The reliability of the isotropic fractionator method for counting total cells and neurons
The reliability of the isotropic fractionator method for counting total cells and neurons
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DOI：
10.1016/j.jneumeth.2019.108382
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发表时间：
2019-10-01
影响因子：
3
通讯作者：
Mota, Bruno
中科院分区：
文献类型：
--
作者：
Neves, Kleber;Guimaraes, Daniel Menezes;Mota, Bruno
背景：各向同性分馏器是一种测定神经组织细胞组成的方法。它主要用于评估物种间的差异，预计差异将足够大，不会被方法错误所掩盖。然而，如果目标是发现较小的差异，例如在相同物种的比较中，了解该方法中的变异来源是重要的。不同品系的小鼠之间的比较表明，IF足够一致，可以检测到这些差异。然而，该方法的可靠性尚未得到直接检验。方法：在本研究中，我们评估了该方法测定瑞士小鼠细胞和神经元数量的可靠性。我们对同一材料进行了不同实验者的重复细胞计数，以量化不同来源的差异。结果：在总细胞计数中，我们观察到大脑皮层的大部分差异处于相反的水平。对于小脑，大部分差异归因于样本本身。至于神经元，随机误差和免疫染色符合大多数变异，无论是在大脑皮层还是在小脑。重测信度相对较高，尤其是细胞计数。结论：尽管在汇总不同来源的数据时，计数器之间的偏差和染色的随机变异可能存在问题，但我们提出了切实可行的建议，以提高该方法的可靠性。虽然规模很小，但这项研究是朝着更精确地测量大脑细胞成分迈出的最需要的一步。
Background: The Isotropic Fractionator (IF) is a method to determine the cellular composition of nervous tissue. It has been mostly applied to assess variation across species, where differences are expected to be large enough not to be masked by methodological error. However, understanding the sources of variation in the method is important if the goal is to detect smaller differences, for example, in same-species comparisons. Comparisons between different mice strains suggest that the IF is consistent enough to detect these differences. Nevertheless, the reliability of the method has not yet been examined directly.Method: In this study, we evaluate the reliability of the method for the determination of cellular and neuronal numbers of Swiss mice. We performed repeated cell counts of the same material by different experimenters to quantify different sources of variation.Results: In total cell counts, we observed that for the cerebral cortex most of the variance was at the counter level. For the cerebellum, most of the variance is attributed to the sample itself. As for neurons, random error along with the immunostaining correspond to most of the variation, both in the cerebral cortex and in the cerebellum. Test-retest reliability coefficients were relatively high, especially for cell counts.Conclusions: Although biases between counters and random variation in staining could be problematic when aggregating data from different sources, we offer practical suggestions to improve the reliability of the method. While small, this study is a most needed step towards more precise measurement of the brain's cellular composition.