development and validation of assay protocols for use with dried blood spot samples

9
Human Biology Toolkit Development and Validation of Assay Protocols for use with Dried Blood Spot Samples THOMAS W. MCDADE* Department of Anthropology and Institute for Policy Research, Northwestern University, Evanston, Illinois, 60208 ABSTRACT: Dried blood spots (DBS)—drops of capillary whole blood collected from finger stick—represent a mini- mally invasive alternative to venipuncture that facilitates the collection of blood samples from research participants in naturalistic, field-based research settings. But the number of validated assays for quantifying biomarkers in DBS sam- ples is relatively low in comparison with serum or plasma. The objective of this review is to discuss the advantages and disadvantages of DBS sampling, and to outline the steps involved in developing and validating an immunoassay for application to DBS samples. These steps include deciding on reagents, preparing calibration and quality control mate- rial, evaluating elution protocols, optimizing sample quantity, and assessing multiple aspects of assay performance, including intra- and interassay variation, lower limit of detection, accuracy, stability, and agreement between results from matched DBS and plasma samples. The broader goal of this “how-to” approach is to encourage investigators to val- idate, implement, and disseminate assay protocols for DBS samples in order to advance field-based research on human biology. Am. J. Hum. Biol. 26:1–9, 2014. V C 2013 Wiley Periodicals, Inc. Biological anthropology has an established track record of innovation with respect to methods for quantifying hor- mones, immune factors, and other biomarkers in diverse field settings all over the world (Ellison, 1988; James, 1991; McDade et al., 2000b; O’Connor et al., 2003; Salvante et al., 2012; Worthman and Stallings, 1997). Methodological inno- vation has been essential in advancing our understanding of the causes and consequences of human biological varia- tion, and in complementing—and at times challenging— prevailing clinic- and lab-based research paradigms that inform our understanding of human biology and health. In particular, the measurement of biomarkers in blood, saliva, and urine allows us to study the pathways through which social, cultural, and other physical ecological factors “get under the skin” to shape physiological function and health over the life course (Finch et al., 2001; Panter-Brick and Worthman, 1999; Weinstein et al., 2007). The collection of plasma or serum (the liquid fraction of blood that remains after whole blood is centrifuged to remove red and white blood cells) is the current standard for biomarker measurement, but the costs, participant bur- den, and logistics associated with venipuncture blood col- lection are major barriers to community-based research, particularly in remote field settings where access to a cen- trifuge, freezer, or even electricity, may be limited. Saliva and urine are useful alternatives for the subset of bio- markers that enter these fluids in a measurable form (e.g., cortisol, estradiol), but this is not an option for the majority of analytes that are accessible only in blood. Dried blood spots (DBS)—drops of whole blood collected on filter paper following a simple finger stick—represent a low cost, “field-friendly” alternative that allows investiga- tors to collect blood from large numbers of participants in naturalistic settings, and to integrate physiological infor- mation with rich contextual data in ways not possible with clinic-based research designs (McDade et al., 2007; Mei et al., 2001). Biological anthropologists have been using DBS overseas for 20 years (Campbell 1994; Worthman and Stallings, 1994, 1997), and recently more than 35,000 DBS samples have been collected for research purposes in the US, including applications in large surveys like the National Longitudinal Study of Adolescent Health and the Health and Retirement Study (McDade, 2011). Since the majority of biomarker assays are designed for use with serum or plasma, relatively few assays have been developed and validated for DBS samples (for a list of pre- viously validated assays, see McDade et al., 2007). The dearth of validated methods constrains the scientific ques- tions that can be addressed by studies collecting DBS sam- ples. The objective of this review, therefore, is to outline the steps involved in validating an immunoassay for applica- tion to DBS samples so field-based research on human biol- ogy can keep pace with developments emerging from clinic and lab-based approaches. The focus here is on the enzyme immunoassay due to its established utility for quantifying proteins with high sensitivity and specificity, the relatively low costs of instrumentation, and the commercial availabil- ity of assay kits and reagents (Lequin, 2005). However, many of the issues and procedures outlined below are rele- vant to other forms of immunoassay (e.g., radioimmunoas- say, fluorometric immunoassay), as well as other methods for evaluating proteins (e.g., mass spectrometry) or molecu- lar markers (e.g., mRNA, DNA) in DBS samples. ADVANTAGES AND DISADVANTAGES OF DRIED BLOOD SPOTS The filter papers (Whatman #903, GE Healthcare, Pis- cataway, NJ) used for DBS sampling were originally developed in the early 1960s to facilitate the collection of heel prick blood samples from newborns (Guthrie and *Correspondence to: Thomas Mcdade, Department of Anthropology, Northwestern University, Institute for Policy Research, Evanston, IL 60208, USA. E-mail: [email protected] Received 30 July 2013; Revision received 30 August 2013; Accepted 4 September 2013 DOI: 10.1002/ajhb.22463 Published online 15 October 2013 in Wiley Online Library (wileyonlinelibrary.com). V C 2013 Wiley Periodicals, Inc. AMERICAN JOURNAL OF HUMAN BIOLOGY 26:1–9 (2014)

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Page 1: Development and validation of assay protocols for use with dried blood spot samples

Human Biology Toolkit

Development and Validation of Assay Protocols for use with Dried Blood SpotSamples

THOMAS W. MCDADE*Department of Anthropology and Institute for Policy Research, Northwestern University, Evanston, Illinois, 60208

ABSTRACT: Dried blood spots (DBS)—drops of capillary whole blood collected from finger stick—represent a mini-mally invasive alternative to venipuncture that facilitates the collection of blood samples from research participants innaturalistic, field-based research settings. But the number of validated assays for quantifying biomarkers in DBS sam-ples is relatively low in comparison with serum or plasma. The objective of this review is to discuss the advantages anddisadvantages of DBS sampling, and to outline the steps involved in developing and validating an immunoassay forapplication to DBS samples. These steps include deciding on reagents, preparing calibration and quality control mate-rial, evaluating elution protocols, optimizing sample quantity, and assessing multiple aspects of assay performance,including intra- and interassay variation, lower limit of detection, accuracy, stability, and agreement between resultsfrom matched DBS and plasma samples. The broader goal of this “how-to” approach is to encourage investigators to val-idate, implement, and disseminate assay protocols for DBS samples in order to advance field-based research on humanbiology. Am. J. Hum. Biol. 26:1–9, 2014. VC 2013 Wiley Periodicals, Inc.

Biological anthropology has an established track recordof innovation with respect to methods for quantifying hor-mones, immune factors, and other biomarkers in diversefield settings all over the world (Ellison, 1988; James, 1991;McDade et al., 2000b; O’Connor et al., 2003; Salvante et al.,2012; Worthman and Stallings, 1997). Methodological inno-vation has been essential in advancing our understandingof the causes and consequences of human biological varia-tion, and in complementing—and at times challenging—prevailing clinic- and lab-based research paradigms thatinform our understanding of human biology and health. Inparticular, the measurement of biomarkers in blood, saliva,and urine allows us to study the pathways through whichsocial, cultural, and other physical ecological factors “getunder the skin” to shape physiological function and healthover the life course (Finch et al., 2001; Panter-Brick andWorthman, 1999; Weinstein et al., 2007).

The collection of plasma or serum (the liquid fraction ofblood that remains after whole blood is centrifuged toremove red and white blood cells) is the current standardfor biomarker measurement, but the costs, participant bur-den, and logistics associated with venipuncture blood col-lection are major barriers to community-based research,particularly in remote field settings where access to a cen-trifuge, freezer, or even electricity, may be limited. Salivaand urine are useful alternatives for the subset of bio-markers that enter these fluids in a measurable form (e.g.,cortisol, estradiol), but this is not an option for the majorityof analytes that are accessible only in blood.

Dried blood spots (DBS)—drops of whole blood collectedon filter paper following a simple finger stick—represent alow cost, “field-friendly” alternative that allows investiga-tors to collect blood from large numbers of participants innaturalistic settings, and to integrate physiological infor-mation with rich contextual data in ways not possible withclinic-based research designs (McDade et al., 2007; Meiet al., 2001). Biological anthropologists have been usingDBS overseas for 20 years (Campbell 1994; Worthman andStallings, 1994, 1997), and recently more than 35,000 DBS

samples have been collected for research purposes in theUS, including applications in large surveys like theNational Longitudinal Study of Adolescent Health and theHealth and Retirement Study (McDade, 2011).

Since the majority of biomarker assays are designed foruse with serum or plasma, relatively few assays have beendeveloped and validated for DBS samples (for a list of pre-viously validated assays, see McDade et al., 2007). Thedearth of validated methods constrains the scientific ques-tions that can be addressed by studies collecting DBS sam-ples. The objective of this review, therefore, is to outline thesteps involved in validating an immunoassay for applica-tion to DBS samples so field-based research on human biol-ogy can keep pace with developments emerging from clinicand lab-based approaches. The focus here is on the enzymeimmunoassay due to its established utility for quantifyingproteins with high sensitivity and specificity, the relativelylow costs of instrumentation, and the commercial availabil-ity of assay kits and reagents (Lequin, 2005). However,many of the issues and procedures outlined below are rele-vant to other forms of immunoassay (e.g., radioimmunoas-say, fluorometric immunoassay), as well as other methodsfor evaluating proteins (e.g., mass spectrometry) or molecu-lar markers (e.g., mRNA, DNA) in DBS samples.

ADVANTAGES AND DISADVANTAGESOF DRIED BLOOD SPOTS

The filter papers (Whatman #903, GE Healthcare, Pis-cataway, NJ) used for DBS sampling were originallydeveloped in the early 1960s to facilitate the collection ofheel prick blood samples from newborns (Guthrie and

*Correspondence to: Thomas Mcdade, Department of Anthropology,Northwestern University, Institute for Policy Research, Evanston, IL60208, USA. E-mail: [email protected]

Received 30 July 2013; Revision received 30 August 2013; Accepted 4September 2013

DOI: 10.1002/ajhb.22463Published online 15 October 2013 in Wiley Online Library

(wileyonlinelibrary.com).

VC 2013 Wiley Periodicals, Inc.

AMERICAN JOURNAL OF HUMAN BIOLOGY 26:1–9 (2014)

Page 2: Development and validation of assay protocols for use with dried blood spot samples

Susi, 1963). These papers are often referred to as “Guthriecards,” in recognition of Dr. Robert Guthrie’s pioneeringefforts to develop convenient methods to screen for con-genital metabolic disorders (Mei et al., 2001). Dried bloodspots have played a central role in public health surveil-lance efforts in the US ever since, and as a result, the fil-ter papers are certified to meet performance standards forsample absorption and lot-to-lot consistency. The CDC,which maintains an independent quality control monitor-ing program, notes that “The filter paper blood collectiondevice has achieved the same level of precision and repro-ducibility that analytical scientists and clinicians havecome to expect from standard methods of collecting blood,such as vacuum tubes and capillary pipettes” (Mei et al.,2001; p 1631). While this statement does not apply equallyto all analytes in blood, it speaks to the confidence one canhave in DBS-based results following successful assayvalidation.

Advantages of DBS sampling for research applicationsinclude the following:

1. Sample collection is minimally-invasive and low cost.The participant’s finger is cleaned with isopropyl alco-hol, and a sterile, single-use micro-lancet is used todeliver a controlled puncture (Table 1). Up to fivedrops of blood (�50 ml per drop) are applied to filterpaper, allowed to dry, and then stored at room tem-perature or refrigerated before shipment to the labo-ratory. Sample collection is relatively straightforward,but requires attention to important details that canaffect DBS quantity and quality (Table 2). Suppliescost approximately $2/participant, and samples can becollected by non-medically trained interviewers in aparticipant’s home, or in some cases, by participantsthemselves. The ease of sample collection is a boon tofield-based research, and is also advantageous in anysetting for studies with infants, children, and theelderly, for whom venipuncture may be particularlyproblematic. The low burden of sampling also increasesthe feasibility of collecting multiple blood samples fromthe same individual over time (McDade et al., 2012a).

2. Requirements for sample processing are minimal.Unlike venipuncture sampling for serum or plasma,DBS samples do not need to be centrifuged, separated,or immediately frozen following collection. Whole bloodis simply applied to filter paper, allowed to dry, andsamples can be stacked and sealed in gas impermeableplastic bags for storage and shipment. A cold chainfrom the point of sample collection to receipt in thelaboratory is not required since most analytes remainstable in DBS samples stored at room temperature fora week or more (McDade et al., 2007). However, it is

always advisable to refrigerate and freeze sampleswhen possible to maximize future applications, and itis important to protect the samples from exposure toexcessive heat. Since DBS samples are dried, require-ments for shipping are less onerous in comparisonwith serum or plasma: Samples do not have to remainfrozen in transit, dried blood poses a greatly reducedbiohazard risk, and regulations associated with theshipment of DBS are greatly reduced.

3. A single finger prick can provide capillary whole bloodfor spots on filter paper, and for onsite assessmentsusing “point-of-care” instruments. Relatively affordable,portable instruments for the analysis of hemoglobin(e.g., HemoCue), HbA1c (e.g., Bayer A1cNow, DCA Van-tage), and lipid profiles (e.g., CardioChek, Cholestech)are currently available that provide an opportunity tocollect physiological information in real time. Using thesame finger prick sampling procedure detailed above, adrop of blood (or less) can be placed into one of theseinstruments, with subsequent drops applied to filterpaper. By combining these procedures, biomarkerresults can be collected onsite and shared with partici-pants, while DBS samples can be assayed in the labfor a broader range of analytes. In some cases this mayprovide a valuable health screening service, and act asan incentive for research participation.

Advantages of DBS associated with sample collection inthe field need to be weighed against potential disadvan-tages associated with quantification in the lab.

1. The volume of sample is very small. Whereas a typicalvenipuncture blood draw yields at least 5 ml of wholeblood, and easily much more, a DBS sample with fivelarge drops of blood contains approximately 250 ml—avolume of blood that is more than an order of magni-tude smaller. Lancet selection (bigger is better) andadequate training/experience are key to collecting fivelarge drops of blood (Table 2). Otherwise, only two orthree drops will be obtained without a second fingerstick. The constraint of small sample volume places apremium on the efficient use of DBS material, andmay limit the number of analytes that can be quanti-fied. Fortunately, improvements in assay sensitivityhave reduced sample requirements for many analytes,and recent technological innovations are allowing forthe simultaneous quantification of multiple biomarkersin a single volume of sample. For example, Luminex,Meso Scale Discovery, and Quansys Biosciences alloffer multiplex immunoassay platforms (Chowdhuryet al., 2009; Salvante et al., 2012; Skogstrand et al.,2005). Regardless, the number of biomarkers that can

TABLE 1. Protocol for collecting finger stick dried blood spot samples

1. Put on gloves and follow universal precautions for preventing transmission of bloodborne infections (http://www.cdc.gov/niosh/topics/bbp/universal.html).

2. Clean the participant’s finger with isopropyl alcohol. Allow finger to dry.3. Establish a firm grip on the participant’s hand and use your thumb and finger to create a taut surface on the skin of the middle or ring finger.4. Use a sterile, single-use lancet to prick the finger just off the center of the tip of the finger. Immediately dispose of the lancet in a sharps container.5. Wipe away the first drop of blood with a sterile gauze pad. Apply subsequent drops to filter paper (Whatman #903). Allow blood from the participant’s

finger to be drawn onto the paper via capillary action. Do not blot the finger on the paper.6. Collect at least two, and preferably five, drops of blood that fill the borders of the pre-printed circles on the filter paper (approximately 50 ml each).7. After collection, place a bandage on the participant’s finger.8. Allow filter papers to air dry for at least 4 h (or overnight). Do not stack the samples until they are dry.9. Stack filter papers and place in an airtight container (e.g., sealable plastic bag or container) with desiccant.

2 DEVELOPMENT AND VALIDATION OF ASSAY PROTOCOLS

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be quantified in a given DBS sample is likely to bereduced in comparison with venipuncture-basedapproaches.

2. Some analytes cannot be measured in DBS. Althoughthe default assumption should be that anything thatcan be measured in serum or plasma can also bequantified in DBS samples, there are potentialobstacles that may prove to be insurmountable. Forexample, the presence of red and/or white blood cellsmay interfere with some assays. When whole blooddries on the filter paper, cellular fractions rupture andtheir contents are subsequently released into solutionwhen DBS samples are reconstituted. Different assaysystems and specific analytes will vary in their sensi-tivity to potential interference. This is not a commonproblem, but in some cases (e.g., ferritin; Ahluwalia,1998) it may prevent accurate quantification.In addition, analytes in DBS have to come off the fil-ter paper and enter solution in a form suitable foranalysis. While whole blood on filter paper is rela-tively stable and easy to store and transport, the pro-cess of drying may alter the biochemical structure of amolecule and affect the efficiency with which analytesenter solution.Lastly, analytes have to be present in quantities suffi-cient for analysis. Even with improvements in immu-noassay technology that reduce requirements forsample volume, there are limits. Serum- or plasma-based assays that require large volumes of undilutedsample (e.g., 50 or 100 ml) are not likely to translateeasily to use with DBS samples. (Plasma differs fromserum in containing clotting factors, but for mostimmunoassay applications both sample types providesimilar results; for the sake of simplicity in theremainder of the article, I refer to plasma only).

3. DBS samples are not the clinical standard. With theexception of newborn screening, DBS sampling israrely applied clinically and values from the analysisof venipuncture samples are considered the goldstandard. Results from DBS samples represent theconcentration of an analyte in whole blood, which bydefinition will differ from results determined in

plasma. But the correlations across matched plasmaand DBS samples are typically very high, and correc-tion factors can be applied to DBS values to generateplasma equivalents if desired. Such corrections willnot be necessary for within-study comparisons, butwill be important for any attempts to compare data toprior research based on results from plasma samples,or to make use of established clinical cut-points.These issues relate to the broader point that clinicalchemistry is not oriented toward the analysis of DBSsamples, and in some contexts this may pose addi-tional challenges.

Some of the disadvantages of DBS can be overcome byalternative methods for collecting and transporting capil-lary blood, which produce plasma for analysis in the lab.Whole blood from a finger stick can be collected in a capil-lary tube (a thin tube open on both ends) or a microtainerdevice (a small vial with a screw top). Like DBS, the bur-den of sample collection is low, but handling and transportare complicated by the fact that the samples remain in aliquid state: a cold chain is typically needed to maintainthe integrity of the sample, the blood has to be centrifugedand separated before analysis or freezing, and the trans-portation of liquid blood poses greater biohazard risk.Serum separator cards are similar to DBS cards in termsof sample collection, but differ in using a diffusion gradi-ent or membrane that isolates red and white blood cellswhile capturing and drying serum in the filter paper. Thedevice therefore retains the advantages of traditionalDBS sampling, with the added benefit of providing aserum sample that better approximates the clinical stand-ard for most analytes. However, serum separator cardsare more costly, and they pose challenges to precise quan-tification when concentration gradients are present acrossthe cards.

PRINCIPLES OF ENZYME IMMUNOASSAY

The enzyme immunoassay can be applied in manyforms, all of which involve a highly specific interactionbetween antibody and the target of interest (antigen), and

TABLE 2. Tips for successful collection of finger stick dried blood spot samples

1. Heavy items should not be placed on top of the filter papers before they are used. This will compress the papers and prevent them from absorbing bloodevenly. The papers should be transported in plastic containers or boxes for protection. After the samples are collected and the blood has dried, compres-sion is not a concern and papers can be transported in sealable plastic bags.

2. Larger lancets with blades, rather than needles, produce better blood flow, particularly when individuals have calloused hands. The lancets typicallyused by diabetics to monitor blood glucose have small needles designed for frequent sampling, and will produce small volumes of blood.

3. If the participant’s hands are cold, rub the hand to warm it up and increase blood flow. Heating pads, or soaking in warm water, can also be used towarm hands. It may also be helpful to ask the participant to stand, to make a fist, and/or to swing the hand rapidly downward before the finger stick totry and move blood into the fingers.

4. After pricking the finger, frequently use the gauze pad to wipe away blood from the puncture site. If blood is allowed to stay at the puncture site it willbegin to clot, and blood flow will stop.

5. If blood flow is not sufficient, use your fingers to squeeze the finger lightly or to try to move blood from the base of the finger to the tip. Avoid excessivesqueezing or “milking” of the finger as this will dilute the sample. If blood flow is still not sufficient, the skin puncture may be repeated on another fin-ger if the participant is willing.

6. It is absolutely essential that blood from the participant’s finger be drawn onto the paper via capillary action. The finger should never touch the paper.Blotting blood onto the paper will prevent the uniform diffusion of blood across the paper, which is essential to quantification. If samples are collectedcorrectly, the spots should look identical on both sides of the filter paper once the spot has dried. If one side is larger than the other, or if borders areirregular, then the blood was blotted or smeared on the paper.

7. Do not place blood on top of blood. Once blood has been spotted on the card, another drop of blood should not be placed on top of it, even if the previousspot seems small. This will concentrate the sample and defeat the uniform diffusing properties of the paper.

8. If blood is not flowing freely, and five full drops cannot be collected, it is almost always better to collect one or two large drops of blood than four or fivesmall drops. Uniform drops that fill the borders of the pre-printed circles on the #903 papers will provide more accurate results, and allow for the moreefficient and flexible use of a limited quantity of sample.

T.W. MCDADE 3

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an enzymatic label that changes in color in proportion tothe quantity of antigen in the sample (Crowther, 2009).The enzyme-linked immunosorbent assay (ELISA) is aspecific application that uses a 96-well microtiter plate asthe solid phase, where each well is coated with a captureantibody specific to the target. Immunoassay principlesare comparable regardless of sample type. But for the pur-poses of clarity, and to define terms that will be usedbelow in the discussion of assay development and valida-tion, it may be helpful to present a brief example.

To quantify C-reactive protein (CRP) in DBS (Brindleet al., 2010; McDade et al., 2004), eluted sample is addedto identified wells in the assay plate, where CRP binds tothe anti-CRP antibody that has already been coated to thebottom of the well. The CRP in the sample is therefore“captured” by the plate, and remains bound while the restof the sample is removed through a series of wash steps.In a sandwich ELISA, a second antibody is then addedwhich binds to available epitopes on the CRP molecule,the plate is washed to remove excess antibody, and a CRP“sandwich” remains bound to the bottom of each well. Thesecond antibody—or in this case, the detection antibody—has been previously conjugated to an enzyme that cata-lyzes a color change following the addition of the enzyme’ssubstrate (in some cases the second antibody may be con-jugated to biotin, which in turn binds to streptavidinwhich has been conjugated to the enzyme). The intensityof color change is directly proportional to the concentra-tion of CRP in each well, and it can be precisely measuredusing a microplate absorbance reader. The amount ofcolor change specific to bound CRP is also referred to as“signal,” whereas nonspecific color change is considered“background” or “noise.” The concentration of CRP in eachsample is determined by comparing the amount of colorchange in samples (unknowns) with the amount of colorchange generated by material with known CRP concen-tration (calibrators, or standards).

ISSUES TO CONSIDER BEFOREDBS ASSAY DEVELOPMENT

In most cases, the process of developing an immunoas-say for use with DBS samples is relatively straightforwardunder the following conditions: (1) prior work has demon-strated that the analyte of interest can be quantified byimmunoassay in serum or plasma; (2) the presence of lysedred and/or white blood cells does not interfere with quanti-fication; and (3) personnel involved with assay develop-ment have access to the necessary laboratory equipmentas well as prior experience with immunoassay implemen-tation and troubleshooting. The immunoassay is a trulyremarkable tool for gaining insight into key aspects ofhuman biology, but there are technological limits andtradeoffs in assay design. The following issues may be use-ful to consider as one evaluates different assay approachesfor specific applications.

ASSAY PERFORMANCE AND RANGE OF MEASUREMENT

In order to convince ourselves, and our colleagues, thata DBS protocol produces valid results we need to demon-strate a reasonable level of accuracy, precision, and reli-ability in our laboratory measurements. Operationaldefinitions of these performance parameters are providedbelow, but at this point it is sufficient to say that mea-surement error is a reality, and the level of error will

vary across analytes and across measurement protocols.It is therefore essential to identity sources of error, tominimize their impact to the extent possible, and to con-sider acceptable margins of error for particularapplications.

In addition, it is important to consider the range of val-ues that can be quantified, and the level of accuracy, preci-sion, and reliability across this range. In other words, howlow can the assay go? How high? The immunoassay’senzyme signal has a limited dynamic range, and attemptsto optimize quantification at very low analyte concentra-tions will inevitably constrain quantification at higherconcentrations. Determining whether the assay range cor-responds to the range of expected values in a given studyis therefore an important aspect of assay design.

LOWER LIMIT OF DETECTION LIMITVERSUS SAMPLE QUANTITY

When the relevant range of measurement includes val-ues that approach zero, the use of DBS samples posesadditional challenges. Using a larger quantity of samplein an assay is usually an effective way to improve thelower limit of detection (LLD) and enhance precision andreliability at the low end of the assay range, but DBS sam-ples do not provide much material, and we often want toconserve sample for other analyses. A typical drop of bloodwill contain approximately 50 ml of whole blood, and willyield seven 3.2 mm (1/8 in) discs of blood for laboratoryanalyses. A full card of five blood spots will therefore con-tain enough sample for 35 analyses requiring one 3.2 mmdisc, 17 analyses requiring two discs, 11 analyses requir-ing three discs, etc. However, in practice, five perfect bloodspots are rarely obtained, and sufficient sample for 10 to20 3.2 mm discs is a more reasonable expectation for a sin-gle finger prick. Given the tradeoff between assay per-formance and sample volume, DBS assays typically striveto use the smallest amount of material possible, butneed to evaluate the extent to which using additionalmaterial improves the LLD and other aspects of assayperformance.

ASSAY SUPPLIES: OFF-THE-SHELFOR DO-IT-YOURSELF?

Commercially available immunoassay kits—typicallydesigned for serum or plasma—can often be adapted foruse with DBS samples. Advantages of these kits are as fol-lows: (1) all necessary supplies (e.g., coated plates, detec-tion antibody, calibrators, and sample/wash buffers) aretypically included; (2) kit components are known to workwell together (e.g., capture and detection antibodies paireffectively, enzyme and substrate are complementary, buf-fers do not interfere with signal development); and (3)commercial availability increases the likelihood of proto-col implementation in other labs, and comparability ofresults across labs. Disadvantages include the relativelyhigh cost of immunoassay kits ($150 to $1,000 per kit,each of which can analyze �40 samples in duplicate), thepossibility that production of a particular kit may endwithout notice, and the lack of flexibility in modifyingimportant aspects of the protocol. For example, kits typi-cally provide an immunoassay plate that is precoatedwith capture antibody. The convenience of a precoatedplate—almost certainly optimized for use with plasma—precludes increasing the concentration of coating

4 DEVELOPMENT AND VALIDATION OF ASSAY PROTOCOLS

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antibody, which can be an effective way to increasesignal when small quantities of sample are used, as withDBS.

An alternative do-it-yourself approach involves sourc-ing antibodies and calibration material, immunoassayplates, and chemicals for sample/wash buffers directlyfrom various suppliers. The cost savings of designing anassay from scratch are substantial in terms of materials,but these savings should be weighed against the timerequired to assemble the necessary components (e.g.,make buffers, coat plates with antibody), and the exten-sive time required up front to evaluate components duringassay development. In addition, a higher level of technicalexpertise and immunoassay experience is required todevelop protocols from scratch.

INSTRUMENTATION AND EQUIPMENT

There are no special instrumentation requirements forDBS assays, and any lab that is equipped to implementimmunoassay analysis of serum, plasma, saliva, or urineshould have the capacity to implement DBS assays. Theonly exception is the requirement for a hole punch toremove sample from the DBS card, which can be purchasedfrom office supply stores.

Two highly recommended pieces of equipment, whichare often but not always available in the immunoassaylab, include an orbital plate shaker and a microplatewasher. Most immunoassays include extended periods ofincubation, and increasing incubation times and/or rotat-ing assay plates during incubation are often effective waysto increase performance of DBS assays. In addition, micro-plate washers automate the process of washing plate wellsbetween assay steps (e.g., after sample incubation andbefore addition of detection antibody), which is essentialfor proper color development and to reduce backgroundnoise. Manual washing is possible, but it is time-consuming and generally introduces more variation acrosswells that can reduce assay precision and reliability.

Regardless of sample type, enzyme immunoassaysrequire an absorbance microplate reader to quantifythe amount of color change that is proportional to ana-lyte concentration. Basic models that provide accurateendpoint readings are sufficient for the vast majority ofapplications (e.g., BioTek ELx808, Molecular DevicesEMax), but more advanced (and more expensive) multi-mode models (e.g., BioTek Synergy, Molecular DevicesSpetraMax) provide the flexibility to use fluorescenceand luminescence, in addition to absorbance, as modesof detection. If prior work has shown that the analyteof interest can be quantified in plasma at better resolu-tion using modes of detection other than absorption,then it may be worth applying the same mode to proto-cols designed for DBS samples.

SIX STEPS TO DEVELOPING ANIMMUNOASSAY FOR DBS

Recommended procedures for assay development andvalidation are described here in six steps, although itshould be emphasized that the process is iterative.Results from step 5, for example, might lead to a reconsid-eration of procedures evaluated in steps 3 or 4. Flexibility,creativity, and patience early in the process will paydividends later by preventing one from getting locked intoa protocol that could have been better.

Step 1. Decide on reagents

The range of options for commercially available immu-noassay kits will vary widely by biomarker, and the firstkey decision involves whether to consider these kits or topursue a do-it-yourself protocol. The following factors areuseful to keep in mind when evaluating potential kitoptions: cost, required volume of sample for the serum/plasma protocol, and key aspects of assay performance asevaluated by the manufacturer, if available (e.g., lowerlimit of detection, antibody cross-reactivity, dynamicrange). If a clear choice does not emerge, it is probablyworth evaluating two or more kits since it is not alwayseasy to predict which kits will perform best when appliedto DBS samples (customer service representatives canoften be convinced to provide a free or discounted kit forevaluation).

A major determinant of final assay performance is theaffinity and specificity of the antibodies selected, and itcannot be assumed that antibodies that work well withplasma will function similarly in the more complex sam-ple matrix of DBS. Antibodies will differ across kits, andthis will be a major determinant of kit performance.When pursuing a do-it-yourself approach, the selection ofeffective capture and detection antibodies is critical, andit is worth making the initial investment to evaluate mul-tiple potential combinations.

Step 2. Prepare calibration and quality control material

In order to minimize matrix differences and maximizecomparability between calibrators and unknowns, DBScalibration material should be manufactured. This mate-rial is comprised of a known concentration of the analyteof interest in a plasma-like matrix (for examples seeMcDade et al., 2004, b; McDade and Shell-Duncan, 2002;Miller and McDade, 2012; Tanner and McDade, 2007),added to an equal volume of washed red blood cells toapproximate whole blood with a hematocrit of 50%.Washed erythrocytes are obtained as follows: (1) collectwhole blood by venipuncture in EDTA vacutainer tubes,and centrifuge at 1,500g for 15 min; (2) remove plasmaand buffy coat with a Pasteur or other pipet; (3) add anapproximately equal volume of normal saline (0.86 gNaCl/100 ml deionized H2O) to the packed red blood cells;(4) mix gently with inversion for 5 min; and (5) centrifugeas before. Remove saline and any remaining buffy coat,and repeat steps 3 to 5 for a total of three washes. Thesupernatant should be clear after each centrifugation; apinkish tone indicates hemolysis of red blood cells, inwhich case the centrifugal force should be reduced.Remove saline following the final wash so only red bloodcells remain.

Calibrator dilutions should be prepared during the finalwash steps. Calibration material is typically supplied in aconcentrated form, but occasionally immunoassay kitsprovide prediluted calibrators representing concentra-tions across the range of measurement. When using con-centrated material, calibrators should first be diluted in aplasma-like matrix to known concentrations across thedesired range of measurement. Each concentration of cali-brator is then added to an equal volume of washed eryth-rocytes (1:2 dilution). Mix gently with inversion for 5 min,then apply each calibrator to labeled filter paper cards in50 ml drops using a manual pipette. Dry overnight at roomtemperature. Calibrators should then be stored at 230�C

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or lower in gas impermeable plastic bags with desiccant.DBS-based control samples—to be used for assay valida-tion or to monitor between-assay variation—can also bemanufactured using these procedures.

Step 3. Evaluate elution protocols

Since the blood contained in a DBS sample has beendried on filter paper, analytes must be brought into solu-tion before analysis. This is a key step in the use of DBSsamples. A standard hole punch is typically used to cutout discs of whole blood of uniform size, and one or morediscs are placed into an elution buffer for a fixed amountof time. In effect, the dried blood spot is reconstituted ashemolyzed liquid whole blood, and then transferred to theassay plate where the protocol proceeds just as it wouldwith plasma.

Three variables should be considered in designing anelution protocol: (1) type of elution buffer; (2) durationand temperature of elution; and (3) whether to mix duringelution. Phosphate-buffered saline is a good place to startfor an elution buffer, and in some applications results maybe improved with the addition of protein (e.g., bovineserum albumin), polysorbate 20 (i.e., Tween 20, a surfac-tant), or protease inhibitor. The addition of protein orTween 20 may improve assay signal by stabilizing pro-teins as they come into solution, and by blocking nonspe-cific binding sites in the assay plate. When using acommercial kit it is a good idea to evaluate buffers pro-vided with the kit (e.g., sample dilution buffer) as poten-tial elution buffers. This approach has the advantage thatthe buffer is already provided, and it is known to performwell with other components of the kit. Its effectiveness asan elution buffer, however, is uncertain and should beevaluated against potential alternatives. Examples of elu-tion buffers and protocols can be found in previously vali-dated methods (Dowd et al., 2011; McDade et al., 2000a,2004, 2012b; McDade and Shell-Duncan, 2002; Miller andMcDade, 2012; Shirtcliff et al., 2001; Tanner and McDade,2007).

In all of the assays I have developed I apply overnightincubations during elution. Shorter elution periods arepossible (e.g., 2 h, 4 h), but I have found that overnightincubations maximize elution efficiency and improvework flow in the lab. For example, punching out DBS sam-ples in the morning, eluting for 2 or 4 h, and then imple-menting an immunoassay protocol (which typically takes3 or 4 h) makes for a very long day. With overnight elu-tion, samples punched out the previous day can be trans-ferred to the plate first thing in the morning to start theassay, and DBS samples for the next day’s assay can bepunched out during incubations later in the day.

A potential problem associated with longer elution peri-ods is sample degradation. A great advantage of DBS isthat the paper matrix stabilizes the blood sample, butwhen analytes are brought into solution they are moresusceptible to degradation. The extent to which degrada-tion occurs during elution will vary across analytes. Incu-bating samples at 4�C during elution may prevent orattenuate degradation. In general, incubating at roomtemperature or higher (e.g., 37�C), and adding mixing(end-over-end, or orbital) during elution will reduce elu-tion times, but may increase rates of sample degradation.

In evaluating different elution protocols the efficiencyof elution can be formally calculated by dividing the con-

centration of analyte recovered from whole blood elutedfrom filter paper by the concentration of analyte obtainedfrom the same quantity of hemolyzed whole blood in liquidform. Alternatively, results from different elution proto-cols can be compared directly to determine which methodprovides the best signal:noise ratio in the assay.

Step 4. Optimize the quantity of sample

Hole punches for cutting out uniform discs of driedblood come in various sizes, and a larger hole punch is anobvious way to introduce more sample into an assay. But Ihave used a relatively small 3.2 mm hole punch in priorwork because there is more flexibility in removing samplefrom smaller or irregularly shaped drops of whole bloodon the card. Assays developed so far have required 1 to 8discs of whole blood for duplicate measures, and increas-ing the amount of material is typically an effective way toimprove the lower limit of detection, as well as precisionand reliability at the low end of the assay range. The goalof improved assay performance, therefore, is often tradedoff against limited quantity of sample and the desire topreserve sample for other analyses. The impact of samplequantity on assay performance can be evaluated simplyby comparing results when using, for example, 1 versus2 discs in a fixed amount of elution buffer. More discsshould yield higher signal, but there is often a clear pointof diminishing returns, both in terms of assay signal aswell as use of sample.

Step 5. Optimize the assay protocol

Standard immunoassay protocols involve a series oftime-standardized incubations that facilitate antigen-antibody interactions in samples and calibrators. Again,duration of incubations (e.g., increasing from 1 to 2 h orovernight), temperature during incubation (4�C, RT, or37�C), and mixing (e.g., incubation on plateshaker vs.benchtop) are all variables that can be manipulated tooptimize assay performance. Extending incubation timesafter DBS eluates are added to the assay plate, andusing an orbital plateshaker to rotate the assay plateduring incubation, can be effective ways to increasesignal.

When using a do-it-yourself protocol, determining theoptimal concentration of capture antibody and detectionantibody is an important early step in assay development.Higher antibody concentrations typically increase assaysignal, but at a cost to higher non-specific backgroundnoise. Checkerboard titrations (Crowther, 2009) can beparticularly useful at this point because they allow one tooptimize two or three assay components simultaneously.For example, a 96-well plate can be prepared to comparedifferent concentrations of capture antibody (e.g., 2 mg/ml,10 mg/ml, 20 mg/ml, each set up in different columns on theassay plate) and different concentrations of detection anti-body (e.g., 1 ng/ml, 5 ng/ml, 10 ng/ml, set up in differentrows on the assay plate). The “checkerboard” allows one toevaluate all potential combinations of capture and detec-tion antibody concentrations. By setting up the checker-board twice, once on each half of the 96-well plate, one candetermine the optimal signal:noise ratio that is obtainedwith different quantities of DBS sample across antibodyconcentrations (e.g., eluate from 1 disc on the left side ofthe plate, eluate from 2 discs on the right side of theplate).

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Step 6. Evaluate assay performance

Once the protocol is set and the assay optimized, it istime to evaluate the assay for precision, reliability, accu-racy/recovery, lower limit of detection, and agreementwith plasma results (Nexo et al., 2000; Vikelsoe et al.,1974). Quantitative assessment of these aspects of assayperformance is important for identifying sources andmagnitudes of measurement error that may inform studydesign, the interpretation of assay results, and compara-bility with prior research.

INTRA-ASSAY VARIATION

The level of imprecision of an assay can be estimated bycalculating the coefficient of variation (%CV; 100 3 stand-ard deviation/mean) of multiple determinations (usually10 or more) of a single sample, all measured in a singleassay. This is typically done with two or three samplesacross the range of measurable values since imprecisionmay vary, and is often higher at lower analyte concentra-tions. Repeating this analysis across multiple runs willprovide a better estimate of intra-assay variation.

INTERASSAY VARIATION

The day-to-day variation, or reliability, of a method canbe estimated by calculating the CV for multiple determi-nations of a single sample measured on different days. Aswith precision, reliability should be assessed using two orthree samples with values that span the assay range.Acceptable levels of intra- and inter-assay variation willvary across assays and particular applications, but %CV<10 is a good benchmark.

ACCURACY

There are several approaches to evaluating assay accu-racy, including comparison of values derived from goldstandard methods using venipuncture plasma (discussedbelow). Accuracy can also be directly measured by spikingliquid whole blood samples with a known quantity of ana-lyte before spotting on filter paper. Alternatively, a plasmasample with a high concentration of analyte can be seri-ally diluted in a plasma-like buffer (e.g., 1:2, 1:4, 1:8),mixed with an equal volume of washed red blood cells,then spotted onto filter paper. In both cases, the ratio ofobserved to expected analyte concentrations can be usedas a measure of accuracy.

LOWER LIMIT OF DETECTION

The smallest concentration of analyte that can be differ-entiated from zero with confidence is considered theassay’s lower limit of detection (LLD). The calculation ofLLD is based on the amount of signal that is produced byat least 10 determinations of DBS sample that is free ofthe analyte of interest. These “zeros” are typically manu-factured along with calibration material, and includewashed red blood cells and plasma-like buffer in equal vol-umes, spotted onto filter paper. The mean signal providesa measure of central tendency, while the standard devia-tion provides a measure of variation. The absorbancevalue corresponding to two standard deviations above themean represents a reasonable criterion for defining“different from zero,” and by plotting this value on the cal-ibration curve it is possible to estimate the correspondinganalyte concentration that represents the assay’s LLD.

Using a criterion of three standard deviations above themean provides a more conservative estimate of LLD.

DRIED BLOOD SPOT/VENOUS BLOOD COMPARISON

The comparison of DBS-based assay results with thosefrom matched, simultaneously collected plasma samplesusing a previously established, “gold standard” method isan essential validation tool. Attempts to publish theresults of DBS assay development and validation are notlikely to survive peer-review without such an analysis. Areasonable analysis can be achieved with 40 or more sam-ples, although 100 or so would be preferable. It is impor-tant to have sufficient numbers of samples that cover theentire range of likely values (Stockl et al., 1998).

Statistical evaluation of the correspondence betweenDBS and plasma results can be performed with linearregression and correlation (Stockl et al., 1998), and byinspecting residual plots for evidence of bias or inconsis-tent variability across the range of measurement (Blandand Altman, 1986, 1999). Note that Bland-Altman analy-ses typically plot the average result of two methods on thex-axis, and the difference between the results on they-axis. This approach does not work with raw DBS values,since the presence of red blood cells dilutes DBS resultsrelative to plasma. Therefore, the ratio of DBS to plasmaresults—not the difference—should be plotted on they-axis. For example, consider a scenario where a plasmaassay produces CRP values of 2.0 mg/l for sample A, and10.0 for sample B. If we assume a hematocrit of approxi-mately 50 percent, corresponding results in DBS for sam-ples A and B would be 1.0 and 5.0, respectively. Thedifference in results across the methods increases as asimple function of analyte concentration (A: 2.0–1.0 5 1.0;B: 10.0–5.0 5 5.0) and does not reflect increased bias orvariability in DBS results relative to the gold standard. Incontrast, the ratio remains stable regardless of analyteconcentration (A: 2.0/1.0 5 2.0; B: 10.0/5.0 5 2.0), therebyallowing a meaningful analysis of bias and variabilityacross the assay range.

Many protocols produce a very high level of agreement(e.g., R>0.95) between DBS and gold standard, venousblood results. But in some cases investigators may have tosettle for less, and the advantages associated with DBS inthe field will have to be weighed against lower correlationwith the gold standard. Interference from cellular mate-rial in DBS samples, inconsistent or improper applicationof blood to the filter paper, the process of drying andreconstituting the sample, the application of differentantibodies across DBS and plasma protocols, and meta-bolic/circulatory dynamics can all contribute to differen-ces in results based on the analysis of finger stick DBSsamples versus venous blood.

Analysis of matched DBS and plasma samples can alsobe used to generate a conversion formula to deriveplasma-equivalent values from results based on DBS sam-ples (Worthman and Stallings, 1997). Converted valuesmay be particularly useful for comparisons with priorresearch, and for the application of clinically-relevant cut-off values (it should also be noted that converted valuescan be used in the Bland-Altman analyses describedabove, in which case the difference across methods, ratherthan the ratio, should be plotted on the y-axis). However,caution should be used in the application of converted val-ues, since the relationship between DBS and plasma

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values will vary across analytic methods, and may alsovary across populations (e.g., Shirtcliff et al., 2001). If con-version to plasma equivalent values is a priority for a par-ticular study, it is worth dedicating time and resources todevelop a study-specific conversion formula based on DBSsamples that are collected, handled, and analyzed inexactly the same way as participant samples (Buxtonet al., under review).

STABILITY

A major advantage of DBS sampling is the ability to col-lect blood in field settings, and to store and transport sam-ples without refrigeration or freezing. There are, however,limits to the stability of analytes in filter paper stored atambient temperatures. These limits should be determinedempirically for each analyte before sample collection, basedon the range of conditions DBS samples may be exposed toduring sample collection and transport (McDade et al.,2004; Worthman and Stallings, 1997).

In prior work I have used the following protocol to ana-lyze stability across a range of potential conditions. First,collect at least three sets of DBS samples, representinglow, mid, and high levels of analyte concentration. At thispoint it is important to calculate the quantity of sampleneeded for the evaluation, which will vary depending onthe number of stability parameters being considered, andthe quantity of sample required for the assay. If large vol-umes of sample are required it may not be feasible to col-lect finger stick DBS samples; instead, collect a largervolume of whole blood via venipuncture, and then use apipette to transfer whole blood to the filter paper. Second,after samples are allowed to dry, place one set of each intothe freezer (230�C or lower). These samples will be ana-lyzed to represent “baseline” concentrations of the analyteof interest. Third, expose samples to one of three tempera-ture conditions (4�C, room temperature, 37�C) and oneoscillating condition (12 h at 32�C and 12 h at 22�C to rep-resent ambient conditions in tropical environments), inthe presence of desiccant in a gas impermeable bag, forvarying lengths of time up to 4 weeks (humidity is also aparameter that may affect analyte stability, but it canlargely be eliminated as a factor in the field by storingcards in sealed bags before their use, and by placing DBSsamples in sealed bags with desiccant after they havedried).

Samples should be stored in the same freezer as thebaseline samples after their exposure time is completed.Baseline and exposed samples should then be analyzedtogether, on the same assay plate if possible, to maximizecomparability. Samples can be considered stable so longas values remain within a 10% CV range of the initialbaseline values. In most cases there will be a clear lineartrend toward degradation after a certain amount of time,particularly at elevated storage temperatures.

It is also useful to evaluate the stability of analytes inDBS following repeated cycles of freezing and thawing.Since the same set of samples is often used for multipleanalyses, it is possible that removing samples from thefreezer during assay set up may lead to degradation thatadversely affects subsequent analyses. As recommendedabove, three or more samples across the assay rangeshould be used, with baseline samples stored in thefreezer. Freeze/thaw samples should be removed from thefreezer, taken out of their plastic bags, placed on the

benchtop at room temperature for 1 h, and then returnedto the freezer. This protocol should be repeated over 5 dif-ferent days. Baseline and freeze/thaw samples shouldthen be analyzed together to inspect for evidence of degra-dation relative to samples that have never been thawed.The paper matrix of DBS seems to provide considerableprotection against freeze/thaw degradation, but it isworth evaluating this possibility nonetheless.

CONCLUSIONS

Research into the causes and consequences of globalhuman biological variation encourages investigators tocollect data in the field, while studies of biological mecha-nisms tether them to the lab. “Field-friendly” methodslike DBS sampling provide tools that bridge this gap, andencourage an epistemological shift that reframes thestudy of human biology as a holistic, integrative endeavor(Stinson et al., 2012). By bringing our methods to researchparticipants in the community, rather than relying onselect individuals willing to come to the clinic or lab, weare in a much stronger position to obtain generalizableresults, to link data across levels of analysis, and to fore-ground contextual factors that are important determi-nants of human physiological function and health acrossthe life course.

Finger stick DBS sampling has become an importantpart of the human biology toolkit, with applications in agrowing number of studies in the U.S. and internationally(McDade et al., 2007). The same advantages that fosteredthe development of DBS sampling for large-scale neonatalscreening programs—low costs and burdens of blood col-lection, stability of analytes on filter paper, simplifiedlogistics associated with sample handling and transport—greatly facilitate blood collection in field-based settings,with participants of all ages.

Convenience in the field, however, is traded off againstchallenges associated with quantification in the lab. Inmost cases investigators can expect results that are com-parable to those obtained with gold standard, venousblood-based methods, but only after investing consider-able effort in assay development and validation. My hopeis that this review will encourage more investigators to godown this path, and to develop and disseminate DBS pro-tocols that advance research into human biological varia-tion around the world.

USEFUL RESOURCES

SUPPLIES FOR COLLECTING DBS SAMPLES

� #903 filter papers are manufactured by Whatman, andproduced in multiple formats (http://www.whatman.com/903ProteinSaverCards.aspx). The “Protein SaverCard” (#10534612) is compact, and has a flap to coverthe sample once it is dry.

� Lancets come in a variety of styles and sizes. The BDMicrotainer contact-activated lancet contains a blade(rather than a needle) and produces good blood flow(#366594).

� Desiccant comes in many forms. Self-contained poucheswith color-changing indicator are useful for determin-ing when the desiccant is no longer effective (e.g., VWRHumidity Sponge, Indicating, #61161-319)

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ADDITIONAL INFORMATION ON DBS SAMPLING

� For more information on the use of DBS in newbornscreening programs: http://www.cdc.gov/labstandards/nsqap.html

� For guidance on regulations associated with shippingDBS samples: http://www.cdc.gov/labstandards/pdf/nsqap/Bloodspot_Transportation_Guidelines.pdf

� For information from Whatman on assessing blood spotquality: http://www.whatman.com/References/Simple%20Spot%20Check%20Literature%20Piece%20LR%20FINAL%2011.02.09.pdf

� For useful information and videos associated with theapplication of DBS sampling, see the website devel-oped by the Biomarker Network, sponsored by theNational Institute of Aging: http://gero.usc.edu/CBPH/network/index.shtml

LITERATURE CITED

Ahluwalia N. 1998. Spot ferritin assay for serum samples dried on filterpaper. Am J Clin Nutr 67:88–92.

Bland JM, Altman DG. 1986. Statistical methods for assessing agreementbetween two methods of clinical measurement. Lancet i:307–310.

Bland JM, Altman DG. 1999. Measuring agreement in method comparisonstudies. Stat Methods Med Res 8:135–160.

Brindle E, Fujita M, Shofer J, O’Connor KA. 2010. Serum, plasma, anddried blood spot high-sensitivity C-reactive protein enzyme immunoas-say for population research. J Immunol Methods 362:112–120.

Campbell KL. 1994. Blood, urine, saliva and dip-sticks: Experiences inAfrica, New Guinea, and Boston. Ann NY Acad Sci 709:312–330.

Chowdhury F, Williams A, Johnson P. 2009. Validation and comparison oftwo multiplex technologies, Luminex and Mesoscale Discovery, forhuman cytokine profiling. J Immunol Methods 340:55–64.

Crowther JR. 2009. The ELISA handbook. New York: Humana Press.Dowd JB, Aiello AE, Chyu L, Huang YY, McDade TW. 2011. Cytomegalovi-

rus antibodies in dried blood spots: a minimally invasive method forassessing stress, immune function, and aging. Immun Ageing 8:3.

Ellison P. 1988. Human salivary steroids: Methodological considerationsand applications in physical anthropology. Yearb Phys Anthropol 31:115–142.

Finch CE, Vaupel JW, Kinsella KG, editors. 2001. Cells and surveys :Should biological measures be included in social science research? Wash-ington, D.C.: National Academy Press. p 374.

Guthrie R, Susi A. 1963. A simple phenylalanine method for detecting phe-nylketonuria in large populations of newborn infants. Pediatrics 32:338–43.

James GD. 1991. Blood pressure response to the daily stressors of urbanenvironments: Methodology, basic concepts, and significance. YearbPhys Anthropol 34:189–210.

Lequin RM. 2005. Enzyme immunoassay (EIA)/enzyme-linked immuno-sorbent assay (ELISA). Clin Chem 51:2415–2418.

McDade T, Stallings J, Angold A, Costello E, Burleson M, Cacioppo J,Glaser R, Worthman C. 2000a. Epstein-Barr virus antibodies in wholeblood spots: A minimally-invasive method for assessing an aspect of cell-mediated immunity. Psychosom Med 62:560–567.

McDade TW. 2011. The state and future of blood-based biomarkers in theHealth and Retirement Study. Forum Health Econ Policy 14:5.

McDade TW, Burhop J, Dohnal J. 2004. High-sensitivity enzyme immuno-assay for C-reactive protein in dried blood spots. Clin Chem 50:652–654.

McDade TW, Shell-Duncan B. 2002. Whole blood collected on filter paperprovides a minimally invasive method for assessing human transferrinreceptor level. J Nutr 132:3760–3763.

McDade TW, Stallings JF, Worthman CW. 2000b. Culture change andstress in Western Samoan youth: Methodological issues in the cross-cultural study of stress and immune function. Am J Hum Biol 12:792–802.

McDade TW, Tallman PS, Madimenos FC, Liebert MA, Cepon TJ,Sugiyama LS, Snodgrass JJ. 2012a. Analysis of variability of high sensi-tivity C-reactive protein in lowland Ecuador reveals no evidence ofchronic low-grade inflammation. Am J Hum Biol 24:675–681.

McDade TW, Williams S, Snodgrass JJ. 2007. What a drop can do: driedblood spots as a minimally invasive method for integrating biomarkersinto population-based research. Demography 44:899–925.

McDade TW, Woodruff TK, Huang YY, Funk WE, Prewitt M, KondapalliL, Gracia CR. 2012b. Quantification of anti-Mullerian hormone (AMH)in dried blood spots: Validation of a minimally invasive method forassessing ovarian reserve. Hum Reprod 27:2503–2508.

Mei JV, Alexander JR, Adam BW, Hannon WH. 2001. Use of filter paperfor the collection and analysis of human whole blood specimens. J Nutr131:1631S–1636S.

Miller EM, McDade TW. 2012. A highly sensitive immunoassay forinterleukin-6 in dried blood spots. Am J Hum Biol 24:863–865.

Nexo E, Engbaek F, Ueland P, Westby C, O’Gorman P, Johnston C, Kase B,Guttormsen A, Alfheim I, McPartlin J et al.,. 2000. Evaluation of novelassays in clinical chemistry: Quantification of plasma total homocys-teine. Clin Chem 46:1150–1156.

O’Connor KA, Brindle E, Holman DJ, Klein NA, Soules MR, Campbell KL,Kohen F, Munro CJ, Shofer JB, Lasley BL et al.. 2003. Urinary estroneconjugate and pregnanediol 3-glucuronide enzyme immunoassays forpopulation research. Clin Chem 49:1139–1148.

Panter-Brick C, Worthman CM, editors. 1999. Hormones, health, andbehavior. Cambridge: Cambridge University Press.

Salvante KG, Brindle E, McConnell D, O’Connor K, Nepomnaschy PA.2012. Validation of a new multiplex assay against individual immunoas-says for the quantification of reproductive, stress, and energetic metabo-lism biomarkers in urine specimens. Am J Hum Biol 24:81–86.

Shirtcliff EA, Reavis R, Overman WH, Granger DA. 2001. Measurement ofgonadal hormones in dried blood spots versus serum: verification ofmenstrual cycle phase. Horm Behav 39:258–266.

Skogstrand K, Thorsen P, Norgaard-Pedersen B, Schendel DE, SorensenLC, Hougaard DM. 2005. Simultaneous measurement of 25 inflamma-tory markers and neurotrophins in neonatal dried blood spots by immu-noassay with xMAP technology. Clin Chem 51:1854–1866.

Stinson S, Bogin B, O’Rourke D, editors. 2012. Human biology: An evolu-tionary and biocultural perspective. New York: Wiley-Blackwell.

Stockl D, Dewitte K, Thienpont LM. 1998. Validity of linear regressionin method comparison studies: Is it limited by the statistical modelor the quality of the analytical input data? Clin Chem 44:2340–2346.

Tanner S, McDade TW. 2007. Enzyme immunoassay for total immunoglob-ulin E in dried blood spots. Am J Hum Biol 19:440–442.

Vikelsoe J, Bechgaard E, Magid E. 1974. A procedure for the evaluation ofprecision and accuracy of analytical methods. Scand J Clin Lab Investig34:149–152.

Weinstein M, VAupel JW, Wachter KW, editors. 2007. Biosocial surveys.Washington, D.C.: The National Academies Press. p 414.

Worthman CM, Stallings JF. 1994. Measurement of gonadotropins in driedblood spots. Clin Chem 40:448–453.

Worthman CM, Stallings JF. 1997. Hormone measures in finger-prickblood spot samples: New field methods for reproductive endocrinology.Am J Phys Anthropol 104:1–22.

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