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. 2019 Aug;475(2):191-199.
doi: 10.1007/s00428-019-02595-9. Epub 2019 Jul 1.

Impact of delayed and prolonged fixation on the evaluation of immunohistochemical staining on lung carcinoma resection specimen

Maartje van Seijen  1   2 , Luka Brcic  3 , Atilio Navarro Gonzales  4 , Irene Sansano  5 , Matyas Bendek  6   7 , Iva Brcic  3 , Birgit Lissenberg-Witte  8 , H Ibrahim Korkmaz  1 , Thomas Geiger  9 , Rosita Kammler  9 , Rolf Stahel  9   10 , Erik Thunnissen  11 ; ETOP
Affiliations

Impact of delayed and prolonged fixation on the evaluation of immunohistochemical staining on lung carcinoma resection specimen

Maartje van Seijen et al. Virchows Arch. 2019 Aug.

Abstract

Pre-analytical factors, such as fixation time, influence morphology of diagnostic and predictive immunohistochemical staining, which are increasingly used in the evaluation of lung cancer. Our aim was to investigate if variations in fixation time influence the outcome of immunohistochemical staining in lung cancer. From lung resections, specimen with tumor size bigger than 4 cm, 10 samples were obtained: 2 were put through the standard fixation protocol, 5 through the delayed, and 3 through the prolonged fixation protocol. After paraffin embedding, tissue microarrays (TMAs) were made. They were stained with 20 antibodies and scored for quality and intensity of staining. Samples with delay in fixation showed loss of TMA cores on glass slides and deterioration of tissue quality leading to reduction in the expression of CK 7, Keratin MNF116, CAM 5.2, CK 5/6, TTF-1, C-MET, Napsin A, D2-40, and PD-L1. Prolonged fixation had no influence on the performance of immunohistochemical stains. Delay of fixation negatively affects the expression of different immunohistochemical markers, influencing diagnostic (cytokeratins) and predictive (PD-L1) testing. These results emphasize the need for adequate fixation of resection specimen.

Keywords: Fixation; Immunohistochemistry; Lung adenocarcinoma; Pre-analytical.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Example of a CK 7 (Monosan) staining after normal fixation (a) and after 96 h delay in fixation (b). A is scored as sufficient and B as insufficient quality, due to prominent non-specific (background) staining (for both objective × 15)
Fig. 2
Fig. 2
The distribution of PD-L1 (E1L3N (XP)) staining divided in 4 categories is shown for samples with delay in fixation. Of note, the number of cases with positive PD-L1 staining (1–49% and ≥ 50%) is lower after delay in fixation
Fig. 3
Fig. 3
Presentation of PD-L1 staining in a tumor sample after normal fixation (a), after 6 h (b), 48 h (c), and 96 h (d) of delay in fixation (for all objective × 20). Note: deterioration of membrane staining in 48+ h delayed fixation and increase of non-specific staining
See this image and copyright information in PMC

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