Automatic Identification of Paraffin Pixels on FTIR Images Acquired on FFPE Human Samples

The transfer of mid-infrared spectral histopathology to the clinic will be possible provided that its application in clinical practice is simple. Rapid analysis of formalin-fixed paraffin-embedded (FFPE) tissue section is thus a prerequisite. The chemical dewaxing of these samples before image acquisition used by the majority of studies is in contradiction with this principle. Fortunately, the in silico analysis of the images acquired on FFPE samples is possible using extended multiplicative signal correction (EMSC). However, the removal of pure paraffin pixels is essential to perform a relevant classification of tissue spectra. So far, this task was possible only if using manual and subjective histogram analysis. In this article, we thus propose a new automatic and multivariate methodology based on the analysis of optimized combinations of EMSC regression coefficients by validity indices and KMeans clustering to separate paraffin and tissue pixels. The validation of our method is performed using simulated infrared spectral images by measuring the Jaccard index between our partitions and the image model, with values always over 0.90 for diverse baseline complexity and signal-to-noise ratio. These encouraging results were also validated on real images by comparing our method with classical ones and by computing the Jaccard index between our partitions and the KMeans partitions obtained on the infrared image acquired on the same samples but after chemical dewaxing, with values always over 0.84.

Automated summary

A new automatic and multivariate methodology utilizing EMSC regression coefficients, validity indices, and KMeans clustering was proposed to separate paraffin and tissue pixels in infrared spectral images. Validation on simulated and real images showed high effectiveness of the method.