Gamma-H2AX biodosimetry for use in large scale radiation incidents: comparison of a rapid '96 well lyse/fix' protocol with a routine method.
Gamma-H2AX biodosimetry for use in large scale radiation incidents: comparison of a rapid '96 well lyse/fix' protocol with a routine method.
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作者：
Moquet J;Barnard S;Rothkamm K
在辐射事故发生后，通过γ-H_2AX焦点分析作出的初步剂量估计可以补充基于临床症状的早期伤员分类。当需要对许多个体进行快速评估时，样品处理时间非常重要。因此，开发了一种用于高样本吞吐量的方案，该方案需要少于0.1毫升的血液，因此有可能实现手指刺伤采样。这项技术将红细胞溶解和白细胞固定在96孔板上一步完成，这与常规方案不同，常规方案通常通过Ficoll密度梯度离心法分离较大血容量的淋巴细胞，然后进行洗涤和固定步骤。快速的‘96井裂解/修复’方法将96个样品的估计样品处理时间从使用常规方法的15小时减少到大约4小时。然而，快速方法制备的96个样本中20个细胞的评分时间比常规方法长(零剂量下3.1h比1.5h；照射后样本7.0h比6.1h)。两种方案的病灶产额相近，照射剂量分别为0、0.2、0.6、1.1、1.2、2.1和4.3Gy250 kVPX射线，照射剂量为0.5Gymin，孵化2 h后，常规和Lyse/FIX方法的估算剂量与实际剂量的线性回归系数分别为0.87±0.06(R2=97.6%)和0.85±0.05(R2=98.3%)。因此，LYSE/FIX方案可促进用于大规模辐射事故的γ-H2AX生物剂量测定的高通量处理，但代价是病灶评分时间稍长。
Following a radiation incident, preliminary dose estimates made by γ-H2AX foci analysis can supplement the early triage of casualties based on clinical symptoms. Sample processing time is important when many individuals need to be rapidly assessed. A protocol was therefore developed for high sample throughput that requires less than 0.1 ml blood, thus potentially enabling finger prick sampling. The technique combines red blood cell lysis and leukocyte fixation in one step on a 96 well plate, in contrast to the routine protocol, where lymphocytes in larger blood volumes are typically separated by Ficoll density gradient centrifugation with subsequent washing and fixation steps. The rapid ‘96 well lyse/fix’ method reduced the estimated sample processing time for 96 samples to about 4 h compared to 15 h using the routine protocol. However, scoring 20 cells in 96 samples prepared by the rapid protocol took longer than for the routine method (3.1 versus 1.5 h at zero dose; 7.0 versus 6.1 h for irradiated samples). Similar foci yields were scored for both protocols and consistent dose estimates were obtained for samples exposed to 0, 0.2, 0.6, 1.1, 1.2, 2.1 and 4.3 Gy of 250 kVp X-rays at 0.5 Gy/min and incubated for 2 h. Linear regression coefficients were 0.87 ± 0.06 (R2 = 97.6%) and 0.85 ± 0.05 (R2 = 98.3%) for estimated versus actual doses for the routine and lyse/fix method, respectively. The lyse/fix protocol can therefore facilitate high throughput processing for γ-H2AX biodosimetry for use in large scale radiation incidents, at the cost of somewhat longer foci scoring times.
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DOI：
10.1073/pnas.0830918100
发表时间：
2003-04-29
影响因子：
11.1
作者：
Rothkamm, K;Löbrich, M
通讯作者：
Löbrich, M
影响因子：
3.7
作者：
Horn S;Barnard S;Rothkamm K
通讯作者：
Rothkamm K
影响因子：
3.4
作者：
Turner HC;Brenner DJ;Chen Y;Bertucci A;Zhang J;Wang H;Lyulko OV;Xu Y;Shuryak I;Schaefer J;Simaan N;Randers-Pehrson G;Yao YL;Amundson SA;Garty G
通讯作者：
Garty G
影响因子：
19.7
作者：
Rothkamm, Kai;Balroop, Sheena;Goh, Vicky
通讯作者：
Goh, Vicky
影响因子：
2
作者：
Valente, M.;Voisin, P.;Roch-Lefevre, S.
通讯作者：
Roch-Lefevre, S.