Martin M. M. Pahlplatz

Hue-Saturation-Density (HSD) Model for Stain Recognition in Digital Images From Transmitted Light Microscopy

BACKGROUND Transmitted light microscopy is used in pathology to examine stained tissues. Digital image analysis is gaining importance as a means to quantify alterations in tissues. A prerequisite for accurate and reproducible quantification is the possibility to recognise stains in a standardised manner, independently of variations in the staining density. METHODS The usefulness of three colour models was studied using data from computer simulations and experimental data from an immuno-doublestained tissue section. Direct use of the three intensities obtained by a colour camera results in the red-green-blue (RGB) model. By decoupling the intensity from the RGB data, the hue-saturation-intensity (HSI) model is obtained. However, the major part of the variation in perceived intensities in transmitted light microscopy is caused by variations in staining density. Therefore, the hue-saturation-density (HSD) transform was defined as the RGB to HSI transform, applied to optical density values rather than intensities for the individual RGB channels. RESULTS In the RGB model, the mixture of chromatic and intensity information hampers standardisation of stain recognition. In the HSI model, mixtures of stains that could be distinguished from other stains in the RGB model could not be separated. The HSD model enabled all possible distinctions in a two-dimensional, standardised data space. CONCLUSIONS In the RGB model, standardised recognition is only possible by using complex and time-consuming algorithms. The HSI model is not suitable for stain recognition in transmitted light microscopy. The newly derived HSD model was found superior to the existing models for this purpose.

Image and flow DNA cytometry of small cell carcinoma of the lung

Both image and flow DNA cytometry were performed in isolated nuclei from paraffin‐embedded tumor tissue of patients with small cell carcinoma of the lung (SCCL). In 14 patients tissue was obtained by surgery from the primary tumor. From 14 patients tissue was taken by autopsy. From two patients tissue obtained by both surgery and later autopsy were available. From the autopsy patients tissue was taken only from the primary tumor (n = 6), from a metastasis (n = 1) and from the primary tumor and distant metastases (n = 7). Twelve of the tumors obtained by surgery were diploid, and two multiploid (two stem lines present). This was found both with image and flow cytometry. The group of patients could clearly be subdivided in short survivors (< 9 months, n = 6) and long survivors (> 16 months, n = 8); since in both groups one multiploid and the remainder diploid cases were present, ploidy did not seem to be a good prognosticator for survival. In most (n = 26) of the tissues measured from the autopsy patients, again, a good correlation between image and flow DNA cytometry was obtained, the histograms being either (near) diploid or multiploid. In six cases, however, flow cytometry showed multiploidy whereas image showed aneuploidy (one single peak clearly deviating from diploidy). This discrepancy is caused because normal diploid (nonneoplastic) cells in the preparations could not be discarded from the flow cytometry measurements. Using the image cytometry data of the primary tumors, five diploid, three aneuploid, and four multiploid tumors were found. In five of the seven patients of whom tissue was obtained from the primary tumor and multiple metastases, differences between the histograms were found, mostly showing two malignant cell populations in one tissue and only one of them in another. Of one of the two patients of whom tissue was obtained by surgery and later autopsy, a change in histogram pattern was observed. It is concluded that although there is a high similarity between image and flow DNA cytometry, for an optimal interpretation of the histogram pattern, image measurements are more reliable. Ploidy determination does not seem to be of use in prediction of survival, and care should be taken in interpreting DNA histograms of metastases in SCCL patients because of the variability in histogram pattern.

DNA ploidy patterns in cervical intraepithelial neoplasia grade III, with and without synchronous invasive squamous cell carcinoma: Measurements in nuclei isolated from paraffin-embedded tissue

This study presents the results of cytophotometric (CPM) and flow cytometric (FCM) DNA ploidy measurements in cervical tntraepithelial neoplasias grade III (CIN III) with and without synchronous invasive squamous cell carcinoma. Hysterectomy and biopsy material from 21 patients 35 years of age or younger and from 18 patients age 50 years or older was studied. The DNA analysis was performed in nuclei isolated from specific areas of paraffinembedded tissue. There were significant differences in the distribution of DNA patterns between the two age groups. About 80% of CIN III lesions in women 50 years of age or older, with or without a coexisting invasive cancer were aneuploid. In the group of younger women a diploid DNA pattern was found in about 60% of CIN III with concomitant invasive cancer. In the absence of an invasive cancer, CIN III lesions were mostly polyploid. The DNA pattern of invasive cancers was generally identical with the adjacent CIN, thus suggesting that the two lesions were related. Although the prognostic value of DNA ploidy measurements in cervical intraepithelial lesions in women in these two age groups has to be further evaluated, these results are at considerable variance with previously published data on DNA values in CIN and invasive carcinoma. In four CIN III lesions without invasive cancer, in women of the group of 35 years of age or younger, human papilloma virus common antigen could be demonstrated by immunochemical procedure. In three of these cases a polyploid DNA pattern was present; the fourth case showed a bimodal aneuploid pattern.

The use of light green and organge II as quantitative protein stains, and their combination with the feulgen method for the simultaneous determination of protein and DNA

SummaryThe protein dyes Light Green and Orange II were studied separately and in combination with the Feulgen-Pararosanilin(SO2) and-Thionin(SO2) method for the simultaneous determination of DNA and protein. — With polyacrylamide modelfilms the pH dependency, specificity and stoichiometry of Light Green and Orange II have been investigated. The results of both staining methods with different biological objects have been compared. — In addition, the Feulgen-Thionin(SO2) method was studied with model films with respect to its specificity and stoichiometry. In biological objects it has been compared with the Feulgen-Pararosanilin(SO2) method. — When combining the Light Green staining with the Feulgen-Pararosanilin(SO2) procedure and the Orange II staining with Feulgen-Thionin-(SO2), both Feulgen-DNA stainings, which were first applied, proved to be unaffected by the following protein staining procedure. When the Feulgen procedure was carried out without the dye, followed by Light Green staining, the latter became reduced when a sulfite water rinse was included but was unaffected when a running tap water rinse was used. In the case of the Orange II staining a serious reduction in dye binding capacity was found in both situations. — When the Feulgen-Pararosanilin(SO2) Light Green procedure was carried out on isolated nuclei with all dyes present, a decrease of protein dye binding was observed, similar to that found with the well-known Feulgen-Pararosanilin(SO2) Naphthol Yellow S combination. It is concluded that in spite of this reduction the latter two combinations can be used for the cytophotometric analysis of DNA and protein in the same object.

Image DNA-index (ploidy) analysis in cancer diagnosis.

DNA measurements performed with image cytometry in nuclei isolated from paraffin-embedded tissue of (pre)malignant lesions are described. DNA histograms are obtained in which one or more peaks are present, the latter representing one or more (pre)malignant cell populations. The DNA index [modal DNA peak value of the (pre)malignant cells divided by that of normal cells] can be computed and is comparable with that obtained with flow cytometry, a widely accepted technique to perform DNA cytometry. Quality of the histograms can be improved by eliminating the nonrelevant tissue in advance, and examples are shown of histograms of morphologically different regions in the same tissue. The use of DNA index in the clinical application on dysgeminomas of the ovary and small-cell lung cancer is discussed and its use for the characterization of cervical intraepithelial neoplasms grade III and metastases of small-cell carcinomas of the lung.

Improved prediction of metastasis in tongue carcinomas, combining vascular and nuclear tumor parameters

Predicting the presence of metastasis, based on tumor or tumor‐related characteristics is of utmost importance. The authors studied the significance of tumor DNA features and tumor‐related angiogenesis to predict the occurrence of metastasis in squamous cell carcinomas (SCCs) of the tongue.

Two methods for analyzing pleural smears for the presence of abnormalities

Abstract Two methods of designing a cell classifier as a first stage in a pleural sample classification procedure, where reliable a priori cell class labeling is not available, are presented. Cluster analysis is shown to be capable of taking into account structural information at the cell level, which may be lost in other methods.