ORIGINAL ARTICLE

Utility of the Sputum Cytology Applying MGG and Pap stains in

Monitoring Sudanese Patients Complaining of Bronchial Asthma
ANASS M ABBAS1,2, MANAR G SHALABI1, ABD EL GHANY A MOUSTAFA4, JEREMY MILLS5, ABOZER Y
ELDERDERY1,3, ASAAD MA BABKER 6
1
Clinical Laboratory Sciences Department, College of Applied Medical Sciences- Jouf University, KSA.
2
Medical Laboratory Sciences Department, School of Medicine ,Alyarmouk Medical College, Khartoum, Sudan.
3
Faculty of Medical Laboratory Sciences, Department of Hematology, University of El Imam El Mahdi, Sudan.
4
Histology Department, Faculty of Medicine, Al-Azhar University, Damietta, Egypt.
5
School of Pharmacy and Biomedical Sciences, University of Portsmouth, UK.
6
Department of Medical Laboratory Sciences, College of Health Sciences, Gulf Medical University, Ajman, United Arab Emirates.
Corresponding Author: Asaad MA. Baker, Associate prof. of Hematology, Department of Medical Laboratory Sciences, College of Health
Sciences, Gulf Medical University, Ajman, United Arab Emirates.
Email id - azad.88@hotmail.com, Contact number – 00971527900035

ABSTRACT
Objectives: This is a case control study aimed at detecting cytological changes in diagnosed asthmatic patients,
using sputum samples collected and stained by cytological methods.
Materials and methods: The study was carried out on 80 individuals, of whom 30 were normal as controls, and
50 were asthmatic patients. Along with clinical data cytological, patterns in asthmatic patients were mapped using
two stains, Papaniclauo (PAP) and May-Grunwald Giemsa (MGG).
Results: Inflammatory response changes were reported in 18 cases (36%) compared with 4 controls (13%).
Inflammatory responses with metaplasia and metaplasia changes without inflammatory response were seen
within these 18 cases, with asthmatic patients with 27 cases (54%) and 2 cases (4%) respectively, (p < 0. 01).
Inflammatory response, both with and without metaplasia, was reported in 32 (64%;p <0.05) asthmatic patients
suffering from environmental allergens with respiratory co-infections. Inflammation with metaplasia was also found
to be significantly higher (p < 0.05) in females. The PAP stain gave excellent and clear results for screening of
sputum.
Conclusion: Sputum cytology is a valuable tool. The PAP stain is suggested to be used as a cytological stain
over the MGG stain. Further research is needed to confirm our findings and then to determine whether the
assessment of cytological patterns in sputum of asthmatic patients is useful or not for the routine monitoring of
asthmatic patients.
Keywords: Metaplasia, Allergy, inflammatory airways, Eosinophilic asthma, environmental allergens.

INTRODUCTION sputum compared to sputum eosinophils cationic protein

Asthma is a chronic disease of the respiratory system in (ECP) reinforces the role of cytological analysis in
which the airway constricts, becomes inflamed and lined diagnosis of chronic stable bronchial asthma [7] . A
with excessive amount of mucus, often in response to one Japanese study suggested that the participation of damage
or more triggers such as exposure to allergens, exercise, to the respiratory epithelium in the development of airway
emotional stress and (commonly in children) viral illness [1]. hyper-responsiveness in bronchial asthma, and the
Globally, more than 300 million patients have asthma with detection of desquamated respiratory epithelium (creola
approximately 250,000 asthma-associated deaths [1-3]. The bodies) in sputum is straightforward for estimation of airway
immunopathologic features include denudation of airway hyper-responsiveness [8]. Sputum sampling is a less
epithelium, collagen deposition beneath basement invasive method, and most previous studies used sputum
membrane, edema, mast cell activation and inflammatory to evaluate certain treatment or treatment strategy using
cells infiltration neutrophils (in sudden onset, fatal asthma sputum eosinophils count only without investigating the
exacerbations), eosinophils and lymphocytes [4]. Asthma cytological changes. To our knowledge there are no
can be diagnosed based on patient's history and previous studies reporting cytological patterns with regard
examination. Other methods used are peak flow meter to the cytological assessment of asthma [9].
which tests airway restriction, lung function tests,
radiological tests such as X-ray or computerized SUBJECTS AND METHODS
tomography (CT) scan which are required to exclude the This is a cross-sectional, case-control study which was
possibility of other lung diseases, and blood tests including carried out at Al-shaab chest hospital and Bahri teaching
eosinophil count [5]. hospital, Khartoum, Sudan. Fifty asthmatic patients and
Changes in sputum eosinophil count predicts loss of thirty healthy individuals were matched and used as
asthma control, suggesting firstly that sputum is a controls were enrolled in this study: a total of eighty
potentially useful marker in predicting loss of asthma participants. Age ranged from 15 to 71 years (mean 37
control reflected by loss of airway function and secondly years). Each case and control were provided with sputum
that sputum eosinophils seem to be valid markers for container and asked to collect early morning sputum before
detecting asthma in populations of patients with airway [6] . food is eaten or toothpaste used to eliminate
Moreover, the higher diagnostic accuracy of eosinophils in contamination. Sputum was collected from asthmatic

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Utility of the Sputum Cytology Applying MGG and Pap stains in Monitoring Sudanese Patients Complaining of Bronchial Asthma

patients from all age groups, both genders, all being frequency at 2 (4%) in female asthmatic cases and absent
nonsmokers. in male patients, although this was not a significant
The Institutional Ethical Committee of Ministry of difference (Table 2). 6% of asthmatic cases had no
Health approved the study and written consent was cytological changes
obtained from all the participants involved. All the clinical Inflammation and inflammation/metaplasia profiles
data and history of triggers factors were taken from the were significantly more common in 15–45-yearolds (28%
patient records and 34% respectively) than in those over 45 (8% and 20%
Sample preparation and staining: The clinical respectively, p<0.05). This suggests that the age may play
specimen was decanted into a Petri dish in which the a protective role in reducing asthma in older people.
sputum can be teased out using disposable sticks so that Furthermore, the metaplasia profile was found in 4% of the
bloodstained, discolored and purulent areas can be patients aged 15–45 as opposed to 0% of the over-45
selected and then placed on two glass slides. The material patients. No cytological changes were observed in the
was spread evenly over the slides. One smear was fixed in older age group with 6% of the 15–45 aged asthmatic
95% alcohol for 30 min to be stained in PAP stain and the patients also having no cytological changes.
other allowed to air dry followed by fixation in methanol for Stratifying the cytological changes by occupation
10 min. shows that this way the housewife group has the highest
For PAP staining, the smear was hydrated in 70% incidence at (42%), with students and workers (at ~22%
alcohol for 2 mins and rinsed in water for 1 min. After that it and 14% respectively) are slightly more likely to be
was stained in Harris' hematoxylin for 5 mins, rinsed in diagnosed with asthma than doctors, nurses and
water for 1 min, differentiated in 0.5% aqueous hydrochloric merchants (Table 3). There is an increased incidence of
acid for approximately 10 seconds and again rinsed in the inflammation and inflammatory metaplasia profiles in
water for 2 mins. Bluing was the second step, done in the groups (26% and 14% housewife, 8% and 12%
Scott's tap water substitute for 2 mins and then rinsed in students and 10% and 4% for workers respectively) Other
water. Next, the smear was dehydrated in 70% and two occupations show low but varying incidence of
changes of 95% alcohol respectively for 2 mins each. After inflammation and inflammatory metaplasia.
dehydration it was stained in Orange G 6 dye (OG6) for 2 With regards to trigger factors reported by the
mins and rinsed in two changes of 95% alcohol for 2 mins asthmatic patients, they were categorized into three types:
each. Finally, it was stained in Eosin Azure 50 (EA50) for 3 environmental allergens (smoke, pollen, dust, animals, bird
mins and rinsed in 95% alcohol for 1 min. remnants and cold) with respiratory system co-infections,
For the MGG staining, the fixed smear was stained in environmental with physiological stress, and environmental
the diluted May-Grunwald solution for 10 mins and then with food allergens. Inflammatory response profiles, both
rinsed in pH 6.8 buffer for 10 mins, then rinsed in the with and without metaplasia, were reported within 32
diluted Giemsa solution for 30 mins, washed and patients (64%) and were significantly higher (p<0.05) in
differentiated in pH 6.8 buffer for 5-20 mins. Finally, the asthmatic patients suffering from irritation due to the
smear was allowed to dry and mounted in DPX [0]. environmental allergens with respiratory co-infections
The sputum smears were examined by the (Table 4). With 40% and 20% showing
investigators, and then by two independent expert inflammatory/metaplasia and metaplasia alone profiles
cytopathologists to verify the results. The staining results respectively versus 10% and 12% in those patients in the
were evaluated, and statistical analyses were carried out environmental with physiological stress group. No
using SPSS software. cytological change was found in just 6% of all asthmatic
patients, with them showing no distinct cytological profile.
RESULTS The treatment of asthmatic patients can be by
In this study cytological patterns were categorized into inhalation, tablets, injection, or a combination of therapeutic
three microscopic findings: inflammatory response smear, agents. Within this study population, the inhalation route
inflammatory response with metaplasia changes and was reported to be the most frequently used by 35 patients
metaplasia changes only. When comparing our findings (70%), of whom the patients demonstrating inflammatory
within sputum form the asthmatic patients and matched response with metaplasia, inflammatory response only, and
controls, the inflammatory response profile was reported in metaplasia changes without inflammation were at 34%,
18 cases (36%) compared with 4 controls (13%), while 28% and 2% respectively. The combination treatment with
inflammatory response with metaplasia and metaplasia inhalation and tablets was used by 14% asthmatic patients
changes only profiles were observed in 27 (54%) asthmatic followed by 8% using injections (Table 5).
patients and 2 (4%) matched controls respectively, A high number of cases had a family history of
(p<0.01). Within the 18 inflammatory response profile case asthma at 29 (58%), while 21 (42%) have no family history.
group smears were mostly eosinophilic in cellularity with Cases with family history demonstrated a higher significant
considerable smears of eosinophilic / neutrophilic infiltrates (P<0.05) inflammatory response and inflammatory
and few smears contained a mixed acute and chronic response with metaplasia. Metaplasia appeared on those
inflammatory cell compared with only neutrophilic who have or have no family history, (see Table 2).
inflammatory smear in control group. See Table 1 and A high number of cases had a family history of
Figure 1 asthma at 29 (58%), while 21 (42%) have no family. Cases
Interestingly, the presence of inflammation/metaplasia with family history demonstrated a higher significant
was found to be significantly higher (p< 0.05) in females (P<0.05) inflammatory response and inflammatory
compared to males. Metaplasia was found with low response with metaplasia profile whereas the metaplasia

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 Anass M Abbas, Manar G Shalabi, Abd El Ghany A Moustafa et al

alone profile appeared on those who have or have no Considering that inflammatory response can be
family history. attributed to many factors other than asthma, cases
Of the Cytological stains used PAP stain gave better belonging to the age groups between 15 and 45 have more
results than MGG stain in the demonstration of cytological contact with the external environment making them more
patterns with significant value (0.036), While MGG was susceptible to developing recurrent infections; this may
observed to provide excellent visualization of inflammatory lead to metaplasia. Family history is a major predisposing
cells. factor to asthma assuming that the same factors may
Of the cytological stains used PAP stain gave better increase the propensity of an asthmatic patient to
results than MGG stain in the demonstration of cytological experience an attack, those concluding that for over 300
patterns with significant value (p=0.036 While MGG was million asthmatics worldwide and roughly 250,000 asthma-
observed to provide excellent visualization of inflammatory associated deaths, the family history is one of the major
cells. risk factors with other types of allergies, such as allergic
rhinitis or eczema [1,3,5].
DISCUSSION In regard to the trigger factors and allergens; our
Advances in Knowledge: Most previous studies have findings showed the inflammatory response only and
used sputum to evaluate asthma status or treatment inflammatory response with metaplasia demonstrated
strategy via eosinophil count without reporting full a mostly in patients suffering from irritation due to
cytological profile. To our knowledge there are no previous environmental factors with respiratory co-infections. This
studies reporting full cytological patterns as reported here finding in accordance with the review study of Wilmore and
Application to Patients Care: Sputum cytology using Pap David who’s concluded that infections as mediated risk
and MGG stains are a valuable tool in monitoring factor for induction and exacerbation of asthma
[17].Moreover, this finding is reinforced by studies reporting
Sudanese patients complaining of bronchial asthma.
Moreover, the sputum cytology can be introduced as a sensitization to staphylococcal enterotoxins associated with
follow up protocol in asthmatic patient's management and an increased subsequent risk of severe asthma and
treatment. asthma exacerbations [18].Interestingly, regarding use of
Comparing cases and controls, the inflammatory therapeutics and treatment route in the asthmatic
response changes reported mostly in cases, while population, our study found patients use inhalation
inflammatory response with metaplasia and metaplasia treatment most frequently, and show higher cytological
changes was reported only in the cases. Our findings are findings as inflammatory response with metaplasia,
supported by Revez, et al. who reported the dramatic inflammatory response only, and metaplasia changes. To
increase in sputum inflammatory cells subtype in asthma our knowledge, there is no previous study reporting any
after exposure to the allergen [11].Another study detected cytological changes and the type of therapy used .Finally,
the squamous cell metaplasia changes among asthmatic what the study showed about the better quality of PAP
smokers' group more than non-smokers. Moreover, an stain and its more preferable in sputum cytology is
interventional study aimed at control and treatment of reinforced by other studies. These concluded that PAP
severe refractory asthma in patients using bronchial stain is excellent for the nuclear details. MGG stain showed
thermoplasty showed that the presence of squamous less air-drying artifacts compared to wet fixed smears and
metaplastic epithelium with other histopathologic has better optimal cytoplasmic feature [19-21]. However,
parameters was significantly improved and its percentage Arul, et al.stated; no differences between the stains used in
reduced after this type of control [12]. detecting micronucleus in the exfoliated buccal epithelial
In this study, the younger patient group demonstrated Cells [22].
the higher proportion of inflammatory changes (28%, 14 Limitations and Recommendations: This work can act as
patients), inflammatory with metaplasia (34%, 17) and a pilot study for further research to confirm our findings and
metaplastic changes (4%, 2) compared with the >45 age determine whether the demonstration of different
group. These findings are in accordance with a study which characteristic cytological patterns in sputum of asthmatic
found severe asthma patients aged ≥65 years had patients is useful or not for the diagnosis of asthma in the
improved symptom control, better asthma quality of life first place.
and, in the last year, fewer emergency visits and rescue
oral steroid courses [3 (1–6) versus 5 (2–7), p < 0.001] than CONCLUSION
severe asthmatics aged <65 years ref?. Blood eosinophils Sputum cytology is a valued tool for monitoring, follow-up
were also lower in the elderly group. Patients with severe and diagnosis of asthma. The PAP stain gives excellent
adult-onset asthma had similar symptom control, lung and clear results for screening of sputum and we
function and health-care utilization compared to severe recommend that it be used as a cytological stain over the
childhood-onset asthma. Adult-onset asthmatics had higher MGG stain. We recommend researchers be aware of all
blood eosinophils and were less atopic [13].Furthermore, hazards accompanied by sample collection and
there are other studies which report severe outcomes and preparation; they must follow all safety precaution.
sputum cytology inflammatory response; which is mostly Acknowledgement: Our special thanks and appreciation
with eosinophilic and neutrophilic cellular predominates to all those who helped us through their assistance and
were found in younger patients (< 21 years) and the guidance in performing our study. We would like to thank
childhood asthmatic patients group, without comparing their specially to staff of Al-shaab chest hospital and Bahri
findings with older age group [14-16]. teaching hospital, Khartoum, Sudan.

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Coflict of Interest: The authors declared no conflict of [11] Revez J, Killian K, O'Byrne P, et al. Sputum cytology during
interests. late‐phase responses to inhalation challenge with different
Funding: None. allergens. Allergy 2018, 73:1470-8.
[12] Pretolani M, Bergqvist A, Thabut G, et al. Effectiveness of
bronchial thermoplasty in patients with severe refractory
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FIGURE LEGENDS:
Figure 1. Cytological changes in patients with asthma
(A)Pap stain from sputum of asthmatic cases showing eosinophilic type inflammation (X400) , (B) MGG Stain of sputum
from asthmatic cases showing mixed inflammatory cells with squamous cells metaplasia (X400), (C) MGG stain from
sputum cytological preparation showing squamous cells metaplasia (X400), (D) Pap stain sputum cytological preparation
showing metaplastic squamous cells with numerous Inflammatory cells (X400), (E) MGG stain of sputum cytological prep
showing mixed acute and chronic inflammatory cells from chronic attack asthma patient (X400).

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 Anass M Abbas, Manar G Shalabi, Abd El Ghany A Moustafa et al

Figure 1. Cytological changes in patients with asthma

a b
Z
Z
Z
Z
Z
Z
Z
Z
Z
d Zc
(A)Pap stain from sputum of asthmatic cases Z
showing eosinophilic type inflammation (X400) Z ,
(B) MGG Stain of sputum from asthmatic cases Z
showing mixed inflammatory cells Zwith
squamous cells metaplasia (X400), (C) MGG Z
stain from sputum cytological preparation Z
showing squamous cells metaplasia (X400),Z(D)
Pap stain sputum cytological preparation Z
showing metaplastic squamous cells Zwith
numerous Inflammatory cells (X400), (E) MGG Z
e stain of sputum cytological prep showing mixed Z
acute and chronic inflammatory cells from Z
chronic attack asthma patient (X400).
Z
Z
Table 1. Comparing of the Cytological patterns between cases and control group Z
Study group No Cytological Cytological patterns Total
changes Z
N (%) Inflammatory /metaplasia Inflammatory Metaplasia N (%)
N (%) N (%) N (%) Z
Control 26 (86.6) (a)
0(0) 4 (13) 0(0) Z
30(100)
Cases 3 (6) 27(54%) (a) 18 (36%) 2 (4%) Z
50(100)
Total 29 27 22 2 80Z
Key: Significance determined by comparison between groups. (a) = P<0.05.*Statistical significance shown in parenthesis.Z
Z
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Z
Z
Utility of the Sputum Cytology Applying MGG and Pap stains in Monitoring Sudanese Patients Complaining of Bronchial Asthma

Table 2. Cytological patterns among asthmatic patients stratified by gender, age and family history
Gender No cytological All changes through gender Total
Change Inflammatory/metaplasia Inflammatory Metaplasia
No (%) No (%) No (%) No (%) No (%)
Male 2 (4) 10 (20) 10 (20) 0 (0) 22 (44)
Female 1 (2) (c) 17 (34) (a) 8 (16) 2 (4) 28 (56)(a)
Total 3 (6) 27 (54) 18 (36) 2 (4) 50 (100)
Changes Age Group Total
15 – 45 Yrs >45 Yrs
N (%) N (%) N (%)
Inflammatory 14 (28) (a) 4 (8.0) 18 (36)
Inflammatory/metaplasia 17 (34) (a) 10 (20.0) 27 (54) (a)
Metaplasia 2 (4) 0 (0.0) 2 (4)
No change 3 (6) 0 (0.0) 3 (6)
Total 36 (72) 14 (28.0) 50 (100)
Family history No Cytological Cytological patterns with family history cases Total
changes Inflammatory /metaplasia Inflammatory Metaplasia
N (%) N (%) N (%) N (%) N (%)
Present 1(2%) 15(30%) 12(24%) 1(2%) 29(58%)
Absent 2(4%) 12(24%) 6(12%) 1(2%) 21(42%)
Total 3(6%) 27 18 2 50
Key: Significance determined by comparison between groups. (a) = P<0.05.

Table 3. Relationship between cytological findings and patients' occupation

Cytological changes Total
Occupation No change Inflammatory/metaplasia Inflammatory Metaplasia
No (%) No (%) No (%) No (%) No (%)
Housewife 0 (.0) 13 (26)(a) 7 (14)(a) 1 (2) 21 (42)
Student 1 (2) 4 (8.0)(a) 6 (12)(a) 0 (.0) 11 (22)
Doctor 1 (2) 1 (2) 1 (2) 0 (.0) 3 (6)
Nurse 1 (2) 2 (4) 0 (.0) 1 (2) 4 (8)
Worker 0 (.0) 5 (10)(a) 2 (4) 0 (.0) 7 (14)
Merchant 0 (.0) 2 (4) 2 (4) 0 (.0) 4 (8)
Total 3 (6) 27 (54) 18 (36) 2 (4.0) 50 (100)
Key: Significance determined by comparison between groups. (a) = P<0.05.

Table 4. Relationship between trigger factors and cytological findings

No Cytological changes
cytological Inflammatory/metaplasia Inflammatory Metaplasia Total
Triggers
change N (%) N (%) N (%)
N (%) N (%)
Environmental/infections 1 (2.0) 20 (40.0) (a) 10 (20.0)(a) 1 (2.0) 32 (64.0)
Environmental /
1 (2.0) 5 (10.0)(a) 6 (12.0)(a) 0 (0.0) 12 (24.0)
physiological stress
Environmental/ food type
1 (2.0) 2 (4.0) 2 (4.0) 1 (2.0) 6 (12.0)
allergen
Total 3 (6.0) 27 (54.0) 18 (36.0) 2 (4.0) 50 (100)
Key: Significance determined by comparison between groups. (a) = P<0.05. *Statistical significance shown in parenthesis.

Table 5: Relationship between type of treatment route and cytological findings

Treatment No cytological Cytological changes
Total (n%)
changes(n%) Inflammatory/metaplasia(n%) Inflammatory (n%) Metaplasia(n%)
Inhalation 3 (6) 17 (34)(a) 14 (28)(a) 1 (2) 35 (70)
Tablets 0 (0) 1 (2) 1 ( 2) 0 (0) 2 (4)
Injection 0 (0) 3 (6) 0(0 .0) 1. (2) 4 (8)
Inhalation/ 0 (0)
5 (10) 2 (4) 0 (0) 7 (14)
Tablets
Inhalation/Injectio 0 (0)
0 (0) 1 (2) 0 (0) 1 (2)
n
Inhalation/ 0 (0)
1 (2) 0 (0) 0 (0) 1 (2)
Tablets/ Injection
Total 3 (6) 27 (54) 18 (36) 2 (4) 50 (100)
Key: Significance determined by comparison between groups. (a) = P<0.05.*Statistical significance shown in parenthesis.

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