Immunoisolation of pancreatic epithelial cells from endoscopic ultrasound-guided fine needle aspirates with magnetic beads for downstream molecular application

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Abstract

Background Evaluation of pancreatic mass is routinely performed by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). However, molecular analyses of the tumor cells on FNA samples are limited by the significant cellular heterogeneity. The goal of the current study is to evaluate a magnetic immunoconcentration technique in isolating pancreatic epithelial cells from needle aspirates, and to demonstrate that the isolated cells could be utilized for molecular analysis. Methods Pancreatic EUS-FNA specimens were processed and stored at -80C. Based on the cytopathological diagnosis, 17 adenocarcinoma, 3 lymphoproliferative, and 3 benign cases were retrieved from the collection for further analyses. Epithelial cells were isolated using antihuman epithelial cell specific antibody-bound magnetic beads, and the isolated cellular component was confirmed cytologically. Genomic DNA was extracted, quantitated, and evaluated with methylation-specific PCR for cyclin D2 in 8 adenocarcinoma cases. Results After optimization, malignant epithelial cells were successfully isolated from all adenocarcinoma cases. Normal pancreatic ductal cells were isolated from three benign cases. No cells were retrieved after immunomagnetic isolation in all three lymphoproliferative cases. DNA yields were 5-2646 ng, with a mean of 357 ng. Methylation-specific PCR for cyclin D2 on the 8 carcinoma cases showed methylated state at the promoter region, demonstrating feasible evaluation of the methylation status. Conclusion The magnetic immunoconcentration of pancreatic EUS-FNA specimen described here is a practical method of isolating pancreatic epithelial cells from needle aspirates. Isolated cells were sufficient for performing subsequent molecular analysis.

Original languageEnglish (US)
Pages (from-to)32-38
Number of pages7
JournalDiagnostic Cytopathology
Volume44
Issue number1
DOIs
StatePublished - Jan 1 2016

Fingerprint

Needles
Endoscopic Ultrasound-Guided Fine Needle Aspiration
Epithelial Cells
Cyclin D2
Methylation
Adenocarcinoma
Polymerase Chain Reaction
DNA
Genetic Promoter Regions
Carcinoma
Antibodies
Neoplasms

Keywords

  • EUS-FNA
  • immunoconcentration
  • magnetic beads
  • pancreatic adenocarcinoma

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology

Cite this

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title = "Immunoisolation of pancreatic epithelial cells from endoscopic ultrasound-guided fine needle aspirates with magnetic beads for downstream molecular application",
abstract = "Background Evaluation of pancreatic mass is routinely performed by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). However, molecular analyses of the tumor cells on FNA samples are limited by the significant cellular heterogeneity. The goal of the current study is to evaluate a magnetic immunoconcentration technique in isolating pancreatic epithelial cells from needle aspirates, and to demonstrate that the isolated cells could be utilized for molecular analysis. Methods Pancreatic EUS-FNA specimens were processed and stored at -80C. Based on the cytopathological diagnosis, 17 adenocarcinoma, 3 lymphoproliferative, and 3 benign cases were retrieved from the collection for further analyses. Epithelial cells were isolated using antihuman epithelial cell specific antibody-bound magnetic beads, and the isolated cellular component was confirmed cytologically. Genomic DNA was extracted, quantitated, and evaluated with methylation-specific PCR for cyclin D2 in 8 adenocarcinoma cases. Results After optimization, malignant epithelial cells were successfully isolated from all adenocarcinoma cases. Normal pancreatic ductal cells were isolated from three benign cases. No cells were retrieved after immunomagnetic isolation in all three lymphoproliferative cases. DNA yields were 5-2646 ng, with a mean of 357 ng. Methylation-specific PCR for cyclin D2 on the 8 carcinoma cases showed methylated state at the promoter region, demonstrating feasible evaluation of the methylation status. Conclusion The magnetic immunoconcentration of pancreatic EUS-FNA specimen described here is a practical method of isolating pancreatic epithelial cells from needle aspirates. Isolated cells were sufficient for performing subsequent molecular analysis.",
keywords = "EUS-FNA, immunoconcentration, magnetic beads, pancreatic adenocarcinoma",
author = "Afify, {Alaa M} and Huang, {Eric C} and Matthew Jeong and Shiro Urayama",
year = "2016",
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T1 - Immunoisolation of pancreatic epithelial cells from endoscopic ultrasound-guided fine needle aspirates with magnetic beads for downstream molecular application

AU - Afify, Alaa M

AU - Huang, Eric C

AU - Jeong, Matthew

AU - Urayama, Shiro

PY - 2016/1/1

Y1 - 2016/1/1

N2 - Background Evaluation of pancreatic mass is routinely performed by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). However, molecular analyses of the tumor cells on FNA samples are limited by the significant cellular heterogeneity. The goal of the current study is to evaluate a magnetic immunoconcentration technique in isolating pancreatic epithelial cells from needle aspirates, and to demonstrate that the isolated cells could be utilized for molecular analysis. Methods Pancreatic EUS-FNA specimens were processed and stored at -80C. Based on the cytopathological diagnosis, 17 adenocarcinoma, 3 lymphoproliferative, and 3 benign cases were retrieved from the collection for further analyses. Epithelial cells were isolated using antihuman epithelial cell specific antibody-bound magnetic beads, and the isolated cellular component was confirmed cytologically. Genomic DNA was extracted, quantitated, and evaluated with methylation-specific PCR for cyclin D2 in 8 adenocarcinoma cases. Results After optimization, malignant epithelial cells were successfully isolated from all adenocarcinoma cases. Normal pancreatic ductal cells were isolated from three benign cases. No cells were retrieved after immunomagnetic isolation in all three lymphoproliferative cases. DNA yields were 5-2646 ng, with a mean of 357 ng. Methylation-specific PCR for cyclin D2 on the 8 carcinoma cases showed methylated state at the promoter region, demonstrating feasible evaluation of the methylation status. Conclusion The magnetic immunoconcentration of pancreatic EUS-FNA specimen described here is a practical method of isolating pancreatic epithelial cells from needle aspirates. Isolated cells were sufficient for performing subsequent molecular analysis.

AB - Background Evaluation of pancreatic mass is routinely performed by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). However, molecular analyses of the tumor cells on FNA samples are limited by the significant cellular heterogeneity. The goal of the current study is to evaluate a magnetic immunoconcentration technique in isolating pancreatic epithelial cells from needle aspirates, and to demonstrate that the isolated cells could be utilized for molecular analysis. Methods Pancreatic EUS-FNA specimens were processed and stored at -80C. Based on the cytopathological diagnosis, 17 adenocarcinoma, 3 lymphoproliferative, and 3 benign cases were retrieved from the collection for further analyses. Epithelial cells were isolated using antihuman epithelial cell specific antibody-bound magnetic beads, and the isolated cellular component was confirmed cytologically. Genomic DNA was extracted, quantitated, and evaluated with methylation-specific PCR for cyclin D2 in 8 adenocarcinoma cases. Results After optimization, malignant epithelial cells were successfully isolated from all adenocarcinoma cases. Normal pancreatic ductal cells were isolated from three benign cases. No cells were retrieved after immunomagnetic isolation in all three lymphoproliferative cases. DNA yields were 5-2646 ng, with a mean of 357 ng. Methylation-specific PCR for cyclin D2 on the 8 carcinoma cases showed methylated state at the promoter region, demonstrating feasible evaluation of the methylation status. Conclusion The magnetic immunoconcentration of pancreatic EUS-FNA specimen described here is a practical method of isolating pancreatic epithelial cells from needle aspirates. Isolated cells were sufficient for performing subsequent molecular analysis.

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