Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Improved methods using the reverse transcriptase polymerase chain reaction to detect tumour cells.

Author(s): Burchill SA, Lewis IJ, Selby P

Publication: Br J Cancer, 1999, Vol. 79, Page 971-7

PubMed ID: 10070899 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the sensitivity and reproducibility of RT-PCR-based detection of circulation tumor cells (CTCs) in blood processed by four different methods. Two milliliters of whole blood collected in EDTA tubes was spiked with 0, 1, 10, 10², 10³, or 10⁴ IMR-32 cells (a neuroblastoma  cell line) and processed by four different methods: 1) red blood cells were lysed following addition of 2 ml of lysing buffer and isolation of total RNA by Ultraspec; 2) white cells were isolated after addition of 3 mL of Lymphoprep, centrifugation for 20 min at 600 x g, isolation of mononuclear cells from the interface, and isolation of total RNA by Ultraspec; 3) isolation of total RNA from whole blood by Ultraspec; or 4) poly-A+ RNA was isolated using oligo(dT)25 beads isolated on a magnetic particle concentrator (MPC) from total RNA isolated by Ultraspec after  beads were heated at 65°C for 5 min to elute poly-A+ RNA. A 180 bp TH fragment was amplified by RT-PCR and visualized by acrylamide gel electrophoresis. To assess the sensitivity of RT-PR in detecting CTCs in whole blood, 4 mL of blood was collected in EDTA tubes from nine healthy volunteers and 15 patients diagnosed with advanced stage 4 neuroblastoma with catecholamine-secreting tumors that expressed TH mRNA. Blood samples were divided into 2 mL aliquots and TH mRNA was assessed by RT-PCR using either total RNA or poly-A+ RNA.

   Summary of Findings:

   The sensitivity of TH mRNA detection in specimens spiked with various numbers of IMR-32 cells (0, 1, 10, 10², 10³, or 10⁴/2 mL blood) was highest (100%) when poly-A+ RNA was isolated from whole blood with slightly lower sensitivities observed when total RNA was isolated from whole blood (92% with 100% detection when specimens were spiked with 10 or more cells and 75% when specimens were spiked with only one cell). When total RNA was isolated from the mononuclear cell fraction the sensitivity of TH detection was 70% (100% for 10³ and 10⁴ cells, 75% for 10² cells, 50% for 10 cells, and 25% for 1 cell), while the lowest sensitivity (60%) was observed when total RNA was isolated following red cell lysis with 100% detection for 10², 10³, and 10 cells/2 mL blood but 0% detection for 1 and 10 cells/2 mL blood. Differences in sensitivity for the number of cells detected in spiked blood specimens were significant between each method (P<0.001 for all). Six out of 15 (40%) specimens from neuroblastoma patients were positive for TH mRNA when 1 or 5 µg of total RNA or poly-A+ RNA was used for reverse transcription; however, while only 4 out of 15 (27%) were positive when 10 µg of total RNA was analyzed, 9 out of 15 (60%) patients tested positive for TH mRNA when 10 µg of ploy-A+ RNA was used. The authors concluded that sensitivity was highest when poly-A+ RNA was isolated from whole blood due to lower amounts of cDNA produced from tRNA and rRNA and reduced levels of contaminating DNA.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Blood and blood products	Whole blood Buffy coat
   Biospecimen Aliquots and Components	Biospecimen components	number of cancer cells added/2 mL blood
   Biospecimen Aliquots and Components	Blood processing method	whole blood minus red cells
   Analyte Extraction and Purification	Analyte isolation method	Total RNA extraction polyA+ RNA extraction
   RT-PCR Specific	Targeted nucleic acid	tyrosine hydroxylase (TH)

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