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Method for preparing freeze-dried platelets and application thereof

A platelet and preparation method technology, applied in the application, preparation of test samples, preservation of human or animal bodies, etc., can solve the problems of low positive rate, lack of protection of platelet surface antigens, and easy aggregation of platelets

Active Publication Date: 2020-08-14
TIANJIN DEXIANG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the preservation of platelets has the following problems: 1. Platelets are very prone to aggregation and lose their antigenicity in a liquid environment, and the preservation period is short; 2. Freeze-dried platelets are all for transfusion and treatment (wound healing), and lack For the protection of platelet surface antigens, the platelet surface antigens are greatly damaged during the freeze-drying process, resulting in many problems in the application of platelet antibody screening, such as low sensitivity and low positive rate.

Method used

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  • Method for preparing freeze-dried platelets and application thereof
  • Method for preparing freeze-dried platelets and application thereof
  • Method for preparing freeze-dried platelets and application thereof

Examples

Experimental program
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preparation example Construction

[0032] The method for preparing freeze-dried platelets provided herein may include the following steps.

[0033] step one:

[0034] Platelet pretreatment: Wash the platelets 3 times with basic buffer to remove components such as plasma, and resuspend them with pretreatment solution to a cell concentration of 10 8 -10 10 cells / mL suspension, add 1 / 10-1 / 4 volume of fixative according to the volume of the suspension, react at room temperature for 0.5-1 hour, and fix the suspended cells.

[0035] Basic buffer: phosphate buffer, containing anticoagulants such as EDTA or citric acid, pH6.5-7.5

[0036] Pretreatment solution: phosphate buffer (or HEPES buffer, citric acid buffer), pH6.5-7.5, containing 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

[0037] Fixative: a solution containing 10% (v / v) glutaraldehyde and 80% ethanol (v / v)

[0038] Step two:

[0039] After fixation, wash the platelets 2-5 times with basic buff...

Embodiment 1

[0045] Example 1 Preparation and rehydration of freeze-dried platelets

[0046] 1.1 Preparation of the main solution

[0047] Basic buffer solution: weigh Na with an analytical balance 2 HPO 4 .12H 2 O, 3.581g; KH 2 PO 4 , 0.2450g; NaCl, 8.0067g; KCl, 0.2013g; EDTA, 1.8g; dilute to 1L with ultrapure water; adjust the pH value to 7.2.

[0048] Pretreatment solution: 1L10mM HEPES buffer solution, add NaCl 8g, trehalose 20g; Lysine 25g; Collagen 15g (derived from fish skin, Beijing Suo Laibao Technology Co., Ltd., item number C8090); Sorbitol 5g; Tween -20 2mL, EDTA 1.8g. Adjust the pH to 7.2

[0049] Fixative: Add 200mL of 50% glutaraldehyde to 800mL of absolute ethanol.

[0050] Freeze-dried preservation solution: 1L of 10mM HEPES buffer, to which 40g of human serum albumin; 12g of PVP; 18g of mannitol were added. Adjust the pH to 7.2.

[0051] 1.2 Preparation of freeze-dried platelets

[0052] 1.2.1 Preparation of platelets

[0053] 1) Centrifuge at 4000rpm for 1...

Embodiment 2

[0066] The recovery rate detection of embodiment 2 freeze-dried platelets

[0067] Each group of freeze-dried samples prepared in Example 1 was stored at room temperature for 1 day and then rehydrated. The rehydration solution is physiological saline, add 1.5mL rehydration solution to each sample and shake gently until completely dissolved.

[0068] Calculation of platelet recovery rate: count the platelets before freeze-drying and after rehydration, and calculate the recovery rate of platelets. The formula for calculating the recovery rate is:

[0069]

[0070] Six samples were randomly selected from each group for rehydration, and platelet counts were performed. The calculation results are shown in Table 2 below.

[0071] Table 2 Rehydration recovery rate of freeze-dried platelets in each group

[0072]

[0073] The results showed that there was no significant difference between the control group 1, 2 and 3 and the experimental group, but there was a significant d...

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Abstract

The invention provides a method for preparing freeze-dried platelets. The method comprises the following steps: 1) enabling platelets to be in contact with a pretreatment solution and a stationary solution in sequence; 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, thestationary liquid comprises glutaraldehyde and ethanol, and the freeze-drying preserving fluid comprises serum albumin, PVP and mannitol. Also provided herein are lyophilized platelets prepared by themethod and uses of lyophilized platelets. The freeze-dried blood platelets provided by the invention can keep the activity of blood platelet antigens during long-term storage, and a solution is provided for application of anti-screening blood platelets and standardized application of a blood platelet antibody detection kit.

Description

technical field [0001] This article relates to a method for preparing freeze-dried platelets, especially a method for preparing freeze-dried platelets that can be used for platelet antibody screening. Background technique [0002] Platelet antibodies are antibodies produced by the body against platelet surface or related antigens, and the antigens that stimulate platelet antibody production can be self-identified or allogeneic. There are two types of platelet antibodies, one is mainly antibodies against human histocompatibility antigen (HLA) class I antigens, that is, platelet surface-associated antibodies (PAIgG), and the other is platelet-specific antibodies against GP antigens (PB IgG) . GP antigens and HLA-I antigens stimulate the body to produce alloimmune reactions, resulting in the destruction of exogenous platelets, shortening the lifespan and changing the function of platelets, which is an important reason for the ineffectiveness of platelet transfusion. With the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/42A01N1/00
CPCG01N1/42A01N1/00
Inventor 王丽王秀柱黄志刚王伟权潘大地
Owner TIANJIN DEXIANG BIOTECHNOLOGY CO LTD
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