Recognition and reduction of artifacts from autolysis in paraffin-embedded tissue using DNA/nuclear protein flow cytometry

Alan Pollack, Gaetano Ciancio, N. H A Terry, Norman L Block

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Artifacts from autolysis can be a problem in retrospective flow-cytometric analyses of DNA content in paraffin-embedded tissues. Autolyzed tissue from rat liver, human liver, and rat spleen were stained for DNA and nuclear protein to determine if this technique would be useful in identifying partially degraded cells. After the tissue was deparaffinized and rehydrated, the nuclei were isolated using 0.5% pepsin. Propidium iodide (PI) and fluorescein isothiocyanate (FITC) were used to stain DNA and nuclear protein. When unfixed rat liver tissue was allowed to undergo autolysis at 4°C for 24-48 b before fixation, there was a progressive broadening of the G1 and G2M DNA peaks and a slight increase in the average DNA contents of these peaks. Nuclei that stained more intensely with PI also stained more intensely with FITC. Similar results were obtained using human liver and rat spleen. Sometimes the increased PI staining resulted in a false aneuploid peak. The distinctive skewing of the DNA/nuclear protein histograms from autolysis was reduced by increasing the incubation of the tissue in 0.5% pepsin from 0.5 h to 1.5 h during the nuclei-isolation step. The DNA/nuclear protein method provides a means for identifying artifacts from autolysis, whereas the extended pepsin treatment provides a means for reducing these artifacts.

Original languageEnglish
Pages (from-to)565-568
Number of pages4
JournalCytometry
Volume14
Issue number5
StatePublished - Jan 1 1993

Fingerprint

Autolysis
Nuclear Proteins
Paraffin
Artifacts
Flow Cytometry
DNA
Propidium
Pepsin A
Liver
Fluorescein
Spleen
Aneuploidy
Coloring Agents
Staining and Labeling

Keywords

  • autolysis
  • flow cytometry
  • nuclear protein
  • paraffin-embedded tissue

ASJC Scopus subject areas

  • Biophysics
  • Cell Biology
  • Endocrinology
  • Hematology
  • Pathology and Forensic Medicine

Cite this

Recognition and reduction of artifacts from autolysis in paraffin-embedded tissue using DNA/nuclear protein flow cytometry. / Pollack, Alan; Ciancio, Gaetano; Terry, N. H A; Block, Norman L.

In: Cytometry, Vol. 14, No. 5, 01.01.1993, p. 565-568.

Research output: Contribution to journalArticle

@article{4ef06ccb6f404746b69dcb5a8b470c75,
title = "Recognition and reduction of artifacts from autolysis in paraffin-embedded tissue using DNA/nuclear protein flow cytometry",
abstract = "Artifacts from autolysis can be a problem in retrospective flow-cytometric analyses of DNA content in paraffin-embedded tissues. Autolyzed tissue from rat liver, human liver, and rat spleen were stained for DNA and nuclear protein to determine if this technique would be useful in identifying partially degraded cells. After the tissue was deparaffinized and rehydrated, the nuclei were isolated using 0.5{\%} pepsin. Propidium iodide (PI) and fluorescein isothiocyanate (FITC) were used to stain DNA and nuclear protein. When unfixed rat liver tissue was allowed to undergo autolysis at 4°C for 24-48 b before fixation, there was a progressive broadening of the G1 and G2M DNA peaks and a slight increase in the average DNA contents of these peaks. Nuclei that stained more intensely with PI also stained more intensely with FITC. Similar results were obtained using human liver and rat spleen. Sometimes the increased PI staining resulted in a false aneuploid peak. The distinctive skewing of the DNA/nuclear protein histograms from autolysis was reduced by increasing the incubation of the tissue in 0.5{\%} pepsin from 0.5 h to 1.5 h during the nuclei-isolation step. The DNA/nuclear protein method provides a means for identifying artifacts from autolysis, whereas the extended pepsin treatment provides a means for reducing these artifacts.",
keywords = "autolysis, flow cytometry, nuclear protein, paraffin-embedded tissue",
author = "Alan Pollack and Gaetano Ciancio and Terry, {N. H A} and Block, {Norman L}",
year = "1993",
month = "1",
day = "1",
language = "English",
volume = "14",
pages = "565--568",
journal = "Cytometry Part B - Clinical Cytometry",
issn = "1552-4949",
publisher = "Wiley-Liss Inc.",
number = "5",

}

TY - JOUR

T1 - Recognition and reduction of artifacts from autolysis in paraffin-embedded tissue using DNA/nuclear protein flow cytometry

AU - Pollack, Alan

AU - Ciancio, Gaetano

AU - Terry, N. H A

AU - Block, Norman L

PY - 1993/1/1

Y1 - 1993/1/1

N2 - Artifacts from autolysis can be a problem in retrospective flow-cytometric analyses of DNA content in paraffin-embedded tissues. Autolyzed tissue from rat liver, human liver, and rat spleen were stained for DNA and nuclear protein to determine if this technique would be useful in identifying partially degraded cells. After the tissue was deparaffinized and rehydrated, the nuclei were isolated using 0.5% pepsin. Propidium iodide (PI) and fluorescein isothiocyanate (FITC) were used to stain DNA and nuclear protein. When unfixed rat liver tissue was allowed to undergo autolysis at 4°C for 24-48 b before fixation, there was a progressive broadening of the G1 and G2M DNA peaks and a slight increase in the average DNA contents of these peaks. Nuclei that stained more intensely with PI also stained more intensely with FITC. Similar results were obtained using human liver and rat spleen. Sometimes the increased PI staining resulted in a false aneuploid peak. The distinctive skewing of the DNA/nuclear protein histograms from autolysis was reduced by increasing the incubation of the tissue in 0.5% pepsin from 0.5 h to 1.5 h during the nuclei-isolation step. The DNA/nuclear protein method provides a means for identifying artifacts from autolysis, whereas the extended pepsin treatment provides a means for reducing these artifacts.

AB - Artifacts from autolysis can be a problem in retrospective flow-cytometric analyses of DNA content in paraffin-embedded tissues. Autolyzed tissue from rat liver, human liver, and rat spleen were stained for DNA and nuclear protein to determine if this technique would be useful in identifying partially degraded cells. After the tissue was deparaffinized and rehydrated, the nuclei were isolated using 0.5% pepsin. Propidium iodide (PI) and fluorescein isothiocyanate (FITC) were used to stain DNA and nuclear protein. When unfixed rat liver tissue was allowed to undergo autolysis at 4°C for 24-48 b before fixation, there was a progressive broadening of the G1 and G2M DNA peaks and a slight increase in the average DNA contents of these peaks. Nuclei that stained more intensely with PI also stained more intensely with FITC. Similar results were obtained using human liver and rat spleen. Sometimes the increased PI staining resulted in a false aneuploid peak. The distinctive skewing of the DNA/nuclear protein histograms from autolysis was reduced by increasing the incubation of the tissue in 0.5% pepsin from 0.5 h to 1.5 h during the nuclei-isolation step. The DNA/nuclear protein method provides a means for identifying artifacts from autolysis, whereas the extended pepsin treatment provides a means for reducing these artifacts.

KW - autolysis

KW - flow cytometry

KW - nuclear protein

KW - paraffin-embedded tissue

UR - http://www.scopus.com/inward/record.url?scp=0027319525&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027319525&partnerID=8YFLogxK

M3 - Article

C2 - 8354130

AN - SCOPUS:0027319525

VL - 14

SP - 565

EP - 568

JO - Cytometry Part B - Clinical Cytometry

JF - Cytometry Part B - Clinical Cytometry

SN - 1552-4949

IS - 5

ER -