Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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CTC-mRNA (AR-V7) Analysis from Blood Samples-Impact of Blood Collection Tube and Storage Time.

Author(s): Luk AWS, Ma Y, Ding PN, Young FP, Chua W, Balakrishnar B, Dransfield DT, Souza P, Becker TM

Publication: Int J Mol Sci, 2017, Vol. 18, Page

PubMed ID: 28498319 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated effects of blood collection tube type and up to 48 h processing delay on the recovery of spiked-in prostate tumor cells in whole blood from healthy female participants. The effects of tube type, processing delay, and extended proteinase K treatment during RNA extraction on levels of AR-V7, total AR, and EpCAM of spiked-in cells were also examined. Blood was collected from four healthy female participants into four K₃EDTA, four ACD, four Streck Cell-free DNA BCT, four Streck Cell-free RNA BCT, and four Streck Cyto-Chex BCT; spiked with 100–200 cultured human prostate cancer cells, and stored at room temperature for 0, 24, 30, or 48 h. Peripheral blood mononuclear cells (PBMCs) were isolated using SepMate tubes and spiked-in EpCAM positive-cells were separated using immunomagnetic microbeads on an AutoMACS Pro Separator and then separated into two aliquots. One aliquot was kept on ice until enumeration with a CellCelector microscope and mean cell recovery numbers for each tube type were compared to the EDTA specimens. The other aliquot was centrifuged at 400 x g for 10 min and the pellet frozen at -80°C until RNA extraction using the Total RNA Purification Micro Kit. To examine the effects of extended proteinase K treatment, 38 mL blood from two healthy donors was drawn into one each of EDTA, Citrate, DNA BCT, and RNA BCT tubes; PBMCs were isolated after 48 h at room temperature; and then EpCAM positive-cells were separated using the process described above and frozen at -80°C until RNA extraction with or without an additional 2 h proteinase K treatment. cDNA was synthesized with the SensiFAST cDNA Synthesis Kit and gene expression levels of total AR, AR-V7, and EpCAM were measured by droplet digital PCR (ddPCR).

   Summary of Findings:

   Mean recovery of spiked-in cells ranged from 13%-44% across all time points and tube types. Mean cell recovery was comparable for all tube types to that of the EDTA specimen with the exception of the RNA BCT specimen which had a higher recovery than EDTA at each time point (P<0.05). Cell recovery was significantly lower from blood processed after 24 and 30 h compared to blood processed immediately (P<0.05) but there was no significant difference between mean cell recovery at 0 and 48 h, which the authors attribute to the difficulties of spiking exact cell numbers due to a strong tendency of cell clustering. While total AR, AR-V7, and EpCAM mRNA levels from spiked-in cells from citrate blood were similar to those from EDTA blood, evaluated mRNA levels were significantly lower in cells from all other tube types compared to the EDTA specimens across all timepoints (P<0.01, all). Increased proteinase K digestion resulted in a decrease in measurable copy numbers of all three mRNAs in cells recovered from EDTA and citrate tubes compared to the standard RNA extraction protocol but a small, statistically insignificant increase in the detection of total AR and AR-V7 mRNA in cells from DNA BCT and RNA BCT, respectively.

   Biospecimens

   • Bodily Fluid - Blood
   • Cell - White Cells

   Preservative Types

   • Streck/BCT
   • Other Preservative

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Digital PCR
   Cell count/volume	Fluorescent microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	ACD tube EDTA tube Streck Cell-free DNA BCT Streck Cell-free RNA BCT Streck Cyto-Chex BCT
   Digital PCR Specific	Targeted nucleic acid	Total AR AR-V7 EpCAM
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Storage	Time at room temperature	0 h 24 h 30 h 48 h

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2. Study Purpose

   This study investigated effects of delay to processing of up to 48 h on total AR, AR-V7, and EpCAM levels in circulating tumor cells (CTCs) isolated from EDTA blood of prostate cancer patients. Blood was collected into three K₂EDTA tubes from three prostate cancer patients, stored at room temperature, and PBMCs were isolated using SepMate tubes within 4, 24, or 48 h. EpCAM positive-cells were separated using immunomagnetic microbeads on an AutoMACS Pro Separator and then frozen at -80°C until RNA extraction using the Total RNA Purification Micro Kit. cDNA was synthesized with the SensiFAST cDNA Synthesis Kit and gene expression levels of total AR, AR-V7, and EpCAM were measured by ddPCR.

   Summary of Findings:

   Total AR, AR-V7, and EpCAM mRNA levels varied widely in CTCs isolated from the three CRC patient blood specimens (49-11,392 copies/mL blood, 3-210 copies/mL blood, and 3-100 copies/mL blood; respectively). EpCAM mRNA levels were unaffected by a 24 or 48 h delay to processing in CTCs from CRC patients. While AR-V7 and total AR levels decreased with increased delay, the differences were not statistically significant.

   Biospecimens

   • Cell - White Cells
   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Digital PCR Specific	Targeted nucleic acid	Total AR AR-V7 EpCAM
   Storage	Time at room temperature	≤4 h 24 h 48 h

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