Strategies to overcome hematocrit and volume bias in dried blood spot analysis

Mingas, Panagiotis-Dimitrios (author), Cirar, Matjaž (author), Grabnar, Iztok (author), Drobne, David (author), Vovk, Tomaž (author)

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 (r$^2$> 0.994), with acceptable accuracy (90.3–102.2%) and precision (<7.4%). Moreover, the conductivity method was linear (r$^2$ = 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.

• Language: English
• Keywords: dried blood spots, DBS, microsampling, blood volume, hematocrit, image analysis, conductivity, spectrometry, bias
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FFA - Faculty of Pharmacy; MF - Faculty of Medicine
• Publication status: Published
• Publication version: Version of Record
• Year: 2026
• Number of pages: 17 str.
• Numbering: Vol. 19, iss. 3, art. 403
• PID: 20.500.12556/RUL-180124
• UDC: 615.38
• ISSN on article: 1424-8247
• DOI: 10.3390/ph19030403
• COBISS.SI-ID: 270022147
• Publication date in RUL: 03.03.2026

Record is a part of a journal

• Title: Pharmaceuticals
• Shortened title: Pharmaceuticals
• Publisher: MDPI, Molecular Diversity Preservation International
• ISSN: 1424-8247
• COBISS.SI-ID: 517582617

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: posušene krvne kapljice, DBS, mikrovzorčenje, volumen krvi, hematokrit, analiza slike, prevodnost, spektrometrija, pristranskost, krvne komponente, terapevtska uporaba

Projects

• Funder: ARIS - Slovenian Research and Innovation Agency
• Project number: P1-0189
• Name: Farmacevtska tehnologija: od dostavnih sistemov učinkovin do terapijskih izidov zdravil pri otrocih in starostnikih

• Funder: ARIS - Slovenian Research and Innovation Agency
• Project number: J3-4499
• Name: MEHANIZMI ODZIVA NA IL 12/23 ZAVIRALCE PRI ULCEROZNEM KOLITISU - NAPROTI PERSONALIZIRANI MEDICINI