Журнал аналитических и биоаналитических методов

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An On-card Approach for Assessment of Hematocrit on Dried Blood Spots which Allows for Correction of Sample Volume

John H Miller IV, Philip A Poston, Sarah C Rutan and Thomas Karnes H

One of the biggest concerns with quantitative analysis of dried blood spots (DBS) is the uncertainty related to sample volume from a predefined punch. Blood with a high hematocrit will not diffuse as far as blood with a lower hematocrit when spotted on filter paper, resulting in differences in sample volume per unit area. Quantifying the hematocrit of blood would allow for correction of the sample volume, improving the quantitation in DBS analyses. Therefore, we have developed a novel approach using reflectance to measure hematocrit that will allow for sample volume correction. DBSs prepared at various levels of hematocrit from 25% to 75% were analyzed by UV-visible reflectance from 210 nm to 1000 nm using an Ocean Optics reflectance fiber optic probe. A linear correlation between the percent reflectance at 980 nm and hematocrit was found, with a correlation coefficient (r) of 0.9933. Quantitative analysis of sample volume was conducted by fortification of samples with known concentrations of acylcarnitines (Free carnitine, C2, C3, C4, C5, C6, C8, C10, C12, C14, C16 and C18). Analysis was carried out using LC/MS/MS on a Waters Premier triple quadrupole mass spectrometer. Hematocrit and sample volume showed a linear relationship with a correlation coefficient (r) of 0.9929. We validated our model using donor samples at various levels of hematocrit by first analyzing the samples by NIR reflectance to determine hematocrit and then correcting for sample volume. Our results showed improved quantitation of various donor samples with accuracies between 81% and 115% compared to not correcting for sample volume, which showed accuracies from 67% to 136%. This is the first on-card approach used to determine sample hematocrit allowing for improved quantitation by correcting sample volume. In addition, the reflectance format used allows for direct, automatable volume corrections on the card, without the need for off-line procedures to test whole blood.