Christophe Stove (Presenter)
Ghent University
Bio: Since February 2014 I have taken up a tenure-track position as a assistant-professor in Toxicology at the Laboratory of Toxicology at the Faculty of Pharmacy, Ghent University, Belgium. From October 1st 2014 on, I have taken over the lead of this Laboratory. One of my current research focuses -with different research lines running- is the development of alternative strategies for the GC-MS and LC-MS/MS-based measurement of endogenous molecules and drugs (of abuse) in biological matrices, with particular interest for the use of microsampling and dried blood spots for therapeutic drug monitoring and for toxicology purposes. I graduated as a pharmacist in 1999 and obtained my PhD at the Faculty of Medicine of Ghent University, being active in the field of fundamental cancer research, studying the role of growth factors and cell adhesion molecules in melanoma and breast cancer. This work wa
Authorship: Sara Capiau, Leah Wilk, Maurice Aalders, Christophe Stove
Laboratory of Toxicology, Faculty of Pharmaceutical Sciences, Ghent University (Belgium) and Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam (The Netherlands)
| Short Abstract The hematocrit (Hct) effect is generally considered as one of the most crucial issues in dried blood spot (DBS) based quantitation, as it is known to impact the accuracy of the results and hence, correct interpretation. As an improvement to a previously developed Hct prediction method, based upon the potassium content of a DBS extract, we now developed non-contact methods that enable Hct prediction in mere seconds, allowing complete sample preservation. The methods are based on the hemoglobin content of DBS, measured via noncontact diffuse reflectance spectroscopy, either by using the reflectance spectrum between 500 and 700 nm or by using reflectance at a single wavelength (589 nm). The methods were thoroughly validated, used to predict the Hct from DBS and applied to correct for the Hct-induced bias seen in DBS-based LC-MS/MS analysis of two model compounds, caffeine and paraxanthine. |
Long Abstract
INTRODUCTION:
The hematocrit (Hct) effect is generally considered as one of the most crucial issues in dried blood spot (DBS) based quantitation, as it is known to impact the accuracy of the results and hence, correct interpretation. Recently, our group demonstrated that the Hct of a DBS can be predicted based on the potassium content of a DBS extract [1]. Moreover, a correction algorithm was set up that allows to correct for the hematocrit bias in the quantitative analysis of caffeine and paraxanthine, based on the potassium content of the DBS [2]. Unfortunately, this potassium-based method suffered from some practical drawbacks, as it was destructive and required an additional analysis. Therefore, we now developed two non-contact methods that enable Hct prediction in mere seconds. This way the entire sample is preserved and the regular sample work-up is barely disturbed.
METHODS:
The developed nondestructive methods are based on the hemoglobin content of DBS, which can be measured via noncontact diffuse reflectance spectroscopy. The principle is based on the fact that this technology allows to distinguish between the different hemoglobin forms (oxyhemoglobin, methemoglobin and hemichrome) that are formed during ageing of a DBS and whereby the total amount of measured hemoglobin remains constant over time. More specifically, a first method encompassed the application of an algorithm that was applied on the light reflectance in the spectral range of 500 – 700 nm [3]. In a second, further simplified method, only the reflectance at one wavelength (589 nm, corresponding to a quasi-isosbestic point) was used to predict the Hct. Both methods were thoroughly validated.
RESULTS:
Linear calibration curves were set up and the bias, intraday and interday imprecision of quality controls at three hematocrit levels and at the lower and upper limit of quantitation (0.20 and 0.67, respectively) met acceptance criteria (15%). In addition, the influence of storage and the volume spotted was evaluated, as well as DBS homogeneity. Application of the method to venous DBSs prepared from whole blood samples from over 200 patients revealed a good correlation between the actual and the predicted hematocrit, the single-wavelength method even yielding somewhat better results. Acceptable limits of agreement were obtained after Bland and Altman analysis and incurred sample reanalysis demonstrated good method reproducibility. Lastly, the reflectance-based Hct prediction was successfully used to correct for the Hct-induced bias seen in DBS-based LC-MS/MS analysis of two model compounds, caffeine and paraxanthine.
CONCLUSION:
Mere scanning of a DBS suffices to derive its approximate hematocrit, one of the most important variables in DBS analysis.
References & Acknowledgements:
[1] Capiau S, Stove VV, Lambert WE, Stove CP. Prediction of the hematocrit of dried blood spots via potassium measurement on a routine clinical chemistry analyzer. Anal Chem. 2013, 85(1):404-10.
[2] De Kesel PM, Capiau S, Stove VV, Lambert WE, Stove CP. Potassium-based algorithm allows correction for the hematocrit bias in quantitative analysis of caffeine and its major metabolite in dried blood spots. Anal Bioanal Chem. 2014, 406(26):6749-55.
[3] Capiau S, Wilk LS, Aalders MC, Stove CP. A Novel, Nondestructive, Dried Blood Spot-Based Hematocrit Prediction Method Using Noncontact Diffuse Reflectance Spectroscopy. Anal Chem. 2016, 88(12):6538-46.
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