Hematocrit, blood volume, and surface area of dried blood spots – a quantitative model

Abstract

The use of the dried blood spot (DBS) sampling technique has extended the scope of clinical research, particularly in children. The effects of different hematocrit levels (25–55%) and different blood volumes (7.5–30 μL) on the surface area of the blood spots were investigated using ImageJ® software. Variation in hematocrit levels between patients and inaccuracies in blood volumes applied to Guthrie cards can have a marked effect on analyte concentrations measured in DBS samples. The current study presents a validated model that links blood volume and hematocrit to the surface area of the blood spot. The final model showed that both factors affect the blood spot surface area, however, the positive effect of blood volume is higher than the negative effect of hematocrit. The measurement of surface area could be added as an additional quality control step in clinical studies that have adopted fixed volume DBS sampling for the quantification of the analytes. This approach can be used in estimating the hematocrit if this is not known for a patient or estimating the volume in spots that are visually different in size from the norm, i.e. technical error.

Analysis of six different homologues of phosphatidylethanol from dried blood spots using liquid chromatography–tandem mass spectrometry

“…The influence of Hct in DBS testing has been discussed in detail and is considered as one of the most important issues in this field 6,18,19 . In 2019 the International Association for Therapeutic Drug Monitoring and Clinical Toxicology published a review for the development and validation of DBS‐based methods 20 .…”

“…Whereas analytical procedures including one, 3,13 two, 14,15 The influence of Hct in DBS testing has been discussed in detail and is considered as one of the most important issues in this field. 6,18,19 In 2019 the International Association for Therapeutic Drug…”

“…In addition, the alternative preparation of DBSs directly from capillary blood could be a more convenient way (a prick on the fingertip instead of a puncture of a vein) of sampling in PEth, as it is less invasive and there is no need for a phlebotomist. One of the most challenging analytical issues in the context of DBSs is the hematocrit (Hct) effect: spot formation, drying time, spot homogeneity, and size are primarily influenced by blood Hct (in terms of blood viscosity); in addition, serious impact on analyte recovery, matrix effects (MEs), ruggedness, and reproducibility of the assay can occur 6 . The aim of the present work was to establish a validated liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for monitoring several PEth homologues simultaneously from (volumetrically defined) DBS samples, which is suitable for routine analysis and will further enable the investigation of the kinetics of PEth homologues (eg, formation, elimination, and interindividual differences) related to alcohol consumption in future studies.…”

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

“…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 52 . A more advanced application of non‐destructive HCT measurement during an anti‐doping study has been presented by Lange et al using NIR 53 .…”

“…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. 52 A more advanced application of non-destructive HCT measurement during an anti-doping study has been presented by Lange et al using NIR. 53 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.…”