| Staining | Optimal fixation |
| Antigen retrieval—citrate pH 6.0, but test higher pH buffers if suboptimal staining |
| Antibody validated against biochemical assay |
| Positive control with range of staining; choose test tissue with normal breast included if possible |
| Quality assurance, internal and external |
| |
| Assessment | Nuclear staining only; cytoplasmic staining may be due to excess antigen retrieval |
| Only invasive cancer assessed |
| Strong relationship with grade, so if grade I low/negative repeat |
| Use a recognised scoring system. Allred score easy to use and identifies low positive cases |