Spectral cytology

In order to gain more insight into FTIR cervical pathology, a detailed investigation into the spectral cytology of the various cell types that comprise the cervical transformation zone, which surrounds squamous epithelium and glandular endothelium, was deemed necessary so that the origin of the major spectral types observed in cervical exfoliates and the spectral characteristics of abnormality could be understood. 

Earlier efforts to estabhsh spectral cytology [10, 11] generally failed because the heterogeneity of samples was not understood, and measurements were attempted on cell pellets containing upwards of 1000 or 10000 cells. This was due in part to... 

NC, USA) and Cytyc (Marlboro, MA, USA). When several different spectral cytology fixatives were tested, the fixation of individual exfoliated cells was shown to cause relatively small spectral changes, in contrast to earlier reports where there was a heavy dependence on fixation [14]. (These results are presented in Section 5.3.1.) As the present cytology procedures involved use of the SurePath method, a SurePath preservative fiuid was used as fixative this was an aqueous solution containing ca. 25% ethanol and a few percent of isopropanol and methanol. 

This section contains spectral results collected from entire individual cells, with the specific aim of differentiating between cell types that occur in samples of cells. The long-range goal of these studies has been the development of a completely automatic method to perform spectral cytology-that is, to analyze a sample of exfoliated cells (e.g., cervical cells obtained during the Pap cervical cancer screening procedure) for the presence of abnormal cells. 

Figu re 5.5 Urine-borne cells that could not be diagnosed via classical cytology due to incomplete cytoplasm (A) or poor staining (B,C), but that could be diagnosed easily via spectral cytology. 

An increase in the number of applications for single cell analysis is therefore foreseen and this will undoubtedly foster the development of Raman and IR as innovative approaches for spectral cytology. They can be automated into techniques for earlier detection of diseases with enhanced resolution, sensitivity and specificity. 

Schubert, J.M. (2011) Spectral cytology of human oral and cervical samples, in Chemistry and Chemical Biology, Northeastern University, Boston, MA. 

Cytology, that is, the visual inspection of exfoliated cells, is most commonly carried out for smears of cells, which are deposited ( smeared ) directly from brushes, spatulas or other exfoliation devices onto microscope slides. Such smears are unsuitable for spectral analysis, since they contain clumps of cells, cellular debris, erythrocytes and other contamination. However, better methods of cell slide preparations have been introduced into cytology, among them the ThinPrep methods developed by Cytyc, Inc. (see ref. 7), and spin centrifugation deposition techniques. These methods are very good for real exfoliated cell samples,7 since they permit the purification of the cell exfoliate, enrichment in the cells desired for analysis and produce good monolayers for visual cell inspection. 

The cervical cells were obtained via a routine gynecological examination, and stored on the collection devices in fixative until a satisfactory cytological reading was established (these samples otherwise would have been discarded). Consent to participate in this project was obtained from each patient. In aU of the studies reported below, storage of the fixative was shown not to cause any spectral degeneration of the cells. 

As mentioned earlier, this review article will concentrate mostly on SCP results from the authors laboratory since the LSpD has contributed to this field more than that of the other fields in spectral diagnostics. Also, the size of the data sets in terms of patient numbers (>200 each for oral and cervical cytology) exceeds by far any other data sets investigated by other groups. 

For the initial studies of oral mucosa cells, normal cytological samples were harvested from laboratory volunteers, whereas clinical samples were collected at TMC, as described in Section 5.2.1.1, and immediately inserted into a vial filled with SurePath fixative. Data acquisition was carried out via the PapMap approach as discussed in Section 5.2.3.2 thus, for the first time, thousands of cellular spectra were available for statistical analysis. This large number of cells allowed several ancillary studies, for example, the immediate effects of smoking and over-the-counter medication on the spectra of oral cells, as well as the dependence of spectral characteristics on the site of cell collection. It was found that cells from the top of the tongue, the mouth floor, and the inside of the cheeks had slightly different spectral characteristics, which could be attributed to some different expression of proteins in these cells. Cells from the tongue, for example, had shghtly different... 

Figure 5.13 shows the results of PCA analysis of cells exfoliated from normal patients and patients diagnosed with LSIL/HSIL [50]. Here, the results of the oral cytology are reproduced in that most of the cells from patients with dysplasia exhibit spectral abnormalities, although the cell morphology is normal (see cell images in Figure 5.13). Even more surprising is the fact that the cells from a patient with a prior diagnosis of HSIL, and subsequent treatment, still exhibit abnormal spectral patterns and cluster with the abnormal spectra.

Schubert, J.M. et al. (2010) Spectral cytopathology of cervical samples detecting cellular abnormalities in cytologically normal cells. Lab. Invest., 90, 1068-1077. 

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