Finger-prick Dried Blood Spots Research Articles

Trends in body mass index for people with and without HIV: Pooled analysis of nationally-representative health surveys from 10 countries and 173,800 adults in Africa.

It remains unclear if and how body mass index (BMI) levels have changed over time in HIV endemic regions. We described trends in mean BMI and prevalence of overweight between 2003-2019 in 10 countries in Africa including people living with (PLWH) and without (PLWoH) HIV. We pooled Demographic and Health Surveys (DHS) from countries where ≥2 surveys >4 years apart were available with height/weight measurements and HIV tests. HIV status was ascertained with a finger-prick dried blood spot (DBS) specimen tested in a laboratory. The DBS is taken as part of the regular DHS procedures. We summarized age and socioeconomic status standardized sex-specific mean BMI (kg/m2) and prevalence of overweight (BMI ≥25 kg/m2) by HIV status. We fitted country-level meta-regressions to ascertain if changes in ART coverage were correlated with changes in BMI. Before 2011, women LWH (22.9 [95% CI: 22.2-23.6]) and LWoH (22.6 [95% CI: 22.3-22.8]) had similar mean BMI. Over time, mean BMI increased more in women LWH (+0.8 [95% CI: 0.7-0.8] BMI units) than LWoH (+0.2 [95% CI: 0.2-0.3]). Before 2013, the mean BMI was similar between men LWH (21.1 (95% CI: 20.3-21.9)) and LWoH (20.8 (95% CI: 20.6-21.1)). Over time, mean BMI increased more in men LWoH (+0.3 [95% CI: 0.3-0.3]) than LWH (+0.1 [95% CI: 0.1-0.1]). The same profile was observed for prevalence of overweight. ART coverage was not strongly associated with BMI changes. Mean BMI and prevalence of overweight were similar in PLWH and PLWoH, yet in some cases the estimates for PWLH were on track to catch up with those for PLWoH. BMI monitoring programs are warranted in PLWH to address the rising BMI trends.

Seroprevalence of SARS-CoV-2 antibodies among children receiving primary care in Toronto, Ontario.

Characterizing the seroprevalence of SARS-CoV-2 antibodies in children is needed to optimize the COVID-19 public health response. We quantified the seroprevalence of SARS-CoV-2 infection-acquired antibodies and vaccine-acquired antibodies among children receiving primary care in Toronto, Canada. We conducted a longitudinal cohort study between January 2021 and November 2022 in healthy children aged 0-16years receiving primary care in Toronto. The primary and secondary outcomes were seroprevalence of SARS-COV-2 infection-acquired antibodies and vaccine-acquired antibodies ascertained from finger-prick dried blood spots. Samples were tested using an enzyme-linked immunosorbent assay for antibodies to full-length spike trimer and nucleocapsid. We explored sociodemographic differences with Firth's penalized generalized estimating equations. Of the 475 participants, 50.1% were girls and mean age was 6.4years (SD = 3.2). We identified 103 children seropositive for infection-acquired antibodies, with a crude seroprevalence that rose from 2.6% (95%CI 1.39-4.92) from January to July 2021 to 50.7% (95%CI 39.5-61.8) by July to November 2022. Seroprevalence of vaccine-acquired antibodies was 45.2% by July to November 2022 (95%CI 34.3-56.58). No differences in sociodemographic factors (age, sex, income, or ethnicity) were identified for infection-acquired antibodies; however, children with vaccine-acquired antibodies were more likely to be older, have mothers with university education, and have mothers who had also been vaccinated. Our results provide a benchmark for seroprevalence of SARS-CoV-2 antibodies in children in Toronto. Ongoing monitoring of the serological status of children is important, particularly with the emergence of new variants of concern, low vaccine coverage, and discontinuation of PCR testing.

COVID-19 vaccine immunogenicity and safety surrounding fourth and subsequent vaccine doses in patients with hematologic malignancies

BackgroundImmune response to COVID-19 vaccine is diminished in patients with hematologic malignancy. There is limited data regarding response to vaccine doses in these patients. PurposeTo quantify the humoral immune response engendered by 4th and subsequent doses of SARS-CoV-2 vaccination as measured by anti-Spike (anti-S) antibody levels, based on dried blood spot (DBS) testing, in patients with hematologic malignancies. Anti-S binds to the spike protein of the SARS-CoV-2 virus and is indicative of vaccine immunogenicity. MethodsWe conducted a prospective study of hematologic malignancies between August 2021 and January 2023 at 12 sites across Canada. Participants were followed longitudinally and submitted finger-prick DBS cards at set intervals associated with vaccination. Samples were processed via high throughput ELISA assay to detect serum antibodies against nucleocapsid (N) and spike (S) proteins. ResultsWe obtained 3071 samples on 790 unique patients. Of these, 372 unique participants with 1840 samples had anti-S results available post-4th, 5th or 6th COVID-19 vaccine dose and were included for analysis.Three hundred thirty-three patients of the 372 participants submitted a DBS sample post 4th dose. Of these, 257 patients (77.2%) had a positive anti-S antibody. A total of 198 patients had paired samples pre- and post-dose 4, of which 59 (29.7%) had a negative anti-S antibody pre-dose 4. Of these, 20 (33.4%) developed positive anti-S antibody post-dose 4. One hundred forty-nine patients submitted a DBS sample post-dose 5. Of these, 135 patients (90.6%) had positive anti-S antibody. A total of 52 had paired samples pre- and post-dose 5. Six (8.7%) had a negative anti-S antibody pre-dose 5, of which two (33.3%) developed positive anti-S antibody post-dose 5.Of these 372 patients, 123 (34%) reported COVID-19 infection and 4 (1%) had a COVID-19 related hospitalization. There were no reported deaths from COVID-19. ConclusionsThis prospective cohort study showed that humoral immune response improved with subsequent doses of COVID-19 vaccines.

Factors associated with SARS-CoV-2 infection in unvaccinated children and young adults

Background and objectivesPediatric COVID-19 cases are often mild or asymptomatic, which has complicated estimations of disease burden using existing testing practices. We aimed to determine the age-specific population seropositivity and risk factors of SARS-CoV-2 seropositivity among children and young adults during the pandemic in British Columbia (BC).MethodsWe conducted two cross-sectional serosurveys: phase 1 enrolled children and adults < 25 years between November 2020-May 2021 and phase 2 enrolled children < 10 years between June 2021-May 2022 in BC. Participants completed electronic surveys and self-collected finger-prick dried blood spot (DBS) samples. Samples were tested for immunoglobulin G antibodies against ancestral spike protein (S). Descriptive statistics from survey data were reported and two multivariable analyses were conducted to evaluate factors associated with seropositivity.ResultsA total of 2864 participants were enrolled, of which 95/2167 (4.4%) participants were S-seropositive in phase 1 across all ages, and 61/697 (8.8%) unvaccinated children aged under ten years were S-seropositive in phase 2. Overall, South Asian participants had a higher seropositivity than other ethnicities (13.5% vs. 5.2%). Of 156 seropositive participants in both phases, 120 had no prior positive SARS-CoV-2 test. Young infants and young adults had the highest reported seropositivity rates (7.0% and 7.2% respectively vs. 3.0-5.6% across other age groups).ConclusionsSARS-CoV-2 seropositivity among unvaccinated children and young adults was low in May 2022, and South Asians were disproportionately infected. This work demonstrates the need for improved diagnostics and reporting strategies that account for age-specific differences in pandemic dynamics and acceptability of testing mechanisms.

Four-dimensional trapped ion mobility spectrometry lipidomics for high throughput clinical profiling of human blood samples

Lipidomics encompassing automated lipid extraction, a four-dimensional (4D) feature selection strategy for confident lipid annotation as well as reproducible and cross-validated quantification can expedite clinical profiling. Here, we determine 4D descriptors (mass to charge, retention time, collision cross section, and fragmentation spectra) of 200 lipid standards and 493 lipids from reference plasma via trapped ion mobility mass spectrometry to enable the implementation of stringent criteria for lipid annotation. We use 4D lipidomics to confidently annotate 370 lipids in reference plasma samples and 364 lipids in serum samples, and reproducibly quantify 359 lipids using level-3 internal standards. We show the utility of our 4D lipidomics workflow for high-throughput applications by reliable profiling of intra-individual lipidome phenotypes in plasma, serum, whole blood, venous and finger-prick dried blood spots.

RF15 | PMON304 Utilizing LC-MS/MS for the clinical quantitation of testosterone using dried blood spot and how it compare to serum

Abstract Introduction Hormones are the chemical messengers of your body. Steroid hormones like testosterone are a specific class of hormone that are produced predominantly in the gonads (sex steroids) and less so in the adrenal glands (corticosteroids). When too little or too much of testosterone is produced, adverse symptoms and conditions can occur. In order to restore the vital balance of hormones, detailed and accurate measurement of hormones is needed. Serum currently represents the gold standard when it comes to hormone testing. However, there are other body fluids such as saliva, urine, and capillary blood (finger prick) where the hormone levels correlate well with serum. A significant advantage of these alternative fluids is that they are non-invasive, or semi-invasive, can be collected "on the go" by the patient at home, and don't have the strict requirements for cold-chain shipping. Objectives The primary objective of this work is to show that finger-prick dried blood spot blood is quantitatively equivalent to serum when compared by LC-MS/MS testing in patients only producing endogenous testosterone. We will also show that topically delivered testosterone increases in a dose-dependent fashion, whereas serum levels of testosterone increase very little regardless of topical testosterone dosing. Methods Serum samples were collected by a physician and sent into the lab and analyzed for testosterone on a Siemens Immulite 2000 autoanalyzer. Dried blood spots were collected by the patient and sent into the lab and analyzed on an AB Sciex 5500 triple quadrupole mass spectrometer. Results Patients who don't supplement with topical testosterone show a strong correlation between endogenous venipuncture serum and DBS testosterone levels. In sharp contrast, samples from topical testosterone users show remarkably different results with hormone levels 10-100×higher in the DBS than serum results. Conclusion These results are significant because it gives a glimpse into which forms of testing are most appropriate for different patients based on the mode of steroid hormone delivery. Without using the proper body fluid for testosterone detection, patients potentially could be overdosed if serum results are used to guide therapeutic dosing. Also, venipuncture serum will fail to show "sports-doping" with exogenous testosterone, which easily can be identified with capillary finger stick blood. This is also significant as the World Anti-doping Agency has finally begun accepting these data as fact and have listed dried blood spot as an appropriate form of collection and analysis for drug testing. This can help find athletes using performance enhancers that would normally have skirted by the system (1). WADA. (2021, May 21) WADA Executive Committee approves Technical Document for innovative dried blood spot testing method. https://www.wada-ama.org/en/media/news/wada-executive-committee-approves-technical-document-for-innovative-dried-blood-spot. Presentation: Sunday, June 12, 2022 12:36 p.m. - 12:41 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Detection of neutralizing antibodies against multiple SARS-CoV-2 strains in dried blood spots using cell-free PCR

An easily implementable serological assay to accurately detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies is urgently needed to better track herd immunity, vaccine efficacy and vaccination rates. Herein, we report the Split-Oligonucleotide Neighboring Inhibition Assay (SONIA) which uses real-time qPCR to measure the ability of neutralizing antibodies to block binding between DNA-barcoded viral spike protein subunit 1 and the human angiotensin-converting enzyme 2 receptor protein. The SONIA neutralizing antibody assay using finger-prick dried blood spots displays 91–97% sensitivity and 100% specificity in comparison to the live-virus neutralization assays using matched serum specimens for multiple SARS-CoV-2 variants-of-concern. The multiplex version of this neutralizing antibody assay, using easily collectable finger-prick dried blood spots, can be a valuable tool to help reveal the impact of age, pre-existing health conditions, waning immunity, different vaccination schemes and the emergence of new variants-of-concern.

Analysis of dried blood spots is a feasible alternative for detecting ephedrine in doping control.

Dried blood spot (DBS) testing allows fast, easy and minimally invasive collection of microvolumes of blood. In an anti-doping context, DBS testing has particular relevance for substances prohibited in-competition only such as ephedrine, which is currently detected by urine analysis, because DBS can add information about the blood drug concentrations during the in-competition period. Several collection methods and devices exist for DBS collection from different anatomical sites. Thus, agreements between concentrations of target analytes in DBS samples from different sampling sites, along with between DBS and those in conventional venous plasma samples, need to be evaluated. Herein, we collected matched upper-arm DBS, fingerprick DBS and venous plasma samples from eight healthy male subjects in an 8-h period following oral administrations of 20 mg ('low dose') and 60 mg ('high dose') of ephedrine. We show that the use of alternative sampling sites and matrices is a feasible possibility for ephedrine analysis in doping control. We observed very good agreement between collection sites and that specificity and sensitivity can be upheld despite use of an alternative collection site. However, potential concentration differences between DBS and venous plasma should be considered, and distinct threshold might be necessary if implementing both blood matrices in ephedrine analysis.

A national survey integrating clinical, laboratory, and WASH data to determine the typology of trachoma in Nauru.

The epidemiology of trachoma in several Pacific Islands differs from other endemic settings, in that there is a high prevalence of clinical signs of trachoma, particularly trachomatous inflammation-follicular (TF), but few cases of trichiasis and limited evidence of ocular chlamydial infection. This so-called "Pacific enigma" has led to uncertainty regarding the appropriate public health response. In 2019 alongside Nauru's national trachoma population survey, we performed bacteriological and serological assessments of children to better understand the typology of trachoma and to determine whether there is a need for trachoma interventions. We used two-stage cluster sampling, examining residents aged ≥1 year and collecting household-level water, sanitation, and hygiene (WASH) variables. Children aged 1-9 years provided conjunctival swabs and finger-prick dried blood spots to investigate the presence of Chlamydia trachomatis nucleic acid and anti-Pgp3 antibodies, respectively. In 818 participants aged 1-9 years, the age-adjusted TF prevalence was 21.8% (95% CI 15.2-26.2%); ocular C. trachomatis prevalence was 34.5% (95% CI 30.6-38.9), and anti-Pgp3 antibody prevalence was 32.1% (95% CI 28.4%-36.3%). The age- and gender-adjusted prevalence of trichiasis in ≥15-year-olds was 0.3% (95% CI 0.00-0.85), but no individual with trichiasis had trachomatous scarring (TS). Multivariable analysis showed an association between age and both TF (OR per year of age 1.3 [95% CI 1.2-1.4]) and anti-Pgp3 positivity (OR 1.2 [95% CI 1.2-1.3]). There were high rates of access to water and sanitation and no WASH variable was associated with the presence of TF. TF, nucleic acid, and age-specific antibody prevalence collectively indicate that high levels of C. trachomatis transmission among children present a high risk of ocular damage due to trachoma. The absence of trichiasis with trachomatous scarring suggest a relatively recent increase in transmission intensity.

A new dried blood spot LC-MS/MS method for therapeutic drug monitoring of palbociclib, ribociclib, and letrozole in patients with cancer

Therapeutic drug monitoring (TDM) is strongly suggested to define the proper drug dosage to overcome inter- and intra-patient variability in drug exposure, which is typically observed with oral anticancer agents, such as palbociclib (PALBO), ribociclib (RIBO) and letrozole (LETRO), all approved for the treatment of HR+, HER2- locally advanced or metastatic breast cancer (BC). Optimal TDM implementation requires a blood sampling organization that can be hampered by limited availability of health and laboratory personnel. Dried Blood Spot (DBS) sampling is proposed to overcome such limitations. The aim of this work was the development of a new LC-MS/MS method to analyze DBS samples containing PALBO, RIBO, and LETRO. Analytes extraction from DBS was performed by adding a methanolic solution containing the corresponding internal standards. LC-MS/MS analysis was performed using a LC Nexera (Shimadzu) system coupled with an API 4000 QTrap (SCIEX) mass spectrometer. The chromatographic separation was performed on a Luna Omega Polar C18 column (Phenomenex). The method was applied to 38 clinical samples collected by finger prick. The influence of hematocrit and spot size, sample homogeneity, stability, and correlation between finger prick and venous DBS measurement were assessed. The analytical validation was performed according to EMA and FDA guidelines.The analytical range of the method was 1 to 250 ng/mL for PALBO, 40 to 10000 ng/mL for RIBO, and 2 to 500 ng/mL for LETRO, where linearity was assessed, obtaining mean coefficients of determination (R2) of 0.9979 for PALBO, 0.9980 for RIBO, and 0.9987 for LETRO). The LC-MS/MS method runtime was 6.6 min. Incurred sample reanalysis demonstrated reproducibility, as the percentage difference between the two quantifications was lower than 20% for 100% of PALBO, 81.8% of RIBO, and 90.9% of LETRO paired samples. Intra- and inter-day precision (CV (%)) was lower than 11.4% and intra- and inter-day accuracy was between 90.0 and 106.5%. DBS sample stability at room temperature was confirmed for 2.5 months. A positive correlation was observed between DBS and plasma concentrations for the 3 drugs, Lin’s concordance correlation coefficients obtained by DBS normalization applying a selected strategy were 0.958 for PALBO, 0.957 for RIBO, and 0.963 for LETRO. In conclusion, a fast, easy, and reproducible DBS LC-MS/MS method for the simultaneous quantification of palbociclib; ribociclib and letrozole was developed to be used in clinical practice.

Urine Phenylacetylglutamine Determination in Patients with Hyperphenylalaninemia

Phenylketonuria (PKU), an autosomal-recessive inborn error of phenylalanine (Phe) metabolism is the most prevalent disorder of amino acid metabolism. Currently, clinical follow-up relies on frequent monitoring of Phe levels in blood. We hypothesize that the urine level of phenylacetylglutamine (PAG), a phenyl-group marker, could be used as a non-invasive biomarker. In this cross-sectional study, a validated liquid chromatography coupled to tandem mass spectrometry (LC-MS) method was used for urinary PAG quantification in 35 participants with hyperphenylalaninemia (HPA) and 33 age- and sex-matched healthy controls. We have found that (a) PKU patients present higher urine PAG levels than healthy control subjects, and that (b) there is a significant correlation between urine PAG and circulating Phe levels in patients with HPA. In addition, we show a significant strong correlation between Phe levels from venous blood samples and from capillary finger-prick dried blood spot (DBS) samples collected at the same time in patients with HPA. Further research in order to assess the potential role of urine PAG as a non-invasive biomarker in PKU is warranted.

Genetic diversity and distribution patterns of PfEMP1 in Plasmodium falciparum isolates along the Thai-Myanmar border.

Duffy binding-like domain (DBL) and cysteine-rich interdomain region (CIDR) domain genes of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) encode malaria virulence proteins. The variants of these genes have been reported to be associated with severe/complicated malaria. The present study investigated the prevalence and distribution patterns of DBLα0.6/9, DBLα1.1, DBLα1 not var3 genes, DBLα2/α1.1/2/4/7, DBLβ12 & DBLβ3/5, DBLε8, CIDRα1.4, and CIDRα1.6 of P. falciparum isolates along the Thai-Myanmar border. The association between PfEMP1 variants and parasite density was also investigated. Two hundred and thirteen finger-prick dried blood spot (DBS) or whole blood samples were collected in 2007 and 2015, from patients with acute uncomplicated P. falciparum in Tak, Kanchanaburi, and Ranong provinces. Analysis of the variant genes was performed using polymerase chain reaction (PCR). The DBLs variant which was found at the highest and lowest frequencies in the three provinces were DBLα1 not var3 (72.77%), and DBLε8 (17.37%). The two CIDR domain variants were found at relatively lower frequencies compared with DBL domain variants (9.9% and 30.1%). P. falciparum isolates carrying the four PfEMP1 variants, i.e., DBLα0.6/9, DBLα1.1, DBLα2/α.1.1/2/4/7, and DBLε8 were found to be significantly associated with low parasitemia. Both DBLα0.6/9 and DBLα2/α1.1/2/4/7 variant genes which were present at high frequencies in this border area could be potential candidate markers for predicting P. falciparum hyperparasitemia and in this border area. Furthermore, the information could be exploited as candidate proteins for the development of an effective malaria vaccine in specific malaria-endemic areas.

Abstract OT-09-07: Winpro: A window of opportunity study of endocrine therapy with and without prometrium in postmenopausal women with early stage hormone receptor-positive breast cancer

Abstract Background: There is bidirectional interplay between PR and ER in human breast cancers (Lim, Endo Rel Can 2016). Evidence in breast cancer cell lines suggests that dual treatment with estrogen and progesterone compared to estrogen alone leads to reprogramming of ER chromatin binding sites via regulation of 470 genes (Mohammed, Nature 2015). Importantly, there was an additive anticancer effect in preclinical breast cancer models when natural progesterone was combined with standard endocrine therapy; we hypothesize that this combination has activity in women with breast cancer.Trial design: We are conducting a phase II multi-center, randomised, open-label, three-arm study in which 200 postmenopausal women with early-stage ER-positive (ER ≥10%), PR-positive (PR ≥10%), HER2-negative breast cancer will be randomised 1:1:1 to letrozole 2.5mg daily (arm 1); letrozole 2.5mg and prometrium 300mg PO daily (arm 2); or tamoxifen 20mg and prometrium 300mg PO daily (arm 3). Surgery will occur on day 14 after treatment initiation. Eligible subjects must have tumor size ≥10mm on imaging, no history of uterine cancer or venous thromboembolism, and no receipt of other preoperative therapies. The primary objective is to assess the reduction in proliferative marker Ki67 following treatment in either combination arm compared to letrozole alone.Methods: Blood will be collected at baseline and at end of treatment. Tissue samples will be collected from the diagnostic biopsy and at the time of surgery. The primary endpoint is geometric mean reduction of centrally assessed Ki67 expression after two weeks of treatment compared with baseline. Given the expected geometric mean reduction of 76% for aromatase inhibitor alone (Dowsett, J Natl Can Inst 2007) and allowing 4% dropouts, 200 patients provides 80% power to detect an improvement in Ki67 suppression to 92% in either experimental arm with p-value 0.025 for each comparison.The secondary endpoint of safety and tolerability will be assessed (NCI-CTCAE v4.0). Translational endpoints include definition of a predictive gene set biomarker for Ki67 reduction; tumor biomarkers after treatment, including apoptotic markers Bcl-2 and cleaved-caspase3, as well as protein and mRNA expression of ER, PR, AR, FoxA1, and CyclinD1; and levels of estrone, estradiol, E2, progesterone, testosterone, DHT, and DHEAS in serum and finger prick dried blood spot after treatment.Accrual: Enrolment commenced in February 2018 and 70 patients have been randomized from 7 sites to date (1 July 2020). Target accrual is 200 patients enrolled from 8 sites.Contact information: This study is led at The Kinghorn Cancer Centre, St Vincent’s Sydney Hospital Sydney, Australia, and funded by the Cancer Council of NSW and the NHMRC Translational Breast Cancer Project grant. Contact Elgene Lim MBBS FRACP PhD at e.lim@garvan.org.au. Citation Format: Brandon Lau, Davendra Segara, Andrew Ong, Janne Bingham, Belinda Kiely, Emma-Kate Carson, Julia Chen, Cindy Mak, Sanjay Warrier, Mun Hui, Kate Middleton, Andrew Parker, Bruce Mann, Geoffrey Lindeman, Wayne Tilley, Elgene Lim. Winpro: A window of opportunity study of endocrine therapy with and without prometrium in postmenopausal women with early stage hormone receptor-positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-09-07.

A serological assay to detect SARS-CoV-2 antibodies in at-home collected finger-prick dried blood spots

Accurate surveillance of coronavirus disease 2019 (COVID-19) incidence requires large-scale testing of the population. Current testing methods require in-person collection of biospecimens by a healthcare worker, limiting access of individuals who do not have access to testing facilities while placing both patients and healthcare workers at risk of exposure to infection. We report the development and validation of a at-home finger-prick dried blood spot collection kit and an analysis method. We demonstrated 100% sensitivity and specificity using at-home collected specimens across the US. Such methods may facilitate the conduct of unbiased serosurveys within hard to reach populations and help reduce the sample collection burden of serological testing on both health care systems and individuals alike.

Protocol for a national probability survey using home specimen collection methods to assess prevalence and incidence of SARS-CoV-2 infection and antibody response

PurposeThe U.S. response to the SARS-CoV-2 epidemic has been hampered by early and ongoing delays in testing for infection; without data on where infections were occurring and the magnitude of the epidemic, early public health responses were not data-driven. Understanding the prevalence of SARS-CoV-2 infections and immune response is critical to developing and implementing effective public health responses. Most serological surveys have been limited to localities that opted to conduct them and/or were based on convenience samples. Moreover, results of antibody testing might be subject to high false positive rates in the setting of low prevalence of immune response and imperfect test specificity. MethodsWe will conduct a national serosurvey for SARS-CoV-2 PCR positivity and immune experience. A probability sample of U.S. addresses will be mailed invitations and kits for the self-collection of anterior nares swab and finger prick dried blood spot specimens. Within each sampled household, one adult 18 years or older will be randomly selected and asked to complete a questionnaire and to collect and return biological specimens to a central laboratory. Nasal swab specimens will be tested for SARS-CoV-2 RNA by RNA PCR; dried blood spot specimens will be tested for antibodies to SARS-CoV-2 (i.e., immune experience) by enzyme-linked immunoassays. Positive screening tests for antibodies will be confirmed by a second antibody test with different antigenic basis to improve predictive value of positive (PPV) antibody test results. All persons returning specimens in the baseline phase will be enrolled into a follow-up cohort and mailed additional specimen collection kits 3 months after baseline. A subset of 10% of selected households will be invited to participate in full household testing, with tests offered for all household members aged ≥3 years. The main study outcomes will be period prevalence of infection with SARS-CoV-2 and immune experience, and incidence of SARS-CoV-2 infection and antibody responses. ResultsPower calculations indicate that a national sample of 4000 households will facilitate estimation of national SARS-CoV-2 infection and antibody prevalence with acceptably narrow 95% confidence intervals across several possible scenarios of prevalence levels. Oversampling in up to seven populous states will allow for prevalence estimation among subpopulations. Our 2-stage algorithm for antibody testing produces acceptable PPV at prevalence levels ≥1.0%. Including oversamples in states, we expect to receive data from as many as 9156 participants in 7495 U.S. households. ConclusionsIn addition to providing robust estimates of prevalence of SARS-CoV-2 infection and immune experience, we anticipate this study will establish a replicable methodology for home-based SARS-CoV-2 testing surveys, address concerns about selection bias, and improve positive predictive value of serology results. Prevalence estimates of SARS-CoV-2 infection and immune experience produced by this study will greatly improve our understanding of the spectrum of COVID-19 disease, its current penetration in various demographic, geographic, and occupational groups, and inform the range of symptoms associated with infection. These data will inform resource needs for control of the ongoing epidemic and facilitate data-driven decisions for epidemic mitigation strategies.

Development and validation of an ELISA for a biomarker of thyroid dysfunction, thyroid peroxidase autoantibodies (TPO-Ab), in dried blood spots

BackgroundThe prevalence of allergic and autoimmune conditions has been steadily increasing in wealthy nations over the past century. One hypothesis put forward to explain this is the Old Friends Hypothesis, which posits that increased hygiene, urbanization, and lifestyle changes have reduced our exposure to parasites and microbes that we co-evolved with, resulting in immune dysregulation. However, research in traditionally living populations, who are exposed to greater parasite and pathogen loads such as those encountered during our evolution, is limited, in part due to a lack of minimally invasive, field-friendly biomarkers of autoimmune disorders. We therefore developed an ELISA to assess positivity for thyroid peroxidase autoantibody (TPO-Ab), an indicator of autoimmune thyroid disease, based on dried blood spot (DBS) samples.ResultsWe used the Accubind anti-thyroid peroxidase test system to screen our validation samples comprising matched fingerprick DBS, venous DBS, and plasma samples from 182 adults. After confirming that we had TPO-Ab-positive individuals in our validation sample (n = 12), we developed an indirect ELISA to measure TPO-Ab levels from one 3-mm DBS punch. The sensitivity and specificity of our assay for DBS samples ranged from 91.7–100% and 98.2–98.8%, respectively, using a cut-off value of ≥ 26 IU/mL. Intra-assay reliability for duplicate quality control DBS punches was 5.2%, while inter-assay reliability ranged from 11.5–24.4% for high, medium, and low DBS controls. Dilutional linearity ranged from 80 to 120%, and spike and recovery experiments indicated that the DBS matrix does not interfere with the detection of TPO-Ab. TPO-Ab levels remained stable in DBS samples stored at − 28 °C or − 80 °C, but decreased over time in DBS samples kept at 22 °C or at 37 °C.ConclusionsWe developed an in-house, kit-independent indirect ELISA assay to determine individuals’ TPO-Ab positivity based on dried blood spots, representing a cost-effective method with potential applications in a range of research settings.

Proteomic Profiling and Monitoring of Training Distress and Illness in University Swimmers During a 25-Week Competitive Season.

PurposeTo evaluate relationships of proteomics data, athlete-reported illness, athlete training distress (TDS), and coaches’ ratings of distress and performance over the course of the competitive season.MethodsThirty-five NCAA Division II swimmers were recruited to the study (male n = 19, female n = 16; age 19.1 ± 1.6 years). Athletes provided fingerprick dried blood spot (DBS) samples, illness symptoms, and TDS every Monday for 19 of 25 weeks in their season. Coaches monitored performance and rated visual signs of distress. DBS samples were analyzed for a targeted panel of 12 immune-related proteins using liquid chromatography/mass spectrometry (LC/MS).ResultsThirty-two swimmers completed the protocol. The data were grouped in 2–3 weeks segments to facilitate interpretation and analysis of the data. TDS scores varied between athletes, and were highest during the early fall conditioning ramp up period (8.9 ± 1.6 at baseline to a peak of 22.6 ± 2.0). The percent of athletes reporting illness was high throughout the season (50–78%). Analysis of TDS using Principle Component Analysis (PCA) revealed that 40.5% of the variance (PC1) could be attributed to illness prevalence, and TDS scores for the athletes reporting illness and no illness were different across the season (P < 0.001). The coaches’ ratings of swim performance and swimmer’s distress, sex, and racing distance (sprinters, middle distance, long distance) were not correlated with PC1. Linear Discriminant Analysis (LDA) analysis of the data showed a separation of the baseline weeks from exam weeks with or without competitions, and with competitions alone (p < 0.001). Seven of the 12 proteins monitored over the course of training were upregulated, and the addition of the protein data to LDA analysis enhanced the separation between these groups of weeks.ConclusionTDS and illness were related in this group of 32 collegiate swimmers throughout the competitive season, and expression of immune proteins improved the statistical separation of baseline weeks from the most stressful weeks. TDS data provided by the swimmers did not match their coaches’ ratings of distress and swim performance. The importance of the immune system in the reaction to internal and external stress in athletes should be an area of further research.

Volumetric absorptive microsampling and dried blood spot microsampling vs. conventional venous sampling for tacrolimus trough concentration monitoring

Objectives Monitoring tacrolimus blood concentrations is important for preventing allograft rejection in transplant patients. Our hospital offers dried blood spot (DBS) sampling, giving patients the opportunity to sample a drop of blood from a fingerprick at home, which can be sent to the laboratory by mail. In this study, both a volumetric absorptive microsampling (VAMS) device and DBS sampling were compared to venous whole blood (WB) sampling. Methods A total of 130 matched fingerprick VAMS, fingerprick DBS and venous WB samples were obtained from 107 different kidney transplant patients by trained phlebotomists for method comparison using Passing-Bablok regression. Bias was assessed using Bland-Altman. A multidisciplinary team pre-defined an acceptance limit requiring >80% of all matched samples within 15% of the mean of both samples. Sampling quality was evaluated for both VAMS and DBS samples. Results 32.3% of the VAMS samples and 6.2% of the DBS samples were of insufficient quality, leading to 88 matched samples fit for analysis. Passing-Bablok regression showed a significant difference between VAMS and WB, with a slope of 0.88 (95% CI 0.81-0.97) but not for DBS (slope 1.00; 95% CI 0.95-1.04). Both VAMS (after correction for the slope) and DBS showed no significant bias in Bland-Altman analysis. For VAMS and DBS, the acceptance limit was met for 83.0% and 96.6% of the samples, respectively. Conclusions VAMS sampling can replace WB sampling for tacrolimus trough concentration monitoring, but VAMS sampling is currently inferior to DBS sampling, both regarding sample quality and agreement with WB tacrolimus concentrations.

Paper-based molecularly imprinted-interpenetrating polymer network for on-spot collection and microextraction of dried blood spots for capillary electrophoresis determination of carbamazepine.

Carbamazepine is an antiepileptic drug with a narrow therapeutic index, which requires an efficient method for blood level monitoring. Finger-prick dried blood spot (DBS) collection is an alternative microsampling technique, which is less invasive than conventional venipuncture. Paper-based molecularly imprinted-interpenetrating polymer networks (MI-IPN) were developed as blood collection devices, which allowed for selective on-spot microextraction of carbamazepine from DBS. A hybrid of homogeneous polystyrene and silica gel polymer was synthesized and coated on a Whatman® Grade 1 filter paper. Proteins and other interferences in the blood samples were eliminated by using the MI-IPN collection devices, and the resulting DBS extracts were suitable for direct injection into the capillary electrophoretic instrument. The lower limit of quantitation of 4μg/mL in capillary blood was achieved by the sweeping-micellar electrokinetic chromatography method using a KCl-containing matrix, which was sufficient for the therapeutic drug monitoring purposes. Method accuracies were in the range of 88.4 ± 4.5% to 94.5 ± 2.7% with RSD values ≤ 5.1%. The developed paper-based MI-IPN provided superior extraction efficiencies (92.2 ± 2.5%) in comparison with commercially available DBS collection cards, i.e., Whatman® 903 protein saver card (59.8 ± 2.8%) and GenCollect™ 2.0 card (47.2 ± 1.4%). The paper-based MI-IPN devices for DBS collection and on-spot extraction were characterized by simple fabrication, low costs, disposability, and reduction in sample preparation steps, and their further developments might open new perspectives in clinical applications, such as in therapeutic drug monitoring. Graphical abstract.

Development and validation of LC-MS/MS method for imatinib and norimatinib monitoring by finger-prick DBS in gastrointestinal stromal tumor patients.

The introduction of imatinib, an oral tyrosine kinase inhibitor, as first-line standard therapy in patients with unresectable, metastatic, or recurrent gastro-intestinal stromal tumor (GIST), strongly improved their treatment outcomes. However, therapeutic drug monitoring (TDM) is recommended for this drug due to the large inter-individual variability in plasma concentration when standard dose is administered. A Cmin higher than 760 ng/mL was associated with a longer progression free survival. Thus, a LC-MS/MS method has been developed and fully validated to quantify imatinib and its active metabolite, norimatinib, in finger-prick dried blood spot (DBS). The influence of hematocrit, sample homogeneity, and spot size and the correlation between finger-prick and venous DBS measurements were also assessed. The method showed a good linearity (R2 > 0,996) between 50-7500 ng/mL for imatinib and 10-1500 ng/mL for norimatinib. Analytes were extracted from DBS samples by simply adding to 3 mm-discs 150 μL of acidified methanol containing IMA-D8. The collected extract was then injected on a LC Nexera system in-house configured for the on-line cleanup, coupled with an API-4000 QT. The chromatographic separation was conducted on a Synergi Fusion-RP column (4 μm, 2x50 mm) while the trapping column was a POROS R1/20 (20 μm, 2x30 mm). The total run time was 8.5 min. DBSs stored at room temperature in plastic envelopes containing a silica-gel drying bag were stable up to 16 months. The proposed method was applied to 67 clinical samples, showing a good correlation between patients' finger-prick DBS and plasma concentrations, measured by the reference LC-MS/MS method, internally validated. Imatinib and norimatinib concentrations found in finger-prick DBS were adjusted by hematocrit or by an experimental correction factor to estimate the corresponding plasma measurements. At the best of our knowledge, the proposed LC-MS/MS method is the first analytical assay to measure imatinib and norimatinib in DBS samples.