Correction for the Hematocrit Bias in Dried Blood Spot Analysis Using a Nondestructive, Single-Wavelength Reflectance-Based Hematocrit Prediction Method

Abstract

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.

Dried blood spots for anti‐doping: Why just going volumetric may not be sufficient

“…More straightforwardly, strategies for the assessment of HCT post‐sampling from DBS can be established in a centralized laboratory. Thereby, the HCT assessment is either destructive (requiring the extraction of a DBS) 45,46 or non‐destructive (measuring the DBS's surface) 47–51 . As each collected sample is precious, the main focus of the HCT strategy should be on non‐destructive HCT determination.…”

“…However, they concluded that differences in temperature or humidity might influence the molecular vibrations of the DBS matrix, thus negatively influencing the outcome of the used DBS NIR spectrometry measurement. The use of reflectance‐based DBS HCT measurement does not have this limitation 48,49 . An application of fully automated DBS analysis with reflectance‐based HCT correction has been realized (unpublished).…”

“…Thereby, the HCT assessment is either destructive (requiring the extraction of a DBS) 45,46 or non-destructive (measuring the DBS's surface). [47][48][49][50][51] As each collected sample is precious, the main focus of the HCT strategy should be on non-destructive HCT determination. A simple option to estimate the HCT is to correlate the scanned surface area of the spot in combination to the known applied volume of whole blood.…”

“…The use of reflectance-based DBS HCT measurement does not have this limitation. 48,49 An application of fully automated DBS analysis with reflectance-based HCT correction has been realized (unpublished).…”

“…After the careful assessment of the DBS hematocrit bias during the analytical process, correction and compensation models can be applied if necessary. 46,48 These models should be subject independent, reliable, and scientifically justified.…”