1 Oregon National Primate Research Center, 505 NW 185th Ave., Beaverton, OR 97006, 2 Department of Pathology, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA and 3 University of Edinburgh, 49 Little France Crescent, Old Dalkeith Road, Edinburgh EH16 4SB, UK
4 To whom correspondence should be addressed. e-mail: brennerr{at}ohsu.edu
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Abstract |
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Key words: endometrium/Ki-67/mitotic index/mitotic protein monoclonal antibody 2/phosphorylated histone H3
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Introduction |
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Direct counting of mitotic cells in haematoxylin and eosin (H&E)-stained sections is a time-consuming process that requires highly skilled observers (Hall and Levison, 1990). It is especially difficult to distinguish between cells in prophase and cells undergoing pycnosis or apoptosis in H&E-stained sections. Relatively high magnifications have to be used to distinguish the cycle phases and large numbers of cells must be counted to obtain statistical validity.
The goal of the current work was to assess the value of two antibodies, known to be markers of mitosis, for determining the mitotic index by immunocytochemistry in paraffin sections of human and macaque endometrium, and to determine whether such counts reflected the cyclic and hormonally induced changes that occur in these tissues. These antibodies were: mitotic protein monoclonal 2 (MPM-2) and anti-phosphoylated histone H3 (Phospho H3), a rabbit polyclonal antibody. MPM-2 recognizes several proteins that become phosphorylated during mitosis (Davis et al., 1983), including cdc25 (Kuang et al., 1994
), microtubule-associated proteins (Vandre et al., 1991
) and an M phase-specific H1 kinase (Kuang et al., 1991
). The Phospho H3 antibody recognizes histone H3 after it becomes phosphorylated on serine 10 when the chromosomes condense during prophase (Chadee et al., 1999
; Strahl and Allis, 2000
; Cunningham, 2002
). Histone H3 remains phosphorylated until telophase, at which time it becomes dephosphorylated by specific phosphatases. The Phospho H3 antibody has been used to identify chromosomes during mitosis in cultured cells or whole mounts, but to our knowledge, has not been used to identify mitotic figures in formalin-fixed, paraffin-embedded tissue sections of endometrium. The MPM-2 antibody has been used in paraffin-embedded sections, but a comparison with Phospho H3 has not been made, and the suitability of either marker for computer-assisted counting in sections of endometrium under different hormonal conditions or during the human menstrual cycle has not been evaluated.
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Materials and methods |
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Animals
Archived endometrial tissues in paraffin blocks were available from rhesus macaques (Macaca mulatta) used in studies at the Oregon National Primate Research Center (ONPRC). These animals had been ovariectomized and either treated with Silastic implants of estradiol (E2) for 28 days to create an extended proliferative phase (n = 3), or sequentially treated with E2 for 14 days followed by E2 + progesterone to create an artificial secretory phase (Rudolph-Owen et al., 1998). Secretory phase samples were collected on day 3, 7 and 14 of E2 + progesterone treatment (n = 3/day). Animal care was provided by the veterinary staff of the Division of Animal Resources in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
Human samples
Endometrial tissue was collected from 21 adult women (age range: 3848 years) undergoing hysterectomy. Written informed consent was provided by all subjects and ethical approval for tissue collection granted by the Lothian Research Ethics Committee. All women reported regular menstrual cycles (2535 days) and had not received exogenous hormones or used an intrauterine device in the 3 months prior to inclusion in the study. After the uterus had been removed, a wedge of tissue from the lumen to the muscular myometrial layer that included superficial and basal endometrium as well as myometrium was taken. These tissues were fixed overnight at 4°C in 4% paraformaldehyde (PFA), rinsed and stored in 70% ethanol before routine processing into paraffin wax. The stage of the menstrual cycle was consistent with the patients reported last menstrual period and histological dating using the criteria of Noyes et al., (1950
). Cases with severe uterine pathology, e.g. polyps or fibroids, were excluded. Serum samples taken at the time of hysterectomy were used for determination of circulating progesterone and estradiol levels by radioimmunoassay. All were found to be consistent with the designated cycle stage based on morphological criteria and circulating progesterone concentrations were significantly lower in the late secretory phase compared with the early and mid-secretory phases. Proliferative and secretory endometrial samples were classified as early, mid or late. For statistical analysis, data from the early and mid-proliferative phases were pooled as early and compared with all other cycle stages. Paraffin sections of human endometrial adenocarcinoma were obtained from previously approved studies at Oregon Health and Sciences University and had been archived as formaldehyde-fixed, paraffin-embedded blocks.
Antigen retrieval
All samples compared in this study were fixed with 4% buffered paraformaldehyde and embedded in paraffin by standard methods. Consecutive 5 µm sections were cut and mounted on Superfrost/Plus (Fisher Scientific, USA) or poly-L-lysine-coated slides. The slides were deparaffinized in xylene and then rehydrated stepwise in ethanol and rinsed briefly in deionized H2O. Antigen retrieval was performed by heating sections in citrate buffer (BioGenex, USA) for 10 min (as recommended by Biogenex) in a household-type pressure cooker. The antigen-retrieved slides were allowed to cool to room temperature, rinsed once in PBS and then immediately used for immunocytochemistry.
Immunocytochemistry
The slides bearing antigen-retrieved sections were incubated in 3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidases, rinsed once in deionized H2O and then three times in phosphate-buffered saline (PBS). Unless otherwise indicated all the rinsing steps were three times for 3 min each. The sections were incubated with the appropriate species serum (Vector Laboratories Inc., USA) for 20 min to block non-specific binding. As standard in our laboratory, sections were incubated overnight with primary antibody at room temperature in a moist chamber; shorter times at moderately elevated temperatures would undoubtedly be appropriate but these were not tested. Primary antibodies were diluted from stock in 1% BSA:PBS and a variety of dilutions was assessed in preliminary studies as follows: 0.05, 0.1 and 1 µg/ml of Phospho H3, and 5.2, 2.6, 1.3, 0.65, 0.325 and 0.16 µg/ml of MPM-2. The best compromise between signal to noise (specific staining versus background) for Phospho H3 was 0.1 µg/ml and for MPM-2 was 2.6 µg/ml. All subsequent staining was done with these concentrations; antibodies from other vendors were not tested. For Ki-67 staining, mouse MIB-1 primary antibody was used in prediluted form as provided by Zymed (200 µg/ml) and incubated similarly overnight; subsequent steps were similar for all antibodies. After overnight incubation with primary antibody the slides were rinsed twice in PBS (10 min each). Sections were incubated again with normal serum (20 min) and then with biotinylated secondary antibody for 30 min. The slides were rinsed in PBS, incubated in ABC solution (Vector Laboratories) for 60 min and then re-rinsed in PBS followed by Tris buffer (38 mmol/l Tris, pH 7.60). Slides were incubated in 0.025% diaminobenzidine (Dojindo Inc.) in Tris buffer, for 1015 min, and rinsed again in Tris buffer followed by deionized H2O. Slides were then placed in 0.026% osmium tetroxide for 60 s, and rinsed with deionized H2O. Sections were lightly counterstained with Meyers haematoxylin, dehydrated and mounted with Permount. A set of consecutive sections was strongly stained with H&E to facilitate identification of mitotic cells, dehydrated and mounted. Digital images were captured with an Optronics DEI-750 CCD camera (Optronics, USA) and analysed with Image Pro-plus (Media Cybernetics Inc., USA). Minor adjustments to contrast and sharpness were made with Adobe Photoshop (Adobe Systems Inc., USA) before printing.
Direct manual counting
Technique
Mitotic figures in sections stained only with H&E and immunopositive cells stained with either MPM-2, Phospho H3 or MIB-1 were counted through the microscope at a magnification of x400 by a trained technician with a mechanical tabulator. Non-overlapping fields were selected with the aid of an ocular grid. Negative (unstained or non-mitotic) background cells (12005000 cells per animal) were also counted and the percentage of positive cells [(X positive/Y total count)x100] was calculated for each stain. The abundance of positive or mitotic cells stained by the methods above was assessed by analysis of variance and mean values compared by Fishers protected least significant difference test (Petersen, 1985).
Observer variation
Others have suggested that the major source of variation in direct counting of stained nuclei in sections is inter-observer variation (Darj et al., 1995). In order to assess whether counting error varied with the different nuclear stains, four different observers counted the same section on four separate days for H&E, MPM-2, Phospho H3 and MIB-1. The counts made by each observer on each day were summed and averaged, and both the within-observer coefficient of variation (CV) and between-observer CV were calculated. Also, counts from all observers for all days were summed and averaged and the CV calculated as a measure of overall precision.
Computer-assisted counting
The specimens stained with the Phospho H3 antibody, but not those stained for MPM-2 (or MIB-1) were of sufficient homogeneity, high contrast and low background to allow computer-assisted thresholding and subsequent computer-assisted counting. For this analysis, digital images were captured at x25 original magnification (x2.5 objective). The images were imported into Image Pro-plus, the endometrial glands were traced and defined as regions of interest (ROI), their area was measured, and the number of Phospho H3 positive cells per unit area was determined and expressed as positive cells per 10 000 µm2. To assess the precision of computer-assisted counting, one section was counted six times and the CV calculated.
To assess the degree of correlation between the computer-assisted counting of Phospho H3-labelled mitotic figures and the direct, manual counting of H&E-stained mitotic figures, we plotted the results of direct counting versus computer-assisted counting of the functional layer from the same animals as a scatter plot, and analysed the points by least squares linear regression (Origin Ver 5.0; OriginLab Corp., USA). The macaque data included counts from animals treated with E2 for 14 and 28 days and with E2 + progesterone for 3, 7 and 14 days, which provided a range of high and low values for the correlation analysis. A similar correlation analysis was performed on data from the human endometrium samples taken throughout the menstrual cycle.
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Results |
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Direct manual counting
The direct counts of mitotic figures in endometrium of macaques that had been treated with E2 for 28 days were as follows: H&E (1.5 ± 0.25%), Phospho H3 antibody (1.02 ± 0.23%), and MPM-2 antibody (0.69 ± 0.17%). The MIB-1 stained cells were, as expected, the most numerous (19.7%). Aside from the MIB-1 data, which was significantly higher than any of the other endpoints, there were no statistically significant differences among the H+E, Phospho H3 and MPM-2 indices; however, the MPM-2 positive cells were numerically lowest. Consequently, the Phospho H3 index is a valid equivalent to the mitotic index. The differences in direct counting due to observer variation are shown in Table I. Among the direct stains of mitotic figures (H+E, MPM-2 and Phospho H3) the coefficient of variation was lowest for the Phospho H3 index.
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Discussion |
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There were a few initial concerns about the method. First, because histone H3 is dephosphorylated during telophase, one might expect direct counts of mitoses in Phospho H3-stained sections to be lower than direct counts of mitotic figures in H&E-stained sections, because a microscopist doing H&E counts would count telophases. But the microscopist generally misses many prophases, as these are difficult to discern in H&E-stained sections, whereas prophases are easy to detect with the Phospho H3 stain. In any event, when direct counts of Phospho H3-stained cells were compared with direct counts of H&E-stained mitotic figures there was no statistically significant difference.
Second, during computer-assisted analysis of Phospho H3-stained samples, image thresholding would identify cells during late anaphase as two mitotic figures, because the chromosomes are cleanly separated, while a microscopist performing direct counts would count them as one cell in anaphase. The computer-assisted Phospho H3 index could therefore be higher than the directly counted mitotic index. However, this difference is probably balanced by the lower number of telophases that are included in the Phospho H3 index. The tendency to overcount anaphases is also counterbalanced by the fact that late anaphase is a rapid component of the mitotic cycle and tends to be rare in sections. In any event, there was a very high correlation (R2 = 0.90; Figure 6) between the computer-assisted, Phospho H3 index and the direct, mitotic count (expressed as a percentage) in H&E-stained samples of macaque endometrium. Consequently, the Phospho H3 index is a valid equivalent of the mitotic index, whether determined by direct or computer-assisted counting.
Because of the high contrast of the Phospho H3 stain, the computerized technique can sample large fields at low magnifications (see Figure 5), while the higher magnifications that are required to precisely identify mitotic figures in H&E-stained specimens necessarily limits the size of the microscopic field. The larger sample size obtained during computer-assisted counting reduces experimental error.
In addition to marking mitosis, phosphorylation of histone H3 on serine 10 may occur during chromatin modifications associated with cellular differentiation (Juan et al., 1999; Salvador et al., 2001
) and oncogene-induced transformation (Chadee et al., 1999
). For example, phosphorylation of histone H3 at serine 10 is involved in FSH-stimulated differentiation of rat ovarian granulosa cells and is mediated by protein kinase A (Salvador et al., 2001
). This phenomenon was observed in cell culture under serum-free conditions in the absence of proliferation. Whether this reaction would interfere with the use of a Phospho H3 antibody to evaluate mitotic figures in the primate ovary remains to be determined.
Additionally, the nuclei of human monocytes have relatively high levels of phosphorylated histone H3 during interphase, and differentiation of HL-60 (human myelogenous leukaemia) cells along the monocytic lineage is accompanied by a rise in H3 phosphorylation (Juan et al., 1999). The phosphorylated sites are distributed in a speckled, punctate manner throughout the monocyte nucleus. This punctate pattern is strikingly different from the more homogeneous pattern seen throughout the chromosomes in mitotic nuclei after Phospho H3 staining. Although monocytes and macrophages are numerous in the primate endometrium, no speckled nuclear staining was observed in these cells with our current Phospho H3 staining protocol. Therefore, interphase phosphorylation of histone H3 in monocytes should not interfere with the use of the Phospho H3 antibody to evaluate mitotic indices in the glandular epithelium of the primate endometrium.
During the early, pioneering work on the endometrium, the proliferative state of endometrial specimens was assessed by direct microscopic counting of mitotic cells in H&E-stained sections (Noyes et al., 1950). More recently, detection of the Ki-67 antigen (Gerdes et al., 1983
), which is expressed in all phases of the cell cycle (G1, S, G2 and M) has been extensively used as a measure of proliferative activity in various tissues (Scholzen and Gerdes, 2000
; Brown and Gatter, 2002
) including endometrial biopsies (Jurgensen et al., 1996
). However, the time cells spend in G1 is highly variable, and may be affected by the hormonal or neoplastic state of the tissues (Hall and Levison, 1990
). Also, staining for proteins that are expressed throughout several phases of the cell cycle cannot provide a precise measure of the rate of proliferation at the time of sampling (Hall and Levison, 1990
; Hall and Woods, 1990
). For a staining technique to give the most accurate estimate of proliferative rate, the period of time during which the cells are positive should be short. Because mitosis is one of the shortest and least variable phases of the cell cycle, the mitotic index provides the most precise estimate of the proliferative rate.
Consequently, although the Ki-67 antigen is a highly useful marker to detect proliferating populations, a count of mitotic figures adds information on the number of cells that actually complete the cell cycle, thus enhancing and complementing the information obtained from the Ki-67 marker. In sum, the Phospho H3 antibody adds a powerful new tool to our armamentarium for the analysis of cell proliferation in archived paraffin-embedded sections.
Potential clinical relevance
There are various clinical situations in which analysis of the endometrial Phospho H3 index would be valuable. Endometrial biopsies are commonly performed in pre- and post-menopausal women with abnormal uterine bleeding to exclude neoplastic endometrial disease. One important component of the pathological diagnosis of such biopsies is a determination of the proliferative state of the tissue, typically done by identifying mitotic figures in H&E-stained tissue sections. However, in pathological endometrial tissues stained with H&E, necrotic and apoptotic cells frequently resemble prophase nuclei, complicating assessment of the mitotic index. The Phospho H3 stain, because of its chromosomal specificity and high contrast, greatly facilitates such determinations and also aids in identifying abnormal mitotic figuresa pathological feature associated with neoplastic disease.
Several common womens reproductive health disorders, including endometriosis and polycystic ovary syndrome (PCOS), are estrogen-dependent conditions. In PCOS there may be overexpression of endometrial estrogen receptor co-activators which would enhance estrogen sensitivity of this tissue (Gregory et al., 2002). Androgen receptors are elevated in PCOS endometria (Apparao et al., 2002
) and these may act to counteract such estrogenic effects. Women with PCOS are therefore exposed to counteracting influences that could affect endometrial proliferative rates.
The management of women with PCOS with amenorrhoea or oligomenorrhoea includes administration of cyclic progestin therapy to induce endometrial withdrawal bleeding in order to prevent endometrial hyperplasia (Balen, 2001). In addition, clinical management of such cases includes a transvaginal ultrasound scan to assess endometrial thickness as an aid to predicting endometrial hyperplasia (Cheung, 2001
). However, those subjects with an increased endometrial thickness on ultrasound require an endometrial biopsy to provide a definitive assessment of the histological and proliferative aspects of the tissues, because fluid content can affect the thickness measurement obtained by ultrasound. Phospho H3 provides an excellent additional marker to detect excessive mitotic activity in such samples. Computer-assisted determinations of mitotic activity in such non-neoplastic endometrial disease conditions should permit a more precise assessment of the effect of steroid hormone therapy on endometrial proliferation.
Because of the clinical importance of endometrial hyperplasia and adenocarcinoma, this paper has focused on the endometrium, but the method should be applicable to quantification of mitotic activity in clinical specimens from formalin-fixed, paraffin-embedded samples of other tissue types. Also, the Phospho H3 antibody works well in cryosections (data not shown) and should work well in double-labelling studies with any antibody that requires frozen sections. In sum, immunocytochemical detection of the phosphorylated histone H3 marker should facilitate analysis of how steroid hormones, steroid antagonists and antineoplastic drugs affect endometrial mitotic activity. Phospho H3 is a valuable tool that should improve our understanding of the mechanisms involved in the pathogenesis of female reproductive disorders.
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Acknowledgements |
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Submitted on January 30, 2003; accepted on February 28, 2003.