RU2804668C1 - Method of obtaining platelet-enriched fraction of autoplasma from blood - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a method of obtaining platelet-enriched autoplasma from blood. A method is proposed for obtaining platelet-enriched autoplasma of blood, which includes collecting 7–9 ml of venous blood into a vacuum tube with an anticoagulant — sodium citrate, located above two separation media: a polyester gel and a Ficoll solution underneath, mixing the blood with the anticoagulant by inverting the tube 8–10 times, followed by a single centrifugation of the blood tube for 10–15 minutes at 700–1000 g and sampling with a needle 1 ml of the interphase layer of the supernatant fraction of platelet-rich plasma.

EFFECT: proposed method ensures the production of a platelet-rich autoplasma fraction with a high, more than fivefold content of platelets by single centrifugation from a small amount of blood, while the sorption of platelets to the separation gel is reduced or absent.

1 cl, 1 dwg

Description

Field of technology to which the invention relates

The invention relates to the field of medicine, namely to regenerative medicine, and can be used to stimulate tissue regeneration in various diseases and degenerative conditions. It can be used, for example, in surgery, orthopedics, urology, gynecology, dentistry, and cosmetology.

The use of platelet-enriched fractions of autologous blood plasma is currently widespread in medicine. Platelets contain factors necessary to activate hemostasis, promoting migration, division, growth, and differentiation of cells; stimulation of angiogenesis and tissue regeneration.

These factors, along with plasma hemostasis factors, are the substrate for the therapeutic and regenerative effect of the platelet-enriched fraction of autoplasma in the blood /1/ .

State of the art

The present invention is characterized by the use of a device containing an anticoagulant - sodium citrate, located above two separation media: a polyester gel and a Ficoll solution located underneath it, to obtain a platelet-enriched autoplasma fraction.

There is a known method for obtaining platelet-rich plasma, which includes taking blood, mixing the blood with an anticoagulant and placing it in a cylindrical glass having a trap pocket for platelets and plasma, centrifuging the glass at a speed gradually decreasing every three minutes from 4500 rpm. up to 2000 rpm. and further up to 1000 rpm. (Patent RU 2428995 C1). It should be noted that a special centrifuge is required to centrifuge a beaker of a special shape. In addition, high centrifugation speeds increase the risk of decreased platelet viability in platelet-rich plasma obtained by the claimed method /2/ .

There is a known method for obtaining autologous platelet-rich plasma, which includes collecting venous blood into a double syringe tube, followed by centrifugation of the device in modes from 10 minutes at 112 g to 2 minutes at 447 g. Next, the red blood cells are separated and the device is centrifuged again for 10 minutes at 1000 g to concentrate platelets, which are then separated from the blood plasma. (US Patent 2015/OO64687 A1). It should be noted the complexity of this device, the multi-stage nature of the method and the need for special equipment for centrifugation of double syringe tubes.

There are known methods for obtaining autologous platelet-rich plasma, including collecting venous blood into a syringe connected to a catheter with a pre-drawn anticoagulant in an amount of 1.5 ml, then the syringe is closed, the contents are mixed and injected into a “YCELLBIO-KIT” tube (Patent RU 2713772 C1), or “A sterile ARM tube for processing whole or diluted blood” (Patent RU 2698723 C1). Centrifugation is carried out at room temperature with a rotation speed of up to 4000 rpm for 4-5 minutes, the layer of platelet-rich plasma is extracted through the central hole in the lid of the tube. The authors of the inventions (Patent RU 2713772 C1; Patent RU 2698723 C1) propose to centrifuge blood, including at a speed of 4000 rpm for 4-5 minutes on an “Armed”: 80-2S centrifuge, which corresponds to a relative acceleration of 2130 g and is disadvantage of this method. With such acceleration, a decrease in the number of platelets in the finished product should be expected because according to Jo et al 2013, at accelerations above 1500 g, platelet yield decreases, probably due to their destruction /2/ .

There is a known method for producing autoplasma with a rich content of platelets using vacuum tubes with heparin and a separation gel, called Plasmolifting™. Blood is taken into a test tube, which is then centrifuged at a speed of 1000 rpm for 10 minutes (Patent RU 2606338).

The authors of the invention (Patent RU 2606338) indicate an increase in platelet concentration by 3-3.5 times from the original, which does not meet the modern generally accepted criteria for therapeutically effective platelet-rich plasma, according to which the platelet concentration should be at least five times the original /1/ . In addition, it is impossible to exclude the sorption of platelets to the separating gel, which can lead to a decrease in their number in the finished product.

The closest to the claimed method is the method of blood separation (US Patent 4818418), which includes placing two separation media in a test tube.

Next, blood mixed with an anticoagulant is placed into the test tube, the tube is centrifuged at a speed of 600-2000 g, 5-20 minutes. The authors of the invention (US Patent 4818418) used it to isolate mononuclear cells from peripheral blood, however, by adapting centrifugation modes, an alternative use of this invention is possible to obtain a platelet-enriched autoplasma fraction.

The use of such technology can eliminate a possible drawback of the invention (Patent RU 2606338), namely the sorption of platelets on the separation gel, thereby increasing their quantity in the finished product - the platelet-rich autoplasma fraction of the blood.

The technical problem is the development of a method for obtaining a platelet-enriched autoplasma fraction with a high (i.e., more than fivefold) platelet content by single centrifugation from a small amount of blood, due to a decrease or complete absence of platelet sorption to the separation gel.

Disclosure of the Invention

The technical result to which the claimed invention is aimed is to obtain a platelet-enriched autoplasma fraction with a high (i.e., more than fivefold) platelet content by single centrifugation from a small amount of blood, due to a decrease or complete absence of platelet sorption to the separation gel. This method is simple and reproducible on laboratory centrifuges with standard rotors, which provides the prospect of its wide application.

The technical result is achieved by implementing a method for obtaining platelet-enriched autoplasma of blood, which includes collecting 7-9 ml of venous blood into a vacuum tube with an anticoagulant - sodium citrate, located above two separation media: a polyester gel and, underneath, a Ficoll solution, mixing the blood with the anticoagulant by turning the tube over 8-10 times, centrifuging the tube with blood once for 10-15 minutes at 700-1000 g.

During centrifugation, the polyester gel ensures the separation of platelet-enriched autoplasma and red blood cells, and the Ficoll solution creates an additional density gradient that prevents the sorption of platelets on the separation gel and promotes the separation of the platelet-enriched autoplasma fraction of the blood.

Next, the interphase layer of the supernatant fraction of platelet-rich plasma is collected, which is a strip between the overlying plasma and the Ficoll solution.

This layer contains the maximum gradient concentration of platelets in the entire supernatant fraction of platelet-rich plasma and is withdrawn with a needle in an amount of 1 ml to ensure a high platelet concentration.

Brief description of drawings

The invention is illustrated by illustrative material, where the figure shows general schematic images:

A. test tubes with sequentially located from top to bottom: anticoagulant - sodium citrate (1), polyester gel (2), Ficoll solution (3), so that the overlying anticoagulant (1) is separated by polyester gel (2) from the underlying Ficoll solution (3 )

B. tubes of blood before centrifugation, in which blood mixed with sodium citrate (4) is placed over polyester gel (2) and underlying Ficoll solution (3)

B. tubes with blood after centrifugation, in which the blood is separated into fractions of platelet-rich plasma (5), located above the Ficoll solution (3), separating it from the underlying polyester gel (2) and the red blood cell fraction located below it (6)

D. position of the syringe needle (7) when collecting 1 ml of the interphase layer containing the maximum gradient concentration of platelets (8) of the platelet-enriched autoplasma fraction from the test tube (D).

Carrying out the invention

Obtaining platelet-enriched autoplasma from blood includes the following steps.

1. Collecting the patient’s venous blood into a vacuum tube with an anticoagulant - sodium citrate, located above two separation media: a polyester gel and a Ficoll solution underneath.

2. Mixing blood with anticoagulant by inverting the tube 8-10 times.

3. Single centrifugation of a tube with blood for 10-15 minutes at 700-1000 g.

4. Using a needle, take 1 ml of the interphase layer of the supernatant fraction of platelet-rich plasma, containing the maximum concentration of platelets along the gradient.

Laboratory studies confirmed an increase in the platelet concentration in the platelet-enriched autoplasma obtained in the above manner by 5 times the initial platelet concentration.

Bibliography

1. Everts P, Onishi K, Jayaram P, Lana JF, Mautner K. Platelet-Rich Plasma: New Performance Understandings and Therapeutic Considerations in 2020. Int J Mol Sci. 2020 Oct 21; 21(20):7794. doi: 10.3390/ijms21207794. PMID: 33096812; PMCID: PMC7589810.

2. Jo CH, Roh YH, Kim JE, Shin S, Yoon KS. Optimizing platelet-rich plasma gel formation by varying time and gravitational forces during centrifugation. J Oral Implantol. Oct 2013; 39(5):525-32. doi: 10.1563/AAID-JOI-D-10-00155. Epub 2011 Apr 11. PMID: 21480780.

Claims (1)

A method for obtaining platelet-enriched autoplasma of blood, including collecting 7-9 ml of venous blood into a vacuum tube with an anticoagulant - sodium citrate, located above two separation media: a polyester gel and a Ficoll solution underneath, mixing the blood with the anticoagulant by inverting the tube 8-10 times , subsequent single centrifugation of the tube with blood for 10-15 minutes at 700-1000 g and collection of 1 ml of the interphase layer of the supernatant fraction of platelet-rich plasma with a needle.