A Novel, Nondestructive, Dried Blood Spot-Based Hematocrit Prediction Method Using Noncontact Diffuse Reflectance Spectroscopy

Abstract

Dried blood spot (DBS) sampling is recognized as a valuable alternative sampling strategy both in research and in clinical routine. Although many advantages are associated with DBS sampling, its more widespread use is hampered by several issues, of which the hematocrit effect on DBS-based quantitation remains undoubtedly the most widely discussed one. Previously, we developed a method to derive the approximate hematocrit from a nonvolumetrically applied DBS based on its potassium content. Although this method yielded good results and was straightforward to perform, it was also destructive and required sample preparation. Therefore, we now developed a nondestructive method which allows to predict the hematocrit of a DBS based on its hemoglobin content, measured via noncontact diffuse reflectance spectroscopy. The developed method was thoroughly validated. A linear calibration curve was established after log/log transformation. The bias, intraday and interday imprecision of quality controls at three hematocrit levels and at the lower and upper limit of quantitation (0.20 and 0.67, respectively) were less than 11%. In addition, the influence of storage and the volume spotted was evaluated, as well as DBS homogeneity. Application of the method to venous DBSs prepared from whole blood patient samples (n = 233) revealed a good correlation between the actual and the predicted hematocrit. Limits of agreement obtained after Bland and Altman analysis were -0.076 and +0.018. Incurred sample reanalysis demonstrated good method reproducibility. In conclusion, mere scanning of a DBS suffices to derive its approximate hematocrit, one of the most important variables in DBS analysis.

Improved Dried Blood Spot-Based Metabolomics: A Targeted, Broad-Spectrum, Single-Injection Method

“…This was true if 60 µL, 70 µL, or 80 µL of blood was applied. The hematocrit effect is another potential obstacle to DBS application [26]. Our results showed that hematocrits between 31% and 50% did not play a significant role as a source of variation in broad-spectrum metabolomics results ( Figure S6 and Table S12).…”

Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide and non-peptide doping agents by means of LC-HRMS

“…desiccant, the differences in temperature or humidity might influence the molecular vibrations of the DBS matrix that are crucial for the NIR spectrum calculation. A time-dependent change in total hemoglobin was not assumed as others have already shown its stability in DBS[36]. A considerably slower drying or freezing of remaining moisture of the DBS matrix at reduced temperatures would be in accordance with this observation, suggesting a prolonged drying…”

Dried blood spots in therapeutic drug monitoring and toxicology

“…Third, automated processing of DBS can be made fully compatible with fully automated LC-MS/MS workstations that are about to enter clinical laboratories, allowing 'from card to report, with no manual intervention'. These systems may be equipped with tools for automated recognition of correctly deposited DBS and for coping with variables like hct, for example, via the integration of reflectance spectroscopy for hct prediction [11]. This tailoring to current (and future) workflows of routine clinical laboratories, focusing on highthroughput, is essential for more widespread implementation [12].…”

“…In recent years, many efforts have been made to facilitate correct dried blood sampling, to improve DBS bioanalysis and to cope with challenges related to DBS analysis. New sampling devices are entering the market, automated analysis leads to a higher efficiency, sample throughput and reproducibility and different strategies to cope with the hct effect have been put forward [10,11]. Innovations include whole-spot analysis of volumetrically applied DBS, development of alternative dried blood sampling devices, introduction of special types of filter paper, as well as cards for generating dried plasma spots, and setup of approaches for hct prediction of DBS, allowing the correction of hct-skewed results [11,12].…”