Cytomorphometric and cytomorphologic analysis of oral mucosa in children with sickle cell anemia.

BACKGROUND: Sickle cell anemia (SCA) is an autosomal recessive genetic disorder, characterized by chronic hemolytic anemia, episodic painful crises, and pathologic involvement of many organs, consequence of vaso occlusive phenomenon and vasculopathy. Several forms of the chronic anemia, consequence of hemolysis, can be associated with oral epithelial cells changes. Exfoliative cytology can be used to detect real changes in the oral mucosa in SCA. AIMS: To evaluate morphometric and morphological changes in oral epithelial cells by exfoliative cytology in children with SCA.
MATERIALS AND METHODS: Oral smears were collected from clinically normal-appearing mucosa by liquid-based exfoliative cytology in 20 SCA children (SCA group) and 20 healthy children (C group), matched for age and gender. The slides were prepared and stained by the Papanicolaou technique. Cell morphology and cellularity were analyzed and compared by Chi-square test (P < 0.05). Images of 50 cells per slide were captured and the nuclear area (NA) and cytoplasmic area (CA) were analyzed using an image analysis system. The nucleus-to-cytoplasmic area ratio (NA/CA) was calculated. To compare the means of groups SCA and C, the Student's t-test (P < 0.05) was applied to NA and CA; test non-parametric Mann Whitney U (P < 0.05) was used to compare NA/CA.
RESULTS: MEAN VALUES FOR SCA AND C GROUPS WERE: NA (69.38 and 59.63 μm²; P = 0.01); CA (2321.85 and 2185.60 μm²; P = 0.24); NA/CA (0.03 and 0.02; P = 0.13), respectively. A significant increase in NA for SCA group (P = 0.01) was seen. No morphological differences were found between the groups. There was a predominance of nucleated cells of the superficial layer in the smears of both groups. Class I smears were predominant in both groups.
CONCLUSIONS: This study revealed that SCA was able to induce significant changes on nuclear area of the oral epithelial cells.

Introduction

Sickle cell anemia (SCA) is an autosomal recessive genetic disorder characterized by homozygous hemoglobin S.[1] The clinical findings related to anemia affecting mainly the oral mucosa are atrophy, pale mucosa, depapillation of the tongue, and occasionally recurrent aphthous ulceration.[23] Studies using exfoliative cytology or liquid-based exfoliative cytology have demonstrated that the nuclear and cytoplasmic areas of the epithelial cells in oral mucosa may be modified due to systemic conditions, such as anemia,[3] diabetes,[4] or in response of local agents such as tobacco,[56] alcohol,[7] and crack.[89] The purpose of this study was to evaluate quantitative changes in oral epithelial cells by exfoliative cytology in children with sickle cell anemia.

Materials and Methods

The experimental protocol of the present study was approved by Committee of Ethics in Research at the University. The patients and/or their parents or guardians were informed about the objective and other aspects of the research and they have signed the terms of agreement.

Subjects

The experimental group was diagnosed with sickle cell anemia at Clinical Hospital at the Hematology Ambulatory Care Service and the control group was from School of Dentistry. Name, age, relevant medical history, and drugs in use were recorded.

Twenty patients with SCA and 20 healthy patients (C) aged from 1 to 13 years old, paired regarding sex and age, were considered for the study. Children who utilized mouthwashes or orthodontic braces as well as presenting lesions in their oral mucosa were excluded from this sample, in order to minimize the possible effects of such conditions upon the epithelial cell morphology. In control group, children with any systemic disease were also excluded from the sample.

Cells collection

Exfoliated cells of the clinically normal oral mucosa were obtained by oral liquid-based exfoliative cytology. The squamous epithelial cells were collected using Universal Collection Medium (UCM) kit of DNA-Citoliq SystemTM (Digene, São Paulo, Brazil). The UCM brush provides an adequate and representative number of superficial and intermediate epithelial cells of oral mucosa.

Cytological preparations

The DNA-Citoliq SystemTM allows thin-layer preparations to be provided through a filtration process. An aliquot of 200 μL of UCM was filtered through Filtrogene polycarbonate membrane filtersTM (Digene, São Paulo, Brazil), pore size 5 μm, diameter 25 mm placed in Prepgene pressTM (Digene, São Paulo, Brazil) attached to glass slides. Glass slides were immediately fixed in absolute alcohol for 20 min. Smears were then stained with routine Papanicolaou stain.

Cytomorphometric analysis

Each slide was assessed using the light microscopy by binocular Olympus BX 50 microscopyTM (Olympus, Japan). Fifty randomly selected cells were measured in a stepwise fashion. Cells images were captured by Olympus DP 25 microscope digital cameraTM (Olympus Latin America Inc, Miami, USA) at ×400 magnification. Nuclear area (NA) and cytoplasmic area (CA) were obtained by drawing around the nuclear and cell boundaries using the digitizer cursor and measuring mode[4] of AnalySIS image systemTM (Olympus Soft Imaging Solutions GmbH, Münster, Germany) for Windows XPTM 2008.

The NA/CA ratio of the epithelial cells of each sample was calculated. All smears were analyzed by the same person, who was previously trained to make the morphometric evaluation.

Cytomorphological analysis

Each slide was assessed using the light microscopy by binocular Olympus BX 50 microscopyTM (Olympus, Japan). All cellular features were coded according to Papanicolaou classification.[10] Papanicolaou staining is used as a routine method for the analysis of cytological aspects and permits the identification of basic inflammatory, dysplastic, or malignant alterations.[11] Additionally, the type of predominant cell (cellularity) in each smear was analyzed too.

Statistical analysis

All data were tabulated and statistical tests were performed with SPSS for Windows 13.0 (SPSS Inc., Chicago, IL, USA). Tests of normality Kolmogorov-Smirnov, Shapiro Wilk test, and Levene's homogeneity of variances were applied for all variables. Significant statistical differences of the mean values of variables between groups were examined using Student's t-test or Mann Whitney U test. Chi-square test was used to verify if there was difference in morphological characterization and predominance of cells of oral smears according to groups. Differences were considered statistically significant when P < 0.05.

Results

The screened patients included 12 males and 28 females. The mean age for SCA and C group was 6.2 years old (1-13). Drugs in use, number of patients who were taking drugs, and their percentages in SCA group (n = 20) is described as follows: Folic acid (20-100%); hydroxyurea (2-10%); penicillin (7-35%); amoxicillin (1-5%); paracetamol (1-5%); dipyrone (1-5%); valproic acid (1-5%); and fluticasone propionate (1-5%). A total of nine patients had received one or more blood transfusions. The mean levels of total hemoglobin in SCA group were 9.2 g/dL.

A total of 2,000 epithelial cells were assessed. The values for the NA, CA, and NA/CA ratio are illustrated in Table 1. The normality test of Kolmogorov-Smirnov and homogeneity of variance Levene's test revealed that data showed a normal distribution and homogeneous variances for NA and CA between groups (P > 0.05). Student's t-test revealed a significant increase in NA for SCA group (P = 0.01) [Figure 1]. There was no statistically significant difference in the mean values of the CA (Student's t-test; P = 0.24) and NA/CA (Mann Whitney U test; P = 0.13) for both groups.

Table 1

Mean and standard deviation of NA, CA, NA/CA in SCA and C groups

Figure 1

Epithelial cells exhibiting nuclear enlargement in oral smear of sickle cell anemia (Pap, ×400)

There was no statistically significant difference between groups in morphological analysis of oral smears cells. According to Papanicolaou classification there was a predominance of class I smears (cells with normal morphology, absence of atypical or abnormal cells) in both groups [Figure 2a, Table 2]. Class II smears (cells with normal morphology and inflammatory changes) were also observed in a lower percentage in both groups [Figure 2b, Table 2]. There were no smears from class III (cells with the presence of dysplastic changes and some criteria of malignancy, but with minor alterations), IV (smears with cells alterations strongly suspected malignancy and number of abnormal cells), and V (smears with cells alterations consistent with the presence of malignancy) in SCA and C groups.

Figure 2

Oral smear of sickle cell anemia individuals classified as Class 1 (a) and Class 2 (b) (Pap, ×400)

Table 2

Morphologic characterization of oral smears according to Papanicolaou's system classification in SCA and C groups

No nuclear aberrations such as ovoid and double-nucleus in the epithelial cells of the oral mucosa in SCA group were found.

Table 3 shows the type of predominant cell in each smear. There was a predominance of nucleated cells of the superficial layer and no statistically significant difference between groups was observed.

Table 3

Predominant cells in smears of SCA and C groups

Discussion

SCA is among the most common genetic diseases in the world, and the number affected in the U.S. may approach 100,000, even when accounting for the effect of early mortality on estimations.[12] Most individuals with SCA are healthy at birth and symptoms usually start appearing within the first 6 months of life, with anemia and vasculopathy being the hallmarks of the disease.[1]

Nuclear aberrations (enlargement, ovoid, and double- nucleus) of epithelial cells in oral mucosa have been demonstrated in patients with SCA, pernicious, and iron-deficiency anemia. However, these findings may not be pathognomonic for a specific type of anemia or for any anemia.[13]

Morphometric changes were observed in this study, with a highly significant increase in NA in SCA group. Due to the effect that folic acid has on the nucleus of epithelial cells in individuals with anemia,[14] it was expected that individuals with SCA treated on a daily basis with folic acid, show a normal nuclear size. Unfortunately, it was not possible to perform a metabolic profile to demonstrate the folic acid deficiency in both groups. Probably, in the present study the increase of the NA is not associated only with folic acid deficiency, but it is likely to be directly associated with chronic anemia.

The oral mucosa epithelium is composed mostly of keratinocytes that are arranged in the basal, spinous, granular, and superficial layers. Oral keratinocytes proliferate and differentiate along these layers until exfoliated. The keratinocytes of the spinous layer have a nuclear increase, as they pass into the granular layer to the surface layer, the nucleus naturally decreases. In epithelial atrophy, the thickness of the epithelial layers is reduced. Furthermore, keratinocytes do not exhibit a normal maturation process and are exfoliated before the decrease of the nucleus occurs. This would justify a hypothesis that the nucleus increase in keratinocytes of oral mucosal epithelium of individuals with sickle cell anemia. However, this hypothesis only could be proved if a histopathological study had been developed.

The folate deficiency occurs in all types of hemolytic anemia.[15] Moreover, deficiencies of B12 vitamin are associated with megaloblastic anemia.[15] Megaloblastic crisis in sickle cell anemia patients which results from sudden arrest of erythropoiesis by folate depletion often happens. Chronic erythroid hyperplasia imposes a drain on folate reserve and biochemical evidence of mild folate deficiency can be demonstrated with a high frequency in the subjects with SCA.[16]

Hemolytic anemia is a chronic lifelong condition, hard to manage in SCA. The criteria used by the World Health Organization (WHO) to determine anemia, differ by age as follows: For children 6 months to 5 years of age anemia is defined as a hemoglobin (Hb) level < 11 g/dL, for children 5-11 years of age Hb < 11.5 g/dL.[17] Severe anemia is defined as Hb < 7.0 g/dL.[17] The mean levels of total hemoglobin in SCA group was 9.2 g/dL, showing that they were considered anemic. Thus, in the present study we suggest that keratinocytes are undergoing an adaptive response of atrophy. The atrophy of oral mucosa epithelium may contribute to increase susceptibility to infection and possible damage caused by physical and chemical agents.

It is well established that reduction of blood supply, inadequate nutrition, and the loss of innervations are the most common causes of oral epithelium atrophy.[18] SCA patients are more susceptible to develop oral epithelium atrophy due to the pathophysiology of the disease that includes anemia and vasculopathy, resulting in microvascular obstruction by sickle cells, preventing blood flow to tissue. Oral epithelium atrophy may affect the balance between protein synthesis and degradation, giving rise to abnormalities in cell structure and keratinization pattern of the oral epithelium. Other cause of oral epithelial atrophy is the deficiency of B12 vitamins[18] that is necessary for the synthesis of red blood cells.

Children with SCA require a combination of wide range therapies to control the main symptoms of the systemic condition. Besides adequate dietary intake of folic acid supplementation, the SCA therapy include: Prophylactic antibiotics (to reduce risk of infection and sepsis); up-to-date immunizations (to reduce risk of bacterial and viral infection); iron chelation therapy (to prevent blood iron overload); red blood cell transfusion (to replace blood volume lost by hemorrhage and splenic sequestration or to increase the ability of carrying oxygen in cases of anemia exacerbations); hydroxyurea and butyrate derivatives (to increase the concentration of fetal hemoglobin and to reduce painful vaso occlusive crises).[1920]

The effect of hydroxyurea on oral epithelium cells remains unknown until now. Only two patients in this study were using this medication and no studies have demonstrated oral collateral effects induced by hydroxyurea. Hydroxyurea (antineoplastic agent), butyric acid, and butyrate derivatives have demonstrated rapid stimulation of fetal globin expression. These drugs increase the concentration of fetal hemoglobin and decrease the proportion of HbS, leading to a reduction of the painful attacks rate in sickle-cell disease. A total of nine patients had received one or more blood transfusions in this study. The blood transfusion may be necessary to replace blood volume lost; however, periodical transfusions can cause sensitization and should be used with caution, in addition to iron overload and risk of infection.

In general, these treatments improve the overall systemic condition and quality of life of patients with SCA and this gain probably is also reflected in the oral mucosa.

Morphologically, all smears of both SCA and C groups were classified as class I of Papanicolaou system for cytology. In the original Papanicolaou classification system, class I is defined as the absence of atypical or abnormal cells.[1011] This result was expected because all smears were obtained from the region of clinically normal mucous membrane (posterior upper buccal mucosa).

The study suggests that individuals with sickle cell anemia, regardless of clinically visible oral lesions, show cytological changes in oral mucosal epithelium.

Further clinical and cytological studies should be performed simultaneously to investigate whether children with sickle cell anemia have an increased susceptibility to develop oral lesions due to atrophy of the epithelium cells of the oral mucosa.

Conclusion

In conclusion, this study revealed that sickle cell anemia was able to induce significant changes on NA of oral epithelium cells.