# Strategies to Overcome Hematocrit and Volume Bias in Dried Blood Spot Analysis

## TL;DR

This paper introduces methods to correct biases in dried blood spot analysis caused by hematocrit and blood volume differences, improving accuracy in drug and biomarker measurements.

## Contribution

The study proposes and validates new strategies using image analysis, hemoglobin quantification, and conductivity to correct hematocrit and volume biases in DBS samples.

## Key findings

- Image analysis and hemoglobin measurements accurately estimated hematocrit with high linearity and precision.
- Conductivity measurements provided precise and accurate blood volume determination in DBS samples.
- Correction strategies for HCT- and volume-related biases were validated using patient samples.

## Abstract

Background/Objectives: Dried blood spot (DBS) sampling, a technique for collecting capillary blood samples, is widely used in therapeutic drug monitoring, pharmacokinetic and toxicology research, newborn screening, and population health because it enables simple, non-invasive sampling across large cohorts. However, it presents several challenges, mainly due to the effect of hematocrit (HCT), which can influence the quantification of analytes. Methods: A combination of methods was developed to estimate the HCT and blood volume in DBS samples. Image analysis and hemoglobin (Hb) quantification using UV-VIS spectrometry were used for HCT estimation, and conductivity was used to determine blood volume. DBS samples from five donors were prepared with HCT between 0.2 and 0.6 and were used to prepare calibrators and quality control samples. The developed methods were applied to 23 samples obtained from ten adult patients with inflammatory bowel disease. Results: The methods for HCT determination using image analysis or Hb measurements were linear (r2 > 0.994), with acceptable accuracy (90.3–102.2%) and precision (<7.4%). Moreover, the conductivity method was linear (r2 = 0.999) and enabled accurate (96.8–100%) and precise (<5.65%) determination of blood volume in DBS samples. All three methods were in good agreement with the reference values in patient samples. Finally, strategies to correct HCT- and volume-related bias in DBS samples were proposed for analytes with different blood cell-to-plasma partition coefficients. Conclusions: We accurately and precisely estimated HCT in DBS samples using image analysis and Hb determination, and the volume of blood in DBS using conductivity measurement. We evaluated different approaches and derived an optimal procedure for HCT-bias correction.