, Kursk and having as comorbidities of the metabolic syndrome were examined in a planned manner.

Upon examination revealed that the patient regularly takes selected antihypertensive therapy with amlodipine 5 mg 1 time per day and Diroton 10 mg 1 time per day), which ensures the stability of blood pressure within the normal figures. At the time of examination of complaints, the patient was not found.

At objective examination revealed obesity on Android type IIA Art. and the offset of the left border of the heart to the left of 1.0 see Electrocardiographically revealed the presence of the patient hypertrophy of the left ventricle. Ophthalmologist diagnostical the patient the hypertensive angiopathy of the retina. Laboratory examination revealed she had impaired glucose tolerance, hyperlipidemia type IIB with mild hypercholesterolemia (5.6 mmol/l).

Platelet aggregation with a combination of inductors, the patient had no abnormalities. The combination of inductors: ADP + epinephrine, ADP + collagen, adrenaline + collagen, ADP + collagen + epinephrine, ADP + collagen + thrombin evoked platelet aggregation with minimal vzaimootmenyaemy aggregation concentrations in the case of two inductors ADF - 10-6M, collagen - 1:4 dilution of the primary suspension of collagen, adrenaline - 2,5·10-7M, thrombin - 0,100 IU/ml, in the case of three inductors: And the f - 5·10-7M, for collagen - 1:5 dilution of the primary suspension, adrenaline - 10-7M, thrombin - 0,090 u/ml lower concentrations of inducers never had examined platelet aggregation. Thus, the development of platelet aggregation in response only to vzaimootmenyaemye dose inductors typical of healthy humans suggest that the normal function of platelets in patients.

The patient was recommended to continue to strictly control blood pressure and reduce body weight due to the low-calorie diet and physical exercise.

Method study aggregation of platelets on glass, including blood sampling, stabilization, separation of platelet-rich plasma and its application on a glass slide, adding aggregation inductor, characterized in that in hypertensive patients with metabolic syndrome investigate platelet aggregation with several inductors using article concludes that the minimum concentration of the aggregation inductors, which are double the combination of inductors ADP - 5,0·10-8M, adrenaline 3,0·10-9the collagen is the main breeding suspension 1:8, the thrombin of 0.075 u/ml; a triple combination of inductors ADF - 10-9M, adrenaline 10-9the collagen - breeding is basically the suspension 1:9, thrombin to 0.060 u/ml, the development of aggregation speaks of platelet activation in hypertensive patients with metabolic syndrome.



 

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1 dwg, 3 tbl, 2 ex

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1 cl, 1 ex, 1 tbl

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1 tbl

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FIELD: medicine, diagnostics.

SUBSTANCE: the present innovation deals with blood sampling, separating plasma against erythrocytes, moreover, in plasma on should detect activity of antithrombin III, proteins C and S, XIIa-dependent fibrinolysis and concentration of plasminogen obtained results should be expressed as relative units followed by calculating integral parameter that characterizes the state of anticoagulant-fibrinolytic potential (IPAFP) by the following formula: IPAFP = [(C1 + C2)/(C3 + C4)] x 100, where C1 - the ratio of observed value of antithrombin III activity to the value of inferior border of the range of analogous parameter norm; C2 - the ratio of observed value for the activity of proteins C and S system to the value of inferior border of the range of this parameter norm; C3 - the ratio of the value of inferior border of plasminogen concentration under normal conditions to observed value of analyzed parameter; C4 - coefficient calculated with the help of regression equation: C4 = 0.9 + (0.01 x X), where X - terms of lysis of patient's euglobulin clot/min, and at IPAFP value of 101.4 U and higher one should state anticoagulant-fibrinolytic blood potential to be in norm, in interval of 64.8 - 101.3 -as insufficient, and at 64.7 and below - as critical. The present method simplifies the procedure of evaluating the state of endogenous anticoagulants and activity of XIIa-dependent fibrinolysis.

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3 ex, 1 tbl

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3 ex, 1 tbl

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