RU2720487C1 - Additional solution for storage of thrombocyte concentrate - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to solutions for resuspension and storage of components of harvested donor blood (additive solutions), and particularly for resuspension and storage of thrombocyte concentrate (thrombocyte concentrate). Additional solution for storing the thrombocyte concentrate includes sodium, potassium and magnesium chlorides, mono- and disubstituted sodium phosphates and a nutritional component containing sodium citrate and additionally sodium fumarate, wherein the components are taken in the following ratio in millimoles per 1 liter of the solution: 61.0±3.0 sodium chloride, 4.9±0.25 potassium chloride, 1.45±0.25 magnesium chloride, 17.0±0.1 sodium hydrophosphate, 6.65±0.15 sodium dihydrophosphate, 10.75±0.25 sodium citrate and 17.0±5.0 sodium fumarate.

EFFECT: obtaining additional solution for storage of thrombocyte concentrate, which allows preserving the amount and functional activity of thrombocytes, as well as their survival rate during storage and compensation in vivo.

1 cl, 2 tbl

Description

The invention relates to medicine, namely to solutions for resuspension and storage of components of the prepared donated blood (additional solutions), and in particular for resuspension and storage of platelet concentrate. Transfusion of platelet concentrate is a necessary measure for the correction of thrombocytopenia, primarily in oncological and hematological practice.

Despite the high clinical demand, providing patients with platelet concentrate creates certain difficulties for the blood service and for clinical practitioners. This is due both to the short shelf life of it, not exceeding 5-7 days, and the duration of the preparation process, and the likelihood of rapid development of clinically significant thrombocytopenia in patients. The creation of a sufficient supply of platelet concentrate of the necessary blood groups also limits its high cost.

The main requirement for the platelet concentrate used during transfusion is to maintain platelet activity when they enter the vascular bed during transfusion. The quality of the platelet concentrate is due to a number of factors, among which the environment in which it is stored is a plasma or an additive solution. The natural environment for platelets is the blood plasma of the donor, but it is also the best environment for the life of bacteria, and, in addition, contains donor antibodies that can damage the recipient during transfusion of platelet concentrate.

The use of additional solutions as a storage medium allows one to minimize the frequency of adverse reactions caused by transfusion of platelet concentrate, to use platelets that are not identical in blood group with a lower titer of hemagglutinins, simplifies the determination of bacteria in contaminated platelet concentrates, and allows photochemical treatment to inactivate bacteria and other pathogens in platelets using certain techniques, improves storage conditions and increases asset yield s platelets. Reducing the amount of plasma transfused with platelets also allows you to save plasma for further processing.

A platelet concentrate prepared by hardware using an additional platelet storage solution usually contains about 30% native plasma and about 70% additional solution.

Studies on the search for platelet preservation media began abroad in the 80s of the last century. Platelet storage supplements created abroad are designated by the abbreviation PAS - platelet additive solutions. The first such PAS-A solution contained sodium chloride, citrate and sodium phosphate, as well as potassium ions. Further developments led to the creation of multicomponent salt media, including sodium, potassium and magnesium chlorides, as well as acetate, citrate, phosphate and sodium gluconate, differing in the composition and concentration of the included components [Gulliksson N. Platelet storage media // Vox sang. - 2014 .-- Vol. 107, N 2. - P. 205-212].

The most famous and widely used in our time solution SSP + (other names PAS-III M and PAS-E) contains 69.3 mmol / L sodium chloride, 10.8 mmol / L sodium citrate, 32.5 mmol / L sodium acetate, 28.2 mmol / L mono / disodium phosphate, 5.0 mmol / L potassium chloride and 1.5 mmol / L magnesium chloride [Gulliksson N. Defining of optimal storage conditions for the long-term storage of platelets // Transfus. Med. Rev. - 2003. - Vol. 17. - P. 209-215]. The shelf life of platelets in a medium containing this solution can reach 7 days.

An additional platelet storage solution is also known, including 4.21-5.14 g of sodium chloride, 0.84-1.26 g of sodium bicarbonate, 0.37-0.45 g of magnesium chloride per 1000 ml, 3.97-4 85 g of sodium citrate, 1.85-2.25 g of sodium acetate, 0.45-0.75 g of sodium dihydrogen phosphate, 0.34-0.42 g of potassium chloride, 22-35 mg of L-arginine and 1.80 3.60 g glucose [CN 101485886 (A), IPC A61K 47/26, 2009].

However, according to our data, the addition of arginine to an additional platelet storage solution accelerates the consumption of glucose remaining in the solution with native plasma; accelerated consumption requires the addition of exogenous glucose to the solution, which was done by the authors. However, the introduction of an additional amount of glucose increases the risk of bacterial contamination of harvested platelets, which is undesirable; in addition, the addition of glucose creates additional technological difficulties when sterilizing the additive solution.

The closest in technical essence and the achieved effect to the claimed solution is an additive solution for storing platelets [US 10271541 (B2), IPC A01N 1/02; A61J 1/10, 2019], including electrolytes - sodium, potassium, magnesium and calcium chlorides, - components of the buffer mixture - dihydrogen phosphate and sodium hydrogen phosphate, - sodium citrate, “nutrient component” - sodium acetate and glucose, - and an additional 0.01-100 mmol of β-galactosidase inhibitor and 0.01-100 mmol of sialidase inhibitor. As an inhibitor of β-galactosidase, for example, 1-deoxyhalactone neimycin, N- (n-butyl) deoxyhalactone neimycin, etc. were used; fetuin, 2,3-dehydro-2-deoxy-K-acetylneuraminic acid (DANA), ethyl (3R, 4R, 5S) -5-amino-4-acetamido-3- (pentan-3-yloxy) are available as a sialidase inhibitor ) -cyclohex-1-en-1-carboxylate) and others. According to the data given in the prototype, in the presence of galactosidase and sialidase inhibitors, platelets retain their properties for 9 days of storage.

US 10271541 provides data on the positive effect of these inhibitors on the state of platelets, but does not provide data on their effect on the recipient receiving transfusion of the platelet mass prepared in this solution. It cannot be excluded that the introduction of the substances indicated in the prototype as inhibitors can lead to undesirable reactions in the clinical use of platelets, since these substances are not physiological, that is, they are not participants in the processes of natural metabolism. This argument is especially weighty when using platelet concentrate in hematologic practice. In addition, this solution has the disadvantages associated with the additional introduction of exogenous glucose.

The result, the achievement of which the claimed invention is directed, is to preserve the number and functional activity of platelets during their storage using a metabolic component of a physiological nature in an additive solution.

This result is achieved in that the additive solution for storing platelets, including sodium, potassium and magnesium chlorides, monosubstituted and disubstituted sodium phosphates and a nutrient component including sodium citrate, additionally contains sodium fumarate in the composition of the nutrient component in the following ratio of components in millimoles (mmol ) per 1 liter of solution:

sodium chloride 61.0 ± 3.0 potassium chloride 4.9 ± 0.25 magnesium chloride 1.45 ± 0.25 sodium hydrogen phosphate 17.1 ± 0.1 sodium dihydrogen phosphate 6.65 ± 0.15 sodium citrate 10.75 ± 0.25 sodium fumarate 17.0 ± 5.0

The solution may include 46 ± 1 mmol / L mannitol, which is introduced to adjust and maintain the osmolarity of the solution within 300-330 mosm / l.

The inventive solution is obtained by dissolving the components of the solution in 1000 ml of double-distilled water; the solution was filtered through a millipore filter, poured into vials and sterilized by autoclaving.

In an experiment on animals (rats, rabbits) it was found that the claimed solution is not toxic and pyrogen-free.

To obtain platelet concentrate, whole blood was collected in a disposable polymer container for collecting, storing and transporting TERUMO blood, constructed with a volume of 450/450/450 ml, CPD / SAGM, Top & Bottom, Japan. Donor blood was harvested on a Transwaag KL stirring balance (Transmed Medizintechnik GmbH & Co, Germany). Optimum donation time up to 12 min.

After keeping the preserved blood for 2 hours at a temperature of + 22 ° С, the initial “hard” centrifugation was carried out on a Cryofuge 5500i refrigerator centrifuge (Heraeus, Thermo Electron Corporation, USA). A 45-60 ml volume automatic blood component extraction system (LMB Tehnologie GmbH, Germany) was used for blood fractionation with further obtaining erythrocyte suspension, plasma and a standard dose of a leukotrombolayer (LTS) in a volume of 45-60 ml. The received doses of LTS were stored at a temperature of 22 ± 2 ° С for up to 24 hours from the moment of blood preparation without stirring.

After culling, individual doses of LTS were combined according to group compatibility in a container for separation, storage and transportation of blood components TAKSI PL KIT, Terumo. Next, extraction was carried out by dilution with the inventive additive solution or SSP + solution.

To separate platelet concentrate from other cellular elements of the pool, a “soft” centrifugation mode was used on a specialized TACSI centrifuge separator (Belgium). Platelet extraction occurred during centrifugation. The press of the system unit under the control of the microprocessor and optical sensors "squeezed" the platelet pool through the leukocyte filter into a bag for long-term storage. At the same time, the storage medium contained 30% autologous (native) plasma and 70% of an additional solution.

The change in platelet safety indicators during storage in the inventive solution for 9 days was studied; SSP + solution was taken for comparison.

The metabolic activity of platelets was determined by a decrease in the content of native glucose in the samples and an increase in the lactate content and by a change in pH. The content of glucose, lactic acid (lactate) and the pH value in platelet concentrate samples were studied using a Radiometer ABL-800 biochemistry analyzer (Denmark). To determine platelet concentration and cell volume, a SISMEX hematology analyzer, KX 21N (Japan) was used. The test results are presented in Table 1.

The functional state of platelets was assessed by the degree of platelet aggregation induced. The study of platelet aggregation properties was carried out according to the Born method on a 4-channel platelet aggregation analyzer AT-2. The temperature in the thermostatically controlled cell under study is 37 ° C, and the rotation speed of the magnetic stirrer is 750 rpm.

To assess platelet aggregation, the most informative value was used - the maximum amplitude of aggregation (MA). 100% light transmission (control) was set using a mixture of platelet-free plasma and buffer solution in a ratio of 3: 2 (0.3 ml of plasma + 0.2 ml of buffer).

The number of platelets in the studied plasma influenced the measurement result, and for the study, the plasma was standardized to obtain a platelet concentration of 200-250 × 10 ⁹ / L. Platelet concentration was determined in platelet counting mode on a SISMEX analyzer. If the platelet concentration in the plasma was above a predetermined level, then it was diluted by adding platelet-free plasma.

The studied platelet suspension was diluted with a platelet-free donor plasma of group IV to increase the protein fraction and normalize the platelet count to 200 × 10 ⁹ / L. A cuvette with 450 μl of the test suspension was placed in an aggregometer, 50 μl of inducer was added, and platelet aggregation was recorded.

CHRONO-PAR reagents were used: adenosine diphosphate (ADP), collagen, ristocetin from CHRONO-LOG. Reagents were packaged in Eppendorf type microtubes and stored in a refrigerator or freezer. Before starting the study, the reagents were diluted with buffer solution. The measurement results are presented in Table 2.

A comparative study of platelet safety during storage in the inventive solution and in the SSP + solution showed (Table 1) that the inventive solution creates better conditions for platelet storage compared to the SSP + solution. The number of platelets in the inventive solution by the 9th day of storage is 93.3 ± 4.0% of the initial amount, in the SSP solution + 87.6 ± 6.1%.

The amount of glucose in two solutions at the beginning of storage was almost the same: 3.87 ± 0.30 mmol / L in the inventive solution and 3.73 ± 0.23 mmol / L in the SSP + solution, but by the 6th day of storage 0 remained in the inventive solution , 97 ± 0.63 mmol / L glucose, and in the solution SSP + 0.16 ± 0.04 mmol / L, and by the 8th day of storage 0.57 ± 0.14 mmol / L glucose remained in the inventive solution, and in the solution SSP + 0.10 ± 0.00 mmol / L, the difference is statistically significant.

The amount of lactate at the beginning of storage was different in solutions and amounted to 6.09 ± 0.74 mmol / L in the inventive solution, in the SSP solution + 7.87 ± 0.76 mmol / L; when stored in the inventive solution by the 6th day of storage is 9.87 ± 1.09 mmol / l, in the SSP solution + 14.45 ± 0.44 mmol / l, and by the 9th day of storage it is in the inventive solution.

Thus, with the same initial values of the glucose content in solutions of the inventive solution provided a more economical consumption. In this case, 11.9 ± 0.1 mmol / L, in a solution of SSP + 14.2 ± 1.7 mmol / L, the difference is statistically significant.

The increase in lactate concentration in the presence of sodium fumarate is slower than in the SSP + solution. Lower values of the lactate content in combination with a higher glucose content at the end of platelet storage in the inventive solution indicates the ability of this solution to support aerobic oxidation processes, allowing for more efficient ATP production. In the SSP + solution, judging by the results, anaerobic glycolysis reactions prevail, indirectly reflecting the state of platelet energy deficiency.

The pH during storage of platelets in two solutions did not change significantly: in the inventive solution from 7.16 ± 0.02 at the beginning of storage to 7.09 ± 0.02 by day 9, and in a solution of SSP + from 7.07 ± 0.04 up to 7.13 ± 0.06 at the same time. And, although the dynamics of pH values during the observation period in both solutions was different, the pH values themselves changed very slightly, which indicates a sufficient compensatory ability of buffer systems in solutions.

According to the literature, with an increase in the shelf life of platelets in additional solutions, their volume increases. According to our observations, the average platelet volume increased slightly with increasing shelf life in both solutions: from 8.0 ± 0.2 μm at the beginning of storage to 8.9 ± 0.6 μm by day 9 in the inventive solution, and in SSP + from 8.4 ± 0.2 to 9.8 ± 0.2 μm at the same time. Identified changes in platelet size fit within the physiological norm.

However, the observed difference is statistically significant and indicates a better preservation of platelets in the inventive solution compared to the SSP + solution.

A study of the aggregation ability of platelets with various inducers (Table 2) during storage showed that at the beginning of storage, the maximum aggregation amplitude (MA) when using ADP was 11.4 ± 1.8% for the inventive solution and 15.9 ± 2.3 % for the SSP + solution, however, by the 9th day of storage, these figures were 3.6 ± 1.5% and 0.4 ± 0.4, respectively. With a higher initial aggregation ability of the platelets harvested in the SSP + solution, by the end of the cell storage, it dropped to zero, while the platelets in the inventive solution retained some aggregation ability. The same trend was observed in the case of using ristocetin and collagen inducers.

To be clinically effective, platelets must return to circulation after transfusion. According to the literature [N.M. Rinder, E.L. Snyder, J.B. Tracey et al II Transfusion. - 2003. - Vol. 43, N 9. - P. 1230-1237; S.J. Slichter, D. Bolgano, M.K. Jones et al. // Transfusion. - 2006. - Vol. 46, N 10. - P. 1763-1769] none of the in vitro tests correlates with the compensation and survival of platelets, although they provide important information about the physiology of platelets and the degree of decrease in their functions during storage. In vivo recovery and survival are indispensable indicators in assessing platelet quality.

To determine these indicators, model experiments were performed on rabbits. By the method of massive bloodletting in animals, about 40% of the circulating blood volume was removed, thereby causing severe thrombocytopenia. 24 hours after the bloodletting, the number of platelets in the bloodstream of rabbits was 13-15% of the initial values. The introduction of a therapeutic dose of platelet concentrate, prepared on the claimed solution, led to an increase in the platelet content to the original values, and sometimes to exceed the initial level. In rabbits that did not receive platelet transfusion, spontaneous restoration of the platelet count in the bloodstream at the same observation time did not exceed 60%.

Thus, the claimed additive solution containing as a metabolic component sodium fumarate - one of the substrates of the Krebs cycle - allows you to significantly preserve the metabolic and functional activity of platelet concentrate, as well as their survival during storage and compensation in vivo.

Claims (3)

1. An additive solution for the storage of platelet concentrate, including sodium, potassium and magnesium chlorides, mono- and disubstituted sodium phosphates and a nutrient component including sodium citrate, characterized in that it additionally contains sodium fumarate as part of the nutrient component in the following ratio of components in millimoles (mmol) per 1 liter of solution: sodium chloride 61.0 ± 3.0 potassium chloride 4.9 ± 0.25 magnesium chloride 1.45 ± 0.25 sodium hydrogen phosphate 17.1 ± 0.1 sodium dihydrogen phosphate 6.65 ± 0.15 sodium citrate 10.75 ± 0.25 sodium fumarate 17.0 ± 5.0 2. The solution according to claim 1, characterized in that it additionally contains mannitol at a concentration of 46 ± 1 mmol / L.