Dried Blood Spots: Analysis and Applications - Analytical Chemistry

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Dried Blood Spots: Analysis and Applications Plamen A. Demirev Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac303205m • Publication Date (Web): 21 Nov 2012 Downloaded from http://pubs.acs.org on November 27, 2012

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Analytical Chemistry

Dried Blood Spots: Analysis and Applications

Plamen A. Demirev, Johns Hopkins University Applied Physics Laboratory, Laurel, MD 20723, USA

[email protected], phone: 443-778-7712

This literature review highlights recent advances and challenges in applying dried blood spots (DBS) as a cumulative technique for sample acquisition, transport, archiving, and prospective/retrospective bioanalysis on a large scale. The technique is minimally invasive, requiring microliters of typically peripheral blood, it is inexpensive, and easy to multiplex and automate. DBS handling and logistics, e.g., storage and shipment at room temperature, allow its deployment even in resource-poor settings. DBS samples are compatible with a large number of bioanalytical methods, among them chromatography, mass spectrometry, DNA and immunoassays. The range of established and emerging DBS applications is vast, to name only a few: large scale neonatal screening, preclinical drug development for lead validation, toxicokinetic (TK) and pharmacokinetic (PK) studies, clinical pharmacology, targeted and non-targeted metabolic profiling, therapeutic drug monitoring (TDM), forensic toxicology, doping or environmental contaminant control, microbiological and epidemiological disease surveillance.

ACS Paragon Plus Environment

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Guthrie introduced DBS samples for wider use exactly 50 years ago for the neonatal screening of inborn errors of metabolism (initially phenylketonuria, PKU1). Since then, large scale neonatal DBS analysis has included more than thirty disorders, and has saved and changed many lives in the industrialized world. The first report of blood spots on filter paper as a laboratory technique, was published almost a century ago by Ivar Bang, considered to be the father of modern clinical microanalysis.2 DBS-related research has been growing exponentially, and the list of published accounts has exploded in the last two years or so (Figure 1). Only select analytical approaches and methods, most introduced since 2010 and likely to impact current and future DBS applications, will be discussed here. Recent review papers on particular topics, covered here in less detail, are also cited. Regulatory, economic, legal, and ethical aspects, although important in further expanding DBS applications,3-8 are beyond the scope of this review.

DBS sample handling and processing

While the small sample volume is a major advantage for DBS, it also poses challenges in sensitivity and quantitation, particularly for trace analytes.9,10 Therefore, sample processing is an important step in DBS assay development. Several companies manufacture robotic end-to-end hardware for DBS handling - extraction, purification, concentration, and introduction into the analysis system. Efficient quantitative extraction procedures from a complex matrix, containing hundreds of denatured proteins, depend on the chemical properties and total amount of the analyte, filter paper used, analysis method.11,12 Another important issue to consider in DBS handling is analyte


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modifications during storage due to enzymes or other chemicals present (although stability is typically enhanced compared to liquid blood, stored under similar conditions). Analyte stability is influenced by such factors as filter paper type, temperature, humidity, storage time. Autoradiography was used to study analyte lateral distribution in DBS deposited on different filter paper types.13 Similar patterns - radially-decreasing concentration and unevenly-distributed speckles across the spot - were observed independent of compound and paper type studied. It is argued that autoradiography can be a helpful tool for further optimizing DBS sampling as a function of spotting volume, punch radius, temperature, humidity, in TK and PK studies. A major technical challenge in the regulatory acceptance of DBS for drug development is the accurate quantitation of analytes. This difficulty stems mostly from the uncertainty in blood volume deposited on the spot. In DBS analysis, a fixed area is punched out of the DBS sample for analysis. However, the non-uniform analyte/blood distribution across the spot and changes in hematocrit levels contribute to blood/plasma volume uncertainty.14 In addition, there is a potential of contamination (carry-over from sample to sample) during punching. To alleviate some of these problems, perforated DBS (PDBS) were introduced as a microsampling method and compared with conventional (punched) DBS for TK analysis.15-16 In PDBS, accurate and predetermined (5 to 10 ul) microsampling volume is achieved by spotting the blood with a micropipette on precut ¼” filter paper disk. The PDBS is inserted into a well of a 96-well plate for on-line extraction, concentration and subsequent HPLC MS/MS analysis. Several drugs in whole blood were successfully quantified by the PDBS method. PDBS provide several advantages over conventional DBS, e.g., no need for punching (thus saving time and increasing throughput),


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eliminating sample carry-over, ease of automation, utilization of the entire sample, and better quantitation (known sample volume, hematocrit assessment). Another method employs two-layered polymer filter membrane that separates plasma from whole blood to form a dried plasma spot (DPS).17 The lower surface with the DPS is first removed, and analyte plus internal standard are eluted from it by online solid phase extraction (SPE) followed by HPLC MS/MS. DPS is demonstrated as a DBS alternative that overcomes hematocrit issues, does not require centrifugation and is also easy to automate.

A challenge in quantitation by DBS is analyte loss that may result from incomplete and inefficient extraction and/or analyte degradation, particularly for unstable analytes. Accounting for these effects would improve the accuracy in analyte quantitation in DBS for TK, PK and TDM analyses. A reliable approach for estimating analyte stability in DBS, that involves consistent sample processing and a suitable positive control, was developed.18 A drug, typically degrading during drying, was initially stabilized by rapid lowering of the pH of the spotted blood. The drug was stable in the DBS for at least 48 days at room temperature, suggesting that the approach could be utilized for other drugs known to be stabilized at lower pH.

Screening assays for several inherited metabolic disorders (among them Fabry, Gaucher, Krabbe, Niemann-Pick A/B, and Pompe) are based on measuring activities of lysosomal enzymes in DBS either fluorometrically or by tandem MS.19 Therefore, maintaining enzyme stability during collection, storage and handling of DBS is of primary importance for successful assay performance.20 It was pointed out in this study that elevated heat and


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humidity reduced enzyme activity. Other detrimental factors for stability are DBS prepared from poorly mixed blood, and heparin. It is therefore recommended that EDTA be used as an anti-coagulant. Synthetic 4-methylumbelliferone derivatives are typical substrates for fluorescence-based lysosomal enzyme activity assays. However, fluorescence signal is quenched in DBS thus reducing considerably assay sensitivity. It was found21 that hemoglobin precipitation with trichloroacetic acid prior to analysis increased light signal intensity up to eight fold, providing a clear separation of control versus affected blood for ten different DBS lysosomal enzyme assays. Effects of temperature (up to 37oC) and humidity on the stability and changes in the levels of 34 markers, recommended for newborn screening in the US, were thoroughly investigated.22 Low humidity and temperature in DBS are essential factors in preserving sample integrity. Biomarkers for 26 newborn DBS screening assays were compared for the same blood samples deposited on different FDA-registered filter papers.23 Biomarker analyte recovery has been similar for all tested paper types, suggesting that papers can be interchanged between devices and DBS assays.

Effects of DBS storage and filter paper type on global metabolic profiling from DBS by UPLC/TOF/MS were investigated.22 Application of DBS for this type of bioanalysis is again limited by analyte stability, which is considerably enhanced when the samples are stored below -20oC. Also, untreated filter paper generates considerably less background chemical noise. An online sample dilution, enrichment, and cleanup protocol was utilized for enhancing the sensitivity of LC/MS/MS analysis of DBS.24 Analytes were extracted with water from DBS punches (3 mm diameter), concentrated in a trap column, in-line


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with an analytical HPLC /MS/MS. In that study, signal-to noise ratio of 40 was observed from 0.1 ng/mL lansoprazole concentration in whole blood. Three distinct DBS sample preparation procedures for LC/MS/MS in TK studies were evaluated.25 It was found that protein precipitation and liquid-liquid extraction were more effective in eluting the analyte, compared to pure organic solvent extraction. A liquid extraction-based surface sampling system that uses a chip-based infusion nanoelectrospray system was developed.26 In it, a robotic pipettor, controlled by appropriate software, forms and withdraws a liquid microjunction for sampling from individual discrete spots.

The

system was tested on DBS. Its qualitative and quantitative performance is comparable to other liquid extraction-based methods. A commercial TLC-MS interface was evaluated for its suitability for drug quantitation in DBS.27 Both direct introduction MS and HPLCMS were compared to the “conventional” manual extraction after punching out a disk. The TLC device provides better sensitivity, linearity, accuracy, and precision at physiologically relevant concentrations, compared to the manual procedure. An important step in the DBS preparation for quantitative analysis is the introduction of an internal standard. A novel piezoelectric system was used to spray methanol solution with dissolved internal standard over the DBS - Figure 2.28 To evaluate the system two other protocols for internal standard introduction - addition to the whole blood prior to spotting, and addition of the standard to the extraction solvent - were compared to the spray method. No significant differences were found using either method. The spray method can be easily included into an automated system for DBS analysis. An LC/MS/MS comparison of DBS and whole mouse blood, collected by an automated sampling system for PK studies, was conducted.29 The results indicate that DBS are stable for at least 34


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days at room temperature with more than 90% recovery upon extraction, provide full PK profiles from individual mice and reduce the use of animals from 27 to 3.

DBS analysis methods Chromatography/Mass Spectrometry. Chromatography combined off-line or online with (tandem) MS has been the major tool for DBS analysis for decades.30-37 The capabilities of this general method have been further expanded in the last two years, often times coupling directly the DBS sample processing device to the LC system, and/or utilizing two-dimensional (2D) UHPLC.38-42 Improved separation efficiency, sensitivity, quantitation, accuracy, precision, matrix and carry-over effects, and expanding the classes of compounds to be analyzed in DBS characterize most such methods. For example, simultaneous analysis of polar and non-polar pharmaceuticals was described using an online DBS extraction device coupled with LC/MS/MS.43 It is achieved by 2D separation of the polar and non-polar fractions on a zwitterionic-hydrophilic interaction (HILIC) column linked to a reverse phase (RP) C18 column, on which the non-polar fraction is eluted. The method was successfully applied to monitor in vivo buprenorphine metabolism in rats. 2D strong cation-exchange (SCX) RP LC/MS/MS for online enrichment, separation and detection of basic polar compounds, e.g., clonidine hydrochloride in monkeys, was described.44 Nearly all DBS sample values (ranging from 0.1 to 50 ng/ml) agreed with observations obtained by other methods. Combined analysis of twelve acylcarnitines and seven amino acids for newborn screening was performed utilizing HILIC amide column UHPLC/tandem MS (in multiple reaction monitoring, MRM, mode). Spiking the samples after extraction with isotopically-labeled internal


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standards demonstrated precision within 10% and accuracy within 15% for all analytes.45 A study of orotic acid (OA), a marker of hereditary aciduria, by HILIC LC/MS/MS, provided the age-related OA reference intervals in DBS samples from healthy controls.46 Combining multidimensional chromatography and tandem FTMS instruments with ultrahigh resolving power and mass accuracy requires novel bioinformatics software for clinical diagnostics applications (including DBS sample analysis).38

Direct mass spectrometry. Direct MS allows automated handling of DBS without any treatment prior to MS analysis.47, 48 When interfaced to tandem MS instruments, rapid identification and quantitation can be obtained in less than 3 min total run time. Thus direct MS can provide the high throughput capabilities required for, e.g., large screening, TK and PK studies. Desorption electrospray ionization (DESI) MS49 was successfully employed for direct analysis of xenobiotics in DBS without sample preparation and separation.50 Sensitivity down to 10 ng/mL and standard internal standard calibration curve linearity from 10-10 000 ng/mL was demonstrated. Comparison of different types of commercial filter paper shows that untreated papers are better substrates for DBS analysis by DESI MS. DESI is the precursor of a large number of ionization methods that allow samples to be directly analyzed in atmosphere, simplifying the instrumentation as well as improving significantly the throughput.51 One such ambient ionization method, introduced by Cooks and coworkers, that is particularly well suited to analysis of DBS and dried spots of tissue homogenates is paper spray.52 In paper spray, high voltage applied to a paper tip, containing the sample, generates gas-phase ions subsequently analyzed by MS (Figure 3). The tip is wetted with less than 10 uL solvent immediately


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prior to analysis, and the capillary action transfers the analytes towards the sharp end of the tip. The sample is spotted onto the paper (e.g., DBS) or blotted from another surface. It can also be deposited with the wetting solution. The time for sample preparation to analysis is less than 30 s. Paper spray is applicable to the MS analysis of a wide range of compounds - from small organics to large proteins (hemoglobin). Reported limits of detection for small molecules in blood are routinely less than 1 ng/mL, e.g., a chemically diverse set of therapeutics, including hydrophobic and weakly basic drugs (sunitinib, citalopram, verapamil).53 Drug concentrations were determined quantitatively over the entire therapeutic range with 10% accuracy by prespotting an internal standard onto the paper prior to application of the blood sample. Protein-drug interactions in DBS analysis by paper spray were studied for two drugs - propranolol and atenolol - with widely disparate protein binding properties.54 These studies suggest that protein binding does not affect analyte signal. Introduction of internal standards by three different methods pretreating the paper, doping into the spray solvent, and adding to a punched out DBS section - was compared. Less than 8% variance is observed when adding standard to the paper either before or after adding blood, a factor of 2 lower than the variance when standard is added to the eluent. Matrix effects result in 10 times lower ion signal when the drug is sprayed from a drop of whole blood drop as compared to neat solution. Paper spray analysis of less than 100 pg heroin or pesticides, swiped from surfaces (including fruit peels) was demonstrated. Compounds can be also derivatized on the tip to increase sensitivity and specificity, as illustrated for cholesterol in human serum. Furthermore, paper spray is easily interfaced with various mass spectrometers, from high resolution FT and tandem machines to portable instruments. The latter combination would allow rapid


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and sensitive analysis directly at point-of-care facilities, even in resource-limited environments.55 Effects of paper and wetting solution types on analyte release, diffusion through the substrate and ion formation, were studied with the aim to optimize paper spray for TDM.56 Silica-coated paper (commercially available) and dichloromethane/ isopropanol as wetting solvent were identified as optimal for analysis of various drug types in DBS, compared to, e.g., chromatography paper and methanol/water.

The

recovery efficiency as well as the limit of quantitation are 5 to 50-fold improved with the silica-coated paper. Paper spray MS was applied to direct and quantitative analysis of a mixture of ten underivatized acylcarnitines in serum and whole blood at various concentration and without any sample pretreatment, separation, or derivatization.57 Methanol/water/formic acid spray solvent was found to be optimal for paper spray of serum, while acetonitrile/water was much more suitable for DBS. The limits of detection and quantitation are much lower than respective clinical values in fatty acid oxidation disorders, thus demonstrating the potential of paper spray MS (and MS/MS) for large scale, high throughput neonatal screening.

Hemoglobinopathies are the most common single gene disorder afflicting humans. However, neonatal screening performed by isoelectric focusing (IEF), coupled to HPLC, is technically more challenging than screening for other inborn disorders. Recently, MALDI TOF MS was investigated as a first-tier universal profiling method for hemoglobinopathy screening in neonathes.58 MALDI MS offers several practical advantages: high throughput, cost-effectiveness, sensitivity and ability to automatically analyze data for anomalous hemoglobin peaks. The assay takes around 1 min. per DBS


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sample. In a retrospective study of more than 800 individual Guthrie card, a very high degree of correlation with the reference was obtained. Liquid microjunction surface sampling of DBS26 and subsequent direct introduction in a high-resolution tandem FTMS is the basis of another very accurate method for characterization of variant hemoglobin (Hb) a- and b- chains.59 Top-down proteomics with complementary collisionally-induced dissociation/ electron transfer dissociation (CID/ETD) precursor ion excitation results in increased (typically > 60%) sequence coverage of the intact proteins. Combined with the high resolving power (~ 100k) capability of the mass spectrometer, the tandem mass spectra allow the detection and successful diagnosis of Hb variants from 5 out of 6 clinical samples marked as “unknown” after analysis by HPLC (accepted for routine neonatal screening in the UK). Top-down proteomics provides several well-known advantages over bottom-up proteomics (the latter has already been applied for Hb variant characterization60). Among these is the increased sensitivity of the former, the reduction in sample preparation/analysis time, the “conservation” of sequence/amino-acid connectivity information, direct detection of anomalies.

A direct laser desorption (LD) TOF MS method for detection of malaria in spots of diluted whole blood, deposited on metal substrates, was described several year ago.61 The method is based on the sensitive and specific detection of heme, sequestered by the parasite in malaria pigment crystals in red blood cells. The LDMS method was compared to PCR and microscopy smear analysis of samples from pregnant women.62 The LDMS method is more rapid and potentially more sensitive than microscopy and comparable to PCR. While the initial LDMS equipment cost may be higher than PCR, LDMS sample


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preparation and analysis per patient is much less expensive, and it can be easily automated. A novel laser diode thermal desorption (LDTD) atmospheric pressure chemical ionization (APCI) interface was applied for the rapid and reproducible direct MS/MS analysis of drug mixtures, extracted from DBS.63 The commercially-available LDTD device in a 96-well format can be optimized for sensitivity and selectivity in an automated fashion, providing simultaneously high throughput capabilities.

Inductively-coupled plasma (ICP) MS provides a method for direct elemental analysis of DBS. The method requires no sample pretreatment (e.g., digestion or filtering), and is amenable to automation for rapid screening of large number of samples. A feasibility study for Pb quantitation in DBS by laser ablation (LA) ICP TOF/MS was published several years ago.64 Less than 2% of the sample is consumed in LA, thus the DBS can be stored for further analysis. Using LA ICP/MS, thirteen elements, present in whole blood (Be, Mn, Co, Ni, Tl, Bi, Sb, Pb, Cu, Zn, Ba, Mg, and Cd) have been simultaneously quantified.65 No additional sample pretreatment was done, thus increasing the throughput of 5 minutes per sample. Within-run precision less than 10% and good reproducibility has been obtained.

DNA-based assays. DNA amplification, e.g., PCR, has become one of the major molecular level methods for clinical diagnostics and screening of infectious diseases as well as genetic disorders. Therefore, the applications of PCR and similar nucleic acid amplification techniques for DBS analysis are constantly expanding.12 The influence of extraction and PCR protocols on human cytomegalus virus (HCMV) detection in DBS


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was studied recently.66 HCMV congenital infection affects up to 2% of newborns. Detection of DNA from HCMV in DBS allows discrimination between congenital and postnatal infection. Optimizing extraction with phenol-chlorophorm and qPCR a sensitivity of 200 copies HCMV DNA/ml has been achieved. The accuracy of one- or two-primer real-time PCR assays for newborn HCMV screening from DBS was compared to saliva rapid culture.67 The conclusion is that the PCR sensitivity, compared to the rapid culture, has lower sensitivity, limiting its value as a screening test. A similar conclusion was drawn from a pilot study, in which DBS PCR was found not sufficiently sensitive as a screening tool compared to throat swabs.68 A more recent study involved neonates born with congenital HCMV symptoms or born to mothers with a history of primary infection during pregnancy.69 It compares two PCR DBS tests to a gold standard assay (PCR in urine). The sensitivity and specificity of both HCMV PCR DBS assays are very high in this high risk neonate population, thus suggesting that DBS can still be considered for congenital HCMV screening.

The diagnosis of HIV in infants younger than 18 months cannot be performed by the standard antibody tests. Therefore, diagnosis in that age group depends on HIV nucleic acid detection. Further, earlier diagnosis is important for initiating antiretroviral treatment as early as possible thus reducing infant mortality. Several recent studies compare the performance of PCR-based diagnostic kits for extracted DBS samples to plasma or whole blood samples.70, 71 Under specific conditions, DBS are appropriate for HIV screening and therapeutic monitoring in resource-limited settings.71 Three commercially available PCR assays were compared in HIV detection from serial DBS samples, collected at birth


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and at 2- 4- and 6-weeks from HIV-exposed infants (in utero).72 For all assays, the conclusion has been that testing at birth is most successful in detecting the infection, compared to later time-points. Simultaneous detection of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) in DBS was achieved by a qualitative multiplex real-time RT-PCR.73 Specific viral amplicons are detected from 2500 copies/ml of HCV and 400 copies/ml of HIV-1, suggesting the assay can be applied in large scale screening even in resource-poor settings. The effects of three commercial filter paper types for HIV-1 detection by PCR of DBS were evaluated.74 DBS were stored at three different temperatures (25oC, 37oC and -20oC) for up to 12 weeks. HIV-1 DNA was detected successfully on the three DBS cards, regardless of storage conditions and PCR assay type. Two newly developed low-cost RT-PCR assays - one targeting the HIV-1 integrase gene and a “Long Terminal Repeat” assay - were compared with good results for both.75 The sensitivity of the second assay is approx. 600 HIV copies/ml), after introduction of an internal standard, making it a good candidate for implementation in resource-limited settings. Another HIV-1 detection assay for resource-limited areas includes manual DNA extraction from DBS and isothermal amplification and melt-curve analysis.76 The manual extraction protocol consistently demonstrates sensitivity down to 470 copies/ml, comparing it favorably to commercial extraction kits. Effects of DBS storage temperature and time on the performance of PCR assays for monitoring HIV drug resistance in patients were studied, with the conclusion that for DBS stored for 3 months at 37oC HIV could be genotyped at 5000 copies/mL.77 DBS extraction protocols and paper type on PCR-based assays for HIV drug resistance surveillance were examined.78 Interrogating multiple DBS from the same patient provides the necessary sample amount


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for detection of minor (i.e., drug-resistant) HIV variants. Further it is recommended that for highest sensitivity DBS are stored at sub-ambient (+4oC to -20oC) temperatures.

Severe combined immunodeficiency in infants can be detected by quantitation of a specific functioning T-cell DNA marker - the T-cell receptor excision circle (TREC). A sensitive multiplex qPCR assay with internal control was developed for TREC quantitation in DBS, that could be applied for large-scale newborn screening.79 Manual and automated column-based extraction protocols combined with qPCR for measuring TREC in DBS were compared.80 The automated protocols provide lower error rates, avoid contamination and reduce sample handling times.

High resolution DNA melting analysis was applied for detection of 20 Fabry disease mutations from DBS cards of newborn females, considered until recently only asymptomatic carriers.81 While female carriers are not detected by enzyme assays, the DNA assay was successful in identifying heterozygous and hemizygous patients, suggesting that it may become a reliable and sensitive rapid screening method. DBSbased spinal muscular atrophy (SMA)-diagnosing and screening system was developed.82 DNA, extracted from the DBS stored at room temperature for up to 4 years, is subjected to PCR-restriction fragment length polymorphism analysis. The results from a DBS assay are concordant with results from fresh blood specimens, demonstrating the reliability of the DBS sampling for SMA screening


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Enzyme activity assays. Screening assays for several inherited metabolic disorders (Fabry, Gaucher, Krabbe, Niemann-Pick A/B, and Pompe) are based on measuring activities of the respective lysosomal enzymes (e.g., alpha-glucosidase, alphagalactosidase, beta-glucocerebrosidase, sphingomyelinase, and galactocerebrosidase) in DBS fluorometrically or by tandem MS. A direct multiplexed assay for all five enzymes uses synthetic substrates and internal standards to quantify substrate degradation products by tandem MS in MRM mode.19 In the assay, which can be readily automated, rehydrated DBS are incubated with the substrates, followed by liquid-liquid extraction with ethyl acetate, drying, SPE and purification before flow injection analysis (FIA) by MS/MS. For all five diseases, samples from patients with the disorder showed lower enzyme activity, compared to samples from healthy or heterozygous controls. The performance of the assay was improved by incorporating a single buffer for simultaneous measurement of the activities of three lysosomal enzymes (alpha-glucosidase, alpha-galactosidase A, and alpha-L-iduronidase).83 The triplex assay can indicate “problematic” DBS, e.g., insufficient blood deposited, if enzyme activity values are low for all enzymes (which is not clinically observed). The assay, evaluated on more than 5000 DBS samples, reduces both amount of chemicals and processing time, and thus it can be adapted for high throughput neonatal screening. Four different sample handling methods - two HPLC and two FIA - and respective hardware were evaluated for the triplex enzyme assay using fast liquid chromatography.84 In one method, assay quenching with acetonitrile to precipitate blood proteins was followed by HPLC/MS/MS analysis by switching between two parallel columns for increased throughput. A triplex sample analysis takes 1.5 min, matching the throughput of the method based on liquid-liquid extraction into ethyl acetate


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and flow injection.19 The former also produces excellent results (Figure 4). The other methods involved liquid-liquid extraction into ethyl acetate followed by HPLC/MS/MS and acetonitrile quenching followed by direct flow injection. It is concluded that HPLC/MS/MS is a viable alternative for high throughput multiplex neonatal screening for lysosomal disorders, using DBS. A fluorometric assay was used to compare alphaglucosidase enzyme activity determined in DBS versus lymphocytes, obtained from Pompe disease patients.85 The observed DBS activity values provided reliable diagnosis from all samples, suggesting that the assay could be adapted for neonatal screening.

Immunoassays.

Two

commercially-available

immunoassays

were

tested

and

experimental conditions were optimized for the detection of HCV antibodies in DBS.86 The effects of DBS long-term storage temperature (ambient versus -20oC) and time, type of elution buffer, sample volume and incubation time were evaluated for both DBS and serum samples. Accurate results were obtained with both commercial kits from DBS stored for more than 100 days, and samples stored at -20oC had lower result variations. The feasibility of using a competitive ELISA for quantitation of biologics in DBS was addressed on the example of exenatide (a 4.1 kDa peptide drug for diabetes).87 Extraction protocols were optimized by varying solutions (with or without protease inhibitors) and incubation times. The assay was linear for 100-5000 pg/mL exenatide blood concentrations. Exenatide on DBS was stable for storage at temperatures from 4o to 700C. ELISA, combined with DBS, was evaluated for quantification of an anti-CD20 monoclonal antibody drug from DBS.88 The assay range of the anti-CD20 drug standards in DBS was 100-2500 ng/mL. The drug was stable for one week in DBS stored at room


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temperature as well as after 3 freeze/thaw cycles. High humidity and temperature (55oC) as well as direct sunlight degraded the drug. Both these studies demonstrate the potential of the ELISA-DBS combinations for quantitation of large molecule drugs or biomarkers.

A high sensitivity ELISA for quantitation in DBS of C-reactive protein (CRP) - a biomarker of morbidity and mortality risk as an objective indicator of acute infection was developed.89 Readily available monoclonal antibodies allow reproducible CRP quantitation at physiologically relevant concentrations in DBS as well as other blood specimen types. Concentration CRP in serum was on average 1.6 times (SD 0.37) higher than in DBS. Contrary to other reports, it was found that CRP degraded quickly upon DBS storage at room temperature, and even at -20oC upon longer-term storage. Thus, ELISA-DBS as an inexpensive and efficient tool can still be used for large-scale population health screening, provided that the storage time is shortened. This was demonstrated in a recent successful study of the specificity and sensitivity of the ELISADBS method for quantitation of CRP and alpha 1-acid glycoprotein to diagnose episodes of acute infection among children.90 The need for novel assays for neonatal screening for primary immunodeficiency disease stems from the fact that the PCR assays for TREC79 are not applicable to the most common forms of the disease. To assess the applicability of IgA as a biomarker for the disease, DBS from three different sources (patients with various forms of the disease, healthy newborns, and healthy newborns with IgA-deficient mothers) were analyzed by ELISA.91 Surprisingly, it was found that IgA in neonatal DBS had both maternal and fetal origin. This finding precludes the use of IgA in DBS as a reliable biomarker of primary immunodeficiency diseases.


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Microfluidics. The “lab-on-a-chip” with its nano- to picoliter sample volumes, minimized energy and material consumption, miniature size, automation and multiplexing capabilities, and reduced cost for mass production is revolutionizing many aspects of experimental science - from materials and synthetic chemistry to cell and molecular biology.92-95 Microfluidics holds promise to become the next generation technology for DBS screening for a wide range of applications.96-98 A digital microfluidics platform was developed for rapid, multiplexed enzymatic assays of acid alpha-glucosidase (GAA) and acid alpha-galactosidase to screen for Pompe and Fabry disorders.97 The assay has fully automates all liquid-handling operations in an inexpensive overall system. Its performance was compared to standard fluorometric methods. The incubation time from a single DBS punch was reduced from 20 h for the standard assay down to 1 h for the microfluidics assay, with 3 times higher mean measured GAA enzymatic activity for the latter. Clear separation between samples from control and diseased individuals was achieved. A similar digital microfluidics approach was successfully used for the development of a rapid, single-step assay for Hunter syndrome in DBS.99 Digital microfluidics, coupled to off-line or on-line MS/MS, was employed in a prototype system for amino acid quantitation in DBS.98 Analytes are extracted, mixed with internal standards, derivatized, and reconstituted on the chip in an automated fashion. A similar digital microfluidics chip for DBS sample extraction and derivatization with feedback droplet control was evaluated for quantitation of succinylacetone (tyrosinemia marker).100 The feedback allows high-fidelity droplet manipulation without manual intervention. A nanoelectrospray capillary emitter, sandwiched between two substrates of the chip, is the


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interface to a MS instrument for on-line analysis (Figure 5). The demonstrated performance is comparable to the currently used clinical laboratory methods. A multilayer paper, glass fiber and plastic device for performing five-step isothermal enzymatic amplification of HIV DNA was designed and tested.101 DNA is amplified and then detected with commercially available lateral flow strips in 15 minutes with detection limits of 10 HIV copies. Combined with extraction from DBS, the device can be deployed for early HIV diagnosis at point-of-care facilities in resource-limited settings.

Select applications and challenges. The constantly growing range of DBS applications includes both traditional, e.g., large scale neonatal screening, microbiological and epidemiological disease surveillance, and more recent ones, e.g., preclinical drug development - lead validation, TK, PK and TDM, clinical pharmacology, targeted and non-targeted metabolic profiling, forensic toxicology, doping or environmental contaminant control. In fact, DBS may follow us from “the cradle to the grave”, as pointed recently.102 Only a few recent applications, not already indicated in conjunctions with DBS analysis methods, will be listed below.

Patient screening. The most widely used application of DBS in the clinical laboratory, with greatest societal impact, is neonatal screening of inborn errors of metabolism. As pointed out almost a decade ago, more cases of 31 different inborn metabolic disorders are diagnosed by DBS screening than clinically.103 Practical approaches and screening methods, routinely utilizing tandem MS, were thoroughly reviewed.30,31,34,104 A recent study discusses the effects of contamination during intravenous feeding of premature


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infants on DBS-based MS/MS metabolic profiling.105 Observation of background peaks from dextrose, a major component of the parenteral nutrition solution, is an indication of possible contamination with amino acids. The study concludes that the combination of dextrose markers, very high elevations of amino acids and their unusual molar ratios is an indication that the sample was collected improperly and the observation should be rejected (rather than declaring it false positive). MS/MS had been evaluated as a tool for newborn screening for mucopolysaccharidosis IVA106 and mucopolysaccharidosis II107. The assays determine the N-acetylgalactosamine-6-sulfate sulfatase and iduronate-2sulfatase in DBS, respectively, by using appropriate synthetic substrate for each enzyme. Both assays showed high degree of discrimination between diseased (enzyme-deficient) and normal samples. Both require minimal number of sample-preparation steps, and they can be automated and multiplexed, making them good candidates for implementation for large scale patient screening. Development of high-throughput methods for analysis of serum N-glycans have been prompted by their potential use as biomarkers for diseases, e.g., cancer(s), Alzheimer’s disease, cirrhosis, fibrosis and diabetes. A method for qualitative N-glycan profiling in DBS was reported recently.108 DBS punches were reconstituted in N-glycan release buffer, proteins were denaturation by DTT, and glycans were released by PNGase. After ethanol precipitation and graphitized carbon SPE, samples were loaded on a nano-HPLC-chip-TOF instrument for MS and MS/MS analysis. Commercially-available software was used for identification of around 450 Nglycan structures can be monitored, originating from 44 N-glycan compositions with good repeatability. N-glycans were stable upon DBS storage at room temperature. The observed N-glycan profiles from a DBS and a respective serum sample (the classical


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matrix for N-glycan profiling) were very similar (Figure 6). High (>100k) resolution single stage MS has been presented as an alternative to HPLC/MS/MS for large scale metabolomic profiling.109 More than 400 individual compounds, including acylcarnitines, amino acids, organic acids, carbohydrates, lipids, could be routinely identified and quantified from DBS extracts, containing internal standards. Fully automated chip-based nanoelectrospray in both positive and negative ion modes reduced total sample analysis time on an FTMS instrument to less than 2 min. While the dynamic range for some compounds

was

lower,

comparison

with

traditional

HPLC/MS/MS

methods

demonstrated lower false-positive rates for, e.g., PKU screening thus reducing the need for subsequent second-tier sample analysis.

Preclinical drug development. A DBS method for acetaminophen quantitation at physiologically-relevant concentrations (0.1-50 ug/ml), utilizing reversed phase HPLCMS/MS, was developed.110 DBS (15 uL blood) were extracted with methanol and electrosprayed for MRM analysis. The analyte was stable for at least ten days in DBS stored at room temperature. The results of this pioneering study - reproducibility, accuracy - were compared to traditional analysis methods involving much larger volumes of whole blood. These results demonstrate convincingly that DBS can be used for TK and PK studies. An issue in expanding the applications of DBS for such studies is the validity the monitored drug/metabolite levels when compared in the solid versus the liquid. While in DBS the total drug concentration is measured, in liquid blood it is the amount of unbound drug (dissolved in the plasma) that is monitored.111 The ratio of unbound-tobound drug in blood, its variations with overall concentration as well as in time, all need


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to be explored and addressed in vitro prior to the start of PK and TK efforts. TK studies of pioglitazone using DBS and whole blood in two different laboratories were reported.112 Comparable (within 3%) concentrations are obtained from DBS and whole blood samples. Further, results for DBS samples, analyzed in two different laboratories by different methodologies, are also comparable indicating that DBS sampling is robust and can be used widely for TK and PK studies. A viable strategy to expand the use of DBS and LC/MS/MS in discovery toxicology was compiled.113 Standard curves, generated from DBS without dilution, were linear from 78 nM over four decades, and in three different studies of different analytes. Utilizing DBS circumvents the need for sample dilutions, thus reducing the potential for error and variability, simultaneously with reduced analysis time. The overall importance of accurate drug quantitation in conjunction with DBS assays for development of better treatment strategies for children was stressed recently.114 It stems from the significantly reduced availability of TK and PK data for children, compared to adults.

Therapeutic drug monitoring. TDM can be accomplished either by monitoring the drug, the biomarker or their metabolites directly on the DBS or, as in the case of viral infections, monitoring the viral load after administering the drugs. Monitoring drug efficacy ensures early detection of emerging drug resistance in, e.g., HIV, and signals a transition to a different drug. The advantages and pitfalls of DBS-based methods for TDM were reviewed.115 It is argued that DBS offers both benefits and potential for errors, as well as further need for assay standardization and quality assurance. A recent example of DBS for TDM is based on LC MS/MS.116 Using MRM mode, in which ion


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fragmentation channels specific for a particular analyte are detected, four metabolites and a drug in blood of patients suffering from type 1 tyrosinemia are simultaneously monitored. The assay is rapid - approximately 30 min., including sample preparation, and quantitative - utilizing isotopically-labeled internal standards. It allows drug efficacy optimization while simultaneously minimizing side effects. The DBS assay results are compared to measurements in plasma, with good correlation between the two sets of measurements. A similar protocol was also successfully tested for the quantitation of ertapenem (a beta-lactam antimicrobial agent) in DBS.117 MALDI is an ionization technique that can provide MS analysis of DBS either directly or with minimal sample pretreatment. An ultrafast and high-throughput MALDI MS assay for TDM of antiretroviral drugs (lopinavir and ritonavir) in pediatric HIV-1 treatment was reported.118 DBS extracts (acetonitrile/water) and spotted on MALDI slides are analyzed by triple quad MS/MS. The assay was successfully cross-validated with an HPLC-UV assay.

Banned substances/doping. All recently-reported studies demonstrate same reliability of results obtained from DBS, compared to whole blood, as required by regulatory and law enforcement agencies. Detection of 26 banned drugs from different classes was achieved at relevant regulatory and clinical concentrations (less than 0.5 ng/mL) with one generic sample preparation of DBS.119 In this method, DBS organic solvent extracts are separated by UHPLC and analyzed for the target compounds by electrospray tandem MS on an instrument that combines a quadrupole mass filter, a higher collision dissociation energy cell and a ultrahigh resolution FT detector. A full scan in positive and negative mode is followed by single-product ion scans in data-dependent analysis mode (employing an


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inclusion list) for preselected target compound precursor ions appearing at a specified retention time. GHB (gamma-hydroxybutyric acid) – a date-rape drug – was quantified in DBS by punching, on-spot derivatization and subsequent GC/MS analysis.120 The method is independent of blood hematocrit and is linear within the forensically-important range of 2-100 ug/ml, as specified by US and European regulatory agencies. In another study, successful on-line extraction HPLC/MS/MS for the simultaneous determination of a large number of illicit drugs in DBS was reported.121 The illicit drugs tested - five opiates, five, and five amphetamines - were all recovered at least up to 80%. Analytes were stable for up to 6 months on DBS stored at -20oC.

A MS-based assay for detection in DBS of large molecules, banned for use in sports, was developed and validated on the example of peginesatide.122 Peginesatide is a commercially-available pegylated erythropoietin-mimetic peptide (MW 45 kDa), approved for anemia treatment, but banned as a performance-enhancing drug. Utilizing bottom-up proteomics protocols, DBS is first extracted, the extract is then subjected to proteolytic digestion and purification by cation-exchange and finally analyzed by HPLC/MS/MS. The assay is specific, sensitive (limit of detection 10 ng/mL) and precise (relative standard deviations below 18 %).

Large (> 4 kDa) biomolecules. There are intense efforts in the pharmaceutical industry for development of large biological molecules as drugs (biologics). These efforts in turn require validated and accurate methods for bioanalysis. A procedure for qualitative analysis of biologics in DBS was developed.123 It is similar to the LC/MS/MS bottom-up


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proteomics protocol for peginesatide analysis described above.122 Two structurally different therapeutic proteins - pegylated Adnectin-1, MW 11.1 kDa, and Fc-fusion protein, MW 67 kDa - were selected for the method development in this study. Both proteins were stable in DBS stored at room temperature for at least two weeks. The authors argue that the issues for practical application of DBS analysis in TK and PK studies of biologics are analogous to those for small molecules: analyte stability in DBS, blood volume, hematocrit levels, extraction efficiencies, etc. It is anticipated that with further advances in quantitative proteomics in particulars, the applications of DBS in the analysis of large molecules (biologics as well as disease-specific biomarkers59) will be expanding.

Health/disease surveillance. DBS as a minimally-invasive technique provide an ideal sampling method for large scale disease outbreak and prevalence surveillance, mass screening for initial and chronic infections and treatment progress.124 The logistical ease of transport and storage, the constantly improving sensitivity and specificity of the analytical methods turn DBS into a universal tool for world-wide public health data collection and integration. Large retrospective studies of congenital virus infection in newborns using stored DBS have already been performed. A literature review on DBS for diagnosis, monitoring and epidemiological studies of CMV, HBV, HCV, HAV, HEV, HTLV, EBV, HSV, measles, rubella and dengue, was published recently.125 The application of DBS for HIV load quantitation for drug resistance monitoring and treatment in resource-limited settings was also reviewed.126 PCR was successfully introduced for genotyping of Plasmodium (the malaria-causing parasite) in DBS more


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than 20 years ago.127 During that time, DBS extraction protocols, PCR sensitivity and assay throughput have improved significantly. Equally important, the costs per assay have dropped, allowing its introduction for parasite detection and control even in resource-poor settings. RT-PCR, targeting the parasite lactate dehydrogenase gene, and conventional microscopy were compared for malaria detection in DBS or smears from peripheral blood.128 RT-PCR offers more reliable parasite detection at low parasitemia levels than conventional microscopy, which is still more affordable and easy to deploy in the field. Semi-nested multiplex PCR performance for Plasmodium detection using DBS was compared to 2 ml frozen venous blood samples.129 The assay has higher specificity but lower sensitivity for DBS compared to the frozen blood samples. To reduce costs, a strategy for pooling individual DBS prior to PCR-based detection of malaria parasites was proposed.130 Reliable detection of a single low-parasitemic sample (100 parasites/ul) in pool sizes up to 50 has been achieved.

Perspective Applications of DBS sampling and analysis will continue to expand and close the circle from health monitoring and rapid diagnosis through drug development to personalized point-of-care therapy. With exponential reduction in cost, it is anticipated that DNAbased analysis of DBS would be expanding. Microfluidics coupled with DBS sampling would play an increasing role for, e.g., targeted TDM at point-of-care facilities, as well as large scale screening in resource-poor settings. The analyses of dried spots from other bodily fluids and tissue homogenates will be expanding as well, in parallel with their applications in diverse areas, that include preclinical drug development, diagnostics and


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treatment. Furthermore, the advent of next-next generation sequencing technology would provide in the not-so-distant future the entire sequence of an individual’s genome from a single drop of blood. Exhaustive microbiome and metabolome profiles for an individual will also be obtained from a single DBS. Combining this wealth of data with the help of bioinformatics, the medical practitioner will utilize it for more rapid and accurate patient diagnosis and personalized treatment.


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Plamen A. Demirev is Principal Staff at the Applied Physics Laboratory, Johns Hopkins University, Laurel, Maryland. He received his MS in Physics and PhD in Chemistry degrees from the University of Sofia and the Bulgarian Academy of Sciences, respectively. From 1990 to 1998 he has been on the faculty of Uppsala University, Sweden. His current research includes physical methods, sensor systems and informatics for rapid detection of chemicals and pathogens in complex environments. He has developed a laser desorption mass spectrometry method for rapid malaria detection from dried blood spots.


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Figure captions

Figure 1. Number of publications per year for the last 20 years that contain the term “dried blood spot”. More than 2300 papers have been published since 1936, while their number in the last three years alone (since 2010) is more than 1200 (data from ThomsonReuters Web of Science® bibliographic database). A Google search with the same term returns more than 1.5 million hits.

Figure 2. Experimental setup for automated deposition of chemicals, e.g., internal standards, on spots of dried blood. Reprinted with permission from ref.28. Copyright 2011. American Chemical Society.

Figure 3. A. Principle of paper spray ionization. B. Paper spray DBS mass spectrum and tandem mass spectrum (insert) of a drug (atenolol, in two different amounts in 0.4 ul blood, spotted directly on the paper tip). The spray solution is 1:1 methanol/water (10 ul). Adapted with permission from ref.52. Copyright 2010. American Chemical Society.

Figure 4. GAA activity distribution for 4 Pompe patients and 31 normal controls, determined by HPLC/MS/MS after acetonitrile precipitation of proteins. Diseased from healthy subjects are readily discriminated. Reprinted with permission from84. Copyright 2011. American Chemical Society.


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Figure 5. A. Digital microfluidics device (mounted on a 37.5mmx25mm glass plate) for direct analysis by nanoelectrospray of amino acids in dried blood. B. Representative mass spectrum of amino acids in blood. Adapted with permission from ref.100. Copyright 2012. American Chemical Society.

Figure 6. Qualitative and quantitative comparison of N-glycan classes obtained from DBS and serum, respectively. The areas are correlated with the relative N-glycan abundance within each class, the numbers indicate the individual identified structure within each class. Adapted with permission from ref.108. Copyright 2012. American Chemical Society.


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450 400 350

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300 250 200 150 100 50 0 1990

1995

2000

2005

2010

2015

YEAR ACS Paragon Plus Environment

DBS_Figure 1


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DBS_Figure 2

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A.

B.


DBS_Figure 3


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DBS_Figure 4

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A.


Blood spot Nano ES Fp

B.


DBS_Figure 5


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DBS_Figure 6

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Health monitoring, diagnosis

Disease surveillance

Dried blood spot analysis

Drug development

Therapy


DBS_TOC Graphic

Dried Blood Spots: Analysis and Applications - Analytical Chemistry

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