Formalin fixation and paraffin embedding is a timeless, cost-efficient, and widely adopted method of preserving human tissue biospecimens that has resulted in a substantial reservoir of formalin-fixed, paraffin-embedded blocks that represent both the pathology and preanalytical handling of the biospecimen. This reservoir of specimens is increasingly being used for DNA, RNA, and proteomic analyses.
To evaluate the impact of preanalytical factors associated with the formalin fixation and paraffin embedding process on downstream morphological and molecular endpoints.
We surveyed the existing literature using the National Cancer Institute's Biospecimen Research Database for published reports investigating the potential influence of preanalytical factors associated with the formalin fixation and paraffin embedding process on DNA, RNA, protein, and morphological endpoints.
Based on the literature evidence, the molecular, proteomic, and morphological endpoints can be altered in formalin-fixed, paraffin-embedded specimens by suboptimal processing conditions. While the direction and magnitude of effects associated with a given preanalytical factor were dependent on the analyte (DNA, RNA, protein, and morphology) and analytical platform, acceptable conditions are highlighted, and a summary of conditions that could preclude analysis is provided.
Formalin fixation and paraffin embedding are part of a globally applied method of tissue preservation; however, they also represent a multistage process that is far from standardized. A recent review article1 published by our office identified 15 preanalytical factors associated with formalin fixation and paraffin embedding tissue processing that have documented effects on immunohistochemistry (IHC) efficacy and many more that were unaddressed or underaddressed in the scientific literature. While technological advancements afford the molecular analysis of formalin-fixed, paraffin-embedded (FFPE) biospecimens, efforts have achieved varying levels of success, which may be a result of differences in FFPE processing regimens or extraction techniques. In the present review, we summarize reported effects of FFPE processing factors on molecular and morphological endpoints, explore differences between analytes, and underscore evidence-based and analyte-specific recommendations for specific preanalytical factors when possible. It is our aim that this review will serve as a resource both for the evaluation of archival FFPE specimens and as a guideline for the collection of new FFPE specimens. Although additional sources of preanalytical variability, including extraction methods, antigen retrieval techniques, and patient-related factors, may be capable of influencing analytical endpoints, the scope of the present review was limited to evidence available for FFPE fixation and processing factors.
Also see p. 1426.
MATERIALS AND METHODS
Potential sources of preanalytical variability associated with the procurement, fixation, processing, and storage of human FFPE tissue biospecimens were identified based on the experience of the authors, data contributed to the Biospecimen Research Network (http://biospecimens.cancer.gov/researchnetwork), and literature evidence and are summarized in Table 1. Targeted surveys were conducted for each preanalytical factor. Relevant peer-reviewed, primary research articles that used human FFPE tissue biospecimens were located through the Biospecimen Research Database (http://biospecimens.cancer.gov/brd), a free online resource from the National Cancer Institute's Biorepositories and Biospecimen Research Branch, and through the National Institutes of Health's PubMed database (http://www.ncbi.nlm.nih.gov/pubmed), as well as by cross-referencing. Studies that failed to adequately report methodology or that used animal models or cell or tissue cultures for study were omitted from the meta-analysis. Because of the prevalence of analyte-specific effects, meta-analysis findings and evidence-based recommendations were organized by both analyte and preanalytical factor (Table 2).
ACCEPTABLE FFPE TISSUE HANDLING CONDITIONS FOR DNA ANALYSIS
The available literature indicates that postmortem interval (PMI), cold ischemia (defined as the time between biospecimen removal from the body and its preservation), specimen size, and decalcification method can each affect subsequent DNA analysis of FFPE specimens. However, differences in recorded or presumed warm ischemia time (the time a biospecimen remains at body temperature after its blood supply has been restricted) have not altered DNA integrity2 or polymerase chain reaction (PCR) success rates3,4 in FFPE biospecimens, although further investigation is warranted because time points were limited to 20 and 80 minutes2 or were unreported for biopsy and surgical resection comparisons.3,4 Importantly, additional prefixation factors have not been addressed in the literature for their potential effects on DNA endpoints.
Based on published findings, detectable effects of a delay to preservation may be dependent on the platform used for DNA analysis. DNA extracted from FFPE tissue that was subjected to a cold ischemia time of 1 hour displayed reduced fluorescent in situ hybridization (FISH) signals,5 although a cold ischemia time of 24 hours did not alter PCR amplification success rates (data not shown).6 Similarly, FISH signals were reduced after a PMI of 48 hours compared with a PMI of 1 hour in FFPE biospecimens,7 while PCR success remained unaffected after a PMI of 4 days8 or 8 days.9
Investigations of the potential influence of biospecimen size before fixation found that PCR success rates were highest when DNA was extracted from specimens that were 3 to 10 mm in diameter as opposed to smaller specimens (multiple sections were needed) or larger specimens (a higher background was encountered).10 Importantly, extraction method4 and amplicon size3 have been shown to influence PCR success rates when different-sized FFPE biospecimens were examined because optimization was required for extraction from 2-mm2 biopsy biospecimens compared with larger surgically resected biospecimens (16 × 18 mm),4 and PCR amplification was unsuccessful for a 435–base pair fragment in the smallest biopsy examined (0.36 mm2).3
DNA analysis was also reportedly affected by decalcification method. Reports agree that decalcification using ethylenediaminetetraacetic acid (EDTA) as opposed to acid-based methods was beneficial because it allowed for amplification of longer PCR products,11 reduced background staining and stronger FISH signals,12–14 and superior determination of loss and gain of sequences for comparative genomic hybridization.12 However, a study14 reported superior results when EDTA was used in conjunction with ultrasound compared with EDTA alone. When different concentrations of formic acid were compared, decalcification with 5% formic acid for 12 to 18 hours produced FISH signals, while decalcification in a 10% formic acid solution for 7 to 10 days abolished the signal.15
Preanalytical factors pertaining to biospecimen fixation that have documented effects on downstream DNA analysis include whether or not the formalin was buffered, what the time and temperature of fixation were, and whether formalin penetration of the tissue was expedited by ultrasound or microwave irradiation.
In general, when unbuffered formalin and neutral buffered formalin (NBF) were compared, the literature is in agreement that DNA extracted from NBF-fixed specimens gave greater yields16 and in situ hybridization (ISH)17 and genotype determination18 success rates. However, reports are divided as to whether PCR success rates were superior10,16,19–23 or equivalent19,21 for NBF-fixed specimens compared with those fixed in unbuffered formalin.
Most reports agree that fixation time can affect DNA analysis and that less than 72 hours in formalin was preferable to longer durations for DNA integrity24 and yield,25 as well as PCR,6,14,21,24,26–29 ISH,7,30 and single-nucleotide polymorphism detection assay performance.18 Amplification success after prolonged fixation was reportedly influenced by the target sequence,27 amplicon length,6,14,21,31 and tissue type.21 In contrast, long-term fixation had no effect on successful PCR amplification of nuclear DNA,32,33 sequencing,32,33 DNA yield,24 or viral8 and mitochondrial DNA21 amplificability.
Studies comparing fixation temperatures at and above ambient reported reductions in DNA yield and integrity25 and PCR success22 at elevated temperatures (37°C or 60°C). However, it remains unclear whether fixation at ambient or 4°C is preferable because fixation at 4°C increased the yield of high-molecular-weight DNA and PCR success,34 while fixation at ambient temperature increased amplification efficiency.22 Yet another study35 reported equivalent DNA integrity and PCR amplification success rates between the 2 fixation temperatures.
Little information is available on the potential impact fixation acceleration methods may have on DNA. However, there is some evidence that microwave-accelerated or ultrasound-accelerated fixation can improve the yield of high-molecular-weight DNA36,37 and PCR success rates36,38–40 from FFPE tissue.
Processing and Storage
Potential influences of dehydration, clearing, and paraffin reagents and conditions on DNA endpoints have remained largely unaddressed in the literature. Evidence is limited to a single study19 that highlighted the importance of using pure paraffin versus a mixture of paraffin and beeswax because of the latter's adverse effects on PCR amplification of some genes.
Long-term storage of paraffin blocks has received considerably more attention, and although some studies have reported that FFPE blocks can be stored for several years with minor effects on subsequent DNA analysis,3,41–48 others have shown that the length of amplifiable DNA29 and whole-genome–amplified fragments49 decreased with block storage even when coupled with optimized DNA extraction procedures. We were able to locate only a single study50 that investigated the effect of storage duration of slide-mounted FFPE sections on downstream DNA analysis: notably, while storage of FFPE sections for 10 years before DNA extraction and analysis was detrimental to PCR success rates, shorter storage durations have not been investigated.
While the literature suggests that a range of paraffin block sizes can be appropriate for DNA extraction and analysis51 and that even fractions of a standard 5-μm section can be successfully analyzed,28 DNA extraction from FFPE sections rather than cores generated higher yields52 and better PCR success rates.10 Similarly, analysis of sections rather than isolated nuclei led to more intense FISH signals.7
As highlighted by meta-analysis evidence, when FFPE specimens are to be used for DNA analysis, PMI and cold ischemia times should be limited to 48 hours and 1 hour, respectively, when DNA is analyzed by FISH and 4 days and 24 hours, respectively, for PCR analysis. Optimally, FFPE biospecimens should be 3 to 10 mm3, decalcified with EDTA when necessary, fixed in NBF for less than 72 hours at ambient temperature or 4°C, and embedded in paraffin free of beeswax. In addition, evidence suggests that microwave-accelerated and ultrasound-accelerated fixed specimens are amenable to DNA analysis. While blocks stored for years have been used successfully for DNA analysis, it is important to note that the length of amplifiable gene fragments may decrease over time. Extraction of DNA from cores, isolated nuclei, or FFPE sections that have been stored for 10 years should be avoided (Table 2).
ACCEPTABLE FFPE TISSUE HANDLING CONDITIONS FOR RNA ANALYSIS
The available literature indicates that suboptimal fixation delays and decalcification methods can affect RNA integrity, amplification success, and ISH staining in FFPE biospecimens. We were unable to locate articles that investigated the potential effects of other prefixation factors on RNA analysis in FFPE specimens, which are summarized in Table 1.
RNA degradation was detectable by Northern blot after a PMI of 4 to 6 hours in FFPE specimens.53 Conversely, neither ISH53 nor reverse transcriptase (RT)–PCR54 success was affected by PMIs of comparable length (6 hours and 4–10 hours, respectively). RNA integrity numbers (RIN) were comparable between FFPE specimens subjected to a cold ischemia time of 0 hours or 2 hours,55 and the relative expression of 6 transcripts did not differ among FFPE specimens subjected to 0 hours or 12 hours of cold ischemia.56 Decalcification of FFPE specimens with EDTA or ultrasound rather than an acid-based method led to stronger and more intense ISH staining57,58 and successful RT-PCR amplification of longer fragments.14,59
Published findings demonstrate several factors capable of altering RNA endpoints in FFPE biospecimens. These include the buffer used in the fixation solution, temperature and duration of fixation, and methods aimed at accelerating the penetration of formalin into the biospecimen.
Although evidence was limited to a single study,60 real-time quantitative RT-PCR cycle threshold values were reduced for biospecimens fixed with NBF compared with those fixed in unbuffered formalin. In terms of acceptable fixation durations for RNA analysis, most studies agree that 8 to 48 hours were suitable for ISH57,61 and RT-PCR57,60–66 analyses but that prolonged fixation (range, 21 days to 11 years) was detrimental to RNA analysis.24,54,64,67 However, there is some disagreement as to whether a shorter fixation duration of 6 hours produced inferior61,66 or comparable57,62 ISH and RT-PCR results, and transcript-specific differences in RT-PCR amplicon levels were reported by 2 studies68,69 for biospecimens fixed for 24 or 48 hours. While fixation at 37°C resulted in poor RNA quality compared with ambient fixation,25 reports conflict as to whether fixation at 4°C generated superior70,71 or equivalent35 RNA quality and levels of amplifiable RNA compared with fixation at ambient temperatures. The performance of accelerated fixation techniques is method-specific for RNA analyses investigated to date, although reductions in processing time continue to be a shared benefit. Ultrasound-accelerated fixation resulted in more intense and uniform ISH staining,39 which better matched protein distribution patterns61 and yielded longer amplicons40,61 and higher levels of amplifiable RNA39,40 compared with immersion fixation. In contrast, cycle threshold values were equivalent in biospecimens preserved by microwave-accelerated fixation and conventional immersion.38
Processing and Storage
To the best of our knowledge, studies investigating effects attributable to different dehydration, clearing, and embedding parameters on RNA analysis in FFPE tissue have not been published. However, the impact of paraffin block storage on RNA endpoints has received considerably more attention. While a few studies observed that 5 to 21 years of paraffin block storage had no effect on amplification of messenger RNA39,56,67 or microRNA and ribosomal RNA72 levels, most studies we identified reported decreased messenger RNA amplification efficiency and success,41,59,60,73–78 as well as reduced RNA integrity, as determined by RNA integrity number or electrophoresis,60,74,76 after storage for 2 to 20 years compared with those stored for 1 year or less. The occurrence and magnitude of effects attributable to storage duration appear to be influenced by amplicon size,59,74,78 as well as primer or probe location within the transcript, because 3′ ends were generally more intact than upstream regions after fixation and embedding.75 Effects associated with the storage of slide-mounted tissue sections on RNA analysis have received less attention, although a single study79 reported greater real-time quantitative RT-PCR success for FFPE sections stored for 90 days or less at ambient temperature compared with those stored for longer durations or at 4 or −80°C.
Based on the available literature, if FFPE tissue is to be used for RNA analysis, PMI and cold ischemia time should be less than 4 and 12 hours, respectively, and specimens should be decalcified with EDTA or by ultrasound when necessary. Time in fixative should be limited to 8 to 48 hours in NBF at ambient temperature or 4°C. The FFPE blocks should be analyzed within 1 year, and sections should be stored at room temperature for no longer than 3 months (Table 2).
ACCEPTABLE FFPE TISSUE HANDLING CONDITIONS FOR PROTEIN ANALYSIS
The reported effects of preanalytical factors associated with FFPE fixation, processing, and storage on IHC have been reviewed in detail previously.1 Those effects are summarized herein and provide a framework and context for the effects of preanalytical factors on other proteomic applications.
Evidence from the literature indicates that cold ischemia, specimen size, and decalcification method can each affect protein analysis. However, many effects are antigen-specific, and the focus of many such investigations has been on IHC.
Published reports agree that cold ischemia time should be minimized to less than 12 hours to avoid significant negative effects on IHC staining.5,80–83 While the shortest cold ischemia duration for which an effect has been reported was 1 to 2 hours, the reported decline in the percentage of estrogen receptor–immunopositive and progesterone receptor–immunopositive cells was nonsignificant.5 Large specimens (1.2–3.5 mm3) were preferred over smaller punch biopsy specimens (0.7 mm3) for Western blot analysis because of the lower abundance of high-molecular-weight proteins reported in the latter.84 Placing specimens in any one of a number of gelatinous solutions before fixation or using specimens that were frozen and cryosectioned before fixation led to decreased immunostaining for some antigens.85–87 Investigations on the potential effects of decalcification method on protein endpoints were limited to IHC, for which tissue-specific and antigen-specific effects on immunostaining were reported87–89 ; therefore, based on available literature, there is not a preferred universal decalcification method for IHC analysis of FFPE specimens.
Literature evidence concerning the effects of fixation-associated preanalytical factors on protein endpoints was limited to IHC analysis and included fixative composition, fixation duration and temperature, and methods used to expedite formalin penetration. Conversely, the tissue to fixative ratio (1:1 to 1:20) did not reportedly alter IHC results for multiple antigens, although data were limited to a single study.90
IHC staining was reported to be optimal when tissue biospecimens were fixed in a buffered86,87 10% to 15%91,92 formalin solution with a neutral pH,90,93 although antigen-specific exceptions were also reported for several tissue types.94 In general, fixation at ambient temperature for 6 to 24 hours led to acceptable immunostaining,61,86,87,90,91,95–102 while prolonged fixation times for 3 days or more were detrimental.86,87,95,101 For proteomic applications, fixation durations of 6 to 24 hours were favored compared to longer durations. Compared with biospecimens fixed for 1 day, fixation for 2 days resulted in fewer observable mass spectrometer spectra, and fixation for 4 days yielded fewer identifiable protein groups.103 A fixation duration of 24 to 144 hours compared with 6 hours resulted in progressive reductions in both total protein yield, quantified by the Bradford protein assay, and specific levels of 4 proteins targeted by Western blot.104
While conventional fixation traditionally occurs at ambient temperature, several studies93,105,106 have reported superior IHC staining at 4°C. The literature also indicates that fixation durations should be adequately shortened at temperatures higher than ambient to avoid overfixation.90
Several studies have shown that formalin injection,107 perfusion,37,108 ultrasound-acceleration,39,61 heat-acceleration,109 or microwave-acceleration37,110 yielded IHC results superior to conventional immersion fixation for several antigens, although equivalent IHC results have also been reported for the same delivery methods.36,37,40,111 Analysis by Western blot revealed a greater abundance of total and specific proteins for specimens fixed by ultrasound acceleration compared with conventional immersion.61 While some of the effects associated with fixative delivery method may be antigen-specific,37 antigen retrieval techniques may be an additional confounding variable.
According to our survey of the literature, suboptimal dehydration, clearing, and embedding reagents and conditions can have detrimental effects on protein analysis by IHC, although each of these preanalytical factors is associated with antigen-specific effects, confounding the identification of ideal and deleterious reagents and conditions. The FFPE processing–related factors that have documented effects on IHC endpoints include dehydration reagent, temperature and duration, clearing agent and temperature, and embedding reagent and temperature. To the best of our knowledge, data on the potential influence of these factors on protein analytical platforms other than IHC have not been published.
The use of isopropanol for dehydration resulted in superior immunostaining for surface membrane glycoproteins compared with other alcohols in one study,93 but equivalent estrogen receptor immunostaining was reported for a variety of dehydration solutions in another study,112 suggesting that effects may be antigen-specific. The total duration of dehydration did not adversely affect immunostaining when it was limited to 5 to 10 hours90,93,112 ; however, dehydration for 24 hours required trypsin digestion before IHC.113 Although it is clear from the literature that the temperature of dehydration can impact IHC results, reports conflict over the preferred temperature. Superior IHC results have been reported in specimens dehydrated at 4°C,93,114 −20°C,93 and 45°C90 compared with ambient temperature controls. Furthermore, dehydration at an elevated temperature may accelerate the process because specimens dehydrated in 100% alcohol for 5 to 20 minutes at 67°C, followed by 5 to 20 minutes in 100% isopropanol at 74°C, produced IHC results equivalent to those of specimens subjected to dehydration in a graded alcohol series for 2.5 to 5 hours at 40°C.115
Multiple studies report equivalent IHC results with several different clearing agents, including the following: (1) xylene, chloroform, Clearene (Surgipath, St Neots, England), and xylene substitute90 ; (2) chloroform, Inhibisol (methyl chloroform containing a patented inhibitor; Bestobell Chemical Products Ltd, Mitcham, Surrey, England), and xylene if sections were digested with trypsin before IHC116 ; or (3) xylene, isopropyl alcohol–chloroform (1:1), chloroform, and chloroform-xylene (1:1).112 Still, other studies identify xylene and chloroform93 or just chloroform (compared with xylol after tissue digestion with trypsin)113 as superior clearing agents. Reports conflict as to the preferred temperature of clearing for IHC. Compared with specimens that underwent clearing at ambient temperature, 45°C improved the intensity of immunostaining,90 while 4°C reduced background staining.114
While several studies have investigated the potential IHC impact associated with the use of various embedding reagents, different commercial products were compared in each study, which precludes product-to-product comparisons. However, immunostaining was superior with low-melting-point (45°C) polymer paraffin as opposed to high-melting-point (65°C) polymer paraffin93 and with nonpolymer paraffin compared with polymer paraffin,113 although equivalent IHC results were reported for polymer, polymer with dimethyl sulfoxide, nonpolymer, and microcrystalline paraffin.90
Regarding the temperature and duration of impregnation, both conventional (56–60°C for 1–2 hours)112,115 and rapid microwave-assisted (15–20 minutes)115 paraffin impregnation reportedly yielded adequate IHC results. However, another study90 reported that the optimum duration of paraffin embedding for IHC may be antigen-dependent.
In general, long-term storage of paraffin blocks does not significantly impact IHC staining because studies have reported stable antigenicity of freshly cut sections from blocks that were 4 to 68 years old,117–124 with the notable exception of a decline in proliferating cell nuclear antigen immunostaining after storage for 3 years or longer.125 Conversely, for other protein applications that require extraction, long-term storage of FFPE blocks should be avoided. The amount of extractable protein decreased by approximately 50% in FFPE specimens stored for 14 or 20 years compared with those stored for less than 1 year.84,104 Reports conflict as to whether archival FFPE blocks are suitable for Western blot analysis. One study126 reported weak or no immunoblotting signals from FFPE specimens from blocks stored for 6 months to 4 years, while another study84 found detectable levels of all antigens investigated in blocks stored for 1 to 20 years. Proteome profiles generated by capillary isotachophoresis–reversed-phase liquid chromatography–mass spectrometry were highly correlated among FFPE blocks stored for 6, 11, and 18 years, despite modest declines in yield and total and distinct peptides at the 18-year time point.121 Similarly, Sprung et al103 observed no significant differences in liquid chromatography–tandem mass spectrometry protein groups or spectral counts in FFPE specimens stored for up to 10 years compared with those stored for 1 year.
Section thickness, slide type and adhesive, and section storage can each impact IHC success. The clarity of immunostaining was reportedly superior when sections 2 to 4 μm thick were used compared with 8 μm sections,113 but the effects of section thickness on other protein applications have not been published. The literature suggests that for most antigens, slide type127 and the type of slide adhesive used128–130 do not impact IHC results, although the use of protected isocyanate129 and Mayer albumin128 as adhesives has been reported to increase section retention rates compared with slides coated with aminosilane, poly-L-lysine, and polysine or with Para Pen (Zymed, San Francisco, California), respectively. While a wide range of slide drying temperatures can yield adequate IHC results,90,93,131 temperatures higher than 68°C have been shown to diminish staining intensity.131 IHC staining was affected by storage of slide-mounted FFPE sections for most antigens surveyed,* with adverse effects observed after 1 to 3 weeks of storage,118,130,132,135,137 although the threshold of effect was ultimately antigen-dependent.†
Based on the literature available, when FFPE specimens are to be used for protein analysis, cold ischemia time should be limited to less than 12 hours. Optimally, specimens should be 1.2 to 3.5 mm3 in size and should not have undergone prior freezing, cryosectioning, or gel positioning. Specimens should be immersed in 10% to 15% NBF for 6 to 24 hours at ambient temperature or 4°C. FFPE blocks can be stored for several years for IHC, but block storage should be minimized for protein platforms that require extraction. Slide-mounted FFPE sections (2–4 μm thick) should be dried at a temperature below 68°C and analyzed within 1 week.
ACCEPTABLE FFPE TISSUE HANDLING CONDITIONS FOR MORPHOLOGICAL ANALYSIS
Cold ischemia time, gel positioning, prefixation cutting, and the method of decalcification each have reported impacts on morphological analysis of FFPE biospecimens. To the best of our knowledge, other prefixation variables have not been addressed by the literature.
Reports conflict as to whether macroscopic and microscopic degradation is observable in FFPE specimens following cold ischemia. Two studies reported that tissue quality declined, evidenced by the subjective measure of poor cellular morphology in hematoxylin and eosin–stained slides, with progressive cold ischemia times of 1 hour to 1 day at ambient temperature or 4°C139 and 1 hour to 4 days at 4°C.91 However, reports conflict as to whether mitotic counts are140,141 or are not80,139 significantly affected by a cold ischemia time of 6 hours.
Specimen positioning before fixation may improve morphological results, but the benefits and detriments to morphology appear to be tissue specific. Orienting prostate core needle biopsies between nylon mesh in tissue cassettes improved both diagnosis142 and the histologic yield143 by stretching cores and reducing the number of tissue sections needed. Suspension in 3.5% agar gel before immersion in formalin facilitated whole-mount sectioning and preserved the 3-dimensional conformation of breast tissue.85 On the other hand, uterine specimens that were fixed intact before opening demonstrated superior preservation of gross morphology and smaller gap size, which can impact prognostic dimensions, compared with those that were opened before fixation.81
Most articles14,89,144,145 we examined agreed that several decalcification solutions were acceptable and performed similarly for morphological analysis. However, Walsh et al58 and Sanjai et al146 concluded that decalcification with EDTA was preferable to other decalcification solutions, including formic and nitric acid, which resulted in loss of morphological detail and abnormal hematoxylin and eosin staining. In addition, DECAL (Decal Chemical Corp, Pomona, New York) and Cal-Ex (Fisher Scientific Co, Fair Lawn, New Jersey) have been shown to inhibit Mayer hematoxylin staining.144 Regardless of solution, standard incubation times should be used (days for formic acid and EDTA and hours for nitric acid and Plank-Rychlo solution) because longer incubations can lead to tissue deterioration when coupled with trypsin digestion.89 Interestingly, it was reported that ultrasonic decalcification permitted easier sectioning compared with nonultrasonic decalcification, regardless of decalcification solution.14
Published reports indicate that the concentration of formalin, duration of fixation, and use of accelerated fixation methods are capable of influencing the morphological quality of FFPE specimens. We were unable to locate published reports on the potential influence of other fixation-associated preanalytical factors.
Immersion fixation in 4% formaldehyde yielded morphology superior to that of lower concentrations (1%–2%),113 but microwave-accelerated fixation in 0.5% or 1% buffered formalin was superior to that in 7% buffered formalin.147 In general, fixation durations between 1 hour and 1 month31,100,148 or even 1 year149 failed to introduce morphological differences, with a few notable exceptions. One study14 reported an influence of fixation duration on morphology and specified that 24 hours was superior to 12 hours, 48 hours, 1 week, or 3 weeks, although details were not provided. Furthermore, AgNOR staining decreased as fixation duration increased from 15 minutes to 4 days, but statistical significance was not evaluated.150 Prolonged fixation for 6 to 42 years led to fixation artifacts in brain tissue, including granular neutrophil changes.149
Studies conflict as to whether accelerated fixation techniques yield results that are comparable or superior to those obtained by conventional immersion. To illustrate, results equivalent to those with immersion fixation were achieved with microwave-accelerated fixation,110,111 ultrasound-accelerated fixation,40 or perfusion,151 while results superior to those of immersion fixation were also reported for microwave,147 ultrasound,39,61 and perfusion methods.37,108,152 Notably, accelerated fixation with a microwave or vacuum oven produced superior morphology when the end temperature was 60 to 63°C as opposed to 55°C.153
Processing and Storage
Published reports explicitly investigating the effects of FFPE processing-related and storage-related preanalytical factors on morphological endpoints are sparse. The available literature suggests that accelerated processing methods can affect biospecimen morphology and that prolonged FFPE block storage can affect 4′,6-diamidino-2-phenylindole (DAPI) staining. However, it is important to note that none of the IHC studies reviewed above reported morphological differences when processing variables or FFPE block or section storage durations were compared.
A few studies have shown that microwave-assisted tissue processing, along with the various reagent substitutions and altered time lines and temperatures, did not detrimentally affect specimen morphology.154–158 Furthermore, it led to easier tissue sectioning159 and fewer edge effects154 than conventional processing.
Storage of FFPE blocks for 48 years resulted in decreased DAPI staining compared with those stored for 1 year.124 However, morphological integrity was not addressed.
Effects of section thickness on morphology per se have yet to be reported. However, according to the literature, thicker sections (5 μm) were associated with incomplete deparaffinization, which resulted in retained paraffin drops that were not present with sections 2 to 3 μm thick.160 While studies investigating the retention rate of FFPE sections to slides after IHC report superior retention rates when Superfrost Plus slides128,129 (VWR, West Chester, Pennsylvania; Lomb Scientific, Australia) were used, another study reported instances of section folding and paraffin retention when Superfrost Plus (Fisher Scientific, Pittsburg, Pennsylvania) and poly-L-lysine–coated slides were used, although issues were more frequent among tightly organized tissue types and thicker sections (5 versus 3–4 μm).160
Based on published evidence, the cold ischemia time for morphological analysis of FFPE specimens should be limited to less than 6 hours. The effects reported for biospecimen manipulation before fixation are method and tissue dependent, offering the benefits of increased visible surface area and improved preservation of 3-dimensional conformation but having the drawback of increased biospecimen shrinkage. Decalcification in EDTA, or a number of other solutions, was preferable to DECAL, Cal-Ex, and acid-based solutions. Fixation by conventional immersion should be in a solution containing 4% formaldehyde and limited to 1 year or less. While rapid microwave-assisted fixation and processing have reportedly generated superior morphological detail compared with conventional immersion, optimization, including time and temperature, remains an important consideration. While a specific time line for FFPE block storage cannot be extracted from the literature, no adverse effects on morphology have been reported for blocks stored for several years.
As detailed above, the following preanalytical factors have published effects on the analysis of at least 1 type of analyte in FFPE tissue: PMI, cold ischemia time, specimen size, fixative buffer, fixative delivery method, fixative temperature and duration, decalcification, block storage, section thickness, and section storage (Table 1). Importantly, these parameters can influence the analysis of nucleic acids, proteins, and morphology in different ways and to different extents. After a careful examination of the literature, we failed to identify an extensively investigated preanalytical factor that did not affect downstream molecular, proteomic, or morphological analysis. We identified several preanalytical factors that, to date, have not been evaluated in the literature for their potential impact on any of the analytes targeted in our meta-analysis, including the use of pathology ink, postfixation washing parameters, alcohol storage, equipment and conditions related to sectioning, and FFPE section transfer to slides (Table 1).
For instances when more than 1 analyte is to be investigated using the same FFPE specimen, the more stringent conditions (Table 2) should be used as a guideline. To illustrate, human FFPE tissue specimens of 3 mm3 in size that were subjected to a PMI of less than 4 hours or a cold ischemia time of less than 1 hour and were preserved in NBF for 8 to 24 hours at room temperature or 4°C should yield acceptable DNA, RNA, protein, and morphology data if blocks are stored for less than 1 year and if slide-mounted tissue sections are stored for less than 1 week. However, thresholds of effect for a specific preanalytical factor were often unique to each analyte and in some cases to each analytical platform.
Additional sources of preanalytical variability, including extraction methods, antigen retrieval techniques, and uninvestigated variables, were not addressed in this review and may serve as additional confounding variables. A comprehensive list of preanalytical factors, including patient-related factors, with the potential to influence downstream analysis of FFPE specimens has recently been published and is the result of a College of American Pathologists–sponsored working group.161 A review article that is in preparation by our office will focus on the effects associated with nucleic acid extraction, enrichment, and analytical techniques, in addition to assessing the reported accuracy of molecular data generated with FFPE specimens compared with fresh or frozen controls (S.R.G., K.B.E., B.P.B., and H.M.M., unpublished data).
Overall, we must weigh the benefits versus the consequences of using archival FFPE tissue when the handling, fixation, processing, and storage parameters for a specimen are unknown. The use of FFPE tissue that has been handled improperly may not generate data or may yield data that are representative of the FFPE processing conditions applied rather than the biospecimen donor's disease condition. With a concerted effort and attention to detail, accuracy, and awareness, FFPE biospecimens can serve as an important resource to clinical and research communities.
We thank Jim Rob, MD, for useful discussions pertaining to the information included in Table 1, and Jim Vaught, PhD, for his insightful review of the manuscript.
This study was supported by the Biorepositories and Biospecimen Research Branch of the National Cancer Institute.
The authors have no relevant financial interest in the products or companies described in this article.