2023 ISSN 1757-6180 10.4155/BIO.13.156 2013 Future Science Ltd Bioanalysis (2013) 5(16), 20232041REVI EWBackgroundDriedbloodspot(DBS)samplingisamicro-samplingtechnique,inwhichadropofcapil-lary blood, derived from a nger or heel stick, is collected on special lter paper. This approach has been used successfully in newborn screening since it was introduced in the 1960s by Guthrie todeterminephenylketonuriainneonates[1]. During the last decade, thanks to the develop-mentofmoresensitiveanalyticaltechniques, DBSsamplinghasgaineditsplaceinalotof other elds, such as preclinical and clinical stud-ies,epidemiologicalresearch,toxicologyand therapeutic drug monitoring [25].The increased use of DBS sampling is the logi-cal consequence of the many advantages associ-atedwiththissamplingtechnique,whichare summarized in BOX 1A. Indeed, DBS sampling is a very easy and inexpensive way of taking a rep-resentative sample, which can even be performed by the patient himself in his home-environment, eliminating the need for a trained phlebotomist [68].Moreover,thedriedmatriximprovesthe stability of most compounds [912], enabling more cost-effective transport and storage; DBS samples aregenerallytransportedviaregularmail[8,13] and can often be stored at ambient temperature for prolonged periods of time. Since only small volumes of blood, typically between 10 and 80 l,arecollected,theDBSsamplingtechnique ishighlysuitableforthecollectionofsamples during preclinical and TK studies involving ani-mals.DBSsamplingcertainlyconnestothe principlesofthe3Rs(replacement,reduction andrenement),asfeweranimalsareneeded and sampling procedures are rened using DBS [14,15]. In addition, the advantage of small blood volumes,combinedwiththepossibilitytocol-lectsamplesbyaminimallyinvasivengeror heel prick, creates a high potential for the use of DBS in pediatric studies as well [16,17]. Another benetofusingDBSinsteadofliquidsamples isthatpathogensthatmaybepresentinblood aredeactivatedupondrying,stronglyreducing theriskofinfection[18,201].Furthermore,from the analytical point of view, the process of gen-erating DBS can also be considered as a simple samplepreparationprocedure[19,20].Indeed, many analytes can selectively be extracted from thecards,oftenallowingstraightforwardand automatableprocessingandana lysis[2126].In a typical workow for DBS ana lysis, a 36 mm diameter disc is punched from the DBS, followed by extraction of the disc with a mixture of water and organic solvent and injection of the (diluted) extractintotheanalyticalsystem,typicallyan LCMS/MSconguration.Mostoften,isoto-picallylabeledanaloguesareusedasIS,which areeitheraddedtotheextractionsolvent(the most used approach) or to the DBS punch [27], although other approaches have been described as well, for example, spraying [28,29] or application to the DBS card prior to spotting [28,30,31].Despite the numerous applications using DBS andthemanyadvantagesassociatedwiththis samplingprocedure,DBSsamplingstillfaces someimportantchallenges,assummarizedin BOX 1B. A rst concern is if and how venous and capillary blood concentrations of an analyte cor-relate [32]. Obviously, this is an aspect that needs Hemato-critical issues in quantitative ana lysis of dried blood spots: challenges and solutionsDried blood spot (DBS) sampling for quantitative determination of drugs in blood has entered the bioanalytical arena at a fast pace during the last decade, primarily owing to progress in analytical instrumentation. Despite the many advantages associated with this new sampling strategy, several issues remain, of which the hematocrit issue is undoubtedly the most widely discussed challenge, since strongly deviating hematocrit values may signifcantly impactDBS-basedquantitation.Inthisreview,anoverviewisgivenofthedifferentaspectsofthehematocrit problem in quantitative DBS ana lysis. The different strategies that try to cope with this problem are discussed, alongwiththeirpotentialandlimitations.Implementationofsomeofthesestrategiesinpracticemayhelpto overcome this important hurdle in DBS assays, further allowing DBS to become an established part of routine quantitative bioana lysis.Pieter MM De Kesel, Nele Sadones, Sara Capiau, Willy E Lambert & Christophe P Stove*Laboratory of Toxicology, Ghent University, Faculty of Pharmaceutical Sciences, Harelbekestraat 72, 9000 Ghent, Belgium *Author for correspondence: Tel.: +32 9 264 81 36 Fax: +32 9 264 81 83 E-mail: christophe.stove@ugent.beThese authors contributed equallyFor reprint orders, please contact reprints@future-science.comREVI EW |De Kesel, Sadones, Capi au, Lambert & StoveBioanalysis (2013) 5(16) 2024 future science groupto be evaluated on a case-by-case basis and may depend on the aim of the study (e.g., setting up a TK prole versus measurement of trough levels). This concern, which is inherent to any nonvenous sampling technique, has been addressed in several recent publications [6,3338] and, although of key importance when deciding to set up DBS-based methods, is beyond the scope of this review and hence will not be discussed further here. Other concerns associated with DBS-based sampling are the acquisition of correctly obtained samples, con-tamination risk, sensitivity issues in case of ana-lytesatverylowconcentrations,thechromato-graphic or volcano effect and the site of punching, the inuence of the volume of blood spotted, and the hematocrit (Hct) effect [2,3,39,40]. The latter is undoubtedly the most widely discussed DBS-related problem, which is clearly reected by the factthatinDBSpublicationsthewordhema-tocritisalmostinvariablylinkedtoissueand problem. This is clearly exemplied by a selec-tion of quotes that appeared the last few years in the literature (BOX 2) [23,4144].It needs to be remarked, though, as Xu et al. recentlynicelypointedoutinaPerspective inthisjournal[45],thatthehematocritprob-lemisinfactatwofoldproblem.Whilethe rstproblemisanalyticalinnature,affecting mainly the accuracy and possibly also the pre-cisionoftheanalyticalmethodologyinse,the second is physiological in nature and relates to the blood/plasma ratio of an analyte, as detailed further in the following sections.HematocritThe Hct is dened as the volume fraction of the blood that is taken in by red blood cells (RBCs). It is determined by the amount and the size (vol-ume) of these cells. Although reference ranges are available at the population level, important inter-aswellasintra-individualdifferences exist.AmongthefactorsdeterminingtheHct are age, sex, health and nutritional status. Refer-ence ranges for men and women lie at approxi-mately0.410.50and0.360.44,respectively [39].HigherHctvaluesaretypicallyobserved in newborns, people living at high altitudes, as well as in persons suffering from, for example, polycythemia (primary) or chronic obstructive pulmonarydisease(secondary).Patientswith anemia have lower Hct values. Examples include patients receiving chemotherapy and immuno-compromised patients, in which Hct values of 0.200.30 are not rare [46]. FIGURE 1 shows the Hct distribution of a hospital population. This gure, representing over 200,000 Hct measure-ments can be considered as a worst-case scenario: patients with strongly deviating Hct values are likely to be followed up more closely, thus lead-ing to an overrepresentation in this distribution prole. Still, 95% of the measurements lie in the Hctrangeof22.747.5,therange0.190.63 coveringover99.5%ofthemeasurements. Hence,whenaimingatevaluatingtheimpact of Hct (except when aiming at evaluating very specic populations), it is not useful to extend the range to even lower or higher Hct values. Nowadays,automatedhematologyanalyz-ersdeterminetheHctofliquidblood(typi-callyK2-EDTA-anticoagulated)usingelec-tricalimpedance.Inthisprocedure,thecells inaliquidbloodsampleowinaconductive uidthroughanarrowchannelbetweentwo electrodes.Whenacellpassestheelectrodes, theconductivityoftheuidwilllower.Since largecellsdisplacemoreconductiveuid,the increaseinelectricalresistancewillbedepen-dentonthecellularvolume.Thisway,RBCs canbedistinguishedfromthelargerwhite bloodcellsandsmallerplatelets.Intheend, theincreaseinelectricalresistancewillgener-ate an electrical pulse. When all pulses derived Box 1. Advantages and challenges associated with dried blood spot sampling. A. Advantages

Ease of sampling, enabling sampling at home

Cost-effective sampling, transport and storage of samples

Improved compound stability

Small blood volumes

Minimally invasive sampling

Reduced risk of infection

Simplication of sample preparation procedures

Suitability for automation of sample processing and analysisB. Challenges

Correlation between venous and capillary blood concentrations

Adequate sampling

Contamination risk

Lack of sensitivity

Chromatographic effect and inuence of the site of punching

Inuence of spotted blood volume

Hematocrit effectKey TermsDried blood spot sampling: Micro-volume sampling technique where 100 l of whole blood is collected as a spot on a (cellulose) flter paper.Hematocrit: Volume percentage of red blood cells in blood.Hemato-criti cal issues i n quantitati ve ana l ysis of DBS| REVI EWwww.future-science.com 2025future science groupfrom RBCs are summed, the nal Hct can be derived, based upon the number of RBCs and their volume [202]. An alternative way to deter-mine the Hct is to ll an anticoagulant-coated capillary with blood and subject it to centrifuga-tion for an established period of time [47,48]. The resultsobtainedbytheimpedance-basedpro-cedure will differ slightly from those obtained by microcentrifugation, since the former is not accompanied by plasma sequestration between the RBCs [47]. Indirect measurement of the Hct isalsopossiblebymeasuringthehemoglobin (Hb) content. More specically, the rule of 3 can be applied, by which the Hct (in % 3%) canbecalculatedbymultiplyingtheHbcon-tent(g/dl)withanarbitraryconversionfactor of (approximately) three [49].The Hct effect: impact of Hct on quantitative DBS analysisAs observed by many authors, the Hct may have a large impact on the result in quantitative DBS Box 2. Selected quotes illustrating the hematocrit problem in dried blood spot analysis.

Current thinking is that this issue (hematocrit) needs to be addressed before practical application of DBS analysis can progress to the next level, and any direct analysis technique needs to be compatible with this solution [23]

In summary, we found that most metabolites used for newborn screening depend on hematocrit and on position of the disk. The effects of hematocrit and position of the disk were sometimes additive and sometimes even synergistic [41]

Hematocrit is currently identied as the single most important parameter inuencing the spread of blood on DBS cards, which could impact the validity of the results generated by DBS methods, affecting the spot formation, spot size, drying time, homogeneity and, ultimately, the robustness and reproducibility of the assays [42]

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 [43]

The future of DBS in clini cal bioanalysis is dependent on eliminating or limiting the so-called hematocrit effect, that is, inaccuracy caused by hematocrit variability [44]DBS: Dried blood spots.0.100.110.120.130.140.150.160.170.180.190.200.210.220.230.240.250.260.270.280.290.300.310.320.330.340.350.360.370.380.390.400.410.420.430.440.450.460.470.480.490.500.510.520.530.540.550.560.570.580.590.600.610.620.630.640.650.660.670.680.690.700.71Hct range0123456Relative frequency (%)Figure 1. Relative frequency of the hematocrit values measured over the course of 1 year at the clinical laboratory of Ghent University Hospital (Ghent, Belgium), measured via a Sysmex XE-5000 analyzer. From the data, representing over 200,000 data points, it is obvious that two populations are present, consistent with the nature of this population (i.e., a hospital population), with maxima at Hct values of approximately 0.31 and 0.40.Hct: Hematocrit.REVI EW |De Kesel, Sadones, Capi au, Lambert & StoveBioanalysis (2013) 5(16) 2026 future science groupana lysis.Asalreadymentioned,theimpactof the Hct can be divided into an analytical and a physiological aspect. The analytical impact of deviating Hct valuesThemostobviousimpactoftheHctisthat itdeterminestheviscosityofthebloodand willthereforehaveanimpactonthespread-ingofthebloodonlterpaper.Onclassical cellulose-basedpapersubstratesusedforDBS samplecollection,suchasWhatman903, FTA DMPK-C (GE Healthcare) or Ahlstrom 226 (PerkinElmer), blood with higher Hct will spread less than blood with lower Hct. In other words, following application of the same blood volume,DBSwithasmallerdiameterwillbe formedbybloodwithahigherHct[39].Con-sequently, when a xed-diameter partial-punch (e.g., 3 mm) is taken from these DBS, punches from DBShigh Hct will contain larger volumes of blood than punches from DBSlow Hct. This posi-tive correlation between the blood volume in a xed punch taken from a DBS and the blood Hct has been described by several authors[39,5052]. Denniff and Spooner, for example, found 35% difference in blood volume across the Hct range of 0.200.80 [39]. Consequently, if a calibration lineisestablishedinbloodwithintermediate Hct,thismayresultinanunderestimationin the case of DBSlow Hct and an overestimation in the case of DBShigh Hct. TheimpactthatdivergingHctvaluesmay haveonthequantitativeresultobtainedfrom a DBS has been evaluated for small molecules, traceelementsandproteins.Thevastmajor-ityofstudiesevaluatingtheHctimpacton theanalyticalresultdealwiththedetermina-tionofsmallmoleculesinDBS.Numerous authorsreportedontheeffectofHctonthe assayaccuracywhenanalyzingxed-diameter punchesfromDBS.TheinuenceofHcton -hydroxybutyricacidconcentrationsmea-sured on Whatman 903 paper was examined by Ingels et al. [53]. These results are summarized in FIGURE 2, the sample with a Hct of 0.44 being usedfornormalization.Whilelittleinuence wasobservedintheHctrangeof0.390.51, inaccuracybecamemorepronouncedwhen the Hct range was extended, with biases up to -15 and 11.1% at Hct values of 0.34 and 0.56, respectively[53].Holubetal.investigatedthe effectofHctontheana lysisofaminoacids, acylcarnitinesandguanidinoacetateonS&S 2992paper[41].Mostcompoundsshoweda signicant increase in measured concentration with increasing Hct over a range of 0.200.60. Fortyrosine,aspartateandsomeacylcarni-tines, however, no signicant correlation with Hctwasfound[41].Wilhelmetal.,quantify-ing immunosuppressants in DBS on Whatman 903 paper, demonstrated biases of -12 and 14% for cyclosporine A, at Hct levels 0.20 and 0.72, when normalized to the sample with 0.35 Hct, and of -11 to 19% for mycophenolic acid in the Hct range of 0.220.45 (normalization to 0.36 Hct)[54].Vuetal.foundhighassaybiasesfor thedeterminationofmoxioxacininsamples withvaryingHctvaluesonWhatman31ET CHRpaper;morespecically,adifferenceof approximately40%betweenconcentrations measured in samples with Hct of 0.20 and 0.50 was seen. After correction for Hct (see later), the biases were below 15% [55]. For the ana lysis of thiorphan on DMPK-A cards, Mess et al. also observed increasing assay biases with increasing Hct values, ranging from -4.8 to 11.2% in the 0.250.65 Hct interval, when normalized to the 0.45 Hct sample [56]. A similar effect of Hct in the range 0.200.50 was described for losartan anditsactivemetabolitelosartancarboxylic acid on Whatman FTA cards by Rao et al. [57]. Peng et al. found large biases for the determina-tion of succinylacetone on 903 paper over the Hct range 0.300.60. For samples with a Hct of0.30thebiaswas-45%,whileforsamples with a Hct of 0.60 a bias of 24% was observed [58]. Shah et al. examined the inuence of Hct onthedeterminationofantiepilepticdrugs (levetiracetam,lamotrigine,phenobarbital, carbamazepineanditsactivemetabolitecar-bamazepine-10,11epoxide)onWhatman903 paper. For all ve compounds, the bias of mea-suredconcentrationswasbelow5%atHct levelsof0.30and0.55,whennormalizedto the samples with 0.425 Hct [59]. The inuence ofthepapersubstrateontheHct-dependent biaswasevaluatedbyDenniffandSpooner. Theseauthorsquantiedacetaminophenand sitamaquineinDBSonthreedifferentpaper substrates (Whatman 903, FTA and FTA Elute) overaHctrangeof0.200.80andobserved considerable variation in assay bias [39]. On FTA paper,thebiaswentfromnegativetopositive for both compounds with increasing Hct. On 903paper,aswellasonFTAElutepaper,a similar trend was observed for acetaminophen withaHctbetween0.20and0.60;however, for Hct values between 0.60 and 0.80 the bias decreased again. A possible explanation for the Hemato-criti cal issues i n quantitati ve ana l ysis of DBS| REVI EWwww.future-science.com 2027future science grouplatter might be a negative effect on recovery of the analyte at high Hct levels, as has been dem-onstrated for other compounds (see later in this article).Remarkably,thebiasforsitamaquine on 903 paper was not signicantly affected over the entire Hct range, while on FTA Elute, the biashadarathercomplexcourse[39].Hence, theimpactofHctonthebiasdependedboth onthepapersubstrateandontheanalyte.In ordertoexploretheroleofvariousanalyte parameters, OMara et al. selected a set of ve compounds with varying physicochemical and bindingcharac teristicsandanalyzedthisset on four different paper types (Whatman FTA, FTA Elute, FTA DMPK-C and Ahlstrom 226) overaHctrangeof0.250.75[40].Measured concentrationswerecomparedwiththoseat the reference Hct level of 0.45. All compounds displayedanegativebiasatHctlevelsbelow 0.45 and a positive bias above this reference Hct value. Again, the degree of bias was inuenced bybothcompoundcharacteristicsandpaper type [40].Consideringtheana lysisoftraceelements inDBS,mostattentionhasbeengiventothe determination of lead in DBS [4]. There seems to be no real consensus regarding the effect of deviating Hct on the measured lead concentra-tions.WhileCarteralreadyreportedin1978 onincreasedbloodleadvalueswithincreas-ingHct[60],morerecentstudiesfoundlittle ornoinuenceofdifferentHctlevelsonlead concentrations in DBS [61,62].In addition to small molecules and trace ele-ments,DBShavealsobeenusedinanalytical methods for the determination of peptides and proteins,largemoleculespredominantlyresid-ing in the plasma [52]. Here, xed-size punches originatingfromDBShighHctwillcontainless plasma than punches from DBSlow Hct. This effect may counteract the above-described Hct effect (i.e.,punchesfromDBShighHctcontainmore bloodthanpunchesfromDBSlowHct).Hence, the net result will be determined by two oppos-ing factors: on the one hand the spreading of the DBS (with more concentrated spots in the case ofDBShighHct);on theother handthepresence of plasma in the DBS punch (with less plasma in the case of DBShigh Hct). Hoffman et al. inves-tigatedtheinuenceofvaryingHctonthe concentrationofprostate-specicantigenover theHctrange0.240.61.Asprostate-specic antigen concentrations decreased with increas-ing Hct, it can be concluded that the decreased presenceofplasmainDBShighHctprevailedin this case. More specically, the bias ranged from 18to-10%overthetestedHctinterval,when normalized to the sample with a Hct of 0.45 [63].The above-mentioned data clearly demonstrate that the impact of Hct on assay bias differs from casetocaseandmaydependon,forexample, papertype,differentialspreadingofblood, compound characteristics (e.g., blood cell afn-ity, plasma binding, molecular weight, pKa and hydrophobicity),extractionsolventsandsoon. Thus, there is a clear need to dene a Hct interval in which the impact of the Hct on the accuracy of the analytical result is still acceptable. Conse-quently, evaluation of the impact of the Hct on the accuracy should be part of the validation pro-cedure for any DBS-based bio-analytical method. In general, the acceptance criteria are set at 15% (% bias); at the LLOQ of 20%. Important to note as well is that a distinction should be made between absence of a Hct effect, no signicant impact and the Hct effect fell within the accep-tance limits of 15%. Whereas all of these state-ments have been used in literature, they do not always imply the same. In many cases where no Hcteffectwasdescribedthisactuallyrefersto the fact that the impact of deviating Hct on the accuracydidnotexceed15or20%.However, even when during validation, a Hct interval has beendescribedinwhichthebiasisacceptable, one does not know whether the Hct of the blood usedtoprepareagivenDBSactuallydoeslie within this interval. Strategies to cope with this problem are outlined further in this review.-20-15-10-505101520Hct 0.34 Hct 0.39 Hct 0.46 Hct 0.51 Hct 0.56Average % deviation fromnormalized sample (Hct 0.44)5 g/ml100 g/mlFigure 2. Inuence of hematocrit on the -hydroxybutyric acid concentration measured in dried blood spot samples (n = 5), using GCMS in selected ion monitoring mode. For QCs at two concentration levels (5 and 100 g/ml), the average percentage deviation from the -hydroxybutyric acid concentration obtained for the sample with a Hct of 0.44 is shown. Hct: Hematocrit. Data adapted with permission from [53] Future Science Group (2011). REVI EW |De Kesel, Sadones, Capi au, Lambert & StoveBioanalysis (2013) 5(16) 2028 future science groupApart from the differential spreading of blood with low and high Hct on lter paper, the Hct may also have an impact on the extraction ef-ciencyand,consequently,ontherecoveryof an analyte. DBShigh Hct may, upon drying, form some kind of barrier from which it may be more difcult to extract the analyte of interest, when comparedwithextractionfromDBSsamples with a normal or low Hct, resulting in an under-estimation of the analyte concentration. A Hct-dependenteffectontherecoveryofnaproxen from DBS was observed by Youhnovski et al. [64]. Since these authors extracted the entire spotted blood volume, at least theoretically, no signi-cant bias from varying Hct levels was expected. However,measurednaproxenconcentrations decreased with increasing Hct values. Biases of -9.2 and -11.1% at 0.75 Hct were observed for FTAandDMPK-Ccards,respectively,when compared with the 0.45 Hct sample. Post column infusion proles, as well as a stable response for theIS,revealednodifferencesinionsuppres-sionorenhancementbetween0.45and0.75 Hct samples. The hypothesis that the observed negative bias could be owing to a lower recovery of the analyte at high Hct levels was conrmed by the fact that optimization of the extraction procedureeliminatedthiseffect.Mlleretal. described a similar effect on the determination of peginesatide, a peptide of approximately 45 kDa, in whole DBS. While the assay bias showed little variationbetween0.45and0.55Hct,asteep decline in measured concentration was observed at 0.60 Hct, when compared with the 0.40 Hct sample. Again, as the IS response showed no Hct dependency,itislikelythatthiseffectisalso causedbydecreasedextractionefciency[65]. Likewise, Cox et al., quantifying IGF-1 in DBS, observed a negative bias at 0.60 Hct, when com-pared with a 0.40 Hct reference[66]. This bias was most pronounced at lower concentrations. Although the above-mentioned examples did not establish any apparent effect of the sample Hct onion-suppresionorenhancement,eachDBS sample with a different Hct can be considered as having a different matrix, with the potential of giving rise to a Hct-dependent matrix effect. This matrix effect can signicantly impact the accuracyofananalyticalresult,leadingtoan under- or over-estimation, depending on the Hct of the sample. Furthermore, varying recoveries andmatrixeffectsinfunctionofthesample Hct may also affect the precision of an analyti-calmethod.Therefore,duringmethodvalida-tion,itcouldbevaluabletoevaluaterecovery and matrix effects using blood samples covering a broad range of Hct values.To conclude, the above-described phenomena explain how Hct levels may have a large impact on the accuracy of an analytical result. This in turn may inuence the diagnostic decision based onthoseresults.Inparticular,whenaperson hasastronglydeviatingHct,thismayleadto seemingly elevated or lowered levels of the deter-minedcompound,resultinginafalse-positive orfalse-negativediagnosis[58].Importantto note, however, is that the effect of Hct is not an isolated factor in the ana lysis of xed-diameter punchestakenfromDBS.Otherfactors,such as spotted blood volume and/or punch location, could interact with the Hct effect. For example, the impact of taking a peripheral versus a cen-tralpunch,insomecases,appearstobemore pronounced at lower Hct levels [40,41]. Overall, the bias originating from different punch loca-tionmaycounteract,aswellasenhance,the bias caused by deviating Hct [40]. In conclusion, thecombinedeffectofHct,volumespotted and site of punching should be evaluated on a case-by-case basis when using partial-cut DBS. The physiological impact of deviating Hct valuesBesides the effect of the Hct on the accuracy of an analytical result, it may also have a signicant inuence on the interpretation of a DBS result that in itself may be correct. Indeed, currently applied reference intervals that are used to evalu-ate whether a result is normal are mostly based uponplasmaorserumconcentrations.When comparingDBSresultswiththesereference intervals, one has to be aware that no straightfor-ward conversion of one value to another is pos-sible. Concerning the correlation between DBS andplasmaconcentrations,animportantfac-tor is the blood-to-plasma concentration ratio (B/P ratio), which is, according to Emmons and Rowland, expressed by the following equation: Cb/Cp = (1-Hct) + Hct**fuEQUATION 1 whereCb isthetotalbloodconcentration,Cp the total plasma concentration, r the blood cell-to-unboundplasmaconcentrationratio,fu the unbound fraction in plasma and Hct the hema-tocrit [67,68]. From this equation it can be deduced that Hct is one of the prime factors determin-ing the B/P ratio. Compounds that do not, or onlytoalimitedextent,entererythrocytes, willdisplaylowB/Pratios,andconsequently Key TermsBlood-to-plasma concentration ratio: Ratio between the concentration of a specifc compound measured in blood and the concentration measured in plasma. The blood-to-plasma concentration ratio refects the partitioning of this compound between erythrocytes and plasma. Bridging studies: Studies in which both dried blood spots and venous blood/plasma/serum samples are collected in order to evaluate the correlation between the concentrations measured in both sample sets.Hemato-criti cal issues i n quantitati ve ana l ysis of DBS| REVI EWwww.future-science.com 2029future science groupthepresenceoferythrocytesmaybeseenasa dilution of the plasma fraction of whole blood. In this situation, Hct is an important parameter inuencing the B/P ratio. Typical examples of such analytes, exclusively residing in the plasma, are proteins or peptides. When not considering the impact of Hct on the spreading of a DBS, theoretically, for a given plasma concentration, the B/P ratio of these molecules will lower with increasingHctvalues.Formoleculesthatdo partitionintoerythrocytes,andbindtoboth cellular and plasma proteins, both r and fu are of importance. When blood cell afnity is more important than plasma binding, the B/P ratio willbehigherandHctisafactorinuencing that ratio. In this case, higher Hct levels will lead tohigherB/Pratios.Othercompoundsfreely entererythrocytesbutdonotbindtocellular proteins, nor to plasma proteins. In this case the B/P ratio will approximate one and almost no impact of Hct on B/P ratio is expected [45,6769]. Itneedstoberemarkedthatinallscenarios, irrespective of the B/P ratio, the impact of Hct on spreading of the DBS plays a role.These data indicate that Hct, amongst other factors, needs to be taken into account when a correlation or conversion is to be made between DBSandplasmaconcentrations,especiallyin populations with deviating Hct levels, such as newbornsorpatientssufferingfromanemia. Currently, such conversion is often based on the assumption that all patients Hct correspond to whatisconsiderednormalforthepopulation they belong to. However, for clinical validation ofthecorrelationbetweenDBSandplasma measurements,invivobridgingstudiesare essential[45].Inthesestudies,DBSandcor-respondingplasmasamplesarecollected,and thecorrelationbetweenbothisevaluated statistically.Strategies to cope with the Hct effectIt needs to be remarked that the following strat-egies only overcome or assist in overcoming theissueofdifferentialspreadingofblood withdifferentHct.Althoughtheimpactof diverging Hct values on other parameters, for example,matrix effects and recovery, may be alleviatedbyoptimizingtheextractionand chromatographic conditions [64], these valida-tion parameters still need to be investigated, as outlined above. Furthermore, if a comparison is to be made with plasma or serum concentra-tions, one ideally still needs to know the actual Hct of the sample. Avoid the Hct effectGenerate volumetrically applied DBS, followed by whole DBS ana lysisWithoutashadowofadoubt,theeasiestway ofovercomingtheHctproblemistoavoidit byanalyzingcomplete,volumetricallyapplied spots, instead of using discs generated by punch-ingDBS.Roughly,twostrategieshavebeen followedintheliteratureforwhole-spotana-lysis;intherstapproach,thecompleteDBS ispunchedaftervolumetricapplicationofthe blood [3,27,38,43,70,71]; in the second approach the blood is volumetrically applied on pre-punched discs[64,7274].Thelatterapproachexistsin different formats. In the concept of perforated DBS, paper discs with diameters of several mil-limeters are punched out from the lter paper andplacedbackintothatlterpaperbefore DBSsampling[72,73].Then,alimitedamount of blood can be applied volumetrically on these discs.Inthecaseofpre-cutDBS,theperfo-ratedlterpaperdiscsareattachedonasup-portsysteminsteadofbeingplacedbackinto the sheet of lter paper [64]. A variant of the lat-ter is dried matrix on paper discs, a whole-cut DBS microsampling technique with a dedicated support system, described by Meesters et al. [74]. Although these approaches may seem very con-venient, we recommend to always reect on the context in which sampling will need to be done eventually.Additionally,thechoicebetween apartial-andwhole-cutDBSmethodshould ideally be made before setting up the method, as validation of both methods differs in several aspects, as outlined above. When the aim is to establish a method for ana-lysis of DBS obtained from, for example,ani-mals, patients in a hospital environment or from postmortem matrices, volumetric application by dedicated staff (e.g., an experienced animal tech-nician, a dedicated nurse or laboratory staff) may befeasible.Inthesecases,sampleapplication canbedonebyaccuratepipettingorbyusing anticoagulant-coated microcapillaries [75]. Such capillaries can be used for direct spotting or can beusedinconjunctionwithdevicesallowing repeatedvolumetricdispensing.Aprototype of an incremental dispenser device that facili-tates the process of volumetric spotting has been developed by Drummond Scientic Company. This device allows the precise and accurate gen-eration of multiple volumetric spots from a single blood-lled microcapillary [43,73,76]. Li et al. also found Microsafe pipettes (Safe-Tec, PA, USA) suitable for accurate microsampling [73]. These REVI EW |De Kesel, Sadones, Capi au, Lambert & StoveBioanalysis (2013) 5(16) 2030 future science groupare one-time-use plastic pipettes that draw blood via capillary forces and from which an accurate volume (e.g., 5 l) can be delivered on lter paper by squeezing an attached bulb. In the context of forensic toxicology, when dealing with postmor-tem matrices, volumetric application should be the rule, as blood from a postmortem source is often lyzed and spreads differently on lter paper than fresh blood. Here, the process of spotting a matrix (e.g., blood) on paper can actually be considered as an alternative sample preparation technique [19].Whensamplingistobeperformedby non-experiencedindividuals(e.g.,patientsat home), all of the above-mentioned whole-DBS approachesmaybedifculttosustain.Inthis scenario, the requirement for accurate volumet-ricapplicationofthebloodconstitutesanon-negligibledisadvantage,anddirectapplication of a blood drop from the ngertip onto the paper maybethemostrealisticandfeasibleoption. Althoughthisnonvolumetricapplicationmay berelativelyeasyandstraightforward,careful instructionoftheindividualisstillneededas incorrect sampling will inevitably lead to erro-neous results. Some instructions to be given to self-sampling patients are indicated in BOX 3 [2]. Thesecanbegivenvia,forexample,ahands-ondemonstration,ayer,avideo,adedicated website or a combination of these.Use dried plasma spots instead of DBSAnother way of avoiding the Hct problem may be to use dried plasma spots (DPS) instead of DBS, as this matrix does not comprise RBCs. This way, the convenienceofsampletransport,handling, storage and ana lysis may be maintained. Several publications have described the use of DPS, dem-onstrating a close correlation between the results obtainedfromDPSandthoseobtainedfrom plasma [7783]. However, in all but one of the DPS reports, plasma was prepared by centrifugation of whole blood, which is not feasible for sampling at home.Agood-to-excellentcorrelationbetween concentrations measured in DPS and plasma has been reported for paroxetine [77], iothalamate [78], triazole antibiotics [80] and nevirapine [81]. Like-wise, Ruhwald et al., evaluating the chemokine interferon-inducibleprotein10(IP-10)[79,82] andFlowersandCook,measuringferritinand transferrinreceptorforironstatusassessment [83], found an excellent agreement between DPS andplasmameasurements.Whiletheprevious authors prepared DPS from plasma obtained from venous blood, recently, several reports described thepreparationofplasma,startingfromcapil-lary microsamples [8486]. For PK and TK stud-ies in plasma from rats and mice, Jonsson et al. applied capillary microsampling of blood [8486]. Plasma was prepared from the blood by sealing the microcapillary at one end and centrifuging it. Next, after cutting it with a ceramic cutter above the blood cell phase, an exact volume of plasma was collected by holding an exact volume capillary (4 or 8 l) end-to-end to the rst capillary. The only centrifugation-independent method to pre-pare DPS was described recently by Li et al., who used an interesting plasma separator device [87], depicted in FIGURE 3. This device, originally devel-oped to allow homocysteine monitoring in DPS via patient self-sampling, was used for DPS-based monitoring of guanfacine. DPS are generated by applying blood drops onto a multilayered poly-meric lter membrane, consisting of a collection membrane, a separation membrane and a remov-able top layer. While the plasma can ow through the separation membrane to the bottom collec-tion membrane, the cellular fraction remains lying ontop.Next,afterwaitingfor5min,thistop layer, on which RBCs are trapped, can be peeled Box 3. Instructions to be given to self-sampling patients in a dried blood spot study (not limiting).

Required size of the dried blood spots (DBS); this can be indicated by using custom pre-printed circles

Contact of the ngertip with the DBS card should be avoided (to avoid contamination and smearing of the DBS)

Only one drop of blood should be applied per DBS

The rst blood drop should be wiped off (may contain interstitial uid)

Too much milking of the ngertip should be avoided

There should be minimal manipulation of the DBS card (to avoid contamination)

The disinfectant used to clean the skin should be completely dry before puncture (may interfere with the blood spreading)Data from [2].Hemato-criti cal issues i n quantitati ve ana l ysis of DBS| REVI EWwww.future-science.com 2031future science groupoff.AlthoughanissueofDPShomogeneityis mentioned by the authors, the proof-of-concept appearspromising.Moredata,coveringmore analytesanddemonstrating,forexample,the correlationbetweenplasmaconcentrationsand concentrations in these DPS are needed. Minimize the Hct effectSpot onto a special type of flter substrateSeveral companies have tried to reduce the Hct effectbydevelopingspecialltermaterialor speciallterpaperformats.Forinstance,Agi-lent Technologies has developed a special type of noncellulose lter paper, Bond Elut Dried Matrix Spotting, which is acclaimed to be less inuenced by the Hct. In other words, different Hct values lead to less variation in DBS diameters compared with the classically used cellulose lter paper [203]. Inourhands,however,thereducedrigidityof thispaper,ascomparedwithcellulosepaper, makes it less suitable for reproducibly punching out discs (at least, via manual punching). More-over, we found that blood did not always spread outevenlyonthistypeofpaper,alsocompro-misingreproduciblepunching.HemaFormTM, a fan-shaped lter paper with eight blades pro-trudingfromacenter,distributedbySpotOn Sciences, is also claimed to have a reduced Hct effect,althoughnodatahavebeenpublished [204].Likely,otherdevelopmentswillfollowin the near future, as also exemplied by a possibly promising,supposedlyHct-independent,mate-rial developed by Mengerink and colleagues [88]. However,moredataareneededasnopublica-tions are available yet. Other recently developed nonpaper substrates are glass paper lters, [89] and alginateandchitosanfoams[90].Thelatterare porous,swollenhydrophilicmatricesthatcan absorb water-containing matrices, such as blood, andcanbedissolvedindiluteacidicsolutions. The impact of Hct on the spreading of blood on these matrices remains to be evaluated.Use calibrators with a Hct close to the range of the target populationAs outlined above, it is necessary to evaluate the impactofHctontheaccuracy,aswellason theprecision,ofanyDBS-basedbioanalytical methodandtoestablishanintervalinwhich this impact remains acceptable. A good starting pointbeforesettingupavalidationprocedure forquantitativeDBSana lysisistorstreect on the population onto which the procedure will beapplied:newborns,elderlypeople,healthy individuals, patients with a specic disease, and soon.ThiswaytheimpactoftheHctcanbe minimized for a large part of the study popula-tion. The box plots in FIGURE 4 depict the median Hct and central 50% range of Hct values mea-sured in distinct hospital populations, the ags indicatingtherangecontaining95%ofthe Hct values. From these data, it is obvious that, dependingonthepopulationunderinvestiga-tion, one may opt to prepare calibration lines in blood with Hct close to the range of the target population [2,3,40,43,52,9193]. For example, when aiming at quantitative monitoring of biomarkers in healthy newborns (in which Hct values may exceed 0.50), a different (higher) calibration line shouldbeusedthanwhenaimingatmonitor-ing these same biomarkers in immunosuppressed patients (often having Hct values around or even lowerthan0.30).Suchanapproachwasfol-lowed by Sadilkova et al., who prepared calibra-tion and QC material in blood with a Hct corre-sponding to the median level of Hct (0.300.35) ofthetargetpopulation,transplantpatients receivingimmunosuppressants[94].Likewise, Ingels et al., monitoring -hydroxybutyric acid in, predominantly, male drug abusers, opted to SeparationmembraneCollectionmembraneBlood sampleappliedTwo membranesseparatedRed blood cells trapped onthe upper membranePlasma collected onthe lower membraneFigure 3. Multilayered membrane ltration device utilized by Li et al. for the generation of dried plasma spots. Adapted with permission from [87] John Wiley & Sons, Ltd (2012).REVI EW |De Kesel, Sadones, Capi au, Lambert & StoveBioanalysis (2013) 5(16) 2032 future science groupprepare calibrators in blood with a Hct of 0.44 [53].Whenthepopulationis(expectedtobe) heterogeneous, an intermediate calibration curve (preparedinbloodwithHctofapproximately 0.40) may be a good option. Alternatively, two calibrationcurvesmaybesetup,oneatthe higher end and one at the lower end, both with intervalsinwhichtheimpactofHctonaccu-racyisstillacceptable.Obviously,thisfurther extends the amount of work.EvenwhenaHctintervalhasbeendened duringthevalidationprocedure,onestillcan-not know whether the Hct of the blood to pre-pareagivenDBSactuallydoesliewithinthis interval. Also, in the case where two calibration lines would be set up, knowledge of the Hct of the blood that was used to prepare the DBS is necessary, in order to assign a given DBS to one of both calibration curves. As outlined further in this review, recently a strategy has been devel-oped that allows approximate prediction of the Hct from a DBS [95]. Measure or estimate the Hct of a DBSRather than focusing on strategies that avoid or minimize the Hct effect, another possible strat-egy that may be followed to cope with the Hct problem is to get an idea of the Hct of a given DBS sample. This may allow two things: rst, it can be conrmed that the Hct of a given DBS does lie within the range of a given validated bio-analytical procedure; second, knowledge of the Hct may allow the introduction of a correction factor compensating for the impact of the Hct. The approaches followed in literature, as well as potential future approaches, are discussed below.Acquire an extra blood sample to measure the HctTaking an extra blood sample in conjunction with the capillary blood sample has been sug-gestedasalogicalwaytoknowtheHct[43]. However,whenthisextrasampleisavenous sample, again dedicated staff are necessary and one may still wonder what the added-value of the capillary sample is. Indeed, except in cases where, because of stability issues, direct appli-cation onto (pretreated) lter paper is required, the most straightforward way may be to analyze the venous blood right away or prepare venous DBS from it. DBS prepared from venous blood maybeusefulinthescenariowhereclassical Hctdeterminationispossibleinthetreating hospital,butanalyticalmeasurementsareto bedoneelsewhereand,thus,requiretrans-port.Moreover,itneedstoberemindedthat differencesmayexistbetweencapillaryand venousblood,alsowithrespecttotheHct [96,97]. Hence, if an analyte is to be measured in capillary DBS, one should ideally know the HctofthecapillaryDBS.Therefore,asan alternative for an extra venous blood sample, a small volume of capillary blood can be sampled using a calibrated anticoagulant-coated capil-lary,ofwhichtheHctcanbereadoutafter centrifugation[48].However,hereoneofthe main advantages associated with DBS sampling therelativeeaseoftheprocedureisalso lost. Indeed, both of the above-mentioned sce-nariostoacquireanextrabloodsamplejust toknowtheHctarenotonlyimpossiblein the case of patient self-sampling at home, but will undoubtedly also complicate the sampling procedure in other circumstances. A more fea-sible and convenient alternative may be to use point-of-care-tests, such as those developed by HemoCue [98,99]. These are relatively simple to handle, require only 10 l of blood and allow fast(withinaminute)determinationofHb, from which the Hct can be calculated, as out-linedabove.Althoughwhenconsideringthe context of patient self-sampling, cost is likely to be a limiting factor(it is unlikely that every patientwouldhaveadevice).However,in 0.00.10.20.30.40.50.6Overallhospitalpopulation(n = 201847)Vaccinationcenter(n = 529)HematocritNeonatalcare(n = 6101)Emergencyservice(n = 14845)Burn woundcenter(n = 1837)Intensivecare(n = 24995)Figure 4. Box plots depicting the distribution of hematocrit values measured in distinct hospital population subsets. Indicated are the median, the central 50% interval (boxed), and the 2.5 and 97.5% percentiles (ags). The number of measurements underlying these data are indicated. It should be noted that this number is distinct from the number of patients, and that patients with (strongly) deviating hematocrit (Hct) and/or residing for a long time in the hospital will provide a bias to the result. A clear distinction can be seen between on the one hand the normal population, represented here by the patients at the emer-gency service and those having visited the vaccination center, and on the other hand, the ill patients, represented here by patients at the burn wound center and at the intensive care unit. Whereas a mixed picture is obtained in the overall hospital population and in patients from neonatal care, it is clear that the latter population contains more patients with higher Hct values, in line with higher Hct values in healthy newborns.Hemato-criti cal issues i n quantitati ve ana l ysis of DBS| REVI EWwww.future-science.com 2033future science groupremoteareaswithcentralizedDBSsampling and/or in centers specializing in DBS sampling frompatients,thesedevicesmayrepresenta simple means to know a patients Hct.Calculate the Hct using physical characteristics of DBSSeveral authors have proposed to estimate the Hct based upon some physical characteristics oftheDBS,suchasdiameter,surface,color and/or frustum volume [39,55,205]. For example, Denniff and Spooner took photographs of DBS to measure the spot area on different types of paper [39]. However, as the blood did not spread homogenously through all types of lter paper, it was not always easy to dene the edges of the spot. The frustum volume, which is calculated from the front diameter, the back diameter and the thickness of the lter paper, correlated lin-early with the Hct [205]. Vu et al. also introduced a correction factor, based on the diameter of the punch, the blood volume and the DBS area [55]. These authors, however, observed that despite thecorrectionforHct,thebiasmaynotbe totallyeliminated.Inaddition,allcharacter-isticsbaseduponthedimensionsoftheDBS primarily depend on the amount of blood spot-ted.Therefore,thisapproachisonlyfeasible whenusingvolumetricapplication,whichis, aspreviouslypointedout,notanidealsitua-tion. However, it needs to be remarked that it is possible to know the approximate volume of blood spotted on lter paper by weighing the DBS [55,100]. Theoretically, the volume deduced from this weight could be combined with the surfaceoftheDBStoreadtheapproximate Hctfromacalibrationcurve.However,this approach may be both impractical and impre-cise. Lastly, while the color of a DBS darkens as the Hct of blood increases [39], this aspect is not suited for the purpose of estimating Hct as the DBS color changes upon aging of the DBS, as described by Bremmer et al. for blood stains (cfr infra) [101,102].Predict the Hct by measuring endogenous compoundsAs readily suggested by Denniff and Spooner inthisjournal,anapproachcouldbeto normalizethemeasuredconcentrationsinthe puncheddisctoanotherendogenouscompo-nent of blood that also varies with Hct [39]. In orderforsuchanendogenouscompoundto be a suitable candidate it has to fulll certain criteria (BOX 4).PotassiumAt rst glance, potassium as a correlate of Hct mayseemsomewhatsurprising.However,the choiceforthiselectrolyteislessofasurprise when considering the following: Erythrocytes are the predominant cells in the blood,outnumberingwhitebloodcellsbya factor of approximately 5001000. When also considering the points below, erythrocytes can be seen as the major contributors to the total blood potassium concentration; Potassium is primarily located intracellularly in all cells of the body: its intracellular con-centration(~140mM)isapproximately 35-foldhigherthanitsextracel lular concentration [103]; Potassium levels are under tight physiological control,withnormalserumorplasmalevels rangingbetween3.5and5mM,andrepre-sentingonlyalowpercentageoftotalblood potassium [103]. As a consequence, physiologi-calvariationsinplasmaorserumpotassium levelsareexpectedtohaveonlyminimal,if any, impact on the total blood potassium con-centration.Inaddition,eveninanemicor inamedpatients,erythrocytesstillgreatly outnumber other cell types in the blood; Potassiumcanreasonablybeindicatedasa stable analyte;Box 4. Criteria to be fullled by a potential hematocrit marker.

Correlation with hematocrit (Hct): a good Hct marker should correlate with the number and size of red blood cells.

Universal applicability: a good Hct marker should be measurable in every member of a population, independent of, for example, age, race or sex.

Minimal inter-individual non-Hct-related variation: a good Hct marker should correlate with the Hct, independent of a variety of disease conditions.

Stability: a good Hct marker should be measurable in both freshly prepared and old dried blood spots.

Easy to determine: ideally, a good Hct marker is easy to determine (simple and inexpensive sample preparation), starting from small (e.g., 3 mm) punches, leaving enough of the dried blood spots to determine the actual analyte of interest.REVI EW |De Kesel, Sadones, Capi au, Lambert & StoveBioanalysis (2013) 5(16) 2034 future science group High-throughput,low-costmeasurementof physiologicalpotassiumconcentrationsin serumisroutinelyperformedbyclinical laboratories worldwide.As, theoretically, potassium appeared to fulll all criteria we had put forward, we investigated whether potassium concentrations could indeed be used as a correlate for the Hct of a DBS, start-ingfroma3mmDBSpunch[95].Potassium could be efciently extracted from 3 mm DBS punches by soaking the discs in ultrapure water for 5 min in a thermoshaker at room tempera-ture. Since we opted to determine the potassium concentrationviaaroutineclinicalchemistry analyzer,wereadilyadded2.50mMKClto the extraction buffer so that the nal potassium concentrationofallextracts(fromDBSfrom blood with low and high Hct) would lie within thevalidatedrangeofthechemistryanalyzer. Thevalueofthisblankwasthensubtracted from the measured value to know the contribu-tionoftheDBS-derivedpotassium.toAddi-tion of 50 l, twice, to the DBS punch enabled recovery of 90 l of extract in total, which was transferredtoamicrocupandanalyzedwitha Cobas 8000 routine clinical chemistry analyzer. This approach turned out to be successful: the developed procedure was validated, and its appli-cation on real patient samples demonstrated its practical applicability and potential to provide a good Hct prediction [95]. Moreover, the applied procedureis verysimple,fast,comes atalmost no cost and may be universally implementable, as virtually every clinical laboratory even remote ones measure potassium on a routine basis. The limited sensitivity of the clinical chemistry ana-lyzer necessitates the addition of potassium to the extraction solvent, which inextricably results in an additional error and, hence, additional uncer-tainty in the calculated Hct. Consequently, there mayberoomforimprovementofthecurrent procedure, for example, by using more sensitive instrumentation,overcomingtheneedtoadd potassiumtotheextractionsolvent.However, this may be at the expense of the simplicity and the low cost. Moreover, it still has to be evaluated whether Hct prediction by itself may effectively assist in overcoming the Hct problem for a given DBS-based analytical procedure in real practice.HemoglobinAnotherobviouscandidateforHctprediction isHb,aniron-bindingproteinthattransports oxygen through the body and that is exclusively presentinRBCs.Asreadilyoutlinedabove, Hb is already used to calculate the Hct of fresh blood. However, whether Hb determination can effectively be used for accurate Hct prediction of the blood used to prepare DBS remains to be demonstrated. Orsini et al. have suggested that a HPLCUV method, originally aimed at qual-itativedetectionofHbvariantsinanewborn screeningforhemoglobinopathies,mayallow semi-quantitativeHctprediction[104].These authorsreasonedthat,sinceeachpeakinthe chromatogram corresponds to a Hb variant, the total area of all peaks should be an indication of the total Hb concentration. While a calibration line could be set up successfully, these authors didnotincludeacomparisonwithdirectHct measurement. Instead, a comparison was made withHctprediction,baseduponanotherin-house developed Hb-based colorimetric method. Hence, it is not surprising that a promising trend couldbeobserved.Inaddition,itneedstobe remarked that a very large part of the evaluated population newborns had Hct values of over 0.65. Even in newborns, however, which indeed havehigherHctvaluesthanadults,valuesof over 0.65 are the exception rather than the rule. Thisworkprovidesaninteresting,butvery preliminary proof-of-concept of using Hb as a Hct-predictor. Animportantfactorthatneedstobekept inmindwhenstudyingHbisthattheform inwhichitispresentchangesintime,which isalreadyevidentbythechangingcolorof DBS upon aging. This has important implica-tions when Hb is measured using, for example, routineclinicalchemistryanalyzers,which typicallyonlymeasureabsorptionatcertain wavelengths. The consequence is that a higher signal is obtained for Hb eluted from fresh, as compared with older DBS (FIGURE 5A). Whether thistrulyrepresentsalowertotalHbconcen-tration ormaybetheresultofa conversionto another Hb form, which absorbs less at the uti-lized wavelength(s), has not been established yet. Anyway, this implies that the Hb concentration from fresh DBS cannot be calculated straightfor-wardly using calibration curves generated by Hb measurements from older DBS (and vice versa). Interesting in this context are the recent ndings by Bremmer et al. [101,102,105], working on dried blood stains rather than DBS. Using spectromet-ric techniques, these authors demonstrated that in dried blood stains the original form present in blood, oxy-Hb, is oxidized to met-Hb, which isfurtherconvertedintothedenaturedform, Hemato-criti cal issues i n quantitati ve ana l ysis of DBS| REVI EWwww.future-science.com 2035future science grouphemichrome, which is the most prominent Hb formafterlongerstorage[101,102,105].Wealso observed a conversion in DBS, as measured by CO-oximetry performed on DBS extracts, albeit met-Hbwasthemainspeciesweobservedin extractsfromolderDBS(FIGURE5B).Itneeds to be remarked, though, that no denitive con-clusionscanbedrawnfromourresults,asit isnotclearif,ortowhatextent,hemichrome contributes to the signal of oxy- and met-Hb in the CO-oximeter, or, alternatively, may renature tomet-Hbupondissolution.Inconclusion,if total Hb would remain constant in DBS and if it would be possible to quantitatively determine the different Hb species in DBS, Hb-based Hct determination from DBS may remain possible. As a side note it needs to be remarked that the correlation between Hb and Hct is not absolute: a near-normal Hct may be present, with below-normal Hb values, as may occur in hypochromic RBCs, associated with iron-decient anemia [46].Other candidatesRather than focusing on an electrolyte in RBCs, suchaspotassium,Erhardtetal.focusedon another tightly regulated electrolyte sodium fornormalizationpurposes[100].Theseauthors determined retinol in DBS, which, like sodium, primarily resides in the plasma. The sodium con-tent in a DBS extract, measured via ame pho-tometry, was used to calculate the plasma content of the examined specimen, assuming a value of 140mMsodiuminplasma.Asimilarstrategy couldbefollowedtodeterminechloriderather thansodium.Aprerequisitefortheusefulness ofotherphysiologicalelements(e.g.,calcium, magnesium, iron, copper and zinc) is that these show minimal non-Hct-related variation and are concentrated either in the RBCs or in the plasma, allowing estimation of the Hct or the plasma con-tent, respectively. Iron, being complexed with Hb in RBCs, but also zinc, occurs at higher levels in RBCs. However, since the levels of these elements may uctuate more than, for example, those of potassium or sodium, a higher intra- and inter-individualnon-Hct-relatedvariationmaybe expected. Furthermore, calcium and magnesium concentrationsinblood,whicharebothunder tight physiological control, have been suggested to have potential for normalization [106]. However, experimental evidence for this is currently lacking.Apart from trace elements, amino acids may represent candidate markers to predict the Hct of DBS. Using a metabolomics approach, Wang etal.demonstratedthattheplasmaconcen-trationsofvebranched-chainandaromatic amino acids in fasting subjects may be predic-tiveoftheriskofdevelopingfuturediabetes [107].Whiletheseauthorsprimarilyfocused upon amino acids differentially present in sub-jects,theirndingsalsoimplythatthelevels ofvariousaminoacidsremainfairlyconstant between individuals. Focusing on these amino acids may help to trace back the Hct of the blood to prepare DBS. This hypothesis has been put forwardbyHilletal.,whoalsodemonstrated agoodcorrelationbetweenplasmaandRBC concentrationsofaminoacids[204].Alsohere, 050100150200250300Fresh DBS Old DBS0 5 10 15 20 25 30 35010203040506070Time (days)Fraction of total Hb (%)[Hb] (mg/dl)Oxy-HbMet-HbA BFigure 5. (A) Hemoglobin concentration following extraction of dried blood spots and (B) conversion of hemoglobin in dried blood spots as a function of time. (A) Hb concentration in the extracts of fresh DBS and old DBS, respectively processed 2 h and 2 days postspotting. Ultrapure H2O was used for extraction. The two series of bars each represent three experiments (n = 6). Indicated are mean DS. (B) Fraction (%) of oxy- and met-Hb, measured in function of time in extracts from DBS. Measurements were done using a GEM OPL CO-oxymeter (Instrumentation Laboratory, MA, USA).DBS: Dried blood spot; Hb: Hemoglobin. Data taken with permission from Capiau et al. [Capiau et al. Unpublished Data].REVI EW |De Kesel, Sadones, Capi au, Lambert & StoveBioanalysis (2013) 5(16) 2036 future science grouptheabsoluteamountofcertainaminoacids maycorrelatewiththeplasmacontent,rather than with the RBC content of the blood. Addi-tionally,adifcultynottobeunderestimated maybetheinuencethatfoodconsumption, physical activity or pre-analytical variables may haveonthelevelsofcertainaminoacids,for example,necessitatingDBStobetakenfrom fasting individuals [108]. Theoretically, the erythrocyte proteome could be exploited to search for RBC-specic markers [109,110]. These may constitute both membrane-associatedmarkers(e.g.,surfacemarkers)and cytoplasmic proteins. Not surprisingly, globins constitute the most abundant group of erythro-cyte-specicproteins,althoughRBCmarkers suchasglycophorins(majorsialoglycoproteins present at the RBC surface), as well as other pro-teins,maybevaluablecandidatesaswell[110]. However, the use of such protein markers would requirethatexpressionissubjecttominimal inter-individualvariation.Moreover,determi-nationofproteinsmaybemorecumbersome, because of possible issues of stability. If suitable antibodiesareavailable,determinationofthe RBCmarkersmaybefeasibleviaimmunoas-says (e.g., ELISA), which are, however, typically associated with a signicant cost. Theoretically, quantitative determination via proteomics may beanalternativeoption;however,application ofsuchsophisticatedmethodologymayrepre-sent overshooting considering the aim (i.e., Hct prediction).DNA-basedprocedures(e.g.,PCR)arenot suited for Hct prediction as erythrocytes anucle-ateduringmaturation,thus,asaconsequence matureerythrocytesdonotpossessnuclear DNA.Asmatureerythrocyteshavealsolost organelles such as mitochondria, they also lack mitochondrialDNA.Consequently,themajor DNAcontributorsofthebloodarethenucle-ated,mitochondria-containingnon-RBCs.As for RNA, Kabanova et al. reported the presence oftranscriptsofover1000differentgenesina >99.9999%pureerythrocytepreparation[111]. Althoughitremainstobeestablishedwhether there are true RBC-specic housekeeping tran-scripts among these, mRNAs encoding Hb might be a candidate, as these are the most abundant mRNAs in whole blood extracts, with reticulo-derived globin mRNA constituting up to 70% of totalmRNAfromwholeblood [206].However, even if there would be a correlation between glo-bin mRNA and Hct of a DBS, a limiting problem maystillbethesensitivityofmRNAtodecay, which may give rise to differences between DBS withthesameHct,dependingonthelength andconditionsofstorage[112,113].Inaddition, mRNAdeterminationrequiresconversionto cDNA, again lengthening and complicating the procedure.ConclusionAs generally recognized, DBS sampling offers sev-eral advantages over classical wet sampling. On the other hand, despite the progress made during the last decade, DBS ana lysis still faces consider-able challenges. Among these, the effect of Hct on the measured concentration is unquestionably the most addressed problem. From an analytical point of view, although analyte-dependent, dif-ferentialspreadingofbloodwithdifferentHct mainly affects the accuracy of an analytical result, possiblyleadingtounacceptableassaybiasesat extremeHctlevels.AtelevatedHctlevels,this effect may in some cases be partially counteracted byreducedextractionefciencyoftheanalyte. Hence, a Hct interval in which the impact of the Hct on the analytical result falls within accept-able limits needs to be dened for any DBS-based bioanalytical method. From a physiological point ofview,theHctisanimportantfactortobe taken into account when DBS results are to be compared with reference plasma values. DifferentstrategiestocopewiththeHct-dependentspreadingofbloodonpapersub-strateswerediscussed.TheHcteffectmay simplybeavoidedbyanalyzingwhole,volu-metricallyappliedDBS,insteadofxed-size partial DBS punches. The volumetric approach, however, excludes direct application of a drop of blood from the ngertip onto the paper card andmaybedifculttosustainwhenenvisag-inghome-sampling.TheuseofDPSinstead of DBS may offer another way of avoiding the Hct problem. On the other hand, the effect of Hct may be minimized by using special types of substrates that are less inuenced by the Hct, orbypreparingthecalibrationlineinblood withHctclosetotheexpectedHctrangeof the target population. Apart from avoiding or trying to minimize the Hct effect, another pos-siblestrategytocopewiththiseffectmaybe tomeasureorestimatetheHctoftheblood usedtoprepareDBS.Here,severalstrategies arepossible,suchasdirectHctmeasurement, Hct estimation based upon the physical charac-teristics of DBS and Hct prediction based upon endogenouscompounds.Withrespecttothe latter, potassium measurement in extracts from Hemato-criti cal issues i n quantitati ve ana l ysis of DBS| REVI EWwww.future-science.com 2037future science groupDBSpuncheshasbeendemonstratedtobea promising strategy. Future perspectivesTheHctproblemisagenerallyacknowledged theme in DBS ana lysis, and continuous efforts to further understand and tackle this issue will likely be made in the future. The effect of the Hct on quantication of an analyte in DBS samples is a compound-dependent matter and is inuenced by many factors. It is primarily an issue when deal-ing with nonvolumetrically applied spots, where discs are punched from the DBS. An interesting area for future research may be to further identify the role and the importance of distinct analyti-cal, physiological and compound parameters in the overall Hct effect in DBS ana lysis. To more deeply comprehend the impact of Hct on DBS measurements, attention should also be paid on how, and to what extent, varying Hct levels affect the recovery and matrix effects of DBS methods. Inaddition,noveland/orimprovedstrate-gies to cope with the Hct effect will be required tosupportthegrowingandwidespreaduseof DBSinbioana lysis.Developmentofinnova-tivematerials/substratesthatarelessaffected by Hct-dependent spreading of blood and allow more homogenous distribution across spots will contribute to this. Another focus may lie in the explorationofthepotentialofvariousendog-enous compounds to function as surrogate Hct markers.Asoutlinedinthisreview,currently onlypotassiumhasbeendemonstratedtobe agoodHctcorrelate,thepotentialofother candidatesremainstobeexplored.Estimation or prediction of the Hct of a DBS allows one to know whether the Hct of the blood used to make the DBS approximately fell within the accepted Hctintervalornot.Thiswayitispossibleto determinewhichanalyticalresultsarereliable, andwhichrepresentanunder-orover-estima-tion. Consequently, those DBS samples that can-not be accurately determined can be eliminated or, alternatively, the analyte concentration might be corrected, taking into account the deviation of the Hct. Such correction would require a suf-ciently accurate Hct prediction and submission of the obtained result into an established algo-rithm. This way, all DBS samples could be ana-lyzed with any DBS method, without leading to distorted results and/or incorrect interpretations.AcknowledgmentsWe wish to acknowledge V Stove and A Slabbinck for pro-vidingdataontheHctdistributionindifferenthospital populations. We wish to apologize to those authors whose interesting work could not be cited here. Financial & competing interests disclosureTheauthorshavenorelevantafliationsornancial involvement with any organization or entity with a nan-cial interest in or nancial conict with the subject matter ormaterialsdiscussedinthemanuscript.Thisincludes employment, consultancies, honoraria, stock ownership or options,expertt estimony,grantsorpatentsreceivedor pending, or royalties.No writing assistance was utilized in the production of this manuscript.Executive summary

Both from an analytical and physiological point of view, the hematocrit (Hct) may have an impact on the analytical result.

Whole dried blood spot (DBS) analysis of volumetrically applied DBS may overcome the Hct impact associated with differential spreading of blood with different Hct.

Dried plasma spots obtained by centrifugation of whole blood or by a plasma separator device may avoid the Hct problem.

A special type of substrate or a calibration line in blood with Hct close to the range of the target population may minimize the Hct effect.

Currently, there is only one endogenous compound, potassium, which allows prediction of the approximate Hct from nonvolumetrically applied DBS. ReferencesPapers of special note have been highlighted as:

of interest

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