896
DOI 10.1002/prca.201400042
Proteomics Clin. Appl. 2014, 8, 896–900
REVIEW
The utility of dried blood spots for proteomic studies: Looking forward to looking back Vera Ignjatovic1,2 , James Pitt2,3 , Paul Monagle2,4 and Jeffrey M. Craig1,2 1
Murdoch Childrens Research Institute, Royal Children’s Hospital, Victoria, Australia Department of Paediatrics, The University of Melbourne, Victoria, Australia 3 Victorian Clinical Genetic Services, Royal Children’s Hospital, Victoria, Australia 4 Department of Clinical Haematology, Royal Children’s Hospital, Victoria, Australia 2
The possibility to detect biomarkers of adult disease in early life and particularly in newborns holds enormous promise for early disease detection and prevention. Early detection of disease or potential for future disease would allow for prevention or amelioration of disease before overt symptoms develop, by lifestyle modifications, appropriate medication and monitoring. It is now increasingly important to develop the technologies that allow dried blood spots (DBS) to be utilized for protein-based studies. The use of DBS in proteome wide association studies (PWAS) may in turn allow for detection of major diseases of adulthood at the earliest possible time. This review focuses on the utility of DBS in proteomics, the main challenges, as well as the latest approaches for overcoming those, facilitating the use of DBS for detection of major diseases of adulthood at the earliest possible time.
Received: April 7, 2014 Revised: August 4, 2014 Accepted: September 3, 2014
Keywords: Disease prevention / Dried blood spots
1
Protein biomarkers of complex disease
A biomarker is any biological characteristic: molecular, physiological, or biochemical—that can be measured objectively and act as a predictor or indicator of a normal or a diseased state. The search for protein biomarkers of complex diseases such as cardiovascular disease (CVD), cancer and diabetes has seen significant investment in the past 10–15 years. However, despite this investment in technology and an increase in proteomics-based studies in a variety of clinical settings, the discovery of protein biomarkers has not advanced at the expected rate [1]. In fact, there are very few disease biomarkers that have been discovered using a proteomics-based approach.
Correspondence: Associate Professor Vera Ignjatovic, Haematology Research Laboratory / Murdoch Childrens Research Institute, Flemington Road, Parkville, Victoria, 3052, Australia E-mail: [email protected] Abbreviations: CVD, cardiovascular disease; DBS, dried blood spots; EWAS, Epigenome wide association studies; MRM, multiple reaction monitoring; PEA, proximity extension assay; PWAS, proteome wide association studies C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2
Challenges of current proteomics approaches
The methodological approach in proteomics studies varies significantly not only based on the choice of samples, but more importantly based on the specific methods utilised. At the sample level there is a significant variation in sample preparation and fractionation techniques. For example, serum and plasma samples are widely used; however, there is significant variation in the concentrations of proteins present within each of these. Plasma is separated from whole blood using centrifugation, while serum is obtained from whole blood by a two-step process that involves formation of a blood clot and subsequent centrifugation, to separate the serum from the clot and other cellular elements. In plasma, for example, there is a ten order of magnitude difference in concentration of the least and most abundant proteins [2]. In turn, proteins of interests may be difficult to detect against a background of high abundance proteins such as albumin and immunoglobulins. Therefore, the preparation of plasma and sera for proteomic studies may involve a variety of depletion techniques (e.g., albumin, IgG, or depletion of six or 12 major proteins) and/or fractionation techniques (i.e. multi lectin affinity chromatography, cation exchange). When it comes to the analysis of tissue-specific proteomes (i.e. kidney, tumor), the variation lies in the way the tissue extracts are prepared (i.e. direct lysis, enrichment of particular cells such as tumor www.clinical.proteomics-journal.com
897
Proteomics Clin. Appl. 2014, 8, 896–900
cells, density gradient centrifugation). It is important to realize that sample preparation can potentially distort the proteome through nonspecific binding or failure to extract hydrophobic (membrane-bound) proteins, which could lead to the loss of informative biomarkers. Comprehensive studies may therefore require more than one sample preparation technique to ensure wider coverage of the proteome. Once prepared, the proteome of a specific biological sample can be further analyzed by using multiple approaches such as gel electrophoresis (1 or 2D), 2DE, as well as enzymatic proteolytic digestion, all of which are followed by the use of MS. The MS component of the proteomics studies can be performed using a variety of approaches and instruments, such as LC-MS, LC-MS/MS, and MALDI-TOF. Each of these technologies has strengths and limitations with respect to parameters such as analytical sensitivity, imprecision, and specificity as well as sample throughput, ease of use, and costs [3]. The rationale for choosing any particular method over another in a given circumstance needs considerable validation and standardization. Such a process would require a focused effort of the proteomics community in concert with translational research environments that are focused on solving the health challenges that face our society. In this effort to overcome the challenges of current proteomics approaches, a different research target might need to also be considered, where the focus is consciously shifted from the differences between disease and health, to early detection and prediction of disease.
priate medication, and monitoring. Reducing the incidence of major diseases such as CVD, cancer, and diabetes would improve the quality of life for individuals, as well as reduce the health care related costs and burden on governments and individuals alike. Epigenome-wide association studies (EWAS) are an example where this early detection approach has been utilized successfully. Epigenetics describes the chemical tags that associate with DNA, over and above the primary sequence, to control gene activity. These tags are stable through cell division but can change during early development and react to environmental change. In a typical EWAS, cases and controls are analyzed on a genome scale for epigenetic marks such as DNA methylation and associations adjusted to levels of genome-wide significance [10]. Samples sizes have ranged from 200; most have focused on a single time point. However, notable studies have included longitudinal analysis, providing potential biomarkers of disease risk in asymptomatic individuals (e.g., type 1 diabetes in children) [11] or markers of disease progression (e.g., type 2 diabetes in adults) [12]. EWAS can also be performed within a group of individuals for a particular environmental exposure, such as maternal smoking in pregnancy [13]. If birth samples are analyzed, both types of EWAS could be performed on the same set of samples, as has been done for candidate-based epigenetic analysis [14]. Birth samples may be collected prospectively [15] or accessed retrospectively, in the form of dried blood spot (DBS) samples, the so-called “Guthrie cards” [16–19].
4 3
Early life detection of biomarkers of adult disease
The majority of proteomic biomarker studies to date have relied on protein-based differences that can be detected in individuals with disease compared to healthy individuals, specifically in the adult setting. Anderson has suggested a set of 177 protein biomarkers from human plasma that could be useful in the setting of CVD [1]. Cardiac troponin I has been shown to be a strong predictor of mortality following cardiac surgery [4]. Differences in the abundance of apolipoprotein E, afamin, isoform 1 of interalpha trypsin inhibitor heavy chain H4 (ITIH4), alpha-2-macroglobulin and ceruloplasmin have been demonstrated to be associated with breast cancer compared to age-matched controls [5]. Developmental Origins of Health and Disease, previously known as the Fetal Origins of Adult Disease, hypothesizes that major diseases such as coronary heart disease, stroke, hypertension, and type 2 diabetes originate in utero and during early infancy [6–9]. The possibility to detect biomarkers of adult disease in early life and particularly in newborns holds enormous promise for early disease detection and prevention. Early detection of disease or potential for future disease would allow for prevention or amelioration of disease before overt symptoms develop, by lifestyle modifications, appro C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The utility of DBS samples in proteomics
Dried blood spot sample represents a unique individual sample collected within a few days postbirth that was originally designed for screening of inborn errors of metabolism and other congenital disorders. Examples of diseases currently screened include phenylketonuria, congenital hypothyroidism, cystic fibrosis, and sickle-cell disease. From the late 1990s, newborn-screening laboratories also began to introduce multiplex testing for approximately 25 additional inborn errors of metabolism using tandem MS analysis. Four blood spots of up to 10 mm in diameter are collected onto a high-grade paper card for each individual. It should be recognized that there are analytical limitations to DBS studies due to the variable nature of the sample, paper, and the circumstances under which samples are collected. Newborn screening samples are usually collected from a heel prick by nursery staff with varying degrees of training. Squeezing the heel may cause alterations to capillary blood composition and contamination of samples from urine, faeces or incomplete removal of topical antiseptics may not be obvious. Variations in hematocrit cause differences in the spreading of samples [20] and chromatographic effects can lead to varying concentrations across the blood spot. Even with careful control of manufacturing processes, paper characteristics can vary www.clinical.proteomics-journal.com
898
V. Ignjatovic et al.
somewhat between batches and significant differences occur between manufacturers. For these reasons, there is an inherent variability of the order of 10% in DBS assays. Studies should be carefully designed and controlled to prevent false discoveries caused by the above factors. Storage practices vary but in many countries the cards are archived indefinitely at ambient temperatures while some programs store the cards in freezers. The blood sample is usually subsampled using a manual hole-punch and placing the punched piece into a microplate well for subsequent elution of the sample from the card. This method allows for recovery of between 10 [21] and 69% of the total protein in the sample that was initially blotted onto the card [22]. The extraction of analytes from cards that have been in long-term storage may present a problem and more stringent extraction protocols (e.g. extended proteolysis or treatment with detergents) may be required. Proteins are amenable to measurement from DBS. Well-known examples include thyroid-stimulating hormone, immunoreactive trypsinogen and the measurement of hemoglobins, as well as some more recent examples such as ceruloplasmin (Wilson’s disease) and pancreatitis-associated protein (second tier cystic fibrosis screening). Immunoassay is a well-established and viable targeted approach when a relatively small number of proteins are to be studied. On the other hand, MS is typically used when large numbers of proteins are to be studied. Dried blood spots have been used in the setting of proteomics where the MALDI-TOF-MS approach has been used to diagnose haemoglobin disorders, including sickle cell disease [23]. Specifically, this study demonstrated the applicability of the MALDI-TOF-MS method for high throughput, low cost, automated screening for sickle cell disease. Despite such investigation, the use of DBS in the setting of proteomics is limited.
5
Overcoming the problem of protein quantity
A significant limitation for proteomic investigation in the setting of samples collected on DBS could be the quantity of protein that can be recovered from such samples. The use of DBS limits the sample volume to about 10 L, compared to multiple milliliters that are used in most proteomics approaches. Unlike genetic-based studies where amplification provides the solution for limitation of sample quantity, proteomic studies have to date relied on the quantity of protein that is actually present in the sample. However, this has recently changed with the invention of the proximity extension assay (PEA) that takes advantage of quantitative real-time PCR technology to amplify the signal from the initial protein quantity found in the sample. This method is based on paired antibodies linked to oligonucleotides that have affinity for each other. Once these two antibodies are bound to the protein target, their close proximity allows for the two oligonucleotides C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics Clin. Appl. 2014, 8, 896–900
to be extended by a DNA polymerase, with the new sequence acting as a unique and surrogate marker for the bound protein [24, 25], and one that can be quantified by real-time PCR. The specificity of the antibody interaction in the PEA is an advantage that allows for extensive multiplexing and in fact there are already panels of up to 96 proteins available that use as little as 1 L of sample [26]. The majority of proteomic approaches allow for the study of proteomes at the femtomole level and open the way for studies of low abundance proteins such as hormones and signaling molecules in DBS. In addition, targeted proteomic methods such as multiple reaction monitoring (MRM, see below) provide a sensitive and more quantitative approach that is extremely advantageous. However, achieving the ultimate sensitivity is still reliant on good sample preparation methods.
6
Stepping up to the next level
In order for researchers and clinicians to be able to have full access to the proteomic-based information contained within DBS, there needs to be a significant investment in the technology that allows us to reliably measure proteins across a wide concentration range in a nontargeted or semitargeted fashion while increasing the throughput and providing automation. This will in turn allow population-wide studies involving vulnerable populations such as infants born prematurely, and the proteome wide association studies (PWAS) can make such studies possible. However, this new concept of PWAS must be mirrored by advancements in technology to facilitate such studies. The main question is whether the automated approach utilized for metabolomic studies from DBS can be adapted for proteomic studies? The speed with which MS technology is advancing, allowing the identification and quantitation of proteins from smaller sample volumes, makes PWAS possible. A potential exploratory approach would be to utilize a relatively recent and extremely popular MS technique of MRM. This technique is highly specific and sensitive and is used to quantitate predefined peptides of interest. In the setting of PWAS, the MRM approach could be used to determine the newborn concentration of proteins that have been defined to be important in disease states in adults. The MRM technique allows for absolute quantification/monitoring many different transitions/peptides and has recently been used in the setting of DBS for targeted measurement of 60 proteins [27]. Other studies have shown comparable protein distributions in plasma and DBS and the nontargeted detection of up to 253 proteins using high-end MS instruments [28, 29]. The top-down proteomics approach can be extremely useful in the setting of DBS as it analyses intact proteins and does not need to deal with the issue of protein loss that is a feature of bottom-up approaches [30]. The top-down approach has been combined successfully with liquid microjunction surface sampling for identification of hemoglobin variants www.clinical.proteomics-journal.com
899
Proteomics Clin. Appl. 2014, 8, 896–900
from DBS without the need for sample preparation [31–33]. Importantly, this robust top-down approach has been shown to be a viable option in cases that could not be diagnosed with standard methods [33]. The PEA approach as a semidirected approach focusing on 96 markers has been shown to perform well with DBS and to be highly compatible in this setting [26]. In this recent study the PEA DBS approach achieved high precision up to 6.4CV% across all assays, and surprisingly, for six assays there was an increased level of protein detected in the EDTA DBS compared to the EDTA plasma. This difference was attributed to cell disruption and additional release of analytes from blood cells following addition to the paper, a finding that is not uncommon in the setting of DBS [34]. However, this new approach has yet to be tested in the setting of DBS that have been stored for an extended time period. The use of the antibody-based approach in the setting of DBS is an effective method for protein profiling, biomarker discovery, and disease screening for large population-based studies. This has been confirmed in a recent study that investigated the utility of a semiquantitative and quantitative sandwich based antibody arrays for detection of 60 and 20 serum markers, respectively [35]. Bead-based multiplex assays also provide an effective approach and one that has recently been used to study nine acute phase proteins in DBS of almost 700 individuals the setting of psychosis [36].
7
Overcoming limitations
PWAS studies will be aided by better understanding of the normal protein constituents of plasma and a global initiative, led by the Human Proteome Organisation (HUPO, http://www.hupo.org/) is currently underway to document the human plasma proteome and provide web-based tools to assist researchers (see http://www.plasmaproteomedatabase. org/). Concerted EWAS and PWAS approaches are essential for breakthroughs in health improvement and prevention of major diseases. In order for this to happen we must first solve some of the issues associated with DBS, such as ethical issues, collection variables, and hematocrit variation. Once we overcome these limitations we can take full advantage of the benefits that the proteomics approach utilizing DBS offers. The ethical issues focus around consent for the use of DBS, especially in population-based studies that do not have an explicit consent for research on the previously collected spots. There are understandable public concerns surrounding the use of stored DBS from newborn screening in research [37]. An extreme example of this was the 2010 decision to destroy more than 5 million stored samples from the Texas newborn screening program following legal action by a public lobby group concerned at the use of deidentified samples for research when explicit consent for that research had not been obtained [38]. In cases where explicit consent C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
was not obtained, the option exists to use retrospective consent, which is essential for any research involving identified individuals. The ethical issues can be largely overcome for future studies with explicit consent at the time of the collection. One approach is to obtain consent for future deidentified research and some programs have even established biobanks of samples for which this consent has been obtained (see http://www.mnbb.org/). Access to such samples will be extremely useful in determining control ranges, changes with storage or regional differences.
8
Conclusions
The use of DBS has been taken to a new level when it comes to epigenetic-based studies. It is now increasingly important to develop the technologies that allow DBS to be utilized for protein-based studies. This approach may in turn allow for detection of major diseases of adulthood at the earliest possible time. The authors have declared no conflict of interest.
9
References
[1] Anderson, L., Candidate-based proteomics in the search for biomarkers of cardiovascular disease. J. Physiol. 2005, 563, 23–60. [2] Anderson, N. L., Anderson, N. G., The human plasma proteome: history, character and diagnostic prospects. Mol. Cell. Proteomics 2002, 1, 845–867. [3] Wasinger, V. C., Zeng, M., Yau, Y., Current status and advances in quantitative proteomics mass spectrometry. Int. J. Proteom. 2013, 2013, 180605. [4] Bignami, E. L., Crescenzi, G., Gonfalini, M., Bruno, G. et al., Role of cardiac biomarkers (troponin I and CK-MB) as predictors of quality of life and long-term outcome after cardiac surgery. Ann. Card. Anaesth. 2009, 12, 22–26. ˚ [5] Opstal-van Winden, A. W., Krop, E. J. M., Karedal, M. H., Gast, M. C. W. et al., Searching for early breast cancer biomarkers by serum protein profiling of pre-diagnostic serum: a nested case-control study. BMC Cancer 2011, 11, 381–391. [6] Barker, D. J., Osmond, C., Infant mortality, childhood nutrition, and ischaemic heart disease in England and Wales. Lancet 1986, 1, 1077–1081. [7] Barker, D. J., Winter, P. D., Osmond, C., Margetts, B. et al., Weight in infancy and death from ischaemic heart disease. Lancet 1989, 2, 577–580. [8] Barker, D. J., Gluckman, P. D., Godfrey, K. M., Harding, J. E. et al., Fetal nutrition and cardiovascular disease in adult life. Lancet 1993, 341, 938–941. [9] Hales, C. N., Barker, D. J., Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis. Diabetologia 1992, 35, 595–601. www.clinical.proteomics-journal.com
900
V. Ignjatovic et al.
Proteomics Clin. Appl. 2014, 8, 896–900
[10] Rakyan, V. K., Beyan, H., Down, T. A., Hawa, M. I. et al., Identification of type 1 diabetes-associated DNA methylation variable positions that precede disease diagnosis. PLoS Genet. 2011, 7, e1002300.
[25] Gullberg, M., Gustafsdottir, S. M., Schallmeiner, E., Jarvius, J., Bjarnegard, M. et al., Cytokine detection by antibodybased proximity ligation. Proc. Natl. Acad. Sci. USA 2004, 101, 8420–8424.
[11] Rakyan, V. K., Down, T. A., Balding, D. J., Beck, S., Epigenome-wide association studies for common human diseases. Nat. Rev. Genet. 2011, 12, 529–541.
[26] Assarsson, E., Lundberg, M., Holmquist, G., Bjorkesten, J., Bucht Thorsen, S. et al., Homogenous 96-Plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability. PLoS One 2014, 9, e95192.
[12] Toperoff, G. D., Aran, D., Kark, J. D., Rosenberg, M. et al., Genome-wide survey reveals predisposing diabetes type 2-related DNA methylation variations in human peripheral blood. Hum. Mol. Genet. 2012, 21, 371–383. ˚ [13] Joubert, B. R., Haberg, S. E., Nilsen, R. M., Wang, X. et al., 450K Epigenome-wide scan identifies differential dna methylation in newborns related to maternal smoking during pregnancy. Environ. Health Perspect. 2012, 120, 1425–1431. [14] Godfrey, K. M., Sheppard, A., Gluckman, P. D., Lillycrop, K. A. et al., Epigenetic gene promoter methylation at birth is associated with child’s later adiposity. Diabetes 2011, 60, 1528–1534. [15] Martino, D., Loke, Y. J., Gordon, L., Ollikainen, M. et al., Longitudinal, genome-scale analysis of DNA methylation in twins from birth to 18 months of age reveals rapid epigenetic change in early life and pair-specific effects of discordance. Genome. Biol. 2013, 14, R42. [16] Beyan, H., Down, T. A., Ramagopalan, S. V., Uvebrant, K. et al., Guthrie card methylomics identifies temporally stable epialleles that are present at birth in humans. Genome. Res. 2012, 22, 2138–2145. [17] Hollegaard, M. V., Grauholm, J., Nørgaard-Pedersen, B., Hougaard, D. M., Grauholm, J., DNA methylome profiling using neonatal dried blood spot samples: a proof-of-principle study. Mol. Genet. Metab. 2012, 108, 225–231. [18] Joo, J. E., Wong, E. M., Baglietto, L., Jung, C. H. et al., The use of DNA from archival dried blood spots with the Infinium HumanMethylation450 array. BMC. Biotechnol. 2013, 13, 23. [19] Cruickshank, M. N., Pitt, J., Craig, J. M., Going back to the future with Guthrie-powered epigenome-wide association studies. Genome Med. 2012, 4, 83. [20] de Vries, R., Barfield, M., van de Merbel, N., Schmid, B. et al., The effect of hematocrit on bioanalysis of DBS: results from the EBF DBS-microsampling consortium. Bioanalysis 2013, 5, 2147–2160. [21] Carchon, H. A., Nsibu Ndosimao, C., Van Aerschot, S., Jaeken, J., Use of serum on Guthrie cards in screening for congenital disorders of glycosylation. Clin. Chem. 2006, 52, 774–775. [22] Zurfluh, M. R., Giovannini, M., Fiori, L., Fiege, B. et al., Screening for tetrahydrobiopterin deficiencies using dried blood spots on filter paper. Mol. Genet. Metab. 2005, 86, S96–103. [23] Hachani, J., Duban-Deweer, S., Pottiez, G., Renom, G. et al., MALDI-TOF MS profiling as the first-tier screen for sickle cell disease in neonates: matching throughput to objectives. Prot. Clin. Appl. 2011, 5, 405–414. [24] Fredriksson, S., Gullberg, M., Jarvius, J., Olsson, C. et al., Protein detection using proximity-dependent DNA ligation assays. Nat. Biotech. 2002, 20, 473–477. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[27] Chambers, A. G., Percy, A. J., Yang, J., Camenzind, A. G., Borchers, C. H., Multiplexed quantitation of endogenous proteins in dried blood spots by multiple reaction monitoringmass spectrometery. Mol. Cell. Proteomics 2013, 12, 781–791. [28] Chambers, A. G., Percy, A. J., Hardie, D. B., Borchers, C. H. Comparison of proteins in whole blood and dried blood spot samples by LC/MS/MS. J. Am. Soc. Mass. Spec. 2013, 24, 1338–1345. [29] Martin, N. J., Bunch, J., Cooper, H. J., Dried blood spot proteomics: surface extraction of endogenous proteins coupled with automated sample preparation and mass spectrometry analysis. J. Am. Soc. Mass. Spectrom. 2013, 24, 1242–1249. [30] Cui, W., Rohrs, H. W., Gross, M. L., Top-down mass spectrometry: Recent developments, applications and perspectives. Analyst 2011, 136, 3854–3864. [31] Van Berkel, G. J., Kertesz, V., Koeplinger, K. A., Vavrek, M., Kong, A.-N. T., Liquid microjunction surface sampling probe electrospray mass spectrometry for detection of drugs and metabolites in thin tissue sections. J. Mass Spectrom. 2008, 43, 500–508. [32] Edwards, R. L., Creese, A. J., Baumert, M., Griffiths, P. et al., Hemoglobin variant analysis via direct surface sampling of dried blood spots coupled with high-resolution mass spectrometry. Anal. Chem. 2011, 83, 2265–2270. [33] Edwards R. L., Griffiths, P., Bunch, J., Cooper, H. J., Top-down proteomics and direct surface sampling of neonatal dried blood spots: diagnosis of unknown haemoglobin variants. J. Am. Soc. Mass Spectrom. 2012, 23, 1921–1930. [34] Matsuda, K. C., Hall, C., Baker-Lee, C., Soto, M., Scott, G. et al. Measurement of in vivo therapeutic mAb concentrations: comparison of conventional serum/plasma collection and analysis to dried blood spot sampling. Bioanalysis 2013, 5, 1979–1990. [35] Jang, W., Mao, Y. Q., Huang, R., Duan, C. et al., Protein expression profiling by antibody array analysis with use of dried blood spot samples on filter paper. J. Immunol. Meth. 2014, 403, 79–86. [36] Gardner, R. M., Dalman, C., Wicks, S., Lee, B. K., Karlsson, H., Neonatal levels of acute phase proteins and later risk of non-affective psychosis. Transl. Psychiatry 2013, 3, e228. [37] Botkin, J. R., Goldenberg, A. J., Rothwell, E., Anderson, R. A., Lewis, M. H. Retention and research use of residual newborn screening bloodspots. Pediatrics 2013, 131, 120–127. [38] Lewis, M. H., Goldenberg, A., Anderson, R., Rothwell, E., Botkin, J., State laws regarding the retention and use of residual newborn screening blood samples. Pediatrics 2011, 127, 703–712.
www.clinical.proteomics-journal.com
DOI 10.1002/prca.201400042
Proteomics Clin. Appl. 2014, 8, 896–900
REVIEW
The utility of dried blood spots for proteomic studies: Looking forward to looking back Vera Ignjatovic1,2 , James Pitt2,3 , Paul Monagle2,4 and Jeffrey M. Craig1,2 1
Murdoch Childrens Research Institute, Royal Children’s Hospital, Victoria, Australia Department of Paediatrics, The University of Melbourne, Victoria, Australia 3 Victorian Clinical Genetic Services, Royal Children’s Hospital, Victoria, Australia 4 Department of Clinical Haematology, Royal Children’s Hospital, Victoria, Australia 2
The possibility to detect biomarkers of adult disease in early life and particularly in newborns holds enormous promise for early disease detection and prevention. Early detection of disease or potential for future disease would allow for prevention or amelioration of disease before overt symptoms develop, by lifestyle modifications, appropriate medication and monitoring. It is now increasingly important to develop the technologies that allow dried blood spots (DBS) to be utilized for protein-based studies. The use of DBS in proteome wide association studies (PWAS) may in turn allow for detection of major diseases of adulthood at the earliest possible time. This review focuses on the utility of DBS in proteomics, the main challenges, as well as the latest approaches for overcoming those, facilitating the use of DBS for detection of major diseases of adulthood at the earliest possible time.
Received: April 7, 2014 Revised: August 4, 2014 Accepted: September 3, 2014
Keywords: Disease prevention / Dried blood spots
1
Protein biomarkers of complex disease
A biomarker is any biological characteristic: molecular, physiological, or biochemical—that can be measured objectively and act as a predictor or indicator of a normal or a diseased state. The search for protein biomarkers of complex diseases such as cardiovascular disease (CVD), cancer and diabetes has seen significant investment in the past 10–15 years. However, despite this investment in technology and an increase in proteomics-based studies in a variety of clinical settings, the discovery of protein biomarkers has not advanced at the expected rate [1]. In fact, there are very few disease biomarkers that have been discovered using a proteomics-based approach.
Correspondence: Associate Professor Vera Ignjatovic, Haematology Research Laboratory / Murdoch Childrens Research Institute, Flemington Road, Parkville, Victoria, 3052, Australia E-mail: [email protected] Abbreviations: CVD, cardiovascular disease; DBS, dried blood spots; EWAS, Epigenome wide association studies; MRM, multiple reaction monitoring; PEA, proximity extension assay; PWAS, proteome wide association studies C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2
Challenges of current proteomics approaches
The methodological approach in proteomics studies varies significantly not only based on the choice of samples, but more importantly based on the specific methods utilised. At the sample level there is a significant variation in sample preparation and fractionation techniques. For example, serum and plasma samples are widely used; however, there is significant variation in the concentrations of proteins present within each of these. Plasma is separated from whole blood using centrifugation, while serum is obtained from whole blood by a two-step process that involves formation of a blood clot and subsequent centrifugation, to separate the serum from the clot and other cellular elements. In plasma, for example, there is a ten order of magnitude difference in concentration of the least and most abundant proteins [2]. In turn, proteins of interests may be difficult to detect against a background of high abundance proteins such as albumin and immunoglobulins. Therefore, the preparation of plasma and sera for proteomic studies may involve a variety of depletion techniques (e.g., albumin, IgG, or depletion of six or 12 major proteins) and/or fractionation techniques (i.e. multi lectin affinity chromatography, cation exchange). When it comes to the analysis of tissue-specific proteomes (i.e. kidney, tumor), the variation lies in the way the tissue extracts are prepared (i.e. direct lysis, enrichment of particular cells such as tumor www.clinical.proteomics-journal.com
897
Proteomics Clin. Appl. 2014, 8, 896–900
cells, density gradient centrifugation). It is important to realize that sample preparation can potentially distort the proteome through nonspecific binding or failure to extract hydrophobic (membrane-bound) proteins, which could lead to the loss of informative biomarkers. Comprehensive studies may therefore require more than one sample preparation technique to ensure wider coverage of the proteome. Once prepared, the proteome of a specific biological sample can be further analyzed by using multiple approaches such as gel electrophoresis (1 or 2D), 2DE, as well as enzymatic proteolytic digestion, all of which are followed by the use of MS. The MS component of the proteomics studies can be performed using a variety of approaches and instruments, such as LC-MS, LC-MS/MS, and MALDI-TOF. Each of these technologies has strengths and limitations with respect to parameters such as analytical sensitivity, imprecision, and specificity as well as sample throughput, ease of use, and costs [3]. The rationale for choosing any particular method over another in a given circumstance needs considerable validation and standardization. Such a process would require a focused effort of the proteomics community in concert with translational research environments that are focused on solving the health challenges that face our society. In this effort to overcome the challenges of current proteomics approaches, a different research target might need to also be considered, where the focus is consciously shifted from the differences between disease and health, to early detection and prediction of disease.
priate medication, and monitoring. Reducing the incidence of major diseases such as CVD, cancer, and diabetes would improve the quality of life for individuals, as well as reduce the health care related costs and burden on governments and individuals alike. Epigenome-wide association studies (EWAS) are an example where this early detection approach has been utilized successfully. Epigenetics describes the chemical tags that associate with DNA, over and above the primary sequence, to control gene activity. These tags are stable through cell division but can change during early development and react to environmental change. In a typical EWAS, cases and controls are analyzed on a genome scale for epigenetic marks such as DNA methylation and associations adjusted to levels of genome-wide significance [10]. Samples sizes have ranged from 200; most have focused on a single time point. However, notable studies have included longitudinal analysis, providing potential biomarkers of disease risk in asymptomatic individuals (e.g., type 1 diabetes in children) [11] or markers of disease progression (e.g., type 2 diabetes in adults) [12]. EWAS can also be performed within a group of individuals for a particular environmental exposure, such as maternal smoking in pregnancy [13]. If birth samples are analyzed, both types of EWAS could be performed on the same set of samples, as has been done for candidate-based epigenetic analysis [14]. Birth samples may be collected prospectively [15] or accessed retrospectively, in the form of dried blood spot (DBS) samples, the so-called “Guthrie cards” [16–19].
4 3
Early life detection of biomarkers of adult disease
The majority of proteomic biomarker studies to date have relied on protein-based differences that can be detected in individuals with disease compared to healthy individuals, specifically in the adult setting. Anderson has suggested a set of 177 protein biomarkers from human plasma that could be useful in the setting of CVD [1]. Cardiac troponin I has been shown to be a strong predictor of mortality following cardiac surgery [4]. Differences in the abundance of apolipoprotein E, afamin, isoform 1 of interalpha trypsin inhibitor heavy chain H4 (ITIH4), alpha-2-macroglobulin and ceruloplasmin have been demonstrated to be associated with breast cancer compared to age-matched controls [5]. Developmental Origins of Health and Disease, previously known as the Fetal Origins of Adult Disease, hypothesizes that major diseases such as coronary heart disease, stroke, hypertension, and type 2 diabetes originate in utero and during early infancy [6–9]. The possibility to detect biomarkers of adult disease in early life and particularly in newborns holds enormous promise for early disease detection and prevention. Early detection of disease or potential for future disease would allow for prevention or amelioration of disease before overt symptoms develop, by lifestyle modifications, appro C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The utility of DBS samples in proteomics
Dried blood spot sample represents a unique individual sample collected within a few days postbirth that was originally designed for screening of inborn errors of metabolism and other congenital disorders. Examples of diseases currently screened include phenylketonuria, congenital hypothyroidism, cystic fibrosis, and sickle-cell disease. From the late 1990s, newborn-screening laboratories also began to introduce multiplex testing for approximately 25 additional inborn errors of metabolism using tandem MS analysis. Four blood spots of up to 10 mm in diameter are collected onto a high-grade paper card for each individual. It should be recognized that there are analytical limitations to DBS studies due to the variable nature of the sample, paper, and the circumstances under which samples are collected. Newborn screening samples are usually collected from a heel prick by nursery staff with varying degrees of training. Squeezing the heel may cause alterations to capillary blood composition and contamination of samples from urine, faeces or incomplete removal of topical antiseptics may not be obvious. Variations in hematocrit cause differences in the spreading of samples [20] and chromatographic effects can lead to varying concentrations across the blood spot. Even with careful control of manufacturing processes, paper characteristics can vary www.clinical.proteomics-journal.com
898
V. Ignjatovic et al.
somewhat between batches and significant differences occur between manufacturers. For these reasons, there is an inherent variability of the order of 10% in DBS assays. Studies should be carefully designed and controlled to prevent false discoveries caused by the above factors. Storage practices vary but in many countries the cards are archived indefinitely at ambient temperatures while some programs store the cards in freezers. The blood sample is usually subsampled using a manual hole-punch and placing the punched piece into a microplate well for subsequent elution of the sample from the card. This method allows for recovery of between 10 [21] and 69% of the total protein in the sample that was initially blotted onto the card [22]. The extraction of analytes from cards that have been in long-term storage may present a problem and more stringent extraction protocols (e.g. extended proteolysis or treatment with detergents) may be required. Proteins are amenable to measurement from DBS. Well-known examples include thyroid-stimulating hormone, immunoreactive trypsinogen and the measurement of hemoglobins, as well as some more recent examples such as ceruloplasmin (Wilson’s disease) and pancreatitis-associated protein (second tier cystic fibrosis screening). Immunoassay is a well-established and viable targeted approach when a relatively small number of proteins are to be studied. On the other hand, MS is typically used when large numbers of proteins are to be studied. Dried blood spots have been used in the setting of proteomics where the MALDI-TOF-MS approach has been used to diagnose haemoglobin disorders, including sickle cell disease [23]. Specifically, this study demonstrated the applicability of the MALDI-TOF-MS method for high throughput, low cost, automated screening for sickle cell disease. Despite such investigation, the use of DBS in the setting of proteomics is limited.
5
Overcoming the problem of protein quantity
A significant limitation for proteomic investigation in the setting of samples collected on DBS could be the quantity of protein that can be recovered from such samples. The use of DBS limits the sample volume to about 10 L, compared to multiple milliliters that are used in most proteomics approaches. Unlike genetic-based studies where amplification provides the solution for limitation of sample quantity, proteomic studies have to date relied on the quantity of protein that is actually present in the sample. However, this has recently changed with the invention of the proximity extension assay (PEA) that takes advantage of quantitative real-time PCR technology to amplify the signal from the initial protein quantity found in the sample. This method is based on paired antibodies linked to oligonucleotides that have affinity for each other. Once these two antibodies are bound to the protein target, their close proximity allows for the two oligonucleotides C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics Clin. Appl. 2014, 8, 896–900
to be extended by a DNA polymerase, with the new sequence acting as a unique and surrogate marker for the bound protein [24, 25], and one that can be quantified by real-time PCR. The specificity of the antibody interaction in the PEA is an advantage that allows for extensive multiplexing and in fact there are already panels of up to 96 proteins available that use as little as 1 L of sample [26]. The majority of proteomic approaches allow for the study of proteomes at the femtomole level and open the way for studies of low abundance proteins such as hormones and signaling molecules in DBS. In addition, targeted proteomic methods such as multiple reaction monitoring (MRM, see below) provide a sensitive and more quantitative approach that is extremely advantageous. However, achieving the ultimate sensitivity is still reliant on good sample preparation methods.
6
Stepping up to the next level
In order for researchers and clinicians to be able to have full access to the proteomic-based information contained within DBS, there needs to be a significant investment in the technology that allows us to reliably measure proteins across a wide concentration range in a nontargeted or semitargeted fashion while increasing the throughput and providing automation. This will in turn allow population-wide studies involving vulnerable populations such as infants born prematurely, and the proteome wide association studies (PWAS) can make such studies possible. However, this new concept of PWAS must be mirrored by advancements in technology to facilitate such studies. The main question is whether the automated approach utilized for metabolomic studies from DBS can be adapted for proteomic studies? The speed with which MS technology is advancing, allowing the identification and quantitation of proteins from smaller sample volumes, makes PWAS possible. A potential exploratory approach would be to utilize a relatively recent and extremely popular MS technique of MRM. This technique is highly specific and sensitive and is used to quantitate predefined peptides of interest. In the setting of PWAS, the MRM approach could be used to determine the newborn concentration of proteins that have been defined to be important in disease states in adults. The MRM technique allows for absolute quantification/monitoring many different transitions/peptides and has recently been used in the setting of DBS for targeted measurement of 60 proteins [27]. Other studies have shown comparable protein distributions in plasma and DBS and the nontargeted detection of up to 253 proteins using high-end MS instruments [28, 29]. The top-down proteomics approach can be extremely useful in the setting of DBS as it analyses intact proteins and does not need to deal with the issue of protein loss that is a feature of bottom-up approaches [30]. The top-down approach has been combined successfully with liquid microjunction surface sampling for identification of hemoglobin variants www.clinical.proteomics-journal.com
899
Proteomics Clin. Appl. 2014, 8, 896–900
from DBS without the need for sample preparation [31–33]. Importantly, this robust top-down approach has been shown to be a viable option in cases that could not be diagnosed with standard methods [33]. The PEA approach as a semidirected approach focusing on 96 markers has been shown to perform well with DBS and to be highly compatible in this setting [26]. In this recent study the PEA DBS approach achieved high precision up to 6.4CV% across all assays, and surprisingly, for six assays there was an increased level of protein detected in the EDTA DBS compared to the EDTA plasma. This difference was attributed to cell disruption and additional release of analytes from blood cells following addition to the paper, a finding that is not uncommon in the setting of DBS [34]. However, this new approach has yet to be tested in the setting of DBS that have been stored for an extended time period. The use of the antibody-based approach in the setting of DBS is an effective method for protein profiling, biomarker discovery, and disease screening for large population-based studies. This has been confirmed in a recent study that investigated the utility of a semiquantitative and quantitative sandwich based antibody arrays for detection of 60 and 20 serum markers, respectively [35]. Bead-based multiplex assays also provide an effective approach and one that has recently been used to study nine acute phase proteins in DBS of almost 700 individuals the setting of psychosis [36].
7
Overcoming limitations
PWAS studies will be aided by better understanding of the normal protein constituents of plasma and a global initiative, led by the Human Proteome Organisation (HUPO, http://www.hupo.org/) is currently underway to document the human plasma proteome and provide web-based tools to assist researchers (see http://www.plasmaproteomedatabase. org/). Concerted EWAS and PWAS approaches are essential for breakthroughs in health improvement and prevention of major diseases. In order for this to happen we must first solve some of the issues associated with DBS, such as ethical issues, collection variables, and hematocrit variation. Once we overcome these limitations we can take full advantage of the benefits that the proteomics approach utilizing DBS offers. The ethical issues focus around consent for the use of DBS, especially in population-based studies that do not have an explicit consent for research on the previously collected spots. There are understandable public concerns surrounding the use of stored DBS from newborn screening in research [37]. An extreme example of this was the 2010 decision to destroy more than 5 million stored samples from the Texas newborn screening program following legal action by a public lobby group concerned at the use of deidentified samples for research when explicit consent for that research had not been obtained [38]. In cases where explicit consent C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
was not obtained, the option exists to use retrospective consent, which is essential for any research involving identified individuals. The ethical issues can be largely overcome for future studies with explicit consent at the time of the collection. One approach is to obtain consent for future deidentified research and some programs have even established biobanks of samples for which this consent has been obtained (see http://www.mnbb.org/). Access to such samples will be extremely useful in determining control ranges, changes with storage or regional differences.
8
Conclusions
The use of DBS has been taken to a new level when it comes to epigenetic-based studies. It is now increasingly important to develop the technologies that allow DBS to be utilized for protein-based studies. This approach may in turn allow for detection of major diseases of adulthood at the earliest possible time. The authors have declared no conflict of interest.
9
References
[1] Anderson, L., Candidate-based proteomics in the search for biomarkers of cardiovascular disease. J. Physiol. 2005, 563, 23–60. [2] Anderson, N. L., Anderson, N. G., The human plasma proteome: history, character and diagnostic prospects. Mol. Cell. Proteomics 2002, 1, 845–867. [3] Wasinger, V. C., Zeng, M., Yau, Y., Current status and advances in quantitative proteomics mass spectrometry. Int. J. Proteom. 2013, 2013, 180605. [4] Bignami, E. L., Crescenzi, G., Gonfalini, M., Bruno, G. et al., Role of cardiac biomarkers (troponin I and CK-MB) as predictors of quality of life and long-term outcome after cardiac surgery. Ann. Card. Anaesth. 2009, 12, 22–26. ˚ [5] Opstal-van Winden, A. W., Krop, E. J. M., Karedal, M. H., Gast, M. C. W. et al., Searching for early breast cancer biomarkers by serum protein profiling of pre-diagnostic serum: a nested case-control study. BMC Cancer 2011, 11, 381–391. [6] Barker, D. J., Osmond, C., Infant mortality, childhood nutrition, and ischaemic heart disease in England and Wales. Lancet 1986, 1, 1077–1081. [7] Barker, D. J., Winter, P. D., Osmond, C., Margetts, B. et al., Weight in infancy and death from ischaemic heart disease. Lancet 1989, 2, 577–580. [8] Barker, D. J., Gluckman, P. D., Godfrey, K. M., Harding, J. E. et al., Fetal nutrition and cardiovascular disease in adult life. Lancet 1993, 341, 938–941. [9] Hales, C. N., Barker, D. J., Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis. Diabetologia 1992, 35, 595–601. www.clinical.proteomics-journal.com
900
V. Ignjatovic et al.
Proteomics Clin. Appl. 2014, 8, 896–900
[10] Rakyan, V. K., Beyan, H., Down, T. A., Hawa, M. I. et al., Identification of type 1 diabetes-associated DNA methylation variable positions that precede disease diagnosis. PLoS Genet. 2011, 7, e1002300.
[25] Gullberg, M., Gustafsdottir, S. M., Schallmeiner, E., Jarvius, J., Bjarnegard, M. et al., Cytokine detection by antibodybased proximity ligation. Proc. Natl. Acad. Sci. USA 2004, 101, 8420–8424.
[11] Rakyan, V. K., Down, T. A., Balding, D. J., Beck, S., Epigenome-wide association studies for common human diseases. Nat. Rev. Genet. 2011, 12, 529–541.
[26] Assarsson, E., Lundberg, M., Holmquist, G., Bjorkesten, J., Bucht Thorsen, S. et al., Homogenous 96-Plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability. PLoS One 2014, 9, e95192.
[12] Toperoff, G. D., Aran, D., Kark, J. D., Rosenberg, M. et al., Genome-wide survey reveals predisposing diabetes type 2-related DNA methylation variations in human peripheral blood. Hum. Mol. Genet. 2012, 21, 371–383. ˚ [13] Joubert, B. R., Haberg, S. E., Nilsen, R. M., Wang, X. et al., 450K Epigenome-wide scan identifies differential dna methylation in newborns related to maternal smoking during pregnancy. Environ. Health Perspect. 2012, 120, 1425–1431. [14] Godfrey, K. M., Sheppard, A., Gluckman, P. D., Lillycrop, K. A. et al., Epigenetic gene promoter methylation at birth is associated with child’s later adiposity. Diabetes 2011, 60, 1528–1534. [15] Martino, D., Loke, Y. J., Gordon, L., Ollikainen, M. et al., Longitudinal, genome-scale analysis of DNA methylation in twins from birth to 18 months of age reveals rapid epigenetic change in early life and pair-specific effects of discordance. Genome. Biol. 2013, 14, R42. [16] Beyan, H., Down, T. A., Ramagopalan, S. V., Uvebrant, K. et al., Guthrie card methylomics identifies temporally stable epialleles that are present at birth in humans. Genome. Res. 2012, 22, 2138–2145. [17] Hollegaard, M. V., Grauholm, J., Nørgaard-Pedersen, B., Hougaard, D. M., Grauholm, J., DNA methylome profiling using neonatal dried blood spot samples: a proof-of-principle study. Mol. Genet. Metab. 2012, 108, 225–231. [18] Joo, J. E., Wong, E. M., Baglietto, L., Jung, C. H. et al., The use of DNA from archival dried blood spots with the Infinium HumanMethylation450 array. BMC. Biotechnol. 2013, 13, 23. [19] Cruickshank, M. N., Pitt, J., Craig, J. M., Going back to the future with Guthrie-powered epigenome-wide association studies. Genome Med. 2012, 4, 83. [20] de Vries, R., Barfield, M., van de Merbel, N., Schmid, B. et al., The effect of hematocrit on bioanalysis of DBS: results from the EBF DBS-microsampling consortium. Bioanalysis 2013, 5, 2147–2160. [21] Carchon, H. A., Nsibu Ndosimao, C., Van Aerschot, S., Jaeken, J., Use of serum on Guthrie cards in screening for congenital disorders of glycosylation. Clin. Chem. 2006, 52, 774–775. [22] Zurfluh, M. R., Giovannini, M., Fiori, L., Fiege, B. et al., Screening for tetrahydrobiopterin deficiencies using dried blood spots on filter paper. Mol. Genet. Metab. 2005, 86, S96–103. [23] Hachani, J., Duban-Deweer, S., Pottiez, G., Renom, G. et al., MALDI-TOF MS profiling as the first-tier screen for sickle cell disease in neonates: matching throughput to objectives. Prot. Clin. Appl. 2011, 5, 405–414. [24] Fredriksson, S., Gullberg, M., Jarvius, J., Olsson, C. et al., Protein detection using proximity-dependent DNA ligation assays. Nat. Biotech. 2002, 20, 473–477. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[27] Chambers, A. G., Percy, A. J., Yang, J., Camenzind, A. G., Borchers, C. H., Multiplexed quantitation of endogenous proteins in dried blood spots by multiple reaction monitoringmass spectrometery. Mol. Cell. Proteomics 2013, 12, 781–791. [28] Chambers, A. G., Percy, A. J., Hardie, D. B., Borchers, C. H. Comparison of proteins in whole blood and dried blood spot samples by LC/MS/MS. J. Am. Soc. Mass. Spec. 2013, 24, 1338–1345. [29] Martin, N. J., Bunch, J., Cooper, H. J., Dried blood spot proteomics: surface extraction of endogenous proteins coupled with automated sample preparation and mass spectrometry analysis. J. Am. Soc. Mass. Spectrom. 2013, 24, 1242–1249. [30] Cui, W., Rohrs, H. W., Gross, M. L., Top-down mass spectrometry: Recent developments, applications and perspectives. Analyst 2011, 136, 3854–3864. [31] Van Berkel, G. J., Kertesz, V., Koeplinger, K. A., Vavrek, M., Kong, A.-N. T., Liquid microjunction surface sampling probe electrospray mass spectrometry for detection of drugs and metabolites in thin tissue sections. J. Mass Spectrom. 2008, 43, 500–508. [32] Edwards, R. L., Creese, A. J., Baumert, M., Griffiths, P. et al., Hemoglobin variant analysis via direct surface sampling of dried blood spots coupled with high-resolution mass spectrometry. Anal. Chem. 2011, 83, 2265–2270. [33] Edwards R. L., Griffiths, P., Bunch, J., Cooper, H. J., Top-down proteomics and direct surface sampling of neonatal dried blood spots: diagnosis of unknown haemoglobin variants. J. Am. Soc. Mass Spectrom. 2012, 23, 1921–1930. [34] Matsuda, K. C., Hall, C., Baker-Lee, C., Soto, M., Scott, G. et al. Measurement of in vivo therapeutic mAb concentrations: comparison of conventional serum/plasma collection and analysis to dried blood spot sampling. Bioanalysis 2013, 5, 1979–1990. [35] Jang, W., Mao, Y. Q., Huang, R., Duan, C. et al., Protein expression profiling by antibody array analysis with use of dried blood spot samples on filter paper. J. Immunol. Meth. 2014, 403, 79–86. [36] Gardner, R. M., Dalman, C., Wicks, S., Lee, B. K., Karlsson, H., Neonatal levels of acute phase proteins and later risk of non-affective psychosis. Transl. Psychiatry 2013, 3, e228. [37] Botkin, J. R., Goldenberg, A. J., Rothwell, E., Anderson, R. A., Lewis, M. H. Retention and research use of residual newborn screening bloodspots. Pediatrics 2013, 131, 120–127. [38] Lewis, M. H., Goldenberg, A., Anderson, R., Rothwell, E., Botkin, J., State laws regarding the retention and use of residual newborn screening blood samples. Pediatrics 2011, 127, 703–712.
www.clinical.proteomics-journal.com
