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首页> 美国卫生研究院文献>Cells >Normal Distribution of CD8+ T-Cell-Derived ELISPOT Counts within Replicates Justifies the Reliance on Parametric Statistics for Identifying Positive Responses
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Normal Distribution of CD8+ T-Cell-Derived ELISPOT Counts within Replicates Justifies the Reliance on Parametric Statistics for Identifying Positive Responses

机译：复制物中CD8 + T细胞衍生的ELISPOT计数的正态分布证明了依靠参数统计来确定阳性反应是合理的

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摘要

Accurate assessment of positive ELISPOT responses for low frequencies of antigen-specific T-cells is controversial. In particular, it is still unknown whether ELISPOT counts within replicate wells follow a theoretical distribution function, and thus whether high power parametric statistics can be used to discriminate between positive and negative wells. We studied experimental distributions of spot counts for up to 120 replicate wells of IFN-γ production by CD8+ T-cell responding to EBV LMP2A (426 – 434) peptide in human PBMC. The cells were tested in serial dilutions covering a wide range of average spot counts per condition, from just a few to hundreds of spots per well. Statistical analysis of the data using diagnostic Q-Q plots and the Shapiro-Wilk normality test showed that in the entire dynamic range of ELISPOT spot counts within replicate wells followed a normal distribution. This result implies that the Student t-Test and ANOVA are suited to identify positive responses. We also show experimentally that borderline responses can be reliably detected by involving more replicate wells, plating higher numbers of PBMC, addition of IL-7, or a combination of these. Furthermore, we have experimentally verified that the number of replicates needed for detection of weak responses can be calculated using parametric statistics.
机译：准确评估低频抗原特异性T细胞的阳性ELISPOT反应是有争议的。特别是，仍然不知道复制孔中的ELISPOT计数是否遵循理论分布函数，因此是否可以使用高功率参数统计量来区分正孔和负孔。我们研究了人PBMC中CD8 + T细胞对EBV LMP2A（426 – 434）肽应答的IFN-γ产生的多达120个重复孔的斑点计数的实验分布。在系列稀释液中对细胞进行了测试，涵盖了每种条件下广泛的平均斑点计数范围，每孔只有几个到数百个斑点。使用诊断Q-Q图和Shapiro-Wilk正态性检验对数据进行统计分析，结果表明，在重复孔内ELISPOT整个动态范围内，斑点计数均遵循正态分布。该结果表明，学生t检验和方差分析适合识别阳性反应。我们还通过实验表明，通过涉及更多的重复孔，接种更多数量的PBMC，添加IL-7或这些的组合，可以可靠地检测边界反应。此外，我们已经通过实验验证了可以使用参数统计来计算检测弱响应所需的重复次数。

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