Immunohistochemical Evaluation of the Effects of Paraffin Section Storage on Biomarker Stability

Abstract

Environmental stresses can alter immunoreactivity of biomarkers in stored tissue sections. The effect of temperature and lighting on 49 cellular or microbial antigens was evaluated in 4 serial paraffin sections, cut 12 months, 10 months, 8 months, 5 months, 3 months, 1 month, 3 days, and 1 day before immunohistochemistry. Slides were stored at room temperature (RT) in the dark, at 4°C in the dark, at RT under fluorescent light, or at RT with windowpane exposure to sunlight. Immunohistochemistry was performed simultaneously in an automated immunostainer. Immunoreactivity was compared with that in the corresponding 1-day-old section and scored as 4 (<10% reduction), 3 (10%-25% reduction), 2 (26%-60% reduction), 1(>60% reduction), or 0 (no reactivity). Any loss of immunoreactivity was proportional to the tissue section age and was least in sections stored in the dark. Immunoreactivity was only completely lost in light-exposed sections and as early as 1 month for CD45. Other markers with complete loss of immunoreactivity were bovine viral diarrhea virus, CD18 (only with fluorescent light), CD31, CD68, canine parvovirus, chromogranins, and thyroid transcription factor-1. Markers with complete loss after light exposure also had reduced immunoreactivity when stored in the dark, as early as day 3. Eight markers (Bartonella spp, CD11d, high molecular weight cytokeratins, feline coronavirus, GATA-4, insulin, p63, progesterone receptor) had minimal decrease in immunoreactivity, regardless of treatment. In conclusion, light-induced antigen decay (tissue section aging) is antigen dependent and could explain unexpectedly weak or negative immunohistochemical reactions in stored paraffin sections.

When Tissue Antigens and Antibodies Get Along

“…149 Both light and temperature contribute to the loss of antigenicity that occurs with photo-oxidation of tissue sections. 30,304 Tissue section aging is a rather common problem with nuclear antigens (eg, Ki67, ER, p53); z however, in a study of animal tissues, cytoplasmic membrane antigens were more sensitive than nuclear antigens to degradation from photo-oxidation and/or ambient temperature. 304 Immunoreactivity can also decrease when tissues are suboptimally dehydrated (retained endogenous water) during paraffin embedding or with paraffin sections stored under high humidity.…”

“…30,304 Tissue section aging is a rather common problem with nuclear antigens (eg, Ki67, ER, p53); z however, in a study of animal tissues, cytoplasmic membrane antigens were more sensitive than nuclear antigens to degradation from photo-oxidation and/or ambient temperature. 304 Immunoreactivity can also decrease when tissues are suboptimally dehydrated (retained endogenous water) during paraffin embedding or with paraffin sections stored under high humidity. 420 To reduce antigen degradation due to photo-oxidation, paraffin sections should be used within 1 week of sectioning or at least should be stored in an airtight container in the dark.…”

“…149 Keeping the paraffin sections in the dark under refrigeration also diminishes the loss of antigenicity. 304 In summary, all steps in tissue processing, from acquisition to storage, can affect the quality of the IHC test. 376 When there is decreased intensity or loss of reaction in stored control tissue sections, the IHC test should be repeated with a different stored control tissue section plus a freshly cut control tissue section.…”

Rodent Immunohistochemistry

“…Fixatives can play a major role for providing optimal results. [5][6][7] Tissue macroarrays or microarrays are most helpful provided they contain positive and negative control tissues. Macroarrays are made easily by hand embedding small portions of normal or abnormal tissues into 1 block.…”

“…There have been many review publications and books on IHC that can aid the veterinary pathologist and investigator. 2,[5][6][7][8][9][10][11][12][15][16][17][18][19] We review some of our own methods for IHC interpretation and troubleshooting investigative procedures. Various procedures are used to confirm and optimize specific antigen localization in FFPE mouse tissues.…”