Comparison of [3H]-Thymidine, Carboxyfluorescein Diacetate Succinimidyl Ester and Ki-67 in Lymphocyte Proliferation
BackgroundPatients with T cell deficiency <10% of normal proliferation are indicated to receive immune reconstruction by hematopoietic stem cell transplantation (HSCT). This study aimed to investigate whether non-radioactive assays can be used to quantitatively detect the lymphocyte prolifera...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2022-04-01
|
| Series: | Frontiers in Pediatrics |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fped.2022.638549/full |
| _version_ | 1817992003941564416 |
|---|---|
| author | Hsin-Ju Lee Chun-Chun Gau Chun-Chun Gau Chun-Chun Gau Wan-Fang Lee Wen-I Lee Wen-I Lee Jing-Long Huang Jing-Long Huang Jing-Long Huang Shih-Hsiang Chen Ho-Yu Yeh Chi-Jou Liang Shih-Hang Fu |
| author_facet | Hsin-Ju Lee Chun-Chun Gau Chun-Chun Gau Chun-Chun Gau Wan-Fang Lee Wen-I Lee Wen-I Lee Jing-Long Huang Jing-Long Huang Jing-Long Huang Shih-Hsiang Chen Ho-Yu Yeh Chi-Jou Liang Shih-Hang Fu |
| author_sort | Hsin-Ju Lee |
| collection | DOAJ |
| description | BackgroundPatients with T cell deficiency <10% of normal proliferation are indicated to receive immune reconstruction by hematopoietic stem cell transplantation (HSCT). This study aimed to investigate whether non-radioactive assays can be used to quantitatively detect the lymphocyte proliferation <10% of normal as radioactive [3H]-thymidine.”MethodsRadioactive [3H]-thymidine, non-radioactive carboxyfluorescein diacetate succinimidyl ester (CFSE), and Ki-67 protein expressions were used to measure the lymphocyte proliferation as calculated using the stimulation index (SI), subtraction percentage, and proliferation index (FlowJo software). Normal references were established for comparison in the absence of parallel healthy controls.ResultsNormal ranges of mitogen-stimulated lymphocyte proliferation were established as a SI of 15–267 (CSFE 47–92%, Ki-67 42–79%) with phytohemagglutinin (PHA) 5 μg/ml stimulation; 19–139 (CFSE 62–83%, 45–74% Ki-67) with concanavalin-A (ConA) 5 μg/ml stimulation; 7–53 (CFSE 6–23%, Ki-67 10–24%) with pokeweed mitogen (PWM) 0.1 ug/ml stimulation; 3–28 (CFSE 4–10%, Ki-67 5–14%) with candida 10 ug/ml stimulation; and 2–27 (CFSE 6–41%, Ki-67 6–30%) with bacille Calmette-Guerin (BCG) 0.02 ng/ml stimulation. The normalized CFSE-proliferation index was between 2.1 and 3.0. Although there was no significant correlation between these three assays in the healthy controls, the SI value for <10% [3H]-thymidine proliferation in those with T cell deficiency was compatible with CFSE- and Ki-67-stained lymphocyte percentages, and validated in patients with IL2RG, RAG1, and ZAP70 mutations. When calculating [3H]-thymidine <10% of normal lymphocyte proliferation, the threshold of parallel controls was more reliable than previously established normal references.ConclusionThe large quantitative value of radioactive [3H]-thymidine was more easily recognizable than that for non-radioactive CFSE and Ki-67. Even though the correlation was not significant, those identified to have <10% of normal proliferation by [3H]-thymidine could be consistently detected by CFSE and Ki-67, and consequently indicated for HSCT. |
| first_indexed | 2024-04-14T01:20:54Z |
| format | Article |
| id | doaj.art-a16ace09b6e54ca2bb72a50c2396c14e |
| institution | Directory Open Access Journal |
| issn | 2296-2360 |
| language | English |
| last_indexed | 2024-04-14T01:20:54Z |
| publishDate | 2022-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Pediatrics |
| spelling | doaj.art-a16ace09b6e54ca2bb72a50c2396c14e2022-12-22T02:20:39ZengFrontiers Media S.A.Frontiers in Pediatrics2296-23602022-04-011010.3389/fped.2022.638549638549Comparison of [3H]-Thymidine, Carboxyfluorescein Diacetate Succinimidyl Ester and Ki-67 in Lymphocyte ProliferationHsin-Ju Lee0Chun-Chun Gau1Chun-Chun Gau2Chun-Chun Gau3Wan-Fang Lee4Wen-I Lee5Wen-I Lee6Jing-Long Huang7Jing-Long Huang8Jing-Long Huang9Shih-Hsiang Chen10Ho-Yu Yeh11Chi-Jou Liang12Shih-Hang Fu13Division of Allergy, Asthma, and Rheumatology, Department of Pediatrics, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, TaiwanDivision of Allergy, Asthma, and Rheumatology, Department of Pediatrics, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, TaiwanDepartment of Pediatrics, Chang Gung Memorial Hospital, Keelung, TaiwanDivision of Pediatric General Medicine, Department of Pediatrics, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taoyuan, TaiwanDivision of Allergy, Asthma, and Rheumatology, Department of Pediatrics, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, TaiwanDivision of Allergy, Asthma, and Rheumatology, Department of Pediatrics, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, TaiwanPrimary Immunodeficiency Care and Research (PICAR) Institute, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, TaiwanDivision of Allergy, Asthma, and Rheumatology, Department of Pediatrics, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, TaiwanPrimary Immunodeficiency Care and Research (PICAR) Institute, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, TaiwanDepartment of Pediatrics, New Taipei Municipal TuCheng Hospital, New Taipei City, TaiwanDivision of Hematology/Oncology, Department of Pediatrics, Chang Gung Memorial Hospital, Taoyuan, TaiwanPrimary Immunodeficiency Care and Research (PICAR) Institute, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, TaiwanPrimary Immunodeficiency Care and Research (PICAR) Institute, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, TaiwanPrimary Immunodeficiency Care and Research (PICAR) Institute, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, TaiwanBackgroundPatients with T cell deficiency <10% of normal proliferation are indicated to receive immune reconstruction by hematopoietic stem cell transplantation (HSCT). This study aimed to investigate whether non-radioactive assays can be used to quantitatively detect the lymphocyte proliferation <10% of normal as radioactive [3H]-thymidine.”MethodsRadioactive [3H]-thymidine, non-radioactive carboxyfluorescein diacetate succinimidyl ester (CFSE), and Ki-67 protein expressions were used to measure the lymphocyte proliferation as calculated using the stimulation index (SI), subtraction percentage, and proliferation index (FlowJo software). Normal references were established for comparison in the absence of parallel healthy controls.ResultsNormal ranges of mitogen-stimulated lymphocyte proliferation were established as a SI of 15–267 (CSFE 47–92%, Ki-67 42–79%) with phytohemagglutinin (PHA) 5 μg/ml stimulation; 19–139 (CFSE 62–83%, 45–74% Ki-67) with concanavalin-A (ConA) 5 μg/ml stimulation; 7–53 (CFSE 6–23%, Ki-67 10–24%) with pokeweed mitogen (PWM) 0.1 ug/ml stimulation; 3–28 (CFSE 4–10%, Ki-67 5–14%) with candida 10 ug/ml stimulation; and 2–27 (CFSE 6–41%, Ki-67 6–30%) with bacille Calmette-Guerin (BCG) 0.02 ng/ml stimulation. The normalized CFSE-proliferation index was between 2.1 and 3.0. Although there was no significant correlation between these three assays in the healthy controls, the SI value for <10% [3H]-thymidine proliferation in those with T cell deficiency was compatible with CFSE- and Ki-67-stained lymphocyte percentages, and validated in patients with IL2RG, RAG1, and ZAP70 mutations. When calculating [3H]-thymidine <10% of normal lymphocyte proliferation, the threshold of parallel controls was more reliable than previously established normal references.ConclusionThe large quantitative value of radioactive [3H]-thymidine was more easily recognizable than that for non-radioactive CFSE and Ki-67. Even though the correlation was not significant, those identified to have <10% of normal proliferation by [3H]-thymidine could be consistently detected by CFSE and Ki-67, and consequently indicated for HSCT.https://www.frontiersin.org/articles/10.3389/fped.2022.638549/fullCFSE[3H]-thymidineKi-67lymphocyte proliferationprimary immunodeficiency diseasesproliferation index |
| spellingShingle | Hsin-Ju Lee Chun-Chun Gau Chun-Chun Gau Chun-Chun Gau Wan-Fang Lee Wen-I Lee Wen-I Lee Jing-Long Huang Jing-Long Huang Jing-Long Huang Shih-Hsiang Chen Ho-Yu Yeh Chi-Jou Liang Shih-Hang Fu Comparison of [3H]-Thymidine, Carboxyfluorescein Diacetate Succinimidyl Ester and Ki-67 in Lymphocyte Proliferation Frontiers in Pediatrics CFSE [3H]-thymidine Ki-67 lymphocyte proliferation primary immunodeficiency diseases proliferation index |
| title | Comparison of [3H]-Thymidine, Carboxyfluorescein Diacetate Succinimidyl Ester and Ki-67 in Lymphocyte Proliferation |
| title_full | Comparison of [3H]-Thymidine, Carboxyfluorescein Diacetate Succinimidyl Ester and Ki-67 in Lymphocyte Proliferation |
| title_fullStr | Comparison of [3H]-Thymidine, Carboxyfluorescein Diacetate Succinimidyl Ester and Ki-67 in Lymphocyte Proliferation |
| title_full_unstemmed | Comparison of [3H]-Thymidine, Carboxyfluorescein Diacetate Succinimidyl Ester and Ki-67 in Lymphocyte Proliferation |
| title_short | Comparison of [3H]-Thymidine, Carboxyfluorescein Diacetate Succinimidyl Ester and Ki-67 in Lymphocyte Proliferation |
| title_sort | comparison of 3h thymidine carboxyfluorescein diacetate succinimidyl ester and ki 67 in lymphocyte proliferation |
| topic | CFSE [3H]-thymidine Ki-67 lymphocyte proliferation primary immunodeficiency diseases proliferation index |
| url | https://www.frontiersin.org/articles/10.3389/fped.2022.638549/full |
| work_keys_str_mv | AT hsinjulee comparisonof3hthymidinecarboxyfluoresceindiacetatesuccinimidylesterandki67inlymphocyteproliferation AT chunchungau comparisonof3hthymidinecarboxyfluoresceindiacetatesuccinimidylesterandki67inlymphocyteproliferation AT chunchungau comparisonof3hthymidinecarboxyfluoresceindiacetatesuccinimidylesterandki67inlymphocyteproliferation AT chunchungau comparisonof3hthymidinecarboxyfluoresceindiacetatesuccinimidylesterandki67inlymphocyteproliferation AT wanfanglee comparisonof3hthymidinecarboxyfluoresceindiacetatesuccinimidylesterandki67inlymphocyteproliferation AT wenilee comparisonof3hthymidinecarboxyfluoresceindiacetatesuccinimidylesterandki67inlymphocyteproliferation AT wenilee comparisonof3hthymidinecarboxyfluoresceindiacetatesuccinimidylesterandki67inlymphocyteproliferation AT jinglonghuang comparisonof3hthymidinecarboxyfluoresceindiacetatesuccinimidylesterandki67inlymphocyteproliferation AT jinglonghuang comparisonof3hthymidinecarboxyfluoresceindiacetatesuccinimidylesterandki67inlymphocyteproliferation AT jinglonghuang comparisonof3hthymidinecarboxyfluoresceindiacetatesuccinimidylesterandki67inlymphocyteproliferation AT shihhsiangchen comparisonof3hthymidinecarboxyfluoresceindiacetatesuccinimidylesterandki67inlymphocyteproliferation AT hoyuyeh comparisonof3hthymidinecarboxyfluoresceindiacetatesuccinimidylesterandki67inlymphocyteproliferation AT chijouliang comparisonof3hthymidinecarboxyfluoresceindiacetatesuccinimidylesterandki67inlymphocyteproliferation AT shihhangfu comparisonof3hthymidinecarboxyfluoresceindiacetatesuccinimidylesterandki67inlymphocyteproliferation |