CN111537326B - Method for preparing freeze-dried blood platelet and application thereof - Google Patents
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Abstract
Provided herein is a method of preparing freeze-dried platelets, comprising: 1) Contacting the platelets with the pretreatment liquid and the fixing liquid in sequence; and 2) lyophilizing the platelets treated in step 1) in the presence of a lyophilization preservative fluid, wherein the pretreatment fluid comprises lysine and collagen; the fixing liquid comprises glutaraldehyde and ethanol; the freeze-dried 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 platelets provided herein can maintain platelet antigen activity during long-term storage, and provide a solution for the application of anti-sieve platelets and the standardized application of platelet antibody detection kits.
Description
Technical Field
This document relates to methods of preparing freeze-dried platelets, and in particular to methods of preparing freeze-dried platelets useful for platelet antibody screening.
Background
Platelet antibodies are antibodies generated by the body by immunizing the surface of platelets or related antigens, and the antigens for stimulating the generation of platelet antibodies can be autologous or allogeneic. Platelet antibodies are of two classes, one class being antibodies directed mainly against human histocompatibility antigen (HLA) class I antigens, namely platelet surface-associated antibodies (PAIgG), and one class being platelet-specific antibodies (PB IgG) directed against GP antigens. GP antigen and HLA-I antigen stimulate organism to produce alloimmune reaction, and the exogenous platelet damage caused by the same can shorten the life span and change the function of the platelet, which is an important reason for ineffective platelet infusion. As the number and amount of infusions increases, the positive rate of platelet antibody detected increases, and the rate of ineffective infusions and non-hemolytic transfusion reactions increases.
The platelet antibody can be accurately, rapidly and simply detected and identified, and plays an important role in the aspects of diagnosis, treatment and the like of platelet immune diseases. The main method of clinical platelet antibody detection is mainly immunological method, but no commercial pure antigen is sold so far because of the complexity and diversity of platelet surface antigen. Platelets themselves are often used in current assays to provide antigens. The following problems exist in the current preservation of platelets: 1. platelets are extremely easy to aggregate and lose antigenicity in a liquid environment, and the preservation period is short; 2, freeze-dried platelets are all used for infusion and treatment (wound healing), and lack of protection for platelet surface antigens, and the platelet surface antigens are destroyed greatly in the freeze-drying process, so that the products have a plurality of problems in platelet antibody screening application, 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 the pretreatment liquid and the fixing liquid in sequence; and
2) Freeze-drying the platelet treated in the step 1) in the presence of a freeze-drying preservation solution,
wherein the pretreatment liquid comprises lysine and collagen; the fixing liquid comprises glutaraldehyde and ethanol; the freeze-dried preservation solution comprises serum albumin, PVP and mannitol.
In some embodiments, the pretreatment liquid further comprises trehalose, sorbitol, tween-20, and EDTA.
In some embodiments, the pretreatment liquid is a buffer comprising the following components: 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.
In some embodiments, the pretreatment liquid is formulated by: to each liter of the buffer was added 8g of NaCl, 20g of trehalose, 25g of lysine, 15g of collagen, 5g of sorbitol, 2mL of Tween-20 and 1.8g of EDTA.
In some embodiments, the volume ratio of glutaraldehyde to ethanol in the fixative solution is 1:8.
in some embodiments, the lyophilized preservation solution is a buffer comprising: 1-6% serum albumin, 0.1-1.5% PVP, and 0.5-5% mannitol.
In some embodiments, the method of formulating the lyophilized preservation solution is: to each liter of buffer was added 40g serum albumin, 12g PVP, and 18g mannitol.
In some embodiments, step 1) comprises: platelets were formulated as 10 with the pretreatment solution 8 -10 10 And adding 1/10-1/4 volume of the fixing solution according to the volume of the first platelet suspension, and reacting for 0.5-1 hour at room temperature.
In some embodiments, the method further comprises washing the platelets with phosphate buffer containing EDTA or citric acid prior to step 1).
In some embodiments, between step 1) and step 2) there is also included washing of the platelets treated in step 1) with phosphate buffer containing EDTA or citric acid.
In some embodiments, step 2) further comprises suspending the platelets treated in step 1) to 10 with the lyophilization preservative fluid prior to the lyophilization 8 -10 10 And a second platelet suspension at a concentration of one/mL.
In some embodiments, the lyophilization in step 2) is performed in a lyophilizer with a cold trap temperature set at-45±5 ℃, a vacuum set at <133mbar, and a vacuum lyophilization for more than 24 hours.
In some embodiments, the lyophilizing in step 2) comprises: transferring the second platelet suspension into a freeze-drying bottle, pre-freezing overnight at the ultralow temperature of-80 ℃, transferring into a pre-cooled freeze dryer for vacuum drying the next day, setting the cold trap temperature to-45+/-5 ℃, setting the vacuum degree to be less than 133mbar, and taking out after vacuum drying for 24 hours.
In another aspect, provided herein are freeze-dried platelets prepared by the above method.
In another aspect, provided herein are assay kits comprising the above-described lyophilized platelets.
In another aspect, provided herein is the use of the above-described lyophilized platelets or the above-described detection kit in platelet antibody screening.
The freeze-dried platelets provided herein can maintain platelet antigen activity during long-term storage, and provide a solution for the application of anti-sieve platelets and the standardized application of platelet antibody detection kits.
Drawings
FIG. 1 shows the results of detection of negative and positive controls in a test kit using a freeze-dried platelet sample of the experimental group in combination with a commercial kit.
Detailed Description
Unless otherwise defined, 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 procedures used herein for cell biology or immunology are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of some terms are provided below.
The term "comprising" means that other steps or components than those specifically mentioned may be added, as long as such additional added steps or components do not affect the function of the original method or composition itself.
The term "platelet" has the meaning commonly understood in the art and refers to a small mass of cells that has been shed from megakaryocytes, without a nucleus, and with an intact cell membrane on the surface. The platelets vary widely in size, mostly in the range of 1-8 microns in diameter. Platelets are capable of movement and deformation, and therefore appear to be polymorphic when viewed by conventional methods. Platelets play an important role in physiological and pathological processes such as hemostasis, wound healing, inflammatory reactions, thrombosis, and organ transplant rejection. Platelets as used herein may be derived from a variety of mammalian, preferably human, platelets. Platelets obtained in various ways, such as apheresis platelets (also known as apheresis platelets) or platelets isolated from whole blood, may be employed. Preferably, the platelets used are fresh platelets, for example, kept 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 typically loses 90%, or even more than 95% of its moisture content in its physiological state. Freeze-dried platelets can be rehydrated (otherwise known as rehydrated) by an aqueous solution prior to use to restore their experimentally or clinically desired function.
The term "buffer" refers to a liquid, typically a weak acid and its salts or a mixed solution of weak bases and its salts, that counteracts, lessens to some extent the effects of an added acid or base on the ph of the solution. Typical buffers are, for example, phosphate buffer, citrate buffer, tris-HCl buffer, HEPES buffer, barbital buffer, acetate buffer, etc. Methods of 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. In addition to reagents for detection purposes, the detection kit may include various buffers, negative or positive control reagents, standards, and the like. In addition, instructions for use of the reagents therein are also typically included in the test kit.
In this context, unless otherwise indicated, the component content in percent is the weight percent content.
The methods of preparing freeze-dried platelets provided herein may include the following steps.
Step one:
platelet pretreatment: washing blood platelet with basic buffer for 3 times to remove components such as blood plasma, and re-suspending with pretreatment solution to cell concentration of 10 8 -10 10 And adding 1/10-1/4 volume of fixing solution into each mL of suspension according to the volume of the suspension, reacting for 0.5-1 hour at room temperature, and fixing the suspended cells.
Base buffer: phosphate buffer solution containing EDTA or citric acid as anticoagulant with pH of 6.5-7.5
Pretreatment liquid: phosphate buffer (HEPES buffer, citric acid buffer), pH6.5-7.5, 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 solution: solution containing 10% (v/v) glutaraldehyde and 80% ethanol (v/v)
Step two:
after fixation, platelets were washed 2-5 times with basal buffer and cells were resuspended to 10 with lyophilized preservative solution 8 -10 10 And each mL.
Freeze-drying preservation solution: phosphate buffer (HEPES buffer, citric acid buffer) with pH of 6.5-7.5 containing 1-6% human serum albumin; 0.1-1.5% polyvinylpyrrolidone (PVP, K90); 0.5-5% mannitol.
Step three:
transferring the platelet suspension obtained in the second step into a freeze-drying bottle, pre-freezing overnight at the ultralow temperature of-80 ℃, transferring into a pre-cooled freeze dryer for vacuum drying the next day, setting the cold trap temperature to be-45+/-5 ℃, and taking out the platelet suspension after vacuum drying for 24 hours, sealing the platelet suspension, and storing the platelet suspension at refrigeration or room temperature.
In the first step, the antigen on the platelet membrane is protected and blocked by using a protective agent of protein antigen, and the platelet antigen and a cell structure are fixed by using a composite fixing agent 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 platelets can be stored for a long time under the condition of room temperature or refrigeration, and further the problems of application and standardization of anti-sieve cells in platelet antibody detection application are solved.
The invention is further illustrated by the following specific examples.
EXAMPLE 1 lyophilized platelet preparation and rehydration
1.1 preparation of the Main solution
Base buffer: respectively weighing Na by analytical balance 2 HPO 4 .12H 2 O，3.581g；KH 2 PO 4 0.2450g; 8.0067g of NaCl; KCl,0.2013g; EDTA,1.8g; the volume is fixed to lL by ultrapure water; the pH was adjusted to 7.2.
Pretreatment liquid: 1L10mM HEPES buffer, to which was added 8g of NaCl and 20g of trehalose; 25g of lysine; 15g of collagen (from fish skin, beijing Soy Bao technology Co., ltd., product number C8090); 5g of sorbitol; tween-20 2mL, EDTA 1.8g. Adjusting pH to 7.2
Fixing solution: 200mL of 50% glutaraldehyde was added to 800mL of absolute ethanol.
Freeze-drying preservation solution: 1L10mM HEPES buffer, to which 40g of human serum albumin was added; 12g 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 platelets by a processor;
2) Discarding the supernatant, adding a basic buffer solution with the volume of 1/2 of the platelet volume, and re-suspending the platelet;
3) Repeating the steps 1) and 2) twice to finish the cleaning of the blood platelets.
1.2.2 platelet lyophilization pretreatment and test grouping
Discarding the supernatant after the last washing and centrifugation of the platelets, re-suspending the platelet cells by using the pretreatment liquid in the experimental group, and adjusting the cell concentration to 10 9 individual/mL; no. 1 control group uses basic buffer directly to re-suspend and adjust cell concentration to 10 without pretreatment solution 9 individual/mL; control group No. 2 was subjected to cell resuspension using pretreatment solution containing no lysine and collagen, and cell concentration was adjusted to 10 9 And each mL. The platelet suspensions of each group were shaken at room temperature for 30min.
Next, 1/8 volume of fixative was added to each group of platelet suspensions, wherein a portion of the suspensions from each of the experimental and control groups, no fixative treatment, was not performed, labeled as control group 3 and control group 4, respectively. Mixing the platelet suspensions, and standing at room temperature for 30min. The differences between the experimental group and each control group are summarized in Table 1 below.
Table 1 description of test groups
* Refers to the resuspension of platelets with a basal buffer solution instead of a pretreatment solution
* Mean that no fixative is added to the platelet suspension
After fixation, platelets were washed 2-5 times with basal buffer and cells were resuspended to 10 with lyophilized preservative solution 9 And each mL.
1.2.3 Freeze-drying treatment of platelets
Transferring the platelet suspension into 10mL penicillin freeze-drying bottles, sub-packaging 1.5mL each bottle, pre-freezing overnight at-80 ℃, transferring into a pre-cooled freeze dryer for vacuum drying the next day, setting the cold trap temperature to-45+/-5 ℃, vacuum degree to be less than 133mbar, taking out after vacuum drying for 24 hours, sealing, and storing at refrigeration or room temperature.
Example 2 detection of recovery of lyophilized platelets
Each set of lyophilized samples prepared in example 1 was rehydrated after being stored for l days at room temperature. Rehydration solution was physiological saline, and 1.5mL of rehydration solution was added to each sample and gently shaken until completely dissolved.
Calculation of platelet recovery: platelets before lyophilization and after rehydration were counted and the recovery of platelets was calculated. The recovery rate calculation formula is:
6 samples from each group were randomly rehydrated and platelet counted, with the results calculated as shown in Table 2 below.
Table 2 recovery of lyophilized platelets from each set
The results show that: control groups 1, 2, 3 were not significantly different from the experimental group, and control group 4 was significantly different from the experimental group. The pretreatment and the fixation treatment have a certain protection effect on the integrity of the platelets in the freeze-drying process, and can reduce the damage of the platelets in the freeze-drying process.
Example 3 platelet surface antigen detection assay
The loss (destruction) of platelet antigens in the experimental and control groups was detected by enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (anti-platelet glycoproteins II b/III a, I a/II a, I b/IX, IV) corresponding to the different surface antigens. The results showed that there was a significant difference in platelet surface antigen after lyophilization in the treated group compared to the control group, and no significant change in the treated group compared to freshly collected platelets.
The specific test method is as follows:
preparing a reaction plate: with PBS buffer (8 g NaCl, 0.2g KCl, 1.44g Na) 2 HPO 4 And 0.24g KH 2 PO 4 The pH of the solution was adjusted to 7.4 with HCl, water was added to a volume of 1L, and the mouse anti-human platelet antibody (GeneTex, cat. No. GTX 60763) was diluted to 10. Mu.g/mL, added to the ELISA plate at 50. Mu.L per well, and coated for 2h at 37 ℃. Plates were washed 2 times, 200. Mu.L of blocking solution (1% BSA in PBS) was added to each well and incubated at 37℃for 2h. The plates were then washed 3 times with wash solution and patted dry.
Platelet coating: the freeze-dried platelets of the experimental group and the control group 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 wash solution.
ELISA detection: adding anti-platelet glycoprotein IIb/III a, I a/IIa, I b/IX, IV, respectively, to the ELISA plateThe cloned antibodies (Santa Cruz, accession numbers SC-7310; SC-53502; SC-166420; and SC-73643) (1. Mu.g/mL, 100. Mu.L/well), respectively), were incubated with normal mouse serum as a negative control for lh at 37 ℃. Wash 5 times with wash solution, add AP-anti-mouse IgG 50. Mu.L/well and incubate L h at 37 ℃. Washing with washing solution for 5 times, adding substrate disodium p-nitrophenylphosphate (pNPP) (50 μL/well) for color development for about 30min, adding 3M NaOH solution (50 μL/well) for stopping color development, and measuring A 405 Values. Record A corresponding to each group of platelets 405 The measurements of the experimental and control groups were compared with the measurements of fresh machine blood collection platelets, respectively.
The results of the detection and comparison of the 4 antibodies are shown in Table 3 below.
Table 3 4 antibody detection results (A 405 )
From the results, it can be seen that the experimental group was closest to the results of fresh machine blood collection platelets, the two experimental groups had no significant differences in results, while the control groups 1-4 had significant differences from the experimental groups.
Example 4 platelet surface antigen detection assay
The freeze-dried platelet cells of the experimental group prepared in example 1 were stored at a temperature of 2-8 ℃ and sampled 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 of the experimental group were stored for 6 months at room temperature or for 12 months at 2-8℃without significant changes in glycoprotein II b/III a, I a/II a, I b/IX, IV.
Table 4 results of detection of each surface antigen after storage of the freeze-dried platelets of the experimental group at room temperature (a 405 )
TABLE 5 detection results of surface antigens after cryopreservation of the freeze-dried platelets of the experimental group (A) 405 )
EXAMPLE 5 use of lyophilized platelet samples in platelet antibody detection (solid phase agglutination method)
The assay plate is prepared according to instructions of a commercial kit such as a platelet antibody assay kit (solid phase agglutination method) (Sankun reagent Co., ltd. In the Netherlands). The lyophilized platelet sample prepared in example 1 (stored under refrigeration for 9 months) was rehydrated using 1.5mL of physiological saline and allowed to stand at room temperature until it was sufficiently dissolved. Platelet coating was performed and the detection procedure was completed according to the kit instructions. The normal yin-yang results indicate that the freeze-dried platelets prepared by the invention can be used for matching commercial antibody detection kits and laboratory or clinical platelet antibody detection. The results of the detection of negative and positive quality controls in the kit with the lysed platelet samples are shown in FIG. 1. Positive results: indicating red blood cells are paved on the bottom surface of the reaction hole, and positive results indicate that the sample contains platelet antibodies. Negative results: indicating that red blood cells form red blood cell aggregation at the center of the bottom of the reaction well, and negative results indicate that the sample does not contain platelet antibodies.
The freeze-dried platelet preparation method provided by the invention uses lysine, collagen, sorbitol and tween-20 to carry out protective treatment on the surface antigen of the platelet, so that the damage to the antigen in the preservation process can be effectively reduced. The freeze-dried blood platelet preparation method provided by the invention uses the combined solution of glutaraldehyde and ethanol to fix the blood platelets, so that antigens can be further protected, and meanwhile, the fixation of the intracellular skeleton is beneficial to the maintenance of a cell structure, so that the loss in the freeze-drying process is reduced.
Therefore, the preparation method of the freeze-dried blood platelets provided by the invention protects and fixes the antigen on the blood platelet membrane before freeze-drying the blood platelets, and simultaneously adds an antigen protecting agent into the freeze-drying liquid, thereby reducing the damage of the freeze-drying process to the antigen on the membrane surface and preserving the integrity of the antigen to the greatest extent. The freeze-dried platelets prepared by the method can be used for antibody screening and other test purposes. At the same time, the freeze-dried platelets may provide a longer shelf life. For example, the freeze-dried platelets can be stored stably for at least 6 months at room temperature (12 months under refrigerated conditions), providing a solution for anti-sieve platelet applications and standardized use of platelet antibody detection kits.
Claims (14)
1. A method of preparing freeze-dried platelets, comprising:
1) Contacting the platelets with the pretreatment liquid and the fixing liquid in sequence; and
2) Freeze-drying the platelet treated in the step 1) in the presence of a freeze-drying preservation solution,
wherein the pretreatment liquid is phosphate buffer, HEPES buffer or citric acid buffer comprising the following components: 0.2-5% trehalose; 1-5% lysine; 0.2-4% collagen; 0.1-2% sorbitol; 0.01-0.5% (v/v) tween-20; 2-30mM EDTA, pH 6.5-7.5;
the fixing liquid comprises glutaraldehyde and ethanol;
the freeze-dried preservation solution comprises serum albumin, PVP and mannitol.
2. The method of claim 1, wherein the pretreatment liquid is formulated by: to each liter of the buffer was added 8g of NaCl, 20g of trehalose, 25g of lysine, 15g of collagen, 5g of sorbitol, 2mL of Tween-20 and 1.8g of EDTA.
3. The method of claim 1 or 2, wherein the volume ratio of glutaraldehyde to ethanol in the fixative solution is 1:8.
4. the method of claim 1 or 2, wherein the lyophilized preservation solution is a buffer comprising: 1-6% serum albumin, 0.1-1.5% PVP, and 0.5-5% mannitol.
5. The method of claim 4, wherein the lyophilization preservative fluid is formulated by: to each liter of buffer was added 40g serum albumin, 12g PVP, and 18g mannitol.
6. The method of claim 1 or 2, wherein step 1) comprises: platelets were formulated as 10 with the pretreatment solution 8 -10 10 And adding 1/10-1/4 volume of the fixing solution according to the volume of the first platelet suspension, and reacting for 0.5-1 hour at room temperature.
7. The method of claim 1 or 2, further comprising washing the platelets with phosphate buffer containing EDTA or citric acid prior to step 1).
8. The method of claim 1 or 2, wherein between step 1) and step 2) further comprising washing the platelets treated in step 1) with phosphate buffer containing EDTA or citric acid.
9. The method of claim 1 or 2, wherein step 2) further comprises suspending the platelets treated in step 1) to 10 with the lyophilization preservative fluid prior to the lyophilization 8 -10 10 And a second platelet suspension at a concentration of one/mL.
10. The method according to claim 1 or 2, wherein the lyophilization in step 2) is carried out by evacuating in a lyophilizer having a cold trap temperature set at-45±5 ℃, a vacuum set at <133mbar, and vacuum lyophilization for more than 24 hours.
11. The method of claim 9, wherein the lyophilizing in step 2) comprises: transferring the second platelet suspension into a freeze-drying bottle, pre-freezing overnight at the ultralow temperature of-80 ℃, transferring into a pre-cooled freeze dryer for vacuum drying the next day, setting the cold trap temperature to-45+/-5 ℃, setting the vacuum degree to be less than 133mbar, and taking out after vacuum drying for 24 hours.
12. A lyophilized platelet prepared by the method of any one of claims 1-11.
13. A test kit comprising the freeze-dried platelets of claim 12.
14. Use of the lyophilized platelets of claim 12 or the detection kit of claim 13 in platelet antibody screening.
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