Citologia Da Mucosa Oral

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Exfoliative Cytology of Oral Mucosa in Kidney Transplant Patients:A Cytomorphometric Study

M. Keles, U. Tozoglu, D. Unal, F. Caglayan, A. Uyanik, H. Emre, K. Cayir, and B. Aydinli

ABSTRACT

Objectives. The aim of this study was to investigate quantitative cytologic changes in oralmucosal smears collected from kidney transplant patients by modern stereologic methods.

Methods. Smears were obtained from the clinically healthy buccal mucosa and floor ofmouth of transplant patients (n 21) and healthy volunteers (n 27). Smears from eachindividual stained by the Papanicolaou method were analyzed using a stereologic method.Nuclear (NV) and cytoplasmic (CV) volumes were evaluated with Stereo Investigator

software.Results. CV values were 123,012.71 15,840.96 fL in the floor of the mouth and133,667.10 26,653.39 fL in the buccal mucosa of the kidney transplant patients. CV

values were 133,746.93 14,210.67 fL in the floor of the mouth and 167,797.78 21,007.70 fL in the buccal mucosa of the control group. NV values were 945.68 65.85 fLin the floor of the mouth and 845.15 81.56 fL in the buccal mucosa of the kidneytransplant patients and 485.17 18.03 fL in the floor of the mouth and 410.25 52.84 m3

in the buccal mucosa of the control group. Significantly higher NV (P .000) and NV/CV(P .000) and lower CS (P .000) values were found in the patient group compared withthe control group.

Conclusion. The findings suggest that there were alterations in oral epithelial cells,detectable by microscopy and cytomorphometry in kidney transplant patients.

Renal transplantation (RT) is an important procedures

in the management of patients with end-stage renal

disease (ESRD). Some drugs, such as prednisolone, myco-

phenolate mofetil (MMF), cyclosporine (CsA), and tacroli-

mus (Tac), are commonly used agents in RT. These drugs

have some side effects in the oral region, such as gingival

overgrowth, fungal infections, hairy leukoplakia, and in-

creased risk of oral cancer. Early detection of oral cancer intransplant patients is very important owing to mortality andmorbidity.1,2

Classic diagnostic methods for precancerous and cancer-ous oral lesions include clinical examinations and histo-pathologic studies of biopsied material.3 The most acceptedclinical technique for the diagnosis of lesions in the oralmucosa is incisional or excisional biopsy.4 Biopsies ofnormal and malignant tissues, or scrapings containing exfo-liated oral cells, have been explored for many years.5

Exfoliative cytology is a simple nonaggressive technique

that is well accepted by a patient and allows a quick andfairly accurate assessment of suspicious lesions of the oral

cavity.6 After the cells have been collected, fixed, andstained, the morphology of surface epithelial cells is ob-served under a microscope.7 This technique to examine the

morphologic and morphometric aspects of the cell may yetbe implemented in public health programs.8

One of the important methods is stereology to makemorphometric assessment. A stereology method that isbased on fundamental principles of geometry and statisticsand is largely concerned with the 3-dimensional interpreta-tion of planar sections of materials or tissues are suggested

From the Department of Nephrology (M.K., A.U.), Department

of Histology and Embryology (D.U.), Department of Medical

Oncology (K.C.), and Department of General Surgery (B.A.),

Faculty of Medicine, Erzurum, Turkey, Department of Oral Diag-

nosis and Radiology (U.T., F.C.), Faculty of Dentistry, Ataturk

University, Erzurum, Turkey; and Department of Nephrology

(H.E.), Faculty of Medicine, Yznc Yl University, Van, Turkey.

Address reprint requests to Mustafa Keles, MD, Department of

Nephrology, Medical Faculty, Atatrk University, 25240 Erzurum,

Turkey. E-mail: [email protected]

2011 by Elsevier Inc. All rights reserved. 0041-1345/see front matter360 Park Avenue South, New York, NY 10010-1710 doi:10.1016/j.transproceed.2011.01.111

Transplantation Proceedings, 43, 871 875 (2011) 871
mailto:[email protected]:[email protected]


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to perform morphometric analyzes. There are some ofmethods in stereology to obtain the volume parameter ofsmaller objects, such as cells: optical fractionator withcontour trace, Cavalieri estimator, surface-weighted star volume, area fraction fractionator method, and 3- or 2-

dimensional nucleator.9,10

The aim of the present study was to investigate thequantitative cytologic changes of oral mucosa smears col-lected from RT patients by modern stereologic methods for3 reasons. First, we have not found any stereologic study inthe literature about morphometric analyses of exfoliativecytology. Second, we sought to examine the methodseffectiveness. Finally, we intended to describe the risk ofpostoperative oral cancer of kidney transplantation patientscompared with healthy control subjects.

PATIENTS AND METHODS

A total of 21 patients with renal transplants and 27 healthy volunteers were recruited from the Department of Nephrology, Ataturk University. All procedures followed the tenets of the

Declaration of Helsinki. A pro forma inventory was completeddetailing name, age, gender, and relevant medical history. Patients

with anemia, diabetes mellitus, radiotherapy, alcohol consumption,and smoking were not included in the study. Patients with glomer-

ular filtration rate (GFR) 60 mL/min and 12 months afterpreemptive RT were included in this study.

Smears were obtained from clinically healthy buccal mucosa and

floor of the mouth of patients with transplant attending the privateclinic and from volunteer control individuals. After clinical exam-ination, the oral mucosa was dried with a gauze swab to remove

surface debris and excess saliva, and smears were taken using acytobrush and transfered to clean dry glass slides. These were then

immediately sprayed with a commercial fixative containing 95%ethyl alcohol. Smears from each individual stained by the Papani-

colaou method were analyzed using the stereologic method of 2dimensional nucleator.

The smears were placed on a motor-driven stage attached to an

microscope, and cells were projected onto the monitor via cameraat 200 magnification. Each clearly defined cell with predominantstaining was examined by systematic sampling in a stepwise man-

ner, moving the microscope stage from left to right and then downand across to avoid measuring the same cells again. The nuclear(NV) and cytoplasmic (CV) volumes were evaluated for each cellusing the software Stereo Investigator (Microbrightfield).

The volume of interesting cells obtained from the buccal mucosaand floor of the mouth of RT patients and healthy volunteers

smears was estimated using the nucleator method11

following theformula:

V(N)4

3ln

3

where Vn is the number-weighted volume, l is length of intercepts,and n the number of nucleator estimates.

Although some requirements for obtaining full randomness inthe section-sampling stage while estimating the volume of the small

objects by the nucleator method have been mentioned in theliterature,1113 we did not implement any requirement associated

with section stage of the sampling procedure, because we used a

smear.

The cytomorphometric data were compared between RT and

control groups by the independent-samples t test. The statisticalanalysis was performed using the statistical software package SPSS(version 10.0; SPSS, Chicago, IL, USA). Levels of significance wereset at P .05.

RESULTS

In our study, the mean age was 36.10 10.52 years in theRT patients (15 men and 6 women) and 37.30 8.37 yearsin the control group (19 men and 8 women). There was nodifference between the 2 groups regarding age and gender(P .05). The mean time of buccal mucosa and floor of themouth smears obtainment was 14.9 3.1 months oftransplantation. Mean GFR was 82.09 14.54 mL/min andmean serum creatinine 1.12 0.23 mg/dL. Ten patientswere using CsA MMF prednisolone and 11 patientsTac MMF prednisolone. No patient had received anymonoclonal or polyclonal antibodies.

Cytomorphometric results showed that NV in buccalmucosa was markedly higher (P .000) in the RT patientgroup (845.15 81.56 fL) than in the control group (410.2552.84 fL), and CV in buccal mucosa was markedly lower(P .000) in the R patient group (133,667.10 26,653.39fL) than in the control group (167,797.78 21,007.70 fL).

In addition, NV in the floor of the mouth was markedlyhigher (P .000) in the RT patient group (945.68 65.85fL) than in the control group (485.17 18.03 fL), and CVin the floor of the mouth was markedly lower (P .017) inthe RT patient group (123,012.71 15,840.96 fL) than inthe control group (133,746.93 14,210.67 fL).

Figures 1 and 2. show histologic views of exfoliative cellsamples.

In this study, we also determined the NV-CV ratio (N/C).These ratios in the floor of the mouth were 0.008 0.001 inRT patients and 0.004 0.000 in the control group. In thebuccal mucosa, these ratios were 0.007 0.001 in the RTpatients and 0.002 0.000 in the control group. The N/Cratio mean was higher in the RT patient group than in thecontrol group (P .000).

DISCUSSION

In this study, we performed microscopic and cytomorpho-metric analyses of the oral epithelium (buccal mucosa andfloor of the mouth) in RT patients, as a result of a lack of

previous works regarding this issue. We found that thereare quantitative cytologic changes in cells in transplantpatients. This may be related to the immunosuppressiveagents.14,15 Immunosupressive medication have negativeimpact on immune surveillance and affect DNA repairmechanisms, leading to irreversible DNA alteration andsubsequent carcinogenesis.2 They may cause gingival over-growth, an increased incidence ofCandida spp. in the saliva,and herpes simplex.16

Increased cancer incidence after organ transplantationhas been demonstrated in several studies.2,17,18 Villeneuveet al.18 reported that the cancer incidence in RT patients was 2.5 times higher than rates observed in the general

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population. After transplantation, a significant excess wasobserved for melanoma, Kaposi sarcoma, non-Hodgkinlymphoma, Hodgkin disease, leukemia, and cancer of thelip, tongue, mouth, salivary gland, esophagus, stomach,colon, anus, liver, gallbladder, lung, connective and othersoft tissue, vulva, cervix, penis, eye, thyroid, and unspec-ified site.2,19 The risk has been especially high in the headand neck sites. Van Leeuwen et al20 suggested that lipcancer risk is associated with the duration of immuno-suppression, specific immunosuppressive agents, and ul-traviolet light exposure.

In our study, the cytomorphometric findings in the oralsmears of transplant patients demonstrated that there was areal increase in the NV, and the CV presented statisticallysignificant differences. The mean N/C was higher in the RTpatient group. These findings suggest that oral cells mayshow malignant transformation in RT patients. Ramaesh etal.21 found that cytoplasmic diameter was highest in normal

mucosa, lower in dysplastic lesions, and lowest in squamouscell carcinomas (SCCs) by using cytomorphometric tech-niques. In contrast, nuclear diameter was lowest in normalmucosa, higher in dysplastic lesions, and highest in SCCs.We think that because increased nuclear size and reducedcytoplasm size may be early indicators of malignant trans-formation, exfoliative cytology may play an important rolefor monitoring clinically suspect lesions and for early de-tection of malignancy in RT patients.

Exfoliative oral cytology examines the characteristics ofcells that flake off, whether naturally or artificially, from theoral mucosa. After the cells have been collected, fixed, andstained with Papanicolaou, the morphology of surface epi-thelial cells is observed under a microscope.7 The basicrequirements for a useful technique should be ease of use inany location, minimum trauma, and providing an adequateand representative number of epithelial cells. Cytobrush isan adequate instrument owing to its ease in sampling and

Fig 1. Histologic view of exfoli-

ative cell samples. Top grid pres-

ents buccal mucosa of healthyvolunteers and transplant pa-

tients. Bottom grid shows exfoli-

ate profiles obtained from the

floor of the mouth. (A, B, C) Con-

trol; (A-1C-3) exfoliate profiles of

transplant patients.

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the quality of the oral cytologic sample.7,22,23 Cytobrushpenetrated the lesions to ensure that representative mate-rial is obtained. The aim is to collect both surface andbasal cells, which makes the sample more representa-tive.7 Brush biopsy is a simple, relatively inexpensive,sensitive, risk-free method of screening for cancer and arelatively painless technique that is readily accepted bypatients.22 This technique, however, should complementthe biopsy, not replace it.3,7

In this study, we showed that there are some quantitativecytologic changes in oral epithelial cells in RT patients thatcan be detected by quantitative cytomorphometric tech-niques that are objective, accurate, reproducible, and basedon evaluation of parameters such as variations in the size ofthe NV and CV and alterations in the N/C ratio. It mayprovide useful information in RT patients to both under-stand and diagnose some diseases earlier than by otherconventional methods.

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3. Acha A, Ruesga MT, Rodriguez MJ, et al: Applications of theoral scraped (exfoliative) cytology in oral cancer and precancer.Med Oral Patol Oral Cir Bucal 10:95, 2005

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11. Gundersen HJ: The nucleator. J Microsc 151:3, 1988

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13. Nyengaard JR, Gundersen HJG: The isector: a simple anddirect method for generating isotropic, uniform random sectionsfrom small specimens. J Microsc 165:427, 1992

14. Proctor R, Kumar N, Stein A, et al: Oral and dental aspectsof chronic renal failure. J Dent Res 84:199, 2005

15. Cerver AJ, Bagn VJ, Soriano YJ, et al: Dental manage-ment in renal failure: patients on dialysis. Med Oral Patol Oral CirBucal 13:419, 2008

16. Spolidorio LC, Spolidorio DM, Massucato EM, et al: Oralhealth in renal transplant recipients administered cyclosporine Aor tacrolimus. Oral Dis 12:309, 2006

Fig 2. Illustration of stereologic

estimation according to the nu-

cleator method first described by

Gundersen.14 In this estimation, it

was used in Stereo Investigator

version 6.

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17. Makitea AA, Lundberg M, Salmela K, et al: Head and neckcancer in renal transplant patients in Finland. Acta Otolaryngol128:1255, 2008

18. Villeneuve PJ, Schaubel SS, Fenton SS, et al: Cancer inci-dence among Canadian kidney transplant recipients. Am J Trans-plant 7:941, 2007

19. Vajdic MC, McDonald SP, McCredie M: Cancer inci-dence before and after kidney transplantation. JAMA 296:2823,2006

20. van Leeuwen MT, Grulich AE, McDonald SP, et al:Immunosuppression and other risk factors for lip cancer after

kidney transplantation. Cancer Epidemiol Biomarkers Prev18:561, 2009

21. Ramaesh T, Mendis BR, Ratnatunga N, et al: The effect oftobacco smoking and of betel chewing with tobacco on the buccalmucosa: a cytomorphometric analysis. J Oral Pathol 28:385, 1999

22. Mehrotra R, Gupta A, Singh M, et al: Application of

cytology and molecular biology in diagnosing premalignant ormalignant oral lesions. Mol Cancer 5:11, 200623. Jones AC, Pink FE, Sandow PL, et al: The cytobrush plus

cell collector in oral cytology. Oral Surg Oral Med Oral Pathol OralRadiol Endod 77:95, 1994

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