PRACE ORYGINALNE Małgorzata Żyła 3 Małgorzata Leśniak 3 Dorota Myszkowska 1 Ewa Dąbal 3 Justyna Lorenc 3 Maria Wojtaszek-Czapla 2 Krystyna Obtułowicz 1 Ewa Czarnobilska 1 Eosinophilic reaction in nasal cytology in patients sensitive to perennial and seasonal allergens Odczyn eozynofilowy w cytologicznym wymazie z nosa u osób uczulonych na alergeny całoroczne i sezonowe 1 Zakład Alergologii Klinicznej i Środowiskowej Katedra Toksykologii i Chorób Środowiskowych Uniwersytet Jagielloński Collegium Medicum Kierownik: Dr hab. Ewa Czarnobilska 2 Oddział Kliniczny Chorób Wewnętrznych Poradnia Alergologiczna Szpital Uniwersytecki w Krakowie Kierownik: Dr hab. Ewa Czarnobilska 3 Studenckie Koło Naukowe, Wydział Lekarski Uniwersytet Jagielloński Collegium Medicum Additional key words: Allergic rhinitis intermittent and persistent rhinitis seasonal and perennial allergens nasal mucosa exfoliative nasal cytology skin prick test Dodatkowe słowa kluczowe: alergiczny nieżyt nosa nieżyt okresowy i przewlekły alergeny sezonowe i całoroczne błona śluzowa nosa cytologia złuszczeniowa nosa test skórny punktowy Adres do korespondencji: Dr Dorota Myszkowska Zakład Alergologii Klinicznej i Środowiskowej CMUJ 31-531 Kraków, ul. Śniadeckich 10 48 12 424 88 98, 48 12 423 11 22; e-mail: dorota.myszkowska@uj.edu.pl Nasal exfoliative cytology is a complementary tool in diagnostics of allergic (AR) and non-allergic (NAR) rhinitis. The aim of the study was to determine the usefulness of the nasal cytology in patients sensitive to common inhalant allergens with positive SPT(+) and negative SPT(-) (Skin Prick Tests) depending on the symptoms of intermittent and persistent rhinitis. The study was performed in a group of 285 patients treated in the Department of Allergology University Hospital in Krakow, suspected on AR in 2008-2010. The patients were made a smear test of inferior nasal concha. The samples were stained using the eosin-hematoxylin method and examined under a light microscope (1000 ). Patients were divided into two groups: SPT(+) (144 patients) and SPT(-) (141 patients). Depending on the percentage of obtained eosinophils each group was divided into three subgroups: 0-2%, 3-20%, >20%. In the most percentage of patients with 3-20% of eosinophils in nasal cytology were found, in both studied groups (SPT)(+) and (SPT)(-), while the highest percentage of eosinophils (>20%) was observed in the bigger group of patients with SPT(+), than with SPT(-). The number of patients with eosinophils >20% in the SPT(+) group was higher in patients with persistent symptoms (NS differences), while in the SPT(-) group, the number of patients with intermittent symptoms in the subgroup >20% of eosinophils statistically prevailed (p<0.001). The mean percentage of eosinophils in both groups was comparable, while the statistically significant differences were found considering the distinguished subgroups. In intermittent SPT(+) group the most sensitizing allergens were pollen grains (birch or grass pollen), while the patients with persistent AR symptoms were mainly sensitive the house dust mites. The mean percentage of eosinophils in an exfoliative cytology correlated significantly with allergic rhinitis symptoms and SPT results, the most evident relationship was found between higher Jednym z narzędzi uzupełniających diagnostykę alergicznego ANN i niealergicznego nieżytu nosa (NNN) jest cytologia złuszczeniowa (eksfoliatywna) błony śluzowej nosa. Celem pracy było określenie przydatności badania cytologicznego błony śluzowej nosa u pacjentów uczulonych i nie uczulonych na typowe alergeny wziewne, w zależności od objawów przewlekłego lub okresowego nieżytu nosa. Badania zostały przeprowadzone u 285 pacjentów skierowanych w latach 2008-2010 do Poradni Alergologicznej Szpitala Uniwersyteckiego w Krakowie z podejrzeniem ANN. U badanych wykonano wymaz z małżowiny nosowej dolnej metodą złuszczeniową. Preparaty z pobranym materiałem wybarwiano metodą hematoksylina-eozyna i oceniano je pod mikroskopem świetlnym (komórki zliczano pod powiększeniem 1000 ). Chorych podzielono na dwie główne grupy pod względem wyników testów skórnych: SPT(+) - 144 pacjentów, u których w skórnych testach punktowych potwierdzono alergię IgEzależną na alergeny całoroczne i/ lub sezonowe oraz grupę 141 pacjentów (z ujemnymi wynikami testów skórnych, SPT(-). Każdą z grup podzielono w zależności od odsetka eozynofilów w wymazie cytologicznym na trzy podgrupy: 0-2%, 3-20%, >20%. Pacjenci z eozynofilią 3-20% w wymazach cytologicznych stanowili generalnie najwyższy odsetek w obu grupach SPT(+) i (SPT)(-), natomiast pacjenci z eozynofilią >20% przeważali w grupie SPT(+) (p<0.001). W grupie SPT(+) liczba pacjentów z eozynofilią >20% była wyższa u pacjentów z przewlekłymi objawami ANN (różnice nieistotne statystycznie), natomiast w grupie SPT(-) wśród pacjentów z eozynofilią >20% przeważali objawy okresowego ANN (p<0.001). Średni odsetek eozynofilów był porównywalny w grupach SPT(+) i SPT(-), jednak w grupie >20% wyższy znamiennie odsetek eozynofili stwierdzono u pacjentów z SPT(+). W grupie pacjentów z okresowym ANN najwięcej pacjentów było uczulonych na pyłek roślin (brzoza lub trawy), a 1038 M. Żyła i wsp.
level of eosinophils and the patients with confirmed AR diagnosis on the basis of positive SPT, manifesting the intermittent symptoms. wśród pacjentów z przewlekłym ANN jako czynniki uczulające dominowały alergeny roztoczy. Odsetek eozynofilów w wymazie jest istotnie skorelowany z występowaniem objawów i wynikami testów skórnych, jednak związek ten jest najbardziej widoczny w grupie pacjentów z podwyższoną eozynofilią w okresowym ANN, potwierdzoną dodatnimi wynikami testów skórnych. Introduction Allergic rhinitis is an inflammatory nasal disorder, in which a range of different cells participates [6]. This type of rhinitis caused by a I type, IgE-mediated allergic reaction between the aeroallergen and the antibody bound up with the surface of the mastocytes [20]. There are two forms of AR: intermittent and persistent according to the European Academy of Allergy Clinical Immunology (EAACI) recommendations [1]. In patients with intermittent AR, symptoms such as sneezing, serous secretion, pruritus and nasal blockade commonly coexist with allergic conjunctivitis [25,27] and sometimes with paroxysmal dyspnea typical for atopic asthma [9,10,27]. Above mentioned symptoms of AR and allergic conjunctivitis are significantly connected with exposure to seasonal aeroallergens [10,19,27], so these manifestations occur every year at the same time [9,27]. Transient character, quick symptoms receding after the isolation of allergen indicates AR caused by pollen allergens [9,27,32]. In patients with persistent AR the clinical symptoms are manifested weaker. Usually patients complain of nasal blockade [1,9]. Allergic conjunctivitis occurs occasionally [1], but a susceptibility to bronchospasm is observed more often [1,9]. Dust mites, animal fur and feathers can provoke the symptoms of persistent AR [10,19,32]. Exacerbation of symptoms occurs during housekeeping, home repairs or being in old, damp, dusty spaces [9]. Skin prick tests (SPT) are the basic tools used in AR in vivo diagnostics. In a roundabout way they prove the relationship between IgE antibodies and mastocytes in skin and the ability for degranulation after specific provocation [1,10]. Nasal exfoliative cytology could be a complementary tool in diagnostics of AR [1,5] and as additional examination is recommended by the Polish Standards of Allergology [2]. It is a test of smear of superficial cells of inferior nasal concha and involves sampling and recovering mucosal surface cells [7]. In opposite to histological examination, cytology is a noninvasive method used in the outpatient clinic [12,29]. It could be taken in every patient, every age and any time. At the first time nasal exfoliative cytology was applied by Eyerman and Hansel in 1927, who examined smears in 92 patients and confirmed the presence of all cells layed in nasal cava [5,15]. When the skin prick test could not be taken, cytology is the first examination if there is a suspicion of AR, but it must be confirmed by the skin prick test later [29]. The occurrence of eosinophils in allergic and non-allergic inflammation is confirmed [4]. The nasal cytology in children could be helpful in finding the difference between AR and bacterial inflammation of nasal mucosa [5]. The increase in percentage of eosinophils with negative skin prick tests and correct specific IgE antibodies serum level could indicates eosinophilic non-allergic rhinitis (NARES) [29]. Exfoliative cytology could be used in monitoring of persistent therapy of AR [5,29]. The aim of the study was to determine the usefulness of the nasal cytology in patients sensitive to common inhalant allergens with positive SPT(+) and negative SPT(-) (Skin Prick Tests) depending on the symptoms of intermittent and persistent rhinitis. Material and methods The study was performed retrospectively in a group of 285 patients (176 female, 109 male) at the age of 8-87 years (average 39) diagnosed in the Department of Allergology University Hospital in Krakow, suspected of AR in 2008-2010 (Table I). The patients subjected to a smear test of the inferior nasal concha and skin prick tests. The SPT was performed using allergen kits of Allergopharma. The positive score was wheal reaction 3 mm (positive control with histamine: 3 mm, negative control: 0 mm). The smear was taken from the inferior nasal concha, about 1 cm from the edge. The samples were fixed by Cytofix and stained using the eosin-hematoxylin method and examined under a light microscope (1000 ).The test results were expressed as a percentage of eosinophils in 100 inflammatory cells considering the number of analysed fields of view. The test results of cytology were compared with the SPT results, defined diagnosis and time of the examination using Excel 2007. Patients were divided into two groups: SPT(+) - 144 patients confirmed IgE-mediated seasonal and/or persistent allergy in skin prick tests and SPT(-) - 141 patients with negative skin prick tests. Depending on the percentage of obtained eosinophils each group was divided into three subgroups: 0-2% (normal level), 3-20%, >20%. It is thought, that the percentage of eosinophils in the persistent rhinitis ranges from 15-20% [18]. Patients were analyzed within the subgroups on the basis of diagnosis (intermittent and persistent AR) and, in case of SPT(+) group the most sensitizing allergens were distinguished. The Chi2 test was used to perform the statistical analysis of the scores (accepted p value < 0.05). To perform the statistical analyses GrafPadPrism packets were used. Results In both studied groups, SPT(+) and SPT(-) the percentage of eosinophils in the subgroup 3-20% was demonstrated in the most patients, while the highest percentage of eosinophils (> 20%) was observed in patients with SPT(+) in 39.58% of patients, than in the patients with SPT(-) in 7.80%, only (Table I, Figure 1). Moreover, the number of patients demonstrated the level of 3-20% of eosinophils in intermittent and persistent AR was comparable in the SPT(+) and SPT(-) groups. In intermittent and persistent AR the number of patients with SPT(+) and SPT(-) in the subgroup 0-2% was the same. The number of patients with eosino- Rycina 1 Liczba pacjentów z objawami okresowego i przewlekłego nieżytu nosa w grupach (SPT)(+) i (SPT)(-), w wyszczególnionych podgrupach w zależności od odsetka eozynofilów w wymazie cytologicznym. The number of patients with intermittent and persistent AR in SPT(+) and SPT(-) groups with regard to the three distinguished subgroups considering the percentage of eosinophils in the nasal cytology. Przegląd Lekarski 2013 / 70 / 12 1039
phils >20% in the SPT(+) group was higher in patients with persistent symptoms (NS differences), while in the SPT(-) group, the number of patients with intermittent symptoms in the subgroup >20% of eosinophils statistically prevailed (p<0.001) (Figure 1). According to the subgroup 3-20% of eosinophils, the number of patients with intermittent rhinitis dominated in both main study groups (p<0.001) (Figure 1). Generally, the mean percentage of eosinophils in both groups was comparable, Tabela I Charakterystyka grup pacjentów z SPT(+) i SPT(-) w odniesieniu do obrazu cytologicznego. Characteristics of the patient groups SPT(+) and SPT(-) in relation to the cytological picture. Subgroups (% of eosinophils) *Female; **Male No of patients SPT(+), n=144 Mean age intermittent AR persistent AR 0-2% 14 (9.72%) 34 ± 10 2 F */ 5 M** 4 F / 3 M 3-20% 73 (50.69%) 32 ± 10 23 F / 23 M 11 F / 16 M >20% 57 (39.58%) 35 ± 12 14 F / 12 M 19 F / 12 M Subgroups (% of eosinophils) No of patients SPT(-), n=141 Mean age intermittent AR persistent AR 0-2% 59 (41.82%) 45 ± 15 16 F/ 11 M 17 F/ 15 M 3-20% 71 (50.41%) 50 ± 14 37 F/ 9 M 14 F/ 11 M >20% 11 (7.80%) 46 ± 17 8 / 1 M 1 F/ 1 M 18.26% and 13.28%, respectively, while the statistically significant differences were found considering the distinguished subgroups. The mean percentage of eosinophils in the nasal cytology was higher in the SPT(+) group, than in the SPT(-) referring to the subgroup >20% (p<0.001), in opposite to the subgroups 0-2% and 3-20% (Figure 2). In the intermittent SPT(+) group the most sensitizing allergens were pollen grains of grasses and birch, depending on the subgroup. On the other hand, the sensitivity to house dust mite allergens dominated in patients with persistent AR in all subgroups (Figures 3,4,5). Discussion The exfoliative cytology is helpful to understand the patomechanisms of symptoms in certain disorders taking part in nasal cavities and sinuses and is able to appraise therapeutical intervention (glucocortycosteroids treatment, immunotherapy) [5,9]. The relationship between symptoms of AR and inflammatory cells was showed in the experimental treatment performed in guinea pig (Cavia porcella), where an exfoliative cytology and washings of nasal mucosa proved the increased number of eosinophils, neutrophils and monocytes [25,30]. In prospective trials of infants with Rycina 2 Średni odsetek eozynofilów w wymazie cytologicznym w trzech podgrupach w ramach grup: SPT(+) i SPT(-). The mean percentage of eosinophils in the nasal cytology in three subgroups, in frame of SPT(+) and SPT(-) groups. Rycina 3 Rozkład uczulających alergenów w podgrupie 0-3% eozynofilów w grupie SPT(+) Distribution of sensitizing allergens in the subgroup of 0-3% eosinophils in the SPT group(+) with intermittent AR. Rycina 4 Rozkład uczulających alergenów w podgrupie 3-20% eozynofilów w grupie SPT(+) Distribution of sensitizing allergens in the subgroup of 3-20% eosinophils in the SPT group(+) with intermittent AR. Rycina 5 Rozkład uczulających alergenów w podgrupie >20% eozynofilów w grupie SPT(+) Distribution of sensitizing allergens in the subgroup of >20% eosinophils in the SPT(+) group with intermittent and persistent AR. 1040 M. Żyła i wsp.
the positive family history of allergy, the connection between the presence of eosinophils and basophiles in smears of nasal mucosa and an allergy was showed. These cells did not occur in cytograms in children at the age of four years without the family history (control group) [9,31]. Pipkorn et al. [26] reported that the increase in number of basophiles in the nasal cytology was noted from the beginning of the pollen season. Within 4-5 days of the pollen exposure the number and the percentage of basophiles increased significantly, but migration of the inflammatory cells was more intensive during the high pollen exposure [26]. In the other study performed in a group of adults with intermittent AR, eosinophils, in the nasal cytology, were observed in 81% of patients, basophiles in 42%, neutrophils in 64% and bacteria in 28% of patients [16]. It was reported, that the percentage of eosinophils in the nasal concha is connected with the natural exposition of inhalant allergens, because after the antigen provocation in patients with allergy, the statistically significant increase in migration cells (neutrophils, eosinophils and monocytes in nasal mucosa was noted [8,13]. The migration of eosinophils could be induced by products of lymphocytes T, granulocytes: IL-5, LTB4, PAF, LTE 4. IL-3, IL-5 or GFR, which could extend the survival and eosinophils activation [4,21]. Meltzer et al. in the multi-subject study on intermittent AR, proved the presence of eosinophils of 64-86% in all patients in a certain age group [16,17]. The occurrence of eosinophils in a nasal swab is connected with the positive skin prick tests [12,23,27]. However, the skin prick tests measure the reaction of skin on histamine influenced by allergen. Skin prick tests could fail in case of using antihistamines, they are of a medium evidence for connection between IgE antibodies and mastocytes and their degranulation. However, SPT is a far trial of the provocation test of the shock organ-nasal concha [9,10]. It could be positive in fact of nonclinical symptoms, such as in adolescent [6]. In this case, it was confirmed that SPT precedes symptoms, because in 80% of these patients the allergy symptoms could be positive in five next years. In the most of atopic inflammations eosinophils are involved in the persistent allergic process [9]. There is a group of patients characterized by the negative family history of allergy, negative SPT, IgE antibodies in the range, swollen of the nasal concha and the nasal blockage felling with remission for a long time. These symptoms are diagnosed as non-allergic eosinophilic rhinitis (NARES) [11]. The positive relationship between the mean percentage of eosinophils and the following symptoms of AR: rhinitis, sneezing, swollen of the nasal concha with the blockage was shown [14,25]. The exacerbation of these symptoms is connected with the presence of basophiles-cells of the late hypersensitivity reaction [3]. Some authors stated, that the higher inflammatory cells migration could be related to more active exposition on the increasing seasonal allergen concentration in intermittent AR and the decreasing persistent allergen count in persistent AR [15]. Ouyang et al. [24] comparing the group of patients sensitive to pollen allergens manifested the seasonal symptoms, with patients with persistent symptoms confirmed the exacerbation of symptoms in the pollen season in both groups. In intermittent AR the symptoms of rhinitis with allergic conjunctivitis were stronger expressed than in persistent AR. The results reported by Obtułowicz et al. [22] indicated, that even at the time of symptoms remission in patients with AR (winter), eosinophils are observed in the nasal cytology, but the highest percentage of these cells was found at the time of symptoms intensity. The seasonal appearance of symptoms in case of intermittent AR is connected with the exposition to plant pollen [10,19,27]. The hypersensitivity to dust mites have a whole year (perennial) character [19,20]. Patients complained of the intensity of symptoms in the autumn-winter period, when central heating is working [28]. Then house dust mite allergens start to circulate indoor, especially in such reservoirs as mattresses beds and bedding [9]. Animal epidermis (fur) could be an intermittent or persistent allergen. It is connected with the sporadic or permanent presence of animal allergen [19]. In the present paper, the sensitivity to house dust mites dominate in patients witch persistent symptoms, both in a group of higher and normal eosinophilic response. On the other hand, in patients with intermittent symptoms, the higher eosinophils level was connected with the sensitivity to grass allergens. Conclusions Regardless of the SPT results, the highest percentage of patients demonstrated the above normal percentage of eosinophils in the nasal cytology on the level 3-20%, especially in patients with the intermittent AR symptoms. negative skin prick tests, rarely generated the local eosinophils response. The higher percentage of eosinophils (>20%) was observed more frequent in patients with positive SPT, what indicates that the intensive eosinophils response can diversify the patients with positive and negative SPT results. In patients with intermittent symptoms in the SPT(+) group, who demonstrated the higher level of eosinophils, the most sensitizing allergens were grasses. Cytology of the nasal mucosa, which is the simple and noninvasive test, can be used as a complementary tool in diagnostics of AR, but the most evident relationship was found between higher level of eosinophils and the patients with confirmed AR diagnosis on the basis of positive SPT, manifesting the intermittent symptoms. Conclusions 1. Regardless of the SPT results, the highest percentage of patients demonstrated the above normal percentage of eosinophils in the nasal cytology on the level 3-20%, especially in patients with the intermittent AR symptoms. negative skin prick tests, rarely generated the local eosinophils response. 2. The higher percentage of eosinophils (>20%) was observed more frequent in patients with positive SPT, what indicates that the intensive eosinophils response can diversify the patients with positive and negative SPT results. 3. 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