Factors affecting immunoreactivity in long-term storage of formalin-fixed paraffin-embedded tissue sections

Abstract

Antigen decay in archival formalin-fixed paraffin-embedded (FFPE) tissue sections for immunohistochemistry is a well-known phenomenon which may have repercussions on translational and research studies and length of storage time appears fundamental. The aim of this study was to evaluate all possible factors which may lead to antigen decay on a prospective standardized collection of human tissues with a panel of 14 routinely used antibodies. Serial slide sections from FFPE control tissues were stored using different methods (routine storage at room temperature, Parafilm(®) protected, paraffin coated and cold stored at 4 °C) and for different time periods: 1, 6, 9, 12, 24 and 36 months. Immunohistochemistry was performed at each time cutoff simultaneously on stored sections and on freshly cut sections using a panel of 14 antibodies. Immunoreactivity was compared with immunoreactions performed at time zero. Reduction in immunostaining was observed for a subset of antibodies (CD3, CD 31, CD117, estrogen and progesterone receptors, Ki67, p53, TTF-1, vimentin) while for others (smooth muscle actin, keratins 7, 20, AE1/AE3, 34βE12), no antigen decay was observed. Loss of antigenicity was proportional to tissue section age and was dependent on mode of storage with cold storage slides being the least affected. All antigens with reductions in immunosignal were nuclear or membranous, and they all required heat pre-treatment for antigen retrieval. In contrast to results from other studies, when pre-analytical factors are strictly controlled and standardized, antigen decay seems to be restricted to nuclear or membrane antigens which require heat antigen retrieval.

Optimum immunohistochemical procedures for analysis of macrophages in human and mouse formalin fixed paraffin-embedded tissue samples

“…Grillo F et al demonstrated that antigen loss or decay was seen in IHC of CD3, CD 31, CD117, estrogen and progesterone receptors, Ki67, p53, TTF-1, vimentin, but not in IHC of smooth muscle actin, keratins 7, 20, AE1/AE3, 34βE12, and they also showed cold storage is suitable methods for long term storage of tissue sections. 19 The importance of cold storage (at minus degrees rather than at 4 o C) was reported by other researchers, 20 and these observations were consistent with our observation of figure 3.…”

Immunohistochemistry on old archival paraffin blocks: is there an expiry date?

“…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.…”

“…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…”

Grade Increases in Gastroenteropancreatic Neuroendocrine Tumor Metastases Compared to the Primary Tumor

“…All sections were used for immunohistochemistry within 1 month of cutting [14]. Immunohistochemistry was performed using the Ventana BenchMark® XT platform (Ventana Medical Systems, Oro Valley, Ariz., USA) automated immunostaining device, according to the manufacturer's protocol, using 3,3′-diaminobenzidine as chromogen.…”