Sputum Eosinophil Level among Healthy Individuals Visiting Medicine Outpatient Department of a Tertiary Care Centre: A Descriptive Cross-sectional Study

ABSTRACT Introduction: Sputum differentia! is the most comprehensive, and non-invasive investigation for evaluating airway inflammation because of its reliability, reproducibility and responsiveness. The interpretation of the results of induced sputum examination depends on knowledge of normal values from a healthy population. So far, the reference values of sputum differential cells in Nepalese population are not reported. Neutrophil and eosinophil are used to assess the inflammatory response of the airway. The aim of our study was to detect the eosinophil level in the sputum of healthy individuals with normal pulmonary function. Methods: A descriptive cross-sectional study was conducted in a tertiary care hospital, from November 1, 2021 to December 31, 2021 after taking ethical clearance from Institutional Review Board (Reference number: 1507202106). Convenience sampling was done. Collected data was entered and analysed using Statistical Package for the Social Sciences version 24.0. Point estimate at 95% was calculated along with mean and standard deviation for continuous data. Results: Among 139 induced sputum samples, 7 (5.03%) had eosinophils present in their sputum (1.40 to 8.67 at 95% Confidence Interval). The age range of the participants was 18 years to 79 years with mean age of 39.5 ± 15.06. Prevalence of eosinophilia (percentage of eosinophil >3) was 4 (2.88%). Conclusions: The prevalence of eosinophilia in our study was found to be similar to published literature. The results of the present study show that there is a paucity of eosinophils with predominance of neutrophils, macrophages and lymphocytes in induced sputum samples of healthy non-smoker adults with normal pulmonary function test.


INTRODUCTION
Induced sputum is an useful investigation for evaluating the presence, type and degree of inflammation in the airways of lungs. 1 It helps to determine various phenotypes of asthma, 2-4 chronic bronchitis, 3 and study the effect of treatment on these airway diseases. [5][6][7] Sputum differential is the most comprehensive, noninvasive examination of airway inflammation because of it reliability, reproducibility and responsiveness. [8][9][10] Neutrophil and eosinophil are used to assess the inflammatory response of airway. 1 The interpretation of the results of induced sputum examination depends on knowledge of normal values from a healthy population but there have been only few such studies. [11][12][13][14][15][16] So far, the reference values of sputum differential cells in healthy Nepalese population are not reported.
The aim of our study was to detect the eosinophil level and to find reference values of normal sputum differential cell percentages in the sputum of healthy individuals with normal pulmonary function tests. Hence, the total sample size was calculated to be 133. However, 139 samples were taken.

METHODS
The participants who fulfilled the inclusion criteria underwent induced sputum collection with the help of inhalation of 3% hypertonic saline. The procedure was stopped if the subject experienced respiratory distress. Those with unsuccessful sputum collection or inadequate sputum after induction were excluded from the study. Treatment with inhaled bronchodilator was given to those subjects who developed respiratory distress during the procedure. Sputum sample was then preceded for examination of cytology. Sputum differential of neutrophil, macrophage, lymphocyte, and eosinophil was obtained from each sample in percentage. Eosinophilia is defined as ≥3% eosinophils in sputum. 19 Statistical Package for the Social Sciences (SPSS) version 24.0 was used for data entry and analysis. The percentage of sputum differential cell counts were expressed as arithmetic mean±SD, median and interquartile range (IQR). The normal range was expressed by 10 th and 90 th percentiles. Point estimate at 95% Confidence Interval was calculated.

RESULTS
Total 139 healthy adults who fulfilled the inclusion criteria were included in the study among which 7 (5.03%) had eosinophils present in their sputum ( The eosinophil count among induced sputum samples was mostly zero percent in 132 (94.96%), two percent in 3 (2.16%), three percent in 3 (2.16%) and eight percent in 1 (0.72%) individuals ( Figure 1). Prevalence of eosinophilia (percentage of eosinophil ≥3) was 4 (2.88%). Neutrophils were predominant cells in induced sputum with a mean of 56.54±16.65% and median of 60% (Table 2).

DISCUSSION
In this study, we found the eosinophil level and normal sputum differential cell percentage in the sputum of healthy non-smoker adults visiting medicine OPD of KMCTH with normal pulmonary function test by studying the sputum characteristics of 139 participants who fulfilled the inclusion criteria. Compared with previous studies, this study includes a larger number of participants and a broad range of all ages (18 to 79 years); moreover, the numbers of subjects are comparable in all ages.
The mean values in our sample are in agreement with the findings with other studies with healthy subjects. [11][12][13][14][15][16] The results of our study show that neutrophils predominate in sputum of healthy adults; macrophages and lymphocytes are less common, while eosinophils are seldom present which is similar to those in literature.
In our study, the percentage of eosinophils ranged from zero percent to 8%. The mean reference value of eosinophils was 0.16% which was comparable to studies conducted by Veras TN, et al. (0.1%), 16 Belda J, et al. (0.4%), 11 and Spanevello A, et al. (0.6%). 12 The reference values of percentage of eosinophil in sputum are lower than 2.0% in most studies, 11,12,14,16 but the values from Canada 13 and Korea 15 are above 2.5%, which may attribute to the inclusion of subjects with allergy history. Therefore, it is reasonable to define the abnormality of eosinophil counts if the percentage is above 2.5%, which might be suggestive of airway eosinophilic inflammatory conditions.
In our study, the proportion of neutrophils is 56.54%, which is higher than that reported in previous studies including Belda J, et al. (37.5%), 11

CONCLUSIONS
The prevalence of eosinophilia in our study was found to be similar to published literature. The present study is the first attempt to determine the eosinophil level of sputum differential counts in healthy Nepalese adults with a normal pulmonary function. The results of the present study show that there is a paucity of eosinophils with predominance of neutrophils, macrophages and lymphocytes in induced sputum samples of healthy nonsmoker individuals with normal pulmonary function. It is recommended to conduct a national multi-center study to establish the reference value of sputum cell counts for the Nepalese population.