Polymerase chain reaction microanalysis of tumors from stained histological slides.

Author(s): Whetsell L, Maw G, Nadon N, Ringer DP, Schaefer FV

Publication: Oncogene, 1992, Vol. 7, Page 2355-61

PubMed ID: 1279500 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects cell capture method, PCR fragment length, storage and deparaffinization on PCR and genotype determination in stained FFPE pancreas, colorectal, lung and liver tumors.

   Summary of Findings:

   Use of a standard pipetman p20 tip to scrape cells from a 1-2 mM area of the section or use of a micromanipulator to scrape 30-100 cells was superior to using a syringe or Pasteur pipette. Using oil to collect the scraping was superior to water, ethanol or xylene. A 128 bp fragment of k-ras was amplified directly from the mineral oil-cell mixture, but efficiency improved slightly with the addition of 5 uL water (92% versus 88%). A 260 bp fragment of h-ras was amplified in 24% of specimens with a single round of amplification and 31% when nested primers were used in a second round of amplification. Amplification success decreased when the fragment was more than 210 bp. PCR success was lower in the specimen stored for 10 years (42%) and the specimen that was not stained or deparaffinized (38%) compared to specimens that were routinely stained and deparaffinized and not stored (88%).

   Biospecimens

   • Tissue - Pancreas
   • Tissue - Liver
   • Tissue - Lung
   • Tissue - Colorectal

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic

   Platform:

   Analyte	Technology Platform
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Cell capture method	Scraping with syringe Scraping with Pasteur pipette Pipetman tip Micromanipulator Ethanol Mineral oil Water Xylene
   PCR Specific	Length of gene fragment	128 bp 174 bp 210 bp 260 bp
   PCR Specific	Targeted nucleic acid	h-ras k-ras
   Analyte Extraction and Purification	Deparaffinization	None Xylene
   Storage	Storage duration	None 10 years
   PCR Specific	Nucleic acid amplification	Single round Nested

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