Comparison of 25-hydroxyvitamin D concentration in chimpanzee dried blood spots and serum.

BACKGROUND: Dried blood spots (DBS) are used in human medicine to measure total 25-hydroxyvitamin D (25-OHD) in the blood. However, this easy and affordable sampling technique has not been evaluated in primates to measure vitamin D concentrations. OBJECTIVES: We aimed to compare 25-OHD measurements in chimpanzee serum at two different laboratories and determine the precision and accuracy of the DBS method by comparing DBS and serum results. METHODS: Blood samples from 17 captive chimpanzees were collected, and 25-OHD3 and 25-OHD2 were measured in serum at two accredited laboratories using liquid chromatography-tandem mass spectrometry. The same analytes were measured on DBS cards, and results were compared with that of serum. Data were assessed using the Spearman correlation, Deming regression, and Bland-Altman analyses. RESULTS: The correlation coefficient between the two measurements in serum was rs  = .51 (P = .04), and the mean bias was -1.25 ± 14.83. When comparing 25-OHD concentrations measured in DBS and serum at the same laboratory, the rs was 0.7 (P = .002), and the mean bias was 1.42 ± 14.58. Estimated intra-assay and inter-assay coefficients of variation for DBS results were 6% and 12.6%, respectively. CONCLUSIONS: Although substantial analytical variability was found in 25-OHD measurements regardless of the sample type, the identification of both constant and proportional error and wider limits of agreement with the DBS technique makes the interpretation of DBS results challenging, especially for values close to clinical cut-off points. The DBS and serum methods were not interchangeable, and further studies are needed to validate DBS samples for vitamin D measurements in chimpanzees.

Veterinary utility of dried blood spots for detailed analysis of chlorinated pesticides and polychlorinated biphenyls by gas chromatography tandem mass spectrometry.

Persistent organic pollutants (POPs) are organic compounds of anthropogenic origin that resist atmospheric and microbial degradation and thus persist in the environment and in food chains for exceptionally long periods of time. Veterinarians and wildlife researchers need simple methodologies for monitoring and measuring such compounds including two large and diverse categories, organochlorine pesticides (OCs) and polychlorinated biphenyls (PCBs), compounds that have been largely banned from production and use except for specific exceptions. We present development of methodologies for detection and quantitation of 22 OCs and 10 PCB congeners by tandem quadrupole gas chromatography-mass spectrometric analysis of Dried Blood Spots (DBS). Development was enabled by (1) optimization of suspension and extraction methodologies for DBS; (2) strategic streamlining and condensation of Multiple Reaction Monitoring (MRM) settings on GC/MS/MS; and (3) improvement of GC settings to accommodate all 32 compounds in a single chromatographic run per sample. The method was validated for parameters of linearity, limits of detection and quantitation, recovery and precision, and results from blood were shown to correlate well with those from DBS despite both being only 50 uL in volume. The method was applied successfully to blood samples from nine avian specimens submitted to the MSU Veterinary Diagnostic Lab, and all were shown to bear the burden of varying levels of OCs and/or PCB compounds.

EVALUATION OF 25-HYDROXYVITAMIN D IN HOFFMANN'S TWO-TOED SLOTHS (CHOLOEPUS HOFFMANNI) USING DRIED BLOOD SPOTS ANALYZED BY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY.

Although biochemical analytes have typically been measured using serum or whole blood samples, an increasing number of assays are validated for measurement of analytes from dried blood spots (DBS) on filter paper. DBS techniques are minimally invasive, require only a small sample volume, and simplify processing, storage, and shipment of samples. These qualities make DBS-based assays ideal for sampling of wildlife species in both captive and field settings. In this study, a liquid chromatography-tandem mass spectrometry assay was evaluated for measurement of 25-hydroxyvitamin D in sloths. Paired serum and DBS samples were collected from nine healthy captive Hoffmann's two-toed sloths (Choloepus hoffmanni). Statistical analysis using Passing-Bablok regression analysis, Bland-Altman plots, and the Wilcoxon signed-rank tests found good agreement between 25-hydroxyvitamin D3 measurements in serum and DBS samples. Constant and proportional bias were absent. Results from this study support the use of DBS samples for the evaluation of vitamin D status in Hoffmann's two-toed sloths and provide a foundation for further studies to validate this technique.

HAIR, WHOLE BLOOD, AND BLOOD-SOAKED CELLULOSE PAPER-BASED RISK ASSESSMENT OF MERCURY CONCENTRATIONS IN STRANDED CALIFORNIA PINNIPEDS.

Mercury (Hg) poses a health risk to wildlife populations and has been documented at relatively high concentrations in many marine mammals, including wild-caught pinnipeds along the central California, US coast. We measured total Hg concentrations ([THg]) in hair and blood of live-stranded harbor seals (HS; Phoca vitulina), California sea lions (CSL; Zalophus californianus), and northern elephant seals (NES; Mirounga angustirostris) in California to quantify species, temporal, and spatial variability in [THg] and assess the relationships between [THg] measured by different methods (blood vs. filter paper) and in different matrices (blood vs. hair). We compared [THg] with toxicologic thresholds of concern to aid in identification of at-risk individuals or groups and better understand how the use of different methods and matrices affects assumed toxicologic risk. There was a wide range of [THg] in blood (<0.01-1.13 µg/g) and hair (0.45-81.98 µg/g), and NES had higher [THg] compared with HS and CSL. All three species had individuals with [THg] that exceeded the lower threshold for one or both matrices, but only HS pups had [THg] exceeding upper thresholds. Spatial differences in [THg] were detected, with higher concentrations in HS pups from areas surrounding San Francisco Bay, but differences were dependent on sampling year and matrix. The relationship between [THg] in blood and filter paper (r2=0.98) was strong, and differences had little influence on comparisons with toxicologic thresholds. Blood and hair [THg] were generally in agreement (r2=0.72), but large mismatches for a few seals underscore the importance of combined sampling in adverse effects studies where accurate assessment of Hg exposure is crucial. The wide range of [THg] in stranded HS pups that exceeded published thresholds of concern makes them a promising candidate for adverse effects studies, particularly because different matrices represent Hg exposure across key developmental stages.

Detection of Antibodies Against Foot-and-Mouth Disease Virus Serotypes A, O and Asia-1 by ELISA in Strip-Dried Samples from Vaccinated Bovines.

Antibodies against foot-and-mouth disease virus serotypes A, O and Asia-1 were detected by ELISA in liquid and strip-dried samples from vaccinated bovines. The results showed high concordance of the results in both types of serum samples (coefficient of correlation varied from 0.89 to 0.96). Stability studies showed that anti-foot-and-mouth disease virus antibodies can be detected in strip-dried serum samples stored at different temperatures on a level of that in native samples. Preliminary study in strip-dried whole blood samples demonstrated good potential of this sample pretreatment procedure for the following anti-foot-and-mouth virus antibodies testing.

Veterinary utility of dried blood spots for analysis of toxic chlorinated hydrocarbons.

Dried blood spots (DBS) on filter paper provide a simple and convenient means of collecting, storing and shipping samples for veterinary diagnostics related to toxin exposures. This paper presents validation data on analysis of DBS for chlorinated persistent organic pollutants, specifically 4,4'-dichloro-diphenyl-trichloroethane (4,4'-DDT) and its breakdown product 4,4'-dichlorodiphenyl-dichloroethylene (4,4'-DDE), lindane and a representative polychlorinated biphenyl (PCB) congener PCB-153. Analysis was by gas chromatography with electron capture detection (GC-ECD). The method required one 12.5 mm diameter spot representing application of 50 µL of blood, and working limits of detection (LOD) for each of the compounds was 5 ppb. Data are presented on development and description of the method, assay precision, LOD and quantitation, linearity, accuracy, specificity, effects of long-term storage and ruggedness. The method was also applied to 27 avian DBS, and 4,4'-DDE was detected in the majority of samples.

Evaluation of Trapper-Collected Nobuto Filter-Paper Blood Samples for Distemper and Parvovirus Antibody Detection in Coyotes (Canis latrans) and Raccoons (Procyon lotor).

Blood samples are often collected from free-ranging wildlife for antibody detection. However, filter-paper (FP) strips are more cost efficient and easy to collect and store. We evaluated trapper-collected FP strips and body-cavity blood for canine distemper (CDV) and parvovirus (CPV-2) antibody detection in raccoons (Procyon lotor) and coyotes (Canis latrans). From 2008 to 2010, licensed trappers near Madison and Milwaukee, Wisconsin, US collected paired samples from harvested animals. Canine distemper antibodies were detected using virus neutralization and parvovirus antibodies were detected using hemagglutination inhibition. Titers ≥ 1:32 for CDV and ≥ 1:25 for CPV-2 were considered evidence of exposure. Using Cohen's kappa test of agreement, FP strip titers agreed with sera for CDV in coyotes (n = 28, K = 0.772) and raccoons (n = 29, K = 0.858) and for CPV-2 in coyotes (n = 40, K = 0.775) and raccoons (n = 70, K = 0.646). However, raccoons determined to be exposed to CPV-2 from sera were unexposed by FP strips in 35% of the samples. Titer results may be affected by quality and volume of blood samples, interval between collection and processing, small sample sizes, and diagnostic testing procedures. Filter-paper strips can be useful for detecting CDV and CPV-2 exposure in coyotes and raccoons with correct field sample collection and appropriate diagnostic testing procedures.

Using dried blood spot sampling to improve data quality and reduce animal use in mouse pharmacokinetic studies.

Traditional pharmacokinetic analysis in nonclinical studies is based on the concentration of a test compound in plasma and requires approximately 100 to 200 µL blood collected per time point. However, the total blood volume of mice limits the number of samples that can be collected from an individual animal-often to a single collection per mouse-thus necessitating dosing multiple mice to generate a pharmacokinetic profile in a sparse-sampling design. Compared with traditional methods, dried blood spot (DBS) analysis requires smaller volumes of blood (15 to 20 µL), thus supporting serial blood sampling and the generation of a complete pharmacokinetic profile from a single mouse. Here we compare plasma-derived data with DBS-derived data, explain how to adopt DBS sampling to support discovery mouse studies, and describe how to generate pharmacokinetic and pharmacodynamic data from a single mouse. Executing novel study designs that use DBS enhances the ability to identify and streamline better drug candidates during drug discovery. Implementing DBS sampling can reduce the number of mice needed in a drug discovery program. In addition, the simplicity of DBS sampling and the smaller numbers of mice needed translate to decreased study costs. Overall, DBS sampling is consistent with 3Rs principles by achieving reductions in the number of animals used, decreased restraint-associated stress, improved data quality, direct comparison of interanimal variability, and the generation of multiple endpoints from a single study.

Dried fluid spots for peste des petits ruminants virus load evaluation allowing for non-invasive diagnosis and genotyping.

BACKGROUND: Active surveillance of peste des petits ruminants (PPR) should ease prevention and control of this disease widely present across Africa, Middle East, central and southern Asia. PPR is now present in Turkey at the gateway to the European Union. In Bangladesh, the diagnosis and genotyping of PPR virus (PPRV) may be hampered by inadequate infrastructures and by lack of proper clinical material, which is often not preserved under cold chain up to laboratories. It has been shown previously that Whatman® 3MM filter paper (GE Healthcare, France) preserves the nucleic acid of PPRV for at least 3 months at 32°C. RESULTS: In this study, we demonstrate the performances of filter papers for archiving RNA from local PPRV field isolates for further molecular detection and genotyping of PPRV, at -70°C combined with ambient temperature, for periods up to 16 months. PPR-suspected live animals were sampled and their blood and nasal swabs were applied on filter papers then air dried. Immediately after field sampling, RT-PCR amplifying a 448-bp fragment of the F gene appeared positive for both blood and nasal swabs when animals were in febrile stage and only nasal swabs were detected positive in non-febrile stage. Those tested positive were monitored by RT-PCR up to 10 months by storage at -70°C. At 16 months, using real time RT-PCR adapted to amplify the N gene from filter paper, high viral loads could still be detected (~2 x 10(7) copy numbers), essentially from nasal samples. The material was successfully sequenced and a Bayesian phylogenetic reconstruction achieved adequate resolution to establish temporal relationships within or between the geographical clusters of the PPRV strains. CONCLUSIONS: This clearly reveals the excellent capacity of filter papers to store genetic material that can be sampled using a non-invasive approach.

Comparative evaluation of serum, FTA filter-dried blood and oral fluid as sample material for PRRSV diagnostics by RT-qPCR in a small-scale experimental study.

Recently, research into alternative sample materials, such as oral fluid or filter-dried blood has been intensified, in order to facilitate cost-effective and animal-friendly sampling of individuals or groups of pigs for diagnostic purposes. The objective of this study was to compare the sensitivity of porcine reproductive and respiratory syndrome virus (PRRSV)-RNA detection by reverse transcription quantitative real-time PCR (RT-qPCR) in serum, FTA filter-dried blood and oral fluid sampled from individual pigs. Ten PRRSV negative pigs were injected with an EU-type PRRSV live vaccine. Blood and oral fluid samples were taken from each pig before, and 4, 7, 14 and 21 days after vaccination. All samples were then analyzed by PRRSV RT-qPCR. In serum, eight often pigs tested RT-qPCR positive at different time points post infection. Absolute quantification showed low serum PRRSV-RNA loads in most samples. In comparison to serum, sensitivity of PRRSV-RNA detection was strongly reduced in matched FTA filter-dried blood and in oral fluid from the same pigs. These results indicate that with low PRRSV-RNA loads the diagnostic sensitivity of PRRSV-RNA detection by RT-qPCR achieved with serum is currently unmatched by either FTA filter-dried blood or oral fluid.

Use of blood collected onto and dried on filter paper for diagnosing pregnancy in cattle.

The measurement of serum or plasma pregnancy-associated glycoproteins (PAGs) is increasingly used to diagnose pregnancy in cattle. This study evaluated whether a dried blood spot (DBS) collected on filter paper could be used as an alternative to serum or plasma for such tests. A total of 37 serum, 68 plasma and 68 DBS samples were collected from cows of known pregnancy status and tested using a commercial ELISA. None of the plasma or serum samples resulted in false positives or false negatives. No false positives (sample-negative (S-N) values >0.3 in non-pregnant cows) were observed with DBS samples, but false negatives were observed (S-N values <0.3 in pregnant cattle). The data suggested that PAGs in DBS samples were diluted during processing as samples from pregnant cattle had lower S-N values (0.111-0.494) than the corresponding serum (1.123-2.665) and/or plasma (0.764-2.042) samples. ROC analysis showed that lowering the cut-off S-N value from 0.3 to 0.1 for DBS samples prevented false negatives without increasing false positives. Modifications to the test protocol significantly increased mean S-N values of DBS samples from pregnant cows while those from non-pregnant cows were not affected. In conclusion, lowering the cut-off and modifying the protocol allowed DBS samples to be used for blood-based pregnancy testing.

Dried blood spots for the enzymatic diagnosis of lysosomal storage diseases in dogs and cats.

BACKGROUND: In people, lysosomal storage diseases (LSD) can be diagnosed by assaying enzyme activities in dried blood spots (DBS). OBJECTIVE: The aim of this study was to evaluate the feasibility of using DBS samples from dogs and cats to measure lysosomal enzymatic activities and diagnose LSD. METHODS: Drops of fresh whole blood collected in EDTA from dogs and cats with known or suspected LSD and from clinically healthy dogs and cats were placed on neonatal screening cards, dried, and mailed to the Metabolic Laboratory, University Children's Hospital, Frankfurt, Germany. Activities of selected lysosomal enzymes were measured using fluorescent substrates in a 2-mm diameter disk (~2.6 µL blood) punched from the DBS. Results were expressed as nmol substrate hydrolyzed per mL of blood per minute or hour. RESULTS: Reference values were established for several lysosomal enzyme activities in DBS from dogs and cats; for most enzymes, activities were higher than those published for human samples. Activities of ß-glucuronidase, N-acetylglucosamine-4-sulfatase (arylsulfatase B), α-mannosidase, α-galactosidase, α-fucosidase, and hexosaminidase A were measureable in DBS from healthy cats and dogs; α-iduronidase activity was measureable only in cats. In samples from animals with LSD, markedly reduced activity of a specific enzyme was found. In contrast, in samples from cats affected with mucolipidosis II, activities of lysosomal enzymes were markedly increased. CONCLUSIONS: Measurement of lysosomal enzyme activities in DBS provides an inexpensive, simple, and convenient method to screen animals for suspected LSD and requires only a small sample volume. For diseases in which the relevant enzyme activity can be measured in DBS, a specific diagnosis can be made.