If you are working in the field of drug discovery, you have probably come across the concept of Biospecimens. In this blog post, we are attempting to give researchers a comprehensive guide, helpful tips, and provide context to the role of ethically collected human biospecimens and corresponding metadata.
First things first, what exactly is a biospecimen – according to the NIH website a biospecimen can be biofluids, urine, blood, tissue, cells, DNA, RNA, or protein, from humans, animals, or plants. Biospecimens may be used for a laboratory test or stored in a biorepository to be used for research.
Tissues and biofluids can be obtained from a variety of sources, including:
- Surgical specimens: Tissues removed during surgery can be used for research.
- Biopsies: Tissues removed from the body using a needle can be used for research.
- Autopsies: Tissues removed from the body after death can be used for research.
- Cadavers: Whole bodies donated for research can be used to obtain tissues and biofluids.
- Biobanks: Biobanks are collections of human tissues and biofluids stored for research.
Uses of tissues and biofluids:
- Study the mechanisms of disease. For example, researchers can use tissues from patients with cancer to study how cancer cells grow and spread. This information can be used to develop new drugs and therapies to treat cancer.
- Test the safety and efficacy of new drugs and therapies. For example, researchers can use tissues from patients with a particular disease to test the safety of a new drug. If the drug is safe, it can then be tested in clinical trials in humans.
- Develop new diagnostic tools. For example, researchers can use blood samples from patients with a particular disease to develop a blood test that can be used to diagnose the disease.
Using human tissues and biofluids for research is essential for developing new drugs and therapies. This research can help improve disease diagnosis, treatment, and prevention.
Downstream applications of human tissues, biofluids, and cells are numerous. They can be used to:
- Study the disease’s molecular and cellular mechanisms. This information can be used to develop new drugs and therapies.
- Test the safety and efficacy of new drugs and therapies. This information can be used to determine if a drug is safe and effective before it is tested in humans.
- Develop new diagnostic tools. This information can be used to diagnose diseases earlier and more accurately.
- Personalised medicine. This information can be used to tailor treatments to the individual patient.
- Enhance the understanding of human health and ageing: Human tissues and biofluids can enhance the mysteries behind ageing and how to reverse the processes involved.
Tissues:
The main formats in which tissues have been used are FFPE and snap-frozen (a.k.a. Flash frozen).
Formalin Fixed Paraffin Embedded (FFPE):
Preparation: using 10% neutral formalin and 18-24 h fixation. Downstream & intended application: FFPE samples have been traditionally used in pathomorphological confirmation of diagnosis in oncology. However, due to advances in precision medicine, fixed tumour tissues are being used in the analysis of various mutations using IHC, FISH and NGS. Factors that can affect tissue fixation and that need to be taken into consideration in order to achieve the best results are: fixation time (18-24 hours), temperature, pH, and specimen thickness. Archived specimens have been shown to degrade in terms of nucleic acids (DNA/RNA). Most FFPE blocks are ok for standard pathology, IHC and FISH-based assays. But when it comes to NGS, multiple factors such as the age of the blocks (less than 3 or 5 years preferred), tumour content on the blocks (above 30-50%) and necrosis content less than 10-20% should be discussed as part of tissue acquisition process with any biobank or CRO.
Snap-frozen (a.k.a. Flash frozen):
Preparation: snap-frozen in Liquid nitrogen (LN2) within 30 minutes after resection. Downstream & intended application: Frozen tissues are used in histology or IHC studies and DNA, RNA and protein extractions. The most important takeaway from frozen tissues is that it’s impossible to determine the tumour and necrosis content unless the tissue is OCT-embedded, which makes it unsuitable for DNA/RNA/Protein extractions. To circumvent this bottleneck, mirrored FFPE blocks can be used to give an approximate picture of what the frozen tumour will look like.
Fresh tumour or normal tissues:
Preparation: 200-400 mg of fresh tumour samples collected in tissue preserving media Downstream & intended application: PDX Models – Patient-derived xenograft (PDX) and Immuno-oncology biomarker discovery. Research tumour in its microenvironment and immune response for IO biomarker discovery.
Viable preserved tumours or tissues:
Preparation: small pieces of tumour samples are prepared using Cureline’s custom viable tissue preparation protocol.
Downstream & intended application: PDX Models – Patient-derived xenograft (PDX) and Immuno-oncology biomarker discovery.
Dissociated Tumor cells (DTCs):
Preparation: Tumour tissue is digested with an enzyme mix and frozen as a viable cell suspension in cryo-preservation medium.
Downstream & intended application: viable alternative to acquiring fresh tumour tissue.
Research tumour in its microenvironment and immune response for IO biomarker discovery.
Biofluids:
Whole blood fresh:
Preparation: can be collected in EDTA, Na Hep., CPT tubes or Streck tubes. Downstream & Downstream application: Fresh blood is used for the preparation of fresh Peripheral mononuclear cells (PBMCs) used in FLOW, cell culturing and Immunoprofiling and fresh plasma preparation.
Frozen whole blood:
Preparation: collected in EDTA or Paxgene DNA/RNA tubes. Downstream & intended application: Research is mainly used for DNA or RNA extraction.
Frozen Bone Marrow Aspirates (Normal & Hematology):
Preparation: Bone marrow (BM) aspirate collected in tubes with EDTA anticoagulant and snap-frozen. Downstream & intended application: RNA/DNA/protein preparation.
Plasma, frozen:
Preparation: Single-spun K2EDTA or Double-spun Streck plasma and Platelet-rich plasma (PRP). Downstream & intended application: Circulating tumour DNA (ctDNA), circulating tumour RNA (ctRNA), circulating microRNAs (miRNAs), exosomes, or other biomolecules that are shed or released by tumour cells into the bloodstream or other body fluids.
Serum, frozen:
Preparation: Blood is collected in SST tubes and processed under standard protocol. Downstream & intended application: Analytical chemistry, serum tumour markers (carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), cytokeratin 19 fragment (CYFRA21-1) and squamous-cell carcinoma-related antigen (SCC-Ag)) evaluation in oncology and serum markers in autoimmune diseases (RA, IBD etc.). Biomarker-related proteomics studies.
Cells:
Peripheral Blood Mononuclear cells (PBMCs):
Preparation: PBMC isolation using Ficoll gradient. Downstream & intended application: FACS, Cell-Based Assays, Proteomics, Genomics.
Bone Marrow Mononuclear Cells (BMMCs):
Preparation: BMMC isolation using Ficoll gradient. Downstream & intended application: FACS, Cell-Based Assays, Proteomics, Genomics.
Dissociated tumour cells:
Preparation: Enzymatic and Mechanical disaggregation of fresh tumour tissue. Downstream & intended application: FACS, Cell-Based Assays, Single Cell Genomics.
Protein Lysates:
Preparation: Tissue lysis using RIPA and Custom buffers. Downstream & intended application: PAGE, Proteomics, Western Blot, Immunoprecipitation.
Nucleic Acids (DNA & RNA)
Preparation: DNA is isolated with a commercial DNA isolation kit (DNeasy Blood and Tissue Kit, Qiagen) and RNA is isolated with a commercial RNA isolation kit (e.g. RNeasy, Qiagen).
Downstream & intended application: Next generation sequencing (NGS), rtPCR Assays, RNA sequencing.
Conclusion:
In conclusion, human specimens are essential resources for biomedical research and drug discovery. They offer a unique opportunity to study human biology and pathology in a more relevant and realistic context than animal models or synthetic systems. Researchers who use human specimens should familiarise themselves with the ethical, legal, and social challenges and also have a complete understanding of the various formats of biospecimen and their subsequent downstream applications, to avoid costly mistakes.
References:
Biospecimen and Biorepository Basics | Patient Corner | BBRB (cancer.gov)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3954467/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7094463/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4077003/
Best Practice Guidance: Specimen and Specimen-Product Storage and Retention (aphl.org)
The New Era of Life Sciences (newsweek.com)
Originally published by Cureline on: https://www.cureline.com/news/blog-a-complete-guide-to-human-specimen-1685353466.html
Caltag Medsystems is the distributor of Cureline products in the UK and Ireland. If you have any questions about these products, please contact us.