Method to make paraffin-embedded breast and lymph tissue mimic fresh tissue in DNA analysis.
Determination of DNA ploidy has been found to be of diagnostic and prognostic value with regard to many solid tumors. Flow cytometric analysis of DNA ploidy is dependent on the binding of fluorescent dyes to DNA. Preserving cell morphology by fixing the tissue in formalin interferes with the binding of propidium iodide (PI) and other fluorescent dyes to DNA. This distortion of DNA content measurement can cause inaccuracies in DNA-ploidy determinations of formalin-fixed tissue specimens and precludes the use of appropriate DNA standards. Therefore, it has been impossible to determine accurately the DNA ploidy of formalin-fixed, paraffin-embedded (FFPE) tissues. Using formalin-fixed cells as a model for FFPE cells, we developed a thermal treatment method to reverse the effect of formalin on the binding of propidium iodide to DNA. Applying this approach to the preparation of FFPE lymph node and breast tissue for DNA analysis, we have developed a method that makes the binding of PI to the DNA of FFPE tissue mimic that of fresh tissue. Following dewaxing, rehydration, and trypsin treatment, the FFPE tissue, resuspended in PBS, was heated to 75 degrees C for 90 min to restore the PI binding to that of fresh cells. This method makes it possible to use fresh, DNA-diploid cells as an internal control and, thus, determine more accurately the DNA ploidy of tumors preserved in formalin and paraffin.
Overton
W R
WR
Department of Pediatrics, Cooper Hospital/UMC, UMDNJ-Robert Wood Johnson Medical School, Camden 08103, USA.
Catalano
E
E
McCoy
J P
JP
D000782:Aneuploidy; D001940:Breast / Q000378:metabolism* / Q000473:pathology; D002918:Chymotrypsin / Q000378:metabolism; D004247:DNA / Q000032:analysis*; D010450:Endopeptidases / Q000378:metabolism; D005260:Female; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D008198:Lymph Nodes / Q000378:metabolism* / Q000473:pathology; D008214:Lymphocytes / Q000378:metabolism* / Q000473:pathology; D016612:Paraffin Embedding; D014357:Trypsin / Q000378:metabolism
D005404:Fixatives; D005557:Formaldehyde; D004247:DNA; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin