Washless double staining of unfixed nuclei for flow cytometric analysis of DNA and a nuclear antigen (Ki-67 or bromodeoxyuridine).

Washless methods for double staining of nuclear antigen and DNA in unfixed nuclei were compared with established methods for staining of fixed cells. The methods were tested on phytohemagglutinin (PHA)-stimulated normal human blood lymphocytes for the double staining of 1) Ki-67 antigen and DNA and 2) bromodeoxyuridine (BrdUrd) and DNA, in continuously BrdUrd-labeled cells. With respect to the discrimination between antigen-positive and -negative subpopulations, there was no statistically significant differences between the results from direct (Ki-67) or indirect (Ki-67 or BrdUrd) washless staining of unfixed nuclei and the results from staining of fixed cells. Washless staining of unfixed nuclei was found to be rapid and simple and resulted in greater precision of the DNA analysis and in less aggregation and loss of cells.     

Combined Ki-67 and feulgen stain for morphometric determination of the Ki-67 labelling index

In this note we present a combined Ki-67 and Feulgen stain for morphometric determination of the Ki-67 labelling index. The immunohistochemical part of this double staining technique is based on the alkaline-phosphatase-anti-alkaline-phosphatase (APAAP) method, visualizing the enzyme activity by the nitro-blue-tetrazolium chloride (NBT)/bromo-chloro-3-indolyl-phosphate (BCIP) technique. The NBT/BCIP complex resists the hydrolytic activity of the Feulgen stain. The staining method presented allows semi-automatic determination of both the total nucleus-area as well as the Ki-67 positive nucleus-area using a morphometric computer system. The Ki-67 labelling index thus achieved is based on the relative nuclear area of Ki-67 positive nuclei and is clearly more precise and efficient than the counting method using an ocular grid.     

Sampling strategy for prostate tissue microarrays for Ki-67 and androgen receptor biomarkers

OBJECTIVE: To develop an optimal sampling strategy for tissue microarrays using automated digital analysis for androgen receptor (heterogeneous expression) and the cellular proliferation marker Ki-67 (homogeneous expression and evaluated by others using nonautomated methods). STUDY DESIGN: Tissue microarrays were constructed from 23 radical prostatectomy specimens and immunostained for androgen receptor expression and cellular proliferation. Automated digital image analysis was used, and the minimum number of cores necessary to capture variance change <3% was determined. Androgen receptor immunostaining was described by percent positive nuclei (PPN) and mean optical density (MOD). RESULTS: Androgen receptor PPN variance measurements showed that 5 cores should be obtained when a single block of a radical prostatectomy specimen contained cancer. If all of 15 blocks contained cancer, 2 cores should be obtained from each of 6 blocks. An optimal sampling strategy was developed for androgen receptor PPN, androgen receptor MOD and Ki-67 PPN. CONCLUSION: The selection of the number of cores to sample is a tradeoff between the number of cores available that contain cancer and the amount of work involved in the analysis. Sampling no fewer than 5 but no more than 12 cores per radical prostatectomy specimen can capture tissue heterogeneity.     

Accuracy of a remote quantitative image analysis in the whole slide images

The rationale for choosing a remote quantitative method supporting a diagnostic decision requires some empirical studies and knowledge on scenarios including valid telepathology standards. The tumours of the central nervous system [CNS] are graded on the base of the morphological features and the Ki-67 labelling Index [Ki-67 LI]. Various methods have been applied for Ki-67 LI estimation. Recently we have introduced the Computerized Analysis of Medical Images [CAMI] software for an automated Ki-67 LI counting in the digital images. Aims of our study was to explore the accuracy and reliability of a remote assessment of Ki-67 LI with CAMI software applied to the whole slide images [WSI]. The WSI representing CNS tumours: 18 meningiomas and 10 oligodendrogliomas were stored on the server of the Warsaw University of Technology. The digital copies of entire glass slides were created automatically by the Aperio ScanScope CS with objective 20x or 40x. Aperio's Image Scope software provided functionality for a remote viewing of WSI. The Ki-67 LI assessment was carried on within 2 out of 20 selected fields of view (objective 40x) representing the highest labelling areas in each WSI. The Ki-67 LI counting was performed by 3 various methods: 1) the manual reading in the light microscope - LM, 2) the automated counting with CAMI software on the digital images - DI , and 3) the remote quantitation on the WSIs - as WSI method. The quality of WSIs and technical efficiency of the on-line system were analysed. The comparative statistical analysis was performed for the results obtained by 3 methods of Ki-67 LI counting. The preliminary analysis showed that in 18% of WSI the results of Ki-67 LI differed from those obtained in other 2 methods of counting when the quality of the glass slides was below the standard range. The results of our investigations indicate that the remote automated Ki-67 LI analysis performed with the CAMI algorithm on the whole slide images of meningiomas and oligodendrogliomas could be successfully used as an alternative method to the manual reading as well as to the digital images quantitation with CAMI software. According to our observation a need of a remote supervision/consultation and training for the effective use of remote quantitative analysis of WSI is necessary.     

Use of selected excitation filters for enhancement of diaminobenzidine photomicroscopy

Enhancement of the diaminobenzidine (DAB) reaction product for light photomicroscopy was investigated using commercially available glass interference filters FITC-495, BG38, and BG12. The oxidized DAB transmission curve between 400-700 nm revealed a broad peak extending mostly through the yellow to red portions of the visible light spectrum, indicating that no single color predominates. Absorption spectra from the interference filters showed that FITC-495 gave total absorbance from 495-650 nm, with a smaller peak at 675 nm; BG38 transmitted at least a percentage of every wavelength up to 700 nm, whereas BG12 absorbed all light above 490 nm. To determine whether these filters could photographically increase DAB reaction product contrast, photographs were taken of corneal endothelial cells 24 hr after freeze injury. At this time, these cells demonstrate increased levels of laminin, as revealed by immunoperoxidase cytochemistry. When photography was performed using either no filter or a standard green filter, DAB contrast relative to background was minimal. However, when photographs were made using either the FITC-495 or the BG12 filter, DAB contrast increased sharply, although background density increased in the former case and decreased greatly in the latter. BG38 by itself did not increase DAB contrast. However, when used in combination with FITC-495 good DAB contrast was achieved and background density was lower than that seen using FITC-495 alone. Therefore, selective interference filters can photographically increase DAB contrast for studies using immunoperoxidase cytochemistry.     

Application of different methods for nuclear shape analysis with special reference to the differentiation of brain tumors

OBJECTIVE: To study the discriminatory power of different methods designed for nuclear shape analysis with reference to the differentiation and grading of brain tumors and the differentiation between proliferating and nonproliferating nuclei. STUDY DESIGN: At least 300 tumor cell nuclei per case were measured by means of a digital image analysis system. Fourier amplitudes no. 1 to 15, moments no. 1 to 7 according to Hu, roundness factor, ellipse shape factor, concavity factor, Feret ratio, fractal dimension and bending energy were determined for each nucleus. The discriminatory power of these parameters was tested in three pairwise comparisons: (1) oligodendrogliomas WHO grade II (n = 13) vs. grade III (n = 11), (2) medulloblastomas WHO grade IV (n = 14) vs. anaplastic ependymomas WHO grade III (n = 12), (3) Ki-67-positive vs. Ki-67-negative tumor cell nuclei in the 14 medulloblastomas. RESULTS: When data from Fourier analysis were included in statistical analysis, cross-validated discriminant analysis led to a 100% correct reclassification for the first and for the second pairwise comparison and to a 75% correct reclassification when comparing Ki-67-positive and Ki-67-negative nucleifrom medulloblastomas. Different combinations of the other shape parameters led to a lower percentage of correctly reclassified cases for all three pairwise comparisons, especially when Fourier analysis was not included in the analysis. CONCLUSION: Fourier analysis provided an optimal statistical discrimination between different brain tumor entities and between data sets from proliferating and nonproliferating tumor cell nuclei. Since nuclear shape is an important criterion for the investigation of tumors, the application of Fourier analysis is therefore recommended for quantitative histologic investigations in neuro-oncology.     

PCNA independence of Ki-67 expression in HPV infection.

12 Infectious Wart lesions were stained using the streptavidin-biotin immunoperoxidase method for PCNA10, MIB-1 (Ki-67 equivalent antigen) and Human Papilloma Virus antigen to study the effect of HPV presence on epidermal proliferation. Using strict methods to avoid observer bias, Ki-67 antigen was found in a high proportion of nuclei in the suprabasal layers together with HPV antigens, in the absence of PCNA staining. This finding indicates that DNA synthesis related, Ki-67 antigen bearing structures can be raised in the human nucleus in the absence of induction of PCNA bearing structures, suggesting also structural independence between the antigen bearing molecules.     

IHC Profiler: An Open Source Plugin for the Quantitative Evaluation and Automated Scoring of Immunohistochemistry Images of Human Tissue Samples

In anatomic pathology, immunohistochemistry (IHC) serves as a diagnostic and prognostic method for identification of disease markers in tissue samples that directly influences classification and grading the disease, influencing patient management. However, till today over most of the world, pathological analysis of tissue samples remained a time-consuming and subjective procedure, wherein the intensity of antibody staining is manually judged and thus scoring decision is directly influenced by visual bias. This instigated us to design a simple method of automated digital IHC image analysis algorithm for an unbiased, quantitative assessment of antibody staining intensity in tissue sections. As a first step, we adopted the spectral deconvolution method of DAB/hematoxylin color spectra by using optimized optical density vectors of the color deconvolution plugin for proper separation of the DAB color spectra. Then the DAB stained image is displayed in a new window wherein it undergoes pixel-by-pixel analysis, and displays the full profile along with its scoring decision. Based on the mathematical formula conceptualized, the algorithm is thoroughly tested by analyzing scores assigned to thousands (n = 1703) of DAB stained IHC images including sample images taken from human protein atlas web resource. The IHC Profiler plugin developed is compatible with the open resource digital image analysis software, ImageJ, which creates a pixel-by-pixel analysis profile of a digital IHC image and further assigns a score in a four tier system. A comparison study between manual pathological analysis and IHC Profiler resolved in a match of 88.6% (P<0.0001, CI = 95%). This new tool developed for clinical histopathological sample analysis can be adopted globally for scoring most protein targets where the marker protein expression is of cytoplasmic and/or nuclear type. We foresee that this method will minimize the problem of inter-observer variations across labs and further help in worldwide patient stratification potentially benefitting various multinational clinical trial initiatives.     

Evolutionary conservation in various mammalian species of the human proliferation-associated epitope recognized by the Ki-67 monoclonal antibody

The human proliferation-associated epitope recognized by the Ki-67 monoclonal antibody (MAb) was detected in proliferating normal and neoplastic cells of many mammalian species (lamb, calf, dog, rabbit, rat) besides human. In contrast, Ki-67 stained proliferating cells from other species weakly (mouse) or not at all (swine, cat, chicken, pigeon). The immunostaining pattern of Ki-67 in animal tissues was identical to that previously described in human: Ki-67 reacted only with cells known to proliferate (e.g., germinal center cells, cortical thymocytes) but not with resting cells (e.g., hepatocytes, brain cells, renal cells); this MAb produced a characteristic nuclear staining pattern (e.g., stronger labeling of nucleoli than of the rest of the nuclei and staining of chromosomes in mitotic figures); and Ki-67 crossreacted with the squamous epithelium in both animal and human tissues. In vitro studies showed that when quiescent (Ki-67-negative) NIH 3T3 fibroblasts or bovine peripheral blood lymphocytes were induced to proliferate, the appearance of Ki-67-positive cells paralleled the induction of cell proliferation caused by addition of fetal calf serum or PHA, respectively, to the cultures, and in both human and rat proliferating cells the Ki-67 expression closely paralleled the incorporation of [3H]-thymidine. These findings indicate that the epitope recognized by the Ki-67 MAb in human and animal species is the same. The widespread evolutionary conservation of the human proliferation-associated epitope recognized by the Ki-67 MAb suggests that it and/or its carrier molecule may play an important role in regulation of cell proliferation.     

Correlation between preoperative magnetic resonance spectroscopic data on high grade gliomas and morphology of Ki-67-positive tumor cell nuclei

OBJECTIVE: To investigate possible statistical correlations between metabolic data from preoperative proton magnetic resonance spectroscopy (1HMRS) and morphology of proliferating tumor cell nuclei in anaplastic gliomas and glioblastomas. STUDY DESIGN: Ki-67-positive tumor cell nuclei in paraffin sections of surgical specimens from 36 patients (7 anaplastic gliomas, World Health Organization grade 3; 29 glioblastomas, World Health Organization grade 4) were investigated by means of a digital image analysis system. Stringent inclusion criteria were formulated for all cases with respect to histologic quality and spectroscopic examination. As morphometric variables, nuclear area, shape variables (roundness factor, size-invariate Fourier amplitudes) and density of Ki-67-positive tumor cell nuclei per reference area were determined. RESULTS: Correlation analysis according to Spearman revealed a significant positive correlation between the total creatine (TCR) peak and nuclear area (P = .005). This correlation was also found within the glioblastoma group (P = .019). There was also a significant negative correlation of nuclear area with the ratio between choline and TCR in all cases (P = .014) and within the glioblastoma group (P = .046). No significant correlation of spectroscopic data was found with nuclear shape or density of Ki-67-positive tumor cell nuclei. CONCLUSION: The results demonstrate a correlation between spectroscopic data and morphology of proliferating tumor cell nuclei (nuclear size) in high grade gliomas. This study is part of a detailed investigation of the interrelationship between preoperative 1HMRS and quantitative histomorphology of gliomas.     

Cell cycle dependent distribution of the proliferation-associated Ki-67 antigen in human embryonic lung cells

The cell cycle-dependent distribution of the proliferation-associated Ki-67 antigen has been evaluated immunocytochemically in L-132 human fetal lung cells. The cells were synchronized and cell cycle phases were determined: G1 = 6.7 h, S = 5.4 h, G2 = 8.5 h and mitosis = 1.3 h. The Ki-67 patterns were strictly correlated with the cell cycle phases. In late G1-phase, Ki-67 antigen was present only in the perinucleolar region. In the S-phase, Ki-67 staining was found homogeneously in the karyoplasm and in the perinucleolar region. G2-phase cells contained a finely granular Ki-67 staining in the karyoplasm with Ki-67-positive specks and perinucleolar staining. In early mitotic cells (pro- and metaphase) an intense perichromosomal Ki-67 staining was observed in addition to a homogeneously stained karyoplasm in prophase, and cytoplasm in metaphase. During ana- and telophase the Ki-67 antigen disappeared rapidly. In resting cells there was no Ki-67 staining.     

Immunohistochemical localization of bovine placental retinol-binding protein.

An immunogold staining method was used in combination with epipolarization microscopic detection to demonstrate the presence of bovine placental retinol-binding protein in bovine extraembryonic membranes. Amnion, chorion and allantois were fixed in Bouin fixation fluid and embedded in polyethylene glycol 1500. Sections (5 mm) were cut and transferred onto Digene silanated slides and immunostained using rabbit antiserum raised against bovine placental retinol-binding protein followed by goat anti-rabbit IgG labeled with 1 nm gold. Gold particles after silver enhancement were viewed and photographed under epipolarization microscopy. Epithelial cells of all three membranes (i.e. amniotic ectoderm, chorionic trophectoderm, and allantoic endoderm) were immunoreactive, while mesodermal cells, collagen, and blood cells were not. These data, together with our previous observation that these three placental membranes synthesize and secrete retinol-binding protein, indicate that epithelial cells lining the amnion, chorion and allantois are the major sources of this protein. The presence of retinol-binding protein in placental membranes and their fluids may be indicative of an important role for retinol in placental differentiation and development.     

Quantitative evaluation of immunohistochemical staining in gastrointestinal stromal tumors

OBJECTIVE: To evaluate the differences among pathologists' interpretations in gastrointestinal stromal tumors (GISTs) and to demonstrate the usefulness of quantitative pathology in the assessment of immunohistochemical staining. STUDY DESIGN: Twenty GISTs were separately evaluated by 4 pathologists by the visual estimation method using a 6-antibody panel. Each case was then quantitatively measured with a computer-assisted image analysis system by 2 pathologists. Cohen's kappa test was performed for statistical analysis. RESULTS: All GISTs showed some degree of expression of CD 117, CD34, SMA and Ki-67. No case was immunoreactive for desmin or S-100 protein. There were remarkable differences in the pathologists' visual estimations. Moreover, the discrepancies between visual and quantitative methods were noteworthy. The differences in interpretations showed the greatest variability for Ki-67, which is known to be related to poor prognosis. CONCLUSION: Quantitative pathology in assessment of immunohistochemical staining of GISTs may improve the consistency in the interpretation of staining results and provide some degree of reproducibility.     

Ki-67 labeling in postmitotic cells defines different Ki-67 pathways within the 2c compartment

Simultaneous quantification of DNA and Ki-67 proliferation-associated antigen was performed using fluorescence image cytometry. In the MCF-7 cell line, the Ki-67 antigen content increases during the cell cycle, and its intranuclear distribution pattern varies. Quantitative evolution of Ki-67 content as a function of nuclear area makes it possible to define several pathways followed by cells going through the 2c compartment. 1) In some cells, the amount of Ki-67 antigen remains constant during G1 (Ki-67 stable pathway), and a characteristic speckled pattern can be observed. 2) In the larger fraction of cells analyzed, there is a postmitotic decrease in the Ki-67 (Ki-67 decrease pathway) content. In this pathway, labeling is located in the nucleoplasm in small nuclei, is located in nucleoli in intermediate-sized nuclei, and is absent from larger nuclei (G0). A progressive increase in Ki-67 content (Ki-67 increase pathway) was observed from intermediate-sized nuclei to S phase nuclei. From these results, we hypothesize that the Ki-67 stable pathway is the G1 phase of newly formed cells going directly to S phase in local optimal conditions of growth and that Ki-67 decrease pathway and Ki-67 increase pathway correspond to cells whose progression to S phase is regulated by extracellular factors.     

Simultaneous staining of exponentially growing versus plateau phase cells with the proliferation-associated antibody Ki-67 and propidium iodide: analysis by flow cytometry

The antibody Ki-67, which detects proliferating cells, was used in combination with propidium iodide, a DNA-specific dye. The double-staining method allowed discrimination of cells in the phases of the cell cycle as well as the recognition of Ki-67 staining characteristics. Suspension cultures of U937 cells were measured in exponential growth and plateau phase in nutritional deprivation. The fraction of Ki-67 positive cells was nearly 100% 2 days after dilution and 46% 7 days after dilution of the cultures. Stathmokinetic measurements with colchicine and flow cytometry measurements with the BrdU-Hoechst technique yielded close to 100% proliferation at day 2 but only 18% and 6%, respectively, at day 7. The discrepancy between Ki-67 results and the results of the two other methods is considered to be a characteristic of nutritionally deprived cells.     

Comparison of monoclonal antibodies PC 10 and MIB 1 on microwave-processed paraffin sections

Abstract. Antibodies against the proliferation-associated nuclear antigen (PCNA) and against the Ki-67 protein are widely used as operational proliferation markers in human tumour diagnostics. The original Ki-67 antibody had the inherent drawback in that it could only be used when fresh-frozen material was available. The antibody PClO was supposed to offer the advantage that it could be applied on formalin-fixed and paraffin-embedded tissues. However, in cases in which the formalin fixation exceeded 4 h, PC10 staining proved to be inconsistent and often failed.
The aim of this study was to compare a recently prepared Ki-67 equivalent monoclonal antibody (MIB 1) and PC10 in routinely fixed histopathological material using antigen retrieval by microwave processing.
Antibody MIB 1 stained the nuclei of cells known to belong to the proliferative compartments in microwave-processing paraffin sections of formalin-fixed tissues. Quiescent cells were consistently negative for MIB 1 staining. In contrast, PC10 was positive in almost all nuclei of different tissues in microwave-treated paraffin sections. Thus, antigen retrieval by microwave processing is beneficial for the detection of the Ki-67 protein in paraffin sections, whereas it is not needed for the detection of the PCNA.     

Cyclin A and Ki-67 with DNA content in benign and malignant prostatic epithelial lesions.

OBJECTIVE: To evaluate the usefulness of immunohistochemical staining of cyclin A and Ki-67 together with DNA content in the classification of benign prostatic hyperplasia, prostatic intraepithelial neoplasm (PIN) and prostatic carcinoma foci and to compare these parameters with each other and with parameters obtained from conventional histopathology. STUDY DESIGN: We selected 37 carcinoma, 18 PIN and 8 hyperplastic foci from prostatectomies done during 1996 and 1997 at Turku University Central Hospital. Cyclin A and Ki-67 staining was assessed by immunohistochemistry and DNA content by image cytometry. RESULTS: The hyperplastic, PIN and carcinoma foci differed clearly in their 2.5c exceeding rates, image cytometric proliferation indices and staining indices for cyclin A and Ki-67. No significant differences were found between these histologic entities in their modal DNA ploidy values. In carcinomas, cyclin A and Ki-67 indices differed between low, intermediate and high Gleason and World Health Organization grading groups. Diploid and tetraploid carcinomas had similar cyclin A and Ki-67 indices, which differed from those of aneuploid carcinomas. CONCLUSION: The 2.5c exceeding rate and image cytometric proliferation index as well as the cyclin A and Ki-67 indices differed significantly between different types of prostatic lesions. Cyclin A and Ki-67 had good correlations with the histologic grade of carcinoma.     

Simultaneous staining of exponentially growing versus plateau phase cells with the proliferation-associated antibody Ki-67 and propidium iodide: analysis by flow cytometry

Abstract. The antibody Ki-67, which detects proliferating cells, was used in combination with propidium iodide, a DNA-specific dye. The double-staining method allowed discrimination of cells in the phases of the cell cycle as well as the recognition of Ki-67 staining characteristics. Suspension cultures of U937 cells were measured in exponential growth and plateau phase in nutritional deprivation. The fraction of Ki-67 positive cells was nearly 100% 2 days after dilution and 46% 7 days after dilution of the cultures. Stathmokinetic measurements with colchicine and flow cytometry measurements with the BrdU-Hoechst technique yielded close to 100% proliferation at day 2 but only 18% and 6%, respectively, at day 7. The discrepancy between Ki-67 results and the results of the two other methods is considered to be a characteristic of nutritionally deprived cells.     

Ki-67 and B72.3 expression in breast cancer: an immunohistochemical study

The present study was performed to evaluate Ki-67 and B72.3 immunostaining in 20 selected cases of breast cancer. In particular, we have examined the intracellular localization of TAG 72 and the tumour growth fraction, identified by Ki-67 antibody, on frozen sections of mammary carcinoma, by immunohistochemical technique (ABC method sec.Hsu). Immunostaining of TAG 72 and Ki-67 antigen was related to histologic subtype, diameter, nodal involvement, and number of positive axillary nodes. The preliminary results suggest that: (a) the presence of Ki-67 nuclear staining appeared to be associated with a poorer degree of differentiation, but no direct relationships were observed with diameter and nodal involvement; (b) no correlation between Ki-67 labelling rates and B72.3 intracytoplasmic immunostaining was observed; (c) myoepithelial cells show weak intracytoplasmic positivities.