RU2431838C1 - Diagnostic technique for thrombocyte hyperaggregation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: pH of thrombocyte-poor plasma, and a diagnostic coefficient D is calculated by formula: D=const1xPPP-const2, where D is a diagnostically significant coefficient, PPP is a pH value of thrombocyte-poor plasma of an examined patient, const1=18.877, const2=139.381. If D is less than 0.25, thrombocyte hyperaggregation is stated reliably, and the value of D exceeding 0.25 makes to state reliably the absence of thrombocyte hyperaggregation signs.

EFFECT: use of the declared technique allows high-accurate and sensitive evaluation of thrombocyte hyperaggregation.

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Description

Application area

The present invention relates to medicine, namely to laboratory diagnostics, and can be used by doctors of other specialties to assess the risk of pathological thrombosis.

Description of the invention

State of the art

Analogs

An analogue of this invention are the methods for studying the functional activity of platelets described in domestic and foreign literature.

The studies of the functional activity of platelets used in clinical practice on the basis of the methodological approach can be divided into two groups: indicative and quantitative.

1. Indicative methods. Direct assessment (visual morphological and functional differentiation in the appearance of cells) and indirect: the functional state of platelets can be assumed from the results of a study of the interaction of blood vessels in vivo with standardized damage to the skin microvasculature. The methods are laborious, subjective, require special equipment, are invasive and psychologically traumatic for the patient.

2. Quantitative methods. The functional activity of platelets is evaluated in the quantitative characteristics of the formation of platelet aggregates under the influence of stimulants. Quantitative methods are more informative both from the point of view of the obtained diagnostic information and from the point of view of the correctness of the quality of the research, but require specific sample preparation, reagents and special equipment for stimulating platelets using aggregation inductors and for registering the aggregation process. In addition to these methods, electron microscopy (the study of platelet ultrastructure), radioisotope (the life of "labeled" platelets), immunological (determination of antiplatelet antibodies) are used. The methods are not widely used (2, 6, 7, 8). All of the above methods for studying platelet aggregation activity are combined with increased complexity in organizing the diagnostic process and laboriousness in execution. The methods are expensive, they require specific equipment, consumables for sample preparation and registration of the aggregation process.

Prototype

The prototype of the proposed method is a unified reference method for determining the functional activity of platelets by fluctuating the light transmission of platelet concentrate according to Born (12) on an aggregometer. The advantage of this method is the study of aggregation in standardized conditions.

The method is expensive and time-consuming to implement, it requires specific equipment for organizing and registering the aggregation process: modeling of special conditions of rheology of the vascular wall in vitro: isolation of “living” platelet concentrate, quantitative adjustment of diagnostic platelet samples in accordance with the requirements of diagnostic equipment manufacturers, acquisition of diagnostic reagent kits .

SUMMARY OF THE INVENTION

The purpose of the invention is to develop a method for the diagnosis of platelet hyperaggregation, which does not require special conditions, and therefore, high material costs, a method that is simple and convenient in practical use, fast in time.

The method is as follows.

The research material is venous blood obtained from the patient’s elbow vein by a standardized method of vacuum venipuncture for hemostasiological laboratory tests using primary test systems with filler (9 NC 0.129M) of the type Vacuteiner, Monovette, Venoject in the volume specified by the manufacturer for this type of test systems.

To obtain platelet-poor plasma, use the standard protocol for the centrifugation procedure for this type of blood plasma (centrifugation at 3000-4000 rpm (1200g) for 15 min). It is possible to use a centrifuge of any model with a sufficient value of the relative centrifugal force of centrifugation of a particular rotor; taking into account compliance with the installation of time and centrifugation conditions (9, 11, 14).

Analyzed samples thus prepared are examined to determine the pH value. (The proven dependence of the functional and morphological changes of cells on the pH (concentration of hydrogen ions) of the surrounding physiological environment is known (1, 3, 4, 5, 10, 13)).

To measure the pH, any analyzer of the acid-base state and gas composition of the blood of the third generation can be used. Platelet-poor plasma in accordance with the instructions for the device is collected in the capillaries of the volume set by the manufacturer. (The volume of capillaries depends on the amount of sample required for the pH analysis, and is indicated in the Instrument Certificate; capillaries for research are attached to the device). The pH measurement procedure is carried out in accordance with the instructions for the device. The interpretation of the results is based on the calculation of the diagnostic index.

The diagnostic index was obtained by mathematical processing of the results of pH-metry and the degree of platelet aggregation by the method of cluster and step-by-step discriminant analysis using the Quasar-Plus application package. Derived Decision Rule

D = const1 × PPP-const2,

where D is a diagnostically significant index,

PPP - pH value of platelet-poor plasma of the studied patient,

const1 = 18.877,

const2 = 139.381.

When D is less than 0.25, a reliable conclusion is made (p <0.001) about platelet hyperaggregation.

With D more than 0.25, a reliable conclusion is made (p <0.001) about the absence of signs of platelet hyperaggregation.

The proposed method gives the probability of correct detection of healthy in 96% and patients in 80% of cases with a significance level of P <0.001.

The sensitivity of the algorithm to the presence of platelet hyperaggregation is 96.0%, specificity 80.3%.

The effectiveness of the method is -88.15%.

Examples

Example 1. Patient G., 32 years old. Somatically VSD hypertonic type. She had a history of early surgical labor at 26-27 weeks of pregnancy in 2009 due to severe preeclampsia. A baby born weighing 970 grams died in 2 months. It was sent for examination to the laboratory of the Federal State Institution "Research Institute of OMM of Rosmedtehnologii" in connection with the planning of pregnancy with an advisory reception by the obstetrician-gynecologist of the scientific advisory clinic of the institute.

A pH study of platelet-poor plasma was carried out, which amounted to 7.331. The diagnostic index D, calculated by the formula D = const1 × PPP-const2 (D = 18.877 × 7.331-139.381), is 0.994; which indicates the presence of platelet hyperaggregation. To verify the result, a study was carried out using a unified Born method on an aggregometer. Platelet aggregation with ADP (2.5 μmol / L): degree 98.7%, rate 98% / min, time 8 min, two-phase curve. Hyperaggregation of platelet link of hemostasis was revealed: This confirms the correctness of the diagnostic index.

To prevent inferior trophoblast invasion and the phenomena of hypercoagulation in the intervillous space, the patient was prescribed antiplatelet therapy.

Example 2. Patient S., 38 years old (medical history No. 489/532). A history of primary infertility for 15 years. Somatically chronic cholecystopancreatitis, chronic bronchitis, chronic pyelonephritis, urolithiasis, common osteochondrosis, polyvalent allergies, chronic nicotine intoxication.

I turned to an advisory reception of the ECO department of the Federal State Institution "Research Institute of OMM of Rosmedtechnology" regarding prolonged infertility.

A pH study of platelet-poor plasma was carried out, which amounted to 7.359.

The diagnostic index D, calculated by the formula D = const1 × PPP-const2 (D = 18.877 × 7.359-139.381), is -0.465, which indicates the presence of platelet hyperaggregation.

To verify the result, a study was carried out using a unified method according to Born on an aggregometer. Platelet aggregation with ADP (2.5 μmol / L): degree 123.5%, rate 97% / min, time 10 min, monophasic curve. Hyperaggregation of platelet hemostasis was revealed. Which confirms the correctness of the diagnostic index. The patient was treated for infertility using assisted reproductive technologies (IVF-PE and ICSI).

To prevent complications and pregnancy outcomes of women with thrombophilia, the patient was selected with adequate anticoagulant, antiplatelet therapy from the first trimester of pregnancy.

Example 3. Patient Z. (medical history No. 3469). I entered the department of the OPB FSI "Research Institute of OMM of Rosmedtehnologii" for a planned re-hospitalization with a diagnosis of pregnancy 23-24 n. A history of habitual miscarriage. In the period (17-18 BC), the cervix was circulated with a mercilene ribbon, the threat of miscarriage. At the time of inspection, the condition is satisfactory, no complaints.

A pH study of platelet-poor plasma was carried out, which amounted to 7.372.

The diagnostic index D, calculated by the formula D = const1 × PPP-const2 (D = 18.877 × 7.372-139.381), is 0.219; which indicates the absence of signs of platelet hyperaggregation.

To verify the result, a study was carried out using a unified method according to Born on an aggregometer. Aggregation with ADP (2.5 μmol / L.): Degree 78.85%, rate 55.4% / min, time 10 min, normal type of curve. The pathology of the platelet link of hemostasis was not identified. Which confirms the correctness of the diagnostic index.

The patient underwent complex therapy of habitual miscarriage. A woman in satisfactory condition with a progressive uterine pregnancy is discharged home.

Thus, the proposed method for the diagnosis of platelet hyperaggregation makes it possible to accurately assess the state of platelet hemostasis and identify a risk group for the development of thrombophilia (it is proved that the development of a vascular catastrophe of any localization is based on increased platelet aggregation ability); allows you to timely resolve the issue of the need for a comprehensive paraclinical examination with subsequent therapeutic correction of the revealed disorders, especially in the initial stages of the disease.

This diagnostic method can be proposed for widespread use.

The method does not require expensive material and technical base, special reagents, it is convenient to use when using the minimum amount of test material, it provides the necessary information within an hour from the moment of blood sampling of the patient, reproduced in the laboratory at any level.

Information sources

1. Abramochkina I.G. Clinical evaluation of the morphological features of urine red blood cells in children with hematuria of various origins: Abstract. dis ... cand. honey. Sciences, - Samara, 1998

2. Barkagan Z.S., Mamot A.P. Diagnosis and controlled therapy of hemostasis disorders, - M .: Newdiamed - AO, - 2001. - P.296.

3. Dawson R., Elliot D., Elliot W., Jones K. Handbook of biochemist - M .: Mir, - 1991. - S.347-373.

4. Georgescu P., Paunescu E. Biochemical methods of diagnosis and research. - Bucharest, - 1963. - 84-106.

5. Delevsky Yu.P. The effect of pH on the agglutinating ability of anti-a-monoclonal antibodies and their inhibition by lipid and protein a-glycoconjugates with different isoelectric properties // Klin. lab. the diagnosis - 2008. - No. 8. - S. 29-32.

6. Ivanov E.P. Diagnostics of hemostasis disorders (Practical guide for doctors) - Мn .: Belarus, - 1983. - P.107-119.

7. Kozlovskaya L.V., Nikolaev A.Yu. A manual on clinical laboratory research methods - 2nd ed. - M .: Medicine - 1984, p. 88-98.

8. Menshikov VV, Delektorskaya L.N., Zolotnitskaya R.P. Laboratory research methods in the clinic: Handbook, ed. V.V. Menshikov - M .: Medicine - 1987. - P.152-154.

9. Moshkin A.V., Dolgov V.V. Quality assurance in clinical laboratory diagnostics - M. - 2004. - P.14-23, 38-39.

10. Kholmanskikh N.A., Pestryaeva L.A. Diagnosis of functional disorders of the vascular-platelet link of hemostasis during pregnancy // Mother and Child: Mat. X anniversary of All-Russia. Forum. - M. - 2009. - P.227.

11. ISO 151189: 2003. Medical laboratories - specific requirements for quality and competence (draft Russian version). Standardization in clinical laboratory medicine. Organizational and metrological aspects - M. - 2005. - P.37-105.

12. Born C.V.R. // Nature. - 1962. - Vol. 189, N 1-2. - R. 927-929.

13. Marchetti P. Et al. // Diabetologia. - 1986. - Vol.29, N10. - P.695-698.

14. NCCLS, document H18-A2. Procedures for the Handing and processing of Blood Specimens, 2nd Ed, Approved Guideline, NCCLS, 1999.

Claims (1)

A method for determining platelet hyperaggregation by centrifugation, characterized in that after centrifugation, the pH of the platelet-poor plasma is determined and the diagnostic index D is calculated by the formula:
D = const1 PPP-const2,
where D is a diagnostically significant index;
PPP - pH value of platelet-poor plasma of the studied patient,
const1 = 18.877,
const2 = l39.381;
and with D less than 0.25, a reliable conclusion is made about platelet hyperaggregation, and with D more than 0.25, a reliable conclusion is made that there are no signs of platelet hyperaggregation.