[1]姜 华,黄 瑛,王 超,等.自制红细胞裂解液对流式细胞术测定外周血T 淋巴细胞亚群的影响研究[J].现代检验医学杂志,2023,38(05):165-170.[doi:10.3969/j.issn.1671-7414.2023.05.031]
　JIANG Hua,HUANG Ying,WANG Chao,et al.Study on the Effect of Homemade Erythrocyte Lysate on Flow Cytometric Determination of Peripheral Blood T Lymphocyte Subsets[J].Journal of Modern Laboratory Medicine,2023,38(05):165-170.[doi:10.3969/j.issn.1671-7414.2023.05.031]
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自制红细胞裂解液对流式细胞术测定外周血T 淋巴细胞亚群的影响研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第38卷
期数:
2023年05期
页码:
165-170
栏目:
研究简报·实验技术
出版日期:
2023-09-15

文章信息/Info

Title:
Study on the Effect of Homemade Erythrocyte Lysate on Flow Cytometric Determination of Peripheral Blood T Lymphocyte Subsets
文章编号:
1671-7414（2023）05-165-06
作者:
姜 华1; 黄 瑛1; 王 超1; 秦 超1; 孙 立1; 韩素芹1; 2; 王三龙1; 文海若1
（1. 中国食品药品检定研究院国家药物安全评价监测中心，药物非临床安全评价研究北京市重点实验室，北京100176；2. 中山大学药学院，广州 510006）
Author(s):
JIANG Hua1;  HUANG Ying1;  WANG Chao1;  QIN Chao1;  SUN Li1;  HAN Suqin1; 2;  WANG Sanlong1;  WEN Hairuo1
（1.Beijing Key Laboratory, National Center for Safety Evaluation of Drugs, National Institutes for Food and Drug Control, Beijing 100176，China；2. School of Phamaceutical Sciences, Sun Yat-sen University, Guangzhou 510006，China）
关键词:
红细胞裂解液; 流式细胞测定法; 外周血T 淋巴细胞亚群; 食蟹猴; SD 大鼠; 荧光标记
分类号:
R446.11
DOI:
10.3969/j.issn.1671-7414.2023.05.031
文献标志码:
A
摘要:
目的 比较实验室配制的红细胞裂解液的裂解时间对流式测定SD 大鼠和食蟹猴外周血T 淋巴细胞亚群的影响。方法 ①取15 只食蟹猴的肝素钠抗凝血各50μl，血样与荧光检测抗体PerCP-CD3/FITC-CD8/PE-CD4 孵育后分别经自制和市售红细胞裂解液裂解一段时间，比较样本经自制与市售裂解液处理不同时间后其 CD3+CD4+，CD3+CD8+ 和CD3+淋巴细胞的平均荧光强度（median fluorescence intensity，MFI）等数值的差异；②取15 只SD 大鼠的EDTA-K2 抗凝血各50μl，血样与荧光检测抗体APC-CD3/FITC-CD4/PE-CD8a 孵育后，分别经自制红细胞裂解液和市售裂解液裂解一段时间，比较样本经自制与市售裂解液处理不同时间后CD3+CD4+ 和CD3+CD8+ 淋巴细胞的MFI 等数值的差异；③取15 只大鼠EDTA-K2 抗凝血各50 μl，血样与荧光检测抗体APC-CD3/PE-CD4 孵育后，分别经自制红细胞裂解液和市售裂解液裂解一段时间，比较样本经自制与市售裂解液处理不同时间后PE-CD4+ 的MFI 差异。结果 ①食蟹猴外周血经自制红细胞裂解液，裂解时间约15min 和5min 的FITC-CD8+ 的MFI 分别为1 745±173 和2 439±253，淋巴细胞百分比分别为47.9%±7.2% 和42.1%±7.6%；裂解时间约15min 和5min 的PE-CD4+ 的MFI 分别为12 924±892 和13 695±984，淋巴细胞百分比分别为38.7%±7.2% 和33.1%±7.2%。裂解时间约15min 比裂解5min 的FITC-CD8+（t=8.779，P<0.000 1）和PE-CD4+（t=2.250，P<0.05）的MFI 降低；淋巴细胞百分比占比（t=2.139，2.162，均P<0.05）增高，差异具有统计学意义。食蟹猴外周血经市售红细胞裂解液裂解15min，FITC-CD8+，PE-CD4+，PerCP-CD3+ 的MFI 分别为2 413±240，12428±1 208 和1 015±123。与自制裂解液裂解5min 比较，市售裂解液裂解15min 的PE-CD4+ 和PerCP-CD3+ 的MFI 降低，差异具有统计学意义（t=3.150，4.862，均P ＜ 0.01）。② SD 大鼠外周血经自制红细胞裂解液，裂解时间5min 和2min的FITC-CD4+ 的MFI 分别为55.72±14.10 和225.08±12.44；裂解5min 与裂解2min 的FITC-CD4+ 的MFI 比较差异有统计学意义（t=34.89，P ＜ 0.001）；SD 大鼠外周血经市售红细胞裂解液，裂解5min 和15min 的FITC-CD4+ 的MFI 分别为277.02±30.36 和310.04±43.56，PE-CD8a+ 的MFI 分别为3 453.43±443.19 和3 211.28±357.85；裂解时间5min 与裂解15min 的FITC-CD4+ 和PE-CD8a+ 的MFI 比较差异有统计学意义（t=2.409，P ＜ 0.01）。③ SD 大鼠经自制红细胞裂解液，裂解5min 和2min 的PE-CD4+ 的MFI 分别为1 766±67, 1 765±51，差异无统计学意义（t=0.045，P ＞ 0.05）。结论 与市售裂解液比较，自制红细胞裂解液可缩短裂解时间。将FITC 荧光抗体更换为PE 荧光抗体后，在一定的裂解时间内可消除对大鼠测定淋巴细胞MFI 的影响；但随着裂解时间延长会造成FITC 和PE 荧光抗体MFI 值的下降。
Abstract:
Objective To compare the effect of the lysis time of lab-prepared erythrocyte lysate on flow cytometric determination of peripheral blood T lymphocyte subsets in SD rats and cynomolgus monkeys. Methods ① About 50 μl of heparin sodium anticoagulated blood from 15 cynomolgus monkeys was taken, the blood sample was incubated with the fluorescent antibodies PerCP-CD3/FITC-CD8/PE-CD4 and lysed by erythrocyte lysate for a period of time, the effect of lysis time on the mean fluorescence intensities (MIF) of CD3+CD4+ and CD3+CD8+ lymphocytes determined by flow cytometry was compared. ② About 50 μl of EDTA-K2 anticoagulant blood from 15 SD rats, the blood sample was incubated with the fluorescent antibodies APCCD3/ FITC-CD4/PE-CD8a and lysed by erythrocyte lysate for a period of time, the effect of lysis time on the MIF of CD3+CD4+ and CD3+CD8+ lymphocytes determined by flow cytometry was compared. ③ About 50 μl of EDTA-K2 anticoagulant blood from 15 SD rats, the blood sample was incubated with the fluorescent antibodies APC-CD3/PE-CD4 and lysed by erythrocyte lysate for a period of time, the effects of lysis time on the MFI of PE-CD4+ lymphocytes determined by flow cytometry was compared. Results ① The peripheral blood of cynomolgus monkeys was lysed by self-made erythrocyte lysate and the MFIs of FITC-CD8+ with lysis time of about 15 min and 5 min were 1 745±173 and 2 439±253 respectively, and the percentage of lymphocytes was 47.9%±7.2% and 42.1%±7.6%. The MFIs of PE-CD4+ with lysis time of about 15 min and 5 min were 12 924±892 and 13 695±984, and the percentage of lymphocytes was 38.7%± 7.2% and 33.1%±7.2%.The MFI of in FITCCD8+ (t=8.779, P<0.000 1), PE-CD4+ (t=2.250, P<0.05) and percentage of lymphocytes(t=2.139, 2.162; all P<0.05) lysed for 15min were significantly lower than those lysed for 5min, and the differences were statistically significant. There was also a difference in the percentage of FITC-CD8+ and PE-CD+ lymphocytes(t=2.131, P<0.05). The peripheral blood of cynomolgus monkeys was lysed by commercially available erythrocyte lysate, and the MFIs of FITC-CD8+, PE-CD4+ and PerCP-CD3+ with lysis time of about 15 min were 2 413±240,12 428±1 208 and 1 015±123, respectively. The MFI of PE-CD4+ and PerCP-CD3+ lysed for 15min by homemade lysate were different from the commercial lysate (t=3.150, 4.862, all P < 0.01). ② The peripheral blood of SD rats was lysed with self-made erythrocyte lysate, the MFIs of FITC-CD4+ with lysis time of 5 min and 2 min were 55.72± 14.10 and 225.08±12.44, and the lysis time of 5 min was significantly different from that of FITC-CD4+ with 2 min lysis（t=34.89, P ＜ 0.001）.The peripheral blood of SD rats was lysed with commercially available lysate, the MFIs of FITCCD4+ for 5 min and 15min lysis were 277.02± 30.36 and 310.04±43.56, and the MFIs for 5 min and 15 min PE-CD8a+ were 3 453.43± 443.19 and 3 211.28 ± 357.85, and the MFIs of FITC-CD4+ and PE-CD8a+ lysed for 5 min were significantly different from those lysed for 15 minutes(t=2.409, P ＜ 0.01). ③ The MFIs of PE-CD4+ lysed by homemade erythrocyte lysate in SD rats were 1 766±67, 1 765±51, and there was no significant change between the two(t=0.045, P> 0.05). Conclusion Compared with commercially available lysates, homemade lysates could shorten the lysate time. When the FITC fluorescent antibody was replaced with PE fluorescent antibody, the influence on the determination of lymphocyte MFI in rats could be eliminated within the same lysis time. However, when the lysis time was extended to 3 times, the MFIs of FITC and PE fluorescent antibody were affected to some extent.

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备注/Memo

备注/Memo:
基金项目： 国家重点研发计划课题（2021YFA1101602）：干细胞及相关治疗产品质量控制和非临床评价关键技术与规范研究；中国食品药品检定研究院关键技术研究基金（GJJS-2022-6-1）：脐带间充质干细胞和CD19 嵌合抗原受体T 细胞非临床有效性评价方法及药效学机制研究。
作者简介： 姜华（1976-），女，大学本科，主管技师，研究方向：药理毒理，E-mail：1828800241@qq.com。
黄瑛（1983-），女，博士，副研究员，研究方向：药理毒理，E-mail：huangying1002@nifdc.org.cn。
通讯作者：文海若（1981-），女，博士，研究员，研究方向：药理毒理，E-mail：wenhairuo@nifdc.org.cn。
更新日期/Last Update: 2023-09-15