As the spreading area of blood onto a filter paper membrane is dependent on the volume percentage of the red blood cells (hematocrit (HCT)), punching DBS using a fixed area is associated with a certain error (hematocrit effect). To compensate for this effect, different strategies evolved, focusing on obtaining a fixed sample volume. Using these devices, a quantitative analysis is performed, referencing the known sample volume. Although the mentioned devices are widely used, they are expensive, sometimes complicated to handle, and not globally registered as a medical device. Furthermore, the sample’s HCT and the HCT impact on the analyte (e.g. plasma to red blood cell analyte distribution ratio) are generally not assessed.
By determining the sample’s HCT and knowing the HCT dependency of a specific analyte within a DBS sample, it is possible to correct for the hematocrit effect without using volumetric sampling devices. This way, cheap and readily available standard filter paper DBS cards can be used for accurate DBS sub-spot analysis strategies over a wide HCT range. Correcting for the HCT effect is especially valuable for the accurate quantification of analytes that show large differences in the distribution between the cellular and the plasma fraction. The strategies to determine the HCT from the DBS may be either invasive (destroy the spot) or non-invasive (measure the spot surface).
To measure the HCT, CAMAG developed a novel, fully automated HCT correction module, which permits correct all HCT related effects.
The HCT analysis process in detail
The determination of the HCT of a DBS in the CAMAG DBS-MS 500 HCT relies on the reflectance measurement of hemoglobin and its derivatives (Oxyhemoglobin etc.) at a specific wavelength. The system is composed of a low-voltage fiber-coupled LED light source that generates the excitation light, with a nominal wavelength at 590 nm. The light is transmitted towards the DBS illumination fibers, and the reflected light is transmitted to a spectrometer by a read fiber, located in the middle of the probe. The end of the fiber optics probe for the HCT reflectance is automatically set to the center of the DBS by the image processing module. The z-position is corrected by a laser distance sensor. The distance measurement is accurate to ±0.1 mm, to ensure the same distance for every measurement. The reflected light is analyzed in a mini-spectrometer.
A measurement is realized by first performing a background (BG) measurement. For the BG measurement, a predefined card position is chosen, where blank filter paper is present. Then the predefined distance of the probe tip to the spot is adjusted using the z-drive in combination with the laser distance measurement. Afterward, the target spot is centered below the probe. The signal processing, from the reflectance spectra to a single value for the HCT, is carried out using a specific algorithm, which processes the BG spectral information and the actual measurement on the DBS (RAW). The resulting value can then be set into relation to the HCT or further processed.
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