|Year : 2014 | Volume
| Issue : 4 | Page : 183-188
|Integrated cervical smear screening using liquid based cytology and bioimpedance analysis
Lopamudra Das1, Tandra Sarkar2, Ashok K Maiti3, Sukla Naskar4, Soumen Das1, Jyotirmoy Chatterjee1
1 School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur, India
2 Department of Radiology, Apollo Gleanengles Hospital, Midnapore, India
3 Department of Pathology, Midnapore Medical College and Hospital, Midnapore, India
4 Department of Pathology, Calcutta National Medical College, Kolkata, West Bengal, India
Click here for correspondence address and email
|Date of Web Publication||10-Feb-2015|
| Abstract|| |
Objective: To minimize the false negativity in cervical cancer screening with Papanicolaou (Pap) test, there is a need to explore novel cytological technique and identification of unique and important cellular features from the perspectives of morphological as well as biophysical properties.
Materials and Methods: The present study explores the feasibility of low-cost cervical monolayer techniques in extracting cyto-pathological features to classify normal and abnormal conditions. The cervical cells were also analyzed in respect to their electrical bioimpedance.
Result: The results show that newly developed monolayer technique for cervical smears is cost effective, capable of cyto-pathological evaluation. Electrical bioimpedance study evidenced distinction between abnormal and normal cell population at more than two order of magnitude difference.
Conclusion: The integration of bioimpedance observation along with the proposed low-cost monolayer technology could increase the efficiency of the cervical screening to a greater extent thereby reducing the rates of faulty diagnosis.
Keywords: Cervical cytology screening; electrical bioimpedance; liquid based technology; morphometric features;
|How to cite this article:|
Das L, Sarkar T, Maiti AK, Naskar S, Das S, Chatterjee J. Integrated cervical smear screening using liquid based cytology and bioimpedance analysis. J Cytol 2014;31:183-8
|How to cite this URL:|
Das L, Sarkar T, Maiti AK, Naskar S, Das S, Chatterjee J. Integrated cervical smear screening using liquid based cytology and bioimpedance analysis. J Cytol [serial online] 2014 [cited 2021 Sep 19];31:183-8. Available from: https://www.jcytol.org/text.asp?2014/31/4/183/151127
| Introduction|| |
The second most common cancer in women worldwide  is the cervical cancer. Since 1940, the conventional pap smear (CPS) has been recognized globally as the primary screening technique for cervical cancer , as it is safe, efficient, well-established and noninvasive. Many advanced technologies involving sampling and smear preparation, or screening quality control have been developed to reduce the false negative cases. ,,, Liquid based cytology (LBC) is a technique employed to improve the smear preparation forming monolayer slides devoid of the blood, mucus and other debris thereby increasing the sensitivity by reducing the rate of false negative compared to CPS. ,, LBC technique successfully reduces the number of inadequate smears and provides scope to detect infectious agents  like human papillomavirus, trichomonas vaginalis etc., if present. However, the LBC prepared by commercially available kit is costly and inaccessible to the common people in India for cervical cancer screening.  In order to make the LBC technique more informative and easily accessible to the common people, an effort has been made to bring down the cost of cervical monolayer by developing an easy and low cost technique which eliminates the mucus, red blood cells (RBC) and reduces the load of inflammatory cells and bacterial flora rather than completely eliminating them. Besides morphological features, biophysical attributes are also gaining importance in cytopathological studies. ,
The electrical properties of biological cells are an area of active interest and showed its potential in extracting information about the morphology and physiology of the cells. , This technology is exploited for label-free detection of diseases by identifying and measuring nonbiological parameters that may carry the disease signature. A cell being an electrically neutral entity opposes current flow when subjected to an electric field and provides its bioimpedance characteristics. In fact, the insulating properties of living cells vary under applied electrical frequency.  It also depicts varying resistance and capacitance  to maintain the required potential difference. Due to dielectric behavior, cells' impedance of the system varies under different cellular activities in static and dynamic conditions.  Cell-based biosensors are employed in studying cellular electrical property.  Impedance cytosensors offer instantaneous and quantitative means to monitor cellular events. ,,,,,, This upcoming technique of bioelectrical property study in real time may be valuable in classifying cells as normal and abnormal one. Thus, in reducing ambiguities in exfoliative cytology based diagnostics, besides morphological parameters, cellular electrical attributes may also be studied. 
Hence, the present study aims at multimodal characterization of cervical exfoliative cytology through development of cost effective LBC for cervical smear and also performs electrical characterization of those cells in suspension.
| Materials and Methods|| |
Total of 150 samples were collected for the study that satisfied the inclusion exclusion criteria over the periods of 2 years from a multispecialty hospital. Out of 150, number of abnormal cases was 26, of which 10 were low-grade squamous intraepithelial lesion and 16 high-grade squamous intraepithelial lesion.
All the cases corresponded to patients who had nonstop bloody or foul-smelling vaginal discharge, abnormal vaginal bleeding after menopause, heavier and longer lasting periods, spotting between periods and bleeding after intercourse. The samples were collected from women with a mean age of the volunteers being 54.6 years.
Sample collection and slide preparation method
The samples were collected from patients covering different zones of the cervix by means of Ayer's spatula and endocervical brush. The cells were then transferred into vials containing polysol (an isotonic aqueous solution of chloride salts of sodium, potassium, calcium and magnesium along with sodium acetate acidified with few crystals of phenol) to retain the intact morphology of the cells for future slide preparation and bioimpedance analysis.
The cervical cells in the polysol solution were vortexed for 30 seconds and then centrifuged at 2000 rpm for 5 min. The supernatant was discarded keeping the cell pellet intact which was then re-suspended in 1 mL of the fresh polysol solution by vortexing. The monolayer smears was prepared by cyto-centrifuge using funnel and filter cards on the slides and dipped in 95% ethanol for 15 min (wet fixation) immediately after monolayer smear preparation.
Staining of the prepared slides were performed manually following the routine protocol and using commercially prepared Harris hematoxylin, OG 6 and EA 36 (Hi Media, India). The slides were cleaned with xylene and mounted by DPX after staining.
Microscopic image grabbing
The images as shown in [Figure 1] were grabbed digitally by Axio-Cam MRc at 1388 × 1040 pixels by a Zeiss Observer.Z1 microscope under 10X (NA 0.21), 20X (NA 0.8) and 40X (NA 0.55) objectives with their respective resolutions being 0.4 µm, 0.31 µm and 0.16 µm.
|Figure 1: Cervical smears prepared by cost effective monolayer technique|
(Pap, (a): ×100, (b): ×200 and (c): ×400)
Click here to view
Semi-automated analysis of morphometric features
Morphological features of the cervical cells were extracted under the guidance of experts, cytopathologists using the software Axiovision (version 4.7.2, Carl Zeiss). Measurements of the morphometric features such as area of the whole cell, area of the nucleus and the cytoplasm were performed for this study. Nucleus-cytoplasm (N/C) ratio was computed using Microsoft Excel. Pap being a differential staining technique imparts red to pink color to the keratinized mature cells and blue to green color to the immature or intermediate cells that are metabolically active. Based on the differential staining principle, four study groups were designed considering the color of stain uptake and nucleus size:
These might be useful in detecting a change in the size of NC with the disease progression. Due to the progression of the disease from normal to precancer to cancer, the nucleus size increases and the cytoplasm decreases accounting for high N/C ratio. The morphometric data obtained were analyzed statistically to find the level of significance.
- Normal mature (NMA) cells which are red to pink in color with pycnotic nucleus.
- Normal immature (NIM) cells, blue to green in color with a large nucleus.
- Abnormal mature (ABMA) cells are red to pink in color with a larger nucleus.
- Abnormal, immature (ABIM) cells blue to green in color with a larger nucleus.
Statistical analysis was performed on measured morphometric data of 100 mature and immature cells respectively in both normal and abnormal cases. An independent sample t-test was applied for comparing the N/C ratio in between four designed study groups using the software SPSS version 7.5. The study groups were further analyzed, using analysis of variance (ANOVA) to obtain their level of significance as represented in [Table 1].
|Table 1: N/C ratio of mature and immature cells in normal and abnormal conditions|
Click here to view
The cervical smear collected from patients was transferred into polysol. The cervical cells in the polysol solution were subsequently vortexed for 30 seconds and the cell suspension was centrifuged at 2000 rpm for 5 min. The supernatant was discarded, and the cell pellet was resuspended in 500 µL of the fresh polysol solution by vortexing. The bioimpedance of the cell suspension was measured using commercially available miniature planar electrode (model no. 8W1E), impedance-based device from Applied Bio-physics, USA and LCR meter (HOIKI, Japan). 500 µµL volume of polysol solution with 10 5 number/mL of suspended cells was poured in the device for bioimpedance measurement.
Frequency sweep of 100 Hz to 1 MHz was given to the sample using the alternating current voltage signal of 10 mV peak to peak, and the corresponding impedance were measured using LCR meter. The bioimpedance of the cells was measured for both normal and abnormal cases. The frequency response characteristic of bioimpedance was plotted by taking the average of impedance values for all the normal and abnormal cases separately with their standard deviation as shown in [Figure 2].
|Figure 2: Variation on of bioimpedance of normal and abnormal cervical cells over the electrical frequency range of 100 Hz to 1 MHz|
Click here to view
| Results|| |
Cervical Monolayer prepared by cost effective technique
The cost effective monolayer slide preparation technique provided a clear background and also retained important cytopathological information (inflammatory cells and microbial flora) aiding in proper diagnosis as shown in [Figure 1]. The images were grabbed at different magnification to show the cells spread and number in the field of view.
Semi-quantitative analysis of cellular morphometric features in newly developed liquid based cytology data
[Table 1] representes the result of an independent sample t-test and ANOVA applied for comparing the N/C ratios in between four designed study groups. The t-test applied for ABIM versus NIM and ABMA versus NMA yielded highly significant result with P < 0.0001*. The high F value obtained also depicted that the obtained N/C ratios of the study groups are highly significant.
Electrical Bioimpedance Analysis
[Figure 2] represents the graph of electrical impedance (Z) versus frequency (f) over the range of 100 Hz to 1MHz for both the normal and abnormal cervical smear. The bio-impedance of the normal samples was much higher compared to abnormal samples throughout the frequency range. In the case of normal samples, the graph showed a steady decrease of bioimpedance with the increase of frequency. However, the impedance value for the abnormal cervical samples decreased with increasing frequency up to 10 4 Hz and thereafter reaches saturation at higher frequency. The results of the bioelectrical properties clearly showed that the electrical signatures of the normal cells were distinctly different from that of the abnormal one.
| Discussion|| |
Early detection is a major step in improving survival rates in any form of malignancy  and cytopathological screening plays a significant role. , Routinely used CPS suffers from high level of false negativity  and so LBC technique gained importance in addressing these fallacies due to better sensitivity.  Though LBC renders a clear background with good cellular spread and removal of mucus, RBC's and inflammatory cell to aid proper diagnosis ,, but it is not well accepted due to total absence of microbial flora and inflammatory cells information including cost. 
The newly developed cost-effective monolayer (NDCM) is cost effective as it did not employ any costly gradient solution and hence low cost compared to the existing LBCs.  This technique was easy, and the necessary manpower may be developed with short training. The NDCM successfully removes the mucus and RBC's present in the sample, and the cells are also well spread to form monolayer smear having very minimum cellular overlapping as depicted in [Figure 1]a-c. Moreover, it preserves microfloral information in respect to pathological status of the cervical mucosa.  Hence, the overall information from these slides were effective for precise diagnostic decision making with improved sensitivity.
The LBC techniques successfully reduce the rate of faulty diagnosis compared to CPS.  But the ambiguities are still prevailing in such cytological screening process due to insufficiency in the accepted cytomorphological features to be considered for diagnosis in eliminating false negativity.  So, semi-automated features have been analyzed and hence as found in the study, the N/C ratios of the NIM and ABIM cells are almost of same size and account for high subjectivity leading to faulty diagnosis. So there is a scope to identify newer cytological features to address the prevailing ambiguities. In this regard, it may be argued that the computer aided disease diagnosis though somewhat successful in eliminating the subjectivity by implementation of statistical tool  yet it could not override the knowledge base developed through ages by the cytopathologists. So to address the prevailing cytomorphological ambiguities, an independent sample t-test analysis was done on the four designed study groups as given in [Table 1] for some selected cytological features. As shown in [Table 1], the highly significant (<0.0001*) result was obtained between the groups ABIM versus NIM and ABMA versus NMA. Thus, it may be useful for value addition to the routinely practiced cytopathology screening.
In minimizing false negativity of screening, an effort has been made for the first time in this study by exploring the feasibility of bioelectrical property study of cervical smear cells. Integration of biophysical attribute such as bio-impedance study along with this cytological examination may result in a better outcome thereby reducing the faulty diagnosis. The voltage across the plasma membrane is due to the presence of different ion channels with specific selectivity and permeability  which plays an important role in cell proliferation and differentiation.  Cancer cells possess distinct bioelectrical properties, and the depolarized voltage across the membrane favors extensive cell proliferation and migration.  The change in biophysical attribute may be observed well ahead of any morphological change.  The altered ion channels during disease progression may result in a change of biophysical signature.  [Figure 2] shows the bioimpedance characteristic of normal and abnormal cervical smear against frequency. From the initial study, it is evident that the impedance of the normal cells is more than two order higher compared to the abnormal one in the entire frequency range. The N/C ratio of the abnormal cells is higher than the normal  and as evident from literature survey, ,,,,,, the impedance magnitude decreases with increase of frequency as the dielectric property of cell membrane impedes the electrical current flow through the membrane at low frequency and at higher frequency, it allows the current to pass through the cells due to lower reactance of cell membrane. Considering the above factors, it is expected that the current will flow mostly through the nucleus area in the abnormal samples depicting much lower impedance of the cells attributed for noncompactness of DNA within the nucleus. Moreover, the ion channels also get altered during cancer development and progression leading to the alteration of membrane potential.  In contrary, the ion channels available at cell membrane in normal samples are generally facilitated to a specific process for ion transportation and thus offer higher cytoplasmic resistivity. Since the electrical conduction path is more in cytoplasm as compared to its nucleus due to its size limitation, the resistance offered by cytoplasm will be higher as illustrated in [Figure 2]. The above experimental findings primarily indicate that the cellular bioelectrical properties alter along with the physiological changes during disease progression. However, detailed and quantitative investigation is required to establish an integrated approach toward clinical screening process.
Thus, this study has developed an integrated approach for cervical smear screening technique for early detection of carcinoma cervix through (NDCM) with improved compatibility for automation and analysis of biophysical attribute for improved classification of normal and abnormal smear.
| Acknowledgments|| |
Financial support from the Department of Information Technology (DIT), Govt. of India, for this study, is greatly acknowledged. We also extend our heartiest thanks to Debnath Das and Krishnabrata Panda of School of Medical Science and Technology, Indian Institute of Technology, Kharagpur for providing technical support in sample collection and staining. We sincerely acknowledge the support, guidance and expert comments provided by Dr. (Mrs) R. Pagey, Dr. (Mrs). S. Kothari and Dr. M. Deshmukh of RST Regional Cancer Hospital and Research Institute, Nagpur during the course of this study.
| References|| |
WHO/ICO Information Centre on Human Papilloma Virus (HPV) and Cervical Cancer. Human Papillomavirus and Related Cancers in India. Summary Report; 2009.
Walter SD, Irwig L, Glasziou PP. Meta-analysis of diagnostic tests with imperfect reference standards. J Clin Epidemiol 1999;52:943-51.
Fahey MT, Irwig L, Macaskill P. Meta-analysis of Pap test accuracy. Am J Epidemiol 1995;141:680-9.
Hartmann KE, Nanda K, Hall S, Myers E. Technologic advances for evaluation of cervical cytology: Is newer better? Obstet Gynecol Surv 2001;56:765-74.
Ku NN. Automated Papanicolaou smear analysis as a screening tool for female lower genital tract malignancies. Curr Opin Obstet Gynecol 1999;11:41-3.
Linder J. Automation of the Papanicolaou smear: A technology assessment perspective. Arch Pathol Lab Med 1997;121:282-6.
Stoler MH. Advances in cervical screening technology. Mod Pathol 2000;13:275-84.
Bernstein SJ, Sanchez-Ramos L, Ndubisi B. Liquid-based cervical cytologic smear study and conventional Papanicolaou smears: A metaanalysis of prospective studies comparing cytologic diagnosis and sample adequacy. Am J Obstet Gynecol 2001;185:308-17.
Nanda K, McCrory DC, Myers ER, Bastian LA, Hasselblad V, Hickey JD, et al.
Accuracy of the Papanicolaou test in screening for and follow-up of cervical cytologic abnormalities: A systematic review. Ann Intern Med 2000;132:810-9.
Park J, Jung EH, Kim C, Choi YH. Direct-to-vial comparison of a new liquid-based cytology system, liqui-PREP versus the conventional pap smear. Diagn Cytopathol 2007;35:488-92.
Davey E, Baratt A, Irwig L, Chan SF, Macaskill P, Mannes P. Effect of study design and quality on unsatisfactory rates, cytology classifications, and accuracy in liquid based verses conventional cervical cytology: A systemic review. Lancet 2006;367:122-32.
Arbyn M, Anttila A, Jordan J, Ronco G, Schenck U, Segnan N, et al.
European guidelines for quality assurance in cervical cancer screening. Second edition - summary document. Ann Oncol 2010;21:448-58.
Ching CT, Sun TP, Huang SH, Hsiao CS, Chang CH, Huang SY, et al.
A preliminary study of the use of bioimpedance in the screening of squamous tongue cancer. Int J Nanomedicine 2010;5:213-20.
Guofeng Qiao, Wei Wang, Wei Duan, Fan Zheng, Sinclair AJ, Chatwin CR. Bioimpedance analysis for the characterization of breast cancer cells in suspension. IEEE Trans Biomed Eng 2012;59:2321-9.
Abdul S, Brown BH, Milnes P, Tidy JA. The use of electrical impedance spectroscopy in the detection of cervical intraepithelial neoplasia. Int J Gynecol Cancer 2006;16:1823-32.
Davey CL, Markx GH, Kell DB. On the dielectric method of monitoring cellular viability. Pure Appl Chem 1993;65:1921-6.
Abdul S, Brown BH, Milnes P, Tidy JA. A clinical study of the use of impedance spectroscopy in the detection of cervical intraepithelial neoplasia (CIN). Gynecol Oncol 2005;99:S64-6.
Brown BH, Tidy JA, Boston K, Blackett AD, Smallwood RH, Sharp F. Relation between tissue structure and imposed electrical current flow in cervical neoplasia. Lancet 2000;355:892-5.
Ohmine Y, Morimoto T, Kinouchi Y, Iritani T, Takeuchi M, Monden Y. Noninvasive measurement of the electrical bioimpedance of breast tumors. Anticancer Res 2000;20:1941-6.
Blad B, Baldetorp B. Impedance spectra of tumour tissue in comparison with normal tissue; a possible clinical application for electrical impedance tomography. Physiol Meas 1996;17 Suppl 4A:A105-15.
Arndt S, Seebach J, Psathaki K, Galla HJ, Wegener J. Bioelectrical impedance assay to monitor changes in cell shape during apoptosis. Biosens Bioelectron 2004;19:583-94.
Kovacs GT. Electronic Sensors with Living Cellular Components. Proc IEEE 2003;91:915-29.
Asphahani F, Zhang M. Cellular impedance biosensors for drug screening and toxin detection. Analyst 2007;132:835-41.
Ceriotti L, Ponti J, Broggi F, Kob A, Drechsler S, Thedinga E, et al
. Real-time assessment of cytotoxicity by impedance measurement on a 96-well plate. Sens Actuators B 2007;123:769-78.
Lee RM, Choi H, Shin JS, Kim K, Yoo KH. Distinguishing between apoptosis and necrosis using a capacitance sensor. Biosens Bioelectron 2009;24:2586-91.
Morgan H, Sun T, Holmes D, Gawad S, Green NG. Single cell dielectric spectroscopy. J Phys D App Phys 2007;40:61-70.
Yin HY, Wang FL, Wang AL, Cheng J, Zhou YX. Bioelectrical impedance assay to monitor changes in aspirin-treated human colon cancer HT-29 cell shape during apoptosis. Anal Lett 2007;40: 85-94.
Hakama M, Chamberlain J, Day NE, Miller AB, Prorok PC. Evaluation of screening programmes for gynaecological cancer. Br J Cancer 1985;52:669-73.
Nieminen P, Kallio M, Hakama M. The effect of mass screening on incidence and mortality of squamous and adenocarcinoma of cervix uteri. Obstet Gynecol 1995;85:1017-21.
Singer A. Cervical cancer screening: State of the art. Baillieres Clinical Obstet Gynaecol 1995;9:39-64.
Peto J, Gilham C, Fletcher O, Matthews FE. The cervical cancer epidemic that screening has prevented in the UK. Lancet 2004;364:249-56.
Lynge E, Poll P. Risk of cervical cancer following negative smears in Maribo County, Denmark, 1966-1982. Lyon: IARC Scientific Publications; 1986. p. 69-86.
Fitzgibbon AW, Pilu M, Fisher RB. Direct least-squares fitting of ellipses, IEEE Trans Pattern Anal Mach Intell 1999;21:476-80.
Prevarskaya N, Skryma R, Shuba Y. Ion channels and the hallmarks of cancer. Trends Mol Med 2010;16:107-21.
Yangand M, Brackenbury WJ. Membrane potential and cancer progression. Front Physiol 2013;4:1-10.
School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur - 721 302, Kolkata, West Bengal
Source of Support: Department of Information Technology (DIT), Govt.
of India, New Delhi, Conflict of Interest: None
[Figure 1], [Figure 2]
|This article has been cited by|
||Novel benchmark database of digitized and calibrated cervical cells for artificial intelligence based screening of cervical cancer
| ||Abid Sarwar,Jyotsna Suri,Mehbob Ali,Vinod Sharma |
| ||Journal of Ambient Intelligence and Humanized Computing. 2016; 7(4): 593 |
|[Pubmed] | [DOI]|
| Article Access Statistics|
| Viewed||2604 |
| Printed||54 |
| Emailed||0 |
| PDF Downloaded||273 |
| Comments ||[Add] |
| Cited by others ||1 |