Proper specimen handling and preparationhas been under-appreciated by clinicians and pathologists alike.How preanalytical variables such as tissue handing and tissue fixation have the potential to significantly and adversely affect the accurate assessment of therapeutic targets such as ER/Her2neu in tumor specimens.,must be understood properly.
Following are the Consensus Recommendations on Estrogen Receptor Testing in Breast Cancer By Immunohistochemistry by Members of the Standardization Ad-Hoc Consensus Committee.
- As soon as they are available from the operating room, breast specimens with a known cancer, or a suspicion of cancer, should be oriented, inked, carefully sectioned at 0.5 to 1 cm intervals and placed in the appropriate volume of fixative with gauze pads or paper towels in between slices to assist with the penetration of fixative into all areas of the tissue.
- In addition,if gross tumor is easily identifiable, the pathologist (or pathology assistant) should remove a 2-mm thick sample of tumor, together with a 2-mm thick sample of benign tissue and place both into the same cassette at the time of the initial evaluation, thus ensuring that normal breast elements are available as appropriate internal tissue controls for subsequent breast marker testing.
- Breast core biopsies should be fixed and processed in an identical manner to excision specimens.
- Only 10% aqueous phosphate-buffered 4% formaldehyde pH 7.0-7.4 (10% phosphate-buffered formalin) should be used as the fixative for breast tissue samples.
- Minimum fixation times of at least 8 hours optimally 24 to 48 hours, not to extend to more than 72 hours are recommended for all laboratories accepting breast tissue samples.
There is a misconception that smaller biopsy samples will fix more quickly than larger resection specimens and therefore require less time in formalin. It is true that formalin will penetrate smaller samples more quickly than larger samples, but actual fixation is a chemical reaction that takes time.Penetration time of formalin is of little importance than the chemical reaction time .For example, a single layer of cells is penetrated by formalin extremely rapidly, however the chemical reaction requires 24 hours to complete. A 4-mm thick slice of tissue is fully penetrated within an hour and requires 25 hours for the chemical reaction to complete.
Having a record of the fixation time for each breast tissue sample is expected to prove valuable for interpreting and troubleshooting aberrant and/or unexpected ER results.One such example would be when an ER IHC assay is found to be negative in a well-differentiated cancer such as a tubular carcinoma or a classic lobular carcinoma. This result would be unexpected, and having access to the details on tissue fixation, such as a long delay before the initiation of fixation or short fixation time in formalin would be valuable in interpreting the validity of the unexpected results for such a patient.
Consensus Recommendations on Estrogen Receptor Testing in Breast Cancer By Immunohistochemistry;Hadi Yaziji et al.Apllied immunohistochem Mol.Vol16,Dec.2008