Journal of Cytology
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ORIGINAL ARTICLE  
Year : 2014  |  Volume : 31  |  Issue : 4  |  Page : 183-188
Integrated cervical smear screening using liquid based cytology and bioimpedance analysis


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

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Date of Web Publication10-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; semi-automated analysis

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 Top


The second most common cancer in women worldwide [1] is the cervical cancer. Since 1940, the conventional pap smear (CPS) has been recognized globally as the primary screening technique for cervical cancer [2],[3] 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. [4],[5],[6],[7] 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. [8],[9],[10] LBC technique successfully reduces the number of inadequate smears and provides scope to detect infectious agents [11] 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. [12] 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. [13],[14]

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. [15],[16] 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. [17] It also depicts varying resistance and capacitance [18] 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. [19] Cell-based biosensors are employed in studying cellular electrical property. [20] Impedance cytosensors offer instantaneous and quantitative means to monitor cellular events. [21],[22],[23],[24],[25],[26],[27] 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. [15]

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 Top


Study sample

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.

Inclusion/exclusion criteria

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.

Pap staining

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)


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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:

  • 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.
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.

Statistical analysis

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

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Bioimpedance measurement

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

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   Results Top


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 Top


Early detection is a major step in improving survival rates in any form of malignancy [28] and cytopathological screening plays a significant role. [29],[30] Routinely used CPS suffers from high level of false negativity [31] and so LBC technique gained importance in addressing these fallacies due to better sensitivity. [3] Though LBC renders a clear background with good cellular spread and removal of mucus, RBC's and inflammatory cell to aid proper diagnosis [8],[10],[11] but it is not well accepted due to total absence of microbial flora and inflammatory cells information including cost. [32]

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. [32] 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. [32] 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. [32] 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. [32] 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 [33] 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 [34] which plays an important role in cell proliferation and differentiation. [35] Cancer cells possess distinct bioelectrical properties, and the depolarized voltage across the membrane favors extensive cell proliferation and migration. [34] The change in biophysical attribute may be observed well ahead of any morphological change. [22] The altered ion channels during disease progression may result in a change of biophysical signature. [34] [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 [32] and as evident from literature survey, [15],[16],[17],[18],[19],[20],[21] 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. [27] 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 Top


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.

 
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Correspondence Address:
Lopamudra Das
School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur - 721 302, Kolkata, West Bengal
India
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Source of Support: Department of Information Technology (DIT), Govt. of India, New Delhi, Conflict of Interest: None


DOI: 10.4103/0970-9371.151127

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