Abstract

Purpose

The cytokinesis-block micronucleus (MN) assay is a widely used technique in basic radiobiology research, human biomonitoring studies and in vitro radiosensitivity testing. Fresh whole blood cultures are commonly used for these purposes, but immediate processing of fresh samples can be logistically challenging. Therefore, we aimed at establishing a protocol for the MN assay on cryopreserved whole blood, followed by a thorough evaluation of the reliability of this assay for use in radiosensitivity assessment in patients.

Materials and methods

Whole blood samples of 20 healthy donors and 4 patients with a primary immunodeficiency disease (PID) were collected to compare the results obtained with the MN assay performed on fresh versus cryopreserved whole blood samples. MN yields were scored after irradiation with 220 kV X-rays (dose rate 3 Gy/min), with doses ranging from 0.5–2 Gy.

Results

The application of the MN assay on cryopreserved blood samples was successful in all analyzed samples. The radiation-induced MN and NDI scores in fresh and cryopreserved blood cultures were found to be similar. Acceptable inter-individual and intra-individual variabilities in MN yields were observed. Repeated analysis of cryopreserved blood cultures originating from the same blood sample, thawed at different time points, revealed that MN values remain stable for cryopreservation periods up to one year. Finally, radiosensitive patients were successfully identified using the MN assay on cryopreserved samples.

Conclusions

To our knowledge, this study is the first report of the successful use of cryopreserved whole blood samples for application of the MN assay. The data presented here demonstrate that the MN assay performed on cryopreserved whole blood is reliable for radiosensitivity testing. Our results also support its wider use in epidemiological, biomonitoring and genotoxicity studies. The presented method of cryopreservation of blood samples might also benefit other assays.

中文翻译：

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冻存全血的胞质分裂阻断微核测定

 抽象的

 目的

胞质分裂阻断微核（MN）测定是基础放射生物学研究、人体生物监测研究和体外放射敏感性测试中广泛使用的技术。新鲜全血培养物通常用于这些目的，但新鲜样本的立即处理在后勤方面可能具有挑战性。因此，我们的目的是建立一种冷冻全血 MN 测定方案，然后对该测定法用于患者放射敏感性评估的可靠性进行彻底评估。

 材料和方法

收集了 20 名健康捐献者和 4 名原发性免疫缺陷病 (PID) 患者的全血样本，以比较对新鲜全血样本和冷冻保存全血样本进行 MN 测定所获得的结果。用 220 kV X 射线（剂量率 3 Gy/min）照射后对 MN 产率进行评分，剂量范围为 0.5-2 Gy。

 结果

MN 测定在冷冻保存的血液样本中的应用在所有分析样本中都是成功的。新鲜和冷冻保存的血培养物中辐射引起的 MN 和 NDI 评分相似。观察到 MN 产量存在可接受的个体间和个体内变异。对来自同一血样、在不同时间点解冻的冷冻保存的血培养物进行重复分析表明，在长达一年的冷冻保存期内，MN 值保持稳定。最后，使用 MN 测定对冷冻保存的样本成功识别出放射敏感患者。

 结论

据我们所知，这项研究是第一份成功使用冷冻保存的全血样本进行 MN 测定的报告。这里提供的数据表明，对冷冻保存的全血进行的 MN 测定对于放射敏感性测试是可靠的。我们的结果也支持其在流行病学、生物监测和遗传毒性研究中的更广泛应用。所提出的血液样本冷冻保存方法也可能有利于其他测定。