Use of Stained Blood/Bone Marrow Smears and Fixed Cell Suspensions for Complex Molecular Oncology Genotyping – A Practical Specimen Alternative for Molecular Testing in Remote Areas
M Cankovic, S Ali, L Whiteley, D Chitale. Henry Ford Hospital, Detroit, MI
Background: Molecular oncologic pathology is a highly specialized and emergent field especially in the era of targeted therapy. For reliable molecular results, proper specimen handling to extract high quality nucleic acid is imperative, however; sometimes this may not be possible in remote areas. Here we investigated the feasibility of performing molecular testing on Wright/Giemsa stained (WGS) peripheral blood (PB) and bone marrow aspirate (BMA) smears from overseas and fixed cell suspensions (FCS) left over from cytogenetic testing.
Design: DNA and RNA were extracted from 19 air-dried WGS smears [8 PB, 11 BMA] and from 18 FCS (12 PB; 6 BMA) left over from FISH testing for BCR/ABL t(9;22). WGS samples were about 8 weeks old, and had been transported on an airplane for 24 hours to simulate international shipment conditions. DNA and RNA were extracted per standard protocol. DNA/RNA yield and purity were evaluated by spectrophotometry, and integrity by PCR amplification of housekeeping genes (mixture of primer pairs yielding fragment sizes of 100bp, 200bp, 300bp, and 400bp for DNA, and beta 2 microglobulin (B2M), a 120 bp fragment, for RNA/cDNA).
Results: WGS slides: Adequate DNA was isolated from 19/19 samples. While 260/280 OD ratio showed inferior DNA purity and total recovery between 0.15 ug to10 ug, the amplified DNA yielded product sizes of 300 bp for 19/19 samples, and 400 bp for 16/19 samples. RNA from WGS smears was of marginal quality, but low level B2M amplifcation was observed in 15/19 samples. Fixed cells: Adequate DNA was recovered from 17/18 samples. Purity (260/280 OD ratios) ranged from 0.94 to 2.79, with majority being 1.66 to 2.15. Total yield ranged from 0.21 to 61.78 ug. Amplified DNA gave product sizes up to 300 bp for 17/17 samples, and 400 bp for 15/17 samples. Adequate RNA was recovered from 9/18 samples. 7/18 tested positive for BCR/ABL t(9;22) translocation, concordant with cytogenetics and fresh sample results. BCR/ABL mRNA vs housekeeping gene mRNA copy number ratios were lower in fixed samples than in fresh. All of the BCR/ABL positive samples were negative for JAK2 V617F mutation.
Conclusions: We were able to extract good quality DNA from WGS PB and BMA smears prepared overseas and shipped over long distances, but RNA was of marginal quality and was definitely not suitable for quantitative RNA testing. Qualitative detection of BCR/ABL mRNA, can be done on FCS, with sensitivity slightly lower compared to fresh samples.
Monday, March 22, 2010 1:00 PM
Poster Session II # 243, Monday Afternoon