We present a study comparing the most popular heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven, microwave with pressure cooker, autoclave, and steamer heating. A panel of 21 antibodies was tested on formalin fixed, paraffin embedded sections using these heating methods and Tris-HC1 buffer, pH 9.5, plus 5% urea as the AR solution. Three observers independently evaluated the intensity of AR immunostaining. All heating methods yielded good results for AR immunostaining. There were only minor differences among the heating methods for AR when the optimal concentration of primary antibody for normal immunostaining was used; however, background staining may occasionally be troublesome if antibodies are not retitrated and diluted further for use on tissues after AR. Significant differences were observed only after further dilution of the primary antibodies: the microwave pressure cooker, extended microwave heating (5 min x 4) and autoclave heating then showed a similar intensity of staining that was stronger than results obtained with the steamer (20 min) or regular microwave heating (5 min x 2). Extension of the steamer heating time, however, yielded equivalent results. This study indicates that different heating methods can yield similar intensities of AR immunostaining if the heating times are adjusted appropriately. It is noteworthy that, in general, the adjusted conditions for maximal retrieval differ from those most widely cited in the literature, or recommended by manufacturers. That several heating devices may provide similar results permits the use of different AR heating methods according to the equipment available. This study also is an early step in standardizing the AR immunostaining protocol by providing uniform conditions for "maximal retrieval" as a common end point for all laboratories.

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4. Laryngoscope, 127:1283-1287, 2017.

The retinoblastoma (RB) gene, which encodes the nuclear RB protein (pRB), is believed to be involved in cell cycle control and cell differentiation. Studies have demonstrated that loss of RB function may play a role in tumour formation and progression of a variety of human tumours, such as bladder, lung, breast, and prostate cancers. The immunohistochemical detection of pRB expression in formalin–paraffin sections of human cancer has potential advantages of convenience, economy, and compatibility with routine surgical pathology practice. In practice, however, results using pRB antibodies on routinely processed, paraffin‐embedded tissue have been inconsistent. In this study, the antigen retrieval (AR) method has been applied to the immunohistochemical detection of pRB in paraffin‐embedded tissues and a ‘test battery’ approach has been developed to identify the principal variables that result in the optimal AR protocol. This approach includes the use of buffered solutions at pH 1, 6, and 10 with three different heating conditions (temperatures 120°C, 100°C, and 90°C). In the example described here with antibody RB‐WL‐1, the low pH solution with the microwave heating at 100°C proved most effective. Both fresh and routinely processed formalin–paraffin tissues of normal and bladder carcinoma were used for a comparison of the pRB immunostaining. The AR method was evaluated by comparing the immunohistochemical staining result on routinely processed formalin–paraffin sections with frozen sections of the same tumour. A consistent intensity of immunohistochemical staining for pRB was achieved using the identified optimal AR protocol on formalin–paraffin sections. All slides showed positive staining of pRB in normal mesenchymal and epithelial tissues. The pattern of pRB localization and intensity of staining was similar to that obtained in frozen sections, though the intensity obtained by AR treatment on paraffin sections was slightly to moderately stronger than that obtained in frozen sections. Once the protocol was identified, it was tested using routinely processed paraffin tissue sections of 245 cases of bladder carcinoma, with consistent pRB immunostaining results. The protocol described is simple to perform and gives reproducible results for evaluation of pRB expression by immunohistochemistry.

Objectives Our objectives were (1) to use in situ simulation to assess the clinical environment and identify latent safety threats (LSTs) related to the management of pediatric tracheostomy patients and (2) to analyze the effects of systems interventions and team factors on LSTs and simulation performance. Methods A multicenter, prospective study to assess LSTs related to pediatric tracheostomy care management was conducted in emergency departments (EDs) and intensive care units (ICUs). LSTs were identified through equipment checklists and in situ simulations via structured debriefs and blinded ratings of team performance. The research team and unit champions developed action plans with interventions to address each LST. Reassessment by equipment checklists and in situ simulations was repeated after 6 to 9 months. Results Forty-one LSTs were identified over 21 simulations, 24 in the preintervention group and 17 in the postintervention group. These included LSTs in access to equipment (ie, availability of suction catheters, lack of awareness of the location of tracheostomy tubes) and clinical knowledge gaps. Mean equipment checklist scores improved from 76% to 87%. Twenty-one unique teams (65 participants) participated in the simulations. The average simulation score was 6.19 out of 16 points. Discussion In situ simulation is feasible and effective as an assessment tool to identify latent safety threats and thus measure the system-level performance of a clinical care environment. Implications for Practice In situ simulation can be used to identify and reassess latent safety threats related to pediatric tracheostomy management and thereby support quality improvement and educational initiatives.