Few studies have focused on antigen preservation in formalin-fixed and paraffin-embedded (FFPE) tissue in old archival material and additional studies are required, especially considering that these samples are an irreplaceable resource for scientific and clinical research. The purpose of this study is to verify antigen preservation in FFPE tissue samples stored for several decades. From the pathology archives, FFPE blocks were selected dating back to the 1960s, 1970s, 1980s, 1990s, 2000s and 2010. A panel of 12 antibodies was applied and immunoreactivities were compared. While cytoplasmic antigens showed no reduction in immunostaining intensity over time, membrane and nuclear antigens presented reduced staining intensity in older blocks. In particular, the nuclear antigen, Ki67 and CD31 showed the most pronounced antigen decay in the oldest archival blocks. In order to test possible antigen recovery, deep sectioning and lengthening of heat pretreatment were applied. Both strategies partially recover antigenicity, but their simultaneous application shows the best results.
- ANTIGEN PROCESSING
- KI 67
- ANTIGEN RETRIEVAL
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Formalin-fixed, paraffin-embedded (FFPE) tissue blocks are the standard archived specimen in the pathology laboratory. As many authors have stated,1–4 unlocking the pathology archive for new techniques has become a daily process with important impact on patient prognosis and therapy and these samples are an irreplaceable resource for scientific research.
Since the 1980s, immunohistochemistry (IHC) has gained enormous ground, mostly due to its contained costs and applicability on FFPE tissue and is now widely used in histopathology for diagnosis, prognosis and biomarker identification.
A number of studies, in the last years, have demonstrated that molecular techniques such as hybridisation in situ5–7 and IHC8 9 can become problematic due to ageing of FFPE tissue blocks or of tissue sections obtained from these blocks and stored over years on glass slides. The main and most explored cause of antigen decay, for IHC, is the storing of FFPE sections on glass slides and several reports have shown that section ageing (delay between cutting sections and IHC staining) and humidity can decrease antigenicity as early as 2 weeks after sectioning,10–12 while cold temperature storage of sections or paraffin blocks seem to be the best method for antigen preservation over time.13
In contrast, few studies have tested the IHC staining of biological markers in stored paraffin blocks.14 15 These studies show that antigenicity in stored paraffin blocks is preserved at least for several decades and if antigen decay happens, it is a very slow process.14 16 The cause of antigen degradation is uncertain, but archival conditions and oxidation processes are probably involved.
Survival has increased in some patients following new therapeutic strategies and for some neoplasms; recurrences may present decades later when new treatment is available and choice is based on biomarkers which may be evaluated on old archival FFPE tissue. A perfect example is the relatively new histological classification of gastro-entero-pancreatic neuroendocrine neoplasms17 for which grade is based on the proliferation index identified by Ki67 immunostaining. In these tumours, grade identification is important for prognostic stratification and is essential in the therapeutic algorithm. As demonstrated in a recent publication,18 reclassification of tumours may be fundamental as patients are long survivors even with metastatic disease; for this, archival tissue must be retrieved and Ki67 IHC must be performed.
The purpose of our study is therefore to verify whether FFPE tissue samples truly retain antigenicity over time, if an ‘expiry date’ can be suggested, if possible antigen decay is antigen dependent and whether methods for recovery of tissue antigenicity are identifiable.
Materials and methods
The study was conducted on FFPE blocks stored from 1960 to 2010; for cases from the last 20 years, only samples from deceased patients were chosen, while older samples were completely anonymised as the original reports were not computerised and all related paper diagnoses had previously been disposed of. A minimum of 10 to a maximum of 15 inclusions were selected from the first year of each decade (eg, 1960, 1970, 1980, etc). The samples stored between 1960 and 1990 were re-embedded in paraffin, because modern rotation microtomes cannot section wood-based/old plastic-based paraffin blocks. Re-embedding consisted of paraffin-melting of the original FFPE block on a warm plate and re-embedding using new paraffin and new biocassette mounts. The FFPE blocks were selected from two different institutions (Pathology Unit, IRCCS AOU San Martino-IST, Genoa and Pathology Unit, S. Andrea Hospital, Vercelli, Italy) and were stored within the archives as follows:
1960: inclusions from IRCCS-San Martino-IST on wood-based supports, stored in plastic bags (in cardboard boxes) in a dry, dark place at room temperature.
1970: inclusions from S. Andrea Hospital, Vercelli, without any supports, stored in torn plastic bags and exposed to light, dust and humidity at room temperature.
1980–1990: inclusions from IRCCS-San Martino-IST on plastic supports, stored in plastic bags until 2000, then transferred in dedicated cardboard boxes in a dry, dark place at room temperature.
2000–2010: inclusions from IRCCS-San Martino-IST on plastic supports, stored in dedicated cardboard boxes in a dry, dark place at room temperature.
All samples, from all ages except for 1970s, had been formalin fixed for a minimum of 24 hours and they were chosen to be of comparable size. No information was available regarding formalin concentration, buffering or types of tissue processing instruments used for all samples before 1985. After this date, 4% buffered formalin and different types of vacuum tissue processors were used. Low melt paraffins were introduced in the early 1990s, while previous embedding was obtained using a mixture of paraffin and bees wax. The 1970s blocks were obtained from a different hospital and for these, no information is available regarding fixative, fixation times and procedures.
From each paraffin block, a H&E stained section was obtained for morphological analysis to identify the tissue and decide the appropriate IHC panel (see later section).
Twelve different antibodies used in routine diagnostic practice were chosen to include nuclear, cytoplasmic and membranous staining (see table 1). The choice of antibody was aimed at the identification of various commonly used tissue antigens, including:smooth muscle actin (SMA), cytokeratins AE1/AE3, cytokeratins CAM 5.2, cytokeratins 5 & 6, cytokeratin 7, vimentin, desmin, CD31, CD34, leucocyte common antigen (LCA), S-100 protein and Ki67. Furthermore, these antibodies were chosen to include antigens which required heat and/or enzymatic pretreatment. For each paraffin block, antibodies were chosen depending on the type of tissue present (eg, myometrium for SMA and desmin, skin for Ck-5 & 6, etc). Each antibody was performed on three different samples from the same decade. All sections were cut on the same day, using the same microtome and by the same laboratory technician. All IHC reactions were performed within a week of sectioning.
Positive and negative controls were chosen from a selection of routinely employed controls used in our IHC laboratory and a section of each was added to every IHC run.
IHC was performed on the automated BenchMark XT immunostainer (Ventana Medical Systems) which permits standardised heat and enzymatic pretreatment. The streptavidin–biotin-based indirect method, iVIEW DAB Detection Kit (Ventana Medical Systems), was used. After deparaffinisation and rehydration of sections, endogenous peroxidase block was performed with 5% H2O2 for 10 min. Haematoxylin was used for counterstaining. IHC protocols for each antibody, including heat and enzymatic pretreatment, are specified in table 1.
All reactions were simultaneously evaluated by two histopathologists (FG and LM), blinded to paraffin block age, by using a multi-arm light microscope. All reactions were compared with positive controls. IHC-specific positivity was graded as follows: +/+ when the intensity was graded equal to positive control; +/− when graded lower than positive control and −/− when no appropriate staining was present.
Recovery of antigenicity
For immunoreactions in which loss of antigenicity was noted, in particular Ki67 and CD31, trials for recovery were carried out by:
comparison between antigenicity in the first superficial section collected from the paraffin block and sections of the paraffin block obtained at 500 μm and 1000 μm depth;
lengthening of heat pretreatment times from 30 min to 60 min to 90 min in both superficial and deeper sections.
Age of paraffin block and antigen decay
A total of 74 paraffin blocks were selected and 216 IHC stains were performed.
For most of the antibodies used (SMA, cytokeratins AE1–AE3, cytokeratins CAM 5.2, cytokeratins 5 & 6, cytokeratin 7, desmin, S-100 protein and vimentin), homogenous and intensely positive immunostaining, regardless of the age of the FFPE blocks, was obtained.
On the other hand, four antibodies (Ki67, CD31, CD34, LCA) showed variable immunoreactivity (16 weak reactions and seven negative reactions—table 2 and figure 1) and this appeared to be archival-time dependent, with older blocks (1960–1970s) showing the more evident immunosignal decay. In particular, blocks from the 1970s showed the worst antigen preservation with weak or mostly negative (seven negative and four weak out of 12 immunoreactions performed) results.
Focusing on the four reactions with variable immunoreactivity, reduction in immunosignal was confined to those antigens which required heat-based antigen retrieval and which were membranous or nuclear in location. Ki67, in particular, showed the most marked immunosignal decay; indeed, only the most recent 2010 sections stained adequately with Ki67.
Conversely, no immunosignal change was observed in antibodies directed against cytoplasmatic antigens, without pretreatment or requiring enzymatic (cytokeratins AE1/AE3, cytokeratins CAM5.2, desmin and SMA) or heat-based antigen unmasking (cytokeratin 7, cytokeratin 5 & 6, S-100 protein and vimentin).
Ki67 proved to be highly problematic (but extremely useful); therefore, recovery strategies were performed on this antigen. Both deeper sections of the paraffin block and longer heat pretreatment protocols progressively improved the intensity of the nuclear immunosignal; indeed, the combination of the two recovery strategies provided the best results with the most intense and easily recognisable, nuclear staining (figure 2). The same recovery protocol was also applied to CD31 and similar results were seen (except for 1970s blocks which remained negative) (figure 2).
While performing antigen recovery, different regions of reactivity were noted with a stronger immunostaining in the periphery of the sections compared with the centre.
The paraffin block archive, present in every Anatomic Pathology Laboratory, stores a wealth of information for the understanding and cure of disease. Problems in archival space and storage, however, lead many laboratories to only store recent FFPE blocks, while older tissue is discarded (some legislations require the storage of FFPE tissue for a limited period, eg, 20 years in Italy). Furthermore, storage conditions may vary as archives are often in damp premises, on the underground floor of laboratory buildings or far away where little control is possible on storage conditions.
Our current knowledge about the conservation of antigens and molecular information in FFPE is mostly limited to few years and little is known about what takes place for long storage periods. This study demonstrates that for cytoplasmic antigens, antigenicity is maintained in FFPE material for 60 years or more.
Slight antigen decay, however, begins after 15–20 years and may become more pronounced in the following decades, especially for antigens requiring heat-based antigen retrieval and localised on the cell membrane.
The only nuclear antigen tested, Ki67 (chosen for its positivity in both neoplastic and normal tissues), was found to be the most problematic antigen of all. Indeed, except for more recent samples (<10 years), a reduction of immunosignal intensity was identified in almost all archival tissues (most positive nuclei were light beige in colour and no dark brown staining was seen). Furthermore, Ki67 evaluation is often performed by counting all positive cells with automated systems where possible miscalculations may be due to difficulties in counting of light, ‘café au lait’, staining,19 which is an evident problem in archival tissue. Following a recent study by our group,18 where decrease in signal intensity for Ki67 was noted when archival tissue was tested, possible methods for antigen recovery from old FFPE blocks were investigated. Similarly to what happens to sections stored on glass slides,13 the most superficial layers of the paraffin block may undergo the same processes of antigen loss. We have shown that antigen recovery may be possible if IHC is performed on sections collected after having discarded the first 500–1000 μm of tissue. In addition, the lengthening of heat pretreatment times further helps in recovering immune intensity; a possible drawback to this is, however, an increase in section detachment.
Although tissue oxidation has been assumed to be the cause of antigen loss, a relatively recent study has proved that water content, both endogenous and exogenous, is also a key player in antigen decay.12 Indeed, the FFPE blocks from the 1970s showed the worst antigen preservation: apart from the passage of time, further explanations for this phenomenon could be the use of fixatives other than formalin or non-standardised procedures. Another possible explanation are the inadequate storage conditions (environmental humidity in damp premises, no temperature control, torn plastic bags subject to dust and light) for these blocks, which probably influence immunoreactivity.
Conversely, endogenous water content, due to inadequate fixation/processing, could explain the zonality in immunosignal recovery seen for Ki67 and CD31, where the periphery of the sections showed stronger immunostaining compared with the central area.
A limitation to this study is that the only nuclear antigen we tested was Ki67 as little was known about the type of tissue present in the oldest blocks and a search for specific types of tissues to test specific antigens was not possible. From the literature, numerous investigators have studied antigen loss of nuclear antigens, such as oestrogen and progesterone receptors in breast tissue or p53, demonstrating analogous problems to those reported for Ki67.20 21 We can therefore presume that other nuclear antigens may show the same immunosignal loss as Ki67 on old archival FFPE blocks, but this will require further testing. Another limitation, which has already been mentioned, is that preanalytical variables, which can greatly influence immunoreactivity, were only partially known.
In conclusion, FFPE blocks are an incomparable resource of information which is still available after many decades for research and clinical purposes. Our study highlights that most antigens are well preserved and available for analysis. Membranous and especially nuclear antigens are more subject to antigen decay, but antigen recovery is possible by deeper sectioning of the paraffin block and modifying pretreatment strategies.
Take home messages
Most antigens in FFPE blocks are well preserved over time (several decades).
Membranous and nuclear antigens (in particular Ki67) are most subject to antigen decay.
Antigen recovery by deeper sectioning of the paraffin block and modifying pretreatment strategies is possible.
Handling editor Runjan Chetty
Contributors FG and LM planned the research, viewed the slides and wrote the manuscript.
SPi and SPr performed immunohistochemistry.
MB and PM identified the cases and provided the FFPE blocks.
RF supervised and critically reviewed the study and manuscript.
Competing interests None declared.
Ethics approval Internal departmental ethics committee.
Provenance and peer review Not commissioned; externally peer reviewed.
Correction notice This paper has been amended since it was published Online First. Owing to a scripting error, some of the publisher names in the references were replaced with ‘BMJ Publishing Group’. This only affected the full text version, not the PDF. We have since corrected these errors and the correct publishers have been inserted into the references.