Deconvolution microscopy: A platform for rapid on-site evaluation of fine needle aspiration specimens that enables recovery of the sample

Context Rapid on-site evaluation (ROSE) optimises the performance of cytology, but requires skilled handling, and smearing can make the material unavailable for some ancillary tests. There is a need to facilitate ROSE without sacrificing part of the sample. Objective We evaluated the image quality of inexpensive deconvolution fluorescence microscopy for optically sectioning non-smeared fine needle aspiration (FNA) tissue fragments. Design A portion of residual material from 14 FNA samples was stained for 3 min in Hoechst 33342 and Sypro (TM) Red to label DNA and protein respectively, transferred to an imaging chamber, and imaged at 200x or 400x magnification at 1 micron intervals using a GE DeltaVision inverted fluorescence microscope. A deconvolution algorithm was applied to remove out-of-plane signal, and the resulting images were inverted and pseudocoloured to resemble H&E sections. Five cytopathologists blindly diagnosed 2 to 4 representative image stacks per case (total 70 evaluations), and later compared them to conventional epifluorescent images. Results Accurate definitive diagnoses were rendered in 45 (64%) of 70 total evaluations; equivocal diagnoses (atypical or suspicious) were made in 21 (30%) of the 70. There were two false positive and two false negative definite diagnoses in three cases (4/70; 6%). Cytopathologists preferred deconvolved images compared to raw images (P < 0.01). The imaged fragments were recovered and prepared into a ThinPrep or cell block without discernible alteration. Conclusions Deconvolution improves image quality of FNA fragments compared to epifluorescence, often allowing definitive diagnosis while enabling the ROSE material to be subsequently triaged.

Automated summary

Inexpensive deconvolution fluorescence microscopy improves the image quality of non-smeared fine needle aspiration tissue fragments, enabling more accurate diagnoses and preserving the material for further testing.