CN111537326A

CN111537326A - Method for preparing freeze-dried blood platelet and application thereof

Info

Publication number: CN111537326A
Application number: CN202010377816.1A
Authority: CN
Inventors: 王丽; 王秀柱; 黄志刚; 王伟权; 潘大地
Original assignee: Tianjin Dexiang Biotechnology Co ltd
Current assignee: Tianjin Dexiang Biotechnology Co ltd
Priority date: 2020-05-07
Filing date: 2020-05-07
Publication date: 2020-08-14
Anticipated expiration: 2040-05-07
Also published as: CN111537326B

Abstract

Provided herein is a method of making freeze-dried platelets comprising: 1) contacting the platelets with a pretreatment solution and a fixative in sequence; and 2) freeze-drying the platelets treated in step 1) in the presence of a freeze-drying preservation solution, wherein the pretreatment solution comprises lysine and collagen; the stationary liquid comprises glutaraldehyde and ethanol; the freeze-drying preservation solution comprises serum albumin, PVP and mannitol. Also provided herein are freeze-dried platelets prepared by the method and uses of the freeze-dried platelets. The freeze-dried platelet provided by the invention can keep the antigen activity of the platelet during long-term storage, and provides a solution for the application of anti-screening platelets and the standardized application of a platelet antibody detection kit.

Description

Method for preparing freeze-dried blood platelet and application thereof

Technical Field

This document relates to methods of preparing freeze-dried platelets, and in particular, methods of preparing freeze-dried platelets useful for platelet antibody screening.

Background

The platelet antibody is an antibody generated by the body to the surface of the platelet or related antigens, and the antigen for stimulating the production of the platelet antibody can be self-body or allogenic. There are two classes of platelet antibodies, one that is predominantly antibodies directed against human histocompatibility antigen (HLA) class I antigens, i.e., platelet surface-associated antibodies (PAIgG), and one that is platelet-specific antibodies (PB IgG) directed against GP antigens. The GP antigen and the HLA-I antigen stimulate the body to generate homogeneous immune response, and the damage of exogenous platelets causes the shortening of the platelet life and the change of the function, which is an important reason for the ineffective platelet transfusion. With the increase of the times and the amount of infusion, the positive rate of the detected platelet antibodies is increased, the ineffective infusion occurs and the reaction rate of nonhemolytic blood transfusion is increased.

The method can accurately, quickly and simply detect and identify the platelet antibody, and plays an important role in the diagnosis, treatment and the like of the platelet immune diseases. The major clinical methods for detecting platelet antibodies are immunological methods, but no commercial pure antigen is available so far because of the complexity and diversity of platelet surface antigens. Platelets themselves are often used in current assays to provide antigens. The preservation of platelets currently presents the following problems: 1. the blood platelets are easy to aggregate and lose the antigenicity in a liquid environment, and the storage period is short; freeze-dried platelets are intended for infusion and therapy (wound healing), lack of protection against platelet surface antigens, and are largely damaged during freeze-drying, so that the products have a plurality of problems in platelet antibody screening applications, such as low sensitivity and low positive rate.

Disclosure of Invention

In one aspect, provided herein is a method of preparing freeze-dried platelets, the method comprising:

1) contacting the platelets with a pretreatment solution and a fixative in sequence; and

2) freeze-drying the platelets treated in the step 1) in the presence of a freeze-drying preservation solution,

wherein the pretreatment solution comprises lysine and collagen; the stationary liquid comprises glutaraldehyde and ethanol; the freeze-drying preservation solution comprises serum albumin, PVP and mannitol.

In some embodiments, the pretreatment solution further comprises trehalose, sorbitol, tween-20, and EDTA.

In some embodiments, the pretreatment solution is a buffer comprising: trehalose 0.2-5%; 1-5% lysine; 0.2-4% collagen; 0.1-2% sorbitol; 0.01-0.5% (v/v) tween-20; and 2-30mM EDTA.

In some embodiments, the pretreatment solution is formulated by: to each liter of buffer solution, 8g of NaCl, 20g of trehalose, 25g of lysine, 15g of collagen, 5g of sorbitol, 202 mL of Tween-and 1.8g of EDTA were added.

In some embodiments, the volume ratio of glutaraldehyde to ethanol in the fixation fluid is 1: 8.

in some embodiments, the lyophilized preservation solution is a buffer solution comprising: 1-6% serum albumin, 0.1-1.5% PVP, and 0.5-5% mannitol.

In some embodiments, the lyophilized preservation solution is prepared by the following method: to each liter of buffer was added 40g of serum albumin, 12g of PVP, and 18g of mannitol.

In some embodiments, step 1) comprises: preparing 10 blood platelets by using the pretreatment liquid⁸-10¹⁰A first platelet suspension, and 1/10-1/4 volumes of the fixative are added according to the volume of the first platelet suspension, and the reaction is carried out for 0.5-1 hour at room temperature.

In some embodiments, the method further comprises washing the platelets with a phosphate buffer containing EDTA or citric acid prior to step 1).

In some embodiments, between step 1) and step 2, washing the platelets treated in step 1) with a phosphate buffer containing EDTA or citric acid is further included.

In some embodiments, step 2) further comprises suspending the platelets treated in step 1) to 10 with the lyophilization preservative fluid prior to the lyophilization⁸-10¹⁰and/mL of a second platelet suspension.

In some embodiments, the lyophilizing of step 2) is performed by vacuum-pumping in a lyophilizer having a cold trap temperature set at-45 ± 5 ℃, a vacuum level set at <133mbar, and vacuum-lyophilizing for 24h or more.

In some embodiments, the lyophilizing in step 2) comprises: and transferring the second platelet suspension into a freeze-drying bottle, pre-freezing at ultralow temperature of-80 ℃ overnight, transferring the second platelet suspension into a pre-cooled freeze-drying machine for vacuum drying the next day, setting the temperature of a cold trap to be-45 +/-5 ℃, setting the vacuum degree to be less than 133mbar, and taking out the second platelet suspension after vacuum drying for 24 hours.

In another aspect, provided herein is a freeze-dried platelet prepared by the above method.

In another aspect, provided herein is a test kit comprising the above-described freeze-dried platelets.

In another aspect, provided herein is the use of the above freeze-dried platelets or the above test kit in platelet antibody screening.

The freeze-dried platelet provided by the invention can keep the antigen activity of the platelet during long-term storage, and provides a solution for the application of anti-screening platelets and the standardized application of a platelet antibody detection kit.

Drawings

FIG. 1 shows the results of testing negative and positive quality controls in a test kit using a panel of lyophilized platelet samples in combination with a commercial kit.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise indicated, the cell biological or immunological procedures used herein are all conventional procedures widely used in the corresponding field. Also, for a better understanding of the present invention, definitions and explanations of some terms are provided below.

The term "comprising" means that other steps or ingredients may be added in addition to those specifically mentioned, provided that such additional added steps or ingredients do not affect the function of the original method or composition itself.

The term "platelet" has its commonly understood meaning in the art, and refers to a small piece of material shed from megakaryocytes, without nuclei, with an intact cell membrane on the surface. The size of platelets varies widely, mostly 1-8 microns in diameter. Platelets behave polymorphically when observed by common methods because they can move and deform. Platelets play an important role in the physiological and pathological processes of hemostasis, wound healing, inflammatory response, thrombosis, organ transplant rejection and the like. Platelets for use herein can be from a variety of mammals, preferably human platelets. Platelets obtained in various ways can be used, such as apheresis (also known as machine-drawn platelets) or platelets isolated from whole blood. Preferably, the platelets used are fresh platelets, e.g. stored for no more than one week, or no more than 3 days, most preferably no more than 1 day prior to use.

The term "freeze-dried platelets" refers to platelets obtained by a freeze-drying process. It usually loses 90%, or even more than 95% of its physiological state of water content. The freeze-dried platelets can be rehydrated (otherwise known as rehydrated) with an aqueous solution prior to use to restore their experimental or clinical desired function.

The term "buffer" refers to a liquid capable of counteracting or reducing the influence of an added acid or base on the pH of a solution to some extent, and is usually a mixed solution of a weak acid and its salt or a weak base and its salt. As the buffer, for example, phosphate buffer, citrate buffer, Tris-HCl buffer, HEPES buffer, barbital buffer, acetate buffer and the like are commonly used. Methods for formulating such buffers are well known in the art.

The term "test kit" refers to a package or other packaged form that includes one or more reagents for a particular test purpose. The reagents therein may be present in separate containers. The detection kit may include various buffers, negative or positive control reagents, standards, and the like, in addition to the reagents used for detection purposes. In addition, instructions for illustrating the methods of use of the reagents therein are also typically included in the test kits.

In this context, unless otherwise indicated, the content of the components expressed in percentages is the content in percentages by weight.

The methods of preparing freeze-dried platelets provided herein can include the following steps.

The method comprises the following steps:

platelet pretreatment: washing platelets with basic buffer solution for 3 times to remove components such as plasma, and resuspending the platelets with pretreatment solution to a cell concentration of 10⁸-10¹⁰Adding 1/10-1/4 volumes of fixing solution according to the volume of the suspension, reacting for 0.5-1 hour at room temperature, and fixing the suspended cells.

Basic buffer solution: phosphate buffer solution containing EDTA or citric acid as anticoagulant, and having pH of 6.5-7.5

Pretreatment liquid: phosphate buffer (or HEPES buffer, citric acid buffer), pH6.5-7.5, and trehalose 0.2-5%; 1-5% lysine; 0.2-4% collagen; 0.1-2% sorbitol; 0.01-0.5% (v/v) tween-20; 2-30mM EDTA

Fixing liquid: solutions containing 10% (v/v) glutaraldehyde and 80% ethanol (v/v)

Step two:

after fixation, platelets were washed 2-5 times with basal buffer and resuspended to 10 cells using lyophilized preservation solution⁸-10¹⁰one/mL.

Freeze-drying preservation solution: phosphate buffer (or HEPES buffer, citric acid buffer), pH6.5-7.5, containing 1-6% human serum albumin; 0.1-1.5% polyvinylpyrrolidone (PVP, K90); 0.5-5% of mannitol.

Step three:

and (3) transferring the platelet suspension obtained in the step two into a freeze-drying bottle, pre-freezing at ultralow temperature of-80 ℃ overnight, transferring the platelet suspension into a pre-cooled freeze dryer for vacuum drying the next day, setting the temperature of a cold trap at-45 +/-5 ℃, setting the vacuum degree at less than 133mbar, taking out the platelet suspension after vacuum drying for 24 hours, sealing the platelet suspension, and storing the platelet suspension in a cold storage or room temperature.

In the first step, a protective agent of the protein antigen is used for protectively sealing the antigen on the platelet membrane, and a composite fixing agent is used for fixing the platelet antigen and the cell structure before the platelet is freeze-dried, so that the structural stability of the antigen and the cell is improved, and the aim of reducing freeze-drying loss is fulfilled. The freeze-dried platelet can be stored for a long time at room temperature or under refrigeration condition, and the application and standardization problems of the anti-sieve cells in the platelet antibody detection application are further solved.

The present invention will be further illustrated by the following specific examples.

Example 1 preparation and rehydration of lyophilized platelets

1.1 preparation of the principal solution

Basic buffer solution: weighing Na by using analytical balance respectively₂HPO₄.12H₂O，3.581g；KH₂PO₄0.2450 g; 8.0067g of NaCl; KCl, 0.2013 g; EDTA, 1.8 g; keeping the volume to lL by using ultrapure water; the pH was adjusted to 7.2.

Pretreatment liquid: 1L10mM HEPES buffer solution, to which 8g of NaCl and 20g of trehalose are added; 25g of lysine; 15g of collagen (from fish skin, Beijing Solebao science and technology Co., Ltd., cat # C8090); 5g of sorbitol; tween-202 mL, EDTA1.8g. Adjusting the pH to 7.2

Fixing liquid: 200mL of 50% glutaraldehyde was added to 800mL of absolute ethanol.

Freeze-drying preservation solution: 1L of 10mM HEPES buffer to which 40g of human serum albumin was added; 12g of PVP; 18g of mannitol. The pH was adjusted to 7.2.

1.2 preparation of lyophilized platelets

1.2.1 preparation of platelets

1) Centrifuging at 4000rpm for 10min, and collecting blood platelet by a processor;

2) discarding the supernatant, adding 1/2 platelet volume of basic buffer solution, and resuspending the platelets;

3) and (3) repeating the steps 1) and 2) twice to complete the platelet cleaning.

1.2.2 platelet Pre-lyophilization treatment and test grouping

Discarding the supernatant after the final washing centrifugation of the platelets, resuspending the platelet cells in the experimental group using the pretreatment solution, and adjusting the cell concentration to 10⁹Per mL; no. 1 control group was resuspended and adjusted to 10 cell concentration using the basic buffer without using the pretreatment solution⁹Per mL; control group 2 was cell-resuspended using pretreatment solution without lysine and collagen, and cell concentration was adjusted to 10⁹one/mL. Each group of platelet suspensions was shaken at room temperature for 30 min.

Next, 1/8 volumes of fixative were added to each group of platelet suspensions, wherein a portion of each suspension from the experimental group and the control group No. 1 was not treated with fixative and labeled as control group No. 3 and control group No. 4, respectively. Mixing the platelet suspensions, and reacting at room temperature for 30 min. The differences between the experimental and control groups are summarized in table 1 below.

Table 1 experimental group description

Means resuspending platelets in basal buffer instead of pretreatment

Not adding fixative to the platelet suspension

After fixation, platelets were washed 2-5 times with basal buffer and resuspended to 10 cells using lyophilized preservation solution⁹one/mL.

1.2.3 platelet lyophilization Process

Transferring the platelet suspension into a 10mL penicillin freeze-drying bottle, subpackaging 1.5mL each bottle, pre-freezing at ultralow temperature of-80 ℃ overnight, transferring the bottle to a precooled freeze-drying machine for vacuum drying the next day, setting the temperature of a cold trap at-45 +/-5 ℃, setting the vacuum degree at less than 133mbar, vacuum-drying the bottle for 24 hours, taking the bottle out, sealing the bottle, and storing the bottle at a cold storage or room temperature.

Example 2 recovery assay of freeze-dried platelets

The groups of lyophilized samples prepared in example 1 were rehydrated after storage at room temperature for one day. The rehydration solution was physiological saline, and 1.5mL of rehydration solution was added to each sample and gently shaken to dissolve completely.

Calculation of platelet recovery: and counting the number of the platelets before freeze-drying and after rehydration, and calculating the recovery rate of the platelets. The recovery rate is calculated by the formula:

in each group, 6 samples were randomly rehydrated and subjected to platelet counting, and the calculation results are shown in table 2 below.

Table 2 rehydration recovery of lyophilized platelets for each group

The results show that: the control groups 1, 2 and 3 have no significant difference from the experimental group, and the control group 4 has significant difference from the experimental group. The pretreatment and the fixing treatment have certain protection effect on the integrity of the blood platelets in the freeze-drying process, and the damage of the blood platelets in the freeze-drying process can be reduced.

Example 3 platelet surface antigen detection assay

The loss (damage) condition of the platelet antigens of the experimental group and the control group is detected by an enzyme-linked immunosorbent assay (ELISA) by using monoclonal antibodies (anti-platelet glycoprotein II b/III a, I a/II a, I b/IX and IV) corresponding to different surface antigens. The results show that the platelet surface antigen of the treatment group and the control group has significant difference after freeze-drying, and the treatment group has no significant change compared with the freshly collected platelets.

The specific test method is as follows:

preparing a reaction plate: using PBS buffer (8g NaCl, 0.2g KCl, 1.44g Na)₂HPO₄And 0.24g KH₂PO₄Adjusting the pH value of the solution to 7.4 by using HCl, adding water to a constant volume of 1L), diluting the mouse anti-human blood platelet antibody (GeneTex, the product number is GTX60763) to 10 mu g/mL, adding 50 mu L of the diluted solution into an ELISA plate per well, and coating for 2h at 37 ℃. The plate was washed 2 times, 200. mu.L of blocking solution (1% BSA in PBS) was added to each well, and incubated at 37 ℃ for 2 h. The plate was then washed 3 times with wash solution and patted dry.

Platelet coating: the experimental group and the control group of freeze-dried platelets prepared in example 1 were rehydrated with physiological saline, respectively, and left to stand at room temperature until they were sufficiently dissolved. Each group of platelet solutions was added to the reaction plate at 50. mu.l per well, centrifuged at 60g for 5min, and washed 3 times with a washing solution.

And (3) ELISA detection: monoclonal antibodies (Santa Cruz, cat # SC-7310, SC-53502, SC-166420, and SC-73643) (1) against antiplatelet glycoproteins II b/III a, I a/II a, I b/IX, and IV, respectively, were added to the microplateμ g/mL, 100 μ L/well) with normal mouse serum as negative control, incubated lh at 37 ℃. Wash 5 times with wash solution, add AP-anti mouse IgG 50 u L/hole, 37 degrees C were incubated L h. Washing with washing solution for 5 times, adding substrate-disodium p-nitrophenylphosphate (pNPP) (50 μ L/well) to develop color for about 30min, adding 3M NaOH solution (50 μ L/well) to terminate color development, and measuring A₄₀₅The value is obtained. Record A for each group of platelets₄₀₅And respectively comparing the measured values of the experimental group and the control group with the measured value of the fresh mechanically-collected platelet.

The results of the 4 antibody tests and comparisons are shown in Table 3 below.

Table 34 antibody test results (A)₄₀₅)

As can be seen from the results, the results of the experimental group are most similar to those of the fresh machine-collected platelets, the results of the two groups of experiments are not significantly different, and the control groups 1 to 4 are significantly different from the experimental group.

Example 4 platelet surface antigen detection assay

The freeze-dried platelet cells of the experimental group prepared in example 1 were stored at room temperature and 2-8 ℃, samples were taken every month for antigen stability study, and the detection method was the same as in example 3. The results are shown in tables 4 and 5 below. From the results, the freeze-dried platelets in the experimental group were stored at room temperature for 6 months or at 2-8 ℃ for 12 months, and the glycoproteins II b/IIIa, I a/II a, I b/IX and IV were not changed significantly.

TABLE 4 detection results of various surface antigens after storage of freeze-dried platelets of the experimental group at room temperature (A)₄₀₅)

TABLE 5 test results of various surface antigens after cryopreservation of freeze-dried platelets in the experimental groups (A)₄₀₅)

EXAMPLE 5 use of lyophilized platelet samples for platelet antibody detection (solid phase agglutination)

The detection plate is prepared according to the instructions of a commercial kit such as a platelet antibody detection kit (solid phase agglutination method) (Sankun reagent Co., Ltd. in the Netherlands). The freeze-dried platelet sample (stored under refrigeration for 9 months) prepared in example 1 was rehydrated using 1.5mL of physiological saline and left to stand at room temperature until it was sufficiently dissolved. Platelet coating is performed according to the kit instructions and the detection process is completed. If the positive and negative results are normal, the freeze-dried platelet prepared by the invention can be used for commercial antibody detection kit matching and laboratory or clinical platelet antibody detection. The results of testing negative and positive quality controls in the kit with lysed platelet samples are shown in FIG. 1. Positive results: indicating that the red blood cells are plated on the bottom surface of the reaction well, and a positive result indicates that the sample contains platelet antibodies. Negative results: indicating that the red blood cells form red blood cell aggregates in the center of the bottom of the reaction well, and a negative result indicates that the sample does not contain platelet antibodies.

The preparation method of the freeze-dried platelet provided by the invention uses lysine, collagen, sorbitol and tween-20 to carry out protective treatment on the surface antigen of the platelet, and can effectively reduce the damage to the antigen in the preservation process. The freeze-dried platelet preparation method provided by the invention fixes the platelets by using the combined solution of glutaraldehyde and ethanol, so that the antigens can be further protected, and meanwhile, the fixation of the intracellular frameworks is beneficial to maintaining the cell structure, and the loss in the freeze-drying process is reduced.

Therefore, the preparation method of the freeze-dried platelet provided by the invention protects and fixes the antigen on the platelet membrane before freeze-drying the platelet, and simultaneously, the antigen protective agent is added into the freeze-drying liquid, so that the damage of the freeze-drying process to the antigen on the membrane surface is reduced, and the integrity of the antigen is preserved to the maximum extent. The freeze-dried platelets prepared by the method can be used for antibody screening and other experimental purposes. At the same time, the freeze-dried platelets can provide a longer shelf life. For example, the freeze-dried platelets can be stably stored at room temperature for at least 6 months (12 months under refrigeration conditions), and a solution is provided for the application of anti-screening platelets and the standardized application of a platelet antibody detection kit.

Claims

1. A method of preparing freeze-dried platelets, comprising:

2. The method of claim 1, wherein the pretreatment solution further comprises trehalose, sorbitol, tween-20, and EDTA.

3. The method of claim 1 or 2, wherein the pretreatment solution is a buffer solution comprising: trehalose 0.2-5%; 1-5% lysine; 0.2-4% collagen; 0.1-2% sorbitol; 0.01-0.5% (v/v) tween-20; and 2-30mM EDTA.

4. The method of any one of claims 1 to 3, wherein the pretreatment solution is formulated by: to each liter of buffer solution, 8g of NaCl, 20g of trehalose, 25g of lysine, 15g of collagen, 5g of sorbitol, 202 mL of Tween-and 1.8g of EDTA were added.

5. The method of any one of claims 1-4, wherein the volume ratio of glutaraldehyde to ethanol in the fixation fluid is 1: 8.

6. the method according to any one of claims 1 to 5, wherein the lyophilized preservation solution is a buffer solution comprising: 1-6% serum albumin, 0.1-1.5% PVP, and 0.5-5% mannitol.

7. The method according to any one of claims 1 to 6, wherein the lyophilized preservation solution is prepared by: to each liter of buffer was added 40g of serum albumin, 12g of PVP, and 18g of mannitol.

8. The method of any one of claims 1-7, wherein step 1) comprises: preparing 10 blood platelets by using the pretreatment liquid⁸-10¹⁰A first platelet suspension, and 1/10-1/4 volumes of the fixative are added according to the volume of the first platelet suspension, and the reaction is carried out for 0.5-1 hour at room temperature.

9. The method of any one of claims 1-8, further comprising washing the platelets with a phosphate buffer containing EDTA or citric acid prior to step 1).

10. The method according to any one of claims 1 to 9, wherein between step 1) and step 2 further comprising washing the platelets treated in step 1) with a phosphate buffer containing EDTA or citric acid.

11. The method according to any one of claims 1 to 10, wherein step 2) further comprises suspending the platelets treated in step 1) to 10 with the lyophilization preservative fluid prior to the lyophilization⁸-10¹⁰and/mL of a second platelet suspension.

12. The method according to any one of claims 1 to 11, wherein the lyophilization in step 2) is performed by vacuum in a lyophilizer with a cold trap temperature set at-45 ± 5 ℃, a vacuum degree set at <133mbar, vacuum lyophilization for 24h or more.

13. The method of any one of claims 1-12, wherein the lyophilizing in step 2) comprises: and transferring the second platelet suspension into a freeze-drying bottle, pre-freezing at ultralow temperature of-80 ℃ overnight, transferring the second platelet suspension into a pre-cooled freeze-drying machine for vacuum drying the next day, setting the temperature of a cold trap to be-45 +/-5 ℃, setting the vacuum degree to be less than 133mbar, and taking out the second platelet suspension after vacuum drying for 24 hours.

14. Freeze-dried platelets prepared by the method of any one of claims 1-13.

15. A test kit comprising the freeze-dried platelets of claim 14.

16. Use of freeze-dried platelets of claim 14 or a test kit of claim 15 for platelet antibody screening.