Application of Flow Cytometry for Biomarker-Based Cervical Cancer Cells Detection

Jian Ling, Ph.D.,1* Urs Wiederkehr, B.S.,2 Spring Cabiness, B.S.,3 Kenneth R. Shroyer, Ph.D., M.D.,4 and J. Paul Robinson, Ph.D.5

The Pap test used for cervical cancer screening is subjective, labor-intensive, and has relatively low sensitivity and specicity for the detection of underlying clinically signicant lesions. The objective of this study is to develop a biomarker/ow cytometrybased approach for cervical cancer screening. Immunouorescence technology to quantify cervical cell expression of two biomarkers p16INK4A and Mcm5 was developed and evaluated by both microcopy and ow cytometry. The capability of using biomarker/ow cytometry approach to detect rare-event dysplastic cells in a large background of benign epithelial and inammatory cells was evaluated. The results indicate that ow cytometry could detect 0.01% dysplastic cells in a background of normal cervical epithelial cells with the combination of the two biomarkers. Thirty-two clinical specimens were used to test the biomarker/ow cytometry-based approach and the results were compared with the liquid-based cervical cytology. The experiment yielded 100% sensitivity and 93% specicity with reference to the liquid-based cervical cytology. This study indicates the promise of using multi-color uorescence ow cytometry for biomarkerbased cervical cancer screening. This molecular-based, potentially high-throughput and automated method is expected to provide an alternative/auxiliary means of cervical cancer screening. Diagn. Cytopathol. 2008;36:7684. ' 2008 Wiley-Liss, Inc. Key Words: cervical cancer, cancer screening; ow cytometry; cancer markers; rare-event detection

Cervical cancer is the second most common cancer in women, with 500,000 new cases reported each year and 250,000 deaths worldwide. Eighty percent of the deaths occur in developing countries1 due to the lack of widespread screening programs. In developed countries, the death rate from cervical cancer has been reduced signicantly through the adoption of population-wide screening programs. According to the American Cancer Society,2 the cervical cancer death rate in the U.S. declined 48% between 1973 and 1993.

Current Screening Methods

The recognized leading tools used in cervical cancer screening programs are the Pap smear, pioneered by Dr. George Papanicolaou in the 1930s, and liquid-based cervical cytology, introduced in the mid 1990s. In both methods, cell specimens are collected by gently scraping the surface of the cervix with a sampling device, such as a plastic spatula or cytobrush. In the Pap smear, cells from the spatula or cytobrush are smeared directly on a slide and then xed and stained using the Papanicolaou stain. In liquid-based cervical cytology, the sample is rst rinsed into a liquid xation solution to preserve the cells and, thin layer or monolayer preparations are prepared using density gradient centrifugation or lter membrane technology using automated systems. Both Pap smears and liquid-based cytology slides are subsequently stained using the Papanicolaou stain. Cervical cytology slides are initially screened by microscopic examination by either a cytotechnologist or pathologist. Federal regulations require that all potentially abnormal specimens be reviewed and diagnosed by a qualied pathologist. Slides that are screened as normal, however, may be reported without requiring pathologist review. Cytologic abnormalities that may reect underlying cervical dysplasia or squamous cell carcinoma are categorized under the Bethesda 2001 system as atypical squamous cells of undetermined signicance (ASC-US),
'

1 Medical System Department, Automation and Data System Division, Southwest Research Institute, San Antonio, Texas 2 Cytolution, Inc., San Jose, California 3 Applied Physics Division, Southwest Research Institute, San Antonio, Texas 4 Department of Pathology, University of Colorado Health Science Center, Denver, Colorado 5 Cytometry Laboratories, Bindley Bioscience Center, Purdue University, West Lafayette, Indiana Contract grant sponsor: Cytolution Inc.; Contract grant sponsor: NIH (National Cancer Institute); Contract grant number: 1R21CA125370-01. *Correspondence to: Jian Ling, Ph.D., Southwest Research Institute, 6220 Culebra Rd., San Antonio, TX 78238. E-mail: jling@swri.org Received 18 June 2007; Accepted 6 October 2007 DOI 10.1002/dc.20763 Published online in Wiley InterScience (www.interscience.wiley.com).

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Fig. 1. Morphological changes (the increase of nucleus-to-cytoplasm ratio) of precursor lesions of cervical carcinoma.

atypical squamous cells suspicious but not diagnostic for a high grade squamous intraepithelial lesion (ASC-H), low-grade squamous intraepithelial lesion (LSIL), highgrade squamous intraepithelial lesion (HSIL), and squamous cell carcinoma (SCC).3 Figure 1 illustrates the morphological changes that are characteristic of the development of precursor lesions. A general feature of the high-grade dysplastic cells is that they typically have high nuclear-to-cytoplasmic volume ratios and this ratio increases as the severity of the lesion increases (Fig. 1). Current guidelines provided by the American Cancer Society recommend screening for women 21 year of age and older. The preferred screening frequency is annual unless there are three consecutive normal, technically satisfactory Pap tests but is often increased to every 36 mo if the Pap test indicates an abnormality.4 About 50 million Pap tests are performed each year in the United States and about 110 million worldwide.5,6 Human papillomavirus (HPV) is the main cause of cervical dysplasia and carcinoma. Although HPV vaccines are likely to be highly effective in preventing infection by HPV vaccine types, cervical cancer screening programs will still play crucially important roles for the detection of cytologic abnormalities in currently infected patients and in the detection of disease associated with nonvaccine types.

ing at healthy cells.8 Fatigue and monotony can reduce the acuity of the screener and increase the chance that rare positive cells could be overlooked.7 Current methods of cervical cancer screening are not only labor-intensive but are also highly subjective and have relatively low sensitivity and specicity for the detection of some high-grade clinically signicant lesions. With the liquid-based Pap test, the sensitivity of cervical screening has increased to about 80% from the 65% in conventional Pap smear,9 resulting in an improvement of the overall clinical, economic, and patient outcomes. However, the specicity of liquid-based Pap test dropped from 95% with conventional Pap smear to about 75%.9 Recently, the FDA approved the use of high risk HPV testing in combination with the liquid-based cervical cytology for primary screening of women over age 30.

Biomarkers for Cancer Screening

With the signicant advances in genomics and proteomics over the last decade, hundreds of articles have been published on the subject of understanding the molecular pathogenesis of cervical cancer.10 Molecular changes have been recognized to be the earliest indication of cell abnormalities. The objective of the current study was to develop a method that can achieve a true molecular measurement using immunouorescence technology and ow cytometry technologies. Such a molecular-based cervical cancer screening method is expected to have higher sensitivity and specicity compared to the current cervical cytology methods. A large number of biomarkers have been identied that are overexpressed in cervical cancer cells.11 Some of the markers that appear to have potential for cervical cancer screening include p16INK4A (a cyclin-dependent kinase inhibitor protein), Mcm (minichromosome maintenance) proteins, Cdc (cell division cycle) proteins, topoisomerase 2 alpha, PCNA, Ki-67, Cyclin E, p-53, and Rb (retinoblastoma) proteins.10,1215 This report is focused on the analysis of two of these markers, p16INK4A 1628 and Mcm5.29,30 p16INK4A protein. The p16INK4A protein has been used as an immunohistochemical and immunocytochemical marker in several studies to detect cervical cancer. In cervical carcinomas, viral DNA integration into the host genome may result in disruption of the E2 open reading frame, resulting in unregulated overexpression of HPV oncogenes E6 and E7, E7-mediated catabolism of pRb, and the reciprocal overexpression of p16INK4A.26 Almost 100% of high-grade cervical dysplasias and invasive cancers have been shown to express very high levels of p16INK4A, whereas normal cervical squamous cells do not test positive for p16INK4A.31,32 Several studies16,17,2628 have demonstrated the successful combination of p16INK4A immunocytochemical assay with the liquid-based Pap test. In studies performed by Bibbo et al.,16 very high levels of p16INK4A
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Limitations of Current Screening Methods

The major challenge for cervical cytology is the need to detect rare-events. A liquid-based cervical cytology specimen contains a minimum of 5,000 normal squamous cells; most samples contain 50,000 or more normal cervical squamous epithelial cells, as well as benign endocervical cells, and inammatory components. High-grade squamous intraepithelial lesions (HSILs), on the other hand, may often be based on the detection of only a very small number of abnormal cells, frequently in the range of 10 100 dysplastic cells/slide. Federal guidelines permit cytotechnologists to screen up to 100 slides in a normal 8-hr workday.7 Assuming a minimum number of 5,000 cells per slide, cytotechnologist would review at least 500,000 cells/day and be required to detect as few as 1050 dysplastic cells in a positive specimen. Since *90% of all cases in most diagnostic practices are negative for cytologic abnormalities, most of the screeners time and energy is expended look-

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were detected in almost 100% of high-grade cervical dysplasias and invasive cancers, whereas no p16INK4A-positive stain was found in normal cervical epithelia using the same antibodies. A study performed by Murphy et al.21 also reported that p16INK4A identied dysplastic squamous and glandular cells of the cervix with a sensitivity of 99.9% and a specicity of 100%. A practical limitation to the use of p16INK4A as a cytologic diagnostic adjunct, however, is that sporadic expression of this marker is also sometimes present in scattered benign endocervical glandular cells and in tuboendometrial metaplasia of the cervical mucosa, which could lead to false positive classication of test results.31 Mcm5 protein. The MCM proteins form a hexameric helicase that denatures DNA at the initiation of DNA replication.10 Mcm5 has been extensively studied as a marker for cellular proliferation expressed during the normal cell cycle and recent studies indicate that Mcm5 may be a marker for the presence of cervical intraepithelial neoplasia and carcinoma but is also expressed in low grade dysplastic lesions and in some normal proliferating squamous cells.21,29,30 Although Mcm5 may be expressed in a lower proportion of high grade dysplastic cells than is typically observed for p16INK4A the expression of Mcm5 has not been reported in benign endocerrvial glandular cells. Thus, dual (multiplex) staining of both Mcm5 and p16INK4A could theoretically increase overall test performance because these two biomarkers are complementary in nature. Flow cytometry for cervical cancer detection. Flow cytometry is an ideal format for the analysis of single-cell suspensions, quantifying cell structural and molecular features, and for the detection of rare events. The potential for the use of ow cytometry for cervical cancer screening began in the 1970s and was widely reported during the 1980s and early 1990s.3340 Most of these studies focused on methods that use uorescent dyes to stain nucleic acids and use ow cytometry to measure DNA content (aneuploidy) as a prognostic indicator of solid tumors. However, the use of DNA content as an independent prognostic indicator is uncertain and remains controversial. This study took a different approach in the use of ow cytometry by including the evaluation of p16INK4A and Mcm5 as sensitive and specic markers for the detection of cervical dysplasia and carcinoma.

that the PreservCyt1 solution will preserve both cell morphology and cellular molecular markers for at least 30 days. PreservCyt1 solution is also known to permeabilize cells so that uorochromes-labeled antibodies can penetrate cells. Clinical cervical specimens. Residual cervical cytology specimens from PreservCyt1 vials were obtained from the cytopathology laboratories at the University of Texas Health Science Center at San Antonio and the University of Colorado Health Sciences Center at Denver, following IRB application approval of the study protocol. These specimens have been reviewed by experienced cytopathologists and classied by Bethesda 2001 terminology as negative, ASC-US, LSIL, or HSIL. The clinical specimens were ltered with 70-lm nylon mesh lter to remove cell clusters before ow cytometry measurement.

Fluorescence Labeling
Antibodies and conjugation with uorochrome. Mouse monoclonal antibodies to p16INK4A (Clone ZJ11) and Mcm5 (Clone CRCT5.1) from Labvison Inc. (Fremont, CA) were selected in the study. These two antibodies were directly conjugated with PE and APC uorochromes using commercially available labeling kits (ProZyme Inc., San Leandro, CA). The conjugates were denoted as p16INK4A-PE and Mcm5-APC antibodies. Corresponding mouse IgG1 and IgG2b isotypes were also obtained and conjugated to PE and APC, respectively, as the isotype control. Immunouorescence staining. Before staining, a sample was washed twice with phosphate buffered saline (PBS) to remove the xation solution. The second wash used a staining buffer (PBS plus 1% bovine serum albumin (BSA) and 0.01% sodium azide) to block the intracellular nonspecic binding sites. The sample was concentrated to 100 lL and then simultaneously stained with a cocktail of p16INK4A-PE and Mcm5-APC antibodies. In immunouorescence imaging, 1 lg/mL concentration of antibody was used to stain the samples. In ow cytometry, the optimal antibody concentration was about 0.1 0.25 lg/mL. Flow cytometry is more sensitive for detection of the uorescence signal owing to the use of a laser as the excitation source and a photomultiplier tube (PMT) as the detector. The staining tube was kept on ice or in a 48C dark refrigerator for 30 min. Then the stained cells were washed twice with the staining buffer to remove the unbound conjugates. The same procedure and same concentration were followed for isotype staining.

Materials and Methods Sample Preparation

Control samples. The cervical cancer-derived HeLa cell line, which has been shown to overexpress both p16INK4A and Mcm5 proteins, was used as the positive control in this study. HeLa cells were xed and preserved with a methanol-based xative (PreservCyt1 solution, Cytyc Corp., Marlborough, MA). A previous study41 has shown 78
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Quantitative Microscopy
Before performing the ow cytometry experiment, microscopic imaging was performed to (1) verify the effectiveness of the uorescence stain, and (2) verify whether the

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overexpression of biomarkers is correlated to the abnormal morphology of dysplastic cervical cells. A Nikon Eclipse TE2000E inverted microscope and computer system was used for the uorescence imaging. Three uorescence lters in the FITC, PE, and APC bands (i.e., the 530-nm, 575-nm, and 660-nm emission bands) were used. During imaging, the microscope was rst set in differential-interference-contrast (DIC) video mode and visually focused on an imaging area which contained multiple nonoverlapping cells. Then, four images (three uorescence images in the FITC, PE, APC bands, and a DIC image) were obtained for each imaging area. The DIC image illustrates the morphology of the cells. The PE and APC images show the expression of p16INK4A and Mcm5 markers in the cells. The FITC image measures cell autouorescence in the FITC band, which is used to correct the autouorescence in the PE and APC bands on a cellby-cell basis (see Data Analysis below).

Fig. 2. PE vs. APC uorescence intensities of the HeLa cells stained with cocktail of isotypes (dark triangular symbol) and stained with cocktail of p16INK4A-PE and Mcm5-APC antibodies (light quadrangular symbol).

Flow Cytometry
A FACS Aria ow cytometer (Becton-Dickinson, San Jose, CA) and an FC 500 ow cytometer (BeckmanCoulter, Miami, FL) were used in the ow experiments. Five parameters were measured: forward-scatter (FS), side scatter (SS), and FITC, PE, and APC uorescence bands. The FS and SS measurements were used to gate out cell debris. The cell autouorescence measured in the FITC band was used to correct the autouorescence in the PE and APC bands on a cell-by-cell basis using a postcompensation method (see Discussion). The remaining uorescence measured in the PE and APC bands reects the expression levels of biomarkers p16INK4A and Mcm5, respectively, in each cell. Before each ow experiment, the ow cytometer was calibrated using uorescence beads to minimize the day-to-day variation of optics. The calibration procedure ensured the measurement of different samples under similar conditions. instead of pulse integral was used to represent the uorescence density or biomarker expression of each cell because pulse peak is not signicantly affected by cell size, as is the pulse integral (see Discussion).

Results Microscopy Imaging Experiment

Comparison of antibody stain and isotype stain. Fixed HeLa cells were stained with a cocktail of p16INK4A-PE and Mcm5-APC antibodies. Matched aliquots of xed HeLa cells were stained with a cocktail of PE and APC labeled isotypes. The dot plot in Figure 2 shows that the antibody-stained cells (denoted by light quadrangular symbol) have signicantly higher stain intensities in both the PE and APC bands than that of the isotype-stained cells. Comparison of normal and dysplastic cervical cells. Eleven cervical cytology specimens, including ve negative and six positive specimens (1 ASC-US, 2 LSIL, and 3 HSIL), were used in a pilot imaging study. Each specimen was divided into two parts. One part was unstained and used to establish autouorescence compensation coefcients, and the other part was stained with the cocktail of p16INK4A-PE and Mcm5-APC antibodies. About 70 cells, including cells with differing morphology, were imaged for each specimen. The average uorescence intensities were computed for each cell in imaging areas. Figure C-1 gives an example of the DIC and uorescence images of a normal and an abnormal HSIL cell from one of the HSIL cervical specimens. The autouorescence intensity of the cells in the PE and APC band has been eliminated based on their autouorescence intensity in the FITC band. Figure C-1 indicates that the average PE and
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Data Analysis
For imaging data, a MATLAB program was developed to quantitatively compare cell-to-cell average stain intensities in uorescent images. The software automatically segments the uorescent images to locate individual cells. The average uorescence intensities or the uorescence density in the FITC, PE, and APC bands were determined for each individual cell. The uorescence density, calculated by normalizing the total staining intensity by cell area, provided a fair comparison of the biomarker expression among different types of cervical cells, which usually have large variation in size (from 25 to 65 lm in diameter). For ow cytometry data, FCS Express (De Novo Software, Thornhill, Canada) was used to perform gating and autouorescence correction. Fluorescence pulse peak

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Fig. C-1C-3. Fig. C-1. DIC (upper left), FITC (upper right), PE (lower left) and APC (lower right) images of a normal cell (a), and a HSIL dysplastic cell (b), from a HSIL cervical specimen. The numbers under the cells are the average uorescence intensities of the cells. Fig. C-2. The dot plot (left) illustrates HeLa cells (red dots) identied and separated from normal cervical cells (blue dots) after staining with p16INK4A-PE and Mcm5-APC antibodies. The scatter plot (right) indicates the linear relationship between the number of spiked and the number of p16INK4A and Mcm5 positive HeLa cells. Fig. C-3. Dot plots of PE (P16INK4A) vs. APC (Mcm5) immunouorescence intensities of the cells in a negative (left) and a positive HSIL cervical specimen (right). Each plot contains about 75,000 cervical cells.

APC intensities of the HSIL cell are signicantly higher than those of the normal cells. The experiment also shows that most cells in the eleven specimens had low stain intensities in both the PE and 80
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APC bands. Only a small number of cells in the ASC-US, LSIL, and HSIL specimens had high stain intensities. This suggests that the biomarker overexpressed cells are rare-events, which is similar to the morphology-based

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detection. The experiment also suggested that the overexpression of both the p16INK4A and Mcm5 biomarkers is closely related to the abnormality of cell morphology.

Table I. Comparison of the Classication of 32 Specimens Between Flow Cytometry and Pap Test Positive in Pap test Positive in ow cytometry Negative in ow cytometry TP 17 FN 0 Negative in Pap test FP 1 TN 14

Flow Cytometry Experiment

Spiking experiment. This experiment was conducted to show the feasibility of biomarker-based ow cytometry in the detection of rare cervical cancer cells among a large number of normal cells in a cervical specimen. Aliquots of normal cells were taken from six Pap test negative specimens and combined to create a normal cervical cell pool. The normal cells were counted and divided among seven tubes, with each tube containing about 77,000 benign squamous cells. One tube was left unstained for reference. The other six tubes were spiked with 10, 20, 50, 100, 500, and 1,000 HeLa cells using the serial dilution method. The spiked samples were stained with the cocktail of p16INK4A-PE and Mcm5-APC antibodies. The dot plot in the left panel of Figure C-2 illustrates an example of the PE versus APC intensities in the 500HeLa-spiked sample. The HeLa cells highly stained with p16INK4A and Mcm5 (red dots) are identied and separated from the normal cervical cells (blue dots). The scatter plot in the right panel of Figure C-2 illustrates the linear relationship between the number of spiked and the number of detected HeLa cells. The discrepancy between the detected cells and the number of spiked cells could be due to the following reasons: (1) the number of HeLa cells that were actually added into each sample varied; (2) cells were lost during the poststaining washing steps; (3) a small portion of the samples could not be measured due to the dead space between pipette and test tube; and (4) some HeLa cells did not have a high staining of p16INK4A and Mcm5. This study indicates that it is possible to use ow cytometry to detect as low as 0.01% cancer cells among a large number of normal cervical cells. The outcome exceeded the expectation of detecting less than 0.1% abnormal cells among normal cells, which is considered by pathologists as being an acceptable limit for a cervical cancer screening method. Clinical specimens experiment. Thirty-two residual cervical specimens from routine cervical cancer screening were involved in this study. They were categorized as 15 negative and 17 positive (2 ASC-US, 1 LSIL, and 14 HSIL) cases. Each specimen was split into two aliquots. One aliquot was unstained and used to measure the cell autouorescence in the three uorescence bands: FITC, PE, and APC. The other aliquot, containing around 75,000 cells, was stained with a cocktail of p16INK4A-PE and Mcm5-APC antibodies and then run on the ow cytometer. Figure C-3 shows two dot plots of a negative and a positive (HSIL) specimen generated from ow measurement. These two plots clearly show that the HSIL speci-

men has signicantly more cells with high intensities in both PE and APC bands than the negative specimen. The high intensity in the PE and APC bands indicates that both biomarkers p16INK4A and Mcm5 are overexpressed. The detection threshold was set arbitrarily in this experiment to maximize the separation between negative and positive (ASC-US) specimens. In Table I, the classication of the thirty-two specimens determined by multiparameter ow cytometry is compared with the classication by liquid-based Pap test. Using the Pap test as the reference, the sensitivity and specicity of the ow cytometry method to classify cervical specimens into negative and positive (ASC-US) was 100 and 93%, respectively.

Discussion Autouorescence Compensation

One of the major challenges in the use of ow cytometry to measure biomarker expression of cervical cancer cells is the autouorescence problem. Fixed cervical cells have very strong autouorescence. Figure 3a shows a ow cytometry plot of FITC versus PE of an unstained sample that was mixed with HeLa and normal cervical cells. The autouorescence is present not only in the green band but also in the yellow and even in red frequency bands. In addition, the cell-to-cell autouorescence intensities can vary over a 1,000-fold range in a specimen. Without correction for such a large variance of autouorescence, it is impossible to detect biomarker signals in PE and APC bands. This is illustrated in Figures 3b and c, the dot plot of the same sample but stained with p16INK4A-PE and Mcm5-APC antibodies. These two gures indicate that the PE and APC staining intensities of HeLa cells are much lower than some of the normal cells that have high autouorescence in the same bands. Although cell-to-cell autouorescence has large variation, the dot plots suggest that the intensities between the autouorescence in the yellow or red band and the autouorescence in the green band are linearly correlated among different cells. Using this feature, the cell autouorescence measured in the FITC band (from the samples not stained with FITC dyes) can be used to correct the autouorescence in the PE and APC bands on a cell-by-cell basis using a postcompensation method. The ratios between the autouorescence in PE or APC bands versus that in FITC band can be determined beforehand from the unstained specimen. Figure 3d illustrates the data in Figure 3b and c after
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Fig. 3. (a) Dot plots of FITC vs. PE for an unstained cervical sample that was mixed with HeLa and normal cervical cells. (b, c) FITC vs. PE and FITC vs. APC of the same sample in (a) but stained with p16INK4A-PE and Mcm5-APC antibodies. HeLa cells were separated from normal cells because of the additional stain intensity onto the autouorescence intensity. (d) PE vs. APC of the stained sample after the autouorescence in PE and APC bands were compensated. HeLa cells, with high stain intensity in both PE and APC bands, were clearly separated from normal cells.

autouorescence compensation. HeLa cells are clearly separated from normal cervical cells based on PE and APC staining intensities.

The Measurement of Cervical Cell Fluorescence Intensity in Flow Cytometry

Another major challenge in the study, which is usually not involved in the hematological application of ow cytometry, is how to correctly measure the biomarker expression levels in ow cytometry when cells of large different sizes are mixed together. Cervical epithelial cells in a sample can vary from 25 to 65 lm in size. The uorescence pulse integral (or area), often used in ow cytometry to measure uorescence intensity, is not appropriate in this case to compare the biomarker expression (indicated by uorescence dyes per unit volume or uorescence density) among cells of different sizes. For 82
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example, the pulse integral of a small-size cell with high biomarker expression may be smaller than a large-size cell but with low biomarker expression. In this study, pulse peak was used to estimate the uorescence density instead of pulse integral. As the size (2565 lm) of cervical cells is larger than the height (9 lm) of the excitation laser beam in the ow cytometer, the pulse integral is more signicantly affected by cell size than the pulse peak. The ow cytometry applications to large-size epithelial cells are different from the applications to blood cells, which are usually smaller than or comparable to the excitation laser beam. A better way of estimating uorescence density is to normalize pulse integral by pulse width (or time-of-ight).42 However, pulse width measurement from most commercially available ow cytometers has a large variance. A slit-scanning system with small focal spot size43 might provide a reliable measurement of pulse width. A system with Coulter volume mea-

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surement may also be used to obtain a better estimation of uorescence density. How to modify ow cytometer for epithelial cells analysis is still a topic needs to be investigated. As pointed out by Dong et al., it is a crucial step to obtain an optimal ow cytometry setting suitable for analysis of epithelial cells.44

Summary and Future Research

This study demonstrated the feasibility of (1) using multiplex detection of p16INK4A and Mcm5 to detect dysplastic cervical cells by immunouorescence, (2) using multiparameter ow cytometry to detect rare-event dysplastic cells from large background of normal cells, and (3) using multiparameter ow cytometry to identify positive cervical specimens. Although the results were based on a limited number of clinical specimens, this experiment demonstrated the promise of using multiparameter ow cytometry for biomarker-based cervical cancer screening. This molecular-based, potentially high-throughput and automated method is expected to provide an alternative/auxiliary means of cervical cancer screening. The method developed for cervical cancer screening in this study can be extended to the diagnosis of other nonhematological cancer. Future studies will make this technology more robust. First, the threshold or gating to detect dysplastic cells was arbitrarily set in this preliminary study. A cell sorting and validation experiment is needed to optimize the threshold setting and to reduce the false negatives and false positives in the detection of abnormal cells. Second, the uorescence contrast between the biomarker positive and negative cells needs to be enhanced, especially for the p16INK4A biomarker. Third, cervical cells tend to cluster together. To provide enough cells in single suspension for ow cytometry measurement, sample preparation must include a procedure of cluster desegregation. The potential solutions to these problems will be investigated in future studies.

Acknowledgment
We thank Elizabeth Branch for her assistance in the preparation of this manuscript.

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