Large scale, complex biobanking of biofluids for immunology
research and testing
Rohit K. Gupta1, Vanitha Sampath2,
Kari C. Nadeau2, Holden T. Maecker3,
1 Biospecimen Resource Program, Office of
Research, University of California, San Francisco, CA, USA.
2 Sean N. Parker Center for Allergy and
Asthma Research at Stanford University and Division of Pulmonary and
Critical Care Medicine, Department of Medicine, Stanford University,
Stanford, CA, USA.
3 Institute for Immunity, Transplantation, and
Infection, Stanford University, Stanford, CA, USA.
Corresponding Author: Prof. Holden Maecker, Department of
Microbiology and Immunology, Fairchild Science Building, room D039, 299
Campus Drive, Stanford, CA 94305-5124, USA, Email:
maecker@stanford.edu,
Phone: (650) 723-1671
Funding: Sean N. Parker Center for Allergy and Asthma Research
at Stanford University and Sunshine Foundation.
Conflict of Interest: Dr. Nadeau reports grants from National
Institute of Allergy and Infectious Diseases (NIAID), Food Allergy
Research & Education (FARE), End Allergies Together (EAT), Allergenis,
and Ukko Pharma; Grant awardee at NIAID, National Institute of
Environmental Health Sciences (NIEHS), National Heart, Lung, and Blood
Institute (NHLBI), and the Environmental Protection Agency (EPA); is
involved in Clinical trials with Regeneron, Genentech, AImmune
Therapeutics, DBV Technologies, AnaptysBio, Adare Pharmaceuticals, and
Stallergenes-Greer; Research Sponsorship by Novartis, Sanofi, Astellas,
Nestle; Data and Safety Monitoring Board member at Novartis and NHLBI;
Cofounded Before Brands, Alladapt, ForTra, and Iggenix; Chief
Intellectual Office at FARE, Director of the World Allergy Organization
(WAO) Center of Excellence at Stanford, Personal fees from Regeneron,
Astrazeneca, ImmuneWorks, and Cour Pharmaceuticals; Consultant and
Advisory Board Member at European Academy of Allergy and Clinical
Immunology (EAACI) Research and Outreach Committee, Ukko, Before Brands,
Alladapt, IgGenix, Probio, Vedanta, Centecor, Seed, Novartis, NHBLI,
EPA, National Scientific Committee of Immune Tolerance Network (ITN) and
NIH Programs; US patents for basophil testing, multifood immunotherapy
and prevention, monoclonal antibody from plasmoblasts, and device for
diagnostics. HM, RG, and VS indicate no conflict of interest.
Author Contributions : All authors wrote and edited the final
manuscript.
Word Count: 978
To the Editor:
Biobanks have evolved from simple localized storage of samples in
individual labs and clinics to large industrialized repositories with
sophisticated sample life cycle infrastructure. By enabling
collaborations between researchers working on different aspects of a
disease, biobanks can bridge the gap between clinical care and research,
accelerating medical care towards precision medicine. The concomitant
advances in trans-omic technologies, big data analytics, and
biorepositories make possible a coordinated, robust systems biology
approach. Biobanks can be envisioned as a central hub responsible for
compliant custodianship of specimens and associated clinical and
biological data. Operationally, biobanks should strive to provide
universal consent, standardized processing, cold-chain management, and
quality control checks. Here, we discuss biobanks with respect to
optimal utilization of biofluid derivatives, such as cells,
supernatants, and genomic material, for immunology research and testing.
A number of parameters need to be considered for specimen optimization
and standardization based on sample type and downstream assays to be
performed. Choices begin with the blood collection tubes to be used. For
DNA and RNA analysis, EDTA anticoagulated blood is most common, as
heparin can inhibit downstream polymerase reactions. However, at least
one source suggests that citrate may provide higher quality RNA and DNA
than other stabilizers.1
For immunoassays, either serum or plasma can be effectively used, but
there are subtle differences for some cytokine
analytes.2 As such, a minimal requirement should be to
use the same matrix (serum or plasma) and same anticoagulant if using
plasma, for all samples to be compared in a study. For metabolome and
lipidome studies, a report by Yin et al suggests EDTA plasma as the
preferred matrix, since clotting in serum tubes activates additional
processes, including the release of metabolites and enzymes from
activated platelets.3
For cellular assays such as flow cytometry, CyTOF, or single-cell
RNAseq, viably cryopreserved peripheral blood mononuclear cells (PBMCs)
or other cells of interest are key. Protocols for cryopreservation are
readily available, but careful attention to both freezing and thawing
protocols is particularly important to maintain viability and recovery.
Traditionally, heparinized blood is used for Ficoll isolation of PBMCs,
but other anticoagulants are generally equivalent for functionality of
cryopreserved PBMC. Concomitant use of the whole blood for other
purposes (e.g., stimulation or DNA isolation) may dictate the optimal
anticoagulant—for example, EDTA would inhibit T cell receptor-based
stimulation, but would be compatible with molecular assays). There are
also variations to traditional Ficoll protocols, using Cell Preparation
Tubes (CPTs) or SepMate tubes.4 These should be
considered, as they can save time and labor and overcome some hurdles
for standardization of the Ficoll procedure. The main drawbacks are a
slight reduction in yield or increase in erythrocyte contamination.
Importantly, training and protocol adherence are still important to
prevent, for example, breakage of CPT from improper centrifuge holders,
inadequate PBMC separation from improper spin speed, or loss of
separation if CPT are shipped in very cold temperatures. Another
variable to be considered is time to processing5,
which is of course highly related to whether samples are shipped prior
to processing (see Figure 1). This is particularly relevant to
functional cellular assays. An alternative to overnight shipping and
PBMC cryopreservation for functional assays is to perform on-site
stimulation and stabilization of whole blood (e.g., Smart Tube Inc.,
http://smarttubeinc.com); however, proper monitoring of cold-chain
storage is critical to ensure frozen specimens are not compromised. For
example, when using the Smart Tube system, biobanks must maintain the
frozen samples at -80°C, as micro-fluctuations in temperature can cause
the specimens to coagulate, rendering them unusable. In any case, there
are a number of potential variables that can be detrimental to
downstream analysis and even reproducibility; biobanks should strive to
harmonize collection, processing, and storage of samples related to
biofluids.
Research institutes often have multiple laboratories, each of which may
be supporting various collections of human specimens. Unfortunately,
most labs have employed their own data solutions to track and search for
specimens, which has led to fragmented processes and inconsistent
ontologies. Utilization of biospecimens that have been collected for
scientific purposes continues to be problematic and may be more
effective when paired with informatics tools that enable researchers to
track, annotate, and interrogate.6 Biobanks should
have a sample management system (SMS) which permits labs to accurately
register, label (Figure 2), and track biospecimen inventory related to
study participants7; in addition, the software should
be configurable to align with lab workflows, while maintaining best
practices for biobanking and ensuring governance can be maintained by
the individual laboratory or institute. Further, for bioinventory
tracking, it is critical to connect de-identified clinical attributes
from electronic health records to biological assays following analysis
of specimens in a central ecosystem; this enables researchers to rapidly
search and request specimens for further analysis.8 To
date, although many solutions have been developed to support virtual
sample catalogs, most require extensive software engineering support in
order to be deployed and require data to be migrated to a central
database; robust and innovative solutions for identifying unused
biospecimens in the life sciences are still desired.
Long thought of as freezer farms, a biobank’s primary role has always
been to provide proper cold-chain storage and logistics related to
biospecimens. While much literature exists on optimal storage conditions
and management,9 biobanks have evolved to now
facilitate research in the life sciences that extend from the physical
management of the sample life cycle to supporting standardized
processing, assay optimization, and modernized data infrastructure. As
compliant use of biospecimens continues to be a major component being
addressed through community engagement, biobanks are poised to play an
important role in medical research with increasing demand for high
quality biospecimens. However, a number of questions and challenges
exist regarding standardization, classification, management,
sustainability, as well as ethical considerations including ownership
and informed consent. Ultimately, improving how biospecimens are
utilized for downstream analysis can accelerate our understanding of
biological mechanisms and fuel a better tomorrow.
Rohit K. Gupta1
Vanitha Sampath2
Kari C. Nadeau2
Holden T. Maecker3
1 Biospecimen Resource Program, Office of
Research, University of California, San Francisco, CA, USA.
2 Sean N. Parker Center for Allergy and
Asthma Research at Stanford University and Division of Pulmonary and
Critical Care Medicine, Department of Medicine, Stanford University,
Stanford, CA, USA.
3 Institute for Immunity, Transplantation, and
Infection, Stanford University, Stanford, CA, USA.