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2
Determinants of the
Research Value of Biospecimens
Pathology is the study of the structural and functional changes brought
about by disease or injury. Pathologists analyze biospecimens for such
changes and thereby attempt to discern their causes. The present com
mittee's statement of task poses a number of questions regarding the future
use of the Joint Pathology Center (JPC) repository's biospecimens collection
in clinical care, education, and research activities. This chapter lays the
groundwork for addressing those questions by providing information on
the means of preserving biospecimens, on methods for analyzing and as-
sessing their research value, and on how the details of preservation, storage,
documentation, and the applications for which they are intended may affect
prospects for their use. It focuses on scientific and technical considerations;
legal and ethical issues are addressed in Chapter 3.
COLLECTION AND PRESERVATION OF BIOSPECIMENS
Three types of biologic material may be collected during pathologic
investigations: tissues and cells removed during surgery or obtained spe-
cifically for diagnosis via biopsy; cytologic material, including that from
fine-needle aspiration biopsy, brushings, or swabs; and whole blood. The
main objective in diagnostic pathology and pathology laboratories is to
provide accurate diagnosis of a disease and additional pathologic informa-
tion needed to define a prognosis and determine appropriate therapeutic
strategies. Clinical data--including information about the patient and her
or his medical history, physical examination, and diagnostic imaging, such
as X-rays, CT scans, and the like--are also collected to inform evaluations.
37
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38 FUTURE USES OF THE DOD JPC BIOREPOSITORY
This section briefly addresses how specimens are handled after collec-
tion, focusing on the types of samples found in the JPC repository. Fig-
ure 2-1 shows the relationship between the various materials collected and
their forms of preservation for pathologic analysis.
The pathology workflow for tissues comprises collection or excision
from the patient; visual examination of the macroscopic specimen (called
a gross specimen); initial stabilization; transfer to a laboratory; selection
of material from the gross specimen for further analysis; fixation; further
visual examination; histopathologic, biochemical, or molecular analyses;
and storage.
After collection or excision and any initial diagnostic evaluation, speci-
mens are typically either frozen or chemically stabilized for transport. The
essential processing steps for laboratory preparation of samples that are not
maintained in a frozen state are summarized below.
Fixation. Fixative solutions stabilize tissue structure and biochemical
constituents by coagulating (cross-linking, denaturing, and precipitating)
proteins and thereby prevent cellular hydrolytic enzymes, which are re-
leased when cells die, from degrading tissue components and rendering
tissues inadequate for microscopy. Fixation also immobilizes fats and car-
bohydrates, reduces or eliminates enzymatic and immunologic reactivity,
Frozen
Tissue
Tissues/Cells Tissue
- Tumor (Biopsy or
- Non-malignant Resection) Gross Histology
Gross
Specimen: Slides
Specimen:
Fixed
Fresh
FFPE Tissue Tissue
Microarrays Digital
Blocks
Images
Cells
Body Fluids FFPE Cell
(Cytology)
Blocks
Patient Cytology
Slides
Cell
Personal
Smears
Information
Clinical Data Diagnosis
History and
Physical Exam
Rx-CT
Images
FIGURE 2-1Relationship between pathologic materials collected and form in
which they are preserved for analysis.
Figure 2-1
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DETERMINANTS OF THE RESEARCH VALUE OF BIOSPECIMENS 39
and kills microorganisms that are present in tissues. The fixative routinely
used in pathology is 10 percent neutral buffered formalin, a buffered aque-
ous solution of formaldehyde. Fixation yields "wet tissue" that is either
stored in an air-tight container or processed further as delineated below.
Embedding. Specimen water (about 70 percent of tissue mass) is re-
placed with paraffin wax, and the specimen is surrounded by paraffin in
a mold to provide support during sectioning and to aid in preservation.
Formalin-fixed paraffin-embedded (FFPE) tissue is one of the predominant
forms in which pathologic specimens are stored.
Sectioning. Sections are cut on a microtome, which has a blade similar
to a single-edge razor blade, that is advanced through a block of paraffin-
embedded tissue. The shavings--about 5 µ m thick, about twice the thick-
ness of a human hair--are placed in water, and the floating shavings are
picked up on 1 × 3-in. glass slides. A given tissue block can be recut many
times, although specific slices may differ in the amounts and types of tissues
(for example, primary tumor vs. normal tissue) present.
Staining. Tissue components can be distinguished with selective absorp-
tion of dyes to facilitate viewing under a microscope. The stain routinely
used in histology is hematoxylin and eosin. There are special methods for
highlighting components (such as microorganisms) that do not stain well
with the customary preparations. Pathologists commonly use tissue sections
prepared in this manner in their analyses.
Researchers use tissue microarrays (TMAs) constructed from diag nostic
blocks of FFPE tissue to permit simultaneous evaluation of expression
of specific pathologic features--proteins by immunohistochemistry, for
example--in hundreds of individual tissue samples from different patients
on a single slide (Rimm et al., 2011; Voduc et al., 2008). TMAs are as-
sembled by using a needle to core an FFPE tissue block and extract a 0.5- to
2.0-mm piece that is placed into a predrilled master paraffin block that may
contain up to 400 cores. Sections from the resulting block may be cut with
a microtome, placed on a slide, stained, and analyzed. In cancer research,
TMAs are used to analyze the frequency of a molecular alteration in dif-
ferent tumor subtypes, as detected by immunohistochemical and molecular
techniques, to enable evaluation of potential diagnostic and prognostic
markers by correlating staining patterns with light microscopy and clini-
cal information, which may also contain outcome measures (Camp et al.,
2008; Kapur, 2011). Advantages of using TMAs include minimal tissue use,
lower reagent costs, faster results, and the ability to define a set of cases that
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40 FUTURE USES OF THE DOD JPC BIOREPOSITORY
have related diseases and clinical annotations and to use many such cases
in direct parallel analysis on the same slide.
Advances in technology also permit high-fidelity capture of the infor-
mation contained on slides and in diagnostic images in digital form. Digiti-
zation removes the opportunity for further biologic analysis but facilitates
storage, sharing, and, if desired, deidentification of specimens.
USES OF BIOSPECIMENS
Educational, clinical consultation, and research uses impose different
demands on the physical state of a specimen and the documentation that
accompanies it. This section presents a brief summary of the considerations
that influence the assessment of fitness for those uses.
Educational Uses
Educational uses have the lowest bar for molecular quality and physi-
cal integrity of material and therefore can, in principle, permit the greatest
variety of samples. However, it is uncommon to keep fresh, frozen, or even
preserved samples in a manner that would allow ready distribution beyond
the location at which they were generated. Indeed, unfixed tissue poses a
risk of infection and should be handled only by persons who are trained in
handling potentially infectious agents. The risk associated with fixed tissues
is much lower, but such agents as prions are not inactivated by normal fixa-
tion methods. Fixed specimens can be encased in plastic to facilitate han-
dling, eliminate the risk of contagion, and enhance the educational value,
but this is not commonly done. For those reasons, in most contexts, gross
specimens are likely to have their most effective and widespread educational
use in image form.
Microscope slides are extremely useful for education, are easy to ship
and return, and carry a very low risk of contagion. They have a long but
finite shelf-life. However, there is little need for samples of extremely rare
entities, except in advanced residency and fellowship training, and slides
of common entities are abundantly available. The current trend is to-
ward archiving of teaching slides digitally and viewing them with virtual
microscopy. That avoids the problems of image or slide degradation and
of slide distribution. Digital microscopy allows a teacher and a student to
collaborate as though they are at the same microscope even though they
may be separated by large distances.
Accompanying clinical information often improves the pedagogic value
of specimens, but it is not always required.
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DETERMINANTS OF THE RESEARCH VALUE OF BIOSPECIMENS 41
Clinical Consultation Uses
Clinical consultation was a major function of the Armed Forces Insti-
tute of Pathology, and the JPC continues to serve this function for the Mili-
tary Health System, the Department of Defense, and other federal agencies.
In the past, it was common to limit the materials for consultation to stained
microscopic sections. Advances in diagnostic procedures now require the
ability to extend the histopathologic description with immunohistochemical
and molecular probes. That may require that unstained slides be included
in the materials for consultation and, less often, that the tissue block or
fresh-frozen tissue be available. Consultation also requires that detailed
clinical information be provided to the consulting physician. Large medi-
cal centers can often carry out the more advanced procedures on their own
and need only send appropriately stained slides to the consultant, whereas
smaller centers might require that the consultant carry out the procedures.
In general, fresher, unfixed tissues give better results in assays than do
samples that have had longer intervals at room temperature or long periods
in fixatives. When consultation is limited to examination of slides, digital
(or "virtual") microscopy via scanning of glass slides (Pantanowitz et al.,
2011) allows rapid, interactive consultation without the need to transport
and retrieve slides.
Research Uses
Research comprises a broad array of activities and a correspondingly
broad array of requirements for pathologic specimens. Case reports and
historical studies might need slides alone and be limited only by the condi-
tion of the slides, but more extensive studies of the mechanism of disease
require the freshest materials possible with cryopreservation, snap freezing
in liquid nitrogen, or relatively brief times in fixatives to obtain the best
results. That means not that older materials or materials that have not been
obtained under those conditions are without value--DNA sequences have
been obtained even from Neanderthal bones--but simply that they make
studies technically more difficult and limited in scope and preclude some
studies as the signal-to-noise ratio tilts toward the noise.
In most cases, the more complete the accompanying clinical informa-
tion is, the more valuable the sample. Nonetheless, many otherwise undocu-
mented samples that have been well characterized histopathologically can
be of value in genetic, microRNA (miRNA), and other functional studies.
Studying diseases that have a high incidence, such as breast cancer, need not
depend on suboptimal tissues or special collections inasmuch as specimens
are readily available, whereas studying rare diseases may require greater
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42 FUTURE USES OF THE DOD JPC BIOREPOSITORY
compromises with respect to sample quality and rely critically on special
collections and repositories.
Limitations on the use of pathologic samples in research are addressed
in greater detail later in this chapter.
TECHNOLOGIES USED TO
MANAGE SPECIMEN ACQUISITION AND MANAGEMENT
Over the last two decades, technology development has created both
challenges to and opportunities for human biospecimen resources. The
sensitivity, specificity, multiplexing capability, and speed of operation of
technologies for molecular analysis of all classes of biomolecules in h uman
specimens have undergone transformative improvements, and further re-
finements are developed at ever-increasing rates. However, this greatly
augmented analytic power has raised the bar for the quality of the bio-
specimens that serve as the source of analytes for the technology platforms,
which are increasingly used in research and clinical care. Biospecimens
acquired in the setting of standard clinical practice that formerly served as
adequate sources of research material despite the varied, undocumented,
and uncontrolled sources of preanalytic variation1 to which they were
exposed are no longer adequate, let alone optimal, for new molecular-
assessment platforms. Technology development specifically directed to the
challenges related to biorepository operation in this environment has been
essential in addressing the gap between the demand for and supply of high-
quality human specimens for molecular research.
Biorepositories, such as the JPC, that comprise clinically derived sam-
ples collected in diverse settings and referred for pathologic consultation on
disease, typically face greater complexities in ensuring that their collections
meet quality standards for sensitive analytic platforms than do biobanks
that were established for the express purpose of collecting specimens for
research. Clinical consultation biorepositories have difficulty in control-
ling, recording, or assessing the sources of preanalytic variation that may
compromise the molecular quality of their collections. Thus, technologic
solutions for controlling processing and environmental variation and for
assessing the molecular quality of processed or stored specimens have been
essential for the continued evolution and usefulness of clinical consultation
biorepositories for biomedical research.
1Preanalytic variation refers to any of the many biospecimen acquisition, handling, or pro-
cessing procedures and environmental characteristics (such as temperature and humidity) to
which a specimen may be exposed before analysis takes place. Preanalytic variation may alter
the molecular quality or composition of a biospecimen and render it unsuitable for a specific
type of analysis.
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DETERMINANTS OF THE RESEARCH VALUE OF BIOSPECIMENS 43
Technologic solutions for the problems in specimen acquisition, pres-
ervation, and management include the following:
· Shipping technologies that maintain specific environments dur-
ing transport of samples and thus help to maintain the molecular
quality of the samples for analysis at remote sites.
· Specimen fixation and other stabilization technologies that preserve
the quality of labile biomolecules, allow optimal molecular pres-
ervation and histopathologic quality, or allow in situ stabilization
of the specimen to preclude preanalytic variation incurred during
specimen acquisition (for example, through surgical resection and
pathologic handling).
· Information technology solutions that allow annotation of speci-
men collections with clinical data about the individual from whom
the specimen is derived; consent to specimen collection, transfer,
storage, and use; authorization of use of protected health informa-
tion; pathologic data on the specimen (such as gross description
and accompanying diagnosis); collection, processing, transporta-
tion, and storage data; quality-control data; specimen analysis
data; radiologic imaging data; inventory tracking; overall (system-
wide) quality-management data; and molecular analysis data.
· Specimen or molecular storage technologies, such as ambient-
temperature ("dry-state") storage.
· Molecular quality-assessment technologies for RNA, DNA, and
proteins.
· Digital imaging and image analysis technologies for precise
structure-based data associated with each specimen.
Those categories of technologies are not all equally developed, but all
continue to improve rapidly and decrease in cost. Nevertheless, the rate of
obsolescence of many of the technologies and the increasing knowledge
of the effects of specific preanalytic factors on molecular analysis data
require continual reassessment of the adequacy and functionality of tech-
nologies that are in place in light of the repository's mission.
TECHNOLOGIES USED TO ANALYZE SPECIMENS
Researchers have several tools at their disposal for deriving clinical
and research information from specimens. This section--which is based on
review articles by West (2010) and Beck and colleagues (2010)--identifies
some of the technologies used in analysis and discusses how preservation
technique influences the ability to perform various types of analysis.
Table 2-1 summarizes the results of some recent research on the influ-
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44 FUTURE USES OF THE DOD JPC BIOREPOSITORY
TABLE 2-1 Recent Research on the Influence of Preservation Method on
Specimen Analysis Outcomes
Collection or Preservation
Methods Tissue Types Attributes
Fresh-frozen (Snap-frozen) Pancreatic cancer RNA integrity was determined
specimens with microcapillary
electrophoresis, using RNA
integrity number (RIN) algorithm
and results of laser-capture
microdissection (LCM).
Various ex-vivo procurement
times (up to 10 min, 1130 min,
3160 min, over 1 h); banked
over three periods (20012004,
20042006, 20062008)
Fresh-frozen Invasive breast Manual method: subjective
cancer tissues evaluation of electropherogram;
ratio method: ratio between 28S
and 18S peaks; RIN
Room temperature, iced, saline Normal tonsil Structural RNA integrity via
solution, RNA-stabilizing Normal colon microchip electrophoresis
buffer, snap-frozen (after 0.5,
1, 3, 6, 16 h)
Snap-frozen (unfixed and Tonsil Microchip gel electrophoresis and
immersed in RNA-stabilizing gene expression level via PCR
buffer), thawed for 0, 5, and
45 min, 1, 3, 6, and 16 h
Snap-frozen, formalin-fixed RNA quality
paraffin-embedded (FFPE)
FFPE Diffuse large B-cell Gene expression
lymphoma
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DETERMINANTS OF THE RESEARCH VALUE OF BIOSPECIMENS 45
Effects Source
·42 percent of human pancreas cancer specimens banked under a Rudloff et al.,
dedicated protocol yielded RNA with a RIN of 7 2010
·Brief warm ex-vivo ischemia times did not adversely affect RNA
quality (percentage of tissue with total RNA with RIN of 7 for
10 min, 42 percent; 1130 min, 58 percent; 3160 min, 33 percent;
>60 min, 42 percent)
·Long-term storage of banked pancreas cancer biospecimens did not
adversely affect RNA quality (total RNA with RIN of 7 banked
in 20012004, 44 percent; 20042006, 38 percent; 20062008,
50 percent); RNA retrieved from pancreatic cancer samples with RIN
of C7 subject to LCM yielded RNA suitable for further downstream
applications
·Fresh-frozen pancreas tissue banked according to a standardized
research protocol yields high-quality RNA in about 50 percent of
specimens and can be used for enrichment with LCM; quality of
tissues in the biobank was not adversely affected by slight variations
in warm-ischemia times or different storage periods
·Comparison between RNA quality (RIN) and gene expression analysis Strand et al.,
shows dense clustering of high-quality samples but weak clustering of 2007
low-quality samples
·Manual and RIN methods are superior to ratio method
·RNA stable in both tissues under all conditions for up to 616 h Micke et al.,
·Expression levels essentially stable when samples kept on ice 2006
·Marked regulation of single genes observed during room-temperature
storage in normal saline and RNA-stabilizing buffer
·RNA from 54 of 47 samples had proper ribosomal peaks
·Nonfixed specimens may be transported on ice for hours with minimal
influence
·Minimal RNA degradation after 30 min Botling et al.,
·Relevant changes in some gene-expression levels at 45 min 2009
·Repetitive thawing cycles had similar effects on RNA integrity
·Incubation in RNA-stabilizing buffer prevents RNA degradation
·Introduced heating into extraction protocol to improve quality; Li et al., 2007
incubation at 70°C for 20 min was applied to disrupt cross-links in
FFPE without compromising RNA integrity
·TaqMan detection influenced by master mix, amplicon size, and use of
preamplification step
·Comparable results in frozen and FFPE tissue
·Provided PCR protocol for gene-expression analysis Votavovį et
·62 of 65 samples "successfully" analyzed al., 2009
continued
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46 FUTURE USES OF THE DOD JPC BIOREPOSITORY
TABLE 2-1 Continued
Collection or Preservation
Methods Tissue Types Attributes
FFPE Parathyroid Proteome quality
Fresh-frozen, FFPE Colon adenoma Proteome quality
Liquid chromatography
Frozen, FFPE Frozen/optimal Proteome quality
cutting temperature Liquid chromatography
(OCT)-embedded
livers (rats)
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DETERMINANTS OF THE RESEARCH VALUE OF BIOSPECIMENS 47
Effects Source
·163 unique proteins identified via mass spectrometry Donadio et al.,
·Similar results via sodium dodecyl sulfate (SDS)-out in gel-free method 2011
·Antigenicity not always preserved in Western blot
·Despite some limitations due to extensive formalin-induced covalent
cross-linking, results suggest that FFPE extracts may be an alternative
source for large-cohort samples when frozen samples are unavailable
·"The major difference between frozen and FFPE proteomes was a Sprung et al.,
decrease in the proportions of lysine C-terminal to arginine C-terminal 2009
peptides observed, but these differences had little effect on the proteins
identified."
·"Analysis of archival colon adenoma FFPE specimens indicated
equivalent numbers of MS/MS spectral counts and protein group
identifications from specimens stored for 1, 3, 5, and 10 years."
·"Analysis of the combined frozen and FFPE data showed a 92 percent
overlap in the protein groups identified. Comparison of gene
ontology categories of identified proteins revealed no bias in protein
identification based on subcellular localization."
·"Archival samples displayed a modest increase in methionine
oxidation, from approximately 17 percent after one year of storage to
approximately 25 percent after 10 years."
·"These data demonstrate the equivalence of proteome inventories
obtained from FFPE and frozen tissue specimens and provide support
for retrospective proteomic analysis of FFPE tissues for biomarker
discovery."
·"Comparable molecular mass representation was found in extracts Scicchitano et
from FFPE and OCT-frozen tissue sections, whereas protein yields al., 2009
were slightly less for the FFPE sample."
·"The numbers of shared proteins identified indicated that robust
proteomic representation from FFPE tissue and LCM [laser capture
microdissection] did not negatively affect the number of identified
proteins from either OCT-frozen or FFPE samples."
·"Subcellular representation in FFPE samples was similar to OCT-
frozen, with predominantly cytoplasmic proteins identified.
Biologically relevant protein changes were detected in atorvastatin-
treated FFPE liver samples, and selected atorvastatin-related proteins
identified by MS were confirmed by Western blot analysis. These
findings demonstrate that formalin fixation, paraffin processing,
and LCM do not negatively impact protein quality and quantity as
determined by MS and that FFPE samples are amenable to global
proteomic analysis."
continued
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54 FUTURE USES OF THE DOD JPC BIOREPOSITORY
cancer-related genes is being applied to DNA extracted from FFPE tumor-
tissue specimens with PCR-based approaches. Recent advances in high-
throughput next-generation sequencing methods permit the analysis of
mutations in 200400 genes from small amounts of FFPE-extracted DNA.
DNA, in general, is much more stable that RNA. However, all nucleic
acids are susceptible to depurination in highly acidic environments, and the
preservation of DNA in archival tissues depends heavily on the quality of
the fixative used. Depurination can lead to strand cleavage, which would
create problems for many of the modern molecular techniques. Thus, if
unbuffered formalin was used originally to fix the archival material--as
might be the case for older specimens in the JPC repository--it could lead
to substantial degradation of the DNA (Akalu and Reichardt, 1999; Bonin
et al., 2010). There are few published data on the quality of reads generated
from high-throughput sequencing of archival DNA.
Elemental and Chemical Studies
Tissues are routinely analyzed for trace minerals in clinical pathology
laboratories for diagnostic purposes. Trace minerals are preserved and can
easily be localized and measured in FFPE tissues. For example, hepatic iron
concentrations are essentially the same whether assessed in fresh tissues
or in paraffin-embedded tissues (Torbenson, 2011). However, the altera-
tions in tissue quality, such as a change in weight due to the replacement of
water with less-dense paraffin, can necessitate correction factors (Bischoff
et al., 2008). The routine fixation process, with numerous alcohol washes
to dehydrate the tissue, results in the removal of many lipophilic small
molecules. Elemental concentrations were studied in the past largely with
atomic absorption spectroscopy or energy-dispersive X-ray analysis, but
new methods are available now (Becker and Jakubowski, 2009; Harrington
et al., 2010; Mizuhira et al., 2000).
LIMITATIONS IN THE USE OF PATHOLOGIC
SAMPLES IN RESEARCH
Several factors influence whether pathologic samples obtained for clini-
cal consultation purposes will be fit for use in research. This section iden-
tifies the major issues that limit the research use of such specimens and
addresses how the details of collection, preservation, storage, and docu-
mentation can affect fitness for research use.
The specimen preservationstabilization process might be inappropriate
for or incompatible with the technologic or scientific demands of the re-
search analysis.
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DETERMINANTS OF THE RESEARCH VALUE OF BIOSPECIMENS 55
The suitability of any specific specimen for analysis depends on the
analyte that is to be assessed and the technique to be used for the a
nalysis--
that is, whether it is "fit for purpose." As previously noted, the most com-
mon stabilization method used in pathologic practice is 10 percent neutral
buffered-formalin fixation followed by dehydration with graded alcohols,
xylene exchange, impregnation by liquid paraffin, and permanent pres-
ervation in paraffin. While the paraffin is maintained in liquid state, the
specimen is spatially oriented in a small, thin rectangular container and
then rapidly cooled until the paraffin is solidified. Adequacy of fixation of
a pathologic specimen is determined largely according to the dimensions
of the tissue (when it is submerged in formalin in the gross state and when
it is sampled for single aliquots to be individually processed into tissue
blocks, formalin penetrates about 1 cm into tissue) and the total time in
fixative. In general, inadequate preservation due to underfixation is far
more deleterious to samples than is prolonged fixation. Both fixation in a
molecular cross-linking fixative, such as formalin, and paraffin embedding
with exposure to high temperatures may compromise the molecular quality
of samples.
Other related issues concern variations in concentration, pH, or buffer-
ing of formalin or the use of fixatives other than 10 percent neutral buffered
formalin. The optimal formaldehyde concentration in a fixative and the
optimal pH of the solution may depend on the biomolecule of interest, espe-
cially in the case of immunohistochemical analysis. Practice is not standard-
ized, and these measures are not always recorded in pathology reports and
thus represent important unknowns in research. In the past, the use of such
fixatives as Bouins solution (which contained picric and glacial acetic acid
with formaldehyde) or special hematopathologic fixatives that contained
metals, such as B5 zinc or B5 mercuric chloride, was common, and the
fixative type was sometimes not recorded as a part of the pathologic record.
Many of those fixatives are incompatible with modern molecular-analysis
platforms. When specimens for research are accrued from multiple institu-
tions that use different stabilization and preservation practices, data from
molecular analyses may be neither reliable nor comparable or institution-
specific batch effects may be seen.
Important variation in pathologic processing also occurs before sta-
bilization. For example, the length of time that elapses between specimen
removal from a patient and specimen fixation ("time to fixation" or "cold
ischemia time") and the conditions to which the specimen is exposed during
this period, such as desiccation or various room temperatures (as opposed
to refrigeration), may seriously alter both the molecular quality and the
molecular content of the specimen and thus create artifacts that may be
misinterpreted as reflecting disease biology. In some cases, the variations
may even compromise histologic quality. Highly labile biomolecules may
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56 FUTURE USES OF THE DOD JPC BIOREPOSITORY
be lost altogether as time to fixation increases. Those factors are neither
controlled nor recorded in common clinical practice. Rapid stabilization of
tissue with cryopreservation of various kinds (for example, immersion in
liquid nitrogen, freezing in the vapor phase of liquid nitrogen before im-
mersion, immersion in dry iceisopentane slush, embedding in a freezing
medium based on polyethylene glycolsucrose,3 and placement in a -80°C
freezer or a cryostat) may be used in exceptional circumstances but are not
the standard of care for all tissues. Thus, if frozen (unfixed) tissue is needed
for research, this requirement often cannot be met pathologic tissue. If
viable cells or tissues are required for research, pathologic samples cannot
fulfill this requirement unless viable aliquots are preserved appropriately
at the moment of acquisition of the specimen and not used as a part of a
retrospective pathologic sample set.
Sample aliquot content might be unknown to the investigator, and too
little of the lesion of research interest might remain in the residual tissue
after clinical workup.
Each paraffin tissue block processed for a case is thinly sliced (at 5-µm
intervals) with a microtome to produce sections that are placed on glass
microscope slides and stained for histopathologic analysis under a light
microscope. As previously noted, the standard histopathologic stain is
hematoxylin and eosin, but a wide variety of stains may be used on adjacent
tissue sections to reveal special features as a part of the pathologic workup.
Unstained sections also are often cut and set aside for or used for immuno-
histochemical analyses of various types required for pathologic diagnosis
and characterization. In many cases, small amounts of tissue may remain in
a block after complete workup, and this can result in inadequate residual
amounts for research. Inadequacy of residual tissue for research is particu-
larly problematic if the original specimen from the patient is very small or
the lesion of interest is small, focal, or both and thus not of sufficient qual-
ity (or, indeed, at present all) in the residual tissue in a block. The lack of
quality control of pathologic tissue blocks made available for research, to
verify the nature and content of the remaining tissue, can detract from their
usefulness for research. It can also skew data from investigational studies if
the residual blocks are assumed to be representative of the overall diagnosis
but are depleted of diagnostic tissue.
The original pathologic diagnosis might be unconfirmed or incorrect.
Unconfirmed or incorrect diagnosis of pathologic material is u
ncommon
(Lind et al., 1995; Ramsey and Gallagher, 1992; Renshaw et al., 2003a,b;
Safrin and Bark, 1993; Wakely et al., 1998), but in cases of rare diseases or
3Also known as optimal cutting temperature (OCT) compound.
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DETERMINANTS OF THE RESEARCH VALUE OF BIOSPECIMENS 57
diseases that have a documented, notoriously high degree of interobserver
variation in diagnostic interpretation, it can be an important issue for both
patient management and research. In such cases, it is considered standard
of care to seek a second, specialty pathologic opinion, such as the consulta-
tive opinions produced for specimens submitted to the JPC repository and
its predecessors. Verification of diagnosis by obtaining a new pathologic
analysis in every case used for research is recommended.
However, it may also be problematic for research if the diagnosis that
accompanies the case is outdated and no longer appropriately classifies
the disease. Outdated diagnostic terminology (correct diagnosis but arcane
language) or outdated diagnostic criteria for identification (classification
according to a schema that is no longer in use) may cause considerable
difficulty in mapping a historical case to a current diagnostic category
accurately. Over the span of decades that the JPC repository has existed,
pathologic classification of disease has evolved substantially. In some dis-
ease categories, such a hematopathologic malignancies, entire disease clas-
sifications have changed repeatedly because knowledge of pathogenesis has
grown. Modern diagnosis of lymphoma and leukemia may require delinea-
tion of specific molecular features that were never tested for in older cases.
Depending on the specific preanalytic variation associated with a historical
case, it may not even be possible to test accurately for the molecular fea-
tures required for diagnostic classification.
The more common problem in all disease categories is the lack of
standardization in diagnostic terminology that was widespread in pathol-
ogy for many years. That has been exemplified both by the use of a given
diagnostic term for different disease entities and by the use of multiple diag-
nostic terms for a given disease entity (Cooper, 2006). A researcher using a
historical case may not have the requisite expertise to interpret the existing
diagnostic terminology accurately and map it to the current diagnostic ter-
minology standard correctly. Failure to reclassify cases correctly according
to current diagnostic standards and current diagnostic terminology for any
of the above reasons may skew research data.
Preanalytic variations related to preoperative or intraoperative factors
may create molecular artifacts.
Many drugs used in preoerative and intraoperative periods and such
surgical events as devascularization or arterial ligation with cessation of
blood supply during resection (called warm ischemia time) may cause
changes in the molecular profiles of resected tumor and normal tissues and
preclude use of specimens for research. Shifts in molecular profiles due to
iatrogenic interventions may not be recognized as artifacts and may be mis-
takenly interpreted as disease signatures. Some drugs used in perioperative
and interoperative periods have powerful molecular effects and are, in fact,
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58 FUTURE USES OF THE DOD JPC BIOREPOSITORY
used specifically for these effects (Juhl, 2010), although little research has
been published on this topic. In addition, the type of surgical procedure,
the individual surgeon performing the procedure, and the use of robotic
instruments are variables that affect the duration of the resection and the
resulting tissue ischemia in the resection specimen. Although some informa-
tion related to the number and types of drugs given before and during an
operation may be available in clinical records, such as the anesthesia report,
it is uncommon to have these data available to a biorepository. The com-
mittee does not know whether or how many specimens in the JPC reposi-
tory are annotated with data related to perioperative variables that may be
pertinent to molecular research on the molecular profiles of tissue samples.
Perhaps the most common preanalytic variable with an important effect
on results of analytic tests, such as immunohistochemical staining, is the
time that elapses after surgical removal of tissue until it is stabilized with
fixation or freezing (cold ischemia time). The process of structural and bio-
chemical tissue degradation occurring during this time is termed autolysis.
The amount of time can be substantial, ranging from minutes to hours,
and is rarely recorded in the pathology report. It may cause gain or loss of
signal on molecular analysis, depending on the molecular entity in question.
An analysis done without knowledge of the duration of the time to fixation
and without an intrinsic control within the same specimen that can serve
as a reference (for example, surrounding normal tissue that is known to
express or not to express the molecule constitutively) may be misleading or
even completely incorrect (Spruessel et al., 2004).
Storage conditions or duration may compromise specimen quality.
Oxidation occurs in both blocks and cut sections, but it is much greater
on cut surfaces exposed to room air. Thus, tissue blocks stored at tem-
peratures below the melting point of paraffin yield cut sections that show
little deterioration of immunohistochemical signal compared with freshly
embedded controls for as little as 2 years or as long as 25 years (Engel and
Moore, 2011). RNAases are active even in FFPE tissues. For many antigens,
immunoreactivity has been shown to deteriorate more rapidly if specimens
are stored as cut sections rather than whole blocks; both the time line and
the magnitude of the effect are antigen-dependent.
On a crude level, paraffin blocks that have been stored under condi-
tions that do not include climate control may lead to melting of paraffin
blocks in warm weather or destruction from other causes. Gross specimens,
albeit formalin-fixed, may undergo dehydration, fungal contamination, and
putrefying deterioration when stored under conditions that expose them to
environmental extremes. All those issues have affected specimens held by
the JPC at some point over the course of the repository's history (Asterand,
2008).
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DETERMINANTS OF THE RESEARCH VALUE OF BIOSPECIMENS 59
Case-matched normal control samples from a tumor patient might be
unavailable.
A normal specimen is needed as a source of a reference genome in
tumor patients in for correct identification of tumor-specific mutations.
Depending on the disease, the diagnostic or therapeutic procedure that
produced the specimen, or the tissue remaining in a case after diagnostic
analysis, it may not be possible to meet this requirement.
The clinical data associated with specimens may be inadequate, inaccu-
rate, or nonstandardized.
Even if biospecimens are of sufficient quantity and quality for a particu-
lar molecular analysis, their value for translational research may be severely
limited by the amount, type, and quality of clinical data that are available
for an individual case and by the consistency of the data on the many cases
that may be required for a study. Clinical data provide the essential func-
tional or biologic behavioral correlations that define the medical relevance
of the molecular data. The lack of relevant clinical data elements limits the
value of the molecular analysis data for prediction and the conclusions that
can be drawn.
The type and amount of clinical data provided by the physician request-
ing consultation was not prescribed in a standardized fashion by the JPC
repository. Furthermore, it did not require that any clinical data submitted
adhere to a standardized format.4 Thus, clinical data associated with cases
varies widely in quality, quantity, and consistency, which may create impor-
tant limitations in the utility of the JPC biospecimens for research studies.
In some cases, it may be possible to acquire missing clinical data from
the medical record in the institution in which the referred case originated.
For some institutions, such as those within the Department of Veterans
Affairs health system or the Department of Defense and many private insti-
tutions, the acquisition of additional data elements for a case may be techni-
cally facilitated by using an electronic medical record (EMR). Nevertheless,
additional technical hurdles--such as the difficulty in combining data from
an EMR with the JPC's information technology and the unreliability or
unavailability of identifiers that would allow data from disparate sources
to be combined--may limit the ease with which the data can be transferred
or may preclude electronic transfer altogether. Acquiring or transferring
additional data by nonelectronic means is labor-intensive, is expensive, and
adds a risk of introducing errors. However, given the number of hospital
mergers and closings in recent years, it may not be easy to trace a patient
4The Contributor's Consultation Request Form used in recent years does require that some
standardized information be submitted. The most recent version of the form is reproduced
in Appendix B.
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60 FUTURE USES OF THE DOD JPC BIOREPOSITORY
record through the original submitting institution. The ability to acquire
additional data may also be constrained by ethical and legal restrictions,
such as privacy laws, as noted briefly below.
Consent from the source might be inadequate for use, permission to re-
contact might be lacking, or recontact might be prohibitively expensive.
Although the acquisition of additional clinical data for a case may be
technically possible, there may be circumstances where it would be prohib-
ited or restricted by ethical or legal issues related to recontact or privacy
laws, including the federal Health Insurance Portability and Accountability
Act of 1996 (PL 104-191, 110 Stat. 1936, and accompanying regulations).
In other situations--especially where a number of years have passed since
the data or specimen were collected--it may require extensive research to
locate the source individual. Those issues may render specific cases unusable
for some types of research. Chapter 3 addresses consent and other ethical,
legal, and regulatory aspects of use of the JPC materials for consultative,
educational, and research purposes.
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