False aneuploidy in flow cytometric DNA analysis of paraffin embedded tissue: Effects of carnoy's fixation

Abstract

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 GO/G1 histogram peak to the left, increase the GO/Gl 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 andlor false elevation of S phase fraction. This reinforces the need for matched negative tissue controls for DNA analysis of paraffin embedded specimens whenever possible. (1,7). This report documents an additional cause of both false aneuploidy and false elevation of S phase fraction (SPF) in paraffin embedded normal tissues. These abnormalities were observed in tissues in contact with Carnoy's solution.Carnoy's solution is a rapidly penetrating fixative composed of absolute ethanol, chloroform and glacial acetic acid (6). In our laboratory, Carnoy's is used to "clear" or render translucent fat from node dissections to optimize lymph node yield. The fat is placed whole in a large volume of Carnoy's for several hours prior to node dissection. The dissected nodes are then placed in cassettes and completely fixed in 10% neutral buffered formalin prior to embedding in paraffin.The first phase of this study documents the presence of false aneuploid peaks on flow cytometric DNA analysis of normal lymph nodes processed in the manner described above. The second phase studies the effect of complete fixation of various tissues in Carnoy's solution compared to formalin fixed controls. The third phase of the study examines the effects of partial fixation in Carnoy's compared to formalin fixed controls. This last phase demonstrates the occurrence of false aneuploidy related to partial fixation in Carnoy's. The second phase suggests a mechanism whereby this occurs.Fixation has previously been reported as a factor affecting flow cytometric DNA analysis of paraffin embedded tissues (2,3,5). False aneuploidy as it relates to partial fixation or exposure to more than one fixative has not previously been emphasized. The implications for flow cytometric DNA analysis of paraffin embedded tissues are discussed. 'This project was conducted through The John S. Sharpe Research Foundation at The Bryn Mawr Hospital.

Statistical considerations in DNA flow cytometry

“…Even the thickness of the slice of tissue can affect the amount of debris present in the cell suspension (Hitchcock and Ensley, 1993). Pres-ence of a false aneuploid peak can be the result of choice of fixative (Carr and Abaza, 1993;Herbert et al, 1989;Pelstring, Hurtubise and Swerdlow, 1990;Shankey et al, 1993). Autolysis can yield poor quality data sets, skewed diploid peaks, false aneuploid peaks, increased debris, artifactual antigen expression and changed chromatic structure which alters the stoichiometry of dye binding to DNA (Alanen, Hitchcock and Ensley, 1993;Joensuu, Alanen, Klemi and Aine, 1990;.…”