The impact of RNA stabilization on minimal residual disease assessment in chronic myeloid leukemia.

Author(s): Thörn I, Olsson-Strömberg U, Ohlsen C, Jonsson AM, Klangby U, Simonsson B, Barbany G

Publication: Haematologica, 2005, Vol. 90, Page 1471-6

PubMed ID: 16266893 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of storage of peripheral blood in EDTA or PAXgene tubes prior to extraction of RNA from PBMC using trizol or from peripheral blood using PAXgene on the RNA yield and amplification success. The temperature of EDTA tube storage for 2-5 h and the temperature and conditions of EDTA tube mail transport were unspecified, but specimens stored in PAXgene tubes were stored for up to 16 days at 4 degrees C. In EDTA specimens that were mailed, RNA extraction was delayed by 20-30 h.

   Summary of Findings:

   The RNA yield was highest when RNA was extracted from peripheral blood after storage in PAXgene tubes for less than 5 days (3.5 ug/mL) or 6-16 days (2.1 ug/mL), and the RNA yield was lower when RNA was extracted from PBMC after peripheral blood was stored in EDTA tubes for 2-5 h (1.51 ug/mL) or mailed (20-30 h delay) in EDTA tubes (0.85 ug/mL). However, the RNA concentration was higher in stored EDTA specimens than in stored PAXgene specimens, and the RNA quality was similar between the two types of specimens. When an equivalent amount of RNA was reverse transcribed and used as template for real-time PCR, the CT values for ABL and GAPDH were higher for PAXgene specimens, regardless of storage duration, than in RNA from stored EDTA specimens (p<0.01). The CT values were higher when peripheral blood was stored in PAXgene tubes for 6-16 days rather than less than 5 days (p<0.001). Further, the CT values were similar in PAXgene specimens stored for less than 5 days and PBMC from mailed EDTA specimens. The BCR-ABL fusion transcript was detected in 55% of EDTA specimens but only in 35% of PAXgene-preserved specimens. There was a high degree of correlation between relative BCR-ABL expression in stored PAXgene specimens and PBMC from stored EDTA specimens (R2>0.80).

   Biospecimens

   • Bodily Fluid - Blood
   • Cell - Blood

   Preservative Types

   • None (Fresh)
   • PAXgene

   Diagnoses:

   • Neoplastic - Leukemia

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	None (fresh) PAXgene Refrigeration
   Storage	Storage duration	2-5 h 0-5 days 6-16 days
   Analyte Extraction and Purification	Analyte isolation method	PAXgene Trizol
   Storage	Between site transportation method	Mailed Not transported
   Real-time qRT-PCR Specific	Targeted nucleic acid	BCR-ABL ABL GAPDH
   Biospecimen Aliquots and Components	Blood and blood products	Peripheral blood Peripheral blood mononuclear cells

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