血小板病毒灭活后深低温保存的可行性研究

郭梓璇; 施琳颖; 李艳辉; 单桂秋

doi:10.16016/j.1000-5404.202103023

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血小板病毒灭活后深低温保存的可行性研究

军事医学 | 更新时间：2026-01-09

- 血小板病毒灭活后深低温保存的可行性研究
- Feasibility of cryopreservation of platelets after virus inactivation
- 第三军医大学学报 2021年43卷第16期页码：1513-1520
- 作者机构：
  
  1.510000 广州, 广州中医药大学研究生院
  2.510000 广州, 南部战区总医院输血医学科
- 作者简介：
  
  [单桂秋，E-mail: rabbit_2007@126.comSHAN Guiqiu, E-mail: rabbit_2007@126.com
- 基金信息：
  
  军队后勤重点科研项目(BGZ15C002);广州市科技计划项目(202102021273)
- DOI：10.16016/j.1000-5404.202103023
  中图分类号： R331.143;R457.12
- 收稿：2021-03-03，
  
  修回：2021-04-26，
  
  纸质出版：2021-08-30
- 稿件说明：
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郭梓璇, 施琳颖, 李艳辉, 等. 血小板病毒灭活后深低温保存的可行性研究[J]. 第三军医大学学报, 2021,43(16):1513-1520.

Zixuan GUO, Linying SHI, Yanhui LI, et al. Feasibility of cryopreservation of platelets after virus inactivation[J]. Journal of Third Military Medical University, 2021, 43(16): 1513-1520.
郭梓璇, 施琳颖, 李艳辉, 等. 血小板病毒灭活后深低温保存的可行性研究[J]. 第三军医大学学报, 2021,43(16):1513-1520. DOI： 10.16016/j.1000-5404.202103023.

Zixuan GUO, Linying SHI, Yanhui LI, et al. Feasibility of cryopreservation of platelets after virus inactivation[J]. Journal of Third Military Medical University, 2021, 43(16): 1513-1520. DOI： 10.16016/j.1000-5404.202103023.

摘要

目的

探讨新鲜血小板经核黄素光化学法病毒灭活后进行深低温保存的可行性。

方法

采集本院O型健康无偿献血者单采血小板共24 U。实验分为4个组: A为新鲜血小板对照组

B为血小板病毒灭活组(核黄素光化学法灭活处理)

C为新鲜血小板的深低温保存组(-80 ℃保存)

D为病毒灭活血小板的深低温保存组(核黄素光化学法灭活处理后-80 ℃保存)。用150 μmol/L的核黄素结合1.5 J/cm

的UVB光源对血小板进行病毒灭活。深低温保存后隔日定义为第1天，取样通过扫描电镜观察血小板超微结构的变化；第1、3、5、7天取样检测血小板数量、质量、功能与代谢相关指标。使用全自动血常规计数仪检测血小板计数(platelet count

PLT)和平均血小板体积(mean platelet volume

MPV)，使用比色法观察低渗休克反应能力(hypotonic shock response

HSR)变化；使用Annexin V-FITC和CD62p-PE试剂盒和流式细胞术检测细胞凋亡和激活情况；使用血栓弹力图试验(thromboelastogram test

TEG)检测功能变化，记录最大振幅(maximum amplitude

MA)值；使用速率法检测葡萄糖、乳酸变化。综合评估血小板病毒灭活后深低温保存的可行性。

结果

与A组相比较，扫描电镜中B、C、D组血小板伸出伪足数量增多，形状不规则，有明显激活现象；4组MPV均随着保存时间的延长而增大，C、D组明显大于A、B组；4组HSR能力均随着保存时间的延长逐渐降低，C、D组明显不如A、B组；4组的CD62p和Annexin V阳性率中均随着储存时间的延长而增加，D组凋亡与激活现象最明显；组间MA值变化没有统计学差异；A、B组葡萄糖水平均随着储存时间的延长呈明显下降；乳酸含量与葡萄糖含量变化基本相反。综合分析，深低温保存对血小板形态和质量指标的有一定的影响，深低温保存过程对血小板形态和质量指标影响明显大于病毒灭活过程，但其功能和代谢指标变化不明显。

结论

核黄素/UVB光化学法病毒灭活的血小板进行深低温保存存在一定可行性。

Abstract

Objective

To investigate the feasibility of deep cryopreservation of fresh platelets after riboflavin photochemical-mediated virus inactivation.

Methods

A total of 24 samples of platelets were collected from O-type healthy free blood donors in our general hospital

with each sample of 200 mL. The platelet samples were subsequently divided into 4 groups: fresh platelets control group (group A)

virus inactivated control group (group B)

fresh platelet cryopreservation group (group C

stored at -80 ℃) and cryopreservation of virus inactivated platelets group (group D

riboflavin photochemical-mediated virus inactivation and then stored at -80 ℃). In group B and D

the platelets were treated with riboflavin photochemistry (150 μmol/L riboflavin solution and 1.5 J/cm

UVB radiation). The next day after cryopreservation was defined as day 1

and the ultrastructural alterations of platelets were observed with scanning electron microscopy. The quantity

quality

functional and metabolism parameters of platelets were determined on days 1

5 and 7

respectively. Platelet count (PLT) and mean platelet volume (MPV) were measured using an automatic blood routine counter; the hypotonic shock response (HSR) of platelets was observed with colorimetry; Annexin V-FITC and CD62p-PE kits and flow cytometry were adopted to examine cell apoptosis and activation; thromboelastogram test (TEG) was conducted to detect the functional changes of platelets for the maximum amplitude (MA) value; and the changes of glucose and lactate content were also tested by rate methods.

Results

As compared with group A

the platelets in groups B

C and D were in irregular shape with more parapodia stretched out

which represented an obvious activated state. All groups showed augmented MPV with time prolonging

and the MPV ualues in groups C and D were significant greater than those of groups A and B (

0.01). The HSR of each group was decreased gradually with elapse of preservation time

with groups C and D presenting more obvious reduction than groups A and B (

0.01). The positive rates of C

D62p and Annexin V in all groups were elevated over time

and the apoptosis and activation were most notable in group D. The MA value had no significant differences among groups. In addition

the glucose content was declined significantly during preservation

with groups A and B more obvious

while the alteration of lactate was opposite. We found that the morphology and quality of platelets were affected by cryopreservation

and the effect was significantly greater than that caused by virus inactivation. However

its effect on platelet function and metabolism was not distinct.

Conclusion

It is feasible for deep cryopreservation of platelets after riboflavin/UVB photochemical-mediated virus inactivation.

关键词

Keywords

references

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