False aneuploidy in flow cytometric DNA analysis of paraffin embedded tissue: effects of Carnoy's fixation

False aneuploidy was detected on flow cytometric DNA analysis of paraffin embedded axillary lymph nodes negative for tumor. It was hypothesized that "clearing" of axillary fat in Carnoy's solution to facilitate lymph node dissection might be responsible for false aneuploidy. Various tissues fixed overnight in Carnoy's were compared to formalin fixed paraffin embedded controls. Under these conditions no false aneuploid peaks were detected, but Carnoy's fixation did shift the G0/G1 histogram peak to the left, increase the G0/G1 CV and increase the S phase fraction relative to formalin fixed controls. It was then hypothesized that partial fixation of nodes in Carnoy's followed by formalin fixation might result in false aneuploid peaks. Twenty-two lymph nodes were partially fixed in Carnoy's for periods ranging from 5 to 60 min followed by complete fixation in formalin. Seven of these nodes did show false aneuploid peaks. By contrast, no aneuploidy was detected in formalin fixed controls. It was concluded that tissues in contact with Carnoy's solution may be a source of false aneuploidy and/or false elevation of S phase fraction. This reinforces the need for matched negative tissue controls for DNA analysis of paraffin embedded specimens whenever possible.