I M P R E S S I O N C Y T O L O G Y ( I C ) I N C L I N I C A L P R A C T I C E T a r ı k Ş a p ç ı , M . D . * / A h m e t K a r a v a ş , M . D . * / H a le O n m u ş , M . D . * * M e l d a K a r a v a ş , M . D . * * * / U ğ u r G ü n t e r A k b u l u t , M . D . * * D e p a r t m e n t o f O t o r h i n o l a r y n g o l o g y , M e a d a n d M e e k S u r g e r y . P T T T r a i n i n g H o s p i t a l , İ s t a n b u l , T u r k e y . ** D e p a r t m e n t o f P a t h o l o g y , S S K G ö z t e p e T r a i n i n g H o s p i t a l , I s t a n b u l , T u r k e y . * * * D e p a r t m e n t o f P u b l i c H e a l t h , S c h o o l o f M e d i c i n e , M a r m a r a U n i v e r s i t y , İ s t a n b u l , T u r k e y . A B S T R A C T
O b je c tiv e : Nasal cytology is the most useful technique in differentiating infectious rhinitis from other types of rhinitis. Various techniques have been described fo r obtaining nasal cytology specim ens, including blowing secretions into wax paper; taking a sm ear by using cotton wool swabs or brushes; or perform ing nasal scraping, lavage, biopsy and impression cytology.
W e aim ed to com pare im pression cytology fin d in g s b e tw een allergic, va so m o to r and infectious rhinitis patients and the control group as fa r as e o sinop hils, neutrophils, basophilic cell, goblet cells and bacteria counts were concerned.
M a te ria l a n d M e th o d : In this study, we used an alternative method, namely Impression Cytology (1C) which is a noninvasive diagnostic procedure allowing the collection of a monolayer of cells from the epithelial or mucous surface. 1C was used in 28 allergic rhinitis, 21 vasom otor rhinitis and 23 infectious rhinitis patients. There were 22 control subjects in this study.
R e s u lts : IC was found to show significantly higher gradings of eosinophil counts and goblet cell counts in a lle rg ic rhinitis patients as
com pared to other groups (p<0.001); whereas neutrophil counts were found to be higher in the infectious rhinitis patients as compared to other groups (p<0.001). In the basophilic cell counts allergic rhinitis and the infectious rhinitis patients w ere found to p re se n t higher gradings as com pared to the control group (p<0.05). Bacteria invasion detected by IC was present only in the infectious rhinitis patients with a percentage of 26.1.
C o n c lu s io n : Our study has shown that IC is a very valuable and practical technique, offering a valuable alternative test for investigating cytology in all rhinitis and other pathologies causing cytologic changes.
K e y W o r d s : Impression cytology, rhinitis, eosinophil, basophilic cell, neutrophil.
IN T R O D U C T IO N
Nasal cyto lo g y is helpful in the differential d iagno sis of nasal co m plaints. V arious te ch n iq u e s have been used for obtaining, processing, evaluating, and interpreting nasal cytology s p e c im e n s (l). The selection of a sampling method depends on factors such as the
age of the p a tient, the need for repeated sampling, sim ultaneous biochem ical analysis.
Im pression cyto lo g y (1C) is a noninvasive diagnostic procedure allowing the collection of a m onolayer of cells from the epithelial or mucous surface. This technique was first adopted by Egbert and Thatcher (2,3) as a simple procedure for the cytological assessm ent of m orphological changes occurring in conjunctival cells and utilizes cellulose acetate filter paper.
In this study, we used an alternative method, namely 1C, instead of difficult and som etim es inaccurate nasal sm ear, nasal lavage and invasive nasal biopsy procedures. W e also aimed to com pare 1C findings between allergic, vasom otor and infectious rhinitis patients and the control group as far as eosinophils, neutrophils, basophilic cell, goblet cells and bacteria counts were concerned.
M A T E R IA L S A N D M E T H O D S
This study was carried out on 72 patients with rhinitis, who were treated in the Departm ent of O torhinolaryngology Head and Neck Surgery at Turkey.
The group of patients consisted of 28 patients with allergic rhinitis, 21 with vasom otor rhinitis and 23 with infectious rhinitis. The control group of the study included 22 volunteers who had no com plaints of rhinitis and none of the clinical criterias of rhinitis. The patients included 29 m ales and 43 fem ales, w ith ages ranging between 18-56 years. Control groups included 7 m ales and 15 fem ales, w ith ages ranging between 19-52 years. There were no statistically sig n ifica n t d iffe re n ce s betw een the allergic rhinitis, vasom otor rhinitis and infectious rhinitis patients and controls in age and sex distribution (p>0.05). All patients had a positive history, positive clinical e xam ination and positive laboratory tests.
Nasal mucosal im pression material was collected with a strip of cellulose acetate paper (0.22 m, M illipore P roducts C atalog G S W P 04700) of approxim ately 5x5 mm in size. Prior to the procedure, the strip was soaked in distilled w ater for 8 hours and dried at room tem perature. It was
then pressed onto the anterior surface of the inferior turbinate using alligator forceps and was held there for about 3 to 5 seconds, after which it was taken off by peeling (4) (Fig 1).
The procedure was carried out without topical anesthesia; once a layer of cells was pulled off, only a pricking sensation as accosionally felt by the patient.
S pecim ens w ere im m e d ia te ly fixed in 95% alcohol/5% water. Each specim en was stained with hem atoxylin and eosin (HE) and periodic a cid -S ch iff (P A S ). C yto lo g ia l change s w ere evaluated under light m icroscopy. All cyotolgical specim ens were exam ined by two independent o bservers. The e o sin o p h ils, neutroph ils, basophilic cells, bacteria and goblet cells seen on the Cytogram were evaluated grading between 0 and +4 as recom m ended by M eltzer (5). The Kolm ogorov-Sim irnow test was used in statistical analysis.
R ESU LTS
Im pression cyto lo g y sp e cim e n s were successfully obtained from inferior turbinates in 72 patients and 22 healthy adults w ithout the use of topical anesthesia. No local or system ic reactions were observed.
In the allergic rhinitis group, the predom inant le u ko cyte s in the nasal sp e cim e n s w ere eosinophils, am ounting to 96.4% (n=27) (Fig 2). In this group, 35.7% (n=10) neutrophils, 42.8% (n=12) basophilic cells and 100% (n=28) globlet cells were identified (Fig.4). In the evaluation of the va so m o to r rh in itis group no bacterial infiltration could be observed, whereas 66.6% (n=14) neutrophils, 42.9% (n=9) eosinophils, 9.5% (n=2) basophilic cells and 80.9% (n=17) goblet cells were identified. In the infectious rhinitis group, the predom inant leukocytes were neutrophils, am ounting to 100% (n=23) (Fig.3). H owever 39.1% (n=9) basophilic cells, 34.8% (n=8) eosinophils, 73.9% (n=17) goblet cells and 26.1% (n=6) bacterial infiltration w ere identified.
In the control group there were 40.9% (n=9) neutrophils, 86.4% (n=19) goblet cells but no e o sin o p h il, b a so p h ilic ce lls and bacterial infiltration were observed (Table 1-5).
IC in clinical practice
1C was found to show a significantly higher grading of eosinophil count in allergic rhinitis patients (96.4% ) as com pared to vasom otor rhinitis (42.9% ) and infectious rhinitis patients (34.8% ) and the control group (0%) (p=2.03x10_1°; p=7.6x10‘7; p= 1 .13x1 O'10).
As far as the neutrophil count was concerned, IC was found to show significantly higher gradings in patients with infectious rhinitis (100%) as com pared to allergic rhinitis (35.7%), vasomotor rhinitis patients (66.6% ) and the control group (40.9% ) (p = 3 .1 4 x 1 0 4; p = 6 .1 7 x 1 0 8 and p=4.12x10-8).
As far as the basophilic cell count was concerned a statistically significant difference was detected only between the control group and the allergic and in fe ctio u s rhinitis patients (p=0.0108, p=0.032). Basophilic cells were not detected in
any subject in the control group, however in 42.8% of the allergic rhinitis patients and in 39.1% of the infectious rhinitis patients, different grades of basophilic cell counts were observed.
When the goblet cell counts on IC technique were compared. Allergic rhinitis patients (100%) appeared to show significantly higher grades than the vasom otor (80.9%) and the infectious rhinitis patients (73.9%) and the control group (86.4% ) (p = 3 .7 7 x1 0 11; p = 5 .9 x 1 0 9; p = 1.96 x10'11).
For bacteria count IC however, the subject distribution did not allow the use fo an appropriate statistical test; only infectious rhinitis patients could be com pared with vasomotor and allergic rhinitis patients and the control group but the results were not found to be statistically significant (p>0.05).
F ig . l Cellulose acetate filter paper applied to the right inferior turbinate surface of the nose.
F lg . 3 . : Infiltration of neutrophils in the nasal mucosa of the Infectious rhinitis patient (HEx100).
F ig . 2 Infiltration of eosinophils in the nasal mucosa of the allergic rhinitis patient (HEx100).
F ig . 4 . : Goblet cells in the nasal mucosa of the allergic rhinitis patient (PASx100).
Table I. Comparison of the grading of eosinophil counts between the patient groups and the control group. C e lls Grade A lle rg ic rh in itis (n=28) V asom otor rh in itis (n=21) Infectio us rh in itis (n=23) Control group (n=22) n % n % n % n % None 1 3.6 12 57.1 15 65.2 22 100.0 Eosinophils 1 - 9 42.9 3 13.0 -2 8 28.6 - - 5 21.8 -3 4 14,3 - - - - -4 15 53.6 - - - - -Total 27 96.4 9 42.9 8 34.8 0 0
Allergic x Vasomotor p=2.03x1 O'10 Allergic x Control p=1.13x10 10
Allergic x Infectious p=7.6x10"7 Vasomotor x Control p=0.019
Table II. Comparison of the grading of neutrophil counts between the patient groups and the control group.
C e lls Grade A lle rg ic rh in itis (n=28) V asom otor rh in itis (n=21) Infectio us rh in itis (n=23) Control group (n=22) n % n % n % n % None 18 64.3 7 33.3 - - 13 59.0 Neutrophil 1 - 3 14.3 - - 3 13.6 2 3 10.7 11 52.4 3 13.0 6 27.3 3 5 17.9 - - 7 30.4 - -4 2 7.1 - - 13 56.5 - -Total 10 35.7 14 66.6 23 100.0 9 40.9
Allergic x Vasomotor p=0.034 Allergic x Control p=0.031
Allergic x Infectious p=3.14 x 10'4 Vasomotor x Control p=4.12x10-8 Vasomotor x Infectious p= 6.17x10-8
Table III. Comparison of the grading of basophilic cell counts between the patient groups and the control group.
C e lls Grade A lle rg ic rh in itis (n=28) V asom otor rh in itis (n=21) Infectio us rh in itis (n=23) Control group (n=22) n % n % n % n % None 16 57.1 19 90.4 14 60.9 22 100.0 Basophilic cell 1 4 14.3 2 9.5 4 17.4 - -2 6 21.4 - - 5 21.7 - -3 2 7.1 - - - -4 • - - - -Total 12 42.8 2 9.5 9 391 0 0 Allergic x Control p=0.0108 Infectious x Control p=0.032
1C in clinical practice
T a b l e I V . Comparison of the grading of goblet cell counts between the patient groups and the control group.
C e lls Grade A llerg ic rh in itis (n=28) Vasom otor rh in itis (n=21) Infectious rh in itis (n=23) Control group (n=22) n % n % n % n %
Goblet cells None - 4 19.0 6 26.0 3 13.6
1 - 5 23.8 - 16 72,7
2 - 12 57.1 14 60.9 3 13.6
3 18 17.9 - 3 130
-4 10 35.7 - -
-Total 28 100.0 17 80.9 17 73.9 19 86.4
Allergic x Vasomotor p=3 77 x 10-11 Allergic x Control p=1.96x10-"
Allergic x Infectious p=5.9x10-9 VasomotorxControl p=0.017
T a b l e V . Comparison of the grading of bacteria counts between the patient groups and the control group.
A llerg ic Vasom otor Infectio us Control
C e lls Grade rh in itis rh in itis rh in itis group
(n=28) (n=21) (n=23) (n=22) n % n % n % n % None 28 100.0 21 100.0 17 73.9 -Bacteria 1 - 3 13.0 2 - - 3 13.0 -3 - - - -4 - * - -Total 0 0 0 0 6 26.1 0 0
* p values that are found statistically significant in Kolmogrow-Simirnov test are shown underneath each table.
D IS C U S S IO N
Nasal cytology is the most useful in differentiating in fe ctio u s from o th e r types of rhinitis. M orphological changes in the nasal mucosa may reflect reactions that are also valid for other parts of the airway. Various techniques have been d e scrib e d fo r o b tainin g nasal cytology specim ens, including blowing secretions into wax paper; taking sm ear by using cotton wool swabs or brushes; or perform ing nasal scraping, lavage, and biopsy(6).
The se le ctio n of m ethod depends on he requirem ents of the specimen. Considerations include the age of the patient, the need for repeated sam pling, the site, depth, and thickness of the nasal mucosa, and the requirem ent for sim ultaneous biochem ical studies (5).
Interest in nasal cytology as a method for evaluatin g d ise a se and treatm ent has progressed. Several factors have, in the past, dim inished its usefulness: the variety of methods for grading results, the fact that eosinophils are not p a th o g n o m o n ic for allergy, the lack of available data on how to interpret findings, and the assum ption that the procedure was not cost- effective (5).
1C te ch n iq u e is a n o n inva sive diagnostic procedure. It allows the transfer of the nasal mucosal epithelium sam ple from the adhesive paper strip to a glass slide, saving time and reducing the cost of staining of the 1C procedure (4).
The normal nasal mucosal cytology of infants, children, and adults co n sists of num erous
e p ithelia l cells in cluding ciliated colum nar, nonciliated colum nar, goblet, and basal cells. An ade q u a te ly sam pled specim en w ill alw ays contain som e ciliated cells and goblet cells. There are usually no eosinophils or basophilic cells w ithin the sup e rficia l layer above the basem ent membrane. A moderate number of neutrophils and a few bacteria can be seen, especially if the specim en is taken from the anterior portion of the inferior turbinate (7-9).
Increased numbers of eosinophils are found in the nasal m ucosa in active allergic disease (4,7,10). The deg re e of nasal e o sin o p h ilia appears to correlate with the extent of allergen exposure and with sym ptom s in allergic rhinitis (5,11). Vasom otor rhinitis is used to describe a n o n im m uno logical, no n in fe ctio u s chronic rhinopathy. The majority of these patients identify physical Irritants and clim a tic changes as precipitants(12). In addition to the scarcity of eosinophils in patients with vasom otor rhinitis, there is also no increase in basophilic cells or plasma cells (13). Nasal cytology is also useful in evaluating patients for bacterial infections. Large num bers of bacteria, esp e cia lly in tra ce llu la r bacteria, increased num bers of neutrophils, lym phocytes, and plasm a cells support the diagnosis of infections (10,14).
In this study the dom inating cells in the allergic rhinitis patients were eosinophils (96.4%) and goblet cells (100.0%) and this group of patients was found to show higher grades of eosinophil and goblet cell count on IC as com pared to other groups of patients and as com pared to the control group (p<0.001). H ow ever in the infectious rhinitis patients the dom inating cell count was neutrophils (100.0% ) with these cells presenting as higher grades in IC as com pared to the allergic rhinitis and vasom otor rhinitis patients and the control group (p<0.001). The basophilic cell count showed to be statistically higher in the allergic rhinitis and the infectious rhinitis patients than in the control group (p<0.05). Bacterial invasion could only be detected in the IC of the infectious rhinitis patients with a percentage of 26.1. M eltzer et al (15) reports sim ilar findings in the study w here they used the scroping technique. Also sim ilar findings w ere found where techniques of nose-blowing, brush, lavage and biopsies were used (16-20). It is reported that the techniques give more or less sim ilar
findings, however, we believe clinicians must choose the m ethod with which the patients will cooperate more and which is the least invasive. Many children and patients with som e nasal disorders cannot produce an adequate secretion specim en, and also m ethods like brush and biopsy techniques are invasive. This is why we used an alternative method, nam ely IC.
To know the localization of cells collected by IC seem s to be very im portant for clinical diagnosis since their cellular detail is not disturbed because they are not desquam ated degenerate cells. This te ch n iq u e a llow s us to see not only the in fla m m a to ry ce lls but th e in tra e p ith e lia l infiltration and the relation with the epithelial cells. In the sam e patient, cells in the nasal secretion and the intraepithelial cells may not be sam e. D eterm ining the clinical stage of the disease evaluation of the intraepithelial cells is e sp e cia lly im p o rta n t. A lso the type of inflam m atory cell gives an indication of the etiology of the disease.
Intraepithelial cell infiltration, which can only be dem onstrated with IC, is a sign of active infection and we suggest that it can also be used in the control of therapy.
W e propose that IC is a quick, painless, non invasive, and in e xp e n sive te ch n iq u e . An additional advantage is that it provides early m orphological inform ation about nasal mucosal surfaces. This technique can also be perform ed on the pediatric population, w ithout too much difficulty. We recom m end that IC be used as a d ia g n o stic tool in p a tie n ts w ith rhinitis, to differentiate between clinical form s of rihinitis.
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