Clin Chem Lab Med 2019; aop Rosita Zakaria, Katrina J. Allen, Jennifer J. Koplin, Nick Crinis, Lidia De Rosa, Peter Roche and Ronda F. Greaves* Determination of haemoglobin derivatives in aged dried blood spot to estimate haematocrit https://doi.org/10.1515/cclm-2018-0753 Received July 18, 2018; accepted December 4, 2018 Abstract Introduction: Dried blood spot (DBS) sample applications now encompass analytes related to clinical diagnosis, epi- demiological studies, therapeutic drug monitoring, phar- macokinetic and toxicokinetic studies. Haematocrit (Hct) and haemoglobin (Hb) at very high or low concentrations may influence the accuracy of measurement quantifica- tion of the DBS sample. In this study, we aimed to predict the Hct of the punched DBS through primary spectropho- tometric 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 individu- al’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 develop- ment 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 posi- tive correlation (r = 0.88) between Hb-drv and the original whole blood Hct. The Bland-Altman difference plot dem- onstrates a mean difference of 2.4% between the calcu- lated 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. Keywords: dried blood spot; haematocrit correction; hae- moglobin; spectrophotometry. Introduction The micro-sampling technique associated with dried blood spot (DBS) samples is considered to be less inva- sive, requiring only a small sample volume (usually capillary) applied to filter paper, that is easy to trans- port and store with a lower risk of infection transmis- sion [1–3]. These advantages have prompted the interest in utilising DBS for clinical assessment outside of newborn screening (NBS). As such, DBS applications now encompass analytes related to clinical diagnosis, epidemiological studies, therapeutic drug monitor- ing, pharmacokinetics and toxicokinetic studies [4]. However, the accuracy of DBS quantitation requires additional considerations over the conventional “liquid” blood sample. This is due to the physical char- acteristics of the blotted whole blood sample affecting the quality of the measurement. In particular, potential irregular errors associated with haematocrit (Hct) span the DBS total testing process: Pre-analytically, the levels of a patient’s haemoglobin (Hb), and therefore Hct, at extreme high or low ends may influence the vis- cosity and fluidity of the spreading sample [4–7]. Hence, there *Corresponding author: Assoc. Prof. Ronda F. Greaves, Victorian Clinical Genetics Services, Murdoch Children’s Research Institute, Flemington Rd, Parkville, Victoria 3052, Australia, Phone: +61 (0)3 8341 6409, E-mail: ronda.greaves@vcgs.org.au; and School of Health and Biomedical Sciences, RMIT University, Bundoora, Victoria, Australia Rosita Zakaria: School of Health and Biomedical Sciences, RMIT University, Bundoora, Victoria, Australia; and Murdoch Children’s Research Institute, Parkville, Victoria, Australia Katrina J. Allen: Murdoch Children’s Research Institute, Parkville, Victoria, Australia; Department of Paediatrics, University of Melbourne, Parkville, Victoria, Australia; and Department of Allergy and Clinical Immunology, Royal Children’s Hospital, Parkville, Victoria, Australia Jennifer J. Koplin: Murdoch Children’s Research Institute, Parkville, Victoria, Australia Nick Crinis: Clinical Biochemistry, Austin Pathology, Heidelberg, Victoria, Australia Lidia De Rosa: Core Laboratory, Pathology, The Royal Children’s Hospital, Parkville, Victoria, Australia Peter Roche: School of Health and Biomedical Sciences, RMIT University, Bundoora, Victoria, Australia Brought to you by | Iowa State University Authenticated Download Date | 1/29/19 9:37 AM