RU2230322C2 - Platelet glow revealing method - Google Patents

Abstract

FIELD: hematology. SUBSTANCE: glow level-enhancing substances, in particular luminol at its final concentration 10^-6 mole/l, are added to initially provided platelet-enriched blood plasma and resulting mixture is placed into glow-registration device, said platelet-enriched plasma being preliminarily diluted with platelet-depleted plasma to concentration of platelets 300000-350000 cell/mm². EFFECT: increased spontaneous platelet glow intensity.

Description

The invention relates to medicine, as well as biochemistry, namely to hemostasiology, and can be used to diagnose platelet dysfunction.

A known method for detecting platelet luminescence, including obtaining platelet-rich plasma (OTP) with the addition of thrombin, placing it in a chemiluminomer cuvette, and recording the luminescence (A.B.Samal, S.N. Cherenkevich, etc. Platelet aggregation: methods of study and mechanisms. .3.8 Platelet chemiluminescence in thrombin-induced aggregation - Minsk: Universitetskoye, 1990, p. 87.

A disadvantage of the known technical solution is that the effect on plasma of thrombin causes increased adhesion (aggregation) of platelets, which dramatically increases their activity and causes a short-term glow, and this affects the function of platelets, distorting the picture of their actual state, which does not allow to obtain reliable information about initial functional state of platelets.

The closest in technical essence to the claimed method and selected as a prototype is a method for detecting platelet chemiluminescence, including obtaining platelet-rich blood plasma, adding a substance that increases the level of luminescence, placing this mixture in the device with subsequent registration of the detected glow, and as a substance increasing the level of luminescence using divalent iron (A.B. Samal, S.N. Cherenkovich and others. Platelet aggregation: study methods and mechanisms. Ch. platelet count. - Minsk: Universitetskoye, 1990, p. 88, Fig. 51).

A disadvantage of the known technical solution is that the effect on the plasma of ferrous iron causes such a short-term luminescence of platelets that practically does not seem to receive reliable information about the initial functional state of platelets.

The objective of the present invention is to obtain reliable information on the functional state of platelets.

The technical result is to increase the time and intensity of spontaneous luminescence of platelets.

This problem is achieved due to the fact that in the known method for detecting platelet luminescence, including obtaining platelet-rich blood plasma, adding a substance that increases the level of luminescence, placing this mixture in a device for subsequent registration of luminescence, according to the invention, the platelet-rich plasma obtained is diluted with poor platelets plasma to a platelet concentration of 300,000-350000 cells / μl, and as a substance that increases the glow, use luminol with a final concentration of 10 ^-6 mol.

The proposed method is a set of techniques that allows you to obtain reliable information about the functional state of platelets in the blood, which makes it possible to judge the presence or absence of pathology of the cell link in humans.

Studies on patent and scientific and technical sources of information have shown that the proposed method is unknown and does not follow explicitly from the studied prior art i.e. meets the criteria of “novelty” and “inventive step”.

The proposed method can be applied in any biochemical laboratory of a medical institution equipped with standard equipment manufactured by domestic or foreign industry.

Thus, the claimed method is affordable, and therefore, practically applicable.

Dilution of platelet-rich plasma with platelet-poor plasma to a platelet concentration of 300,000-350000 cells / μl provides an optimal spontaneous platelet glow, experimentally determined.

At a concentration of less than 300,000 cells / μl, a small yield of free radicals occurs, resulting in a faint glow.

At a concentration of more than 350,000 cells / μl, a large number of platelets begins to interact with each other and the formation of free radicals decreases, resulting in a decrease in luminescence.

The addition of luminol of the declared concentration increases the sensitivity of the method, it oxidizes with free radicals formed by platelets, resulting in an increase in the intensity of their glow.

Thus, a spontaneous intense and sufficiently long luminescence of platelets is ensured, which allows to reliably judge their functional state.

The inventive method is carried out using chemiluminomer XL-003, a recording device, such as a computer monitor and a beaker with a volume of 50 ml.

The inventive method is as follows.

Platelet-rich plasma (OTP) diluted with platelet-poor plasma (BTP) in a concentration of, for example, 320,000 cells / μl in an amount of 1 ml is placed in a beaker and a chemiluminomer is placed in the cuvette chamber, which detects spontaneous platelet luminescence for 10 minutes. The luminosity sum is expressed in arbitrary units relative to the ZhS-19 reference source of radiation containing uranium salt and is equal to 1.231 + 1.18 conventional units

After that, a working solution of luminol with a final concentration of 10 ^-6 mol is added to this solution and the luminescence of the resulting solution, which is 7.71 + 1.18 conventional units, is recorded again, which means an increase in luminescence by 6.26 times.

With this level of platelet glow, one can reliably judge the functional state of platelets.

The intensity of the glow ability is used to judge the functional state of platelets, and the duration of the glow is used to judge their viability. The brighter the glow, the better the functional state of the platelets, the longer the glow, the greater their viability.

Example 1

The study was performed at the biochemical laboratory of the Department of Pathological Physiology of the Chelyabinsk State Medical Academy in May 2001.

From a regional blood transfusion station, blood was obtained from a healthy donor A, stabilized with a 3.8% sodium citrate solution (9: 1) in a plastic tube. After centrifugation for 10 minutes at 1500 rpm, an OTP (platelet-rich plasma) was obtained, which was diluted with BTP (platelet-poor plasma) to a platelet concentration of 300,000-320000 per μl. Samples of OTP in a volume of 1 ml were placed in cuvettes and chemiluminescence was determined by the claimed method:

1. The first test of OTP: revealed a spontaneous glow - registration within 10 minutes.

Platelet count

in OTP, μl Light Sum

300,000 1.44

320,000 1.56

2. The second sample, with the addition of luminol, dependent luminescence, the final concentration of luminol in the sample 10 ^-6 mol registration 10 minutes.

  Light sum

Sample 1 6.78

Sample 2 7.32

3. Luminol-dependent luminescence after 1.5 hour incubation at 37 ° C has a light sum of 7.7 + 1.19, which indicates the normal functional state of platelets. The ability of platelets to maintain their glow at 37 ° C for 1.5 hours also indicates their viability at this time.

The proposed method in comparison with the prototype provides:

1. Reliability in assessing the functional state of platelets.

2. The possibility of reliable diagnosis of the disease according to the state of platelets.

Claims (1)

A method for detecting platelet luminescence, including obtaining platelet-rich blood plasma, adding a substance that increases the level of luminescence, placing this mixture in a device for subsequent registration of luminescence, characterized in that the obtained platelet-rich plasma is diluted with platelet-poor plasma to a platelet concentration of 300,000 to 350,000 cells / μl, and as a substance that increases the level of luminescence, luminol of a final concentration of 10 ^-6 mol is used.