Detection of breast micro-metastases in axillary lymph nodes by infrared micro-spectral imaging

Abstract

We report the ability of infrared micro-spectral imaging, coupled with completely unsupervised methods of multivariate statistical analysis, to accurately reproduce the histological architecture of axillary lymph nodes and detect metastatic breast cancer cells. The acquisition of spectral data from tissue embedded in paraffin provided spectra free of dispersive artefacts that may be observed for infrared microscopic measurements using a 'reflection/absorption' methodology. As a consequence, superior tissue classification and identification of cellular abnormality unattainable for deparaffinised tissue was achieved.

Fig. 1

Panel (a): photomicrograph of a H&E stained axillary lymph node (ca. 5 mm × 5 mm in size) containing a small breast micro-metastasis. The sinuses (1), medullary cords (2) and follicles (3) of the lymph node are indicated. Panel (b): microscopic view at 20× magnification of the area indicated by the red square in panel (a). At this resolution, it is easy to visualise two follicles in the cortex, with distinctly lighter stained germinal centres (1), and what would appear to be histiocytes in both the sinuses and the subcapsular sinus (2). Panel (c): microscopic view at 40× magnification of the area indicated by the red square in panel (b). Note the appearance of a small breast micro-metastasis under the capsule of the lymph node.

Fig. 2

Panel (a): microscopic view of a 1 mm × 1 mm tissue area analysed by infrared micro-spectral imaging. The capsular (1), B-lymphocyte (2), T-lymphocyte (3), vascular (4) and breast micro-metastatic (5) regions are indicated. Panel (b): 6-cluster spectral image, constructed via HCA in the 1800–900 cm⁻¹ spectral range, of the unstained and deparaffinised tissue area shown in panel (a). The red colour co-localises with the breast micro-metastasis apparent in panel (a). Panel (c): 15-cluster spectral image obtained from the same HCA.

Fig. 3

Panel (a): microscopic view of a 1 mm × 1 mm tissue area examined by infrared micro-spectral imaging. The capsular (2), B-lymphocyte (4), T-lymphocyte (3), histiocyte (5), and breast micro-metastatic (1) regions are indicated. Panel (b): 4-cluster spectral image, constructed via HCA in the 1800–900 cm−1 spectral range, of the unstained and deparaffinised tissue area shown in panel (a). Panel (c): 14-cluster spectral image obtained from the same HCA described in panel (a). Panel (d): 6-cluster spectral image, constructed via HCA in the 1350–900 cm⁻¹ spectral range. The dark blue colour co-localises with the breast micro-metastasis apparent in panel (a). Panel (e): 14-cluster spectral image obtained from the same HCA described in panel (d).

Fig. 4

Panel (a): mean absorbance spectra calculated for the dark blue (sparse tissue) and cornflower blue (T-lymphocytes) regions of Fig. 3(c). Panel (b): mean absorbance spectra calculated for the dark green (histiocytes) and white (T-lymphocytes) regions of Fig. 5(b).

Fig. 5

Panel (a): 4-cluster spectral image, constructed via HCA in the 1800–1496 cm⁻¹ and 1350–900 cm⁻¹ spectral ranges, of the unstained and paraffinised tissue area shown in Fig. 3(a). Panel (b): 13-cluster spectral image obtained from the same HCA described in panel (a). Panel (c): 13-cluster spectral image, constructed via HCA in the 1350–900 cm⁻¹ spectral range. Panel (d): mean 2nd derivative spectra calculated for the yellow (capsule), white (T-lymphocyte), royal blue (B-lymphocyte), green (histiocyte), dark blue (breast micro-metastasis, cytoplasmic region) and red (breast micro-metastasis, highly nucleated region) regions of panel (b).

Fig. 6

Mean 2nd derivative spectra calculated for the capsule, T-lymphocytes, and breast micro-metastasis shown in Fig. 3(c) (deparaffinised tissue) and Fig. 5(b) (paraffinised tissue). Panel (a): spectral range 1750–1500 cm⁻¹. Panel (b): spectral range 1350–900 cm⁻¹.

Fig. 7

Panels (a)–(c): H&E stained images captured from 3 tissue regions (1 mm × 1 mm areas) scrutinised by infrared micro-spectral imaging. Panels (d)–(f): semi-transparent (red colour) overlays of areas identified by HCA to contain abnormal tissue. These overlays co-localise extremely well with changes in cell morphology detectable by microscopic inspection. Note the isolated tumour cells identified in panel (c) and detected by HCA in panel (f).

Fig. 8

Mean 2nd derivative cluster spectra of 5 different breast micro-metastases. Spectral data were acquired whilst tissue was still embedded in paraffin. Note the spectral homogeneity.