Conventional diatom testing using strong acid: Notable false-positive results caused by an underestimated contamination source (blind spot)
Conventional diatom testing using strong acid: Notable false-positive results caused by an underestimated contamination source (blind spot)
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DOI：
10.1016/j.forsciint.2021.111131
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发表时间：
2021-12-07
影响因子：
2.2
通讯作者：
Yukawa, Nobuhiro
中科院分区：
文献类型：
--
作者：
Kakizaki, Eiji;Shinkawa, Norihiro;Yukawa, Nobuhiro
溺水者封闭器官中硅藻阳性率较低，这给硅藻检测带来了挑战。非溺水受害者闭合器官的高阳性率也提出了一个重要问题。这些矛盾的发现在 1960-80 年代进行的硅藻测试研究中很常见，但原因仍不清楚。在本研究中，我们确定了与使用强酸的硅藻测试中假阳性结果相关的最重要因素之一。在重复使用的凯氏烧瓶中发现了 1 到 290 个假阳性硅藻，这些烧瓶在第一次硅藻测试后彻底清洗，并在第二次测试前保持无组织。当消化的组织或水样中存在超过约 10,000 个硅藻时，20 例中有 11 例出现假阳性结果。重复使用的烧瓶被发现含有许多常见的硅藻（< 30 μm），包括 Cocconeis、Cymbella、Diatoma、Gomphonema、Navicula 和 Nitzschia，这与在封闭器官中发现的硅藻的报道一致。令人惊讶的是，即使在每次分析中使用没有组织的相同烧瓶进行第六次硅藻测试时，也会出现这种假阳性结果。相比之下，在任何试剂中或与其他玻璃器皿相关的硅藻中均未检测到硅藻。因此，重复使用凯氏定氮烧瓶很容易导致假阳性结果，而使用碱性清洁剂清洁烧瓶并不能完全防止假阳性结果，即使在六次测试后仍检测到硅藻即可证明这一点。我们假设导致假阳性结果的硅藻通过加热部分熔化并固定在烧瓶的内表面玻璃上，因为硅藻壳主要由 SiO2 组成，与玻璃类似。然后，通过在下一次硅藻测试中重新加热，将粘附的硅藻从玻璃上释放出来。这些结果还表明，由于重复使用肺或水样本进行分析，烧瓶中剩余的硅藻数量可以稳定增加。相比之下，在仅使用新烧瓶的分析中，在 20 名溺水者的 20 份肾脏样本中的 4 份、12 份肝脏样本中的 2 份以及 8 份血液样本中仅发现了一到两个硅藻。很难确定这些硅藻是否实际上是通过血液循环携带的，因为在尸检过程和硅藻测试过程中可能会发生一些硅藻的污染。总之，只有新的（未使用过的）凯氏烧瓶才可用于强酸消解的硅藻测试。此外，这些数据表明，溺水者封闭器官中硅藻的数量和频率可能比之前认为的要低得多。 (C) 2021 Elsevier B.V. 保留所有权利。
Low rates of diatom positivity in the closed organs of drowning victims present challenges for diatom testing. High positivity rates in closed organs of non-drowning victims also raise an important issue. These contradictory findings were common in diatom testing studies undertaken during the 1960-80 s, but the reasons remained unclear. In the present study, we identified one of the most important factors associated with false-positive results in diatom testing using strong acid. One to 290 false-positive diatoms were found in reused Kjeldahl flasks that were thoroughly washed after the first diatom testing and kept free of tissue before the second testing. False-positive results occurred in 11 of 20 cases when more than approximately 10,000 diatoms were present in digested tissue or water samples. Reused flasks were found to contain many common diatoms (< 30 mu m), including Cocconeis, Cymbella, Diatoma, Gomphonema, Navicula, and Nitzschia, in agreement with reports of diatoms identified in closed organs. Surprisingly, such false-positive results occurred even at the sixth diatom testing using the same flasks kept free of tissues in each analysis. In contrast, no diatoms were detected in any reagent or associated with other glassware. Thus, reuse of Kjeldahl flasks can readily cause false-positive results that cannot be completely prevented by cleaning the flasks using alkali detergents, as evidenced by detection of diatoms even after six tests. We assume that diatoms causing false-positive results are partially melted by heating and fixed onto the flask's inner surface glass, as the diatom frustule consists primarily of SiO2, similar to glass. Adherent diatoms are then released from the glass by re-heating at the next diatom testing. These results also suggest that the number of diatoms remaining in a flask can increase steadily as a result of repeated reuse for analysis of lung or water samples. In contrast, in analyses using only new flasks, only one or two diatoms were found in 4 of 20 kidney, 2 of 12 liver, and 2 of 8 blood samples from 20 drowning victims. It is difficult to determine whether such diatoms are actually carried via the blood circulation, as contamination with a few diatoms can occur during autopsy procedures and diatom testing. In conclusion, only new (unused) Kjeldahl flasks should be used for diatom testing with strong acid digestion. Moreover, these data suggest that the number and frequency of diatoms present in closed organs of drowning victims may be much lower than previously thought. (C) 2021 Elsevier B.V. All rights reserved.