Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University , Ottergemse-steenweg 460, Ghent 9000, Belgium.
Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam , Meibergdreef 9, Amsterdam 1105 AZ, The Netherlands.
Anal Chem. 2018 Feb 6;90(3):1795-1804. doi: 10.1021/acs.analchem.7b03784. Epub 2018 Jan 17.
The hematocrit (Hct) effect is one of the most important hurdles currently preventing more widespread implementation of quantitative dried blood spot (DBS) analysis in a routine context. Indeed, the Hct may affect both the accuracy of DBS methods as well as the interpretation of DBS-based results. We previously developed a method to determine the Hct of a DBS based on its hemoglobin content using noncontact diffuse reflectance spectroscopy. Despite the ease with which the analysis can be performed (i.e., mere scanning of the DBS) and the good results that were obtained, the method did require a complicated algorithm to derive the total hemoglobin content from the DBS's reflectance spectrum. As the total hemoglobin was calculated as the sum of oxyhemoglobin, methemoglobin, and hemichrome, the three main hemoglobin derivatives formed in DBS upon aging, the reflectance spectrum needed to be unmixed to determine the quantity of each of these derivatives. We now simplified the method by only using the reflectance at a single wavelength, located at a quasi-isosbestic point in the reflectance curve. At this wavelength, assuming 1-to-1 stoichiometry of the aging reaction, the reflectance is insensitive to the hemoglobin degradation and only scales with the total amount of hemoglobin and, hence, the Hct. This simplified method was successfully validated. At each quality control level as well as at the limits of quantitation (i.e., 0.20 and 0.67) bias, intra- and interday imprecision were within 10%. Method reproducibility was excellent based on incurred sample reanalysis and surpassed the reproducibility of the original method. Furthermore, the influence of the volume spotted, the measurement location within the spot, as well as storage time and temperature were evaluated, showing no relevant impact of these parameters. Application to 233 patient samples revealed a good correlation between the Hct determined on whole blood and the predicted Hct determined on venous DBS. The bias obtained with Bland and Altman analysis was -0.015 and the limits of agreement were -0.061 and 0.031, indicating that the simplified, noncontact Hct prediction method even outperforms the original method. In addition, using caffeine as a model compound, it was demonstrated that this simplified Hct prediction method can effectively be used to implement a Hct-dependent correction factor to DBS-based results to alleviate the Hct bias.
血细胞比容（Hct）效应对定量干血斑（DBS）分析在常规环境中更广泛应用的阻碍最大。实际上，血细胞比容可能会影响 DBS 方法的准确性以及 DBS 结果的解释。我们之前开发了一种使用非接触漫反射光谱法根据血红蛋白含量确定 DBS 血细胞比容的方法。尽管分析可以轻松进行（即只需扫描 DBS），且获得了良好的结果，但该方法确实需要一个复杂的算法才能从 DBS 的反射光谱中得出总血红蛋白含量。由于总血红蛋白是由氧合血红蛋白、高铁血红蛋白和正铁血红蛋白这三种在 DBS 老化过程中形成的主要血红蛋白衍生物的总和计算得出的，因此需要对反射光谱进行解混以确定每种衍生物的数量。我们现在通过仅使用位于反射曲线准等色点的单个波长的反射率来简化该方法。在该波长下，假设老化反应的化学计量比为 1:1，反射率对血红蛋白降解不敏感，仅与总血红蛋白量成正比，因此与血细胞比容成正比。该简化方法得到了成功验证。在每个质量控制水平以及定量限（即 0.20 和 0.67）下，偏差、日内和日间精密度均在 10%以内。根据已分析样本的重复性测试，方法重复性非常好，超过了原始方法的重复性。此外，还评估了斑点体积、斑点内测量位置以及储存时间和温度的影响，结果表明这些参数没有产生相关影响。对 233 个患者样本的应用表明，全血中测定的血细胞比容与静脉 DBS 中预测的血细胞比容之间存在良好的相关性。Bland 和 Altman 分析得到的偏差为-0.015，一致性界限为-0.061 和 0.031，这表明简化的非接触式血细胞比容预测方法甚至优于原始方法。此外，使用咖啡因作为模型化合物，证明了这种简化的血细胞比容预测方法可以有效地用于实施基于 DBS 的结果的血细胞比容依赖性校正因子，以减轻血细胞比容偏差。
