The Royal College of Pathologists of Australasia Quality Assurance Program: Immunohistochemistry Breast Marker Audit Overview 2005-2015.

The Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP) Anatomical Pathology provides a comprehensive External Quality Assurance (EQA) exercise to review the reporting of immunohistochemistry (IHC) and in-situ hybridization (ISH) breast markers through an audit of clinical results. The aim of this exercise was to provide information regarding the quality of breast marker testing within clinical laboratories from 2005 to 2015. This comprehensive audit included estrogen, progesterone, and HER2 marker reporting. This was an important quality assurance activity established in response to ongoing difficulties experienced in laboratories in this area of testing.

Microwaves for chromogenic in situ hybridization.

In situ hybridization can be employed in formalin-fixed, paraffin-embedded tissue sections (FFPT) and allows direct visualization of amplified genes and chromosomes in individual cell nuclei. Fluorescence in situ hybridization (FISH) is the most widely employed method, but the fluorescence preparations suffer from the main disadvantages of fading over time and poor visualization, the latter making it difficult to accurately separate invasive from in situ cancer cells. Chromogenic in situ hybridization (CISH) is a viable alternative to FISH in FFPT as it employs a peroxidase reaction to visualize the chromogen thus allowing the convenience of bright field microscopy and the correlation of the visualized gene amplification with cytomorphology. It is relatively less expensive and allows a permanent record, with several studies attesting to its validity. As with FISH, heat pretreatment and enzyme digestion are two critical components of the protocol. We describe a protocol for CISH in which a microwave-induced target retrieval step is introduced as a replacement for heat pretreatment. The same procedure is performed following enzyme digestion to produce consistent signals in amplified and nonamplified cells that are both larger in size and numbers when compared with those produced by the conventional protocol.

Citraconic anhydride: a new antigen retrieval solution.

BACKGROUND: The introduction of heat-induced antigen retrieval has been a major milestone in diagnostic immunohistochemistry, enabling the application of many antibodies to fixed paraffin-embedded tissue sections. A number of important variables affect the preservation of tissue antigens, among which are analytical variables including the antigen retrieval methodology. Temperature of retrieval, duration of heating, source of heat, pH and nature of retrieval solution are among more important variables pivotal to results. Citrate buffer at pH 6.0 has become widely embraced as the universal fixative but some antibodies remain capricious and yield poor staining results. METHODS: This study examines the recent suggestion that citraconic anhydride may be a suitable universal retrieval reagent. Immunostaining of 65 commonly employed antibodies following microwave antigen retrieval in 0.05% citraconic anhydride for 10 minutes at 98 degrees C was compared with consecutive tissues sections subjected to antigen retrieval in citrate buffer at pH 6.0 at the same duration and temperature. RESULTS: Thirty-five of the 65 antibodies examined yielded more intense staining following antigen retrieval in citraconic anhydride, including some capricious antibodies such as MyoD1, myogenin, perforin, TIA-1, Tdt, RET and MiTF, confirming the efficacy of this retrieval solution. CONCLUSION: It is recommended that consideration be given to 0.05% citraconic anhydride as an antigen retrieval solution, particularly for antibodies that fail to work or stain weakly in fixed paraffin-embedded tissue sections.

Microwave enhancement of CISH for HER2 oncogene.

We describe a modification to the prescribed procedure for the Zymed Spot-Light HER2 chromogenic in situ hybridization kit (84-0146, Zymed Laboratories, San Francisco, CA) by substituting the heat pretreatment step with MW irradiation in citrate buffer 10 mmol/L at pH 6.0 at 98 degrees C for 10 minutes and repeating the procedure afterenzyme digestion with time and temperature controlled in the Mega T/ T oven (Milestone s.r.l., Sorisole, Italy). The subsequent procedure leading up to hybridized was as per manufacturer's instructions. Invasive breast carcinoma previously scored by immunohistochemistry for HER2, comprising 18 cases of 3+, 18 cases of 2+, and 12 cases of 1+, were examined by chromogenic in situ hybridization using this modified procedure, with a parallel set of cases examined by the prescribed Zymed method. The introduction of the "MW retrieval" steps resulted in consistently a greater number of hybridization signals in amplified tumor cells with benign epithelial cells and lymphocytes displaying 2 clear dots compared with the weaker and less consistent signals obtained with the standard procedure. MW exposed sections showed larger numbers of large and small clusters that often allowed identification of amplified tumors without having to count single dots with crisp staining and absence of background precipitation.

Refinement of immunohistologic parameters for Her2/neu scoring validation by FISH and CISH.

The conventional method of scoring Her2/neu immunostaining is recognized to result in a high false-positive rate among 2+ cases when compared with results obtained with fluorescence in situ hybridization (FISH); however, costs and convenience dictates that immunohistochemistry remains the screening test for Her2/neu status in patients with breast cancer. We describe refined criteria for scoring of Her2/neu on the basis of anatomic localization rather than the subjective assessment of intensity. The presence of a circumferential tram track pattern that results from the staining of apposing cell membranes in >25% of the tumor cells was necessary for a 3+ score (Her2/neu overexpressed) and the presence of the tram track pattern in <25% was scored 2+ (equivocal); granular and fragmented membrane staining was scored 1+ (negative). The tram track pattern of Her2/neu overexpression showed 100% concordance with gene amplification. FISH and CISH testing in selected cases from the other categories validated the revised scoring method. These criteria reduced the numbers of equivocal staining cases that required FISH testing.

Superheating antigen retrieval.

Heat-induced antigen retrieval in a variety of solutions has been shown to enhance the immunoreactivity of a wide range of antigens in routine formalin-fixed, paraffin-embedded tissues. Accurate time and temperature control is important for standardization and optimization of the procedure but is difficult to achieve. This study used a device to attain precise time and temperature control for antigen retrieval at 120 degrees C under 1.9 bar pressure. It compares the efficacy of this method with antigen retrieval in a conventional pressure cooker, by microwave heating at 98 degrees C, and ultrasound retrieval at 40 and 70 W for 40 and 100 seconds. Multitissue and multitumor blocks containing a spectrum of normal tissues and a variety of tumors, respectively, were used, and 42 routine diagnostic antibodies were applied with a standard peroxidase conjugated streptavidin technique. Sections in which antigen retrieval was not performed served as controls. The three heat-induced methods showed distinctly better immunostaining for all antigens compared with those obtained with ultrasound retrieval. The latter method did not produce consistent staining and intensity, and the extent of staining was only marginally better than sections not subjected to antigen retrieval. Superheating at 120 degrees C produced the best overall results with the exception of antibodies to cytokeratin clones Cam 5.2, AE1/3, and 34BE12 in which superheating resulted in slightly inferior immunostaining compared with heating in a pressure cooker and at 98 degrees C.