BLOOD COLLECTED ON FILTER PAPER FOR WILDLIFE SEROLOGY:

EVALUATING STORAGE AND TEMPERATURE CHALLENGES OF

FIELD COLLECTIONS

Patricia S. Curry,1,7 Carl Ribble,1 William C. Sears,2 Karin Orsel,1 Wendy Hutchins,3

Dale Godson,4 Robbin Lindsay,5 Antonia Dibernardo,5 Mitch Campbell,6 and Susan J. Kutz1

1 Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada2 Department of Population Medicine, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1,Canada3 Faculties of Medicine and Education, University of Calgary, Calgary, Alberta T2N 4N1, Canada4 Prairie Diagnostic Services, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B4, Canada5 Zoonotic Diseases and Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada,Winnipeg, Manitoba R3E 3R2, Canada6 Department of Environment, Kivalliq Region, Government of Nunavut, Arviat, Nunavut X0C 0E0, Canada7 Corresponding author (email: pcurry007@gmail.com)

ABSTRACT: Filter-paper (FP) blood sampling can facilitate wildlife research and expanddisease surveillance. Previous work indicated that Nobuto FP samples from caribou andreindeer (Rangifer tarandus sspp.) had comparable sensitivity and specificity to serum samples($80% for both) in competitive enzyme-linked immunosorbent assays (cELISAs) for Brucellaspp., Neospora caninum, and West Nile virus. The same sensitivity and specificity criteria weremet in indirect ELISAs for Brucella spp., bovine herpesvirus type 1 (BHV-1), parainfluenzavirus type 3 (PI-3), and bovine respiratory syncytial virus (BRSV), with adjusted FP thresholdsused for PI-3 and BRSV. Comparable sensitivity and specificity values to serum were alsoobserved for FP in virus neutralization (VN) assays for bovine viral diarrhea virus types I andII; however, reduced sensitivity is a potential limitation of FP samples in protocols that requireundiluted serum (i.e., VN and N. caninum cELISA). We evaluated the performance of FPsamples from reindeer and caribou in these nine assays after simulating potential challenges ofhigh-latitude field collections: 1) different durations of storage and 2) different processing/storage regimes involving freezing or drying. Sample pairs (serum and FP) were collected fromreindeer and caribou populations in 2007–10 and were tested in duplicate. Comparableperformance to serum was defined as sensitivity and specificity $80%. In the storageexperiments, FP performance was determined after 2 mo of storage dry at room temperature,and after two longer periods (variable depending on assay; up to 2 yr). After 1 yr, compared tofrozen serum stored for the same period, sensitivity was $88% for all but two assays (68%BHV-1; 75% PI-3), and specificity remained .90%. A limited trial evaluated the effect offreezing FP samples as opposed to drying them for storage. There were no observeddetrimental effects of freezing on FP sample performance, but rigorous investigation iswarranted.

Key words: Bovine herpesvirus, Brucella, disease surveillance, Neospora, Nobuto filterpaper, Rangifer, serology, storage time and temperature.

INTRODUCTION

Wildlife sampling tends to be challenging,and the advantages of collecting and shippingblood on filter paper (FP) make this anappealing alternative to conventional bloodsampling using tubes (Curry et al. 2011). Inaddition, FP blood collection by subsistencehunters and other trained laypeople couldexpand coverage of disease-surveillance pro-grams (Brook et al. 2009; Curry 2010). Filter-paper blood testing has been widely applied

in human medicine (McDade et al. 2007),whereas relatively few FP tests have beenvalidated for wildlife (Curry et al. 2014).However, even highly accurate diagnostictests are limited by sample quality, and fieldconditions and logistics are key determinantsof this. Quality assurance and control duringtesting are also considerations. For high-latitude fauna such as caribou and reindeer(Rangifer tarandus sspp.), potential threats tosample integrity include low temperatures,lack of controlled transport conditions from

DOI: 10.7589/2012-06-150 Journal of Wildlife Diseases, 50(2), 2014, pp. 000–000# Wildlife Disease Association 2014

0

remote sampling locations to processing sites,frequent need for long-term storage beforeanalysis, and temperature fluxes and in-transit delays during shipping. To assess therobustness of FP samples for wildlife serol-ogy, and particularly those collected at highlatitudes, we evaluated the performance ofFP samples from reindeer and caribou innine antibody assays after exposure to storageand collection challenges that typify remote,cold-climate field settings.

MATERIALS AND METHODS

Design, animals, and antibody status

The University of Calgary Life and Envi-ronmental Sciences Animal Care Committeeapproved this research. The two-part studyspanned July 2007 through December 2010.In part I, we tested the effects of storage timeon FP test results; in part II, we assessedeffects of processing and storage conditions(Table 1). The assays were competitive andindirect enzyme-linked immunosorbent assays(cELISA and iELISA, respectively) for Bru-cella spp.; cELISAs for Neospora caninum andWest Nile virus (WNV); iELISAs for bovineherpesvirus type 1 (BHV-1), parainfluenzavirus type 3 (PI-3), and bovine respiratorysyncytial virus (BRSV); and virus neutraliza-tion (VN) assays for bovine viral diarrhea virustypes I and II (BVDV-I and -II). Assaymethods are described elsewhere (Curry et al.2011 [Brucella]; Curry et al. 2014 [all othertests]).

The samples for Brucella serology camefrom free-ranging hunter-killed caribou in aCanadian herd with known exposure toBrucella (Curry et al. 2011). Those for N.caninum serology were from a Canadiancommercial reindeer herd (Curry et al.2014). All other samples were from a previ-ously unvaccinated research reindeer herd inCanada (Curry et al. 2014). To obtain samplesfor the bovine virus assays (BHV-1, PI-3,BRSV, BVDV-II, and BVDV-II), we injectedthese reindeer with a primary label dose ofthe killed-virus vaccine TriangleH 4+BVDV-II(Wyeth Animal Health, Guelph, Ontario,Canada) and a booster dose either 4 wk (partI, n512 animals) or 3 wk (part II of study,n53 animals) later. To examine a range ofantibody levels, reindeer were sampled atbaseline (prevaccination) and two later timepoints (Table 1). To obtain samples for WNVtesting, we injected reindeer with a primarylabel dose of the killed-virus equine vaccine

West Nile-Innovator (Fort Dodge, WyethAnimal Health, Fort Dodge, Iowa, USA)followed by a booster 4 wk later. Only adults(10 of the 14 research animals sampled forWNV testing) were vaccinated, and bloodsamples were collected 6 mo after primarydose.

Sampling methods

Nobuto FP strips (Toyo Roshi Kaisha, Ltd.,Tokyo, Japan) were used. At each collection,we obtained matched serum and FP samplepairs from each animal. Detailed descriptionsof these techniques in free-ranging caribouand captive reindeer have been describedpreviously (Curry et al. 2011, 2014).

Processing and treatments

Blood tubes were centrifuged 15 min at3,500 3 G, and serum aliquots were stored in1.5-mL microcentrifuge tubes or 0.5-mL flat-cap PCR tubes (MCT-200-C or PCR-05-C,respectively, Axygen Scientific, Union City,California, USA) at 220 C until analysis. Uponcollection from each animal, FP samples weresubjected to one or more of four possibleprocessing/storage regimes or ‘‘treatments’’(Table 1):

1) Dry-Dry (DD): FP samples were col-lected at ambient temperature andplaced in a paper envelope that wasinserted in a large-size ZiplocH Brand(SC Johnson, Racine, Wisconsin, USA)plastic bag (i.e., FP samples and bloodtubes were maintained above freezingand in the same conditions until process-ing). Upon return to the laboratory 2–8 hrlater, the FP samples were dried in rackson the bench overnight and then storeddry at room temperature (RT; 15–22 C)until analysis (see description of drystorage below).

2) Dry-Freeze (DF): The same procedurewas followed as for DD, except sampleswere stored dry at RT for only 2 wk andthen stored at 220 C until analysis (seedescription of 220 C storage below).

3) Freeze-Freeze (FF): FP samples werecollected as above, and the large ZiplocHbag with FP envelope was immediatelyplaced in a cooler with frozen ice packs.Upon return to the laboratory 2–8 hrlater, the samples were transferred to220 C conditions until analysis.

4) Freeze-Dry (FD): The same procedurewas followed as for FF, except sampleswere stored at 220 C for 2 wk, then

0 JOURNAL OF WILDLIFE DISEASES, VOL. 50, NO. 2, APRIL 2014

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CURRY ET AL.—STORAGE AND TEMPERATURE EFFECTS ON FILTER-PAPER BLOOD SAMPLES 0

thawed/dried overnight, and stored dry atRT until analysis.

For dry storage, multiple FP envelopes(maximum 25) were placed in large-sizeZiplocH plastic bags with desiccant packets(Humidity SpongeTM, VWR InternationalLLC, Missisauga, Ontario, Canada) (Curryet al. 2011). The bags were kept in multi-purpose, non-airtight, clear polypropylenestorage boxes (approximately 60-L size) withsnap lids. Desiccant was checked regularly(2-wk intervals for month 1, and approxi-mately 12-wk intervals thereafter) and re-placed as needed. For 220 C storage, nodesiccant was used, and each FP envelopewas kept in an individual ZiplocH bag in alaboratory-grade 220 C freezer unit withoutautodefrost.

In part I of the study (storage time), all FPsamples were dried immediately and storeddry at RT (DD treatment). Serum-FP pairswere assayed at one initial time point (T1,#2 mo of storage) and at two longer storagetimes (up to 2 yr; Table 1). For the Brucellatesting at 24 mo, it was only possible to assay asubset of 88 of the 184 total serum-FP pairs(i.e., a 96-well test plate of serum samples anda matching plate of FP samples, eachcontaining 88 samples plus eight positiveand negative controls). To ensure the rangeof anti-Brucella reactivity was represented inthe subset, we categorized the T1 (2 mo)serum results for the Brucella cELISA as low,medium, or high reactivity (,20%, 20–40%,and $80% inhibition, respectively) and useda random-number generator to identify 34,21, and 33 samples, respectively (as close toequal numbers for each reactivity level aswere available) from these groups. At T1,these 88 sample pairs were all tested on thesame day using the same reagent lots andcontrol lots. These conditions were applied atthe 24-mo test time as well.

In part II of the study (a limited trial of theeffects of processing/storage conditions), allsamples were tested 12 mo after collection.

Testing

The cELISAs, iELISAs, and VNs per-formed and the respective threshold valuesused are listed in Table 1. Comprehensivedetails regarding Nobuto FP elution meth-ods, shipping, laboratories, assay protocolsand controls, and assay thresholds aredescribed elsewhere (Curry et al. 2011[Brucella]; Curry et al. 2014 [all otherassays]). Briefly, FP eluates produced ac-cording to Nobuto manufacturer instructionswere approximately equivalent to 1:10

serum. The FP samples stored at 220 Cwere thawed/dried overnight in a rack at RTbefore elution. Laboratory technicians ad-justed the initial assay protocol steps asneeded to account for the 1:10 dilutionfactor with FP samples and to optimizecomparison of serum and FP results. Labo-ratories carried out batches of matched FPand serum tests on the same day and inblinded fashion, using sets of positive andnegative controls for each test plate, andusing reagents of the same lot whereverappropriate and possible. Serum-FP pairsfrom each animal were assayed in duplicateusing separate FP samples for run A andrun B.

The WNV assay was a two-step process,each step involving a monoclonal antibody thattargeted a different protein of WNV (Curryet al. 2014). At the time of WNV testing forthis study (part II), the monoclonals in usewere 3.112G-NS-1 and 7H2-Env, which tar-geted nonstructural and envelope proteins,respectively. We followed the same conserva-tive method and rationale described by Curryet al. (2014) for establishing positive ornegative status in this two-test assay.

Analysis

Storage time: We analyzed FP test perfor-mance by 1) comparing FP samples at eachstage of storage to the ‘‘ideal’’ (T1, or shortest-storage) serum results, and 2) comparing thepaired FP-serum results after each storageperiod. EpiTools calculators (Sergeant 2009)were used to determine FP test sensitivity,specificity, serum antibody prevalence, FPantibody prevalence, and positive and negativepredictive values for FP. Clopper-Pearsonconfidence intervals were calculated for esti-mates. Comparability to serum was defined assensitivity and specificity $80%, based onproficiency standards for human diagnosticlaboratories (Astles 2010).

For five of the assays in part I (BHV-1, PI-3, BRSV, BVDV-I, BVDV-II), FP-serumsample pairs were obtained from 12 adultvaccinated reindeer at three time points toexamine FP test performance over a range ofantibody levels (Table 1). To our knowledge,no existing statistical software or coding cansimultaneously accommodate data that arebinomial, matched-pairs, and repeated-measures. For test performance analysis,accommodating binomial data and matchedpairing is essential. These animals’ resultswere, thus, analyzed as independent observa-tions (Curry et al. 2014).

0 JOURNAL OF WILDLIFE DISEASES, VOL. 50, NO. 2, APRIL 2014

Each of the five ELISAs generated contin-uous data (% inhibition, % positivity, orELISA units [EU]). These were plotted toshow FP results at successive storage timesrelative to T1 serum and thresholds. For eachassay, the run A serum results at T1 wereplotted with FP results for each stage ofstorage (the lowest-sensitivity FP run identi-fied during test-performance analysis).

It was necessary to exclude some of theBVDV VN results from test performanceanalysis. On both these assays, the thresholdvalues were titer $1:6 for serum and titer$1:20 for FP (Curry et al. 2014). In thesetests, some of the wells containing the firstdilution of serum (1:6) and the first two FPeluate dilutions (i.e., titer steps up to 1:60)were unreadable because of loss of susceptiblecells resulting from sample toxicity (as op-posed to pathogenic virus effects). The labo-ratory results for these toxic cases were listedas ranges, as opposed to individual titers (e.g.,#1:6 for serum or #1:20, #1:40, or #1:60 forFP), and were, therefore, impossible tocategorize as positive or negative based onthe threshold values specified above. Conse-quently, any sample pair with a toxic result forserum or FP (or both) was excluded.

The number of toxic results in the VNsreduced sample numbers such that no BVDVdata could be analyzed relative to T1 serum(Table 2). The same issue limited the stageanalysis for these assays to two storage periods(1 and 12 mo) instead of three (Table 3).

Processing/storage conditions: Performance inthe WNV cELISA was analyzed as above. Thenumber of animals (n53) in the limited trial offour FP treatments precluded statistical test-ing; however, we plotted each animal’s find-ings for serum and each FP treatment in theBHV-1 and PI-3 iELISAs. We also convertedthese data to categorical results (antibody-positive or antibody-negative according tothreshold) and assessed for agreement in twoways: 1) comparing serum vs. FP results foreach treatment (n59 per serum vs. FPcomparison); and 2) comparing FP vs. FP forall possible pairings of the four treatments ateach antibody level (i.e., ‘‘low’’ or prevaccina-tion collection, ‘‘medium’’ 3 wk later [booster],and ‘‘high’’ 6 wk later; n53 per FP vs. FPcomparison).

RESULTS

Part I: Storage time

Based on the means of SE and SP forruns A and B, FP samples performed

comparably to T1 serum in all eight assaysthat were analyzed for storage time(ranges: sensitivity 89–98%, specificity91–99%; Tables 2, 3). One estimate(50% specificity for BRSV at T1) wasexcluded from the analysis as an outlier.When FP results at 12 or 17 mo storagewere compared to T1 serum, sensitivitywas $81% (comparable to serum) in theBrucella, N. caninum, and BRSV assays,but it was lower in the BHV-1 and PI-3 assays (62% and 54%, respectively;Table 2). In contrast, when the FP resultsat 12 or 17 mo storage were compared totheir same-storage-time serum results,sensitivity was $88% for Brucella, N.caninum, BRSV, and both BVDV assays,and it was 68% and 75% for the BHV-1and PI-3 tests, respectively (Table 3;additional performance statistics in Sup-plementary Material Appendix I). After 12or 17 mo of storage, the average drop insensitivity relative to T1 serum was ap-proximately 15% (range, 3–36%), whereasthe corresponding drop in sensitivityrelative to same-storage serum was 7%

(range, 0–23%).The slopes of the iELISA results for

BHV-1, PI-3, and BRSV (i.e., samplesfrom vaccinated animals) appeared to beshallower crossing the threshold thanthose for the Brucella and N. caninumassays (i.e., samples from naturally ex-posed animals) (Fig. 1).

Part II: Processing/storage conditions

The WNV cELISA performance esti-mates for FP samples after 12 mo in DDand FF conditions were the same (meansfor runs A and B: sensitivity 80% andspecificity 90%; Table 4). Figure 2 showsthe BHV-1 and PI-3 results from the FPtreatments trial. The categorical (positive/negative) test results for the PI-3 iELISAagreed in all treatment comparisons (datanot shown). For the BHV-1 iELISA, allcomparisons except those that involvedFD treatment had complete agreement(data not shown).

CURRY ET AL.—STORAGE AND TEMPERATURE EFFECTS ON FILTER-PAPER BLOOD SAMPLES 0

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0 JOURNAL OF WILDLIFE DISEASES, VOL. 50, NO. 2, APRIL 2014

TA

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lect

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set

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ter

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s.

CURRY ET AL.—STORAGE AND TEMPERATURE EFFECTS ON FILTER-PAPER BLOOD SAMPLES 0

FIGURE 1. a–f. Effects of storage time for enzyme-linked immunosorbent assays (ELISAs). Effects ofstorage time on testing of blood on Nobuto filter-paper (FP) strips. Filter-paper blood samples from caribouand reindeer (Rangifer tarandus sspp.) were dried immediately after collection and stored dry. Plots for thecompetitive and indirect enzyme-linked immunosorbent assays (cELISAs and iELISAs, respectively) showthe results for run A of T1 (shortest-storage) serum (open circles) and the corresponding FP results for threestorage times. For each storage period, the FP run with the lowest sensitivity (see Table 3) was plotted using adistinct symbol. To create the graphs, data were sorted according to ascending serum values at T1. Eachsample pair number on the x-axis has a set of data symbols vertically above it: the T1 serum result and threecorresponding FP results, one for each storage time. Dotted lines denote assay threshold values for serum andFP (identical unless noted separately).

0 JOURNAL OF WILDLIFE DISEASES, VOL. 50, NO. 2, APRIL 2014

DISCUSSION

Storage time

The most important findings for the FPsamples subjected to dry/RT storage were1) the sensitivity of FP testing diminished,but in most cases it remained comparableto serum, with longer storage; specificityremained high; 2) the decline in sensitivitywas less pronounced when FP resultswere compared to results from sera thathad been stored at 220 C for the sameperiod, as opposed to serum from T1; 3) FPperformance at 6 mo tended to be betterthan that observed at 12 mo or longer;however, even after 12 mo or more of dry/RT storage, the sensitivity of FP relative tosame-storage serum was $85% (thus,comparable to serum) in six of the eight

antibody assays evaluated for storage time.Specifically, our results for comparison tosame-storage serum suggest that, evenafter $1 yr of dry/RT storage (and insome cases after as long as 2 yr), NobutoFP serum samples from reindeer andcaribou can have sensitivity and specificitycomparable to serum in the cELISA andiELISA for Brucella, the cELISA for N.caninum, the iELISA for BRSV, and theVNs for BVDV-I and -II. For these sixassays, sensitivity of FP remained $88%

at this stage, and in most cases sensitivityand specificity were both .90% (Table 3).As expected, in comparison to longerstorage, sensitivity and specificity weregenerally, but not always, higher after only6 mo of storage. The poorest FP perfor-mance was in the BHV-1 assay, with an

FIGURE 1. Continued.

CURRY ET AL.—STORAGE AND TEMPERATURE EFFECTS ON FILTER-PAPER BLOOD SAMPLES 0

average estimated sensitivity of 77% at6 mo (Table 3). Curry et al. (2014)reported important considerations for FPuse in the BVDV VN assays.

Filter-paper products have differentspecifications for biomedical use, anddrying whole blood in FP matrices stabi-lizes most analytes (McDade et al. 2007).Nobuto FP strips are made of high-puritycellulose, are highly absorbent and uni-form quality, and are recommended forseveral veterinary serology applications(Toyo Roshi Kaisha, Ltd.). Studies of theeffects of storage on FP samples differconsiderably in design, and some resultsare challenging to compare (e.g., Beardand Brugh 1977; Banks 1985; McDadeet al. 2000); however, evidence fromanimals and humans indicates that anti-bodies in whole-blood FP samples gener-ally withstand storage and temperaturechallenges better than some other analytes(McDade et al. 2007; Trudeau et al. 2007).A drop in test sensitivity after prolongedsample storage could reflect a character-istic of the assay, a technical or reagentissue, or loss of analyte stability. Somedegree of interassay variation is expectedwith any antibody assay that is repeatedafter extended intervals, and reports noteapproximately $6% such variation for FPtesting in ELISAs and other platforms(Clague and Thomas 2002; McDade et al.2007). Although we observed this in our

study (Tables 2, 3), the successivelygreater decreases in sensitivity of FPtesting over longer storage times suggestthat antibodies degraded within theNobuto FP matrix over time. In accor-dance with previous research on variousserum analytes (Woodrum and York 1998;Cray et al. 2009), for most assays we alsoobserved reductions in antibody preva-lence when we retested serum afterextended storage at 220 C (Supplemen-tary Material Appendix I). Thus, there ispotential for some loss of sensitivity notonly with FP samples in serologic tests,but also with serum that is banked at220 C for extended periods.

Vaccination and sampling close to timeof seroconversion might have affected ourELISA results. We found that FP testperformance was generally closer to thatof serum in the ELISAs on samples fromherds with natural immunity (Brucella andN. caninum testing) than in the ELISAson samples from vaccinates (BHV-1, PI-3,and BRSV testing; Fig. 1 and Tables 1, 3).Vaccinates’ results exhibited a differentpattern, and more values were nearthreshold (Fig. 1d, e, f). A large propor-tion of results near threshold can impairFP test performance. Also, samples fromanimals that had recently seroconvertedlikely contained significant primary-response immunoglobulin (Ig) M, whereasone would expect IgG to predominate in

TABLE 4. Test performance of filter-paper (FP) blood samples in the West Nile virus assay after twotreatments: dried immediately and stored dry at room temperature (DD) or frozen immediately and stored at220 C (FF) until analysis. The FP-serum pairs were compared after 12 mo storage, and sensitivity (SE),specificity (SP), and confidence intervals (CI) are shown in duplicate (runs A and B). Results for a larger DDsample set that was stored for 1 mo and tested at the same laboratory are presented for comparison.

FP treatment Estimatea

FP/serum storage time and run (no. sample pairs tested)

1 mo (26)b 12 mo A (14) 12 mo B (14)

DD SE, % (CI) 95.2* (80.7–100) 75.0 (34.9–96.8) 85.7 (42.1–99.6)SP, % (CI) 91.7 (61.5–99.8) 89.1* (60.7–1.0) 90.6* (65.2–1.0)

FF SE, % (CI) n/a 85.7 (42.1–99.6) 75.0 (34.9–96.8)SP, % (CI) n/a 90.6* (65.2–1.0) 89.1* (60.7–1.0)

a Clopper-Pearson 95% exact CIs are given. In cases where the initial estimate derived was 100%, the median unbiasedestimate (denoted with *) was calculated and reported instead.

b Curry et al. (2014). Monoclonals used were 3.112G-NS-1 (as in current study) and C03301-Env.

0 JOURNAL OF WILDLIFE DISEASES, VOL. 50, NO. 2, APRIL 2014

animals with nonrecent natural exposure.Studies of FP samples from humans inELISAs for dengue virus and humanimmunodeficiency virus have revealed lossof sensitivity in samples from patients whoare newly seroconverted, and more rapid

degradation of IgM in samples frompatients with primary infections (Behetset al. 1992; Ruangturakit et al. 1994).Thus, it is possible that the performance ofFP samples from herds naturally exposedto Rangifer-specific relatives of BHV-1,PI-3, or BRSV might have better concor-dance with serum antibody results than wehave estimated for these three ELISAs.The same might hold true for BVDV VN.This consideration is particularly relevantfor FP performance in the BHV-1 and PI-3 iELISAs because sensitivity was lower(i.e., below the 80% defining value forcomparable to serum) in these tests thanin the other assays at 6 and 12 mo of dry/RT storage. Rigorous investigation of thisis a potential direction for future research.

We were unable to access reindeer orcaribou herds that were antibody-positivefor all the respective pathogens; thus,small sample size contributed to the widthand overlapping of confidence intervals(Tables 2, 3; Supplementary Material Ap-pendix I). Assessing test performance induplicate test runs is more robust thandoing so in single runs. Excluding oneoutlier specificity result for BRSV, theduplicate sensitivity and specificity resultsfor all the assays provide evidence that ourestimates are reasonable indicators of FPtest performance.

As noted, for the five bovine-pathogenassays, FP-serum sample pairs were col-lected from 12 vaccinated reindeer atthree time points to capture test perfor-mance over a range of antibody levels(Table 1). Curry et al. (2014) detailed theconsiderations for analysis and interpreta-tion of these data.

Processing/storage conditions

Our WNV test results provide noevidence of a difference between dry/RTand 220 C storage conditions (DD andFF treatments) for FP samples fromreindeer (Table 4). Comparison to previ-ously published FP performance results inthis cELISA for WNV indicates that thedrop in FP sensitivity for this assay after

FIGURE 2. a–b. Trial results: Effects of processing/storage regimes for bovine herpesvirus type 1 (BHV-1)and parainfluenza virus type 3 (PI-3) assays. Effects ofdifferent treatments (dry-dry [DD], dry-freeze [DF],freeze-dry [FD], or freeze-freeze [FF]; details inMethods) on testing of filter-paper (FP) blood samples.Results are from three vaccinated reindeer (Rangifertarandus sspp.) that were sampled on the day ofvaccination and then 3 wk (booster) and 6 wk later.Indirect enzyme-linked immunosorbent assays (iELISAs)for BHV-1 and PI-3 were performed on sample pairs12 mo after collection. Dotted lines denote assaythreshold values for serum and FP (identical unlessnoted separately), and the three animals’ results aredistinguished by different symbols. Analysis of category(positive/negative test result) agreement identified FDtreatment (*) as the only processing/storage regime withany disagreement.

CURRY ET AL.—STORAGE AND TEMPERATURE EFFECTS ON FILTER-PAPER BLOOD SAMPLES 0

1 yr of dry/RT storage may be comparableto declines we observed in the other eightassays over this period of storage (part I);however, the small sample size for WNVtesting meant that there was insufficientpower to reveal statistical differences inthis part of our analysis.

Compared to studies of tropical-climateeffects on FP samples, little has beenreported on the effects of freezing fresh FPsamples prior to drying (Behets et al. 1992).According to observations from our limitedFP treatments trial, freezing blood-soakedFP strips upon collection and storing themfrozen (often necessary when collecting athigh latitudes in winter) might not impairdetection of antibodies to BHV-1 or PI-3(Fig. 2); however, rigorous investigation withlarge sample numbers is needed.

Other implications and recommendations

For serologic applications, the NobutoFP manufacturer recommends avoidingsunlight, ultraviolet light, water, extremetemperatures, and moisture, and to store‘‘in a clean indoor space’’ (Toyo RoshiKaisha, Ltd.). More specifics have beenpublished in blood-spot guidelines andreviews (NCCLS 2003; McDade et al.2007), studies from the tropics (e.g.,Punnarugsa and Mungmee 1991; Ruang-turakit et al. 1994) and elsewhere (e.g.,Corran et al. 2008; Mei et al. 2011), and afew animal reports (e.g., Brugh and Beard1980; Trudeau et al. 2007). However, thevast majority of sources focus on humanblood spots that are dried and analyzedfrom small FP discs, and we found onlyone source that assessed effects of freezing

TABLE 5. Key recommendations for collection, processing, storage, and shipping of Nobuto filter-paper (FP)blood samples from wildlife for use in serologic testing.

Recommendations and cautionsa

Collection, handling Avoid touching the absorbent (long) portion of FP strip; dip/saturate entire length ina pool of clean whole blood (serosanguinous fluid in a body cavity may containantibodies but it is not equivalent to a whole-blood sample); avoid contacting hair,feces, organ contents, other tissues/fluids

Processing Option 1: Dry samples completely at ambient room temperature (15–22 C at leastovernight; timing depends on humidity at location); avoid direct sunlight,temperature .30 C, humidity .30%, formalin (or fumes) exposure

Option 2: Freeze samples (220 C) upon collection and keep frozen at 220 CShort-term storage (,2 yr) Dry FP samples: Store in zip-closure plastic bagsb with (but not directly contacting)

desiccant pouchesb and humidity indicator; check/refresh desiccant regularly;ideally, refrigerate (4 C) for up to 2 yr; alternatively, store dry/room temperaturefor several months (exact duration is assay-specific)

Frozen FP samples: Store in individual zip-closure plastic bagsb, 220 Claboratory-grade freezer (no autodefrost)

Long-term storagec ($2 yr) Dry FP samples: Store in 220 C laboratory-grade freezer (no autodefrost)Frozen FP samples: Store in 220 C laboratory-grade freezer (no autodefrost)

Analysis Dry FP samples prior to eluting; keep dry at room temperature while preparingeluates; test FP eluates immediately or freeze at 220 C (if necessary) until testing

Shipping Dry FP samples: Ship in zip-closure plastic bagsb with desiccantb

Frozen FP samples: *Depends on individual circumstances/risks; can ship frozen inzip-closure plastic bagsb or thaw/dry prior to shipping and ship in zip-closure bagswith desiccantb

All FP samples: Ship separate from any formalin-preserved samplesFP eluates: If eluates must be shipped, keep frozen during transit

a Main sources: NCCLS (2003); McDade et al. (2007); Toyo Roshi Kaisha, Ltd. (Tokyo, Japan); authors’ experience withNobuto FP samples from wildlife, and evidence from the current study.

b See details regarding specific products and usage in Methods and Materials section.c 220 C is specifically recommended by the Clinical and Laboratory Standards Institute (formerly NCCLS) for $2 yr’

storage of dried blood spots for human neonatal screening (NCCLS 2003).

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FP blood samples directly upon collection(Behets et al. 1992).

Table 5 integrates our findings in asummary of key considerations for collect-ing, storing, and shipping Nobuto FPsamples for wildlife serology. The mostimportant step in ensuring FP sampleintegrity—and one that should be com-municated explicitly to hunters and otherlayperson samplers—is to make everyeffort to collect a clean blood sample andavoid bacterial contamination, which candegrade antibodies. It is also crucial thatNobuto FP collectors be trained tosaturate the entire length (the absorbentportion) of the FP strip, as elutionrequires a complete FP strip, and a partialsample can negate testing altogether. Forarchiving of Nobuto FP samples, onealternative to the suggestions in Table 5might be to elute and freeze the eluate,which might reduce antibody degradation.However, FP eluate composition differsfrom that of serum, and, to our knowledge,there is no published evidence that thereis benefit to eluting and freezing. In fact,freeze-thaw studies suggest that keepingsamples in the FP matrix might besomewhat protective if there is any riskof thawing (McDade et al. 2000, 2004).Shipping decisions depend on the individ-ual situation, and the transport of frozenFP samples needs careful consideration. Ifsuch FP samples will be tested shortlyafter arrival at the laboratory, and if thereare risks of delays and temperaturefluctuations during transit, then it maybe best to thaw/dry immediately prior toshipping and ship dry with desiccant. For-malin damages FP samples, and so exposureto this chemical or its fumes should beavoided (Toyo Roshi Kaisha, Ltd.). Thepossible toxicity issue with FP samples (morethan with serum) in VN is a topic for futureinvestigation (Curry et al. 2014); however,given our experience in two studies, andgiven the relatively lower sensitivity of FPsamples in VN (compared to other testplatforms where there is no initial dilutionstep), we recommend using wildlife FP

samples in ELISA platforms rather thanVN if possible. Filter-paper samples need tobe validated for each test in each species.

The FP method offers particular advan-tages for wildlife field collections and haslower per-unit storage-space and storage-costdemands than serum. Our estimates of testperformance for FP samples from reindeerand caribou in these antibody assays afterdifferent storage times and under differentprocessing/storage regimes can help guideusers. The evidence, experience, and multi-sourced recommendations provided in thispaper can help decision makers tailor thebest Nobuto FP practices to their field andlaboratory constraints.

ACKNOWLEDGMENTS

We acknowledge the contributions of all theanimals involved in this research, and weespecially thank Marianne Jorgensen and GregMuench for their care of the University ofCalgary reindeer herd. Caribou hunters AaronEmiktowt, Chris Jones, John Nakoolak, MarkPootoolik, and Greg Ningeocheak from CoralHarbour, Nunavut, as well as Brett Elkin, JaneHarms, and the entire Kutz Laboratory team2007–009 provided invaluable support for samplecollection. We are indebted to Klaus Nielsen’slaboratory at the Brucellosis Centre of Expertise(Canadian Food Inspection Agency). Sincereappreciation is extended to Linda Kelly, MelanieSabourin, Lori Hassard, Laura Bond, RobertaYemen, and Johnathon Pameolik for theirtechnical and logistic expertise. We acknowledgethe Government of Nunavut and our funders:International Polar Year Funding from theNatural Sciences and Engineering ResearchCouncil (NSERC) Special Research OpportunityProgram, Environment Canada/Natural Resourc-es Canada, Nasivvik Centre for Inuit Health andChanging Environments (Canadian Institutes ofHealth Research), Alberta Innovates TechnologyFutures, Northern Scientific Training Program(Indian and Northern Affairs, Government ofCanada), Arctic Institute of North America, theCircum-Arctic Rangifer Monitoring and Assess-ment Network (CARMA, www.carmanetwork.org), and the University of Calgary Faculty ofVeterinary Medicine.

SUPPLEMENTARY MATERIAL

Supplementary material for this articleis online at http://doi:10.7589/xxxx-xx-xxx.

CURRY ET AL.—STORAGE AND TEMPERATURE EFFECTS ON FILTER-PAPER BLOOD SAMPLES 0

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Submitted for publication 3 June 2012.Accepted 28 September 2013.

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