Pathology Quality Control for Multiplex Immunofluorescence and Image Analysis Assessment in Longitudinal Studies.

Immune profiling of formalin-fixed, paraffin-embedded tissues using multiplex immunofluorescence (mIF) staining and image analysis methodology allows for the study of several biomarkers on a single slide. The pathology quality control (PQC) for tumor tissue immune profiling using digital image analysis of core needle biopsies is an important step in any laboratory to avoid wasting time and materials. Although there are currently no established inclusion and exclusion criteria for samples used in this type of assay, a PQC is necessary to achieve accurate and reproducible data. We retrospectively reviewed PQC data from hematoxylin and eosin (H&E) slides and from mIF image analysis samples obtained during 2019. We reviewed a total of 931 reports from core needle biopsy samples; 123 (13.21%) were excluded during the mIF PQC. The most common causes of exclusion were the absence of malignant cells or fewer than 100 malignant cells in the entire section (n = 42, 34.15%), tissue size smaller than 4 × 1 mm (n = 16, 13.01%), fibrotic tissue without inflammatory cells (n = 12, 9.76%), and necrotic tissue (n = 11, 8.94%). Baseline excluded samples had more fibrosis (90 vs 10%) and less necrosis (5 vs 90%) compared with post-treatment excluded samples. The most common excluded organ site of the biopsy was the liver (n = 19, 15.45%), followed by soft tissue (n = 17, 13.82%) and the abdominal region (n = 15, 12.20%). We showed that the PQC is an important step for image analysis and that the absence of malignant cells is the most limiting sample characteristic for mIF image analysis. We also discuss other challenges that pathologists need to consider to report reliable and reproducible image analysis data.

Multiplex Immunofluorescence Tyramide Signal Amplification for Immune Cell Profiling of Paraffin-Embedded Tumor Tissues.

Every day, more evidence is revealed regarding the importance of the relationship between the response to cancer immunotherapy and the cancer immune microenvironment. It is well established that a profound characterization of the immune microenvironment is needed to identify prognostic and predictive immune biomarkers. To this end, we find phenotyping cells by multiplex immunofluorescence (mIF) a powerful and useful tool to identify cell types in biopsy specimens. Here, we describe the use of mIF tyramide signal amplification for labeling up to eight markers on a single slide of formalin-fixed, paraffin-embedded tumor tissue to phenotype immune cells in tumor tissues. Different panels show different markers, and the different panels can be used to characterize immune cells and relevant checkpoint proteins. The panel design depends on the research hypothesis, the cell population of interest, or the treatment under investigation. To phenotype the cells, image analysis software is used to identify individual marker expression or specific co-expression markers, which can differentiate already selected phenotypes. The individual-markers approach identifies a broad number of cell phenotypes, including rare cells, which may be helpful in a tumor microenvironment study. To accurately interpret results, it is important to recognize which receptors are expressed on different cell types and their typical location (i.e., nuclear, membrane, and/or cytoplasm). Furthermore, the amplification system of mIF may allow us to see weak marker signals, such as programmed cell death ligand 1, more easily than they are seen with single-marker immunohistochemistry (IHC) labeling. Finally, mIF technologies are promising resources for discovery of novel cancer immunotherapies and related biomarkers. In contrast with conventional IHC, which permits only the labeling of one single marker per tissue sample, mIF can detect multiple markers from a single tissue sample, and at the same time, deliver extensive information about the cell phenotypes composition and their spatial localization. In this matter, the phenotyping process is critical and must be done accurately by a highly trained personal with knowledge of immune cell protein expression and tumor pathology.

Clinicopathologic characteristics and survival of patients with primary effusion lymphoma.

Primary effusion lymphoma (PEL) is an aggressive B-cell lymphoma confined to body cavities and universally associated with human herpesvirus type 8 infection. The prognosis of this entity remains poor, with a median survival time of 6 to 9 months. To better understand the clinicopathologic features of the disease and identify possible prognostic factors, we performed a systematic review of the literature for cases of PEL, including 2 previously unreported cases from our institution. PEL was more prevalent in men (92%), with a median age at diagnosis of 55 years. The median overall survival for the entire series was 6 months. Peritoneal involvement (HR:1.62; 95% CI:1.06-2.48) and elevated serum lactate dehydrogenase (LDH) levels (HR:2.50; 95% CI:1.21-5.19) were associated with higher risk of death, while pericardial involvement (HR:0.43; 95% CI:0.20-0.94) was associated with lower risk of death. Therefore, effusion site and serum LDH levels are potential prognostic factors in patients with PEL.

Procedural Requirements and Recommendations for Multiplex Immunofluorescence Tyramide Signal Amplification Assays to Support Translational Oncology Studies.

In the development of a multiplex immunofluorescence (IF) platform and the optimization and validation of new multiplex IF panels using a tyramide signal amplification system, several technical requirements are important for high-quality staining, analysis, and results. The aim of this review is to discuss the basic requirements for performing multiplex IF tyramide signal amplification (TSA) in formalin-fixed, paraffin-embedded cancer tissues to support translational oncology research. Our laboratory has stained approximately 4000 formalin-fixed, paraffin-embedded tumor samples using the multiplex IF TSA system for immune profiling of several labeled biomarkers in a single slide to elucidate cancer biology at a protein level and identify therapeutic targets and biomarkers. By analyzing several proteins in thousands of cells on a single slide, this technique provides a systems-level view of various processes in various tumor tissues. Although this technology shows high flexibility in cancer studies, it presents several challenges when applied to study different histology cancers. Our experience shows that adequate antibody validation, staining optimization, analysis strategies, and data generation are important steps for generating quality results. Tissue management, fixation procedures, storage, and cutting can also affect the results of the assay and must be standardized. Overall, this method is reliable for supporting translational research given a precise, step-by-step approach.

Pequeña incisión suprapúbica facilita el consentimiento en necropsias perinatal y pediátrica / Small suprapubic incision facilitates consent in perinatal and pediatric necropsies

Objetivo. Presentar un tipo de incisión de la piel en la necropsia perinatal y pediátrica que permite la autorización de los padres que se hubieran negado con la técnica de incisión habitual. Métodos. Las necropsias se realizaron en 20 fetos, 15 neo natos y 8 infantes en el Hospital Madre Niño "San Bartolomé". Esta técnica consiste en una incisión de la piel transversal, suprapúbica, de 10 cm de longitud aproximadamente, evaluación manual de cavidades y órganos, evisceración de los órganos con técnica Letulle (en bloque) o tipo Virchow (por órganos), y cierre de la piel con sutura subdérmica. Resultados. Esta técnica de incisión de la piel facilitó el consentimiento de necropsia por los padres. Conclusiones. Con el procedimiento reportado se obtuvo la autorización por parte de los padres de las necropsias previamente negadas; se entregó a la madre el cadáver de su niño con una mínima incisión y se aseguró un buen estudio necrópsico.

Objective. To report a new approach lor perinatal and pediatric necropsy that allows parental consent. Methods. Experience with necropsies in 20 stillborn, 15 neonates and 8 inlants is presented with this new approach; the parents had relused the use of the standard body incision. Through a small transversal about 10 cm incision in the suprapubic region, manual evaluation of the abdominal and pelvic cavities was perlormed, and then the viscera were obtained by either the Letulles technique (a single block) or the Virchows technique (organ by organ). The incision is closed by subdermic suture. Conclusions. This technique is well accepted by the parents because the skin surface and the body as a whole appear little disturbed.