Biobanks are crucial resources for biomedical research and the Biobank for Translational and Digital Medicine Unit at the European Institute of Oncology is a model in this field. Here, we provide a detailed description of biobanks’ standard operating procedures for the management of different types of human biological samples.
Biobanks are key research infrastructures aimed at the collection, storage, processing, and sharing of high-quality human biological samples and associated data for research, diagnosis, and personalized medicine. The Biobank for Translational and Digital Medicine Unit at the European Institute of Oncology (IEO) is a landmark in this field. Biobanks collaborate with clinical divisions, internal and external research groups, and industry, supporting patients’ treatment and scientific progress, including innovative diagnostics, biomarker discovery, and clinical trial design. Given the central role of biobanks in modern research, biobanking standard operating procedures (SOPs) should be extremely precise. SOPs and controls by certified specialists ensure the highest quality of samples for the implementation of science-based, diagnostic, prognostic, and therapeutic personalized strategies. However, despite numerous efforts to standardize and harmonize biobanks, these protocols, which follow a strict set of rules, quality controls, and guidelines based on ethical and legal principles, are not easily accessible. This paper presents the biobank standard operating procedures of a large cancer center.
Biobanks are biorepositories aimed at the collection, storage, processing, and sharing of human biological samples and associated data for research and diagnosis. Their role is crucial not only for biomarker discovery and validation but also for the development of new drugs1. Hence, the vast majority of translational and clinical research programs rely on access to high-quality biospecimens. In this respect, biobanks are considered a bridge between academic research and the pharmaceutical/biotechnology industry2,3,4,5. Owing to the unprecedented opportunities provided by big data collection and artificial intelligence, the role of biobanks in cancer research is continuously evolving6.
The broad spectrum of biomaterials handled by biobanks is coupled with clinicopathologic information, including demographic and environmental data, tumor type, histologic grade, stage, presence of lymphovascular invasion, and biomarkers status7,8. The more high-quality specimens and data are available, the faster research will advance and impact healthcare delivery9. There is a strict regulatory framework based on ethical and legal principles that should follow widely adopted SOPs, quality controls, and guidelines (e.g., the U.S. National Cancer Institute, the U.K. Confederation of Cancer Biobanks, and the E.U. International Society for Biological and Environmental Repositories)10,11.
The development of SOPs for all major aspects of biobanks brings several advantages in terms of quality, traceability, consistency, reproducibility, and turnaround times12,13. Another important aspect of SOP implementation is represented by the optimization of biobank management, which allows for better problem-solving and alternative procedures for biobank employees and researchers14. All of these facets are part of the biobank workflow (Figure 1).
Figure 1: Different factors that contribute to the optimization of biobanking. Abbreviation: LIMS = laboratory information management system. Please click here to view a larger version of this figure.
These highly specific and sensitive data require stringent managerial standard procedures in biobanking. A detailed and validated project form should be made available to all researchers who need to get access to the biobank samples and data. The information provided in the request should include the study methodology and design, goals, objectives, and budget. A biobank Technical Scientific Committee should be established with the capital role of the evaluation of applications for research projects. This body should include members from the biobank unit, clinical divisions, research groups, data protection, legal office, and technology transfer office.
The Biobank for Translational and Digital Medicine Unit of the European Institute of Oncology (IEO) is a worldwide reference for biobanks in terms of the quality and quantity of services provided, as well as innovation. This fully certified facility (UNI EN ISO 9001:2015-Certiquality) is an integral part of the BBMRI-ERIC Italian node (i.e., Biobanking and BioMolecular Resources Research Infrastructure) and interacts with both clinical units and research infrastructure.
There is great heterogeneity in the types of biospecimens stored by biobanks. These include tissue samples-either fresh-frozen or paraffin preserved-biofluids (e.g., plasma, serum, blood, urine, stool), cell cultures, and peripheral blood mononuclear cells (PBMCs). Our biobank operates synergistically with the European research infrastructure for biobanking (BBMRI-ERIC), which is one of the largest biobank networks in Europe and provides a portal for access to biobanks and biomolecular resources coordinated by national nodes15. In addition to BBMRI-ERIC, the International Society for Biological and Environmental Repositories (ISBER) has also played an important role in the standardization of operating procedures for biobanking16.
The Biobank Unit, which is part of the Division of Pathology, is committed to the centrality of the patient, support for the development of clinical research, continuous improvement, the enhancement of human resources, international collaboration, support for training events, safety in the workplace, and scientific and technological growth. The common vision is to set up the national and European landmarks for biobanks in terms of the quality and quantity of services and innovation. The collected biological samples are used to identify new biomarkers and new drugs (e.g., to develop increasingly personalized therapies) and to ensure the best available treatment for patients through excellence in research.
Each biological specimen is collected and handled after prior verification for the presence of the Scientific Research Participation Agreement expressed by the patient15. Biological collected specimens are used to conduct research projects or clinical trials and include excess (i.e., not needed for diagnostic purposes) pathologic and non-pathologic surgical samples, liquid biopsies (e.g., blood, serum, plasma, and urine), and other biological specimens. These biomaterials are stored according to dedicated cryopreservation protocols. This paper provides the biobank protocols of a large cancer center.
This protocol focuses on the SOPs used for breast, ovarian, prostate, lung, and colon cancer. All the procedures described here were approved by the Scientific Technical Committee, Ethics Committee (EC), and the Directors of Breast, Gynecology, Urology, Thoracic surgery, and Digestive System Surgery Programs. The study procedures follow the Declaration of Helsinki of 1964, the General Data Protection Regulation (GDPR) act of 2018, and the subsequent amendments. An institutional Research Participation Agreement (RPA), derived from the GDPR act, represents the informed consent obtained from all patients to collect biological samples and personal, clinical, and genetic data. The RPA was obtained from all patients for storing, processing, and using the obtained data for scientific purposes.
1. Prerequisites of biological samples
2. Biobank software
Figure 2: A representative interface of biobank LIMS. The Scientific RPA is electronically updated from the clinical record server. Abbreviations: LIMS = laboratory information management system; RPA = Research Participation Agreement. Please click here to view a larger version of this figure.
Table 1: Biofluid types and corresponding codes. Please click here to download this Table.
Table 2: Tissue sample types and corresponding codes. Please click here to download this Table.
3. Sample collection
Figure 3: Sample hierarchy. From a single patient, several subcategories of episodes are processed and stored in the biobank. Abbreviations: PMBCs = peripheral blood mononuclear cells; LIMS = laboratory information management system. Please click here to view a larger version of this figure.
4. Blood sample collection
Figure 4: Frozen sample materials. (A) 2D barcode tubes, (B) barcode reader for single tube, and (C) tubes' plate for registration and storage. Please click here to view a larger version of this figure.
5. Serum sample collection
6. Peripheral blood mononuclear cell isolation
7. Plasma sample collection
8. Stool and buccal swab sample collection
9. Tissue processing
Figure 5: Biobank workflow for tissue samples. Abbreviations: LIMS = laboratory information management system; OCT = optimal cutting temperature. Please click here to view a larger version of this figure.
10. Tissue freezing
11. Tissue quality control
Table 3: Quality control form of OCT-embedded and frozen tissue sections. Abbreviation: OCT = optimal cutting temperature. Please click here to download this Table.
12. Request and retrieval of specimens for research purposes
Following the SOPs described above, we collected a total of 38,446 annotated biological liquid biopsies and a total of 10,205 tissue samples from April 2012 to December 2021 (Figure 6A). In addition, we analyzed in detail the collected samples from the Divisions of Urology, Gynecology, Senology, as well as the Divisions of Head and Neck, Abdominal-Pelvic, and Thoracic Surgery. The highest number of tissue samples we collected were from breast tumors (Figure 6B,C). Since 2019, we have also started collecting other biological specimens, such as urine, stool, and buccal swabs, following the significantly increased demand over the years (Figure 6D).
As shown in Figure 6A, the amount of collected samples, especially tissues, during 2020-2021 suffered due to the COVID-19 pandemic and the related reduction in oncologic procedures. Importantly, scientific work did not diminish during this period due to the use of properly stored and annotated biobank samples collected in the previous years. Proper collection of biological specimens and associated clinicopathological data allowed us to have a well-structured and competitive retrospective and prospective biobank. To this end, the selection of the surgical specimen must be performed by the pathologist, both to ensure a correct diagnosis and have the opportunity to conduct research with appropriate tissue specimens. In our biobank, specific work procedures are firmly established and followed, so that we apply standardized procedures that comply with the certification ISO 9001 in the context of biobanks for research.
Figure 6: Biobank cumulative collection of biospecimens at the European Institute of Oncology, from 2012 to 2021. Cumulative collection of (A) tissue samples (orange curve) and blood with serum samples (blue curve); cumulative collection of (B) breast tissue samples (red curve); cumulative collection of (C) ovary tissue samples (green curve), prostate (grey curve), lung (light blue curve), and colon tissue samples (yellow curve). From 2019 to 2021, cumulative collection of (D) additional biological samples: feces (blue line), a buccal swab (pink line), plasma (light green line), and urine (violet line). Please click here to view a larger version of this figure.
Although oncology has made tremendous advances, cancer remains a leading cause of morbidity and mortality worldwide20. Understanding tumor heterogeneity, its temporal evolution over time, and the outcomes of targeted treatment are strictly dependent on accurate data collection in the context of routine clinical care21. In this respect, the "multi-omics" approach is gaining momentum in oncologic predictive pathology22. The traditional tissue-based biomarker assessment is being integrated using multiple new bioanalytes, such as blood, plasma, urines, saliva, and stool23,24,25,26. Therefore, biobanks are now recognized as pivotal infrastructures to enhance clinical practice. Looking back at the history of cancer research, we realize that the most impressive and groundbreaking discoveries would never have been possible without direct examination of cancer tissue or liquid biopsies. Over time, the source of cancer tissue and the type of liquid biopsy to be examined has evolved from crude dissections, random "chance encounters", and in some cases, illicit trafficking to organized cancer collections and strategic modern oncology banks. Consideration of many ethical issues has changed considerably both in practice and in the main factors that distinguish modern oncology banks from the oncology collections of the past.
Due to the advances in cancer research and the vast amount of molecular information that is now provided by modern technologies, it is becoming more and more evident that biobanks, particularly those in cancer centers, may face several types of methodological issues. Among these, technology has become a universal challenge that still prevents SOP standardization and harmonization. Another critical aspect for maintaining core biobank activities is to have an integrated LIMS software able to receive and automatically maintain all hospital IDs and all the codified clinical data coming from the hospital's software. It is noteworthy that other valuable software used to manage biobanks and some freeware can be obtained for biobank managing27,28,29,30,31. Another critical step in biobanks is the implementation of the participation pact for all patients and the legal and ethical agreement necessary for the storage of clinical data and biospecimens10,32.
In this regard, this protocol has well-defined guidelines that do not permit the collection and storage of biospecimens in the absence of consent. This is also a critical issue since patients can withdraw their participation even after their samples have been stored; thus, methods to rapidly take such samples out of the biobanking system have been implemented. Biospecimens that arrive from patients recruited by our biobank follow strict protocols for collection and storage. In this regard, several important aspects have been evaluated to monitor this process and are being continuously improved. In particular, ISO9001 certification requires several indicators of performance, such as warm ischemic time, which has to be maintained for under 30 min or 60 min depending on the source of the tissue. Additionally, liquid biopsies and biological fluids are collected using standardized protocols following strict time procedures15,33,34,35,36.
Specific features are of great importance in biobanks' workflows. These include the presence of a certified pathologist, which guarantees the sampling of the tissue for diagnostic reasons, and the collection of tissue for biobanking in a time frame compatible with a high quality of samples (ischemic time is an important indication for some types of research, such as RNA-dependent assays, which require less warm ischemic time). Moreover, managing the space required for specimen storage is of great importance in biobanks. The number of collected liquid biopsies could be driven by the study design. Liquid biopsies may often be collected both during the preoperative and the follow-up period, as defined by each study design.
Owing to screening campaigns for cancer prevention and the early diagnosis of tumors, i.e., small-sized breast tumors at early stages of development, as well as the availability of minimally invasive surgical techniques, have reduced the number of tissues samples available for research (as most tissue samples are always used for diagnostic purposes). The capacity to collect and store biological specimens has improved considerably over the last few years. This could be observed for biological fluids, reflecting the increased capacity of this biobank to support this institute's research groups in the growing demand for patient-derived annotated material. Despite these improvements, we have experienced some limitations for multi-center studies that require coordination between biobanks from different parts of the world, which can be integrated only by implementing similar procedures.
Having ruled out most of the ethical and technical issues regarding biobanking, including the collection of all clinical and demographic information, the next goal is to implement the digitalization of all the histological preparations and staining used for diagnosis and research purposes. This is of fundamental importance for the next generation of studies that will greatly benefit from a fully integrated digital pathology and biobank, which is going to become the standard for the future. Only a large series of patients with integrated data and digital images may fuel multi-center, large artificial intelligence (AI) studies for the improvement of cancer patient care. In conclusion, we believe that good healthcare does not end with diagnosis and treatment. Best practices comprise finding the ways of continuous diagnosis and treatment improvement for any disease, in particular ones that severely affect life expectancy or quality.
The authors have nothing to disclose.
The authors would like to thank all the patients who actively participated during the last decade in our research programs through the donation of their biospecimens. Without them, this research would not be possible. We are also grateful to all the personnel working at IEO, nurses, technicians, biologists, doctors, and the directors of all the clinical and research units. The authors are grateful to Prof. Pier Paolo Di Fiore and Prof. Giancarlo Pruneri for their guidance. Finally, we dedicate this work to Prof. Umberto Veronesi, the founder of IEO, and his pioneering approach to integrating cancer research and patient care.
Blue Max Con Tubes 15 mL | Falcon B.D | 352096 | |
Blue Max Con Tubes 50 mL | Euroclone Spa | FLC352070 | |
Box with 81 position for tissue storage | Ettore Pasquali Srl. | 06.0945.00 | |
cf-DNA/cf-RNA Preservative Tubes | Norgen Biotek | 63950 | Preservation and isolation of both cf-DNA and cf-RNA from a single tube and in particular preserve cf-DNA/ct-DNA for 30 days at ambient temperature and for up to 8 days at 37 °C |
Cryomold Standard (25 X 20 X 5 mm) | Olympus Italia S.r.l. | 4557 | Disposable plastic Cryomold molds create a uniformly shaped, flat-surface specimen block when used with O.C.T |
Dimethyl Sulfoxide Plastic Bottle – 1 L | Vwr International S.R.L. | MFCD00002089 | It acts to preserve the reconstitution of the medium for the storage of frozen cells |
Dpbs 1x W/o Ca And Mg – 500 mL | Microtech Srl | TL1006-500ML | Washing Buffer cell |
Dualfilter T.I.P.S 1,000 µL | Euroclone Spa | 4809 | |
Dualfilter T.I.P.S 200 µL | Euroclone Spa | 4823 | |
Easytrack Barcode Reader for single tube datamatrix | Twin Helix Srl | TH-ETR4400 | 2D barcode tubes reader with USB connection |
Fetal Bovine Serum Origin Brazileu S/fil | Microtech S.R.L | RM10532-500ML | Defrost at +4 °C, usually for two days, and once melted, start decomplementation at 56 °C for 45 min Let it cool down to room temperature, and aliquot it. Refroze them to -20 °C, and remember to defrost them every time the aliquots are needed |
Ficoll Paque Plus (ge) 6 x 500 mL | Euroclone Spa | GEH17144003 | Ficoll is a medium for density gradient, It is sterile and ready for use. It alloes to get peripheral blood mononuclear cells, bone marrow and umbilical cord blood |
Fixing solution Killik of 100 mL (OCT) | Bio-optica Milano S.p.a. | 05-9801 | Gel inclusion medium that solidifies at cold the water-soluble tissue (e.g., biopsies, frustules) |
FLASH-FREEZE | Milestone | n.a. | Freezing appliance |
Forma 8600 Series Chest Freezers (Temperature Range: -50 °C to -86 °C) 85 liters | Thermo Fisher Scientific Srl | 803CV | Orizzontal freezer |
Isopentane 500 mL | Vwr International S.R.L. | 24872260 | Liquid included in theFLASH-FREEZE camera for freezing |
Nautilus Lims Software | Thermo Scientific™ | n.a. | The software implementation is able to track all patients’ biological samples. Receives Personal and Clinical information automatically during registration due to the integration with IEO operating systems. Nautilus is integrated with the web service through three IEO operative systems: BAC – IEO central registry with personal information, wHospital – medical record |
Pasteur pipette 10 mL | Euroclone Spa | CC4488 | |
Pasteur pipette 3 mL | Euroclone Spa | APT1502 | |
PATHOX | Dedalus ItaliTesi Elettronica e Sistemi Informativi S.p.A.a S.p.A. | n.a. | PATHOX – management system for the Pathology unit where several factors are registered for the Biobank, such as the histological samples, the related diagnoses, and biomarkers |
Petri dishes, polystyrene – size 100 mm x 20 mm, slippable | Euroclone Spa | FLC353003 | |
Set of 4 adapters 19 x 5/7 mL vac | Thermo Fisher Scientific Srl | 75003680 | |
Set of 4 adapters 4 x 50 conical | Thermo Fisher Scientific Srl | 75003683 | |
Set of 4 adapters 9 x 15 mL conical | Thermo Fisher Scientific Srl | 75003682 | |
Single-use slide for counting cell | Biosigma S.P.A. | 347143/001 | Specifically used for individual cell count |
Stamps Freezerbondz for tissue boxes, nitrogen-liquid proof , H 9,53 mm x L 25,40 mm | Twin Helix Srl | THT-152-492-3 | |
Thermo Scientific TSX Series Ultra-Low Freezers (-50 °C to -86 °C) 949 liters | Thermo Fisher Scientific Srl | TSX70086V | Vertical freezer |
Thermo Scientific Refrigerated Centrifuge SL16R | Thermo Fisher Scientific Srl | 75004030 | |
Tissue box labels in Permanent | Twin Helix Srl | THT-199-482-3 | |
Tuerks Solution | Merck Life Science S.R.L. | 1092770100 | In light microscopy, it is specifically used as stain for leukocyte |
TX-400 Rotor TX-400 swinging bucket hol | Thermo Fisher Scientific Srl | 75003181 | |
White box for storage | Bio Optica | 07-7300 | |
wHospital Software | wHealth Lutech Group | n.a. | wHospital – medical record management system with personal information, administrative cases, and the informed consent of the patients |