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1. Validation of an enzyme‐linked immunoassay assay for osteocalcin, a marker of bone formation, in dried blood spots

   https://doi.org/10.1002/ajhb.23394  

   Eick, Geeta N. ; Madimenos, Felicia C. ; Cepon‐Robins, Tara J. ; Devlin, Maureen J. ; Kowal, Paul ; Sugiyama, Lawrence S. ; Snodgrass, J. Josh ( February 2020 , American Journal of Human Biology)

   Investigating factors that contribute to bone loss and accretion across populations in remote settings is challenging, particularly where diagnostic tools are scarce. To mitigate this challenge, we describe validation of a commercial ELISA assay to measure osteocalcin, a biomarker of bone formation, from dried blood spots (DBS).

   We validated the Osteocalcin Human SimpleStep ELISA kit from Abcam (ab1951214) using 158 matched plasma and DBS samples. Passing‐Bablok regression analysis assessed the relationships between plasma and DBS osteocalcin concentrations. Dilutional linearity and spike and recovery experiments determined if the DBS matrix interfered with osteocalcin measurement, and intra‐ and inter‐assay coefficients of variation (CVs) were calculated. Limit of detection, analyte stability, and specific forms of osteocalcin measured by the kit were also investigated.

   Mean plasma osteocalcin value was 218.2 ng/mL (range 64.6‐618.1 ng/mL). Linear relationships existed between plasma and DBS concentrations of osteocalcin, with no apparent bias in plasma vs DBS concentrations. There was no apparent interference of the DBS matrix with measurement of osteocalcin in DBS. Intra‐assay CV for DBS was ~8%, while average inter‐assay CV was 14.8%. Limit of detection was 0.34 ng/mL. Osteocalcin concentrations were stable in DBS stored at −28°C and room temperature, but not those stored at 37°C. This ELISA kit detects total osteocalcin.

   Osteocalcin, a bone formation biomarker, can be measured from DBS. Combined with a previously validated DBS assay for TRACP‐5b, a bone resorption biomarker, these assays have the potential to help researchers disentangle the many factors contributing to bone strength.

    

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2. Variability of C‐reactive protein in first‐generation Ecuadorian immigrants living in the United States

   https://doi.org/10.1002/ajhb.23547  

   Shattuck‐Heidorn, Heather ; Eick, Geeta N. ; Kramer, Karen L. ; Sugiyama, Lawrence S. ; Snodgrass, James Josh ; Ellison, Peter T. ( December 2020 , American Journal of Human Biology)

   Establish the variability of C‐reactive protein (CRP) within a population of first‐generation immigrants living in the United States. Prior work has theorized that individuals with high levels of childhood pathogen exposure may have lower CRP levels in adulthood, and therefore that for these individuals, CRP may not be as accurate an index of chronic disease risk related to low‐level inflammation as is presumed based on data from wealthy populations. This potentially has major implications for the interpretation of CRP as a biomarker of chronic inflammation.

   This longitudinal study collected a total of 125 dried blood spot (DBS) samples from 31 participants (median 4 samples each) and CRP levels in these DBS were assayed by enzyme‐linked immunosorbant assay. Surveys were administered to characterize childhood pathogen exposure, and current illness. Variance was estimated using mixed effects regression models.

   On average, participants were adults (mean = 41.9 years old) who had immigrated to the United States nearly 20 years prior to the study and had nearly universally experienced childhood helminth infection and other major pathogen exposures. Median serum‐equivalent CRP was 0.77 mg/L. Individuals reliably differed in subacute CRP levels, and, depending on whether untransformed or log‐transformed CRP was the outcome variable, 45% or 62% of variance in CRP was attributable to between‐individual differences.

   The variability of CRP levels in individuals with relatively high childhood pathogen exposure is comparable to previously reported studies in North America and Europe. However, CRP values are relatively low. CRP is an appropriate measure of subacute inflammation in this sample.

    

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3. A surrogate virus neutralization test to quantify antibody-mediated inhibition of SARS-CoV-2 in finger stick dried blood spot samples

   https://doi.org/10.1038/s41598-021-94653-z  

   Sancilio, Amelia E. ; D’Aquila, Richard T. ; McNally, Elizabeth M. ; Velez, Matthew P. ; Ison, Michael G. ; Demonbreun, Alexis R. ; McDade, Thomas W. ( December 2021 , Scientific Reports)

   Abstract The spike protein of SARS-CoV-2 engages the human angiotensin-converting enzyme 2 (ACE2) receptor to enter host cells, and neutralizing antibodies are effective at blocking this interaction to prevent infection. Widespread application of this important marker of protective immunity is limited by logistical and technical challenges associated with live virus methods and venous blood collection. To address this gap, we validated an immunoassay-based method for quantifying neutralization of the spike-ACE2 interaction in a single drop of capillary whole blood, collected on filter paper as a dried blood spot (DBS) sample. Samples are eluted overnight and incubated in the presence of spike antigen and ACE2 in a 96-well solid phase plate. Competitive immunoassay with electrochemiluminescent label is used to quantify neutralizing activity. The following measures of assay performance were evaluated: dilution series of confirmed positive and negative samples, agreement with results from matched DBS-serum samples, analysis of results from DBS samples with known COVID-19 status, and precision (intra-assay percent coefficient of variation; %CV) and reliability (inter-assay; %CV). Dilution series produced the expected pattern of dose–response. Agreement between results from serum and DBS samples was high, with concordance correlation = 0.991. Analysis of three control samples across the measurement range indicated acceptable levels of precision and reliability. Median % surrogate neutralization was 46.9 for PCR confirmed convalescent COVID-19 samples and 0.1 for negative samples. Large-scale testing is important for quantifying neutralizing antibodies that can provide protection against COVID-19 in order to estimate the level of immunity in the general population. DBS provides a minimally-invasive, low cost alternative to venous blood collection, and this scalable immunoassay-based method for quantifying inhibition of the spike-ACE2 interaction can be used as a surrogate for virus-based assays to expand testing across a wide range of settings and populations. 

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4. Telomere length analysis from minimally‐invasively collected samples: Methods development and meta‐analysis of the validity of different sampling techniques: American Journal of Human Biology

   https://doi.org/10.1002/ajhb.23410  

   Rej, Peter H. ; Bondy, Madison H. ; Lin, Jue ; Prather, Aric A. ; Kohrt, Brandon A. ; Worthman, Carol M. ; Eisenberg, Dan T. A. ( March 2020 , American Journal of Human Biology)

   Telomeres are the protective caps of chromosomes. They shorten with cell replication, age, and possibly environmental stimuli (eg, infection and stress). Short telomere length (TL) predicts subsequent worse health. Although venous whole blood (VWB) is most commonly used for TL measurement, other, more minimally invasive, sampling techniques are becoming increasingly common due to their field‐friendliness, allowing for feasible measurement in low‐resource contexts. We conducted statistical validation work for measuring TL in dried blood spots (DBS) and incorporated our results into a meta‐analysis evaluating minimally invasive sampling techniques to measure TL.

   We isolated DNA extracts from DBS using a modified extraction protocol and tested how they endured different shipping conditions and long‐term cryostorage. We then included our in‐house DBS TL validation statistics (correlation values with VWB TL and age) in a series of meta‐analyses of results from 24 other studies that published similar associations for values between TL measured in DBS, saliva, and buccal cells.

   Our modified DBS extraction technique produced DNA yields that were roughly twice as large as previously recorded. Partially extracted DBS DNA was stable for 7 days at room temperature, and still provided reliable TL measurements, as determined by external validation statistics. In our meta‐analysis, DBS TL had the highest external validity, followed by saliva, and then buccal cells—possibly reflecting similarities/differences in cellular composition vs VWB.

   DBS DNA is the best proxy for VWB from the three minimally‐invasively specimen types evaluated and can be used to expand TL research to diverse settings and populations.

    

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5. Sex-specific effects of calving season on joint health and biomarkers in Montana ranchers

   https://doi.org/10.1186/s12891-022-05979-2  

   Thompson, Matthew A. ; Martin, Stephen A. ; Hislop, Brady D. ; Younkin, Roubie ; Andrews, Tara M. ; Miller, Kaleena ; June, Ronald K. ; Adams, Erik S. ( January 2023 , BMC Musculoskeletal Disorders)

   Agricultural workers have a higher incidence of osteoarthritis (OA), but the etiology behind this phenomenon is unclear. Calving season, which occurs in mid- to late-winter for ranchers, includes physical conditions that may elevate OA risk. Our primary aim was to determine whether OA biomarkers are elevated at the peak of calving season compared to pre-season, and to compare these data with joint health survey information from the subjects. Our secondary aim was to detect biomarker differences between male and female ranchers.

   During collection periods before and during calving season, male (n = 28) and female (n = 10) ranchers completed joint health surveys and provided samples of blood, urine, and saliva for biomarker analysis. Statistical analyses examined associations between mean biomarker levels and survey predictors. Ensemble cluster analysis identified groups having unique biomarker profiles.

   The number of calvings performed by each rancher positively correlated with plasma IL-6, serum hyaluronic acid (HA) and urinary CTX-I. Thiobarbituric acid reactive substances (TBARS), a marker of oxidative stress, was significantly higher during calving season than pre-season and was also correlated with ranchers having more months per year of joint pain. We found evidence of sexual dimorphism in the biomarkers among the ranchers, with leptin being elevated and matrix metalloproteinase-3 diminished in female ranchers. The opposite was detected in males. WOMAC score was positively associated with multiple biomarkers: IL-6, IL-2, HA, leptin, C2C, asymmetric dimethylarginine, and CTX-I. These biomarkers represent enzymatic degradation, inflammation, products of joint destruction, and OA severity.

   The positive association between number of calvings performed by each rancher (workload) and both inflammatory and joint tissue catabolism biomarkers establishes that calving season is a risk factor for OA in Montana ranchers. Consistent with the literature, we found important sex differences in OA biomarkers, with female ranchers showing elevated leptin, whereas males showed elevated MMP-3.

    

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