Frontiers in Pediatrics (Apr 2022)
Comparison of [3H]-Thymidine, Carboxyfluorescein Diacetate Succinimidyl Ester and Ki-67 in Lymphocyte Proliferation
Abstract
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.
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