RU2623083C1 - Method for platelets freezing - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: for platelet freezing, the initial platelet concentrate (PC) is sampled with a platelet concentration of 1×10⁹/ml to 1.5×10⁹/ml containing functionally active platelets. The PC is divided to the platelet-containing part and plasma, followed by plasma separation from the platelet-containing part. A combined cryoprotectant containing 55% DMSO and 5% dextran 40 is prepared by diluting the cryoprotectant with plasma to the final DMSO concentration of 10-15%. The platelet-containing part is resuspended by a diluted cryoprotectant which is injected into the platelet-containing part by 1-3 ml with 1-3 min intervals at constant stirring, until the final DMSO concentration of 5-7% in the platelet suspension is reached. The platelet suspension and plasma are frozen at a rate of 1-3°/min in separate containers in the freezing chamber at temperatures from minus 80 to minus 100°C. The frozen platelets and plasma are stored at a temperature from minus 85 to minus 196°C.

EFFECT: application of the invention allows to improve the quality of platelets after defrosting.

6 cl, 4 tbl, 1 dwg, 1 ex

Description

FIELD OF THE INVENTION

The invention relates to medicine, namely to production and clinical transfusiology, and can be used for harvesting frozen platelets.

State of the art

There is a method of freezing platelet-rich plasma without the use of cryoprotectants [RF patent for the invention "Method for cryopreservation of platelet-rich plasma" authors VA Sukhanov, IM Trofimov, AL Levit, No. 2138162, 1999], according to which the plasma is placed in a container with liquid nitrogen, a container is placed between two metal plates, after which they are subsequently subjected to freezing and storage processes, while the plasma freezing process is carried out at a speed of 2-3 ° C / s. However, this method does not include the assessment of the parameters of structural usefulness and functional activity of human platelets.

A known method of freezing platelets with a DMSO content in frozen platelets of 10% [Khuri S.F., Healey N., MacGregor H. et al. Comparison of the effects of transfusions of cryopreserved and liquid-preserved platelets on hemostasis and blood loss after cardiopulmonary bypass J Thorac Cardiovasc Surg 1999; 117: 172-84]. However, this method involves the use of an endocellular cryoprotectant - DMSO, which does not allow to achieve a high degree of preservation of platelets in a frozen state.

Closest to the claimed solution is a method of freezing platelets with DMSO, followed by storage of cells in a frozen state at a temperature of minus 80 ° C [C. Robert Valery, Gina Rango, and Shukri Khuri Freezing human platelets with 6 percent dimethyl sulfoxide with removal of the supernatant solution before freezing and storage at -80 ° C without postthaw processing // Transfusion 2005; 45: 1890-1898]. The initial platelet concentrates (TK) are obtained by hardware apheresis on Cobe Spectra and Amicus separators, the total volume of TK is 200-300 ml, the platelet content is from 3.5 to 4.0 × 10 ¹¹ cells in 1 dose (concentration 1.3- 2.0 × 10 ⁹ in ml). Assessment of the quality of TK cells is carried out before freezing TK and includes the following parameters: platelet count (10 ⁹ / ml); thromboconcentrate pH, the value of the R-period on the thromboelastogram, platelet aggregation activity (inducers - 50 μg / ml of arachidonic acid and 2 μmol / l ADP); thromboxane B2 production after stimulation with 50 μg / ml arachidonic acid and 2 μmol / L ADP; the content of annexin-positive platelets. The procedure for preparing TC for freezing includes 4 technological stages: the introduction of a cryoprotectant in a dose of TC; centrifugation of a dose of TC in the presence of a cryoprotectant; removal of excess cryoprotectant with acellular plasma after centrifugation; resuspension of TC cells with cryoprotectant. As a cryoprotectant, a saline solution of 27% DMSO (endocyte cryoprotectant) is used, which is added to the TC with constant shaking at a frequency of 180 vibrations per minute to a final concentration of 6% DMSO in the TC. Then, the TCs are centrifuged at 1250g for 10 minutes to remove the supernatant containing excess DMSO. All supernatant solution is removed. This reduces the residual amount of DMSO in frozen TC by at least 95%. The remaining 10-15 ml sediment is resuspended by shaking for 3-5 minutes, the container with the platelet suspension is placed in a protective plastic bag, then in a cardboard box and frozen at the bottom of a mechanical freezer at a temperature of minus 80 ° C.

However, this method of freezing includes methods for the analysis of platelets in the TC: aggregometry, thromboelastography and flow cytometry, which do not reflect the structural integrity of the platelets, which reduces the reliability of the general analysis of platelets before freezing; uses only the endocytic (intracellular) cryoprotectant DMSO, which has a low ability to preserve the outer membrane structures of cells during freezing, which increases the risk of platelet receptor damage; the procedure for preparing TC for cryopreservation includes 4 consecutive technological stages, which in total take 25-30 minutes, which significantly increases the risk of toxic effects of DMSO on platelets.

Disclosure of invention

The objective of the invention is to develop a method of freezing platelets to obtain therapeutic doses of TC long-term storage with high preservation of biologically complete platelets.

The technical result to which the claimed invention is directed is to improve the quality of cryopreserved platelets obtained after thawing due to the complex of technological processes used, the qualitative and quantitative parameters of the cryoprotectant and the regimes that reduce the contact time of platelets with the cryoprotectant, and to preserve a much larger number of biologically complete platelets (in 1.5-1.8 times compared with the technology of the prototype), store thawed TC at room temperature temperature for 4 hours without the threat of losing the structural or functional usefulness of platelets, obtain an osmolarity of a dose of TC corresponding to a physiological norm that does not cause the development of post-transfusion reactions in patients.

The problem is solved in that the method of freezing platelets includes the following steps:

- selection of the initial platelet concentrate (TK) with a platelet concentration of 1 × 109 / ml to 1.5 × 109 / ml containing functionally active platelets;

- separation of TC into a platelet-containing part and plasma, followed by separation of the plasma from the platelet-containing part;

- resuspension of the platelet-containing part by a pre-prepared combined cryoprotectant, which is injected into the platelet-containing part of 1-3 ml at intervals of 1-3 minutes with constant stirring to a final concentration of DMSO in the platelet suspension from 5 to 7%;

- freezing a suspension of platelets and plasma at a speed of 1-3 ° / min in separate containers in the freezer at a temperature of minus 80 ° C to minus 100 ° C;

- storage of frozen platelets and plasma at a temperature of minus 85 ° C to minus 196 ° C;

wherein the combined cryoprotectant containing 55% DMSO and 5% dextran 40, is preliminarily diluted with plasma to a final concentration of DMSO from 10 to 15% before resuspending the platelet-containing part.

Platelets that correspond to the following parameters are considered functionally active in the initial TC: platelets with granules from 40% to 70% and platelets with adhesive activity from 40 to 70%.

In addition, the separation of TC into a platelet-containing part and plasma can be realized by centrifugation at a speed of 1100g to 1400g for 7 to 12 minutes. As a cryoprotectant, DIMETYLSULFOXIDE / DEXTRAN 40 SOLUTION (Cryopreservative Solution) 5 ml can be used. Resuspension of the platelet-containing part by a cryoprotectant is carried out for 9 to 12 minutes.

In a specific embodiment, the initial platelet concentrate is taken from 180 to 220 ml, for the preparation of a combined cryoprotectant with a final concentration of DMSO from 10 to 15%, the initial cryoprotectant is diluted with plasma in a volume ratio of 1: 9, resuspending the platelet-containing part with a diluted cryoprotectant to obtain a platelet suspension with the final concentration of DMSO from 5 to 7% is carried out in a volume ratio of 1: 1.

After thawing, the following platelet parameters are measured: platelet count, platelet count with granules and adhesive platelet count. This information is applied to the container for subsequent selection of the container with the necessary parameters for transfusion to a particular patient, depending on its anthropometric characteristics.

Thus, the problem is solved by introducing into the platelet freezing technology adequate methods for analyzing the structural integrity and functional activity of platelets, using a combined (endocytic and exocytic) cryoprotectant, preliminary separation of TK by centrifugation into the platelet part and platelet-poor plasma, followed by freezing of the platelet part platelet-poor plasma at a temperature of minus 80 ° C, storage of frozen TC at a temperature of minus 8 0 ° C to minus 196 ° C from 180 days to 24 months in order to quarantine platelets and plasma.

The use of an adequate method of morphofunctional analysis of human platelets made it possible to optimize the assessment of the quality of cells in TC at different stages of cryopreservation. As such a technique, a previously developed method for assessing the morphofunctional status of human platelets was proposed [RF Patent for invention No. 2485502 “Method for assessing the morphofunctional status of human platelets”, authors Khubutia M.Sh., Makarov MS, Khvatov VB, Vysochin I.V., Kobzeva E.N., Borovkova N.V., Konyushko O.I., June 20, 2013], based on vital (intravital) staining of platelets with fluorochrome dye based on tripaflavin and acridine orange, followed by their analysis in fluorescence a microscope. This method allows you to simultaneously evaluate the structural integrity and functional activity of platelets, regardless of their concentration in the sample. Thus, using this method, it becomes possible to assess the total content of biologically valuable platelets in the studied TC. The studies show that only platelets in which granules are clearly visible during vital staining are clearly biologically complete (with normal structure and functions) - at least 3 granules with a diameter of more than 300 microns per cell in a fluorescence microscope, the brightness of the cell is at least 40 feet -candle (Makarov M.S., Kobzeva E.N., Vysochin I.V., Borovkova N.V., Khvatov V.B. Morphofunctional analysis of human platelets using vital staining // Bulletin of Experimental Biology and Medicine. - 2013 . - No. 9. - S. 388-391 ) Therefore, the assessment of platelet count with granules can be used to control the quality of TK cells (before and after cryopreservation).

When exposed to platelet-rich plasma (BTP) with DMSO in a final concentration of 0.5 to 20% at room temperature, it was found that in contact with DMSO the biological usefulness of BTP cells decreases, and at the same time, the content of platelets with granules decreases (Table . one). So, at 0.5% DMSO, the content of platelets with granules in the BTP practically did not change for 2 hours at room temperature, after 4 hours it decreased by 7-10%, after 20 hours - by 70%. At a DMSO concentration of 1 to 20%, the dynamics of a decrease in the number of platelets with granules was similar during the first two hours: after 1 hour, the loss of platelets with granules was 10-20%, after 2 hours - 40-50%, after 4 hours - from 60 up to 80%, after 20 hours, platelets with granules in the BTP were completely absent. An even more pronounced decrease in platelet count with granules was at 10-20% DMSO: after 2 hours, platelet loss with granules was 55-65%, after 4 hours - 90-99%. It is worth noting that with a DMSO concentration of 5% or higher, the adhesive activity of platelets with granules decreases much faster than their content. This effect becomes apparent even at a concentration of DMSO of 2% and higher. When using 5% DMSO after 30 minutes of exposure, the adhesive activity of platelets decreased on average by 2 times (table. 2). Thus, after 30 minutes of exposure to BTP with 5% DMSO at room temperature, an average of 50% of platelets with granules did not show functional activity. This indicates that platelet contact time with 5% DMSO at room temperature should be minimized. On the other hand, at a concentration of 0.3-0.5% DMSO, the biological usefulness of platelets is practically not disturbed for several hours. Therefore, the dilution of thawed DMSO-containing TC to a concentration of 0.3-0.5% DMSO will allow them to maintain their structural and functional usefulness for 4-6 hours after defrosting.

In the process of conducting research work, a procedure for freezing TC was developed, in which the contact time of platelets under the influence of DMSO was minimized. A combined sterile cryopreservation solution was selected containing 55% DMSO (endocytic cryoprotectant) and 5% dextran (exocytic cryoprotectant), which allows adequate freezing of cells and intercellular medium. It is proposed to introduce a cryoprotectant into a TC after centrifugation and separation into a platelet-containing part and a platelet-poor plasma. To obtain platelet-poor plasma, TC must be centrifuged at an acceleration of 1250 g. Previous studies have shown that centrifugation of BTP and TC with acceleration from 500g to 2000g does not significantly affect the content of cells with granules [Makarov MS, Kobzeva EN, Borovkova NV, Khvatov VB The effect of centrifugation on the biological usefulness of human platelets // Bulletin of the Russian blood service. - 2015. - No. 1. - S. 41-44]. As in the prototype, the volume of the obtained platelet-containing part was 10-15 ml. However, unlike the prototype, a combined cryoprotectant based on DMSO and dextran was introduced after centrifugation and separation of platelet-poor plasma. The total duration of this procedure is 8-12 minutes, while in the prototype from 25 to 30 minutes. Thus, the claimed technology 2.5-3.0 times reduces the contact time of platelets with a cryoprotectant.

A comparison was made of the quality of platelets in cryopreserved TCs obtained using the prototype technology and using the inventive technology (table 3.). To assess the effectiveness of platelet cryopreservation, it was decided to determine the safety of platelets with granules and the safety of their adhesive activity. It was found that the claimed technology allows you to save a much larger number of biologically complete platelets (1.5-1.8 times) than the prototype technology. Cryopreserved TCs were diluted using acellular plasma of the same donor, which was simultaneously frozen (without a cryoprotectant) and then thawed. The volume of thawed plasma was 90-100 ml, which made it possible to dilute the platelet-containing part of the TC by 10 times, reducing the concentration of DMSO in it to 0.4-0.6%. At the same time, in the prototype, the concentration of DMSO is 2.5-3%, the osmolarity is more than 800 mOsmol / l, which is toxic to human platelets. In this case, for dilution of TC, a 0.9% sodium chloride solution is used, which does not have buffering properties and does not interfere with acidification of the medium. As a result, storage of thawed TC obtained by the technology of the prototype is accompanied by a pronounced decrease in the structural and functional usefulness of platelets. So, after 1 hour of storage in thawed TCs, the adhesive activity of cells decreases by 70%, after 2 hours - by 90%, after 4 hours - by 95-98% (Fig. 1). This indicates the inefficiency of storage of TC (in the prototype) at room temperature. On the contrary, when the platelet-containing part of the TC is diluted with acellular plasma (the proposed technology), the adhesive activity of the platelets decreases after 4 hours only by 10% of FIG. one). Thus, it becomes possible to store thawed TC at room temperature for 4 hours without the threat of losing the structural or functional usefulness of platelets. The osmolarity of the received dose of TC is 360-380 mOsmol / l, which corresponds to the physiological norm and does not cause the development of post-transfusion reactions in patients.

Brief Description of the Drawings

The invention is illustrated in the graph, which shows the dynamics of changes in the adhesive activity of platelets in thawed TC prepared using the technology of the prototype and the inventive technology.

The implementation of the invention

Below is a detailed description of the proposed method of platelet freezing, indicating in some cases specific values of the parameters that do not limit the claimed invention, but are presented only for a better understanding of the invention. The inventive method includes the following steps.

1. The selection of the original platelet concentrate (TC)

The selection of TC obtained by apheresis or from a dose of blood is carried out according to indicators: concentration and morphofunctional status of platelets. The concentration of platelets in the TC is determined on a hemisphere analyzer. The functional activity of platelets is evaluated by vital staining. To do this, prepare a vital dye for platelets by diluting 10 mg of tripaflavin and 20 mg of acridine orange at room temperature in 100 ml of phosphate buffer (pH 7.2-7.4), staining the TC sample at the rate of 200 μl of the finished dye per 1 ml of sample for 2-5 min at room temperature, after which 5 μl of a sample with stained platelets is transferred to a glass slide and covered with a coverslip. A vitally stained preparation is analyzed under a fluorescence microscope (objective × 100, numerical aperture 0.6, excitation λ 450–490 nm, emission λ from 520 nm) in a semi-automatic mode. Analyze images of 100-200 cells, determine the platelet content with granules (in%) and the adhesive activity of platelets (in%). For cryopreservation, TCs are used that contain from 1 × 10 ⁹ / ml to 1.5 × 10 ⁹ / ml platelets, having platelets with granules from 40% to 70% and platelets with adhesive activity from 40% to 70%.

2. Fractionation of TC

To concentrate platelets, TCs are centrifuged at a speed of 1100g to 1400g for 7 to 12 minutes. After centrifugation, a supernatant (platelet poor plasma) and a platelet-containing part are obtained.

3. Preparation (breeding) of cryoprotectant

From 1 to 3 ml of a combined sterile cryoprotectant (DIMETYLSULFOXIDE / DEXTRAN 40 SOLUTION (Cryopreservative Solution) 5 ml) containing 55% DMSO and 5% dextran 40 is collected in a 10 ml syringe. Connect the syringe with cryoprotectant to a container containing TK placed in a TK 10 to 12 ml of plasma is drawn into a syringe containing a cryoprotectant. As a result of dilution of the cryoprotectant, the concentration of DMSO is reduced by a factor of 5.

4. Separation of plasma from the platelet-containing part

A container containing a supernatant (platelet-poor plasma) and a platelet-containing part is placed in a plasma extractor and the plasma is removed in another container for subsequent freezing.

5. Resuspension of platelets by a cryoprotector

The platelet-containing part is resuspended with the prepared cryoprotectant DIMETYLSULFOXIDE / DEXTRAN 40 SOLUTION (Cryopreservative Solution) containing 55% DMSO and 5% dextran, previously diluted with plasma to a final concentration of DMSO 10-15% (1: 4 ratio). The prepared cryoprotectant is administered in 2 ml intervals of 2 minutes with constant stirring of the contents of the container until the final concentration of DMSO in the platelet suspension is from 5 to 7%.

6. Platelet Freeze

Platelets resuspended by a cryoprotectant are frozen at a speed of 1-3 ° / min in a freezer at a temperature of minus 85 ° C to minus 100 ° C.

7. Storage of frozen platelets

Frozen platelets are stored at a temperature of minus 85 ° C to minus 196 ° C.

8. Quality control of thawed platelets.

The quality of thawed platelets is determined by the parameters: volume (from 180 to 220 ml), the safety of platelets is at least 75% of the original, the safety of platelets with granules and platelets with adhesive activity of at least 50% of the original.

Example

To obtain frozen platelets, a platelet concentrate (TC) was prepared by apheresis on a Trima Accel® blood separator. The TC volume was 200 ml, the platelet content was 218 × 10 ⁹ / dose, the platelet content with granules was 51%, and the platelet adhesive activity was 50%. TCs were divided into two equal parts: one part (TC No. 1) was frozen using the prototype technology, the other (TC No. 2) was frozen using the proposed technology. The quality parameters of cryopreserved platelets are presented in table 4.

Analysis of the results of storage of frozen platelets showed that the claimed technology in comparison with the prototype provides significantly greater safety of viable cells in thawed TCs. So after thawing platelets according to the proposed technology, the content of viable cells was 2.5-3 times higher than in the prototype.

Claims (12)

1. The method of freezing platelets includes: selection of the initial platelet concentrate (TK) with a platelet concentration from 1 × 10 ⁹ / ml to 1.5 × 10 ⁹ / ml containing functionally active platelets; separation of TC into a platelet-containing part and plasma, followed by separation of the plasma from the platelet-containing part; preparation of a combined cryoprotectant containing 55% DMSO and 5% dextran 40 by diluting the cryoprotectant with plasma to a final concentration of DMSO from 10 to 15%; resuspension of the platelet-containing part with a diluted cryoprotectant, which is introduced into the platelet-containing part of 1-3 ml at intervals of 1-3 minutes with constant stirring to a final concentration of DMSO in a platelet suspension of 5 to 7%; freezing a suspension of platelets and plasma at a speed of 1-3 ° / min in separate containers in the freezer at a temperature of minus 80 to minus 100 ° C; storage of frozen platelets and plasma at a temperature of minus 85 to minus 196 ° C. 2. The method according to p. 1, characterized in that the functionally active platelets in the original TC are determined by the parameters: platelets with granules from 40 to 70% and platelets with adhesive activity from 40 to 70%. 3. The method according to p. 1, characterized in that the separation of the TC into the platelet-containing part and the plasma is carried out by centrifugation at a speed of from 1100g to 1400g for 7 to 12 minutes. 4. The method according to p. 1, characterized in that as a cryoprotectant use DIMETYLSULFOXIDE / DEXTRAN 40 SOLUTION (Cryopreservative Solution) 5 ml. 5. The method according to p. 1, characterized in that the platelet-containing part is resuspended by a cryoprotectant for 9 to 12 minutes. 6. The method according to p. 1, characterized in that the initial platelet concentrate is taken with a volume of from 180 to 220 ml, for the preparation of a combined cryoprotectant with a final concentration of DMSO from 10 to 15%, the initial cryoprotectant is diluted with plasma in a volume ratio of 1: 9, resuspension of the platelet-containing part diluted cryoprotectant to obtain a platelet suspension with a final concentration of DMSO from 5 to 7% is carried out in a volume ratio of 1: 1.

Images