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 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 composition. 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 Accreditation 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.
OVERVIEW: THE CRITICAL ROLE OF PREANALYTICS IN THE MOLECULAR ANALYSIS OF PATIENT BIOSPECIMENS
Biospecimens 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 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 pathology. 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 uninterpretable, 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 compromised.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 organizations 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 Medical 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 preanalytics 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 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 preanalytical factors are critical to ensuring the molecular integrity of patient biospecimens for molecular analysis. The recommendations developed by the PPMPT are based solely on human biospecimen research data. They are focused on controlling and documenting these variables, for specimens 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 important initial step for the pathology community in demonstrating 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 implementation 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.
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 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 decreasing 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 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 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 undergoing testing has been corrupted by preanalytical factors. Therefore, to assure that analysis results are both reliable and biologically meaningful, the integrity of the biospecimen 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 estimated 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 questionable or unacceptable quality may be performed without the knowledge of the tester, producing data of questionable or unacceptable quality without the knowledge of the interpreter 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 measurands 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, convened a cross-sector national conference to address the critical issue of uneven and unknown quality of human biospecimens in clinical medicine and translational research.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 professional 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 acquisition, 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 biomedicine. 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 Specifically, 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 preanalytical 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. Subsequently, an extensive literature search for recent biospecimen 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 literature 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 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:
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.
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 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 interpretations 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 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 implementation. 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 recommended by nationally and internationally recognized authoritative sources is shown in Table 1.
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 temperature.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 ischemia.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 4°C.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–dependent.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 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 literature.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 biomolecular species as well as morphology. It is relatively inexpensive, 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 (25°C), 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 processes. 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 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 recommendation 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 (25°C).90 Notably, however, because the chemistry of fixation is slowed at temperatures lower than 25°C, 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 increasing fixation time.87 Not only does cross-linking of nucleic acids with histones occur with formalin fixation, but formalin reacts directly with nucleotides, causing molecular 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 immunoreactivity of proteins also may be adversely affected by prolonged fixation,4,168,169 but some studies have shown that protein identifications by multidimensional liquid chromatography–tandem mass spectrometry in FFPE tissue subjected 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 stipulate 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 requirements. 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 compromise. 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 dehydration 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 (including that in the processor) does not exceed the target upper limit recommended here.
Type of Paraffin: Pure Low-Melt Paraffin (Melts at <60°C) 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 25°C) 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 embedding 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 determinants 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 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 flexibility.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 transportation 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
EDTA tubes or cell-stabilization specialty tubes are recommended for either proteomic studies or cell-free DNA analysis.
Lithium heparin and sodium heparin tubes are contraindicated 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 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
Blood culture bottles (contains broth)
Coagulation tube (contains sodium citrate)
Clot activator tube (contains a clot activator)
Clot activator plus serum separator tube (contains a clot activator and separator gel)
Heparin tube (contains the anticoagulant sodium heparin or lithium heparin)
EDTA tube (contains the anticoagulant EDTA)
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 Organization.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 dependent. Maintenance and monitoring of processing temperatures is a key quality factor.172 Processing at room temperature (defined as 18°C–25°C) is recommended unless a validated protocol dictates otherwise. Specimen processing 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 quality.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 concentrations 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 hemoglobin.
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 information about how the samples have been obtained, 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 preanalytical 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) immunohistochemistry 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 deviations.
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 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 stabilization (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, phlebotomists, 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 professionals, 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 recommendations 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 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 recommendation included all clinical trials in which molecular data from biospecimens are integral to patient selection and management, as well as scientific insight from correlative experimental 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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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.