a NeuroTrauma Department, Operational and Undersea Medicine Directorate, Naval Medical Research Center , Silver Spring, MD , USA.
b Department of Surgery , Uniformed Services University of the Health Sciences , Bethesda , MD , USA.
Artif Cells Nanomed Biotechnol. 2017 Feb;45(1):58-62. doi: 10.3109/21691401.2016.1138490. Epub 2016 Mar 2.
Background/aims Hemoglobin-based oxygen carriers (HBOCs) have been previously studied as resuscitation fluids. Due to HBOCs specific molecular conformation, hemoglobin (Hb) and methemoglobin (MetHb) determination is not always possible with automated apparatus. A practical technique was designed that allows simultaneous reading of MetHb and Hb in small volume samples. Methods A spectrophotometric method for measuring low levels of MetHb and Hb from limited volume samples was developed using a 96-well-plate by downsizing the Evelyn-Malloy and Drabkin methods. Either blood or buffer samples were spiked with one of five HBOCs (HBOC-201, M101, MP4CO-NP, Sanguinate and Oxyvita C). After treatment with cyanides, the samples were read at 540, 630, and 680 nm, and Hb and MetHb results were compared to certificate-of-analysis. Results Hb levels ranging from 0.2 to 2.8 g/dl were detected accurately with the 96-well-plate method with HBOC-201. Similarly, this method accurately measured Hb from either plasma or buffer samples containing any of the HBOCs. The MetHb plasma samples with HBOC-201 were also in agreement with ABL results (R = 0.99719). MetHb from all HBOCs in buffer measured with this method was comparable to reference but the accuracy was compromised for HBOCs in blood. Conclusions A useful 96-well-plate method of measuring HBOCs' Hb was designed for small-volume plasma samples. It was accurate for measuring MetHb from samples, that contained M101, MP4CO-NP, Sanguinate, and Oxyvita C diluted in buffer. This well-plate method allows reading of batch samples, multiple replicates, and using small volumes to accommodate limited animal blood collection which would not be otherwise detected by automated instrumentation.
背景/目的 血红蛋白基氧载体（HBOCs）此前曾被研究作为复苏液。由于 HBOCs 的特殊分子构象，血红蛋白（Hb）和高铁血红蛋白（MetHb）的测定并不总是可以用自动化仪器进行。本研究设计了一种实用技术，可在小体积样品中同时读取 MetHb 和 Hb。
方法 通过将 Evelyn-Malloy 和 Drabkin 方法小型化，开发了一种分光光度法，用于从小体积样品中测量低水平的 MetHb 和 Hb。将血液或缓冲液样品与五种 HBOC 之一（HBOC-201、M101、MP4CO-NP、Sanguinate 和 Oxyvita C）混合。用氰化物处理后，在 540、630 和 680nm 处读取样品，并将 Hb 和 MetHb 结果与分析证书进行比较。
结果 用 96 孔板法检测 HBOC-201 时，Hb 水平在 0.2 至 2.8g/dl 范围内检测准确。同样，该方法也可准确测量含有任何 HBOC 的血浆或缓冲液样品中的 Hb。HBOC-201 的 MetHb 血浆样品与 ABL 结果一致（R=0.99719）。用该方法测量缓冲液中所有 HBOC 的 MetHb 与参考值相当，但在血液中 HBOC 的准确性受到影响。
结论 设计了一种用于小体积血浆样品的 HBOCs Hb 测定的有用 96 孔板法。该方法可准确测量 M101、MP4CO-NP、Sanguinate 和 Oxyvita C 稀释在缓冲液中的样品中的 MetHb。这种微孔板方法可进行批量样品读取、多次重复，并使用小体积以适应有限的动物血液采集，否则无法用自动化仪器检测。
