Banking of Fine Needle Aspiration Biopsies for Future RNA Based Molecular Testing.
Amy C Ladd, Emerald O'Sullivan-Mejia, Tasha Lea, Jessica Perry, Catherine I Dumur, Ema Dragoescu, Carleton T Garrett, Celeste N Powers. Virginia Commonwealth University, Richmond
Background: Fine needle aspiration (FNA) biopsy is minimally invasive, cost-effective and also allows for sampling of diseased cells (i.e. metastatic and pre-malignant) that may not be accessible through excisional biopsy. Ancillary molecular testing is becoming routine in Surgical Pathology, but there have been limited reports describing the utility of FNA biopsies for this purpose. Quality control (QC) and test validation requirements for development of molecular tests impose a need for access to preexisting clinical samples. Banking FNA biopsy specimens would allow for innovative genomic research and development of novel molecular assays from cytology specimens. Here, we evaluated cryopreservation of FNA specimens as a method of maintaining cellular morphology and RNA integrity in banked tissues.
Design: FNA specimens were collected from tumor resections received in Surgical Pathology. Syringe contents were immediately deposited into either cryopreservation media (n=25) or as a control, the RNA stabilizing reagent RNAlater (n=13(Applied Biosystems, Foster City, CA)). RNAlater specimens were stored at 4oC for less than one month per manufacturer's instructions. Specimens for cryopreservation were processed using controlled rate freezing and stored for up to 27 weeks in the vapor phase of liquid nitrogen. At varying time intervals cryopreserved specimens were quickly thawed at 37oC then washed in fresh media. A portion of each sample was used for cytospin preparation for morphological evaluation. RNA was extracted from the remaining portion and was assessed for integrity using the Agilent Bioanalyzer and RNA integrity number (RIN) software tool (Agilent Technologies, Inc., Santa Clara, CA).
Results: Cryopreserved specimens showed good cell morphology, and in many cases yielded intact RNA. Factors affecting RNA integrity included prolonged sample processing delays (p<0.001) and sampling of necrotic cells (p<0.05). When samples were processed expeditiously there was no difference in RNA quality between cryopreserved and RNAlater specimens (p=0.6). Additionally, there was no correlation between the total time samples were frozen (tested up to 27 weeks) and RNA integrity (r=0.04).
Conclusions: With careful handling, FNA specimens can be stored in a manner that maintains cellular morphology and RNA integrity necessary for studies in gene expression. Although not tested here, cryopreservation is the only method that might also maintain cell viability. In addition to addressing QC and test validation needs, cytology banks will be an invaluable resource for future molecular research studies.
Wednesday, March 2, 2011 1:00 PM
Poster Session VI # 95, Wednesday Afternoon