Special Article

Preanalytics and Precision Pathology

Pathology Practices to Ensure Molecular Integrity of Cancer Patient Biospecimens
for Precision Medicine
Carolyn C. Compton, MD, PhD; James A. Robb, MD; Matthew W. Anderson, MD, PhD; Anna B. Berry, MD; George G. Birdsong, MD;
Kenneth J. Bloom, MD; Philip A. Branton, MD; Jessica W. Crothers, MD; Allison M. Cushman-Vokoun, MD, PhD; David G. Hicks, MD;
Joseph D. Khoury, MD; Jordan Laser, MD; Carrie B. Marshall, MD; Michael J. Misialek, MD; Kristen E. Natale, DO;
Jan Anthony Nowak, MD, PhD; Damon Olson, MD; John D. Pfeifer, MD, PhD; Andrew Schade, MD; Gail H. Vance, MD;
Eric E. Walk, MD; Sophia Louise Yohe, MD

 Biospecimens acquired during routine medical practice
are the primary sources of molecular information about
patients and their diseases that underlies precision medicine
and translational research. In cancer care, molecular
analysis of biospecimens is especially common because it
Accepted for publication June 4, 2019.
Published online July 22, 2019.
From School of Life Sciences, Arizona State University and Mayo
Clinic School of Medicine, Scottsdale (Dr Compton); Consulting
Pathologist, Boca Raton, Florida (Dr Robb); Versiti Diagnostic Labora-
tories, Milwaukee, Wisconsin (Dr Anderson); Molecular Pathology and
Genomics, Swedish Cancer Institute, Seattle, Washington (Dr Berry);
Anatomic Pathology, Grady Health System, Atlanta, Georgia (Dr
Birdsong); Advanced Genomic Services, Ambry Genetics, Aliso Viejo,
California (Dr Bloom); Gynecologic & Breast Pathology, Joint Pathology
Center, Silver Spring, Maryland (Dr Branton); the Department of
Pathology, Brigham and Women’s Hospital, Boston, Massachusetts (Dr
Crothers); the Department of Pathology and Microbiology, University of
Nebraska Medical Center, Omaha (Dr Cushman-Vokoun); IHC-ISH
Laboratory and Breast Subspecialty Service, University of Rochester
Medical Center, Rochester, New York (Dr Hicks); the Department of
Hematopathology, The University of Texas MD Anderson Cancer Center,
Houston (Dr Khoury); the Department of Pathology and Laboratory
Medicine, Northwell Health, New Hyde Park, New York (Dr Laser); the
Department of Pathology, University of Colorado, Aurora (Dr Marshall);
the Department of Pathology, Newton-Wellesley Hospital, Newton,
Massachusetts (Dr Misialek); the Department of Pathology, Walter Reed
National Military Medical Center, Bethesda, Maryland (Dr Natale); the
Department of Pathology and Laboratory Medicine, Roswell Park
Comprehensive Cancer Center, Buffalo, New York (Dr Nowak); the
Department of Pathology, Children’s Hospitals and Clinics, Minneapolis,
Minnesota (Dr Olson); the Department of Pathology, Washington
University School of Medicine, St. Louis, Missouri (Dr Pfeifer); Lilly
Research Labs, Eli Lilly and Company, Lilly Corporate Center,
Indianapolis, Indiana (Dr Schade); the Department of Medical and
Molecular Genetics, Indiana University School of Medicine, Indianapo-
lis (Dr Vance); Medical & Scientific Affairs, Roche Tissue Diagnostics,
Tucson, Arizona (Dr Walk); and Special Hematology MMC, University of
Minnesota Medical Center, Minneapolis (Dr Yohe).
Dr Schade is a full-time employee and shareholder of Eli Lilly and
Company. Dr Walk is a full-time employee and shareholder of Roche
Tissue Diagnostics. The other authors have no relevant financial
interest in the products or companies described in this article.
Corresponding author: Carolyn C. Compton, MD, PhD, Arizona
State University, School of Life Sciences, 1475 N Scottsdale Road,
Suite 361D, Scottsdale, AZ 85257 (email: Carolyn.compton@asu.
edu).
1346 Arch Pathol Lab Med—Vol 143, November 2019
often determines treatment choices and may be used to
monitor therapy in real time. However, patient specimens
are collected, handled, and processed according to routine
clinical procedures during which they are subjected to
factors that may alter their molecular quality and compo-
sition. Such artefactual alteration may skew data from
molecular analyses, render analysis data uninterpretable, or
even preclude analysis altogether if the integrity of a
specimen is severely compromised. As a result, patient care
and safety may be affected, and medical research dependent
on patient samples may be compromised. Despite these
issues, there is currently no requirement to control or record
preanalytical variables in clinical practice with the single
exception of breast cancer tissue handled according to the
guideline jointly developed by the American Society of
Clinical Oncology and College of American Pathologists
(CAP) and enforced through the CAP Laboratory Accredita-
tion Program. Recognizing the importance of molecular data
derived from patient specimens, the CAP Personalized
Healthcare Committee established the Preanalytics for
Precision Medicine Project Team to develop a basic set of
evidence-based recommendations for key preanalytics for
tissue and blood specimens. If used for biospecimens from
patients, these preanalytical recommendations would ensure
the fitness of those specimens for molecular analysis and
help to assure the quality and reliability of the analysis data.
(Arch Pathol Lab Med. 2019;143:1346–1363; doi:
10.5858/arpa.2019-0009-SA)
OVERVIEW: THE CRITICAL ROLE OF PREANALYTICS IN
THE MOLECULAR ANALYSIS OF PATIENT
BIOSPECIMENS
B iospecimens acquired during routine medical practice
are the primary sources of the molecular information
about patients and their diseases that underlies precision
medicine. These molecular data inform both patient
Also see p. 1300.
Preanalytical Practices Precision Path—Compton et al
management decisions for clinical care and the development
of new diagnostics and therapeutics in translational
research, including correlative science studies within clinical
trials. Although the molecular data from patient specimens
may be put to a wide variety of downstream medical and
scientific uses, it is typically derived from clinical samples
acquired in the course of routine patient care.
Clinical specimens are collected, handled, and processed
according to routine procedures in the practice of
medicine, surgery, interventional radiology, and patholo-
gy. All procedures, practices, and environmental factors to
which the biospecimen is subjected before a laboratory
analysis are known as preanalytical factors. Artefactual
alterations caused by these factors may skew the data from
molecular analyses, render the analysis data uninterpret-
able, or even preclude analysis altogether if the integrity of
a specimen is severely compromised.1–11 As a result,
patient care and safety may be affected12–15 and medical
research dependent on patient samples may be compro-
mised.2,5,7,8,16–18
Despite these issues, there is no requirement to control or
record preanalytical variables in routine clinical practice in
pathology with the single exception of breast cancer tissue
that is to be assayed for estrogen/progesterone receptor
expression and/or HER2 protein overexpression. In that
single context, the preanalytical steps of cold ischemia time,
formalin fixation method, and total time in formalin are
addressed in the guideline jointly developed by the
American Society of Clinical Oncology (ASCO) and the
College of American Pathologists (CAP)19 and are included
in the Accreditation Checklist of the CAP Laboratory
Accreditation Program (LAP).20
Several national and international standards organiza-
tions have published guidelines for the preanalytical steps
that are recognized to be critical for valid, reliable
molecular testing (Tables 1 and 2),21–35 but compliance
with any of these guidelines is voluntary unless they are
part of an accreditation program such as International
Organization for Standardization (ISO) ISO 15189 Med-
ical Laboratory Accreditation (https://www.anab.org/lab-
related-accreditation/iso-15189-medical-labs, accessed May
24, 2019). As a result, existing guidelines for biospecimen
acquisition, handling, and processing are inconsistently
applied, either in clinical practice or research settings.36
Compliance is further reduced because most authoritative
guidelines are proprietary21–23,25–35 and are not freely available
to all members of the biomedical community.
Recognizing the widespread and growing importance of
molecular data derived from patient specimens, especially
those from cancer patients, the Personalized Healthcare
Committee (PHC) of the CAP established the Preanalytics
for Precision Medicine Project Team (PPMPT) to develop a
basic set of evidence-based recommendations for preana-
lytics for both tissue and blood specimens that could be
implemented in routine pathology practice. If such
practices were to be widely used, it is envisioned that
the preanalytical factors having the strongest detrimental
effect on the molecular integrity of patient biospecimens
would be both controlled and documented, and the fitness
for molecular analysis of patient specimens would be
assured.
The PPMPT process was grounded in published human
biospecimen research data that had been previously known
to or identified by PPMPT members and was the basis for a
Arch Pathol Lab Med—Vol 143, November 2019
proposed set of practice recommendations. To ensure that
the recommendations were informed by data that were
current and correct, a review of relevant biospecimen
research literature published from 2013 to 2018 was
undertaken. The review process was limited to primary
data on the effects of preanalytical factors on human
biospecimens and was further limited to preanalytical
factors for either formalin-fixed, paraffin-embedded (FFPE)
tissue or blood/plasma biospecimens that impacted analysis
data for either nucleic acids or proteins. It was reasoned that
these 2 types of patient specimens are the ones most
commonly used for molecular testing and that nucleic acids
and proteins are the most commonly analyzed classes of
biomolecules in clinical practice. New data were compared
against existing data known to the PPMPT and determined
to be either consistent or inconsistent with the proposed
recommendations.
The work of the PPMPT has confirmed from the
published literature that a small number of key preanalyt-
ical factors are critical to ensuring the molecular integrity of
patient biospecimens for molecular analysis. The recom-
mendations developed by the PPMPT are based solely on
human biospecimen research data. They are focused on
controlling and documenting these variables, for speci-
mens from cancer patients as a first step and ultimately for
all specimens destined for molecular analysis. The practice
metrics set forth in the recommendations are concordant
with other preanalytical guidelines from authoritative
national and international sources (Tables 1 and 2) and
are believed to be practicable and attainable in most
pathology practice settings. The recommendations mirror
much of what has already been achieved in implementing
the ASCO-CAP guidelines for breast cancer,19 an impor-
tant initial step for the pathology community in demon-
strating the feasibility of controlling and documenting key
preanalytical factors. It is acknowledged, nevertheless, that
implementation of the recommendations presented herein
may require changes in workflows, schedules, or staffing
and that additional costs may be incurred by the
laboratory.
It is the position of the PPMPT that the reliability of
molecular analysis data is dependent upon the quality of
the source analytes from biospecimens. Fundamentally,
biospecimen quality is an issue of patient safety because its
compromise can alter the molecular data on which key
patient management decisions are based. Additionally, in
translational research, it would be expected to impact the
validity and reproducibility of study data. Systematic
implementation of key, preanalytical procedures in routine
pathology practice would ensure a baseline level of quality
for patient biospecimens where none currently exists and
would provide a new level of confidence in the veracity of
analysis data. Documentation of the preanalytical history
of patient specimens would further enable objective
estimation of the fitness of a given sample for either
real-time or future testing. Ultimately, as with all
procedures that directly contribute to the quality of
pathology practice and are essential to the generation of
valid analysis results, laboratories should support imple-
mentation of these preanalytical practices and strive to
achieve them over time, realizing that routine practice of
even a subset of the standards will improve the baseline
quality of specimens.
Preanalytical Practices Precision Path—Compton et al 1347
 Table 1. Preanalytical Recommendations for Tissue for Molecular Analysis From National and International
Authoritative Sources
Organization
CAP General
CAP-ASCO CLSI ILA28-A2 CLSI MM13 NCI-BBRB Surg Path
2014 2011 2005 2016 2015
Biomolecule/biomarker Breast biomarkers Proteins: IHC DNA, RNA, Molecular analysis General surgical
protein pathology
Preanalytical parameter
Cold ischemia time, min 60 min or less As short as 60 min or less As short as possible, ,12 h
possible (min) optimally less
than 20 min but
no more than 1 h

Handling and processing — — — — Room temperature

temperature (228C–258C)
Total time in formalin, h 6–72 h Greater than 8 h; Not to exceed — A minimum of 6 h;
no longer than 72 h (for a maximum of 48
12–36 h nucleic acids) h (actual time
should be
recorded)
Type of fixative 10% Neutral 10% Neutral 10% Neutral — 10% Neutral
phosphate- phosphate- phosphate- buffered formalin
buffered formalin buffered formalin buffered formalin

Volume ratio of formalin — .10:1 — — 15–20:1

to tissue
Type of paraffin (low melt — 558C–588C — — —
,608C versus high
melt 608C)
Quality of tissue-processing — Changed in a Per manufacturer’s — —
fluids timely fashion specifications
Thickness of tissue section — 2 mm or less — — 4 mm
into cassette
Temperature of block — — — Temperatures below —
storage 808F (278C)

Abbreviations: ASCO, American Society of Clinical Oncology; BBRB, Biorepositories and Biospecimen Research Branch; CAP, College of American
Pathologists; CEN/TC, European Committee for Standardization/Technical Committee; CLSI, Clinical & Laboratory Standards Institute; IHC,
immunohistochemistry; ISO/TC, International Standards Organization/Technical Committee; NCI, National Cancer Institute; Surg Path, Surgical
Pathology.

THE RAPID GROWTH IN DEMAND FOR MOLECULAR
TESTING AND THE RATIONALE FOR CHANGE IN
PATHOLOGY PRACTICE
The concept of ‘‘personalized’’ or ‘‘precision medicine’’ is
based on rational management of therapy tailored to the
unique biomolecular features of both the patient and his/her
disease. It represents disruptive innovation in medical care
that has, historically, been based largely on a generic
approach to both diagnosing and treating disease. In cancer
care, precision medicine entails analysis of a patient’s tumor
in the molecular pathology laboratory and, based on the
results, implementation of a therapeutic strategy tailored to
the tumor’s particular molecular aberrations. For cancer and
other diseases that are inherently heterogeneous, molecular
assessment is used to subclassify patients into more
homogeneous groups according to prognostic implications,
whether or not there is a targeted or immune therapy
available for any given subgroup. Although yet to be proven
broadly across the medical landscape, it is presumed that a
molecularly tailored approach will increase effectiveness and
1348 Arch Pathol Lab Med—Vol 143, November 2019
decrease toxicity of treatment, decrease the costs of care
overall, and increase value in health care delivery.37
The rapid development of powerful molecular analysis
platforms during the past 2 decades has steadily increased
the speed and accuracy of molecular testing while decreas-
ing operational costs, expediting widespread uptake in
cancer medicine. According to the Personalized Medicine
Coalition, there are currently more than 60,000 molecular
genetic tests on the market, with 8 to 10 new products
entering the market every day.38 Multiplex technologies
such as next-generation sequencing for nucleic acids and
mass spectrometry for proteins, once residing solely in the
research domain, have swiftly moved into the clinical care
arena.
The widespread availability of high-throughput molecular
analysis technologies and services, the expanding arsenal of
targeted and immune oncology therapeutics, and the
biomarker tests that aid in predicting treatment success or
toxicity risk have accelerated the demand for molecular
analyses of many types. Molecular test results may guide
clinical decision-making and the appropriate use of specific
Preanalytical Practices Precision Path—Compton et al
 Table 1. Extended

Organization

ISO/TC-212 ISO/TC-212 CEN/TC 140 CEN/TC 140 CEN/TC 140

2016 2016 2015 2015 2015
Isolated RNA Isolated DNA Isolated protein Isolated RNA Isolated DNA

60 min or less Avoid cold ischemia As short as possible Avoid cold ischemia when Avoid cold ischemia when
when possible. (document actual possible (directly into possible (directly into
Document actual cold ischemia time standard buffered standard buffered formalin).
cold ischemia time and warm ischemia formalin). If unavoidable: If unavoidable: as short as
and warm ischemia time) as short as possible. possible. Documentation
time Documentation required required
— Room temperature Room temperature Room temperature (188C– Room temperature (188C–
(188C–258C) (188C–258C) 258C) 258C)
,72 h (old) 24–36 h 12–24 h 12–24 h Less than 24 h, eg, 12–24 12–24 h for tissue thickness of
(revised 2016) h for tissue thickness of 5 mm. Optimal duration
5 mm. Documentation can vary depending on
required tissue type and size.
Documentation required
10% Neutral 10% Neutral buffered 10% Neutral buffered 10% Neutral formalin 10% Neutral formalin solution
phosphate-buffered formalin (pH and formalin (pH and solution pH 6.8–7.2 pH 6.8–7.2 (type of buffer
formalin concentration concentration (type of buffer not not specified)
checked daily before checked regularly) specified)
use and with each [type of buffer not
new batch) [type of specified]
buffer not specified]
— 15–20:1 At least 10:1 4:1 at least; optimal 10:1 4:1 at least; optimal 10:1

— Low melt Low melt Low melt Low melt

Well maintained Per manufacturer’s Per manufacturer’s Per manufacturer’s Per manufacturer’s
specifications specifications specifications specifications
4–5 mm 5 mm 5 mm Max. 5 mm Max. 5 mm

— Room temperature Room temperature Room temperature (188C– Room temperature (188C–
(188C–258C) or lower (188C–258C) or lower 258C) or lower 258C) or lower

therapies. In the case of companion diagnostics, molecular
testing is the sine qua non of therapy allocation.
For many diseases, assessment of diagnostic, prognostic,
and/or predictive molecular markers has become standard
procedure and is encouraged or dictated by practice
guidelines from the CAP,19,39–41 ASCO,42 the National
Comprehensive Cancer Network (NCCN),43 the European
Society for Medical Oncology,44 and other professional
groups. Blood specimen analyses for circulating cell-free
DNA and RNA from tumors, which have become known as
‘‘liquid biopsies,’’ are moving rapidly into clinical use and
are currently under investigation for a wide variety of clinical
decision-making applications.45 Thus, guidelines for the
testing of circulating tumor DNA in cancer patients have
recently been developed by the CAP and ASCO.46
THE SCOPE OF THE PROBLEM AND THE PRESSING
NEED FOR A SOLUTION
As the use of molecular data from patients continues to
expand, the stakes for accurate test results increase.47 For
example, it has been estimated that false-positive and false-
negative HER2 test results alone affect approximately 12,000
patients with breast cancer annually, resulting in total
Arch Pathol Lab Med—Vol 143, November 2019
economic societal costs of nearly $1 billion.48 The accuracy
of a molecular test result may impact patient management
in a variety of ways, and erroneous, false-positive or false-
negative test results may have significant, even catastrophic,
consequences for a patient in the setting of precision
medicine. Therefore, significant effort must be made to
ensure the accuracy of test results generated in the
pathology laboratory.
In routine pathology practice, standards that apply to the
analytical validity of the test itself, the quality of the
laboratory in which the test is performed, and the
proficiency of the professionals performing the test are all
rigorously enforced. Nevertheless, there is considerably less
focus on the fact that the accuracy of the test result still may
be compromised if the quality of the biospecimen under-
going testing has been corrupted by preanalytical factors.
Therefore, to assure that analysis results are both reliable
and biologically meaningful, the integrity of the biospeci-
men must be safeguarded through the critical steps of
acquisition, transport, stabilization, processing, and storage.
According to current data, error in the preanalytical phase
of patient specimen testing is the most common source of all
mistakes occurring in the pathology laboratory. An esti-
Preanalytical Practices Precision Path—Compton et al 1349
 Table 2. Preanalytical Recommendations for Blood for Molecular Analysis From National and International
Authoritative Sources
Organization
ASCO-CAP CLSI GP41 CLSI GP-44-A4 NCI-BBRB CAP: An Introduction
2018 2017 2010 2016 to Phlebotomy 2017
Guideline Liquid Biopsies (cell- Collection of Venous Handling and Best Practices for General Collection of
free DNA from Blood Specimens Processing Blood Biospecimen Blood Samples
plasma) Specimens for Resources:
Common Laboratory Molecular
Tests Analysis of
Biospecimens
Preanalytical parameter
Time to first 6 hours or less if EDTA — Within 48 hours of — —
processing tube used; 48 hours collection
step (min) or less if cell-
stabilization tube
used
Method of EDTA or cell- — — — Correct tube for the test
acquisition: stabilization tube intended
tube type
Method of — Per stated volume in Per stated volume in — Per stated volume in
acquisition: tube manufacturer tube manufacturer tube manufacturer
tube fill instructions instructions: neither instructions: neither
underfilled or underfilled or
overfilled overfilled
Method of — — At least 5–10 times — 8 times excepting
acquisition: excepting sodium sodium citrate tubes
tube inversions citrate tubes (3–4 (3–4 times)
times)
Method of — 1. Blood culture tube — — 1. Blood culture tube
acquisition: 2. Sodium citrate tube 2. Nonadditive discard
draw order 3. Serum tube tube
4. Heparin tube 3. Sodium citrate tube
5. EDTA tube 4. Serum tube
6. Sodium fluoride/ 5. Heparin tube
potassium oxalate 6. EDTA tube
glycolic inhibitor 7. Sodium fluoride/
tube potassium oxalate
glycolic inhibitor
tube
8. ACD tube

Method of Sequential spins at low — Temperature-controlled — —

processing: speed (not defined) centrifugation at
centrifugation then high speed (not 208C–228C at g-force
defined); (one cited determined by
reference specifies manufacturer
2000 rpm 3 2 recommendations for
followed by 13,500 blood collection
rpm 31 spin) device used

Method of Room temperature — Room temperature — —

processing: (228C–258C) (228C–258C) unless
temperature chilled centrifugation
is required (eg,
potassium)

Storage No more than 1 freeze- — No more than 1 freeze- Aliquot specimens —

conditions: thaw cycle: thaw cycle: 1 or to avoid freeze-
freeze-thaw recommend more aliquots in thaw cycles
cycles aliquoting processed polypropylene tubes
plasma before or straws
freezing
Abbreviations: ACD, acid-citrate-dextrose; ASCO, American Society of Clinical Oncology; BBRB, Biorepositories and Biospecimen Research
Branch; CAP, College of American Pathologists; CEN/TC, European Committee for Standardization/Technical Committee; CLSI, Clinical & Laboratory
Standards Institute; ISO/TC, International Standards Organization/Technical Committee; NCI, National Cancer Institute.

1350 Arch Pathol Lab Med—Vol 143, November 2019 Preanalytical Practices Precision Path—Compton et al
 Table 2. Extended

Organization
ISO/TC 20658 World Health CEN/TC 16835-1 CEN/TC 16835-2 CEN/TC 16835-3
2017 Organization 2010 2015 2015 2015
Medical Laboratory - Guidelines on Drawing Isolated Cellular RNA Isolated Genomic DNA Isolated Circulating Cell-
Requirements for Blood from Venous Blood from Venous Blood Free DNA From Plasma
Collection, Transport, (RNA from blood cells) (DNA from blood cells) From Venous Blood
Receipt, and
Handling of Samples

— — As short as possible - As short as possible - As short as possible -

immediately if using immediately if using immediately if using
tubes without cellular tubes without cellular tubes without cellular
RNA stabilizers DNA stabilizers DNA stabilizers
(document actual time) (document actual time) (document actual time)
— — Blood collection tube EDTA (preferable to ACD) or Tube with cellular DNA
with or without ACD tube or tube with stabilizers recommended;
cellular RNA stabilizers cellular DNA stabilizers otherwise use EDTA tube
— — Per stated volume in tube Per stated volume in tube Per stated volume in tube
manufacturer manufacturer instructions manufacturer instructions
instructions

— — Per manufacturer Per manufacturer Per manufacturer

instructions instructions instructions

— 1. Blood culture tube — — —

2. Nonadditive discard tube
3. Sodium citrate tube
4. Serum activator tube
5. Serum separator tube
6. Sodium or lithium
heparin tube
7. Lithium heparin with gel
separator
8. EDTA tubes
9. ACD tube
10. Sodium fluoride/
potassium oxalate
glycolic inhibitor
— — — — When using tubes with
cell-free DNA stabilizers:
follow manufacturer
instructions; when using
EDTA tubes, use
sequential 10-min spins
at low speed (1600g–
2500g) at 28C–88C and
high speed (14,000g–
16,000g) at 28C–88C
— — Per tube manufacturer Per tube manufacturer Centrifugation at 28C–88C;
instructions (temporary instructions (temporary DNA isolation per kit
storage before storage before processing manufacturer
processing at 28C–88C at 28C–88C as long as 3 recommendation
when using tubes days when using tubes
without cellular DNA without cellular DNA
stabilizers) stabilizers if high-
molecular-weight DNA
is needed)
— — — — No more than 1 freeze-
thaw cycle: recommend
aliquoting processed
plasma before freezing

Arch Pathol Lab Med—Vol 143, November 2019 Preanalytical Practices Precision Path—Compton et al 1351
mated 60% to 70% of laboratory-associated errors are due
to preanalytical factors, the most common of which are
mishandling during collection, transport, processing, and
storage of specimens.9,49–52 Although most of the data on
this subject are related primarily to blood rather than tissue
specimens, there is little reason to believe that tissue-related
preanalytical factor problems are less frequent or less
detrimental.
Compounding the issue for tissue specimens is the lack of
any requirement to track or record the preanalytical history
of a biospecimen. The critical preanalytical variables for the
vast majority of patient specimens are both uncontrolled and
undocumented, and the provenance of samples undergoing
molecular analysis is, therefore, typically unknown. As a
result, molecular analysis of patient specimens of question-
able or unacceptable quality may be performed without the
knowledge of the tester, producing data of questionable or
unacceptable quality without the knowledge of the inter-
preter of that data. This is especially problematic for
translational research laboratories. These laboratories do
not routinely perform clinically mandated quality control
that may reveal poor specimen quality before testing.
Alternatively, in either the clinical or the research laboratory,
the data generated may be completely uninterpretable or
inconclusive, and the value of the test is knowingly lost.
The issue of unknown specimen quality profoundly affects
biomedical research as well as clinical practice.2,5,8,16–18,53–55
Most of the biospecimens that fuel translational research
and the correlative science in clinical trials are apportioned
from clinical samples acquired for medical purposes, not
research. This is true of most tumor samples used for
correlative scientific studies in clinical trials and for so-called
discard specimens that are ‘‘left over’’ following diagnostic
evaluation and are then used in discovery research or
product development. Therefore, biospecimens of poor or
unknown quality continue to contribute to the overall
inefficiency, excessive cost, poor reproducibility, and high
rate of failure of translational research, in general, and of
biomarker development, in particular.2,54,55 Recent efforts by
biobanking experts to address this problem after the fact
include the development of batteries of assays for measur-
ands in specimens that are affected by preanalytical factors.
These ‘‘pretests’’ help to classify or disqualify specimens of
unknown provenance for molecular analysis56–62 but are
rarely used in clinical settings.
THE FORMATION OF THE CAP PPMPT FOLLOWS A
NATIONAL ALL-STAKEHOLDERS CONVERGENCE
CONFERENCE ON THE PREANALYTICS CHALLENGE
Given the widespread impact of the issue on diverse
stakeholders, both public and private, the process of
developing a solution was begun with an all-stakeholder
think tank.
In 2014, the National Biomarker Development Alliance
(NBDA),63 a nonprofit organization and think tank, con-
vened a cross-sector national conference to address the
critical issue of uneven and unknown quality of human
biospecimens in clinical medicine and translational re-
search.18,54,55,64,65 The meeting brought together more than
50 experts and thought leaders from molecular pathology,
laboratory medicine, surgery, genomics, proteomics, health
care delivery, analysis platform technology along with
payers, funders, regulators, patient advocacy, and profes-
1352 Arch Pathol Lab Med—Vol 143, November 2019
sional societies that set and enforce standards of care,
including the CAP.
The goal for the group was to come to agreement
regarding the specific variables in patient specimen acqui-
sition, handling, processing, storage, and transport that
cause most of the quality compromise and molecular
alteration in patient tissue and blood specimens and
adversely affect DNA, RNA, and/or protein analysis results.
Input was based on the experience and knowledge of the
stakeholders and published data in biospecimen science.
The effort was focused on tissue and blood, the specimen
types most commonly used for molecular analysis, and
nucleic acids and proteins, the most commonly assayed
biomolecules in both clinical and investigational biomedi-
cine. Next-generation sequencing and mass spectrometry
platforms were chosen as the technology-specific reference
points, but it was agreed, in principle, that the preanalytical
steps that most affect the biomolecules analyzed on these
platforms would be pertinent to virtually all nucleotide or
protein analysis methodologies.
The Pareto Principle or ‘‘80/20 Rule’’ of inputs and outputs
provided a guiding framework for the discussion.66 Specif-
ically, it was assumed that 80% of the problems in the
outputs of a system emanate from 20% of the inputs.
Accordingly, the goal was to identify the 20% of preanalyt-
ical variables that cause most of the variation in molecular
composition and quality that, in turn, cause most of the
problems in downstream molecular analysis.
The group agreed upon a ‘‘top 6 list’’ of key preanalytical
factors for tissue biospecimens and a ‘‘top 6 list’’ for blood
samples and suggested practice metrics for each factor that
would control it in an adequate and workable manner
within the clinical laboratory (Table 3).
The CAP PPMPT Process for Verification of Preanalytical
Parameters
The NBDA and the CAP created a memorandum of
understanding in order to work together in an official
capacity to address the preanalytics issues raised at the
NBDA conference, and to this end, the CAP established the
PPMPT within the PHC. The CAP PPMPT members
included molecular pathology experts from the PHC as well
as experts across multiple specialty areas including both
anatomic and clinical pathology. The PPMPT took as a
starting point foundational publications in the field of
biospecimen science that were pertinent to the top 6 lists for
tissue specimens and blood samples.* These publications
were identified through previous professional experience
and/or from the Biospecimen Research Database established
by the National Cancer Institute (NCI).148 This historical
evidence was the basis for draft practice metrics. Subse-
quently, an extensive literature search for recent biospeci-
men research data was performed by CAP librarian staff,
and the PPMPT systematically analyzed the identified
publications to either affirm or negate the scientific bases
for the benchmark recommendations.
Several PubMed searches of the English-language litera-
ture from January 2013 to November 2016 were performed.
Literature search strategies were developed in collaboration
with CAP medical librarians to locate relevant publications.
The search strategies created used standardized database
* References 1, 4–6, 8–11, 16, 49–53, 67–147.
Preanalytical Practices Precision Path—Compton et al
 Table 3. Essential Preanalytical Factors Affecting Molecular Testing and Recommendations for Pathology Practice
Top 6 Preanalytical Factors for Tissue for the Maintenance
of Nucleic Acid and Protein Quality and Integrity
Time to stabilization (cold ischemia time)
 1 h or less
Method of stabilization
 Fixative: 10% phosphate-buffered formalin, pH 7.0
 Total time in formalin: at least 6 h, not more than 24–36 h
(tissue with high fat content may require 48 h)
 Acid decalcification, before or during stabilization,
is contraindicated for nucleic acid analyses
Method of processing
 Specimen thickness not to exceed 4–5 mm
 Volume to mass ratio 4:1 at a minimum, preferably 10:1,
with tissue completely submerged
Tissue processor variables
 Processor maintenance daily per manufacturer’s
recommendations
 Quality of processing fluids rigorously maintained
 Maintenance of formalin purity and pH
 Attention to water (ie, formalin) contamination of
alcohol baths
 Type of paraffin
 Low-melt paraffin (melts at ,608C)
Storage conditions
 Dry, pest-free conditions at room temperature
(defined as 188C–258C)
Documentation data for the above factors and/or
deviations from the recommendations
Note: Tissue specimens considered unacceptable for
molecular testing include desiccated tissues or those
known to have been improperly collected or stored
Abbreviations: ACD, acid-citrate-dextrose; EDTA, ethylenediaminetetraacetic acid.
terms and text words. The Cochrane search filter for humans
was applied.149
To identify pertinent publications, the titles and abstracts
of all identified articles were reviewed by the PPMPT
according to the criteria below:
1. In scope: publications with original data referable to the
effect of the preanalytical variables listed in Table 3 on DNA,
RNA, and/or protein analysis in human tissue or in blood.
2. Out of scope: review articles; conference abstracts;
comments; editorials; letters; data on cell lines; data on
cytology specimens; data on animal biospecimens; data
on biomolecules other than DNA, RNA, and/or protein
in biospecimens; data on posttranslational modification
of proteins; and data comparing tissue or blood fixatives.
A total of 648 records were identified, 595 from the 2014
Arch Pathol Lab Med—Vol 143, November 2019
Top 6 Preanalytical Factors for Blood/Serum for the Maintenance
of Nucleic Acid and Protein Quality and Integrity
Time to first processing step
 ,60 min (unless EDTA or specialty cell-stabilization tube used)
 4–6 h for EDTA tube
 48 h for cell-stabilization tube
Method of acquisition
 Tube type: specialized for a specific molecule species versus not
 If processing time is .2–3 h, use ACD tube
 EDTA tube for nucleic acid studies, proteomics studies, or
circulating cell-free nucleic acid studies (cell-free nucleic
acid analysis requires rapid stabilization or potentially
specialty cell-stabilization tubes)
 Volume of tube fill
 Complete fill per manufacturer’s recommendation
 Tube inversions per manufacturer’s recommendations (typically 10)
 Draw order
 Culture tube
 Nonadditive tube
 Coagulation tube (sodium citrate)
 Clot activator tube
 Clot activator and serum separator tube (clot activator plus
separator gel)
 Heparin tube (lithium or sodium heparin)
 EDTA tube
 Tubes with other additives (eg, sodium fluoride; ACD)
Method of stabilization
 Tube inversions per manufacturer’s recommendations
Method of processing
 Centrifugation speed/time per validated protocol and
biomolecule being studied
 Temperature: room temperature (defined as 188C–258C)
unless validated protocol dictates otherwise
Storage conditions
 1 freeze-thaw cycle: for nucleic acids and proteins use aliquots
Documentation data for the above factors and/or deviations from
the recommendations
Documentation should be included in the laboratory’s standard operating
procedures and quality control logs
Note: Blood specimens considered unacceptable for molecular testing
include hemolyzed samples or those known to have been improperly
collected or stored
searches and 53 from the 2015–2016 search. Eighteen people
participated in the abstract review for the 648 publications,
and each abstract was reviewed by 2 individuals. The criteria
listed above were used to determine if the article was in scope
and required full review. In cases of disagreement on inclusion
versus exclusion, the 2 reviewers discussed their interpreta-
tions with each other to come to agreement or a third reviewer
acted as an adjudicator. The dual review of abstracts excluded
320 articles as being inapplicable to the top 6 preanalytical
factors for either tissue or blood and identified 328 articles for
full text analysis. Sixteen people participated in the 328 full text
article reviews. Data were collected from the article to
determine if the article referred to tissue or blood, the analyte
being assessed (DNA, RNA, protein), and whether the results
reported in the article agreed with or contradicted the
preanalytical parameters proposed as the top 6 for each of
Preanalytical Practices Precision Path—Compton et al 1353

The process used and reported here has been adapted from: Moher D,
Liberati A, Tetzlaff J, Altman DG; The PRISMA Group. Preferred
Reporting Items for Systematic Reviews and Meta-Analyses: The
PRISMA Statement. PLoS Med. 2009;6(7):e1000097. doi:10.1371/
journal.pmed1000097.
the 2 specimen types (Table 3). After single review, 23 articles
where thought to deviate from the parameters. These 23
articles were then reanalyzed independently by 2 individuals
and were determined on re-review not to deviate. A summary
of the review process is shown in the Figure.
Implementation of the proposed practice metrics is
believed to be feasible in most practice settings. Through
the CAP LAP, accreditation checklist elements may be
developed and piloted where needed to facilitate imple-
mentation. It is anticipated that the resultant change in
routine pathology practice will simultaneously elevate the
molecular quality of human biospecimens for both clinical
practice and translational research.
THE KEY PREANALYTICAL VARIABLES FOR TISSUE
BIOSPECIMENS
The PPMPT practice recommendations for control of
essential preanalytical variables, based on current evidence,
are discussed individually below. Citations for both recent
original data reviewed by the PPMPT, older publications
with pertinent original data contributed by the experts on
the PPMPT, and academic reviews that include references to
published original data before 2013 are all included below.
As a basic reference for comparison, a summary of the
analogous preanalytical parameters for FFPE tissue recom-
mended by nationally and internationally recognized
authoritative sources is shown in Table 1.
1354 Arch Pathol Lab Med—Vol 143, November 2019
Cold Ischemia Time: A Cold Ischemia Time of 1 Hour or
Less Is Recommended
Cold ischemia time, also referred to in the literature as
‘‘time to fixation,’’ ‘‘delay to fixation,’’ ‘‘prefixation delay,’’
or, simply, ‘‘prefixation,’’† is defined as the length of time
between removal of the biospecimen from the patient and
the time the biospecimen is stabilized in formalin (ie, the
biological activity in the tissue is stopped by fixing). The
label ‘‘cold ischemia’’ actually refers to a room temperature
environment, in contrast to ‘‘warm ischemia’’ following
devascularization of the tissue while still at body temper-
ature.80,81 The effects described below may be altered by
refrigeration of the specimen, as noted.
Depending on the biomolecule class and the biospecimen
type, cold ischemia times of several hours (eg, up to 4–5
hours or even longer at room temperature) have minimal or
no detrimental impact on biomolecular quality, extractable
quantity, or molecular test results,14,81,83,150–152 whereas other
molecular analyses, sometimes for the same specimen, are
altered within 60 minutes or less.‡ Molecules may be either
upregulated or downregulated, and most studies show that
gene transcripts are more likely to be upregulated rather
than downregulated within 2 hours of cold ische-
mia.14,152,154,155 Highly labile molecules, such as protein
kinases and phosphoproteins, may be disproportionately
affected after 30 to 45 minutes or less.§
Cold ischemia has been described as ‘‘a complex
physiological perturbation that integrates the effects of
tissue stress, hypoxia, hypoglycemia, acidosis, hypothermia,
and electrolyte disturbance.’’154 Cold ischemia times of 30
minutes have been shown to increase the levels of proteins
in stress-response pathways, including those related to
apoptosis, hypoxia, and proliferation,153 and may not return
to baseline thereafter.1,153 Since these proteins have been
implicated in cancer progression and drug resistance,156,157
their analysis may be clinically important.
In any specific case, however, the significance of changes
linked to cold ischemia depends on the molecular target of
interest and the implications of the test result in clinical
decision-making. Some routine and clinically important
protein assays, such as estrogen receptor or progesterone
receptor immunohistochemistry, have been shown to be
altered by cold ischemia times of 30 to 60 minutes8,11,61,70,73,84
at room temperature, although longer cold ischemia times
may be tolerated if the specimen is maintained at 48C.84,150
In time course studies of broader changes in different
classes of biomolecules in human tissue specimens, it has
been demonstrated that within 15 minutes of cold ischemia
up to 15% of tested genes and proteins change from
baseline levels and up to 20% of all detectable molecules
change within 30 minutes.80,158–161
Current data suggest that the measurable effects of cold
ischemia are, at least partially, cancer type–specific as well as
biomolecule type–specific and analysis platform–depen-
dent.80,151 In addition, studies comparing the effects of cold
ischemia on cancer tissue and normal tissue from the same
organ have demonstrated that tumor tissue is more
vulnerable to the effects of cold ischemia.80,161 Since no
single recommendation for cold ischemia time would be
optimal for all tissues, all classes of molecules, or all analysis
†
References 4, 68, 70, 71, 73–77, 83, 84.
‡
References 4, 11, 14, 48, 61, 68, 70, 71, 73, 83–84, 152–155.
§
References 5, 14, 61, 82–84, 153, 154.
Preanalytical Practices Precision Path—Compton et al
platforms, the recommendation put forward here represents
a compromise that meets most current molecular testing
needs for cancer patient specimens.
A cold ischemia time of 1 hour is regarded as a prudent
and achievable guideline and supported by the litera-
ture.6,8,10,11,22,26 Additionally, actual cold ischemia times or,
at a minimum, deviations from the 1-hour recommendation
should be recorded in the pathology report. A documented
cold ischemia time of 1 hour or less for every cancer tissue
specimen would achieve baseline standardization that
would meet the requirements of the ASCO-CAP guidelines
for breast cancer specimens,19 achieve a safe margin of
control for many other receptor protein and nucleotide
biomarkers, allow a correction factor to be applied for
interpretation of assays for more labile biomolecules, and be
stringent enough to allow more than 1 class of analyte to be
measured from the same specimen.6 Finally, it is both
reasonable and pragmatic to treat every specimen similarly
within a uniform workflow plan rather than customize cold
ischemia times for individual specimens.
Fixative Type: Standardized and Quality-Controlled 10%
pH Neutral Phosphate-Buffered Formalin Is Recommended
This standard fixative preserves a wide range of biomolec-
ular species as well as morphology. It is relatively inexpen-
sive, widely available, and commonly used throughout
anatomic pathology practice. However, strict adherence to
quality control is required to maintain the chemical quality of
formalin. The pH of the formalin should be checked before
use and routinely thereafter, since formalin is inherently
unstable and oxidizes to formic acid.86 This recommendation
mirrors the All Common CAP LAP requirements for the
handling, monitoring, and documentation of laboratory
reagents and the requirements to meet manufacturer’s
specifications when using commercially acquired reagents
(COM 30350 and 40250). If formalin is prepared in the
laboratory, strict adherence to procedures that ensure both
the correct concentration and pH of the solution are
mandatory. If commercially prepared formalin is used,
concentration cannot be measured, but pH monitoring
should be performed. All formalin stock solutions must be
kept in tightly sealed containers to prevent oxidation to
formic acid and dehydration. Strict adherence to the
manufacturer’s recommendations for shelf life of stored
formalin is recommended.
At room temperature (258C), formalin rapidly penetrates
tissue, but fixation, which is caused by cross-linkage of nucleic
acids and proteins via methylene bridges between reactive
chemical groups, is a slower process. Nevertheless, both tissue
penetration and fixation are temperature-dependent process-
es. They are slowed at lower temperatures and accelerated at
higher temperatures,86 but the rate of fixation is slower than
penetration at any temperature.87,88 Tissue penetration is also
profoundly affected by the pH of the formalin.87,89
Fixatives other than formalin may be used electively for
specific analyses, at the discretion of the pathologist, if
appropriately validated, but should be recorded as a
deviation from routine procedure in the pathology report.
The use of acid decalcification, before or during the fixation
process, results in hydrolysis of DNA and RNA and is
contraindicated for molecular analyses of nucleic acids.
Total Time in Formalin: A Total Fixation Time of No Less
Than 6 Hours90,162 and No Greater Than 36 Hours for Most
Tissues or 48 Hours for Tissues With High Fat Content91–93
Arch Pathol Lab Med—Vol 143, November 2019
Is Recommended; Optimal Fixation Time for Protein in
Most Tissues Is 24 Hours,4,6,93 Especially for Proteomic or
Immunohistochemical Applications, Whereas RNA and
DNA May Tolerate Longer Fixation Times6
Total fixation time has been an ongoing issue and concern
in recent years. The following recommendation is made
with that history in mind. We note that the recommenda-
tion does not appear as a requirement in any current CAP
LAP checklist and may even differ from some current CAP
guidelines (Table 1).
Total time in formalin includes the time the tissue is in
formalin in the tissue processor and is based upon fixation
performed at room temperature. Some tissues, especially
those with a high fat content, such as skin or breast tissue,
are an exception, and fixation times up to 48 hours may be
required.94 The actual fixation time for any given tissue
sample should be documented and recorded in the
pathology report. Both underfixation and overfixation of
tissue compromise molecular analysis results.
As described above, formalin penetrates most tissues
rapidly, but the cross-linking process is much slower. Both
processes are temperature dependent.86,87 Adequate tissue
fixation requires a minimum of 6 hours at room temperature
(258C).90 Notably, however, because the chemistry of
fixation is slowed at temperatures lower than 258C,
refrigeration of specimens after immersion in formalin
may provide additional flexibility around this preanalytical
parameter in clinical practice.
In general, the average size of DNA extracted from
tissues fixed in buffered formalin decreases with increas-
ing fixation time.87 Not only does cross-linking of nucleic
acids with histones occur with formalin fixation, but
formalin reacts directly with nucleotides, causing molec-
ular degradation and alteration of sequences.88 There is
evidence that molecular degradation in nonfatty tissue
starts at 24 hours’ fixation time, but after about 36 hours
of fixation at room temperature excessive cross-linking
and molecular damage may begin to become significant.
Nucleic acid degradation, fragmentation, and sequence
alteration have all been reported as a consequence of
overfixation.1,88,91,163 The types of DNA damage known to
occur in FFPE include (1) formaldehyde-induced cross-
links; (2) molecular fragmentation; (3) deamination of
cytosine bases producing C-to-T mutations; and (4)
production of abasic sites.164 This damage interferes with
both polymerase chain reaction (PCR) amplification and
next-generation sequencing.164,165
The range of 6 to 36 hours is reasonable for nonfatty
tissues and may be more achievable in practice; however, a
range of 6 to 24 hours may be better. This range of fixation is
recommended to make tissue fit for most genomic and
proteomic analyses; however, a different period of fixation
may be acceptable for specific tissue types or testing
methods. For example, routine testing for estrogen receptor,
progesterone receptor, and ERBB2 (HER2) performed by
immunohistochemistry in breast cancer specimens may not
be impacted with fixation up to 72 hours as is allowed
within the ASCO-CAP guidelines.19,166,167 For any given
specimen, however, documentation of the actual fixation
time is advised. Alternatively, if the recommended fixation
timeframe cannot be met, the deviation from the guideline
should be recorded.
Total protein recovery for mass spectroscopy and immu-
noreactivity of proteins also may be adversely affected by
Preanalytical Practices Precision Path—Compton et al 1355
prolonged fixation,4,168,169 but some studies have shown that
protein identifications by multidimensional liquid chroma-
tography–tandem mass spectrometry in FFPE tissue sub-
jected to fixation times of up to 2 days may be comparable to
those in frozen tissue.139
Specimen Thickness: Specimen Sample Thickness That
Does Not Exceed 4 to 5 mm Is Recommended
This recommendation will allow rapid and uniform
penetration of formalin from the tissue surface throughout
the tissue.1,163 Nevertheless, because formalin penetration is
a rapid process, and proceeds at a rate of about 1 mm per
hour at room temperature, specimen thickness that slightly
exceeds 5 mm may be tolerated as long as the specimen is
enveloped by the fixative to allow penetration to occur from
all surfaces and the fixation time is adequate.1 An increase in
the surface area of the tissue sample increases the rate of
penetration of the fixative. However, penetration of formalin
also may be affected by the composition of the specimen.
Penetration of an aqueous fixative such as formalin may be
slowed by high fat content in the specimen, whereas muscle
content or abundant intercellular channels such as blood
vessels may speed penetration.87
Ratio of Fixative Volume to Tissue Mass: A Fixative Volume
to Tissue Mass Ratio of at Least 4:1, or Preferably 10:1
When Feasible, With the Tissue Completely Submerged, Is
Recommended163
An adequate volume of formalin will ensure that tissue
penetration occurs from all surfaces and that full-thickness
penetration will be achieved.87 In studies on fixation of small
samples (biopsy samples of diameters from 1 to 1.5 mm),
adequate fixation has been documented for a volume to
mass ratio of as little as 2:194 or even 1:1.129 In contrast, the
recommendations from several authoritative sources stipu-
late a volume to mass ratio of 10:1 or greater (Table 1).1
Therefore, the recommendation of 4:1 represents a prudent
but economical compromise that would accommodate
biopsies as well as larger samples to ensure that all tissue
surfaces are completely enveloped by fixative.
Tissue Processor Maintenance: Strict Adherence to
Manufacturers’ Maintenance Guidelines, Which Have Met
the Requirements of Regulatory Approval, Is
Recommended
Multiple CAP LAP accreditation requirements cover the
topic of tissue processor maintenance in detail (COM.30550,
COM.30660, ANP.231, ANP23120, ANP23130, ANP2330,
ANP23350, and ANP24050), and our recommendation
parallels and reinforces the importance of these require-
ments. The processing of fixed tissue through dehydration
to paraffin infiltration is typically automated, but strict
adherence to standardization of processing reagents and
processor function is needed to avoid specimen compro-
mise. It is critical that reagents of high quality be used and
regularly maintained.
In particular, adequate maintenance of alcohols is critical.
Inadequate maintenance may lead to inadequate dehydra-
tion of tissues with consequent molecular degradation in
blocks in storage due to hydrolysis.1 In addition, ‘‘topping
off’’ of processor chambers with nonstandard solutions
should be strictly forbidden. Timer settings should be
monitored to assure that the total time in formalin
1356 Arch Pathol Lab Med—Vol 143, November 2019
(including that in the processor) does not exceed the target
upper limit recommended here.
Type of Paraffin: Pure Low-Melt Paraffin (Melts at ,608C)
Is Recommended
The use of high-quality paraffin that liquefies at low
temperatures can help avoid molecular damage created by
high temperatures.1,6 Paraffin wax is an alkane, a family of
aliphatic (acyclic) saturated hydrocarbons. The longer the
chain (including branch chains) the higher the melting
point. The use of high-melting-point paraffins is associated
with inadequate deparaffinization, reduced recovery of
biomolecules from the tissue, and reductions in the extent
and intensity of immunostaining.1,93,166,170
Block Storage Conditions: Maintenance of All Paraffin
Blocks in Dry, Pest-Free Conditions at Room Temperature
(Defined as 258C) Is Recommended
Nucleic acids and proteins extracted from FFPE blocks of
human neoplasms collected and processed according to a
standardized, evidence-based protocol and maintained
under these conditions have been demonstrated to be
comparable in quantity and quality over a time span of 1 to
12 years.171 Molecular degradation in stored tissue is most
often due to poor fixation or inadequate processing,
especially inadequate dehydration, before paraffin embed-
ding and storage.1
Documentation Data: Documentation of Compliance With
Critical Preanalytical Parameters or, at a Minimum, the
Recording of Any Deviations From the Recommendations
for Critical Preanalytical Parameters, Should Be Carried
Out for Tissue Biospecimens and the Data Included in the
Pathology Report
Currently, the preanalytical history of most tissue
specimens is unknown, making it difficult to judge their
fitness for molecular testing. Documentation of cold
ischemia time and total time in fixative, important determi-
nants of molecular quality and key factors in the specimen
provenance, is not routine at present. It is standard to
include the fixative used in the gross description. It is not
necessary to include the details of the composition and
quality of the formalin, as this should be included in a
laboratory’s standard operating procedures and quality
control logs; this is also true for processor maintenance,
paraffin type, and block storage conditions. Specimen
thickness and volume to mass ratio of fixative are likely to
be part of standard laboratory procedures and their
monitoring part of the CAP LAP checklist (ANP.10038,
ANP.11716). Ensuring adequate size containers for large
specimens and ample availability of formalin are likely to be
helpful for ensuring the correct volume to mass ratio of
fixative, and checklist item ANP.11250 requires that there be
adequate storage for large specimens.
THE KEY PREANALYTICAL VARIABLES FOR BLOOD
SPECIMENS
The PPMPT practice recommendations for control of
essential preanalytical variables for blood specimens, based
on current evidence, are discussed below. As with tissue
specimens (above), citations include relevant publications
that are not limited exclusively to the publications reviewed
by the PPMPT. Older publications with original data
pertinent to the recommendations herein and academic
Preanalytical Practices Precision Path—Compton et al
reviews that include references to original data published
before 2013 are also cited.
As a basic reference for comparison, a summary of the
analogous preanalytical parameters for blood specimens
recommended by various authoritative sources including the
CAP, Clinical & Laboratory Standards Institute (CLSI), ISO,
European Committee for Standardization, and the NCI is
shown in Table 2.
As with the recommendations for tissue specimens
discussed above, the following recommendations for blood
specimens are focused on blood specimens from cancer
patients who are undergoing analysis for nucleic acids or
proteins.
Time to First Processing Step: The First Step in the
Processing of Blood Specimens Should Begin Less Than 60
Minutes After the Blood Draw Unless EDTA or Cell-
Stabilization Specialty Tubes Are Used
Ideally, processing for any blood specimen should begin
as quickly as possible after the blood draw.172 Some blood
specimens require immediate centrifugation and processing
(eg, blood for amino acid analysis).173 For blood samples
from cancer patients who are undergoing analysis for
circulating cell-free DNA or RNA from tumors (ie, ‘‘liquid
biopsies’’), plasma is judged to be the optimal specimen
type.46 Minimization of the time between the blood draw
and the first specimen processing step (separating the
plasma from the blood) is especially important in controlling
artifact from normal cellular components in the blood. A
timeframe of 60 minutes is applicable to either serum or
plasma processing but is critical if serum samples are used
for this analysis, since cell lysis during clot formation creates
significant contamination within an hour.174 Blood drawn in
ethylenediaminetetraacetic acid (EDTA) tubes for plasma
derivation is more forgiving but should be processed within
6 hours.46,175–178 Specialty tubes that stabilize the cellular
components of the blood permit much greater flexibili-
ty.174,175,177–186 In practice settings in which blood draws
occur at sites distant from the analysis laboratory, the
flexibility in the time-to-processing step provided by cell-
stabilization specialty tubes may be an important option.
Alternatively, special arrangements for immediate transpor-
tation to the laboratory following the blood draw may be
required.
Specimen Acquisition: Factors That Are Recommended to
Be Controlled and Recorded Are (1) Blood Collection Tube
Type, (2) Tube Fill Level, (3) Draw Order, and (4) Number
of Tube Inversions (Adequate Mixing) as Detailed Below;
Documentation of Deviations From Recommended
Parameters Is Advised
Tube Type: Recommendations for Tube Type Selection
Are Listed Below.—
1. EDTA tubes or cell-stabilization specialty tubes are
recommended for either proteomic studies or cell-free
DNA analysis.
2. Lithium heparin and sodium heparin tubes are contra-
indicated for nucleic acid amplification studies.
When using tubes with additives, such as EDTA tubes or
specialty tubes for cell stabilization, the tube fill level of the
tube and the number of tube inversions are critical factors.
These factors alter the final concentration of the additive in
the tube content and the uniformity of distribution of the
Arch Pathol Lab Med—Vol 143, November 2019
additive through the tube content, respectively. Either factor
may alter tube performance and lead to suboptimal analyte
quality and skewed analysis results. Lithium heparin tubes
are not suitable for nucleic acid analysis by PCR because
lithium heparin is a PCR inhibitor.172,187–189
Volume of Tube Fill: The Optimal Tube-Fill Volume per
the Tube Manufacturer’s Recommendation Is Advised.—
Tube additives are calibrated to provide optimal ratios of
blood to additive. Therefore, in tubes with additives, the
tube fill level is an essential quality indicator for the test
sample and should be documented at the time of the blood
draw.190
Draw Order: The Recommended Draw Order Is as
Shown Below (With Consideration to Alterations as
Indicated Clinically) and Is Only Applicable if Multiple
Specimens Are Being Collected at 1 Draw.—Prioritized
Draw Order, First to Last
1. Blood culture bottles (contains broth)
2. Nonadditive tube
3. Coagulation tube (contains sodium citrate)
4. Clot activator tube (contains a clot activator)
5. Clot activator plus serum separator tube (contains a clot
activator and separator gel)
6. Heparin tube (contains the anticoagulant sodium hep-
arin or lithium heparin)
7. EDTA tube (contains the anticoagulant EDTA)
8. Tubes with other additives (eg, tubes containing acid-
citrate-dextrose; oxalate/fluoride; antiglycolytic agent).
The rationale for adherence to a standardized draw order
is based on minimizing cross-contamination of additives
between tubes to ensure the accuracy of the analysis results
from each tube. The draw order above is the standard for the
CLSI (H3-A6; GP 41)191,192 and the World Health Organi-
zation.193 It is also recommended in phlebotomy guidance
from the CAP.173 Although phlebotomy standards have
existed for more than a decade, recent studies have shown
that compliance with the 29 items on the CLSI guidelines in
European countries is unacceptably low36 and that draw
order is 1 of the top 3 major errors made in phlebotomy,
accounting for 21% of all errors.194
Method of Stabilization: Tube Inversions per
Manufacturer’s Recommendations Is Advised
This critical parameter, if not rigorously followed, leads to
inadequate mixing of the blood with the tube additive and
compromise of the key chemical reaction(s) in the tube.195
Method of Processing: Control and Documentation of
Centrifugation Speed, Centrifugation Time, and
Temperature Is Recommended
Centrifugation is the processing step that separates the
serum or the plasma to be analyzed from the cellular
components (and for serum, clotting proteins) of the blood.
Inadequate speed or inadequate numbers of centrifugation
steps may lead to contamination of the plasma/serum
sample with cellular content.175–177 Cells are typically
adequately separated from plasma by centrifugation at
1000g to 2000g for 10 minutes in a refrigerated centrifuge.172
Temperature is a major variable during many preanalytical
steps. Clot formation in the generation of serum samples,
for example, is temperature sensitive. The integrity of some
proteins in the blood may be temperature sensitive, whereas
the enzymatic activity of others may be temperature
Preanalytical Practices Precision Path—Compton et al 1357
dependent. Maintenance and monitoring of processing
temperatures is a key quality factor.172 Processing at room
temperature (defined as 188C–258C) is recommended unless
a validated protocol dictates otherwise. Specimen process-
ing should be standardized for centrifugation speed, time,
and temperature and written into the standard operating
procedures of the laboratory.
Storage Conditions: It Is Recommended That Freeze-Thaw
Cycles on Plasma or Serum Specimens Be Avoided
Altogether by Aliquoting Before Freezing; for Nucleic
Acids and Proteins, No More Than a Single Freeze-Thaw
Cycle Is Acceptable46
Repeated freeze-thaw cycles contribute to biomolecular
degradation and are detrimental to biospecimen quali-
ty.177,196–198 Mechanisms by which the freeze-thaw process
may damage biomolecules in the sample include physical
shearing by ice crystals and microshifts in solute concen-
trations with effects on pH and metal ions that catalyze
oxidative damage.199 The extent of damage with each freeze-
thaw cycle is difficult to quantitate and may vary according
to the biomolecular species of interest. It is prudent to avoid
freeze-thaw altogether by aliquoting serum/plasma samples
before freezing. Peripheral blood specimens should not be
frozen because induced hemolysis can result in PCR
inhibition through the presence of contaminating hemo-
globin.
Documentation Data: Documentation of Compliance With
Critical Preanalytical Parameters or, at a Minimum, the
Recording of Any Deviations From the Recommendations
for Critical Preanalytical Parameters Should Be Carried
Out for Every Blood Biospecimen and the Data Included in
the Laboratory Report
CONCLUSIONS AND NEXT STEPS
It is important to emphasize that the recommendations
herein represent a baseline for tissue and blood specimens
from cancer patients that are handled in routine pathology
practice to ensure that they are generally fit for most
molecular analysis of nucleic acids and proteins while not
affecting other routine uses of the sample (such as histology
and immunohistochemistry). The recommendations are not
optimized for all specimen types, analyses, or analytic
platforms. It is recognized that preanalytical parameters may
need to be customized for specific applications that are less
common.
The fitness of a tissue or blood specimen for molecular
analysis depends on a number of preanalytical factors, any
of which may affect assay results and have the potential to
bias the analysis data or even preclude successful analysis
altogether.2 Documentation of preanalytical factors needs to
be part of the pathology record for every cancer specimen,
so that its fitness for molecular testing can be determined
before analysis and the quality of results evaluated
appropriately.5
Preserved specimens are also used for future testing for
downstream cancer patient care and/or for scientific
investigation. The provenance of a tissue or blood specimen
should be documented in the pathology report in order to
determine its appropriateness for molecular analysis in
either setting. It has been noted that published translational
research using patient biospecimens rarely contains infor-
mation about how the samples have been obtained,
1358 Arch Pathol Lab Med—Vol 143, November 2019
processed, or stored. One survey of 125 biomarker discovery
publications between 2004 and 2009 found that fewer than
half contained information about sample provenance,
making it impossible to determine how specimen quality
may have impacted the study data.200
In the case of blood/serum specimens, analyses for
circulating cell-free nucleic acids from tumors and/or
circulating tumor cells have recently taken center stage in
oncology.201–203 Often called ‘‘liquid biopsies,’’ these blood
analyses are also sensitive to preanalytical factors. In an
effort to confer consistency and comparability among
studies of the various potential uses of liquid biopsies in
clinical decision-making for cancer patients, ASCO and
CAP jointly reviewed the published data and developed a
consensus statement for preanalytical and analytical steps
for circulating cell-free nucleic acid analysis for cancer
patients.46 The recommendations herein mirror these
guidelines, underscore the need for attention to preanalyt-
ical considerations, and draw attention to the gaps in
current knowledge about specific preanalytical factors that
affect analysis results. Although ‘‘liquid biopsies’’ are topical
at present, the same preanalytical challenges exist for blood-
based molecular tests of many sorts. At the point of care,
blood draws may vary according to the setting, the training
of the staff performing the phlebotomy, and many other
highly variable factors.49–51,204
The current paradigm for immunotherapy also raises
issues related to preanalytics in the testing for response
markers already approved by the US Food and Drug
Administration and for those in development.205 Although
programmed death ligand-1 (PD-L1) immunohistochemis-
try has been approved for several immune oncology
indications and has been incorporated into the non–small
cell lung cancer clinical practice guidelines from ASCO and
NCCN,206,207 the impact of preanalytical variables on the test
results for PD-L1 and other immune oncology markers is
only beginning to be understood.208,209 As the immune
oncology field continues its rapid pace of evolution and
development, progress will be dependent on clarifying the
sensitivity of immune biomarkers to preanalytical variables
and the clinical consequences of sample handling devia-
tions.
For all patient biospecimens, blood and tissue specimens
alike, it is also recognized that significant preanalytical
variation may even result from events occurring before
acquisition of the specimen (ie, removal of the specimen
from the patient). For surgically resected tumors, for
example, preacquisition variables are largely related to
preoperative and intraoperative events and cannot be
controlled by the pathologist or the laboratory. These
preanalytical factors include a wide variety of drugs and
infusates, general anesthesia, surgical manipulations, and
devascularization of the tissue before resection.80 One
important intraoperative variable is the elapsed time
between devascularization and removal of the tissue from
the body, known as ‘‘warm ischemia time.’’ This time varies
according to the surgical procedure and the individual
surgeon, but can be recorded as part of the specimen
history, possibly in the anesthesia record. At present, this is
rarely if ever done and is an unknown element in the
provenance of almost all surgical tissue specimens. The
documentation of warm ischemia time and the control of
cold ischemia time will both require the cooperation of
surgeons. The American College of Surgeons has already
Preanalytical Practices Precision Path—Compton et al
demonstrated interest in educating their members on this
issue and becoming part of the preanalytics solution.210
It is in the postacquisition setting that preanalytical
variables are most amenable to control and monitoring by
pathologists. Post acquisition, tissue specimens are still
viable until their biological activity is stopped by stabiliza-
tion (ie, fixation or freezing). Before stabilization, their
molecular composition may change artefactually in response
to biological stresses and physical factors linked to handling
and processing. Molecular degradation also may occur
during this period and may be further compounded by
processing variables after stabilization. Objective quality
assessment methods (quality indicators) can be used to
evaluate specific molecular components of the tissue before
assay (eg, extracted RNA and DNA quality and quantity),
but the overall molecular integrity of the tissue cannot be
retroactively recovered once it is compromised. It must be
safeguarded upfront by control of the most problematic,
quality-compromising preanalytical variables. Furthermore,
at the time of acquisition, it may not be known whether or
not a biospecimen will be undergoing molecular testing,
either as part of immediate patient care or in the future.
Therefore, it is prudent and reasonable to treat all patient
specimens in a uniform manner that safeguards molecular
integrity and ensures their fitness for molecular analysis as a
routine part of patient care. In working toward this goal, a
tiered approach may be considered, beginning with cancer
specimens and specimens in which malignancy may be
suspected, given the rapidly increasing use of molecular
analyses for patient management in oncology.
While this work is focused primarily on the role of the
pathologist in postacquisition preanalytics, it should be
emphasized that many other medical professionals play key
roles in patient specimen acquisition, handling, and
transport. In addition to surgeons, as mentioned above,
these include nursing staff, pathology assistants, phleboto-
mists, endoscopists, and interventional radiologists, among
others. Any of these individuals may be part of the chain of
custody of the specimen. At present, few recognize their role
in the specimen quality continuum, and none have practices
in place to govern their specific role or coordinate with the
practices of others in the chain of custody. Ultimately, the
solution to the overall quality management of patient
biospecimens will, perforce, include all of these profession-
als, not just pathologists.
For pathology practice, the CAP PHC and PPMPT
endorse the practicable set of benchmark practices put
forward here. Compliance with these data-driven practice
metrics is a first and essential step toward achieving a
seamless quality continuum for patient biospecimens, which
is, in turn, a requisite step toward enabling precision
medicine for all patients. Compliance with these recom-
mendations would also ensure a new level of molecular
quality and consistency for tissues used in translational
research and serve to reduce variation and irreproducibility
of analysis results. Both of these endpoints will bring new
benefit to the patients in our care presently and in the
future.
A CALL TO ACTION
The assessment of molecular biomarkers in patient
biospecimens already plays a central and growing role in
precision medicine and is likely to continue to increase, for
cancer as well as many other diseases. It is recognized that
Arch Pathol Lab Med—Vol 143, November 2019
attention to preanalytical issues has lagged behind, a gap
that is now becoming critical for precision medicine. In a
recent report from the National Academies of Science,
Engineering, and Medicine entitled Biomarker Tests for
Molecularly Targeted Therapies: Key to Unlocking Precision
Medicine,211 it was specifically pointed out that enhancing
‘‘specimen handling and documentation to ensure patient
safety and accuracy of biomarker test results’’ is crucial for
the advancement of precision medicine. This recommenda-
tion included all clinical trials in which molecular data from
biospecimens are integral to patient selection and manage-
ment, as well as scientific insight from correlative experi-
mental studies. It is urgent that pathologists and other
medical professionals who are part of the quality chain for
patient biospecimens implement a coordinated, evidence-
based approach to preanalytics to meet the requirements of
precision medicine.
The authors thank the highly knowledgeable and seemingly
tireless expert CAP staff who supported this project during the last
4 years. In particular we thank Patricia Vasalos, BS, technical
manager, Proficiency Testing; Molly Hansen, CT, cytology technical
specialist II, Proficiency Testing; Jill Kaufman, PhD, former director
of the Personalized Healthcare Committee; Tony Smith, MLS-
records and information manager; Brooke L. Billman, MLIS,
medical librarian; Kelly Westfall, BA, former operations specialist,
Surveys; Denise Driscoll, MS, MT, senior director, CAP Laboratory
Accreditation and Regulatory Affairs; and Dawna Mateski, MT,
checklist customization analyst, CAP Accreditation Programs.
Without their expertise and aid, this project could never have been
done.
We offer a special thanks to Debra Leonard, MD, PhD, former
chair of the PHC, who helped establish the PPMPT and supported
its goal from the outset and Michelle O’Connor for her clerical
assistance.
We also thank the CAP leadership, past and present, especially
Jared Schwartz, MD, PhD, Gene Herbek, MD, Richard Friedberg,
MD, PhD, and Bruce Williams, MD, who provided encouragement
for this work and shared the vision of improving pathology practice
everywhere for the benefit of our patients.
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