Multi-Tissue Controls and Multiplex Immunocytochemistry in Pulmonary Cytology

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY-NC 4.0 https://creativecommons.org/licenses/by-nc/4.0/ Multi-Tissue Controls and Multiplex Immunocytochemistry in Pulmonary Cytology © 2024 The Author(s). Published by S. Karger AG, Basel Published version Vuorisalo, Antti; Haapaniemi, Teppo; Kholová, Ivana Vuorisalo, A., Haapaniemi, T., & Kholová, I. (2024). Multi-Tissue Controls and Multiplex Immunocytochemistry in Pulmonary Cytology. Acta Cytologica, 68, 481-493. https://doi.org/10.1159/000540367 2024 Acta Cytologica Techniques Acta Cytologica 2024;68:481–493 DOI: 10.1159/000540367 Received: April 11, 2024 Accepted: July 12, 2024 Published online: July 30, 2024 Multi-Tissue Controls and Multiplex Immunocytochemistry in Pulmonary Cytology Antti Vuorisalo a, b Teppo Haapaniemi b, c Ivana Kholová a, b a Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland; b Department of Pathology, Fimlab Laboratories, Tampere, Finland; c Department of Biological and Environmental Sciences, University of Jyväskylä, Jyväskylä, Finland Keywords Multi-tissue controls ·Multiplex immunocytochemistry · Immunocytochemistry ·Pulmonary cytology Abstract Introduction: The World Health Organization 2021 lung cancer classification highlights the central role of immunohistochemistry (IHC) in diagnostic pathology. Despite traditional IHC being essential, its limitation to one marker per tissue section brings challenges, particularly when facing cytological limitedly sized samples. To overcome these challenges, multiplex immunocytochemistry (mICC) techniques offer the simultaneous detection of multiple markers from a single section. These advances complement the highly complex imaging techniques that enable additional analyses of cellular interactions. Methods: The present study outlines a comprehensive mICC methodology of an automated multiplex immunoperoxidase staining method and multiple tissue hybrid controls for ICC/mICC. Protocols are presented in detail and demonstrate a careful approach to optimizing various markers for diagnostic workup including immunotherapy. Conclusion: Multiplex IHC/ICC emerges as a transformative force in biomedical diagnostics and research. Beyond simultaneous marker detection, it unravels complexities within tissues –unveiling co-localization nuances, deciphering expression patterns, and enhancing understanding of cellular populations. As personalized treatments gain prominence, the study emphasizes the heightened importance of diagnostic tools and sample adequacy. The present methodological study, encapsulating an automated multiplex immunoperoxidase staining method, symbolizes a stride towards precision in pulmonary carcinoma diagnosis. Multitissue controls represent a key element in quality assurance in pathology laboratories. © 2024 The Author(s). Published by S. Karger AG, Basel Introduction Over the past decades, the field of immunohistochemistry (IHC) has made significant progress. The World Health Organization (WHO) 2021 classification of lung cancer incorporated IHC in the classification system [1, 2]. The traditional IHC is a valuable diagnostic tool in surgical pathology, and despite its limitations, such as the labelling of only one marker per tissue section, it remains an essential technique in a diagnostic workup. Examination of all requisite markers may not be possible if the samples are Antti Vuorisalo and Teppo Haapaniemi share 1st authorship. [email protected] www.karger.com/acy © 2024 The Author(s). Published by S. Karger AG, Basel Correspondence to: Antti Vuorisalo, antti.vuorisalo @ tuni.fi This article is licensed under the Creative Commons AttributionNonCommercial 4.0 International License (CC BY-NC) (http://www. karger.com/Services/OpenAccessLicense). Usage and distribution for commercial purposes requires written permission. Downloaded from http://karger.com/acy/article-pdf/68/5/481/4297180/000540367.pdf by Jyväskylän Yliopisto user on 20 November 2024 depleted, especially with samples acquired through minimally invasive methods like core needle biopsies or fine needle aspirations. This is a challenge, particularly in the evaluation of undifferentiated tumours, sarcomas, and lymphomas, which often demand an extensive panel exceeding 12 IHC stains for accurate diagnosis [3, 4]. However, in the era of targeted therapy and immunotherapy, an extensive panel of predictive markers is also required in the diagnostic workup of carcinomas [5]. The introduction of multiplex immunohistochemistry/ immunofluorescence (mIHC/IF) technologies has addressed this limitation and is becoming more popular, enabling the simultaneous detection of multiple markers in a single section. Additionally, advancements in highly multiplexed imaging techniques offer comprehensive analyses of cell composition and interactions, increasing diagnostic potential. The ability to assess multiple markers in one section is especially beneficial for low cellular samples [6, 7]. The mIHC/IF enables the application of several diagnostic markers in a single tissue section. Furthermore, the mIHC/IF provides detailed, quantitative data on the quantity and spatial distribution of various cells including immune cells within tumours, serving as a powerful investigative tool for understanding the immune context of the tumour [6]. Since fixation and other pre-analytical steps can affect antigenicity and quality of the staining, using control samples with different processing methods can lead to unreliable results. Variability in staining patterns caused by incorrect controls can complicate result interpretation in cytological samples processed differently from surgical pathology specimens. When using an inappropriate control type or controls treated differently from the sample, the likelihood of obtaining a false-negative staining result is heightened [8–10]. According to the College of American Pathologists 2024 guideline update “Principles of Analytic Validation of Immunohistochemical Assays,”laboratories should use validation tissues processed with the same fixative and methods as clinical cases whenever possible. The fixation and processing methods can affect certain epitopes in a manner that may alter the reliability of the assay, making consistent processing crucial. However, harvesting control tissues in-house has become increasingly challenging, leading many laboratories to rely on commercial suppliers, which may not replicate the laboratory’s specificfixation and processing methods. Therefore, maintaining the flexibility of using in-house controls remains important. Additionally, laboratories should verify assay performance using at least one known positive and one known negative control tissue when introducing a new antibody lot for an existing validated assay [11]. In surveys conducted by the European Federation of Cytology Societies (EFCS) and the College of American Pathologists (CAP), the importance of positive and negative controls in ensuring accurate staining results was examined. Without proper controls, it is difficult to determine if the staining observed in the patient’s sample is real or false due to an error in the staining process. Control samples with different fixation and procession methods compared to the patient’s sample can lead to wrong interpretation and diagnostic mistakes [8–10]. In the CAP survey, more than half (59.2%) of laboratories used control samples that were processed differently from the patient’s cytology specimens. Only 40.8% of laboratories used control samples that were processed in the same way as the patient’s samples. Validation of nonFFPE (formalin-fixed, paraffin-embedded) fixation methods is expensive and time and labour consuming. Many cytology tests are low volume, meaning they are performed infrequently, which makes validation even less cost effective. The high cost and time investment required for validation create a challenge for many laboratories [8, 9]. Nevertheless, accreditation systems require those quality assurance steps, and the latest ISO 15189 is risk prevention oriented [12]. As treatments become more individualized and case specific, the importance of diagnostics and the adequacy of samples becomes even more crucial, particularly in immunotherapy, which is largely based on accurate patient profiling and biomarker analysis. This methodological study describes an automated multiplex immunoperoxidase staining method using common diagnostic markers for the differential diagnosis and immunotherapy profiling of pulmonary carcinomas. The horseradish peroxidase (HRP) multimer was used for detection for its stability and reproducibility. The distinct feature of the multiplexes in this study is enhanced by the fact that these were made for brightfield microscopy with chromogenic visualization, thus preserving morphology and facilitating interpretation; translucent chromogens have been used for staining, allowing better visualization of the colocalization by highlighting it as a refractive colour; the automated method facilitates multiple protocol steps and does not require dark field microscopy and fluorochromes for visualization. In addition, cytology-specific multi-tissue controls were developed and applied in the present study. Methods Pulmonary samples were ethanol-fixed biopsies obtained from 486 patients who underwent endobronchial ultrasound bronchoscopy (EBUS) between January 2017 and December 2018 and 482 Acta Cytologica 2024;68:481–493 DOI: 10.1159/000540367 Vuorisalo/Haapaniemi/Kholová Downloaded from http://karger.com/acy/article-pdf/68/5/481/4297180/000540367.pdf by Jyväskylän Yliopisto user on 20 November 2024 were retrospectively selected from C5Lims –laboratory information system of the Fimlab Laboratories Pathology Department, Tampere, Finland [13]. Control samples were collected from fresh normal tissues from diagnostic specimens. The study was approved by the Ethical Committee of Pirkanmaa Hospital District and was performed according to the guidelines of the Declaration of Helsinki. This consent protocol was reviewed and the need for written and informed consent was waived by the Ethical Committee of Pirkanmaa Hospital District, decision reference number R17174. Multi-Tissue Hybrid Controls for Immunocytochemistry A multi-tissue control block consisting of both FFPE and ethanol-fixed paraffin-embedded tissue cores was introduced for immunocytochemistry (ICC). Control tissues from the same original specimen were separated for two different fixation processes, ethanol fixation, and formalin fixation (Table 1). The comparison between FFPE and ethanol-fixed paraffin-embedded control blocks was not fully in line, as the hybrid block was intended to contain pancreatic tissue, which could not be utilized in ethanol-fixed form. The block still allowed to control the normal function of the staining, considering insulinoma-associated protein 1 (INSM1) expression levels, and to verify the reliability of the staining. During the grossing step, fresh tissue was dissected into multiple 2–3 mm pieces to ensure similarity to cytological material. Different fresh tissue types (tonsil, placenta, skin, and small intestine) as normal tissue remnants from the grossing laboratory were collected. A 3 mm puncher for ethanol-fixed tissue and a 4 mm puncher for formalin-fixed tissue were used as internal quality assurance and control parameters. By this method, we were able to practically distinguish them from each other with the naked eye during processing and under microscopy, thus reducing misinterpretation. Formalin fixation, for 24 h–48 h, was performed with neutral buffered 10% formalin at room temperature. Ethanol fixation was performed with two different time points: 1-week fixation in 50% ethanol at room temperature and 2-week fixation, respectively. Post-fixation for 4–6 h in formalin was performed for ethanolfixed tissues before tissue processing. The different fixation times are due to the fact that formalin fixes quickly, but ethanol fixes slowly. In our practice, we check the morphology of the cytospins first, and based on the findings, pathologists order the cell blocks (CBs) and ICC later, so the ethanol fixation is longer, and the time interval mimics the actual laboratory flow. In addition, the pre-analytical fixation period may be long as our central laboratory serves a large geographic area of several provinces, and the distances are long, so samples can reach the laboratory in a few days after collection. Formalinand ethanol-fixed tissues were processed using Pathos Delta hybrid processing technology (Milestone Medical, Kalamazoo, MI, USA). Ethanol-fixed material was processed with a short biopsy protocol, mimicking the cytological material process. Formalin-fixed tissues were processed with the routine protocol for histological specimens. Embedded blocks were sectioned and stained with haematoxylin and eosin to ensure the quality and correct orientation of the tissue. Cores were punched using a Kai medical disposable biopsy puncher (Kai Industries Co., Ltd., Osaka, Japan). Ethanol-fixed tissue cores were punched from donor blocks with a 3 mm puncher and formalin-fixed material with a 4 mm puncher, respectively. Tissue cores were embedded with a similar orientation as in the donor block as shown in Figure 1. Multiplex Immunocytochemistry Staining The automated multiplex immunoperoxidase staining methodology was designed utilizing the common diagnostic markers for the differential diagnosis and immunotherapy profiling of pulmonary carcinomas, including adenocarcinoma, squamous cell carcinoma, and small cell carcinoma. Two chromogenic triple staining methods with five different chromogens were designed. The synaptophysin, chromogranin A, and INSM1 combination for the diagnosis of small cell carcinoma and the programmed death-ligand 1 (PD-L1), p40, and thyroid transcription factor 1 (TTF-1) combination for the diagnosis and immunotherapy profiling of pulmonary non-small cell carcinomas were introduced. All primary antibodies were first optimized with a single staining method before combining multiplex staining. The immunohistochemical multiplex staining consists of three sequential staining methods, respectively. Table 1. Control block composition and fixations NTissue fragment Diagnosis Fixation, duration 1 Small intestinal smooth muscle Normal tissue Formalin, 24–48 h 2 Intestinal epithelium Normal tissue Formalin, 24–48 h 3 Skin Normal tissue Formalin, 24–48 h 4 Pancreas Normal tissue Formalin, 24–48 h 5 Tonsil Normal tissue Formalin, 24–48 h 6 Placenta Normal tissue Formalin, 24–48 h 7 Skin Normal tissue Ethanol 50%, 2 weeks 8 Tonsil Normal tissue Ethanol 50%, 2 weeks 9 Intestinal epithelium Normal tissue Ethanol 50%, 2 weeks 10 Tonsil Normal tissue Ethanol 50%, 1 week 11 Small intestinal smooth muscle Normal tissue Ethanol 50%, 2 weeks 12 Placenta Normal tissue Ethanol 50%, 1 week 13 Small intestinal smooth muscle Normal tissue Ethanol 50%, 1 week 14 Intestinal epithelium Normal tissue Ethanol 50%, 1 week Controls and Multiplex Immunocytochemistry Acta Cytologica 2024;68:481–493 DOI: 10.1159/000540367 483 Downloaded from http://karger.com/acy/article-pdf/68/5/481/4297180/000540367.pdf by Jyväskylän Yliopisto user on 20 November 2024 The designed hybrid control sections and EBUS biopsies [13] were sectioned at a thickness of 3 μm on the positively charged TOMO ® slides (Matsunami Glass Ind., Ltd., Osaka, Japan). IHC was performed using a Ventana DISCOVERY ULTRA automated slide stainer (Ventana Medical Systems, Inc., Tucson, AZ, USA). Deparaffinization and rehydration were performed on the instrument. Cell Conditioning 1 (CC1) alkaline antigen retrieval solution (Ventana Medical Systems, Inc., Tucson, AZ, USA) was used as a pretreatment solution for heat-induced epitope retrieval (HIER). HIER was performed only before the first primary antibody incubation step. Endogenous peroxidases were quenched using the DISCOVERY inhibitor (Ventana Medical Systems, Inc., Tucson, AZ, USA) for 8 min. Between the sequential staining steps, the deactivation of previous primary antibodies and the detection systems was performed using the antibody denaturation step and applying Cell Conditioning 2 (CC2) acidic antigen retrieval solution for 8 min at 99°C (Ventana Medical Systems,Inc.,Tucson,AZ,USA).Afterthefinal chromogenic visualization, the slides were counterstained with haematoxylin II, dehydrated, and coverslipped (Fig. 2, 3). The detailed protocols and reagent information are summarized in Tables 2 and 3. In the present study, we tested the effect of antibody sequence in multiplex staining. Vendors do not select the antibodies based on feasibility for ethanol fixation. It was the reason behind we have both formalinand ethanol-fixed material in a control panel. We found that staining is impaired in all clones of ethanol-fixedtissuematerial compared to formalin-fixed material (Fig. 4, 5). This has also been shown in previous studies that ethanol fixation impairs antigenicity [14–20]. All markers were optimized, and multiplex ICC results were compared to formalin-fixed IHC and ethanol-fixed single ICC. The comparison was made with DAB as chromogen, and based on this implementation testing protocols, antibodies, and its optimized protocols were implemented in the multiplex protocols. Antigen depletion was particularly observed in tonsillar follicular macrophages. Reduced staining was also observed in tonsillar epithelial crypt cells. The weakest staining result was obtained specifically using the SP142 clone, which is typically used in an IC algorithm that considers the staining of immune cells in relation to the sample surface area. The clearest staining result and most intense staining were obtained with staining using the SP263 clone. In placental syncytiotrophoblasts, staining was seen at both the apical membrane and the basal membrane. In our study, we found that Ventana DISCOVERY’s purple chromogen and yellow chromogen translucency allowed the colocalization resulting in orange stain colour. This technique allows more accurate analysis and diagnosis of antigens localized in the same cell and the same cell compartment. In the past, multiplex chromogenicity has been more obscuring and has not allowed for more accurate diagnostics. It should be noted that the Ventana DISCOVERY ULTRA is an open immunostaining automation machine, which allows for protocol amplification and optimization for ethanol-fixed sample material. For triple staining, the clone SP263 was selected as the PD-L1 antibody due to its best staining results. The protocol without amplification allows sensitization of the protocol and thus compensation for antigen loss in the intensity of staining. PD-L1-Stained Controls The performance of PD-L1 staining with hybrid controls was tested using pathology laboratory diagnostic protocols (Table 4). Table 4 presents the final optimized PD-L1 protocols for four different clones. In summary, all HIER steps were performed on board with an alkaline antigen retrieval buffer at 99°C. All PD-L1 antibodies were optimized with hybrid block sections with Ventana BenchMark ULTRA instrument for multiplex protocol in Ventana DISCOVERY ULTRA. Clone SP263 was selected according to its robust and sensitive staining pattern during the validation of multiplex staining. Finally, fine-tuning of antibody incubation and detection method was performed. PD-L1 staining with clones SP263 and SP142 were Ventana PD-L1 IHC assays, while 22C3 and 28-8 were laboratoryprepared proprietary tests. Ventana’s protocols are closed and in line with the Pharmacotherapy Recommendation. The 22C3 and 28-8 are laboratory-validated protocols validated on the Ventana BenchMark ULTRA platform. Tyramide amplification was used for staining with SP142, 22C3, and 28-8 as clones. Fig. 1. Haematoxylin and eosin-stained slide section from a hybrid block containing both ethanol-formalin-fixed control tissues (3-mm punches, upper row) and formalin-fixed control tissues (4-mm punches, lower row). aIntestinal smooth muscle, formalin fixed. bIntestinal epithelia, formalin fixed. cSkin, formalin fixed. dPancreas, formalin fixed. eTonsil, formalin fixed. fPlacenta, formalin fixed. gSkin, 50% ethanol fixed. hTonsil, 50% ethanol fixed. iIntestinal epithelia, 50% ethanol fixed. jTonsil, 50% ethanol fixed. kIntestinal smooth muscle, 50% ethanol fixed. lPlacenta, 50% ethanol fixed. mIntestinal smooth muscle, 50% ethanol fixed. nIntestinal epithelia, 50% ethanol fixed. 484 Acta Cytologica 2024;68:481–493 DOI: 10.1159/000540367 Vuorisalo/Haapaniemi/Kholová Downloaded from http://karger.com/acy/article-pdf/68/5/481/4297180/000540367.pdf by Jyväskylän Yliopisto user on 20 November 2024 Toimprovestainingresults,theantibodysequencewasconsidered. We first applied those antigens that need to be counted and quantified (as PD-L1) as tissue is changing with the following subsequent processing sequences, morphology is less visible, and antigenicity weakens. In addition, chromogen characteristics should be considered: first, we use opaque chromogens and after translucent chromogens. Discussion Analysis of several immunohistochemical markers within a single tissue section is a complex task that requires multiplex chromogen or fluorescence detection. The Fig. 3. mICC in pulmonary small cell carcinomas diagnostic workup. Three neuroendocrine markers (INSM1, synaptophysin, chromogranin) were applied as neuroendocrine markers sensitivity and specificity vary. Pulmonary small cell carcinomas are TTF-1 positive. aCase of small cell carcinoma in an EBUS-targeted lymph node in a 76-year-old female. INSM1 nuclear positivity in brown, synaptophysin cytoplasmatic granular positivity in purple, and chromogranin A cytoplasmatic granular positivity in yellow. Note that despite all markers are positive in this case, not all cells express all three markers; so with less sufficient samples, only one to two markers can be present. Multiplex immunostaining for INSM1, synaptophysin, and chromogranin A (×200 magnification). bTTF1 nuclear positivity in red in a small cell carcinoma in an EBUStargeted lymph node shown in a. Note a squamous cell metaplastic fragment with p40 nuclear positivity in green. PDL1 is negative. Multiplex immunostaining for PD-L1, p40, and TTF-1 (×200 magnification). cCase of small cell carcinoma in an EBUS-targeted lymph node in a 68-year-old male. INSM1 nuclear positivity in brown in part of carcinoma cells, synaptophysin cytoplasmatic granular positivity in purple in almost all cells. Chromogranin A is negative. Expression of neuroendocrine markers is variable in various cases but also within the tissue of a single case as case a. showed expression of all three markers but not diffusely. In this case, only two markers were expressed with only one of them to be expressed diffusely. Multiplex immunostaining for INSM1, synaptophysin, and chromogranin A (×200 magnification). Fig. 2. mICC in pulmonary non-small cell carcinomas diagnostic workup and immunotherapy: p40 antibody nuclear positivity distinguishes squamous cell carcinoma from TTF-1 antibody nuclear positivity characteristic for adenocarcinoma. PD-L1isappliedasamarkerforimmunotherapy.aCase of squamous cell carcinoma in an EBUS-targeted lymph node in a 73-year-old male. PD-L1 membranous positivity in brown in 30% of carcinoma cells. Nuclear green positivity of p40 in most carcinoma cells in variably disperse fragments. Background lymphocytes are negative for all three markers. Multiplex immunostaining for PD-L1, p40, and TTF-1 (×200 magnification). bPD-L1 negative squamous cell carcinoma in an EBUStargeted lymph node in a 70-year-old female. PD-L1 is negative in carcinoma cells. Nuclear green positivity of p40 in most carcinoma cells in a tight sheet. Multiplex immunostaining for PD-L1, p40, and TTF-1 (×200 magnification). cAdenocarcinoma in an EBUS-targeted lymph node in a 57-year-old male. PD-L1 membranous positivity in brown in the majority of carcinoma cells (95%). Nuclear red positivity of TTF-1 in most carcinoma cells in tubular and trabecular groups. Background lymphocytes are negative for all three markers. Multiplex immunostaining for PD-L1, p40, and TTF-1 (×200 magnification). Controls and Multiplex Immunocytochemistry Acta Cytologica 2024;68:481–493 DOI: 10.1159/000540367 485 Downloaded from http://karger.com/acy/article-pdf/68/5/481/4297180/000540367.pdf by Jyväskylän Yliopisto user on 20 November 2024 growing popularity of both chromogenic and fluorescent multiplex staining is due to the development of reliable multiplex staining techniques and sophisticated multispectral imaging. The benefits of multiplex staining include the ability to preserve valuable tissue samples or even the use of limited specimens and the ability to co-localize antigens and improve the accuracy of interpretation. This is especially important for the emerging field of immunotherapy as it allows the identification and detection of immune cells, as well as the characterization of response biomarkers [21–27]. The mIHC/IF approaches have become significantly more powerful, providing enhanced insights into disease heterogeneityandtheunderlyingsystemsbiologymechanisms. These approaches also contribute to the preservation of limited tissue material [6]. Tumour heterogeneity and the limited representativeness of small biopsy or cytology samples Table 2. Multiplex protocol of non-small cell carcinoma and small cell carcinoma Step Reagent Time, min Temperature, °C Multiplex: PD-L1 + p40 + TTF-1 Deparaffinization DISCOVERY Wash 12 70 Pretreatment, HIER in CC1 CC1 64 99 Inhibitor CM for quenching endogenous peroxidases Inhibitor CM 8 37 Primary antibody 1: PD-L1 (SP263) Primary antibody 1 32 36 Detection: Anti-Rabbit-HQ DISCOVERY HQ HRP 16 Ambient Detection: Anti-HQ-HRP DISCOVERY HQ HRP 16 Ambient Chromogen 1: DAB DISCOVERY ChromoMap DAB 8 Ambient Denaturation step for deactivation antibodies and HRP CC2 8 99 Primary antibody 2: p40 (BC28) Primary antibody 2 48 36 Detection: OptiView Linker OptiView DAB IHC Detection 20 Ambient Detection: OptiView HRP Multimer OptiView DAB IHC Detection 20 Ambient Chromogen 2: Green HRP Kit DISCOVERY Green HRP Kit 64 Ambient Denaturation step for deactivation antibodies and HRP CC2 8 99 Primary antibody 3: TTF-1 (SP141) Primary antibody 3 48 36 Detection: OptiView Linker OptiView DAB IHC Detection 20 Ambient Detection: OptiView HRP Multimer OptiView DAB IHC Detection 20 Ambient Chromogen 3: RED HRP Kit DISCOVERY RED HRP Kit 64 Ambient Counterstaining with Haematoxylin II Haematoxylin II 8 Ambient Bluing of haematoxylin Bluing reagent 4 Ambient Multiplex: INSM1 + synaptophysin + chromogranin A Deparaffinization DISCOVERY Wash 24 70 Pretreatment, HIER in CC1 CC1 64 99 Inhibitor CM for quenching endogenous peroxidases Inhibitor CM 8 37 Primary antibody 1: INSM1 (A-8), 1:50 Primary antibody 1 60 36 Detection: Anti-Rabbit-HQ DISCOVERY HQ HRP 24 Ambient Detection: Anti-HQ-HRP DISCOVERY HQ HRP 24 Ambient Chromogen 1: DAB DISCOVERY ChromoMap DAB 8 Ambient Denaturation step for deactivation antibodies and HRP CC2 8 99 Primary antibody 2: Synaptophysin (SP11), RTU Primary antibody 2 52 36 Detection: OptiView Linker OptiView DAB IHC Detection 20 Ambient Detection: OptiView HRP Multimer OptiView DAB IHC Detection 20 Ambient Chromogen 2: Purple HRP Kit DISCOVERY Purple HRP Kit 32 Ambient Denaturation step for deactivation antibodies and HRP CC2 8 99 Primary antibody 3: Chromogranin A (LK2H10), RTU Primary antibody 3 60 36 Detection: OptiView Linker OptiView DAB IHC Detection 20 Ambient Detection: OptiView HRP Multimer OptiView DAB IHC Detection 20 Ambient Chromogen 3: Yellow HRP Kit DISCOVERY Yellow HRP Kit 96 Ambient Counterstaining with Haematoxylin II Haematoxylin II 8 Ambient Bluing of haematoxylin Bluing reagent 4 Ambient Dehydration with rising ethanol series, xylene clearing, and coverslipping. 486 Acta Cytologica 2024;68:481–493 DOI: 10.1159/000540367 Vuorisalo/Haapaniemi/Kholová Downloaded from http://karger.com/acy/article-pdf/68/5/481/4297180/000540367.pdf by Jyväskylän Yliopisto user on 20 November 2024 may lead to discrepancies between cytological specimens and/ or core biopsy and the final histologic diagnosis from resection specimens [28, 29]. The mIHC/IF holds significant utility in various aspects of diagnostics and biomedical research. First, it enables the simultaneous detection of numerous markers in a single tissue section, extracting optimal information from limited or precious samples. Second, it allows the exploration of markers co-localization and interaction.Thirdly,studiesbenefit from mIHC/IF by the identification of expression patterns, providing insights into tumour organization and enhancing our understanding of the presence of various populations within the tumour. Lastly, by revealing spatial relationships among cells and tissues and uncovering disease heterogeneity, mIHC/IF contributes to increasing diagnostic and predictive accuracy as well as advances biomedical understanding of the diseases [7, 26, 30]. Duration of the ethanol fixation varies in the laboratory based on the decision for the preparation of the CB. Increasing the duration of the ethanol fixation, the staining intensity gradually fainted for some antibody clones. However, we noticed the same effect using the hybrid block-controlling method for the optimization of PD-L1 Table 3. Multiplex reagents for non-small cell carcinoma and small cell carcinoma (a) Multiplex: PD-L1 + p40 + TTF-1 Primary antibody Clone Dilution Antibody incubation, min Vendor PD-L1 SP263 Prediluted 32 Roche Ventana p40 BC28 Prediluted 48 Roche Ventana TTF-1 SP141 Prediluted 48 Roche Ventana Detections Steps Dilution Incubation(s), min Vendor DISCOVERY HQ HRP hapten-linked multimer detection 2 Prediluted 16 + 16 Roche Ventana OptiView DAB IHC Detection Kit (using only detection part) a 2 Prediluted 20 + 20 Roche Ventana Chromogens Colour Dilution Incubation(s), min Vendor DISCOVERY ChromoMap DAB Kit (HRP) Brown Prediluted 8 Roche Ventana DISCOVERY Green HRP Kit Green Prediluted 32 + 32 Roche Ventana DISCOVERY RED HRP Kit Red Prediluted 32 + 32 Roche Ventana (b) Multiplex: INSM1 + synaptophysin + chromogranin A Primary antibody Clone Dilution Antibody incubation, min Vendor INSM1 A-8 1:50 60 Santa Cruz Bioscience Synaptophysin SP11 Prediluted 52 Roche Ventana Chromogranin A LK2H10 Prediluted 60 Roche Ventana Detections Steps Dilution Incubation(s), min Vendor DISCOVERY HQ HRP hapten-linked multimer detection 2 Prediluted 24 + 24 Roche Ventana OptiView DAB IHC Detection Kit (using only detection part) a 2 Prediluted 20 + 20 Roche Ventana Chromogens Colour Dilution Incubation(s), min Vendor DISCOVERY ChromoMap DAB Kit (HRP) Brown Prediluted 8 Roche Ventana DISCOVERY Purple HRP Kit Purple Prediluted 32 Roche Ventana DISCOVERY Yellow HRP Kit Yellow Prediluted 48 + 48 Roche Ventana a OptiView detection was performed using Ventana user fillable dispensers. Controls and Multiplex Immunocytochemistry Acta Cytologica 2024;68:481–493 DOI: 10.1159/000540367 487 Downloaded from http://karger.com/acy/article-pdf/68/5/481/4297180/000540367.pdf by Jyväskylän Yliopisto user on 20 November 2024 clones. Tissue cores fixed 1 week in 50% ethanol stained more intensively (tonsil, placenta), then 2 weeks fixed tissues, respectively. For standardization of the ethanol fixation, the fixation time should be kept as short as possible, similar to the formalin fixation time. Post-fixation with the formalin could decrease the adverse effect of ethanol fixation, especially if the antibody is designed against the formalin-fixed and linearized epitope. Therefore, one notable advantage of mIHC/IF is the improved accuracy facilitated by image analysis, which utilizes landmark markers to indicate tissue architecture [6, 27]. Nevertheless, interpreting multiplexed stained samples, particularly those using fluorescence, can be challenging. The use of fluorescence may cause multiple targets to blend, complicating resolution and potentially muddling visual assessment. Additionally, in FFPE tissues, there is the potential for tissue autofluorescence, further complicating visual interpretation [6]. Previous studies examined whether staining with a specific antibody in a multiplex protocol is qualitatively comparable to a single staining and concluded that multiplex staining can be qualitatively comparable to single staining. However, achieving this requires standardization, validation, and careful consideration of detection methods and antibody characteristics [31, 32]. In addition, previous research has concluded that multiplex IHC/ICC can quantitatively replicate single staining when thoroughly validated and optimized. It should also be noted that the location of antibodies in the multiplex panel affects all antibodies in the panel, requiring careful optimization to avoid problems of antibody shedding and interference [33–35]. Prior to the possible integration of mIHC/IF technology into clinical applications, it is crucial to establish a standardized and validated workflow that encompasses the entire process. This comprehensive workflow should be capable of supporting multisite trials and aligning with the requirements of clinical laboratory procedures and accreditation requirements [24]. In optimizing tissue availability for molecular testing, it is advisable to undertake a restricted diagnostic workup. Pulmonary cancer diagnosis necessitates a multidisciplinary approach. The landscape of lung cancer therapy is increasingly personalized, considering individual patient factors such as histologic cell type, subtypes, and molecular status. The role and approach of pathologists in diagnosing lung cancer in small biopsies and cytology specimens have undergone significant transformation [29]. Quality Assurance The European Federation of Cytology Societies (EFCS) survey from 245 laboratories highlights the need for standardized protocols and robust quality assurance/quality control (QA/QC) practices in ICC [9]. Similar findings were identified in a CAP survey with 345 respondent laboratories [8], as well as in the UK NEQAS survey [36] and the meta-analysis already performed in the year 2011 [37]. Further research efforts are crucial to develop optimal fixation and processing methods for diverse cytology samples, establish validated ICC protocols applicable to various cytology preparations, and implement standardized QA/QC measures, including positive controls and internal and external quality Fig. 4. Multi-tissue section stained with PD-L1 (SP263) clone. Ethanol-fixed tissues are punched with a 3 mm puncher and formalinfixed tissues with 4 mm, respectively. The staining intensity of the PDL1 is optimal using formalin-fixed tissues and protocol recommendation by the vendor. Placenta and tonsil are good control tissues for quality controlling of PD-L1 staining. Other tissues of the hybrid control are negative control of PD-L1 antigen. Note: staining intensity decreased in ethanol-fixed tissues compared to the formalin-fixed tissues. Staining intensity decreased especially in tonsil tissue. Top row, ethanol-fixed tissue; bottom row, formalin-fixed tissue. Controls from top left. aSkin, 1 week. bTonsil, 2 weeks. cIntestinal epithelium, 2 weeks. dTonsil, 1 week. eSmall intestinal smooth muscle, 1 week. fPlacenta, 1 week. gSmall intestinal smooth muscle, 2 weeks. hIntestinal epithelium, 1 week. Controls from bottom left. iSmall intestinal smooth muscle. jIntestinal epithelium. kSkin. lPancreas. mTonsil. nPlacenta (×5 magnification). 488 Acta Cytologica 2024;68:481–493 DOI: 10.1159/000540367 Vuorisalo/Haapaniemi/Kholová Downloaded from http://karger.com/acy/article-pdf/68/5/481/4297180/000540367.pdf by Jyväskylän Yliopisto user on 20 November 2024