Specificity of seven monoclonal antibodies against p53 evaluated with Western blotting, immunohistochemistry, confocal laser scanning microscopy, and flow cytometry.

p53 immunostaining of histological sections shows inter- and intratumor variability in distribution and staining intensity which are usually scored semiquantitatively. In order to investigate the variation in p53 expression more accurately and its possible relation to other cellular parameters (e.g., DNA content), we have studied the possibility to measure p53 accumulation by multiparameter flow cytometry. To this end we have evaluated seven, commercially available, monoclonal antibodies (MAbs) against p53 (MAbs 1801, 240, 246, 421, 1620, Do1, and Do7) on five tumor cell lines with known p53 gene status: MCF-7 (wild-type p53 gene), COV362.cl4 and T47d (mutated p53 genes), and SAOS-2 and HL60 (no p53 mRNA). Localization of immunofluorescence was investigated with confocal laser scanning microscopy, immunofluorescence signal intensity with flow cytometry, and antibody specificity with Western blotting. Subsequently, single cell suspensions from two breast carcinomas were flow cytometrically analyzed after triple staining for p53, cytokeratin 8/18, and DNA, and compared to immunohistochemical staining. MAbs Do1 and Do7, and to a lesser extent MAb 421, accurately discriminated p53 positive from p53 negative cell lines. Even at high concentrations these MAbs yielded nuclear immunofluorescence, whereas with MAbs 1801, 240, and 246 strong cytoplasmic signals in both the p53 accumulating and p53 negative cell lines were seen. By using lower antibody concentrations the cytoplasmic immunofluorescence disappeared, but simultaneously the nuclear p53 immunostaining intensity in p53 accumulating cell lines decreased, resulting in false negative nuclei. With MAb 1620 only weak intranuclear spots were obtained in all cell lines tested. Western blotting yielded results with MAbs 1801, Do1, and Do7 in the 53 kD region of the p53 accumulating cell lines. The signal intensity obtained with MAb 1801 was much less compared to MAbs Do1 and Do7. Although all three MAbs are also described as wild-type p53 specific, only MAbs, Do1 and Do7 showed bands in the 53 kD region of cell line MCF-7. With MAb 1801 ascites and MAb 1801 supernatant an additional approximately 80 kD band was present in all cell lines tested, including SAOS-2, indicating cross reactivity of this MAb. Immunohistochemical staining of two clinical breast carcinomas confirmed the results obtained in the cell lines. Multiparameter flow cytometric analysis of these breast carcinomas with MAbs Do1 and Do7 showed intratumor heterogeneity for p53 accumulation, which was independent of DNA index heterogeneity. We conclude that MAbs Do1 and Do7 enable quantitative analysis of p53 accumulation in a multiparameter flow cytometric analysis.

Multiparameter absorption measurements in automated microscopy. Simultaneous quantitative determination of DNA and nuclear antigen.

OBJECTIVE: To test a dual DNA-nuclear antigen staining method for multiparameter absorption image analysis. STUDY DESIGN: MCF 7 cells, grown on glass slides, served as a model to test the staining technique. For DNA, Feulgen-based CAS quantitative DNA staining, and for nuclear antigen, alkaline phosphatase-based immunocytochemical staining with CAS Red as the chromogen, were used. MIB-1, estrogen and progesterone receptors were used as examples of nuclear antigen staining. Measurements were performed with the DISCOVERY image analyzer. RESULTS: Scatterplots, in which the nuclear antigen content was plotted against the DNA content, were obtained. Immunostain-positive and -negative populations could be discriminated. These cells were visualized in image galleries. The DNA histograms of the positive and negative cells showed no change in coefficient of variation or integrated optical density ratio of the G0, G1 and G2 + M peaks as compared to single DNA staining. The intensity of the immunostain increased as compared to the single immunostaining result. CONCLUSION: This staining technique allows the simultaneous accurate measurement of costained DNA and antigen within the same nucleus. This opens the possibility for studies in which nuclear antigen expression is monitored during the cell cycle or in cells of different ploidy classes. Identified cells can also be visualized by presentation in an image gallery or by relocation on the slide. This can support the analysis of clinical samples, where cytometric data can be correlated with and confirmed by visual diagnosis.

Detection of immunocytochemically stained rare events using image analysis.

The detection of rare-event cells circulating in peripheral blood using automated image analysis was evaluated using a model system consisting of cells from a breast cancer cell line (SKBR3) seeded in a mononuclear cell suspension. Slides of cells with optimal morphology were prepared according to an optimized preparation procedure based on centrifugal cytology in combination with formalin fixation. SKBR3 cells were immunocytochemically stained for cytokeratin using the cam 5.2 monoclonal antibody and labelled with alkaline phosphatase using CAS-red as substrate. Because, for optimal segmentation of cell images, plain differences in absorption wavelength are required, the red immunostaining was combined with a green nuclear counter-staining based on ethyl green. Slides were automatically screened for cytokeratin-positive SKBR3 cells resulting in a lowest detectable frequency of one positive cell per 1.87 x 10(6) negative cells. A comparison between manual screening and automated screening for cytokeratin-positive cells showed a high level of correlation (0.9998). For the definition of the total number of objects per slide, two counting procedures were evaluated. Results were close to the visual score with a coefficient of variation of 0.47% for the counting procedure used in this study. It is concluded that optimization of preparation and staining procedures for the detection of rare-event cells using automated image analysis results in optimal image contrast and, consequently, in an increase in sensitivity for detecting rare events.

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 (less than 9 months, n = 6) and long survivors (greater than 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 intraepithelial 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 paraffin-embedded 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.

Quantitative description of classic and variant small cell lung cancer cell lines by nuclear image cytometry.

Six small cell lung cancer (SCLC) cell lines were examined using nuclear image analysis to find features characteristic of the classic and the variant type of SCLC. On the basis of their biochemical and biological properties three of these cell lines have been shown to represent the classic types, and three represent the variant type of SCLC. Using a combination of the image-derived run length, density, and geometric features, it was possible to distinguish between the classic and variant SCLC cell lines. The results of this study may be of help in assessing photometric features for the separation of the classic and variant subtypes of SCLC in solid tumors. Because of differences in treatment and prognosis between these two subtypes, such a separation may be of clinical value.

DNA cytometry of pure dysgerminomas of the ovary.

The value of DNA cytometry for predicting the malignant potential of pure dysgerminomas of the ovary was investigated. Feulgen-thionin DNA image cytometry and propidium iodide DNA flow cytometry were performed in isolated nuclei from paraffin-embedded tissue of 25 dysgerminomas. Nineteen were in clinical stage Ia1, and six were in stage III (two in IIIa and four in IIIb). Eight patients showed recurrences during a 5-year follow-up period and one patient died of the disease. The DNA index (DI; the modal DNA value compared with that of normal cells) of the tumor cells was computed from the image and flow DNA histograms. Comparable DI values, ranging from 1.29 and 3.23, were found with both types of cytometry. No correlation was found with the clinical stage or the recurrence state. Furthermore, it was striking that, although values were found strongly deviating from the normal diploid content (DI = 1.0) suggesting an unfavorable prognosis, the survival rate was relatively high. It can be concluded that prediction of the clinical course of pure dysgerminomas by DNA cytometry does not seem feasible.

Evaluation of DNA and nuclear protein features for their use in describing normal and malignant endometrium.

Different feature sets (geometric, densitometric, and textural) derived from DNA and nuclear protein staining were evaluated for their use in describing atrophic, secretory, and proliferative endometrium, and well-differentiated stage I and moderately differentiated stage I adenocarcinomas of the endometrium. It was found that the pattern of significant differences among these groups varied between feature sets, while remaining consistent within a set of features. The DNA density and run-length features were not very effective in describing group mean differences, whereas the co-occurrence features revealed significant differences among most groups. The protein run-length features were the only ones that consistently showed a difference between proliferative endometrium and well-differentiated adenocarcinomas. Analyses repeated on only cells in the G0/G1 DNA region improved the differentiation between moderately differentiated adenocarcinomas and the other groups. It was concluded that the use of DNA and nuclear protein texture features are effective in describing group differences that cannot be described by DNA content only.

Evaluation of contextual analysis for computer classification of cervical smears.

A procedure for automated analysis of cervical smears has been implemented in an image cytometry system. Smears are described exclusively in terms of global and contextual information extracted by pattern-recognition algorithms and represented by a vector of proportions of cellular object types. Linear discriminant functions, based on a Fisher criterion, are derived to classify smears with a cross-section of diagnoses into two broad categories, normal and abnormal. Results obtained from 83 smears indicate 78% correct classification. In contrast to most automated systems, good classification results were obtained in normal smears with benign changes caused by inflammation and with postmenopausal atrophia and in abnormals with mild dysplasia. These findings suggest that contextual analysis may be sensitive to subtle changes in cellular morphology and to progressive patterns of dysplasia. When used with standard isolated cell analysis, contextual analysis may provide additional complementary information for automated cervical prescreening.

Carcinoma in situ specimen classification based on intermediate cell measurements.

In this study the possibility of classifying carcinoma in situ and normal specimens by measuring normal-appearing intermediate cells was explored. Twenty-five histologically verified carcinoma in situ specimens and 99 normal specimens, matched with the abnormal specimens for age and use or nonuse of an oral hormonal contraceptive, were examined. The smears were monolayer preparations stained with Thionin-Feulgen Congo red. Twenty-one nuclear features were measured. A discrimination among the experimental groups could be made on the basis of the relationship between two features, area and average optical density (AOD). A regression of AOD on area for each smear was performed. The correlation, coefficient of variation, slope, intercept, as well as the mean of the AOD level and the age of the subject were used in a discriminant analysis. This resulted in a smear classification with a false-positive rate of 14% and a missed-positive rate of 32%. When contraceptive use was taken into account the overall classification was improved with a false-positive rate of 12% and a missed-positive rate of 20%.

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.

Application of antibodies to intermediate filament proteins as tissue-specific probes in the flow cytometric analysis of complex tumors.

The flow cytometric (FCM) analysis of carcinomas is often hampered by the presence of stromal and inflammatory cells in the cell suspensions obtained from such neoplasms. Therefore, an FCM method was developed to distinguish epithelial from nonepithelial cells by using polyclonal and monoclonal antibodies to (cyto)keratins, the epithelial type of intermediate filament proteins. Using a model system of cultured bladder carcinoma (T24) and leukemia (MOLT-4) cells, we tested our hypothesis and procedures by labeling cell mixtures with these antibodies. After incubation with an appropriate intermediate filament antibody and propidium iodide staining, the DNA content and distribution of T24 cells could be analyzed separately from MOLT-4 cells. When applied to cell suspensions of endometrial carcinomas, bladder carcinomas and Grawitz tumors, only the epithelial (primarily carcinoma) cells were stained for cytokeratin; these cells could thus be analyzed separately from stromal, inflammatory and other nonepithelial cells. In this way, a more accurate FCM analysis of the malignant fraction within a tumor can be achieved.

Extraction of nuclei from selected regions in paraffin-embedded tissue.

A method for the extraction of nuclei from selected regions in paraffin-embedded tissue is described. Fifty-micrometer sections are cut, dewaxed, and rehydrated. For the final handling, the sections are manually transferred from one tray to another. The sections are put on a slide under a dissection microscope and the region of interest is isolated by scraping off the irrelevant region with a scalpel. An optimal number of single nuclei is obtained by incubation in a protease solution with intermediate syringing. The nuclei are washed and can be used for flow cytometry. Resuspension of the nuclei in foetal calf serum and cytocentrifugation results in preparations suited for image analysis. DNA cytometric measurements of nuclei in a carcinoma in situ and an invasive carcinoma region in breast tissue present in the same tissue block and in a severe dysplasia/carcinoma in situ (CIN III) region of the cervix are presented.

DNA and nuclear protein measurement in isolated nuclei of human endometrium.

Feulgen-DNA and nuclear light green-protein measurements have been performed in isolated nuclei of normal (nonmalignant) and malignant human endometrial homogenates. The DNA content of the G0/G1 fraction of malignant endometrium showed much overlap with that of normal endometrium, or was slightly increased. Two of the 18 carcinomas were clearly aneuploid. No correlation was found between the histological grade and the DNA content. The tumors of clinical stage II and higher all had a higher DNA content than that of normal endometrium. The percentage of cells present in the proliferative fraction was higher in proliferative endometrium than in secretory and post-menopausal atrophic endometrium. For malignant endometrium, percentages were found comparable to that of normal endometrium or higher. No correlation was found with the histological grade. Tumors of stage II and higher had intermediate values compared to those of carcinomas below stage II. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. However, for post-menopausal women, most values of the carcinomas exceeded that of normal, atrophic, endometrium. Within the tumor population, no correlation was found with the histological grade. Higher values were found with tumors of clinical stage II and higher.

DNA and nuclear protein measurement in columnar epithelial cells of human endometrium.

Propidium iodide DNA flow cytometry, Feulgen-DNA, and nuclear light green protein scanning cytometry were performed in columnar epithelial cells of normal, nonmalignant human endometrium and endometrial adenocarcinomas. Columnar cells were identified by immunohistochemical staining for cytokeratin 18, an intermediate filament protein specifically present in columnar cell epithelium. DNA measurements derived from flow and scanning cytometry showed comparable results. The DNA content of the G0/G1 fraction of the adenocarcinomas had a considerable overlap with that of normal endometrium, with that of the carcinomas shifted toward higher values. For the carcinomas, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas. Most of the clinical stage I tumors showed a DNA content in the normal diploid region. Three of the four carcinomas of clinical stage II and higher had an increased DNA content. For the carcinomas, the percentage of cells in the proliferative fraction, as determined from scanning cytometric derived DNA histograms, was comparable to that of normal endometrium, or higher. No correlation was found with the histological grade. Tumors of clinical stage II and higher had intermediate values compared to carcinomas of lower stages. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. Within the tumor population, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas, and clinical stage. Based on the aforementioned parameters, no discrimination could be obtained between normal and malignant endometrium. However, when the DNA content of the G0/G1 fraction was combined with the coefficient of variation of the nuclear protein/DNA ratio, a clear discrimination could be obtained with only two false-positive cases.

Automated deposition of cellular material on glass slides: Cytopress.

The present paper describes an automated apparatus called Cytopress that collects cells or cellular material e.g. nuclei out of suspension on a membrane film by filtration. In a second step this material is transferred on to a glass slide by means of standardized pressure-fixation. Thus slides of high quality are made rapidly and easily that are suited for a variety of stainings.

A visible DNA-protein stain: Feulgen-Pararosanilin(SO2) Light Green.

Feulgen-Pararosanilin(SO2) Light Green, a DNA-protein stain, is described that is suited both for visual analysis and quantitative cytochemical measurement. The stain has been applied on cervical cells and quantitative aspects have been studied on chicken erythrocyte and rat liver nuclei. In comparison to single staining, the Feulgen-Pararosanilin(SO2) DNA content of the nuclei remains unaltered after application of the combined staining. The Light Green protein content is reduced considerably, however, dependent on the degree of chromatin condensation in the nucleus.

Cytopress: automated slide preparation of cytologic material from suspension.

This paper describes a new automated system to prepare slides of cytological material from suspension. The system collects material on a filter tape by filtration and transfers it to glass slides by means of pressure-fixation. Using cervical cells as a model, results show that a well-defined cell number is evenly deposited over a standardized area, while a small number of cells is retained on the tape and a negligible number lost in the filtrate. Contamination is very small. Application of the system to other cytological material (fine needle aspirations, monolayer and cell suspension cultures, agar cultures, and isolated nuclei) is shown. In general, more than one slide can be made from one sample. Several histological staining procedures as well as immunofluorescence labeling protocols can be applied to the preparations obtained in this way. This system thus introduces a method that will standardize specimen preparation, is quick, saves operator time, and can be used for both diagnostic and research applications.

Flow cytometric analysis and sorting of human endometrial cells after immunocytochemical labeling for cytokeratin using a monoclonal antibody.

Endometrial cells in suspension were stained with propidium iodide and a monoclonal antibody against a cytokeratin intermediate filament protein specific for glandular and columnar cells (RGE 53). In this way columnar epithelial cells of the normal endometrium and of adenocarcinomas can be distinguished and separated by flow cytometry from non-epithelial cells (fibroblasts and inflammatory cells) and squamous epithelial cells, all of which are negative for RGE 53. This makes it possible to analyse and also sort pure fractions of this particular tissue type for further studies. The use of propidium iodide allows simultaneous DNA flow cytometry of these columnar epithelial cells. Therefore, the use of antibodies to cytokeratin in combination with propidium iodide can be of help in analyzing and sorting pure fractions of both normal and malignant cells. This allows a more refined examination of complex cell mixtures using flow cytometry.

A new disaggregation device for cytology specimens.

A new means of disaggregating cytology specimens in suspension using an immersible rotor device is described. The new rotor is compared to an automated syringing apparatus using cervical samples. Similar results using both devices are obtained for both normal and abnormal specimens.