Simultaneous flow cytometric analysis of surface markers and nuclear Ki-67 antigen in leukemia and lymphoma

The monoclonal antibody Ki-67 identifies an antigen present during the late G1, S, G2, and M phases of the cell cycle, whereas resting cells do not express this antigen. Immunostaining with Ki-67 provides a simple method with which to determine the growth fraction of a malignant cell population without requiring a laborious procedure or use of radioactive materials. Thus far, detection of Ki-67-positive cells by flow cytometry was limited because of nuclear location of the antigen. In this study, periodate-lysine-paraformaldehyde (PLP) fixation of cells in suspension, labeling with Ki-67, and the subsequent flow cytometric analysis of the tumor growth fraction is described. Fixation with PLP at -10 degrees C for 15 min rendered the plasma membrane permeable without destroying cell surface antigens. Thus double immunofluorescence studies using both a surface marker and Ki-67 could be performed. This offers the additional advantage of being able to define the phenotype of proliferating cells. This method was applied to determine the growth fraction in peripheral blood and bone marrow samples of patients with leukemia and non-Hodgkin's lymphoma. The results of Ki-67 studies in 91 patients are shown. A wide variability of individual Ki-67 values was observed within each entity. Use of this flow cytometric procedure substantially facilitates the quantification of proliferating cells in pathological blood and bone marrow samples.     

Flow cytometric multiparameter analysis of proliferating cell nuclear antigen/cyclin and Ki-67 antigen: a new view of the cell cycle

Flow cytometric multiparameter analysis of two proliferation-associated nuclear antigens (proliferating cell nuclear antigen (PCNA)/cyclin and Ki-67) was performed on seven human hematopoietic cell lines. PCNA/cyclin, an S phase-related antigen, was detected using an autoantibody and a fluorescein isothiocyanate-labeled anti-human antibody. The Ki-67 antigen, which in cycling cells is expressed with increasing levels during the S phase with a maximum in the M phase, was detected using a monoclonal antibody and a phycoerythrin-conjugated anti-mouse antibody. In some experiments the PCNA/Ki-67 staining was combined with a DNA stain, 7-amino actinomycin D, and simultaneous detection of the three stains was performed by a single laser flow cytometer. Using this technique four distinct cell populations, representing G1, S, G2, and M, respectively, could be demonstrated in cycling cells on the basis of their PCNA/cyclin and Ki-67 levels. The cell cycle phase specificity could be verified using metaphase (vinblastine, colcemide) and G2 phase (mitoxantrone) blocking agents, as well as by stainings with a mitosis-specific antibody (MPM-2). Also, G0 cells could be discriminated from G1 cells in analysis of a mixture of resting peripheral mononuclear blood cells and a proliferating cell line. This technique can be valuable in detailed cell cycle analysis, since all cell cycle phases can be visualized and calculated using a simple double staining procedure.     

Two-color multiparametric method for flow cytometric DNA analysis of carcinomas using staining for cytokeratin and leukocyte-common antigen

Solid tumors contain heterogenous cell populations, resulting in flow cytometric (FCM) DNA quantitations of a mixture of tumor and host cells. Such mixed populations can result in dilution of the tumor cells by the host cells, in difficulty defining the diploid reference mean and in histogram peak overlap, precluding cell-cycle analysis. In this study, epithelial (tumor) cells and contaminating host cells in 100 consecutively accessioned human mammary and colorectal carcinomas were segregated in a multiparametric two-color FCM DNA analysis of intact, ethanol-fixed cells. These two carcinomas and bladder carcinomas contain a cytoskeleton of simple epithelium that is selectively stained with an FITC-labeled monoclonal antibody (MAb) to cytokeratin (CK: CAM 5.2-FITC). This MAb detects the CK 8, CK 18 and CK 19 consistently present in all layers of normal and neoplastic urothelium, colonic epithelium and mammary epithelium. Gating on CK in these tumors enables the nonstaining leukocytes, stromal fibroblasts and endothelial cells to be excluded from DNA analysis. A separate aliquot of each tumor evaluated was labeled with an MAb to leukocyte-common antigen (LCA-FITC) to serve as a patient-specific intrinsic diploid reference standard. Both the CK-labeled and LCA-labeled cells were then dual labeled for DNA with propidium iodide. This method (1) correctly identified the intrinsic diploid (LCA-positive) channel, allowing an accurate definition of normal cell DNA content for calculation of the DNA index; and (2) resulted in an increased sensitivity in the identification of both diploid and abnormal hyperdiploid tumor cell populations. It also (3) limited DNA cell cycle analysis to urothelial, colonic and mammary epithelial cells, the majority of which were neoplastic in carefully selected tumor samples. In addition, this method (4) clarified near-tetraploid populations that overlap the normal nonepithelial G2M region by diminishing the normal G2M peak and accentuating the aneuploid tetraploid G0G1 peak and (5) deconvoluted overlapping histograms composed of normal host and diploid-range or aneuploid tumor cells by gating on tissue-specific markers. This exclusion of host cells in both classes of tumors resulted in more accurate cell-cycle calculations in the former and allowed calculation of the S-phase fractions in the latter.     

Flow cytometric analysis of proliferation associated nuclear antigens using washless staining of unfixed cells.

The expression of different proliferation associated nuclear antigens was analyzed using a washless double-staining method and flow cytometry. It is a simple and rapid two-step procedure which can be performed on low cell numbers. A series of hematopoietic cell lines and fresh lymphoma cells were tested and the methodology was found to be applicable to a number of nuclear antigens (PCNA, Ki-67, p105, MPM-2, fibrillarin). For PCNA, the detectability was dependent on the type of antibody used. The immunofluorescence pattern observed by microscopy was altered for antigens stained by the washless technique in comparison with the pattern obtained with fixed cells. With the washless method, detailed cell cycle analysis could be obtained by dual parameter analysis of PCNA and Ki-67.     

Intranuclear localization of the Ki-67 reactive antigen in HeLa cells. Flow cytometric analysis

The intranuclear localization of the Ki-67 reactive antigen was immunocytochemically investigated using flow cytometry. HeLa S3 cells were immunocytochemically stained with the monoclonal antibody, Ki-67, after in situ treatments with various kinds of compounds, namely: HCl; NaCl; RNase; Sl nuclease and DNase I. The only treatment that markedly diminished the immunofluorescence intensity of the cells was exposure to DNase I. Nuclear fluorescence was no longer observed in the cells digested with relatively high concentrations of DNase I. These results suggest that the antigen recognized by Ki-67 is closely associated with DNA, but is not directly associated with either the nuclear matrix or histones.     

Intranuclear localization of the Ki-67 reactive antigen in HeLa cells. Flow cytometric analysis

The intranuclear localization of the Ki-67 reactive antigen was immunocytochemically investigated using flow cytometry. HeLa S3 cells were immunocytochemically stained with the monoclonal antibody, Ki-67, after in situ treatments with various kinds of compounds, namely: HCl; NaCl; RNase; S1 nuclease and DNase I. The only treatment that markedly diminished the immunofluorescence intensity of the cells was exposure to DNase I. Nuclear fluorescence was no longer observed in the cells digested with relatively high concentrations of DNase I. These results suggest that the antigen recognized by Ki-67 is closely associated with DNA, but is not directly associated with either the nuclear matrix or histones.     

The proteasome controls the expression of a proliferation-associated nuclear antigen Ki-67

The proteasome is a protease complex responsible for rapid, selective, and irreversible removal of regulatory proteins, as well as many other cellular proteins. In this study, we have demonstrated that a proliferation-associated nuclear protein Ki-67 depended on the proteasome for its rapid degradation. A proteasome-specific inhibitor lactacystin augmented Ki-67 protein levels in pancreatic cancer BxPC-3 cells while repressed the level of steady-state Ki-67 mRNA. Inhibition of the proteasome also led to accumulation of two CDK inhibitors p27(kip1) and p21(cip1) in the BxPC-3 cells. Failed reduction of Ki-67 protein and enhanced levels of the two CDK inhibitors are likely contributing factors for the suppressed BxPC-3 proliferation after proteasome inhibition.     

Comparison of cell proliferation in breast carcinoma using image analysis (Ki-67) and flow cytometric systems.

Cellular proliferation has been implicated as an important predictor of biologic behavior in breast cancer. Cellular proliferation of 95 breast carcinomas was evaluated by comparing Ki-67 immunoreactivity in frozen sections quantitated by image analysis with S-phase and S + G2/M phase fraction determined by flow cytometry on nuclei extracted from fixed, paraffin-embedded sections (modified Hedley's technique). These parameters were correlated with traditional morphologic features of histologic grade, including mitotic count. Ki-67 immunoreactivity correlated with S-phase fraction determined by flow cytometry (r = .41, P = .001) and with S + G2/M phase fraction determined by flow cytometry (r = .29, P = .008). There was also a correlation between histologic grade and Ki-67 immunoreactivity (r = .30, P = .004) and between histologic grade and S-phase fraction (r = .42, P = .0001). Mitotic count correlated with Ki-67 immunoreactivity (r = .25, P = .015) and with S-phase fraction (r = .35, P = .001). Image and flow cytometric analysis systems provide comparable measurements of cellular proliferation; their measurements correlate with histologic grade and mitotic count in breast cancer.     

Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67

The monoclonal antibody Ki-67 detects a nuclear antigen that is present only in proliferating cells. The aim of the present investigation was to clarify whether the Ki-67 nuclear antigen is restricted in its expression to certain phases of the cell cycle. All experiments consistently showed that the Ki-67 nuclear antigen is present in S, G2, and M phase, but is absent in G0. However, the results concerning Ki-67 antigen expression in G1 phase varied: cells passing the early events of mitogen triggered transition from G0 to G1, i.e., G1T and first G1A, lacked the Ki-67 nuclear antigen, whereas G1 cells after mitosis were constantly Ki-67-positive. This result suggests that after mitosis cells might not follow the same metabolic pathways as G0 cells do when entering G1 for the first time. Therefore, we suggest that the early stages of mitogen stimulation represent initial sequences of proliferation and not parts of the cell cycle. Because our data show that the Ki-67 nuclear antigen is present throughout the cell cycle, immunostaining with monoclonal antibody Ki-67 provides a reliable means of rapidly evaluating the growth fraction of normal and neoplastic human cell populations.     

An indirect immunofluorescence double staining procedure for the simultaneous flow cytometric measurement of iodo- and chlorodeoxyuridine incorporated into DNA

In this paper we describe an indirect fluorescence double staining procedure for the simultaneous detection of IdUrd and CldUrd in the same cell nucleus. Two commercially available antibodies were selected for this purpose. A rat anti-BrdUrd monoclonal antibody from Sera-lab was found to bind specifically to CldUrd and BrdUrd. A mouse monoclonal anti-BrdUrd antibody from Becton Dickinson used in a 1:2 dilution binds to all halogenated deoxyuridines but, when the cells were extensively washed with Tris buffer with a high salt concentration, almost no binding to CldUrd was observed. An immunofluorescence procedure was developed, based on these primary antibodies, raised in different species (rat and mouse), in combination with highly purified second antibodies: FITC conjugated goat antirat and Texas-Red conjugated goat antimouse.     

A rapid flow cytometric method for bivariate bromodeoxyuridine/DNA analysis using simultaneous proteolytic enzyme digestion and acid denaturation

This report describes an immunocytochemical procedure for the simultaneous quantification of bromodeoxyuridine (BrdUrd) incorporated into cellular DNA and total DNA content in individual cells in suspension. Improvement of existing methods was achieved by combining acid denaturation and proteolytic enzyme digestion (0.2 mg/ml pepsin in 2N HCl for 30 min at room temperature). Acid denaturation preceded by enzyme digestion resulted in a large amount of debris and the occurrence of naked nuclei. In contrast, the simultaneous denaturation/protein digestion procedure did not damage the cellular structure, is rapid and reproducible, and has cell recoveries of more than 85%. Although experimental conditions were tested on human cultured keratinocytes, this method also appeared applicable to bone marrow cells and cells obtained from solid tissues.     

Accuracy and reliability of flow cytometric DNA analysis using a simple, one-step ethidium bromide staining protocol

Sources of variation and error were investigated for a simple flow cytometric analysis of DNA content of detergent-isolated nuclei stained with ethidium bromide. Using the ploidy classes of mouse liver nuclei, deviations from linearity were assessed for three different instruments. In more extreme settings, the maximum deviations for a FACS instrument were up to 6 to 9%, but in general deviations were around 1% or lower for all instruments. As biological DNA standards, human peripheral lymphocytes and trout erythrocytes appeared to be suitable and easy to store frozen. The erythrocytes had dye-binding characteristics similar to those of human lymphocytes and a 20% lower fluorescence, thus being well suited as an internal standard, as was demonstrated in tumor ploidy analyses performed with varied tissue concentration. Staining homogeneity was improved when staining time was extended to 24 h, at which time male and female lymphocytes were completely separated with an average difference in DNA content of 1.9%. A small difference in fluorescence between mitogen-stimulated and unstimulated lymphocytes was reduced to less than 1% after 24 h of staining. In general, the manipulations of the conditions for the analysis resulted in maximum variations of around 1%, indicating the robustness and reliability of the technique.     

Long-term storage of nuclear suspensions prepared from paraffin-embedded material for flow cytometric DNA analysis

Nuclear suspensions were prepared from archival material of cancer biopsies. DNA content analysis by flow cytometry was performed after storage at -80 degrees C for 24 h and 6 months, compared to 24 h storage at +4 degrees C. No significant variations in CV or DNA indices were observed. Repeated freezing and thawing did not reduce the DNA histogram quality.     

Simultaneous flow cytometric detection of cellular c-myc protein, incorporated bromodeoxyuridine, and DNA

We describe a multivariate flow cytometric technique for simultaneous analysis of specific nuclear protein, bromodeoxyuridine (BrdUrd) incorporated into DNA and DNA content in single cells in suspension. The procedure involves fixation of BrdUrd-exposed cells with paraformaldehyde, heat denaturation of cellular DNA, followed by sequential immunochemical reactions to label incorporated BrdUrd and nuclear protein, and finally staining of total DNA with propidium iodide. The cells are analyzed flow cytometrically and multivariate data acquired in list mode to facilitate analyses of heterogeneous subpopulations. We applied this technique to measure c-myc protein, incorporated BrdUrd, and DNA content in subpopulations present in a recombinant Chinese hamster ovary (CHO) cell line carrying approximately 800 copies of murine c-myc sequences under control of an inducible heat shock promoter.     

Streptavidin-based quantitative staining of intracellular antigens for flow cytometric analysis.

A streptavidin-biotin-based three-step immunolabeling protocol for quantitative staining of intracellular antigens for flow cytometric analysis was evaluated using simian virus 40 (SV40) large T antigen. The concentration as well as the quantity of antibody used required optimization. The optimum labeling conditions varied moderately with cell lines that express T antigen levels over a 40-50-fold range. The procedure resulted in specific fluorescence 2.4 times higher than that using a comparable two-step indirect immunofluorescence technique. The gain in resolution was shown to be greater when staining cells with lower antigen levels. In the analysis of background fluorescence, the principal components were, as for the two-step technique, autofluorescence and propidium spectral overlap. While streptavidin does add to the background, the increase is relatively small. Decreasing the propidium concentration from 50 micrograms/ml to 5 micrograms/ml was found to reduce significantly the level of background from this source. Theoretical aspects of quantitative staining and of resolution versus quantification are discussed.     

Preparation of tissues for DNA flow cytometric analysis

A method for measuring DNA in tissue cells by flow cytometry utilizing a one step combination nuclear isolation-DNA fluorochrome staining procedure is described. A variety of cells and tissues, both in vivo and in vitro, was used to illustrate the universal nature of this technique. These included murine bone marrow, liver testicle, sarcoma brain tumor, rat pancreatic islets, human peripheral blood, colon mucosa, colon cancer, sarcoma and brain tumor tissues. A special nuclear isolation medium, which contained either of the DNA fluorochromes, 4',6-diamidino-2 phenylindole-2 HCl or propidium iodide, was utilized successfully to isolate single suspensions of DNA fluorochrome stained nuclei in a rapid (5-10 min), consistent manner from a variety of tissues and cells. Multiple sampling of the same tissue or comparison between whole tissues and their single cell isolates showed that a representative sample was being obtained.     

Enzymatic amplification staining for flow cytometric analysis of cell surface molecules

BACKGROUND: Flow cytometric analysis is a powerful technique for the single cell assessment of cell surface expression of selected molecules. The major deficiency of flow cytometry has been its relative insensitivity. Only molecules expressed in abundance have been readily observed. METHODS: We have developed an enzymatic amplification procedure for the analysis of cell surface molecules by flow cytometry. Transformed and nontransformed cells expressing MHC class I, CD5, CD3, CD4, CD6, CD7, CD34, CD45, MHC class II, Fas ligand, and phosphatidylserine were assessed. RESULTS: Our enzymatic amplification technology increased the fluorescence signal between 10 and 100-fold for all surface molecules tested. CONCLUSIONS: Enzymatic amplification staining produces a significant enhancement in the resolving power of flow cytometric analysis of cell surface molecules. Using this technique, we have been able to detect the presence of molecules that could not be observed by the standard procedure.     

Measurement of cell proliferation in gastric carcinoma: comparative analysis of Ki-67 and proliferative cell nuclear antigen (PCNA)

Summary Immunostaining to identify nuclear antigens expressed throughout the cell cycle provides a convenient way of assessing proliferating kinetics in tumours. We studied proliferation activity of gastric carcinomas by Ki-67 and PCNA immunostaining and the two methods were compared. The mode of tissue preparation differed, fresh frozen for Ki-67 and formalin-fixed paraffin-embedded for PCNA. Immunostaining with avidin-biotin was used in both. The labelling index (LI) and a semi-quantitative grading of cell proliferation were assessed in both markers. Significant correlation was shown between LI and grading with either Ki-67 and PCNA. However, no correlation was found between PCNA and Ki-67. This lack of relationship between the two markers may be attributed to a number of factors. 1. The most likely is the marked inter- and intra-tumour heterogeneity of gastric carcinomas reflected in high standard deviation values. 2. Preparation of tissue and small size sampling with Ki-67. 3. Long life of PCNA leading to detection of cells that have recently left the cell cycle. 4. One may be observing deregulated expression of DNA as seen in certain tumours. PCNA offers the advantage of being applicable to archival material.     

Ki-67 monoclonal antibody (MAb) reacts with a proliferation associated nuclear antigen in the rabbitOryctolagus cuniculus

Summary The Ki-67 monoclonal antibody which recognizes a human nuclear antigen expressed by cycling cells but not by resting cells was found to react immunohistochemically with tissues from the rabbitOryctolagus cuniculus. Ki-67 immunoreactivity was restricted to the nucleus. A comparative study with bromodeoxyuridine labelling patterns was carried out to study the association with proliferating cells. In lingual, jejunal and appendix mucosa, skin, adrenal gland, thymus, spleen, bone marrow, testis, growth cartilage, periosteum and perichondrium of long bones the distribution of Ki-67 positive and bromodeoxyuridine labelled cells was similar and consistent with the distribution of proliferating cells in these tissues. In tissue from the brain, kidney, skeletal or cardiac muscle and liver no Ki-67 positive or bromodeoxyuridine labelled cells were seen. In cartilage labelled in vivo with tritiated thymidine, all thymidine labelled cells were also Ki-67 positive. These results suggest that the Ki-67 antibody recognizes a nuclear antigen in the rabbit that is associated with cell proliferation and is expressed by cells in S-phase as well as in other phases of the cell cycle.