College of American Pathologists
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  Q and A





cap today



June 2006

Richard A. Savage

Q: Does Bouin fixation of biopsies cause false-negative immunohistochemical reactions with the lymphocytic markers?

A. I do not know of any publications that have thoroughly addressed this question, but my personal experience with such cases indicates a significant problem with false-negative reactions when cases are fixed in Bouin solution. This has been a particular problem in gastric biopsies sent for immunophenotyping to address the possibility of gastric lymphoma. On more than one occasion, the laboratory could not render a confident diagnosis due to the deleterious effects of Bouin fixation. In several of these cases, it was necessary to rebiopsy the patient and fix the tissue in neutral buffered formalin to obtain a definitive diagnosis. In my experience, tissues fixed in Hollande fixative suffer a similar sorry fate when immunostained for lymphoid markers and other markers.

I recommend avoiding Bouin and Hollande fixatives if there is a chance that the tissue may need immunostains to render a definitive diagnosis. You can be assured that patients who require rebiopsy for a definitive diagnosis would not care that some pathologists feel the perceived histologic detail is better in Bouin and Hollande fixatives than in tissue fixed in neutral buffered formalin.

Rodney T. Miller, MD Director of Immunohistochemistry

Member, CAP Immunohistochemistry Committee

Q: CAP anatomic pathology checklist question ANP. 22550 refers to using positive immunohistochemistry controls that have been processed using the same fixation as the patient tissue. Our institution occasionally does immunoperoxidase stains on cell blocks originally fixed in Cytolyt but subsequently put in formalin on the tissue processor. The controls we use are tissue sections fixed and processed in formalin. Is this compliant with the checklist question, since cytology and histology blocks were processed with formalin, or do we need to use new controls for our cell blocks using specimens originally fixed in Cytolyt?

A. It would be ideal if the positive control tissue used was identical to the patient tissue with respect to fixation, processing, and specimen type—for example, small biopsy, large tissue section, cell blocks, cytospin, or decalcified bone. For most laboratories, however, it is impractical to maintain separate banks of control tissues for every fixative and specimen type encountered in routine practice.

If control tissues that are fixed and processed identically to the patient specimen are not available, it is reasonable for the laboratory to use its normal control tissues, provided they exhibit equivalent immunoreactivity. The laboratory can document this by parallel testing, such as testing a small panel of common markers in a formalin-fixed biopsy and a Cytolyt-fixed cell block of the same specimen type. Comparable antigen expression would confirm the validity of using routine control tissues for specimens that are processed differently.

Richard N. Eisen, MD
Greenwich Hospital
Yale New Haven Health System
Greenwich, Conn.
Member, CAP Immunohistochemistry Committee

Patrick L. Fitzgibbons, MD
St. Jude Medical Center
Fullerton, Calif.
Chair, CAP
Immunohistochemistry Committee