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首页> 外文期刊>Cell biochemistry and function >Leucocytes isolated from simply frozen whole blood can be used in human biomonitoring for DNA damage measurement with the comet assay
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Leucocytes isolated from simply frozen whole blood can be used in human biomonitoring for DNA damage measurement with the comet assay

机译：从简单的冷冻全血中分离出的白细胞可用于人类生物监测，通过彗星测定法测量DNA损伤

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Preservation of human blood cells for DNA damage analysis with the comet assay conventionally involves the isolation of mononuclear cells by centrifugation, suspension in freezing medium and slow freezing to ?80?°C—a laborious process. A recent publication (Al-Salmani et al. Free Rad Biol Med 2011; 51: 719–725) describes a simple method in which small volumes of whole blood are frozen to ?20 or ?80?°C; on subsequent thawing, the comet assay is performed, with no indication of elevated DNA strand breakage resulting from the rapid freezing. However, leucocytes in whole blood (whether fresh or frozen) are abnormally resistant to damage by H2O2, and so a common test of antioxidant status (resistance to strand breakage by H2O2) cannot be used. We have refined this method by separating the leucocytes from the thawed blood; we find that, after three washes, the cells respond normally to H2O2. In addition, we have measured specific endogenous base damage (oxidized purines) in the isolated leucocytes, using the enzyme formamidopyrimidine DNA glycosylase. In a study of blood samples from 10 subjects, H2O2 sensitivity and endogenous damage—both reflecting the antioxidant status of the cells—correlated significantly. This modified approach to sample collection and storage is particularly applicable when the available volume of blood is limited and has great potential in biomonitoring and ecogenotoxicology studies where samples are obtained in the field or at sites remote from the testing laboratory.
机译：用彗星测定法保存人血细胞以进行DNA损伤分析通常涉及通过离心，悬浮在冷冻培养基中以及缓慢冷冻至80°C的过程来分离单核细胞，这是一个费力的过程。最近的出版物（Al-Salmani等人，Free Rad Biol Med 2011; 51：719-725）描述了一种简单的方法，其中将少量全血冷冻至20或80℃。在随后的解冻过程中，进行了彗星试验，没有迹象表明快速冷冻会导致DNA链断裂增加。但是，全血中的白细胞（无论是新鲜的还是冷冻的）对H2O2的损害均具有异常的抵抗力，因此无法使用常见的抗氧化剂状态测试（对H2O2的链断裂抵抗力）。我们通过从融化的血液中分离白细胞来完善这种方法。我们发现，洗涤3次后，细胞对H2O2的反应正常。另外，我们使用甲酰嘧啶嘧啶DNA糖基化酶测定了分离的白细胞中的特定内源性碱基损伤（氧化嘌呤）。在一项针对10位受试者的血液样本的研究中，H2O2的敏感性和内源性损伤（均反映了细胞的抗氧化剂状态）之间存在显着相关性。在血液的可用量有限的情况下，这种改进的样本收集和存储方法特别适用，并且在生物监测和生态遗传毒理学研究中具有很大的潜力，在这些研究中，可以在野外或远离测试实验室的地方获取样本。

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