Participant identification and enrollment:
The identification and enrollment of participants into NoCo-COBIO is a joint effort between Colorado State University (CSU), a land grant institution with a coordinated state-wide extension system, and the University of Colorado Health System (UCH). Individuals are eligible for participation if they have had a positive SARS-CoV-2 polymerase chain reaction (PCR) test and are at least 18 years of age. Participants are recruited from the community via the health department screening, local medical clinics, emails, recruitment flyers, web-based announcements, and directly through UCH northern Colorado hospitals, including Poudre Valley Hospital (PVH) in Fort Collins, Medical Center of the Rockies (MCR) in Loveland, and Greeley Hospital in Greeley. The UCH Trauma Research Department (TRD) identifies eligible hospitalized patients through Epic, the EMR platform. UCH clinicians that oversee patients at the northern Colorado hospitals are aware of the biorepository and may refer potential participants to the biobank research team for follow-up. UCH staff directly approach eligible, hospitalized patients on the ward for consent and enrollment. The UCH investigators also conduct frequent EMR searches to recruit recently discharged/diagnosed COVID-19 patients. Additionally, enrolled participants have also assisted via word-of-mouth and personal networks.
This biorepository was approved by CSU’s Research Integrity and Compliance Review Office Institutional Review Board (IRB; protocol ID 20-10063H), as well as UCH IRB (Colorado Multiple IRB 20-6043) and is registered with ClinicalTrials.gov (NCT05603677). All enrolled participants provide written informed consent. Healthy controls without history of SARS-CoV-2 exposure are enrolled for the same specimen collections on a volunteer basis. Participants receive $25 cash compensation at each of the four study visits.
Clinical data:
All data is de-identified and password-protected for analysis. Clinical data obtained from the EMR are stored in Research Electronic Data Capture (REDCap), and each record is assigned a unique identifier. Demographic data that may affect COVID-19 outcomes (23–26), such as socioeconomic status, employment status, ethnicity, race, and clinical data are obtained from participants at their clinic visits. A physician performs a physical exam at each outpatient visit. Participants are categorized as having a mild, moderate, or severe initial infection based on oxygen requirements during their acute illness (no oxygen requirement, 1-5L oxygen requirement, and greater than 5L oxygen use, respectively). During follow-up visits, participants assessed with a survey of 70 symptoms to identify new or persistent sequelae of COVID-19 (Table 2). Patients are defined as having PASC if they experience at least one of the following symptoms commonly associated with PASC: fatigue, dyspnea, joint pain, chest pain, or cognitive impairment, at any follow-up visit (27).
The study team administers the Short Form 36 health survey (SF-36) at two clinical follow-up visits to assess social impacts and quality of life after COVID-19. This survey gives a average maximum score of 100 in 8 different categories: general health, physical functioning, role limitations due to physical health, role limitations due to emotional health, energy level, emotional wellbeing, social functioning, and pain. A total, combined score is also given, with a maximum of 3600. The survey asks questions about the participants’ emotional and physical well-being during the 4 weeks prior to survey administration. This validated instrument (28,29) also compares current health to their health one year prior, as well as reports on the expectations for future health outcomes.
Biospecimen collection and transport:
Participants consent to provide biospecimens at 4 collection time points over an enrollment period of 6 months. The approximate time points for each of the specimen collections are at the time of enrollment, and at 1 month, 3 months, and 6 months after enrollment. The second and third visits may take place in the hospital if the participant remains hospitalized. Follow-up visits take place at designated campus or medical clinic sites. After 6 months, participants may provide additional consent for a one-year follow-up visit.
Figure 1 depicts the trajectory of each sample collected. Participants provide approximately 50 mL of blood, 10 mL of saliva (5 mL in viral transport media (VTM, sterile hanks balanced salt solution (ThermoFischer Scientific) with 2% FBS) for viral propagation and 5 mL without VTM), a stool sample, and a nasopharyngeal swab specimen at each visit. Blood is collected into five 8 mL sodium citrate cell preparation tubes (CPTs) (BD BioSciences, Franklin Lakes, NJ) and one 5 mL serum separator tube (VWR). Saliva samples are collected by expectoration, or a tracheal aspirate specimen is collected if participants are intubated. Samples with VTM are kept on ice during transport. Nasopharyngeal swab specimens are collected with a latticed nasal swab (Resolution Medical). The swab is then placed in VTM and kept on ice for transport. Stool is either self-collected by the participants or collected by the participant’s nurse if they are hospitalized. The stool can be collected prior to the study visit and stored frozen. Breast milk is collected from lactating participants, either fresh in pre-distributed breast milk storage bags, or previously pumped and stored frozen.
Each patient sample is de-identified and labeled with a unique study code identifier. Information linking participant identification to the study codes is stored in the offices of the investigators and will be maintained for up to five years following the cessation of the study. Nasopharyngeal swab and saliva in VTM from acutely infected patients are transported to and processed in CSU’s biosafety level-3 (BSL-3) facilities. Blood, nasopharyngeal samples, and saliva collected from convalescent patients and all fecal samples are transported to and processed in biosafety level-2 (BSL-2) facilities in accordance with CSU's IRB and Centers for Disease Control and Prevention (CDC) recommendations. Transportation of specimens from clinical sites to laboratory facilities is done in accordance with U.S. Department of Transportation hazardous materials regulations.
Whole blood processing protocol:
An aliquot of whole blood is removed from a CPT, incubated with platelet surface markers, and analyzed by flow cytometry with LSR II (BD Biosciences) and FACSAria Cell Sorter (BD Biosciences) instruments. A second aliquot of blood is removed from the CPT and stained for leukocytes and endothelial cells and analyzed by flow cytometry on a Cytek Aurora (Cytek Biosciences) instrument. The CSU Flow Cytometry Core has instruments with the same laser configurations inside and outside the BSL-3 facility to preserve consistent results when analyzing samples in different locations.
The CPTs are then centrifuged according to manufacturer instructions. The plasma is removed, and the PBMCs are separated and washed. A small aliquot of PBMCs is removed for additional antibody staining and flow cytometry analysis. PBMCs are cryostored in freezing media (90% heat inactivated fetal bovine serum (FBS) (Avantor) and 10% dimethyl sulfoxide (DSMO) (Sigma Altrich)). PBMC are first stored in an isopropanol-filled Nalgene® Mr. Frosty (Thermo Scientific) at -80°C for 24 hours and transferred into liquid nitrogen for long-term cryopreservation.
Plasma and serum aliquots:
Following removal from CPTs, plasma is stored in 1 mL aliquots at -80°C. Blood collected in serum separator tubes is allowed to clot for 30 minutes, and then spun at 1000 x g at 4°C for 10 minutes. Serum is removed and stored in 300 uL aliquots at -80°C. Each participant's samples are designated to an individual cryobox and organized by their visit for quick sample retrieval.
Saliva and nasopharyngeal processing:
An aliquot of both saliva and nasopharyngeal sample in VTM are sent for quantitative PCR (qPCR) analysis for SARS-CoV-2. The remaining saliva is centrifuged, the pellet is discarded, and the supernatant is aliquoted for -80°C storage. Nasopharyngeal samples are aliquoted and stored at -80°C for SARS-CoV-2 viral titer analysis using plaque assay.
Stool processing:
Stool samples are aliquoted and stored at -80°C for DNA, RNA, secretory immunoglobulin A, and metabolite analysis.
Statistical analysis: Statistical analysis was performed using Fischer’s Exact Test.