Application of an intracellular assay for determination of tenofovir-diphosphate and emtricitabine-triphosphate from erythrocytes using dried blood spots.
Application of an intracellular assay for determination of tenofovir-diphosphate and emtricitabine-triphosphate from erythrocytes using dried blood spots.
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DOI：
10.1016/j.jpba.2016.01.038
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发表时间：
2016-04-15
影响因子：
3.4
通讯作者：
Anderson PL
中科院分区：
文献类型：
--
作者：
Zheng JH;Rower C;McAllister K;Castillo-Mancilla J;Klein B;Meditz A;Guida LA;Kiser JJ;Bushman LR;Anderson PL
本通讯描述了应用现有的细胞内方法使用干血斑 (DBS) 定量红细胞中的替诺福韦二磷酸 (TFV-DP) 和恩曲他滨三磷酸 (FTC-TP)。通过提取到 70:30 甲醇:水溶液（裂解的细胞基质）中的 3mm DBS 冲头测定浓度。然后对该提取溶液进行先前验证的裂解细胞基质分析程序。 DBS 验证实验使用了研究参与者的复制样品，以证明点体积范围为 10μL 至 50μL 且打孔位置为点边缘或中心的可接受的再现性。对配对 DBS 与纯化红细胞的分析表明，对于观察到的参与者血细胞比容范围（35% 至 50%），3 毫米 DBS 冲头平均含有 1190 万个细胞。完成的大量稳定性测试表明，EDTA 真空采血管中的全血在点样前可以在室温下放置 24 小时，而 DBS 可以在室温下保存长达五天，包括使用 2 天交付在室温下运输。 DBS 在 -20°C 或 -80°C 下的储存稳定性可达 18 个月，并且 DBS 可以经历 4 次冷冻/解冻循环。所描述的方法应用于艾滋病毒预防研究，通过其区分依从性梯度的能力，证明了与艾滋病毒获得的强大关联。
This communication describes the application of an existing intracellular methodology to the quantitation of tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP) from erythrocytes using dried blood spots (DBS). Concentrations were determined from a 3mm DBS punch extracted into a 70:30 methanol:water solution (lysed cellular matrix). This extraction solution was then subjected to a previously validated analytical procedure for lysed cellular matrix. Experiments for DBS validation used replicate samples from study participants to demonstrate acceptable reproducibility with spot volumes ranging from 10μL to 50μL and punch location either from the edge or center of the spot. Analysis of paired DBS with purified red blood cells showed that a 3mm DBS punch contained an average of 11.9 million cells for the observed hematocrit range of the participants (35% to 50%). Numerous stability tests were completed showing that whole blood in an EDTA vacutainer could sit for 24 hours at room temperature prior to spotting, and DBS could remain at room temperature for up to five days including shipment at ambient using 2-day delivery. DBS stability in storage was acceptable up to 18 months at −20°C or −80°C and DBS could undergo 4 Freeze/Thaw cycles. The described method was applied to HIV prophylaxis studies, demonstrating powerful associations with HIV acquisition through its ability to discriminate gradients of adherence.