Abstract
Introduction
Dried blood spot (DBS) sample applications now encompass analytes related to clinical diagnosis, epidemiological studies, therapeutic drug monitoring, pharmacokinetic and toxicokinetic studies. Haematocrit (Hct) and haemoglobin (Hb) at very high or low concentrations may influence the accuracy of measurement quantification of the DBS sample. In this study, we aimed to predict the Hct of the punched DBS through primary spectrophotometric estimation of its haemoglobin-derivative (Hb-drv) content.
Methods
Formic acid solution was used to elute Hb-drv content of 3.2 mm spotted blood from its dry matrix. Direct spectrometry measurement was utilised to scan the extracted Hb-drv in the visible spectrum range of 520–600 nm. The linear relationship between an individual’s Hct percentage and Hb-drv concentration was applied to estimate the Hct level of the blood spot. De-identified whole blood samples were used for the method development and evaluation studies.
Results
The Hb-drv estimation is valid in samples >2 months old. Method validation experiments DBS demonstrate linearity between 82.5 and 207.5 g/L, average coefficient of variation of 3.6% (intra-assay) and 7.7% (inter-assay), analytical recovery of 84%, and a high positive correlation (r=0.88) between Hb-drv and the original whole blood Hct. The Bland-Altman difference plot demonstrates a mean difference of 2.4% between the calculated DBS Hct and the directly measured Hct from fresh whole bloods.
Conclusions
We have successfully developed a simple Hb-drv method to estimate Hct in aged DBS samples. This method can be incorporated into DBS analytical work-flow for the in-situ estimation of Hct and subsequent correction of the analyte of interest as required.
Author contributions: Ronda Greaves conceived the strategy to correct for Hct variation in the DBS samples. Rosita Zakaria performed the studies as part of her PhD project and wrote the first draft of the manuscript. Katrina J. Allen, Jennifer J. Koplin, Ronda Greaves and Peter Roche supervised this project as part of Rosita Zakaria’s PhD candidature. Ronda Greaves and Rosita Zakaria together with input from Nick Crinis developed the protocol for the de-identified patient sample retrieval, and Lidia De Rosa and Rosita Zakaria selected the de-identified samples for inclusion in the validation study. All authors contributed to the writing of the subsequent drafts, reviewed, edited, and approved the final manuscript.
Research funding: NHMRC – Centre of Excellence in Paediatric Food Allergy and Food-related Immune Disorders – Funder Id: 10.13039/501100000925, Grant ID 1041420.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
Financial disclosure: Nothing to disclose.
Guarantor: Ronda Greaves.
Appendix
Hb is an oxygen transport protein in the form of Hb-O2. As soon as fresh whole blood is air exposed outside the body, Hb is completely oxygen saturated and converts into HbO2. Over time, Hb-O2 is auto-oxidised to met-Hb. Met-Hb eventually denatures into HC (known as the most stable Hb derivative) [34]. HC is a common name for group of low spin forms of the ferri-Hb (met-Hb) constituting ferri-alpha/beta subunits. The distinct feature of the met-Hb is its six liganded state of the heme iron that formed through the separate alterations of the globin conformation so that atoms endogenous to protein became bound as a sixth ligand of the heme iron. The abundant majority of HC have a proximal (F8) and a distal (E7) His as fifth and sixth ligands, respectively [39], [40]. Following our observations using direct spectrometry method after week 8 HC is measured
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