Capturing Preanalytical Variability within the Biospecimen Research Database
Kelly B. Engel, Ph.D.Lead Curator for the Biospecimen Research Database
December 7, 2011
The lifecycle of a biospecimen
Patient AcquisitionHandling/Processing Storage Distribution
ScientificAnalysis
Medical/Surgical
Procedures
RestockingUnusedSample
Time 0Post-acquisitionPre-acquisition
AgeGenderDiagnosis
Administered fluids or drugs Warm ischemia time
Cold ischemia timeAnticoagulant
Type and duration of fixation/preservation
Temperature and duration of storage
Transit time and temperature
Analyte extraction methodPlatform sensitivity
Freeze-thaw cycling
Analytical endpoints and the biospecimen lifecycle
• An analytical endpoint reflects a compilation of effects o disease-state o specimen acquisition, handling, preservation, and storage
• Relationships between preanalytical variables existo Fixation temperature and delivery method can shorten or extend
the time required for adequate preservation
• Relationships between preanalytical and analytical variables existo RT-PCR success is influenced by the duration of formalin fixation,
the storage duration of section slides, and the length of the fragment being amplified
How are specimen handling-induced effects identified?
• Identifying effects induced by a specific event is challenging and requires careful experimental design and specimen tracking
• The challenge of identifying and/or minimizing handling-induced effects is further complicated by tumor heterogeneity, and between patient variability, which can overshadow event-induced effects
What does preanalytical variability mean for the patient?
• Expected effects can be mitigated
• Unexpected effects that are physiologically relevant have the potential to influence analytical endpoints and the course of treatment
o The American Society of Clinical Oncology and the College of American Pathologists recently estimated that up to 20% of immunohistochemical analyses worldwide may be inaccurate (false positive or false negative), which has largely been attributed to preanalytical variability
Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, Badve S, Fitzgibbons PL, Francis G, Goldstein NS, Hayes M et al: American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version). Arch Pathol Lab Med 2010, 134(7):e48-72.
What does preanalytical variability mean for the researcher?
• 42% of the tissue specimens that were prospectively collected and banked using an institutionally approved standard operating protocol (SOP) yielded RNA of acceptable quality (RIN ≥ 7)
o 67 pancreatic tumor specimens
oIschemia times were recorded
oSpecimens were coated in OCT and snap-frozen in liquid nitrogen
oStorage was at -80°C within a Cryovial
Rudloff U, Bhanot U, Gerald W, Klimstra DS, Jarnagin WR, Brennan MF, Allen PJ: Biobanking of Human Pancreas Cancer Tissue: Impact of Ex-Vivo Procurement Times on RNA Quality. Ann Surg Oncol 2010
How can we facilitate the procurement of more valuable data ?
• A better system to capture new datao Biospecimen Tracking Systems that capture the lifecycle of each
biospecimen Minimizing between patient variability
o Improving Standard Operating Procedures to minimize preanalytical variability A goal of BRN-driven research
• Mining existing datao Identify steps in the biospecimen lifecycle that can induce
physiologically important and clinically relevant effects
The Biospecimen Research Database
• The Biospecimen Research Database (https://brd.nci.nih.gov)• A joint venture of the OBBR and the NCI’s Center for
Bioinformatics. • A free and publically available database that contains peer-
reviewed literature pertinent to the field of human biospecimen science.
Why not just use PubMed?
• PubMed is the premiere literature database of the National Library of
Medicine
• It had an estimated 2.2 million entries in July of this year.
• It includes published research reports on human to yeast and
everything in between.
• Data relevant to biospecimen science is there…somewhere.
The contents of the BRD
• The BRD contains published peer-reviewed papers and their associated studies. Each study represents an experiment or a group of related experiments with a biospecimen-centric hypothesis.
• Although it began as a MS Access database of 20 papers in 2007 it is
now a fully functioning web database that contains curations of 1031 papers, and that number is growing every day.
• BRD papers were published in more than 205 journals between the years of 1968-2011.
The BRD Curation Process
• A team of three PhD level scientists (currently all biologists) scour the literature, hand-select relevant paperso Targeted literature searcho Cross-referencingo Institutional and individual referrals
• Papers that report results relevant to human biospecimen science are then entered into the BRD.
The BRD Curation Process (con’t)
Experimental Factors
Experimental Factors (con’t)
Hurdles in terminology
• Gene or protein names that have evolved with time o Estrogen receptor: ER, ERα, ERβ, ESR1, ESR2
• Alternate uses of abbreviationso FF: flash-frozen, fresh-fixed, fresh-frozen, formalin-fixed
• Alternative terms for the same step o Clot time, time at room temperature
• Technology platformo PCR, RT-PCR, Hotstart PCR, Quantitative PCR, Quantitative Real-time PCR,
etc
• Differences in definitions o Ischemia time
Hurdles in terminology: Ischemia time
Warm ischemia time
Warm ischemia time
Cold ischemia time
1st incision Blood flow restricted Excision Preservation
37°C 20-25°C or 4°C or 0-4°C37°C
37°C
20-25°C or 4°C or 0-4°C
20-25°C
37°C 20-25°C
Ischemia time
Ischemia time
37°C 20-25°C
Approach
• To address these discrepancieso When definitions conflict between papers we consult
the NCI Best Practices for Biospecimen Resources Glossary and the NCI Dictionary of Cancer Terms
caDSR Preferred Definitions, The College of American Pathologists’ Glossary of Terms
o We also include details in the free text field if any ambiguity surrounds a term
the temperature of cold ischemia time
o Spell out all abbreviations at least once in a written field for each paper, so the user has an on-hand reference
o We make an effort to use caDSR terms when applicable.
o Technology platforms are organized by analyte (of which there are 14)
Developing Terminology
• And we recognize that our effort is a dynamic one…as more topics are covered we find we need to expand or condense EF terminology accordingly o We recently expanded the Experimental Factor value “snap
frozen” o Snap freezing in now represented in greater detail by the new
Experimental Factor “Freezing method/rate”
• We turn to review and status papers on biobanking for guidance when adding new experimental factors
Betsou F, Lehmann S, Ashton G, Barnes M, Benson EE, Coppola D, DeSouza Y, Eliason J, Glazer B, Guadagni F et al: Standard preanalytical coding for biospecimens: defining the sample PREanalytical code. Cancer Epidemiol Biomarkers Prev 2010, 19(4):1004-1011.
• Wiki format commenting will soon be available to the public
• The BRD will expand to accommodate SOPs
The Future for the BRD
Thank You!
• Past and Present Members of the BRD Curation Teamo Sarah Greytak, PhDo Paige Bass, PhDo Andrea Kelly, PhD
• Past and Present Members of the BRD Development Teamo Ian Foreo Andrew Breychako Brent Landero Ye Wuo Laxmi Venkatasatyao Amit Srivastava
• OBBRo Carolyn Compton, MD, PhDo Jim Vaught, PhDo Helen Moore, PhD
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