Managing The Effect of Hematocrit on Dbs Analysis in A Regulated Environment
2012; Future Science Ltd; Volume: 4; Issue: 4 Linguagem: Inglês
10.4155/bio.11.337
ISSN1757-6199
Autores Tópico(s)Clinical Laboratory Practices and Quality Control
ResumoBioanalysisVol. 4, No. 4 EditorialFree AccessManaging the effect of hematocrit on DBS analysis in a regulated environmentLeimin Fan & Jacob A LeeLeimin FanAbbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA. & Jacob A LeeAbbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA Published Online:7 Mar 2012https://doi.org/10.4155/bio.11.337AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: hematocrit effectpaper chromatographypartial spot analysisspreadwhole spot analysisThe majority of preclinical and clinical pharmacokinetic studies in pharmaceutical development are performed under Good Laboratory Practice regulations, ensuring the accuracy, reproducibility and integrity of analytical results. In partial cut dried blood spot (DBS) methods, blood hematocrit is a known variable that can significantly undermine data quality and reliability. Of late, there have been numerous discussions on DBS methodology assessing the sizable impact of blood hematocrit. The recent recommendation from the European Bioanalysis Forum on DBS states, 'Hematocrit is currently identified as the single most important parameter influencing the spread of blood on DBS cards, which could impact the validity of the results generated by DBS methods[1].' Hematocrit effect is clearly a major hurdle to the success of any DBS method and attempting to ignore or avoid it is not an option in a regulated environment.The basic assumption in all DBS partial cut methods is that by utilizing a standard sized punch, all spots are equal. Because the paper disc punched out from each blood spot is of the same size for all samples, the volume of blood sample contained in each paper disc is assumed to be constant. There are, however, many factors that can influence the results obtained from DBS analyses, both positively and negatively impacting concentrations. Thus, the above assumption is not always true.When a drop of blood touches filter paper during the spotting process, the blood travels down through the paper as it soaks (saturates) the thickness of the paper, while simultaneously spreading radially to form a spot. The viscosity of the blood controls how far the blood is able to spread on the filter paper. The viscosity, in turn, is determined by the hematocrit value of the blood sample, which is the percent volume ratio of red blood cells in the blood sample. Denniff et al. reported a linear, inverse relationship between spot area and blood hematocrit [2]. Thus, at a given volume of blood, a higher hematocrit value yields a smaller DBS, while a lower hematocrit value yields a larger DBS. When a fixed-size paper disc is punched from DBS of different size, a disc punched from a smaller spot (e.g., a high hematocrit sample) contains more blood than one punched from a larger spot.Further complicating the matter is the chromatographic effect that occurs during spotting. Chromatographic effect changes the way an analyte moves in the filter paper, with paper acting as the stationary phase and blood acting as the mobile phase. Depending on the structure of a given analyte, chromatographic effect can drive the analyte to the edge of the spot, or concentrate it in the center of the spot. O'Mara et al. showed variations of DBS analysis results between a center punch and a perimeter punch for five different compounds [3]. The mean perimeter-to -enter concentration ratio (P/C ratio) of each compound is not equal to one. The P/C ratio also varies depending on the hematocrit. For the highest hematocrit, the spot is the most homogeneous (the P/C ratio is closest to 1). The issue of hematocrit effect can drastically complicate DBS analysis.The possible approaches for assessing and reducing/eliminating hematocrit effect are listed below.Validate an analytical method with the analyte spiked into blood covering a range of hematocrit valuesAssessing the effect of hematocrit over a range of normal hematocrit values for the matrix of interest [2] during method development and validation is common practice [4]. The typical acceptance criterion is a bias of <15% for quality control samples within the tested hematocrit range. Although this approach seems reasonable, one shortcoming is that the validated hematocrit range may not sufficiently cover all study samples. Some diseases can push a patient's hematocrit out of the normal range, and a patient may also experience induced anemia after a period of exposure to certain drugs. In addition, the individual concentration values of study samples may all be biased relative to the hematocrit of the blood used to prepare the standard curve, albeit within the established 15%.Correct bioanalysis results using each subject's hematocritAs in the previous approach, a method must be validated across a broad range of hematocrit values, but correction factors must be established for each hematocrit value relative to the hematocrit of the blood used to prepare the standard curve. The hematocrit value for each study subject is determined and used to correct concentration values for that subject. While this may improve DBS analysis reliability, to do so would require the collection and management of a large dataset of hematocrit values and the correction of each study sample concentration value.Abandon partial cut DBS & analyze whole DBSIn my opinion, the best way to solve the hematocrit problem is through whole spot analysis. Bias from the hematocrit effect is mostly caused by sample spreading on the filter paper. Partial sampling methods introduce unwanted bias owing to inconsistent spot sizes and possibly nonhomogeneous spots due to chromatographic effect. Whole spot methods eliminate the variation from spreading and nonhomogeneity. They allow for more consistent DBS concentrations, even at different hematocrit levels [5–7]. In this approach, however, the most critical parameter is the accuracy of spotting volume. This complicates matters because study personnel, animal care technicians and nurses, who may not be accustomed to the laboratory technique necessary for such accurate liquid transfer, are responsible for spotting blood samples. While this is an issue with whole spot methods, it can be resolved by equipping all study personnel involved in sample collection with additional training, along with improved tools and instruments. As Neil Spooner wrote in a Linkedin® discussion, "The main issue we need to solve as a community is to get an affordable and simple device for use by relatively inexperienced personnel for accurately collecting the small volumes of blood we require for DBS sampling" [101]. There are products being actively developed to address this very problem: Drummond Scientific Co. Inc. (Broomall, PA, USA) is developing a hand-held pipette that can deliver multiple small volumes of liquid both easily and accurately [102]. The capillary spotting method, which will be described later, is another alternative.Whole spot DBS analysis has evolved from cutting the entire spot from DBS paper to the use of two new techniques: perforated DBS [5] and precut DBS [6]. Perforated DBS methods predefine an area on a DBS card and limit the blood to this area. The entire perforated disc is removed from the card using a punching tool during analysis. In precut DBS methods, discs of a particular size are precut from filter paper and blood sample is then spotted directly onto the precut paper disc. Youhnovski reported a precut DBS method in which a 6.1-mm paper disc is cut from FTA Classic or DMPK-C filter paper and stored on two-sided tape [6].At our laboratory, we have developed a more sophisticated storage device for precut paper discs, coined dried matrix in paper disc (DMPD) cartridges. The cartridges are made of plastic and are designed to hold six precut paper discs. Precut paper discs are loaded in the DMPD cartridges and blood samples can be neatly spotted directly into the cartridges with a pipettor. An innovative method was implemented to aid sample spotting, using a customized capillary tube supplied by Drummond Scientific. Approximately 5 µl of blood is aspirated by a marked capillary tube. When the capillary tube is placed in the sample loading port of a DMPD cartridge, the blood flows from the capillary to the paper disc until it is saturated. The tube's capillary force prevents excess blood in the capillary from wicking onto the spot. This method greatly simplifies the sample spotting process, eliminating the worry of inaccurate volume transfer. In our laboratory, we observed that this technique reduced the effect of hematocrit and have already implemented DMPD cartridges in several toxicokinetic studies, including a few mouse serial sampling studies and a juvenile rat study. Clinical studies utilizing the DMPD cartridges and capillary spotting method are scheduled to begin in early 2012.Understanding the effect of hematocrit may determine the future of DBS methods used in regulated bioanalytical work. It is a complicated phenomenon that is influenced by intrinsic blood and paper substrate properties. Without addressing the effect of hematocrit, DBS methods will struggle to gain acceptance as valid alternatives to traditional liquid sample methods. However, the effect of hematocrit on DBS analysis can be overcome through various means, from more rigorous measures taken during method validation and sample analysis, to utilizing whole spot methods. The progress that has been made in whole spot analysis leads me to believe that the hematocrit problem will soon be resolved, enabling DBS methods to be deemed acceptable for pharmaceutical development by regulatory agencies.AcknowledgementsThe authors acknowledge help from Jeffrey Hall for proof reading of this manuscript.Financial & competing interests disclosureThe authors disclose employment by Abbott Laboratories where the dried matrix in paper disc cartridge mentioned within was invented. A member of Leimin Fan's family is involved in distributing dried matrix in paper disc products. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.References1 Timmerman P, White S, Globig S et al. EBF recommendation on the validation of bioanalytical methods for dried blood spots. Bioanalysis3(14),1567–1575 (2011).Link, CAS, Google Scholar2 Denniff P, Spooner N. The effect of hematocrit on assay bias when using DBS samples for the quantitative bioanalysis of drugs. Bioanalysis2(8),1385–1395 (2010).Link, CAS, Google Scholar3 O'Mara M, Hudson-Curtis B, Olson K et al. The effect of hematocrit and punch location on assay bias during quantitative bioanalysis of dried blood spot samples. Bioanalysis3(20),2335–2347 (2011).Link, Google Scholar4 Reddy TM, Tama CI, Hayes RN. A dried blood spots technique based LC–MS/MS method for the analysis of posaconazole in human whole blood samples. J. Chromatogr. B.879(30),3626–3638 (2011).Crossref, Medline, CAS, Google Scholar5 Li F, Zulkoski J, Fast D et al. Perforated dried blood spots: a novel format for accurate microsampling. Bioanalysis3(20),2321–2333 (2011).Link, CAS, Google Scholar6 Youhnovski N, Bergeron A, Furtado M et al. Pre-cut dried blood spot (PCDBS): an alternative to dried blood spot (DBS) technique to overcome hematocrit impact. Rapid Commun. Mass Spectrom.25,2951–2958 (2011).Crossref, Medline, CAS, Google Scholar7 Dawes M, Liu G, Schuster A et al. Investigation of the hematocrit effect on bioanalytical assay performance using fixed volume and fixed punch size dried blood spot samples. Presented at: DIA Dried Blood Spot Conference. Philadelphia, PA, USA, 4–5 May 2011.Google Scholar101 Dried blood spots (DBS) analysis. www.linkedin.com/groupItem?view=&gid=2875014&type=member&item=71027719Google Scholar102 Drumond Scientific Co., Inc. www.drummondsci.comGoogle ScholarFiguresReferencesRelatedDetailsCited ByNew perspectives for the therapeutic drug monitoring of tacrolimus: Quantification in volumetric DBS based on an automated extraction and LC-MS/MS analysisJournal of Chromatography B, Vol. 1223Fully soluble polymeric foams for in-vial dried blood spot collection and analysis of acidic drugs by capillary electrophoresisAnalytica Chimica Acta, Vol. 1241Development of an LC-MS/MS method to simultaneously quantify therapeutic mAbs and estimate hematocrit values in dried blood spot samplesAnalytica Chimica Acta, Vol. 1189Review of the Preanalytical Errors That Impact Therapeutic Drug MonitoringTherapeutic Drug Monitoring, Vol. 43, No. 5Toward proteome‐wide exploration of proteins in dried blood spots using liquid 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LtdKeywordshematocrit effectpaper chromatographypartial spot analysisspreadwhole spot analysisAcknowledgementsThe authors acknowledge help from Jeffrey Hall for proof reading of this manuscript.Financial & competing interests disclosureThe authors disclose employment by Abbott Laboratories where the dried matrix in paper disc cartridge mentioned within was invented. A member of Leimin Fan's family is involved in distributing dried matrix in paper disc products. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.PDF download
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