The Use of Human Tissues for Research: What Investigators Need to Know

Abstract

While laboratory animals are necessary for some aspects of the development of biomedical advances, including those of precision medicine, human tissues are necessary to explore the findings and ensure that they are relevant to human systems. While many sources of human tissues exist, researchers, particularly those making the transition from animal to human systems, may not be aware of how best to find quality sources of human tissues or how best to use them in their research.

In this article, we discuss the advantages of using human tissues in research. In addition, we highlight some of the major advances made possible by the use of human tissue, describe how human tissue is collected for research, the various types of bioresources that make human tissue available, and how investigators can find and use appropriate bioresources to support their research. The hope is that providing this information can help facilitate the transition from research on animals to research using human tissues as rapidly as practicable.

INTRODUCTION

The purpose of this article is to help researchers transition from the use of animals in their research to the use of human tissues. Using human tissues can help ensure that the findings obtained in animals are generalizable to human systems. As we discuss in this paper, the use of human tissues in research is critical to validate findings obtained in animals and has led to major scientific and medical advancements. However, investigators wishing to transition to the use of human tissues may not know how to identify appropriate high-quality sources of biospecimens or understand the requirements for using them. We discuss these issues in this paper with the hope that a better understanding of these issues will assist researchers in their transition from the use of animals to human tissues and lead to quality findings that will advance our understanding of biology and science and lead to major developments in medicine and technology.

ADVANTAGES OF USING HUMAN TISSUES IN RESEARCH

The goals of most biomedical research are to make improvements in the cures, therapies and management of diseases. Frequently, cell lines and animal models are used to develop preliminary data to achieve these goals. However, the use of cell lines and animal models have disadvantages and the use of human tissues may facilitate reaching these goals more rapidly. For example, cell lines develop and accumulate many molecular changes such as chromosomal instability compared to the original human tissues from which they were developed, so that they may not accurately reflect the original tissue¹. Cell lines can also be misidentified and contaminated by other cell lines². As another example, the genomic responses, immunology, and other biological responses of animals may vary significantly from humans³. Because of this, results from animal models and cell lines will have to be confirmed in human tissues.

ADVANCES IN SCIENCE AND MEDICINE MADE POSSIBLE BY THE USE OF HUMAN TISSUES

Almost all the advances in medical care are based on research with human tissues. The availability of human tissues for research has led to many seminal discoveries in science and major developments in medical care as we describe in detail subsequently.

Human tissues have been critical to the diagnostic characterization and subtyping of most cancers. For example, most breast cancers are morphologically described (lobular, ductal and mixed) as well as molecularly subtyped. Similarly, the diagnosis of lymphomas has recently been changed based on both morphologic characteristics and molecular features⁴. The morphologic subtyping and determination of molecular features are critical to diagnosis and therapeutic approaches to all types of cancers.

Human tissues have been essential to important developments in precision medicine. Precision medicine is a type of medical care in which features of patients are combined with features of their diseases to develop specific focused therapy for their diseases^5,6. Although precision medicine is a relatively new term, this approach to medical care has been used for several decades, specifically to treat breast cancers. Physicians have been targeting their molecular characteristics for treatment, for example, by blocking estrogen receptor (ER). This therapy was developed using breast cancer tissues and the molecular features of a patient’s tissues are evaluated, even currently, to identify the cancers appropriate for anti-ER therapy.

Breast cancer and gastric cancers are the best examples of uses of one type of precision medicine. Human tissues were used to identify the human epidermal growth factor receptor type2 (HER2) as a molecular, poor prognostic factor in breast cancers⁷. Subsequently, HER2 became a target of precision medicine in both types of cancers. Human tissues were used to test the HER2 antibody, Herceptin®, and its subsequently developed biological equivalents, such as Kanjinti® and other anti-HER2 based conjugates with drugs^8,9. Similarly, the identification in gastric cancers of HER2 overexpression and the sensitivity of positive gastric cancers to anti HER2 therapy, permitted additional precision medicine therapeutic approaches for gastric cancers¹⁰.

A more recent tissue-based advance in precision medicine is the development of immunotherapy. Human tissues demonstrated that some cancers such as melanomas and lung cancers develop in an environment in which the immune system is suppressed. Two categories of immunotherapy have been characterized utilizing human tissues. One type involves general stimulation of the immune system and the other blocks the action of immune check-point inhibitors Specifically the programmed cell death protein 1 (PD-1) is expressed on T cells; when the check point inhibitor, PD-1, is activated, PD-1 decreases T lymphocyte activation, cytokine secretion, proliferation and survival of the T cells expressing PD-1. Some cancer cells express the ligand for PD-1 (PD-L1). When PD-L1 binds to PD-1, it inhibits the immunity directed against the cancer cells¹¹. Multiple antibodies have been developed to inhibit the PD-L1 and PD-1 interaction. These have proved to be effective against PD-L1 positive melanomas, lung cancers, and other cancers^{12, 13}. However, some tumors are not very responsive to this type of immunotherapy.

SOURCES OF HUMAN BIOSPECIMENS

Human tissues used in research may involve a variety of cell types and preparations and they may be collected in a number of different ways. In addition to solid tissues, human biospecimens that are often collected and used for research include whole blood and blood components, such as serum, plasma, and buffy coat. They may also include other bodily fluids, such as urine, saliva/sputum, and joint fluids as well as cellular preparations (e.g., fine needle aspirates). Other types of biospecimens often used in research include cellular derivatives, such as DNA and RNA, as well as immortalized cell lines, organoids, and patient-derived xenografts^{14, 15}.

Human biospecimens and associated data that are used for research may be collected in a variety of ways. Often left-over biospecimens (i.e., remnants) that were originally obtained during the course of routine care may be used for research as long as the research use does not affect future care of the patient. Biospecimens may be obtained specifically for research through a specific intervention (such as a blood draw, oral swab, or urine collection). Biospecimens may also be collected for research during a clinical protocol or as part of a clinically necessary procedure. For example, additional biopsy samples needed for research may be taken at the same time as a clinically indicated biopsy procedure. Finally, biospecimens can be obtained for research from cadavers, autopsies or from unused tissues that may have been initially collected for transplant purposes. In all cases, biospecimens for research are obtained following relevant ethical and regulatory requirements, which in most cases requires consent from the donor/research participant^{16, 17, 18}.

WHAT IS A BIORESOURCE AND WHAT TYPES OF BIORESOURCES EXIST?

Biospecimens and associated data are collected and stored and made available for research by entities often referred to as biobanks and biorepositories. As defined by the International Society for Biological and Environmental Repositories, a biorepository or biobank is “An entity that receives, stores, processes, and/or distributes specimens, as needed. It encompasses the physical location as well as the full range of activities associated with its operation”¹⁹. A more recent term used to describe a biobank is the term bioresource²⁰. We prefer this term because it emphasizes the important role of serving as a resource for the research community rather than simply storing and banking biospecimens.

A variety of different models of bioresources provide biospecimens for research. These are discussed in more detail in previous publications^{20, 21}. The most common bioresources serving typical investigators utilize the tissue procurement model, the classic biobanking model, the population-based model and the combination of classic biobanking and tissue procurement models^{21, 22}. As discussed subsequently, each of these models may be better suited to different kinds of research.

In a tissue procurement model, biospecimens and data are procured prospectively specifically to meet the requests of individual researchers. The procedures for the collection of biospecimens are adapted to meet the investigator’s requirements²². In this model, biospecimens are distributed soon after collection and are not held in storage by the bioresource for significant amounts of time. For many requests, biospecimens can be provided in a matter of months. However, if clinical follow-up data are required, the requests could take much longer. In addition, some requests may be very difficult to meet and may take years to fulfill. For example, a request for 30 frozen at least 0.5 gram breast cancer biospecimens from women should be fulfilled in a matter of weeks, but a request for similar biospecimens from males will take many years. Similarly, when multiple requirements are added to a biospecimen request such as requiring a very large minimum size, the time to fulfill the request may increase exponentially. The tissue procurement model is most suitable for basic and developmental studies. While extensive clinical data may not be available in many cases, clinical data may be available for archived paraffin-embedded material making the model suitable for some translational studies as well. An example of a bioresource using the tissue procurement and distribution model is the NCI Cooperative Human Tissue Network^{22, 23} discussed subsequently in this paper.

In the classic biobanking model, the bioresource decides on its focus as to what biospecimens and associated data will be provided and develops standard operating procedures (SOPs) for their collection. Biospecimens and data are collected according to pre-established criteria that are based on the goals and intended use of the bioresource and banked in anticipation of future research requests. The diseases and diagnoses, processing, and storage of biospecimens are all specified in advance by the bioresource²⁴. The time it takes before biospecimens are available from this model will depend upon where the biobank is in its life cycle, as well as the nature of the request. If a biobank is just starting up and their goal is to collect biospecimens and clinical follow up data to store for future use, it may take many years before the bank “matures” and the associated clinical data are available to investigators who request the biospecimens and data. In addition, it will depend the specific nature of the researcher’s request. For a “mature” biobank, the times for completing most straight forward requests should be a matter of weeks because the biospecimens are already in the bioresource’s inventory with adequate follow-up. However, because biospecimens are collected using criteria pre-established by the bioresource and banked for later use, the bioresource may not be able to meet the needs of current researchers who need biospecimens collected in different ways. Furthermore, because the biospecimens are banked, they may degrade over time. The biobanking model is most useful for studies that require extensive amounts of clinical data, large numbers of biospecimens, and biospecimens from patients with rare diseases. Examples of bioresources using the classic biobanking model include many academic bioresources, but some of these have added a prospective component.

The model of a population based/epidemiological bioresource is designed to address questions related to features of populations or subpopulations²¹. In this model, biospecimens and associated data are usually collected from donors of a healthy population or from patients with specific diseases. Most often this type of bioresource collects biospecimens that are bodily fluids. Because this model can provide biospecimens from donors in a healthy population before the development of disease, the bioresource is useful for identifying biomarkers for risk, early detection, diagnosis and prognosis. A population-based model also may be useful to study the effects of environmental exposures or studies of the impacts of diet and nutrition on the population. Examples of population based bioresources include the National Health and Nutrition Examination Survey (NHANES)²⁵, the All of Us program in the US²⁶ and the UK biobank²⁷.

HOW TO FIND AN APPROPRIATE, GOOD QUALITY BIORESOURCE

While numerous bioresources exist, researchers are often unaware of how to find them. A number of tools exist to help resources find suitable bioresources for their work. This includes the NCI Specimen Resource Locator²⁸, the ISBER International Repository Locator²⁹, and the BBMRI-Eric Negotiator³⁰, a communication platform for bioresources and researchers requesting biospecimens and/or data.

One important bioresource supporting basic and developmental researchers who may be wishing to transition from animal models to research on human tissues is the Cooperative Human Tissue Network²³. The CHTN was established in 1987 to provide increased access to human biospecimens to accelerate discoveries in cancer research. However, the Network supports a wide range of research on human tissue, including research on other diseases. CHTN staff work closely with individual researchers to tailor the collection processes and procedures to meet their precise requirements. All biospecimens that are collected must meet strict quality assurance/control standards to ensure that the biospecimens are of high quality and fit for the intended purpose^{22, 23}.

In addition to bioresources established and maintained by academic medical centers, there are multiple national and international commercial companies that provide human tissues to support biomedical research. These companies usually collaborate with medical sites or organizations from which they obtain the remnant tissues they provide. Sometimes these companies specialize in only certain categories of tissue. For example, some companies collect blood and blood products for medical use from paid individuals; based on a fee, these will provide biofluid aliquots to support research. The non-profit Red Cross also may provide selected blood biospecimens for research. Anabios specializes in cardiovascular and neuropathological tissues. A number of companies, (e.g. Bocabio, Anabios, iSpecimen) can supply, or act as a broker to help researchers obtain, biospecimens including biofluids and solid tissues especially as formalin-fixed paraffin-embedded (FFPE) blocks or sometimes as frozen biospecimens. Various extents of annotation also can be provided depending on the company. The main disadvantage is high costs and sometimes inadequate quality control. Commercial companies providing human tissues can be found on the web.

An important aspect of using human tissues in research is tissue quality. Bioresources use a number of approaches to assure high quality and fitness-for-purpose of the biospecimens they provide. This includes establishing a rigorous quality management system (QMS) with demanding quality control (QC). The QMS must utilize a standard operating procedure (SOP) for each activity and frequent audits ensure that the SOPs are being followed. One aspect of the QMS is QC which ensures the diagnosis of each actual biospecimen provided to an investigator as well as the characteristics of each biospecimen. For example, in the case of cancer biospecimens, quality control data such as the % tumor and % tumor nuclei in the biospecimen as well as the % necrosis, % fibrosis and % mucin should be provided^{22,31, 32}.

In order to assure high quality biospecimens and data, bioresources often follow best practices, such as those developed by the International Society for Biological and Environmental Repositories³³ (website). The ISBER Best Practices: Recommendations for Repositories Fourth Edition provide evidence-based or consensus-based best practices for collection, storage and distribution of biospecimens for research^{34, 35}. International standards have also been established, including ISO 20387:2018 General requirements for biobanking^{36, 37} and are beginning to be implemented by bioresources³⁸. In addition, some bioresources may be accredited by the College of American Pathologists Biorepository Accreditation Program³⁹. These best practices, standards and accreditation programs help to ensure that the biospecimens and associated data obtained from bioresources are of high quality and fit for their intended purpose.

SOURCES OF NORMAL TISSUES

Normal tissues may be difficult to obtain. Bioresources can collect normal tissues from autopsies and donated cadavers but tissues affected by comorbidities should be avoided. When tissues are removed surgically, some tissues may be considered “normal;” these are tissues not involved in the disease processes being treated by the surgery. In the case of cancer, the organ affected is not considered normal. For breast cancer, the tissues of neither breast can be considered to be normal but adjacent skeletal muscle can be classified as normal provided it has not been invaded by the breast cancer. Population based studies may serve as good sources of normal tissues. These include the National Health and Nutrition Examination Survey (NHANES) which evaluates the health and nutrition of adults and children in the USA over time²⁵. Not all biospecimens that are collected are typically used by NHANES and may be available to external investigators. Similarly, normal tissues that are not informative to the trial are available in the bioresource associated with the Prostate, Lung, Colon, and Ovary (PLCO) Cancer Screening Trial; these biospecimens likely may be available to investigators at no costs other than shipping⁴⁰.

BIOSPECIMENS FROM RARE DISEASES

Some investigators may need tissues from a rare disease and these biospecimens may not be easily obtained from most bioresources which may not have these biospecimens in their inventories. Advocates for some rare diseases have recognized this problem. Frequently, these advocates for a rare disease have an associated advocacy organization which may fund a bioresource for tissues from this rare disease. An advocacy organization also may request that their members send remnant tissues of the rare disease and matching uninvolved tissues to the bioresource. Examples include the Chordoma Foundation Biobank⁴¹; such advocate bioresources may be found on the web.

REQUIREMENTS FOR ACCESS TO HUMAN BIOSPECIMENS

Bioresources have a variety of requirements that must be met for access to the biospecimens and data that they provide. Some bioresources may restrict who may have access to the biospecimens; they may be “open bioresources” or “closed bioresources”. An open bioresource may provide biospecimens to any investigators who meet its standards and requirements. An open bioresource serves many important roles, such as providing biospecimens to supplement a biospecimen request which another bioresource, open or closed, cannot completely fulfill. On the other hand, a closed bioresource typically provides biospecimens only to investigators at the institution which is financially responsible for the bioresource; however, other investigators may be designated to have access. Thus, a closed bioresource knows its potential users and can tailor its operations including collection of needed biospecimens based on their stated and perceived needs. Operations can be adjusted when there are new investigators or new projects developed by existing investigators. A closed bioresource can meet most of the needs of its investigators for biospecimens with characteristics that are not too restrictive. For complex requests for biospecimens that a closed bioresource cannot adequately fulfill, open bioresources such as the Cooperative Human Tissue Network (CHTN)²³ may supplement local biospecimen needs.

Bioresources often have other requirements for access to the biospecimens and data that they provide to ensure that the biospecimens are used in ways that are ethically appropriate and scientifically sound. This includes a requirement for some sort of review and approval by an ethics review committee, or Institutional Review Board. In addition, an internal review by the bioresource, sometimes by a biospecimen utilization committee, or similar committee, is often needed to ensure that an investigator’s request is an appropriate use of the bioresource and that it is consistent with biospecimen availability. Some bioresources have requirements that the researcher receiving the biospecimens collaborate with bioresource investigators.

As a best practice, researchers and their institutions may be asked to sign an investigator agreement that defines the responsibilities and obligations of the recipient researcher and the bioresource. These agreements may include a Material Transfer Agreement and/or Data Use Agreement. The agreements document the obligations of the researcher to protect the privacy of the individuals from whom the biospecimens are obtained and the confidentiality of their data, to use the biospecimens only for the approved project, and to agree not to share the biospecimens and data with third parties without approval of the bioresource¹⁹. These agreements may include other terms and conditions for use the biospecimens and associated data (e.g. intellectual property rights, etc.). In addition, the agreements may ask investigators to acknowledge the bioresource in any publications arising from the use of the tissue. These requirements help to ensure that investigators are using the biospecimens appropriately and help document the advances made possible from biospecimens obtained from the bioresource. This is important for the bioresource to demonstrate value to funding agencies and sponsors in order to justify continued funding and provide sustainability for the bioresource.

Operating a bioresource is expensive and few bioresources are self-sustainable. Therefore, many bioresources charge a fee for biospecimens to cover the cost of procuring, annotating and distributing them for research. “Selling” biospecimens for profit is generally considered unethical, although charging a fee for cost recovery is considered acceptable⁴². The cost per biospecimen charged by bioresources depends on the efficiency of operations and the number of biospecimens distributed. For example, if the costs of operating a small bioresource is 200,000$ USD and only 100 biospecimens are distributed, the actual cost of collecting, annotating and storing a biospecimen might be something like 2,000$. This obviously is too expensive for an academic investigator to pay. The decision on charge back fees is typically decided based on what the bioresource decides academic investigators can afford to pay; this is possible provided there is financial support to cover bioresource costs not paid by charge back fees. Alternatively, some bioresources can compensate for this financial deficit by distributing extensive numbers of biospecimens to companies at higher fees.

Commercial companies frequently require human tissues to support their research and product development. It is important to provide commercial companies with human biospecimens in that commercial companies play important roles in development of therapeutic approaches to diseases. Typically, many commercial companies use biospecimens not requested by academic investigators, especially “normal” tissues. To ensure that a commercial company “pays its way,” the charge back cost is as a minimum the total cost of collecting, processing, and distributing each biospecimen. At the time this manuscript is written, this is usually about 150$ USD or more.

Requirements for access to biospecimens may vary considerably from one bioresource to the next. Information about the types of biospecimens available from a bioresource, the requirements for access and the application process are frequently found on the bioresource’s website.

WHAT INVESTIGATORS NEED TO KNOW ABOUT USING HUMAN TISSUES IN THEIR RESEARCH

If one decides to use human tissues to replace some of the research currently performed with animals, the approach should be carefully considered and planned. Investigators transitioning from animals to human tissues should consider testing their approach first on a small number of biospecimens in a pilot. Although human tissues may be available, initially, bioresources may be reluctant to provide many biospecimens until a pilot study has demonstrated that the approach is promising. For example, in studies on common tumors, the initial plan should usually rely on small numbers of biospecimens (e.g. 5 – 10). After the experimental results are analyzed and results are encouraging, additional tumor specimens may be requested along with an experimental plan to confirm and expand the initial results.

The availability of tissue from bioresources can be limited if requests are too restrictive. In general, the actual request for a tissue determines its availability. For example, solid tissues may be difficult to obtain if, for example, the request is for:

• tissues from patients whose diseases are not treated by surgery (e.g. diabetes Type 1).

• tissues from patients that have been treated with a specific therapy

• tissue from patients with a narrow age range which is uncommon for the biospecimens requested (e.g., ductal breast carcinoma from women less than 25 years of age)

• an unusual type of diseased tissue (e.g., ductal breast carcinomas from males)

• tissue involving the combinations of characteristics some of which may be almost impossible to obtain (e.g., ductal breast carcinomas from male African Americans);

• large numbers of a specific type of biospecimens (e.g., 500 ductal breast carcinomas);

• multiple large samples of one type (multiple 200 grm ductal breast carcinomas);

• or tumors that are metastatic to local lymph nodes or other metastases (e.g., metastatic biospecimens of ductal breast carcinoma to the brain). Requests for more routine ductal breast carcinomas can be met without difficulty by many biorepositories/bioresources.

Because of these issues, it is highly recommended that investigators needing biospecimens from a bioresource discuss their needs with staff from the bioresource early on in the planning of the project. Staff from the bioresource can help investigators refine their requests for biospecimens and help them through the application process. If a bioresource is unable to fill the investigators request, staff from the bioresource can sometimes refer them to other more appropriate bioresources.

Bioresources undertake a number of approaches to maximize the number of requests that can be filled from their inventories. For example, to increase the tissues available to support research, during processing, solid biospecimens are aliquoted in standard sizes. The QC blocks represent other samples which can provide separate aliquots. Similarly, plasma, serum, buffy coats, and other bodily fluids are aliquoted into standard volumes.

SAFETY ISSUES

While care must be taken when working with animals and human and animal cell lines, research with human solid tissues and bodily fluids requires more attention to safety because some human tissues may be infected with human pathogens. Usually cell lines with human pathogens have been identified; however, for human solid tissues and bodily fluids, this may not be the case and universal precautions are required when working with them. Universal precautions require the use of gloves, masks, safety glasses and coverings, such as aprons, to prevent exposures of skin and clothes to human tissue products. The use of safety equipment is part of a required safety plan which includes appropriate procedures for handling biohazardous materials⁴³.

VARIABLES AFFECTING THE USE OF SOLID HUMAN TISSUES IN RESEARCH

When using human tissues, investigators need to be aware of the variables that may affect their research. Human tissues, like the individuals from whom they are obtained, are quite variable; they vary with age, race, sex, ethnicity and many other variables. From the standpoint of human tissues removed operatively, the variables can be classified as pre-diagnosis, diagnostic, post diagnostic, neoadjuvant therapy, post neoadjuvant therapy, pre-operative, and operative. Variables also include procedures for processing, storage, and distribution. While there are many variables, not all are that important. Nonetheless, they are variables of which investigators must be aware. Otherwise, bias may result in interpretations of experimental results.

Pre-diagnostic variables are numerous. The most important are probably co-morbidities, that are diseases present in tissue donors prior to diagnosis of the operative disease. More common co-morbidities include cancer, type 1 and 2 diabetes, hypertension, renal failure, and liver failure. Other pre-diagnostic variables are diet and environmental and occupational exposures. More detailed information on all variables have been described previously⁴⁴.

Diagnostic and post diagnostic variables usually are related to the tissues obtained after diagnostic biopsy, analysis of biofluids, or imaging. When a biopsy is obtained, tissues surrounding the biopsy site are frequently affected by trauma, tissue damage and associated changes such as scarring and inflammation. Informative studies using human tissues rarely may include an unrecognized portion of a biopsy site which could confuse interpretation of the tissues provided for research.

Neoadjuvant therapy is therapy which is given to a tissue donor prior to definitive surgery. It may include chemotherapy, biologic therapy such as Herceptin and/or radiation therapy. All the tissue effects, especially molecular changes caused by neoadjuvant therapy currently are unknown, so tissues obtained after neoadjuvant therapy may not be useful for research and probably should be avoided.

Variables associated with the definitive surgery include anesthesia, cautery and other damage to tissues and the time of warm ischemia. Utilization of a cautery in surgery is preferred by many surgeons because the cautery coagulates blood vessels and reduces blood in the surgical field and overall blood loss. Investigators should be aware that heat may spread superficially from the surgical cut into some adjacent tissues. This heat and the temperature increase associated with it can affect immunohistochemistry and other molecular assays. Warm ischemia begins when the tissue is partially or completely isolated from the vascular supply and ends when the tissue is removed from the patient’s body. During this period, the tissue is ischemic at approximately body temperature at which enzymes should be functional. Thus, the time at warm ischemia may cause cellular damage. Cold ischemia begins at the time the tissue is removed from the body and ends when tissue processing begins. Significant ischemic damage can occur during both warm ischemia and early cold ischemia (10 min. after surgery^{45, 46}.

Processing typically includes aliquoting the remnant tissue (i.e., tissue no longer needed for clinical care) provided by surgical pathology for research; processing includes preparing the aliquots for distribution or for storage. The storage preparation may include fixation in neutral buffered formalin and processing to paraffin blocks (FFPE), or preparing aliquots for freezing and storage at ultracold temperatures.

The type of biospecimen utilized must fit the type of analyses that will be performed (“fit-for-purpose”). Some assays are best performed on frozen biospecimens; others can use FFPE samples⁴⁷. Of note, compared to frozen tissue, extraction of mRNA from FFPE is not as efficient. Therefore, a larger FFPE biospecimen would be required to extract mRNA. Whether frozen or FFPE samples are most appropriate for the research depends upon the research questions being asked, the cellular or molecular targets and the specific types of analyses that will be performed. Based on these considerations, investigators must decide which assays and biospecimens (including size) are most suitable for their work.

While the pre-analytical variables related to human tissues will not be able to be controlled as rigorously as those in experimental animals, such as diet, there are significant quality control measures that are utilized by bioresources that provide human biospecimens for research. High quality bioresources take care to process the biospecimens rapidly, minimize collection and processing variables and record these to document the impact of pre-analytical variables.⁴⁴ For example, most bioresources maintain records of the times of warm and cold ischemia and follow SOPs which limit fixation variables.^{32, 47} High quality bioresources take precautions to avoid potentially detrimental extended incubations in formalin for example, by avoiding weekend tissue processing runs. In addition, information is documented beyond that recorded by the pathologist. For example, in the Cooperative Human Tissue Network (CHTN), quality control (QC) includes a separate diagnostic description of each biospecimen provided to an investigator; specifically, for each aliquot in the bank, and a matching portion of tissue is processed to a paraffin for block for a QC evaluation. For cancers, this evaluation includes, % cancer in the biospecimen and of the cancer, % malignant nuclei, % necrosis and % stroma, fat, and inflammatory cells.³² Records are maintained as to times of warm and cold ischemia, fixative and time of fixation, and stages of the tissue processor. Also, tissues are collected and processed according to well-established standard operating procedures (SOPs). Annotation of the tissue source includes age, race, sex, and information from the electronic medical record when needed.²²

While bioresources follow SOPs to provide high quality biospecimens, bioresources and/or researchers, when needed, can perform quality control assays such as RIN, DV200, delta Cq qRT-PCR and mass spectrometry-based assays to identify biospecimens that might be compromised in quality for various analytes. However, such assays can be expensive to perform.

VARIABLES AFFECTING THE USE OF HUMAN BODILY FLUIDS IN RESEARCH

Collection, processing and storage are the key variables to consider regarding the use of bodily fluids. Bodily fluids are best collected prospectively from consented patients and processed rapidly. Blood may be collected at clinic/office visits when the blood is not from a fasting individual or before surgery when the patient is fasting. Disparate samples and other assay components should not be mixed or analyzed in the same assay; this includes mixing fasting and non-fasting blood, serum and plasma, and different sample containers.

In the collection of blood and blood products hemolysis should be avoided by the choice of needles and control of the rate of withdrawal. In addition, when storing or transporting whole blood on solid ice care must be taken to avoid freezing the sample to prevent any hemolysis.⁴⁸ Normal blood depends upon the individual from whom the blood sample is collected. Specifically, blood from a cancer patient would not be considered normal. Normal blood is best obtained prospectively from volunteer donors without known major diseases.

SUMMARY AND CONCLUSION

Animals serve as important resources for scientific research. However, research findings in animals must often be confirmed using human tissues. As we have discussed in this paper, the use of human tissues has led to major advances in science, technology and medical care. While numerous sources of human tissues exist, researchers making the transition from research using animals to human tissues may not be aware of how to identify sources of high quality biospecimens and use them effectively in their research.

Many models of bioresources or sources of high-quality biospecimens exist and some may be more appropriate than others for certain types of research. A number of tools exist to help researchers find appropriate sources of high quality biospecimens for their work and distinguish among existing bioresources. Once potential sources of human biospecimens have been identified, it is essential for researchers to discuss their needs with bioresource personnel who can help them refine their requests or seek other, more appropriate bioresources. It is critical that the type of biospecimens requested are “fit-for-purpose” for the research questions and type of analyses to be performed. In order to ensure that the findings obtained from the use of human tissues are accurate, generalizable and free from bias, it is important for researchers to understand the variables that may affect use of human tissues and their impact on the research. Careful consideration of these and other issues in this paper can help researchers transition effectively from research using animals to human tissues and produce findings that can ultimately lead to major scientific and medical advancements.