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 » Introduction
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ORIGINAL ARTICLE
Year : 2016  |  Volume : 53  |  Issue : 1  |  Page : 193-198
 

A new approach to exfoliative cytology: A comparative cytomorphometric study


1 Department of Oral Pathology and Microbiology, A.B. Shetty Memorial Institute of Dental Sciences, Nitte University, Deralkatte, Mangalore; Department of Oral Pathology and Microbiology, K.V.G. Dental College and Hospital, Kurunjibag, Sullia, Karnataka, India
2 Department of Oral Pathology and Microbiology, A.B. Shetty Memorial Institute of Dental Sciences, Nitte University, Deralkatte, Mangalore, India
3 Department of Periodontics, K.V.G. Dental College and Hospital, Kurunjibag, Sullia, Karnataka, India

Date of Web Publication28-Apr-2016

Correspondence Address:
M Shaila
Department of Oral Pathology and Microbiology, A.B. Shetty Memorial Institute of Dental Sciences, Nitte University, Deralkatte, Mangalore; Department of Oral Pathology and Microbiology, K.V.G. Dental College and Hospital, Kurunjibag, Sullia, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-509X.180830

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 » Abstract 

Context: Early detection of oral premalignancy and malignancy using simple screening aids play a promising role in curbing the disease. Aim: The primary aim of this study is to evaluate and the secondary aim of this study is to compare the cytomorphometry and cellular atypia in keratinocytes obtained from oral rinse and conventional exfoliative cytology in normal oral mucosa and clinically diagnosed oral leukoplakia. Materials and Methods: The study comprised of 55 clinically diagnosed cases of leukoplakia and 55 age and sex matched normal controls. Smears were prepared using oral rinse technique followed by the conventional exfoliative cytology. Papanicoloau stained smears were evaluated for atypia and subjected to image analysis. Based on the presence of atypia they were further divided into three groups (Group 1-cases with atypia, Group 2-without atypia and Group 3-normal controls) and analyzed. Statistical analysis used one-way analysis of variance followed by Tukey Honestly Significant Difference test for intergroup analysis and unpaired students t-test to compare the two methods. Results: Smears prepared with both methods demonstrated atypia in 18 cases. The cellular diameter and cellular area (CA) were progressively increased from Group 1 through Groups 2 and 3 in both the smears. Nuclear diameter and nuclear area and nuclear cytoplasmic ratio progressively decreased from Group 1 through Groups 2 and 3. Both the methods showed no significant differences among the cellular parameters except in normal controls. Conclusion: Cytomorphometric analysis of keratinocytes obtained with oral rinse method and wooden spatula can serve as a useful screening aid to detect oral leukoplakia. Oral rinse method being more convenient results in smears of better quality.


Keywords: Cytomorphometry, exfoliative cytology, oral leukoplakia, oral rinse


How to cite this article:
Shaila M, Shetty P, Pai P. A new approach to exfoliative cytology: A comparative cytomorphometric study. Indian J Cancer 2016;53:193-8

How to cite this URL:
Shaila M, Shetty P, Pai P. A new approach to exfoliative cytology: A comparative cytomorphometric study. Indian J Cancer [serial online] 2016 [cited 2019 Oct 20];53:193-8. Available from: http://www.indianjcancer.com/text.asp?2016/53/1/193/180830



 » Introduction Top


Oral squamous cell carcinoma (OSCC) being the sixth most common malignancy is associated with high morbidity and mortality, which is due, at least in part, to late detection.[1] Early diagnosis of OSCC and premalignant lesions are the best interventions for improving survival and quality of life. Though scalpel biopsy has been the gold standard for diagnosis of lesions, it is invasive and involves both psychological implications for the patient and technical difficulties for the health practitioner. Thus, oral exfoliative cytology emerged in the forefront from 1963 onwards.

It has not been widely adopted and has fallen into disrepute in most centers due to poor sensitivity and specificity for identifying dysplasia and malignancy.[2] However it remains as a non-invasive, simple procedure that is still used as a primary screening tool to diagnose oral premalignant and malignant lesions in many developing countries, due to lack of resources. However, it has not been well-accepted by the general dental practitioners and only few possess the armamentarium required for the same.[3] Hence the need for a simpler procedure, which does not require elaborate armamentarium or trained personnel at the site of collection of specimen is the need of the hour. Oral rub and rinse method is one such method that can be used as a better alternative to the conventional method in general dental care settings and primary health-care centers, which lack in armamentarium required for conventional exfoliative cytology. Oral rinse has been used previously as a source of human buccal cell deoxyribonucleic acid (DNA).[4] Hence the same method has been utilized in the present study whereby oral rinse is used after rubbing against the lesion in the oral cavity to obtain representative cells. Quantitative parameters namely, nuclear and Cellular areas (CAs) and nuclear to cytoplasmic ratio have been shown to be significant in the diagnosis of oral lesions.[5] Thus, the present study was undertaken to compare and evaluate cellular atypia and cytomorphometric changes in nuclear diameter (ND), cellular diameter (CD), nuclear area (NA), CA and nuclear cytoplasmic ratio (N:C) of squames obtained from wooden spatula and oral rub and rinse method from clinically diagnosed oral leukoplakia cases and normal controls.


 » Materials and Methods Top


A total of 55 clinically diagnosed cases with oral leukoplakia and 55 age and sex matched controls were selected for the study from the Department of Oral Medicine of the Institute. The study protocol was approved by the Institutional Ethical committee. Patients were informed with regard to the research objectives, methods, possible benefits and potential risks and a written consent was obtained from all participants.

The following criteria were considered for incorporation of leukoplakia cases in the study.

”The term leukoplakia should be used to recognize white plaques of questionable risk having excluded (other) known diseases or disorders that carry no increased risk for cancer”. Leukoplakia is a clinical term and the lesion has no specific histology.

Clinical types

Two main clinical types of leukoplakia were recognized, being homogeneous and non-homogeneous leukoplakia. The distinction of these is purely clinical, based on surface color and morphological (thickness) characteristics. Homogeneous lesions are uniformly flat, thin and exhibit shallow cracks of the surface keratin. Those with mixed white and red plaques are denoted as “erythroleukoplakia.”[6]

History and physical examination were performed. The latter included careful examination of the oral cavity and was followed by smear preparation using oral rinse technique and by the conventional exfoliative cytology.

Oral rub and rinse technique

This technique was used to collect oral cells. Participant was asked to swish his/her mouth with water and expectorate. The suspected oral lesion was rubbed on firmly by the clinician or by the patient himself using their tongue in accessible areas for at least 30 s and to swish vigorously with 10 ml phosphate buffered saline, pH-7.2 for 45 s.[4] Participant was asked to expectorate into a sterile container after rinsing.

Once the sample was obtained, it was labeled and centrifuged at 1,000 rpm for 5 min. Supernatant fluid was discarded and cells were collected with a micropippette and smears prepared. For conventional exfoliative cytology, scrapings were obtained from a standard wooden tongue spatula moistened with normal saline. Scrapings were smeared on labeled glass slides. All the slides were immediately fixed in absolute alcohol and consequently stained with Papanicoloau stain. Two smears were prepared for each technique of the cases and controls.

All the stained smears were observed under a research light microscope. Each smear was assessed in two different ways. All smears were examined independently by two different cytopathologists in a double-blind fashion. Smears were first assessed for atypia and were assessed as with no atypia/atypia. Criteria for assessing the smears with atypia was presence of two or more of the following features: Nuclear enlargement associated with increased N:C, hyperchromatism, chromatin clumping with moderately prominent nucleolation and irregular nuclear borders, bi or multinucleation, increase keratinization and scantiness of the cytoplasm and variations in size and/or shape of cells and nucleus.[7]

Cytomorphometric analysis

Smears with atypia and without atypia were assessed separately using cytomorphometry. Each smear was assessed using the Motic Microscope (Motic image analyzing system, 2003, 2.0 version) with a ×40 objective lens. 50 randomly selected cells were measured in a stepwise fashion. Cellular diameter (CD), ND, cellular area (CA) and NA were measured. CD and ND were measured drawing a line with a digital cursor from one axis to the other along both X and Y axis. The average of X and Y value were taken as final diameter. For measurement of cellular area (CA) and NA, the nucleus and cell outline was traced on the screen and the software automatically calculated the area.[8] N:C was calculated by subtracting NA from the CAand further dividing the NA by the subtracted area.[9] For repeatability 10% of the slides was re-evaluated by the researcher. For reproducibility 10% of the smears were evaluated by a qualified Oral pathologist.

Statistical analysis

Since the data was divided into involved three (cases with/without atypia and controls) groups one way analysis of variance (ANOVA) test followed by Tukey Honestly Significant Difference (HSD) post hoc test was used to compare the means between the groups. P < 0.05 was considered to be significant. Unpaired t-test was used to compare parameters in all the groups of both methods.

Both methods were separately analyzed by dividing them into three groups as follows:

  • Groups 1-3
  • Group 1-Leukoplakia cases with atypia
  • Group 2-Leukoplakia cases without atypia
  • Group 3-Normal controls.



 » Results Top


Characteristics of the subjects are listed in [Table 1]. Two cases of leukoplakia and two cases of normal controls (smears prepared with a wooden spatula) was rejected due to excessive overlapping of cells in three and inadequate cells in one, one of leukoplakia (smears prepared with oral rinse) was rejected due to inadequate cells. Hence 52 cases of leukoplakia and 53 cases of normal controls were statistically analyzed.
Table 1: Characteristics of the subjects included in the study

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[Table 2] presents the clinical details along with the number of smears exhibiting atypia in both the methods. As noted 18 cases with leukoplakia showed atypia in the smears prepared from both the methods. Re-evaluation of 10% of the slides by a qualified Oral Pathologist showed no disparity in the presence of atypia or cytomorphometric values of the cellular parameters. Cytomorphometric values of the cellular parameters were recorded in microns. The box and whisker plots for the values of ND, CD, NA, CA and N:C for all the groups using both methods are shown in [Table 3]. The CD and CA were progressively increased from Group 1 through Groups 2 and 3 in both the smears. ND and NA progressively decreased from Group 1 through Groups 2 and 3. Means for all cellular parameters of different groups were compared, following a significant group effect in the ANOVA using Tukey HSD test as shown in [Table 4]. Student's unpaired t-test was used to analyze differences between the cellular parameters in both the methods separately for each group as shown in [Table 5],[Table 6],[Table 6]. There were no significant differences at P < 0.05 for Groups 1 and 2 [Table 5] and [Table 6], however Group 3 [Table 7] showed significant differences in all the parameters except N:C (P < 0.05).
Table 2: Leukoplakia cases with their cytologic diagnosis

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Table 3: Box and whisker plots for all the cellular parameters in both the techniques

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Table 4: Inter group comparisons using Tukey HSD test at P=0.05

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Table 5: Two sample t test at 95% confidence interval in leukoplakia cases with atypia using both the techniques

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Table 6: Two sample t test at 95% confidence interval in leukoplakia cases without atypia using both the techniques

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Table 7: Two sample t test at 95% confidence interval in normal controls using both the techniques

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 » Discussion Top


Ideally, a diagnostic procedure should be simple, non-invasive and well-tolerated by patients, cost-effective and highly specific and sensitive. Since exfoliative cytology lacks in sensitivity and specificity, it has been indicated mainly for periodic review of high risk patients, as an adjunctive test for periodic review of premalignant lesions, selection of suitable biopsy site in large lesions, when traditional biopsy contraindicated/unavailable, for review of treated cancer cases, periodic screening of family members of patients with OSCC and population screening for oral cancer.[10]

During the 1980s, a cytobrush was introduced for cervical smears in gynecological lesions. This technique improved the process of spreading cells on to slides, compared with smears obtained by using a wooden spatula, wherefore improving the quality of the smears.[11] Yet another brush named OralCDx gained popularity as OralCDx (OralCDx Laboratories, Suffern, NY) is a computer-assisted method for the analysis of transepithelial cellular samples collected by using a patented brush, which has been designed to evaluate any oral epithelial abnormality without an obvious etiology for dysplasia or cancer. It consists of a specialized neural network-based image processing system specifically designed to detect oral precancerous and cancerous cells.[11] Some authors are of the opinion that oral cancer is a poor man's disease and methods of diagnosis should be cheap and accurate, especially in developing countries. Further their experience have shown that when CDx brush when used as indicated by the manufacturer in patients with atrophic lesions, will result in pain much more severe than the pain felt during a scalpel biopsy.[12]

Inadequate cellularity and overlapping of cells are common reasons for unsatisfactory smears in conventional exfoliative cytology, as evidenced in the present study where four conventional cytology smears were rejected in contrast to one oral rinse smear. Liquid based cytology procedures overcame these hurdles. Few authors compared the diagnostic accuracy of Papspin, a liquid based cytologic preparation with surgical biopsy and concluded that Papspin appeared to be an accurate and economical test for detection of high-risk dysplasias and cancers, but the real significance of this method will be its diagnostic accuracy in studies focusing strictly on lesions with a low level of clinical concern.[13]

In yet another study two different preparation methods were investigated namely conventional transfer procedure to glass slides and the commercially available liquid-based SurePath-System (TriPath Imaging, Burlington, NC, USA) for the early detection of OSCCs. SurePath thin layers showed excellent morphology on a clear background, with preservation of 3-dimensional configurations to allow an accurate diagnosis. In contrast the conventional glass slides showed significantly more blood contamination and cell overlapping. Thus they concluded that in oral cytology, thin layers can safely replace other types of wet-fixed preparations, resulting in enhanced specimen quality and diminished false negative rates in a direct to vial procedure.[14] Oral rinse cytologic preparations are liquid based preparations, thus results in a better smear quality.

Continuous exfoliation of the epithelial cells is a part of physiological turnover. Deeper cells, which are strongly adhered in normal conditions, become loose in case of malignancy and exfoliate along with superficial cells.[15] This feature has been the logical basis of the oral rub and rinse method, as evidenced by the presence of atypia in 18 cases. Oral rub and rinse smears result in uniform distribution of cells in a clear background with no debris, blood and overlapping of cells in contrast to the conventional smears [Figure 1] and [Figure 2]. The use of liquid-based cytology can offer improved and repeatable preparations than conventional cytology and reduce the false negative results.[16],[17] Moreover, the residual material can be used for further investigation.[18],[19] However, preparation of smears from oral rinse is technique sensitive.
Figure 1: Photomicrograph of a papanicoloau stained conventional preparation (×400)

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Figure 2: Photomicrograph of a papanicoloau stained oral rinse preparation (×400)

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Image analysis along with oral exfoliative cytology has been used to increase the specificity of the procedure. Ogden et al.,[2] suggested that quantitative techniques, based on the evaluation of parameters such as NA, CA, and nucleus-to-cytoplasmic area ratio, may increase the sensitivity of exfoliative cytology for early diagnosis of oral cancers, since these techniques are precise, objective and reproducible. A similar study concluded that 50 cells were sufficient to provide an indication of malignant changes.[20] Cowpe et al., found that tissues undergoing malignant transformation generally show a reduction in CA before the reduction in NA.[21] Cytomorphometric techniques were used to assess ND and CD in normal oral mucosa, in dysplastic lesions and in squamous cell carcinomas (SCCs).[5] They found that CD was highest in normal mucosa, lower in dysplastic lesions and lowest in SCCs. By contrast, ND was lowest in normal mucosa, higher in dysplastic lesions and highest in SCCs. These studies suggested that reduced nuclear size and increased cytoplasm size are useful early indicators of malignant transformation and thus exfoliative cytology is of value for monitoring clinically suspect lesions and for early detection of malignancy.[22]

In our study, smears collected from both the methods showed no significant differences in Groups 1 and 2, but showed significant differences in ND, CD, NA and CA in Group 3 as shown in [Table 6]. This observation, however, is expected as smears from oral rinse consisted of cells present throughout the oral cavity lining, in contrast to smears from wooden spatula, which are obtained from a specific site. Comparison of the mean values of the cytological parameters showed significant intergroup differences for NA and N:C in smears obtained from wooden spatula and CD, NA and N:C in smears obtained from oral rinse [Table 3]. However, in both the smears a gradual decrease in ND, NA and N:C and gradual increase in CD and CA was noticed from Group 1 through Groups 2 and 3, as evidenced by other studies.

Oral rinse technique has been used mainly for microbiological purposes, especially to analyze oral candidal colonization.[23] However, it has been utilized to detect OSCC. The aberrant methylation of a combination of marker genes present in oral rinse samples was used to detect OSCC with >% sensitivity and specificity.[24] Epidemiological studies conducted earlier concluded that both a 10-ml oral-rinse sample and 2-ml whole-saliva sample provide sufficient DNA quantity and better quality DNA for genetic epidemiological studies than do the commonly used buccal swab and brush techniques.[25]

The present technique is first of its kind which utilizes an oral rinse to collect cells for smear preparation to assess atypia and morphometric parameters using non automatic manual method. Oral rub and rinse method has some distinct advantages over the conventional exfoliative cytology, such as, it results in smears of better quality, thus ensuring easy detection of dysplastic features.


 » Conclusion Top


Oral rub and rinse method is a convenient alternative to exfoliative cytology, for primary screening of oral leukoplakia. Further application of quantitative methods may improve its diagnostic value.


 » Acknowledgments Top


We would like to thank Management, Principal and Staff of K.V.G. Dental College, Sullia, Karnataka for their kind support.

 
 » References Top

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Mehrotra R, Mishra S, Singh M, Singh M. The efficacy of oral brush biopsy with computer-assisted analysis in identifying precancerous and cancerous lesions. Head Neck Oncol 2011;3:39.  Back to cited text no. 1
    
2.
Ogden GR, Cowpe JG, Wight AJ. Oral exfoliative cytology: Review of methods of assessment. J Oral Pathol Med 1997;26:201-5.  Back to cited text no. 2
    
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4.
Heath EM, Morken NW, Campbell KA, Tkach D, Boyd EA, Strom DA. Use of buccal cells collected in mouthwash as a source of DNA for clinical testing. Arch Pathol Lab Med 2001;125:127-33.  Back to cited text no. 4
    
5.
Cowpe JG, Longmore RB, Green MW. Quantitative exfoliative cytology of abnormal oral mucosal smears. J R Soc Med 1988;81:509-13.  Back to cited text no. 5
    
6.
Warnakulasuriya S, Johnson NW, van der Waal I. Nomenclature and classification of potentially malignant disorders of the oral mucosa. J Oral Pathol Med 2007;36:575-80.  Back to cited text no. 6
    
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Ahmed HG, Idris AM, Ibrahim SO. Study of oral epithelial atypia among Sudanese tobacco users by exfoliative cytology. Anticancer Res 2003;23:1943-9.  Back to cited text no. 7
    
8.
Ogden GR, Cowpe JG, Green MW. Quantitative exfoliative cytology of normal buccal mucosa: Effect of smoking. J Oral Pathol Med 1990;19:53-5.  Back to cited text no. 8
    
9.
White FH, Jin Y, Yang L. An evaluation of the role of nuclear cytoplasmic ratios and nuclear volume densities as diagnostic indicators in metaplastic, dysplastic and neoplastic lesions of the human cheek. Histol Histopathol 1997;12:69-77.  Back to cited text no. 9
    
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Sugerman PB, Savage NW. Exfoliative cytology in clinical oral pathology. Aust Dent J 1996;41:71-4.  Back to cited text no. 10
    
11.
Mehrotra R. The role of cytology in oral lesions: A review of recent improvements. Diagn Cytopathol 2012;40:73-83.  Back to cited text no. 11
    
12.
Mathew B, Nabeel SM. The relevance of CDX brush biopsy in diagnosing early oral cancer in developing countries. Oral Maxillofac Pathol J 2011;2:115-6.  Back to cited text no. 12
    
13.
Afrogheh A, Wright CA, Sellars SL, Wetter J, Pelser A, Schubert PT, et al. An evaluation of the Shandon Papspin liquid-based oral test using a novel cytologic scoring system. Oral Surg Oral Med Oral Pathol Oral Radiol 2012;113:799-807.  Back to cited text no. 13
    
14.
Remmerbach TW, Hemprich A, Boecking A. Effectiveness of liquid based cytology (LBC) in minimal invasive oral brush biopsies: A comparison of conventional cytopreparation techniques. Oral Oncol 2007;2:119.  Back to cited text no. 14
    
15.
Sivapathasundhram B, Kalasagar M. Yet another article on exfoliative cytology. J Oral Maxillofac Pathol 2004;8:54-7.  Back to cited text no. 15
    
16.
Hayama FH, Motta AC, Silva Ade P, Migliari DA. Liquid-based preparations versus conventional cytology: Specimen adequacy and diagnostic agreement in oral lesions. Med Oral Patol Oral Cir Bucal 2005;10:115-22.  Back to cited text no. 16
    
17.
Mehrotra R, Madhu, Singh M. Serial scrape smear cytology of radiation response in normal and malignant cells of oral cavity. Indian J Pathol Microbiol 2004;47:497-502.  Back to cited text no. 17
    
18.
Remmerbach TW, Weidenbach H, Pomjanski N, Knops K, Mathes S, Hemprich A, et al. Cytologic and DNA-cytometric early diagnosis of oral cancer. Anal Cell Pathol 2001;22:211-21.  Back to cited text no. 18
    
19.
Gologan O, Barnes EL, Hunt JL. Potential diagnostic use of p16INK4A, a new marker that correlates with dysplasia in oral squamoproliferative lesions. Am J Surg Pathol 2005;29:792-6.  Back to cited text no. 19
    
20.
Cowpe JG, Longmore RB, Green MW. Quantitative exfoliative cytology of normal oral squames: An age, site and sex-related survey. J R Soc Med 1985;78:995-1004.  Back to cited text no. 20
[PUBMED]    
21.
Cowpe JG, Ogden GR, Green MW. Comparison of planimetry and image analysis for the discrimination between normal and abnormal cells in cytological smears of suspicious lesions of the oral cavity. Cytopathology 1993;4:27-35.  Back to cited text no. 21
    
22.
Ramaesh T, Mendis BR, Ratnatunga N, Thattil RO. Cytomorphometric analysis of squames obtained from normal oral mucosa and lesions of oral leukoplakia and squamous cell carcinoma. J Oral Pathol Med 1998;27:83-6.  Back to cited text no. 22
    
23.
Coulter WA, Murray SD, Kinirons MJ. The use of a concentrated oral rinse culture technique to sample oral candida and lactobacilli in children, and the relationship between candida and lactobacilli levels and dental caries experience: A pilot study. Int J Paediatr Dent 1993;3:17-21.  Back to cited text no. 23
    
24.
Nagata S, Hamada T, Yamada N, Yokoyama S, Kitamoto S, Kanmura Y, et al. Aberrant DNA methylation of tumor-related genes in oral rinse: A noninvasive method for detection of oral squamous cell carcinoma. Cancer 2012;118:4298-308.  Back to cited text no. 24
    
25.
King IB, Satia-Abouta J, Thornquist MD, Bigler J, Patterson RE, Kristal AR, et al. Buccal cell DNA yield, quality, and collection costs: Comparison of methods for large-scale studies. Cancer Epidemiol Biomarkers Prev 2002;11:1130-3.  Back to cited text no. 25
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]

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