Form alin-fixed and paraffin-em bedded tissue specim ens o f norm al and dysplastic cervical epithelia (five C IN 1, seven C IN 2, five C IN 3, and five norm al) were assessed by an im m unoperoxidase technique, using the m onoclonal antibody M IB 1 , regonizing a fo rm alin-tix atio n-resistan t epitope on th e cell p ro liferatio n-asso ciated K i-67 an tig en. An im ag e analysis system w as u sed to m easure four p aram eters associated with p ro liferative activity: the K i-67 labelling index (L I), th e num ber of K i-67-positive nuclei per unit length of basem ent m em brane, an d the m axim um value an d 90th percentile o f the relativ e distances of K i-67-positive nuclei from the basem ent m em brane. A ll these four p ro liferatio n-related p aram eters were highly c o rre la te d w ith the grade o f dy splastic change in the epithelium (0*90<0*0001). T he best correlation was found for the 90th p ercen tile of the relative d istan ce and with this p a ra m e te r all C IN lesions could be co rrectly classified. T he m eans an d s ta n d a rd deviations o f the Ki-67 L is in normal epithelium , C IN 1, C1N 2, and C IN 3 lesions w ere 0*07 ± 0*03, 0*16 ± 0-03, 0*25 ± 0*06, an d 0-39 ± 0*06, respectively. T hese findings su p p o rt the theory th a t C IN involves a progressive dysfunction o f the proliferative activ ity o f cervical epithelial cells. Im ag e analysis o f M IB 1 is a prom ising altern ativ e m ethod for th e objective, reproducible, and reliab le classification o f dysplastic changes in cervical epithelium. joey w o r d s-C IN ; p ro liferatio n ; K i-67; M IB 1 ; m onoclonal antibody; im m unohistochem istry; q u an titativ e analysis

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. Cytometry 39:275–284, 2000 © 2000 Wiley‐Liss, Inc.

A retrospective analysis was performed on archival cervical smears from a group of 56 women with cervical intraepithelial neoplasia (CIN), who had received follow‐up by cytology only. Automated image cytometry of Feulgen‐stained DNA was used to determine the differences between progressive and regressive lesions. The first group of 30 smears was from women who had developed cancer after initial smears with dysplastic changes (progressive group). The second group of 26 smears with dysplastic changes had shown regression to normal (regressive group). The goal of the study was to determine if differences in cytometric features existed between the progressive and regressive groups. CIN categories I, II and III were represented in both groups, and measurements were pooled across diagnostic categories. Images of up to 700 intermediate cells were obtained from each slide, and cells were scanned exhaustively for the detection of diagnostic cells. Discriminant function analysis was performed for both intermediate and diagnostic cells. The most significant differences between the groups were found for diagnostic cells, with a cell classification accuracy of 82%. Intermediate cells could be classified with 60% accuracy. Cytometric features which afforded the best discrimination were characteristic of the chromatin organization in diagnostic cells (nuclear texture). Slide classification was performed by thresholding the number of cells which exhibited progression associated changes (PAC) in chromatin configuration, with an accuracy of 93 and 73% for diagnostic and intermediate cells, respectively. These results indicate that regardless of the extent of nuclear atypia as reflected in the CIN category, features of chromatin organization can potentially be used to predict the malignant or progressive potential of CIN lesions.

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