CN111713487B

CN111713487B - Reagent erythrocyte preservation system and preparation method thereof

Info

Publication number: CN111713487B
Application number: CN202010613045.1A
Authority: CN
Inventors: 赵俸涌; 向东; 郭忠慧; 朱自严; 钱呈睿; 刘李栋
Original assignee: SHANGHAI BLOOD CENTER
Current assignee: SHANGHAI BLOOD CENTER
Priority date: 2020-06-30
Filing date: 2020-06-30
Publication date: 2021-10-08
Anticipated expiration: 2040-06-30
Also published as: CN111713487A

Abstract

The invention relates to a reagent erythrocyte preservation system and a preparation method thereof, wherein the reagent erythrocyte preservation system comprises the following components in percentage by weight: 0.400g/L of citric acid-1 water; 6.100g/L of trisodium citrate-2 water; 20.500g/L glucose; inosine 0.350 g/L; 0.143g/L of chloramphenicol; neomycin sulfate 0.143 g/L; NaCl 4.200 g/L; adenine 0.500 g/L; mannitol 4.000 g/L; 3.000g/L of monopotassium phosphate. Experiments prove that the reagent erythrocyte preservation system can effectively preserve 3 percent of reagent erythrocytes for 90 days and 15 percent of reagent erythrocytes for 60 days at the temperature of 2-8 ℃. Its advantages are: the reagent red blood cells obtained by using the storage system have long effective period, normal appearance, high morphological proportion of red blood cells and good antigen stability.

Description

Reagent erythrocyte preservation system and preparation method thereof

Technical Field

The invention relates to the technical field of medicine, in particular to a reagent erythrocyte preservation system and a preparation method thereof.

Background

Red blood cell blood group antigens are typically single or multiple transmembrane glycoproteins. In the expression mode, blood group antigen proteins can be divided into expression proteins and adsorption proteins directly coded by nuclear genes; in connection with cell membrane components, some of these proteins are anchoring proteins constituting the cytoskeleton, some are more stably bound to the membrane by interacting with anchoring proteins, and some are bound to the membrane by relying only on the interaction with phospholipid layers. It has been shown that the structure and stability of erythrocyte membranes and membrane proteins changes with increasing storage time, and blood transfusion laboratories typically identify antibodies in sample plasma/serum/diffusion solutions using reagent red blood cells (e.g., ABO-primed cells, antibody-screened cells, spectral red blood cells, IgG-sensitized red blood cells, enzyme-treated cells, etc.). It has been shown that the structure and stability of erythrocyte membrane and membrane protein change with the increase of preservation time, reagent cell is stored at 2-8 deg.C generally, and the preservation time is about 2-3 months, if the change of structure and stability of erythrocyte blood group antigen leads to the change of property of erythrocyte blood group antigen, the change of spectrum cell antigenicity will affect the result of antibody identification after preservation. Finally, antibody leakage of a blood recipient can be caused, the infusion effect of red blood cells is influenced, and even immune hemolytic reaction is caused.

Similar to the infusion red blood cell preservation solution principle, the reagent red blood cell preservation solution needs proper osmotic pressure to maintain the normal structure of the red blood cell membrane. In addition, since physiological metabolism is still required during storage, such metabolism requires a physiological environment as a basis and an energy substance as a substrate. Therefore, the reagent erythrocyte preservation solution generally adopts inorganic salt ions to provide crystal osmotic pressure, polysaccharide molecules which can not freely pass through cell membranes to provide colloid osmotic pressure, and glucose is used as an energy metabolism substrate.

The evaluation criteria for the reagent erythrocytes are on the one hand calculated by the haemolysis rate, which is evaluated for stability during storage, the higher the haemolysis rate, the more severe the erythrocyte disruption during storage is demonstrated. On the other hand, the stability of the antigen indicates the degree of effectiveness of the reagent erythrocytes, and therefore, the antigen on the surface of the reagent erythrocytes is usually detected by using a labeled antibody, by detecting the reagent erythrocytes with the same labeled antibody at the time of detection, and determining whether the antigenicity is stable or not by determining whether the agglutination strength thereof is decreased or not; meanwhile, due to different storage solution formulas, false positive agglutination may be caused by partial components, and whether the agglutination strength is higher than that of fresh cells or not is also an important consideration standard when the antibody is adopted to detect the reagent cells. In addition, with the advance of automated detection, the number of gel card automated detection methods is increasing, and the change of cell morphology (such as shrinkage and thorn formation) during storage affects the speed of red blood cells passing through the gel micropores, and finally affects the gel card interpretation result, so the cell morphology should also be used as an index for reagent red blood cell evaluation.

According to the principle and the evaluation scheme, the reagent preservation solution is prepared and the preservation effect is evaluated, and the result proves that the reagent red blood cells with the storage concentration of 3% can be effectively preserved for 90 days at the temperature of 2-8 ℃, and the reagent red blood cells with the storage concentration of 15% can be effectively preserved for 60 days.

The reagent erythrocyte preservation system and the preparation method thereof are not reported at present.

Disclosure of Invention

The invention aims to provide a reagent erythrocyte preservation system and application thereof.

Another objective of the invention is to provide a reagent erythrocyte and a preparation method thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

a reagent erythrocyte preservation system comprises the following components in percentage by weight:

preferably, the reagent red blood cell preservation system comprises the following components in percentage by weight:

further, the use of the preservation system as described above for the preparation of a kit for preserving the reagent erythrocytes.

A kit for preserving reagent red blood cells, said kit comprising a preservation system as described above.

In order to realize the other purpose, the invention adopts the technical scheme that:

a reagent red blood cell comprises the preservation system and the red blood cell sample.

Preferably, the red blood cell sample is from washed raw blood.

More preferably, 15% of the reagent red blood cells are preserved for a period of not less than 60 days, and 3% of the reagent red blood cells are preserved for a period of not less than 90 days.

Further, the preparation method of the reagent red blood cells comprises the following steps: preparing a preservation system and adding red blood cells, wherein the added red blood cells are washed in advance and used for removing part of non-red blood cell components in blood.

Preferably, the method for preparing reagent red blood cells further comprises the steps of: evaluation of the effect of preservation of the reagent erythrocytes.

More preferably, the effect evaluation content includes: the appearance of the preserved reagent red blood cells is evaluated, and the stability of the antigen of the reagent red blood cells is evaluated.

The invention has the advantages that:

the invention preferably selects each component and the concentration thereof, each component of the whole system has synergistic effect, glucose is mainly responsible for providing energy for erythrocyte metabolism during the preservation period, mannitol provides colloid osmotic pressure, and the other salt buffer systems are mainly responsible for maintaining crystal osmotic pressure and buffering pH fluctuation caused by metabolites during the preservation period. The components are mutually contained and act synergistically to achieve the purposes of keeping the activity of the erythrocyte and prolonging the preservation period of the erythrocyte, and experimental results show that the erythrocyte with normal appearance obtained by using the preservation system disclosed by the invention has high morphological proportion and good antigen stability, can be well used for identifying the antibody of a recipient, and has wide application prospect.

Drawings

FIG. 1 is a parallel experiment of red blood cell preservation with different preservation solutions, A: three samples of 3% and 15% on day 0 in group 4; b: group I three samples, 3% and 15% on day 0; c: three samples, 3% and 15% on day 0 in group R; d: day 30 3% group appearance; e: day 30 15% group appearance; f: day 60 3% group appearance; g: day 60 15% group appearance; h: 3% group appearance on day 90; i: day 90 15% group appearance; note: D. e, F, G, H, I are 4 groups of 3 tubes, R group of 3 tubes and I group of 3 tubes from left to right.

FIG. 2 shows the morphology of erythrocytes at the end of storage, A: no. 4 preservation solution with 3% preservation concentration for 90 days; b: 3% preservation concentration of Immucor preservation solution is 90 days; c: 3 percent of Shanghai blood biological erythrocyte diluent is preserved for 90 days; d: no. 4 preservation solution with 15% preservation concentration for 60 days; e: preserving Immucor preserving fluid with 15 percent of preservation concentration for 60 days; f: the 15% preservation concentration of Shanghai blood biological erythrocyte diluent is 60 days.

FIG. 3 is a comparison of the hemolysis rates of various concentration groups, wherein A: comparing the hemolysis rate of each group with 3 percent concentration; b: the hemolysis rates of the 15% concentration groups were compared.

FIG. 4 is a comparison of PS valgus at different concentrations for various groups, where A: PS valgus comparison for each group at 3% concentration; b: the PS evaginations were compared for each group at 15% concentration.

FIG. 5 is a graph of the results of a gradient-preserved cell assay, wherein A: 3% cell concentration for 7 days; b: 3% cell concentration for 75 days; c: the cells are preserved for 7 days at 15 percent cell concentration; d: preserving at 15% cell concentration for 75 days; note: the dilution from left to right in each figure is 0.7; 0.8; 0.9; 1; 1.2; 1.5X.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the claims appended to the present application.

EXAMPLE 1 Effect test

Erythrocytes are a special cell, mature erythrocytes have no nucleus, so that their metabolism is entirely powered by own glycolysis, and with increasing preservation time, the metabolism of energetic substances leads to changes in the structure of the erythrocyte membrane. The change of the membrane structure can lead to the change of the permeability of the erythrocyte membrane and the change of the cell morphology on the one hand; on the other hand, spatial conformation changes of membrane proteins are also caused, resulting in antigenic changes. Therefore, this example is intended to evaluate the cell membrane structure and antigenicity of an objective anti-reagent erythrocyte preservation solution and to reflect the effect of the reagent erythrocyte preservation solution on the preservation of the reagent erythrocyte.

1 method of experiment

1.1 reagent erythrocyte preservation solution (reagent erythrocyte preservation solution, hereinafter referred to as solution 4), the preparation method is shown in Table 1.

TABLE 1 composition of reagent erythrocyte preservation solution

1.2 detection

Using 3 parts of single AB type red blood cells, and using 4 solutions (hereinafter represented by 4); immucor reagent Red Blood Cell (RBC) storage Solution,

(hereinafter, represented by I) LOT RR077, having a pot life of 2020-02-11; the dilution (R below) for domestic commercialized blood group analysis, Shanghai blood biological medicine Co., Ltd. (SHPBC), product lot No. 20196201, validity period to 20210422, and two concentration gradients of 3% and 15% were set for the test. The related evaluation indexes are detected on 0 th day, 30 th day, 60 th day and 90 th day after being stored at the temperature of 2-8 ℃.

The detection method comprises the following steps:

the appearance is stored and recorded by photographing, the antigen stability adopts the labeled antibody (labeled to the moderate agglutination strength in the reaction with antigen positive erythrocytes, see table 2) to detect the main blood group antigens of the ABO blood group system, the Rh blood group system, the MN blood group system, the Kidd blood group system and the Duffy blood group system, and the agglutination strength is recorded by adopting a Bio-rad gel column method for detection. The appearance of the red blood cells was recorded by taking a photograph using a 40-fold inverted microscope (biotekrotation). The supernatant hemolysis rate was measured using a hemoglobin measurement kit from BioAssay Systems, according to the kit instructions. The PS valgus detection adopts Annexin V-APC detection kit of the company for detection. And (5) evaluating the effect of the detection result.

TABLE 2 reagent batches and standard dilutions for antigen detection

Lyophilized powder traceable to WHO international standard IgM anti-A, anti-B and anti-D;

SHPBC, Shanghai blood biomedicine Co.

Since the important value of the reagent red blood cells is the identification of the specificity of the antibodies in the serum/plasma sample by the specific blood group antigens present thereon. Therefore, the stability of the reagent erythrocyte antigen has a decisive influence on the whole serological experiment result. Therefore, we used the stability of the reagent erythrocyte antigen as the most important evaluation basis. Because serology experiments are difficult to quantify, a scoring system is adopted in the experiments to evaluate the stability of the antigen. The main method comprises the following steps: a series of antibody reagents (see table 2) were used, labelled, and reacted with erythrocytes of different retention times. The reaction intensity was read by standardized detection conditions. The reaction intensity is expressed by negative, +/-. 4+, and different agglutination plus signs are assigned. During storage, if a specific antigen is weakened, agglutination is weakened continuously, and the agglutination is converted into corresponding values through agglutination plus signs so as to be aligned with each other.

The method comprises the steps of using commercial erythrocyte preservation solution at home and abroad as a reference, respectively adjusting the concentration of the erythrocytes to two concentrations of 3% and 15%, setting 3 multitubules for each concentration of each preservation solution, respectively marking as '1', '2' and '3', and respectively adding fresh erythrocytes from different individual sources into three tubes. The test was conducted after storage at 4 ℃ for "0 day", "30 days", "60 days" and "90" days, respectively. The specific antibody reacts with 3 multitubular erythrocytes respectively, the agglutination strength is converted into numerical values, then the average values are taken for comparison, and the reduction amount of the average values corresponds to the reduction degree of the antigen.

In order to quantitatively estimate the effect of the erythrocyte preservation solution, a stable and reliable microcolumn agglutination card method is used for detecting the preservation of various antigens in research. Including A, B, M, N, which is a relatively representative carbohydrate antigen; representative of protein antigens D, c, e, Fya and Jka were compared. All antibodies and preserved erythrocytes were labeled to 1+ to 3+ intensity and were split and frozen. The antibody used in each test was kept as consistent as possible. The judgment of the agglutination strength is carried out by the same person according to the following standards, and the judgment result is converted into a score value so as to be beneficial to statistical calculation.

TABLE 3 erythrocyte blood group antigen detection evaluation scoring table

In the statistics, the mean value of the agglutination intensity exhibited by the reaction of the erythrocytes of different individuals with the specific antibody on the

day

0 and 3 different storage solutions was used as a standard, and the standard value was subtracted from the reaction intensity of the specific antibody with each group of erythrocytes during the storage period to obtain a measure of the change of the erythrocyte antigen during the storage period. Then, the average values of the detection of the carbohydrate antigen and the protein antigen under the concentration of 3 percent and 15 percent respectively on different days are calculated, so that the T test is carried out, and whether the storage conditions of different types of antibodies in different storage solutions have significant differences or not is judged. The results are shown in tables 4 and 5 below.

2 evaluation of Effect

2.1 preservation appearance:

the preserved appearance of the reagent red blood cells can directly influence result interpretation (hemolysis is also used as positive interpretation in serology experiments), so that the original observation index of the preserved appearance of the red blood cells is used as a primary evaluation standard. According to the appearance photographing record, the following steps are carried out: the initial total volume of each group is the same as the proportion of erythrocytes, so the appearance hemolysis degree can visually express the erythrocyte preservation capability, and the slight hemolysis appears in the group I on day 60 in 3 percent of experimental groups through preservation; slight hemolysis occurred in group R and group I at 90 days, and slight hemolysis occurred in group I at day 30 in 15% of the experimental groups; slight hemolysis occurred in group R and significant hemolysis occurred in group I on day 60; on day 90, 4 groups showed slight hemolysis, R group showed significant hemolysis, and I group showed severe hemolysis, and experiments showed that the hemolysis rate at each detection point was slightly better than that of R group and significantly better than that of I group in 4 groups at 3% and 15% preservation concentrations (see FIG. 1).

Reagent red blood cells can deform along with the prolonging of the preservation time, and the serological result interpretation, especially the gel card result interpretation, can be influenced by the change of the adsorption capacity of the deformed red blood cells. Therefore, the appearance of the red blood cells is taken as an important evaluation index. By comparison we found that: in the aspect of red blood cell morphology preserved by the solution 4, no matter 3% or 15% of groups, the normal red blood cell morphology proportion of each detection node is the highest, which shows that the preservation effect of the red blood cell is superior to that of the other two preservation solutions. See fig. 2.

2.2 reagent erythrocyte antigen stability:

TABLE 4 comparison of stability of 3% concentration preserved erythrocyte blood group antigens

Note: *: with statistical differences

TABLE 5 comparison of stability of antigens of red blood cell blood group stored at 15% concentration

By using paired T test, when 3% of red blood cells are preserved in the patent formula, the preservation of carbohydrate antigens and protein antigens is obviously superior to that of domestic maintenance liquid, and has no significant difference from that of maintenance liquid of international well-known company; when 15% of red blood cells are preserved, the preservation of carbohydrate antigens and protein antigens has no significant difference from the preservation solution of domestic and international well-known companies.

2.3 indexes of the hemolysis rate of the supernatant:

the percentage of erythrocytes which are lysed or which cause an efflux of hemoglobin due to a change in permeability caused by a membrane disruption in an erythrocyte sample, which is a direct reaction reagent for the hemolysis rate of the supernatant, is based on the following principle: (supernatant hemoglobin light absorbance-background absorbance)/total hemoglobin absorbance of the sample ═ lysed cells/total cells%, and in order to quantify the cell preservation effect, we used the index of the supernatant hemolysis rate as an important reference standard for reagent red blood cell preservation. By comparison we found that: in a 3% preservation experiment, the hemolysis rate of the group I on the 30 th day is remarkably higher than that of the group 4 (p is less than 0.05), and the hemolysis rate of the group I and the group R on the 90 th day is remarkably higher than that of the group 4 (p is less than 0.05); in the 15% group preservation experiment, the hemolysis rate I group was significantly higher than 4 groups (p < 0.05) at day 60, and the hemolysis rate I and R groups were significantly higher than 4 groups (p < 0.05) at day 90 (see FIG. 3).

2.4 reagent erythrocyte Membrane stability assay (PS Excipit assay)

The eversion of PS (phosphatidylserine) marks the reduction of the stability of the erythrocyte membrane of the reagent, and the reduction of the stability of the erythrocyte membrane of the reagent can cause the generation of cell hemolysis and nonspecific adsorption, thereby influencing the use of the reagent cells. Therefore, the stability of the erythrocyte membrane of the reagent is finally used as an important reference basis for the preservation effect. By flow cytometry detection using fluorescent antibodies, we found: in 3% preservation experiments, the PS eversion degree of the group I on the 30 th day is obviously higher than that of the group 4 (p is less than 0.01), and the group I and the group R on the 90 th day are respectively obviously higher than that of the group 4 (p is less than 0.01); in 15% group preservation experiments, the PS eversion degree of group I was significantly higher than that of group 4 (p < 0.05) at day 60, and the PS eversion degree of group I and group R was significantly higher than that of group 4 (p < 0.01) at day 90 (see FIG. 4).

EXAMPLE 2 concentration gradient test

1 method of experiment

Respectively preparing 0.7; 0.8; 0.9; 1; 1.2; 1.5X preservation solution (optimum concentration is 1), and 3% cell ratio and 15% cell ratio preservation experiment was performed. Two test nodes were selected at day 7 and at the midpoint of 75 days in the 3% and 15% planned cell storage periods to evaluate the short-term and long-term effects of the gradient on the cells.

2 results

And (4) preserving appearance observation: the appearance of the 1X stock solutions from 7 and 75 days of storage indicated that the 1X stock solutions had the best storage effect, at the best concentration (see figure 5).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.

Claims

1. The reagent erythrocyte preservation system is characterized by comprising the following components in percentage by weight:

2. use of the preservation system according to claim 1 for the preparation of a kit for preserving the reagent erythrocytes.

3. A kit for preserving reagent red blood cells, said kit comprising the preservation system of claim 1.

4. A reagent red blood cell comprising the preservation system of claim 1 and a red blood cell sample.

5. The reagent red blood cell of claim 4, wherein the red blood cell sample is derived from washed raw blood.

6. The reagent red blood cells of claim 4, wherein 15% of the reagent red blood cells are preserved for a period of not less than 60 days and 3% of the reagent red blood cells are preserved for a period of not less than 90 days.

7. The method for preparing reagent erythrocytes according to claim 4, comprising the steps of: preparing a preservation system and adding red blood cells, wherein the added red blood cells are washed in advance and used for removing part of non-red blood cell components in blood.

8. The method of claim 7, further comprising the steps of: evaluation of the effect of preservation of the reagent erythrocytes.

9. The manufacturing method according to claim 8, wherein the content of the effect evaluation includes: the appearance of the preserved reagent red blood cells is evaluated, and the stability of the antigen of the reagent red blood cells is evaluated.