Research ArticlesThe Immunohistochemical Expression of Programmed Death Ligand 1 (PD-L1) Is Affected by Sample OverfixationBarberà, Angels MNurs*,†; Marginet Flinch, Ruth APT†; Martin, Montserrat APT†; Mate, Jose L. PhD†; Oriol, Albert MD‡; Martínez-Soler, Fina PhD*; Santalucia, Tomas PhD*; Fernández, Pedro L. PhD† Author Information *Fundamental Care and Medical-Surgical Nursing Department, School of Nursing, Faculty of Medicine and Health Sciences, Barcelona University †Pathology Department, Germans Trias i Pujol Hospital and Institute, Faculty of Medicine and Health Sciences, Universitat Autònoma de Barcelona ‡Josep Carreras Leukemia Research Institute, Barcelona, Spain The authors declare no conflict of interest. Reprints: Angels Barberà, MNurs, Hospital Germans Trias i Pujol, Carretera del Canyet, Badalona, Barcelona 08916, Spain (e-mail: [email protected]). Applied Immunohistochemistry & Molecular Morphology: January 2021 - Volume 29 - Issue 1 - p 76-81 doi: 10.1097/PAI.0000000000000847 Buy Metrics Abstract Humanized antibodies targeting programmed death receptor 1 (PD-1) or its ligand (PD-L1) have been approved for the treatment of different cancers. Some of these antibodies show a correlation between the tissue expression of PD-L1 and response. Evaluation of PD-L1 expression presents multiple challenges, but some preanalytical issues such as tissue fixation have been scarcely evaluated. With the hypothesis that immunohistochemical staining of PD-L1 may be impacted by the time of specimen fixation, we evaluated differences in its expression in tonsil samples exposed to predefined fixation times. Random nontumoral tonsillectomy specimens were blindly evaluated in tissue microarray slides after staining with SP142 and SP263 antibodies. With fixation times ranging from 12 to 72 hours, between 2.8% and 6.1% of the samples were considered to be suboptimally stained, with no differences between the 2 antibodies within these fixation times. A significantly higher proportion of samples exposed to a fixation time of 96 hours presented suboptimal immunostaining (15.6%, P<0.0001). In addition, suboptimally stained spots were 20.8% using SP142 and 10.4% using SP263 after 96 hours of fixation (P=0.046). In conclusion, the quality of staining for PD-L1 in tonsil samples decreased with overfixation of the specimen at times >72 hours. Samples exposed to formaldehyde for longer periods presented suboptimal results for both clones, but the SP142 antibody presented a significantly lower tolerance to formalin overexposure than SP263. These results indicate the relevance of a controlled preanalytical processing of samples and particularly the length of fixation of tumor specimens. Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.