Comparison of pepsin and trypsin digestion on paraffin-embedded tissue preparation for DNA flow cytometry.
Preparation with enzymes plays an important part in obtaining good results in cellular DNA content measurements from paraffin-embedded tissue using flow cytometry. Therefore, we have compared two groups of DNA histograms obtained with pepsin (Hedley method) and trypsin digestion (Schutte method). Samples of five tumor types were compared: eight lung, seven breast, five thyroid, seven stomach, and six colon cancer cases. All samples were stained with propidium iodide (PI). The incidence of DNA aneuploidy determined by the Schutte method indicated a higher ratio in four tumor types than that determined by the Hedley method. Concerning the coefficient of variation (CV) used to estimate the quality of the DNA histogram, the diploid G1 peak determined by the Schutte method produced a smaller CV in five tumor types than did the Hedley method. This Schutte method had especially good results in lung and thyroid cancer. Furthermore, the amount of debris (background) determined by the Schutte method indicated a decrease in four tumor types compared with the Hedley method. In cell-cycle analysis of ten DNA diploid cases, DNA histograms by the Hedley method showed an increased S-phase fraction due to an overlapping of debris and aggregated cells. These results indicate that preparation by trypsin digestion is a method superior to pepsin digestion when the sample from paraffin-embedded tissue is stained with PI for the purpose of DNA content measurement.
Tagawa
Y
Y
First Department of Surgery, Nagasaki University School of Medicine, Japan.
Nakazaki
T
T
Yasutake
T
T
Matsuo
S
S
Tomita
M
M
D001943:Breast Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000473:pathology; D002453:Cell Cycle; D003110:Colonic Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000473:pathology; D016612:Paraffin Embedding; D010434:Pepsin A / Q000494:pharmacology*; D011003:Ploidies; D013274:Stomach Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D013964:Thyroid Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000473:pathology; D014357:Trypsin / Q000494:pharmacology*
D004273:DNA, Neoplasm; D014357:Trypsin; D010434:Pepsin A