10.14639/0392-100X-N0711
Objective. Characterising the eosinophilic profile represents the main step in chronic rhinosinusitis (CRS) endotyping. The aim of the study is to verify the correlation between different methods for tissue eosinophilia quantification. Methods. 33 CRS patients undergoing endoscopic sinus surgery and 30 controls undergoing non-CRS surgeries were enrolled. Blood venous sampling, nasal biopsy on uncinate process (UP), nasal cytology on inferior turbinate (IT) and middle meatus (MM) were performed.
Results. Differences in eosinophil count in blood (P=0.0001), UP (P&lt;0.0001), IT (P = 0.01) and MM (P = 0.0006) were significant between CRS cases and controls. A weak correlation was found between UP and blood eosinophil count (r = 0.34, P = 0.006) and between UP and IT eosinophil count (r = 0.30, P = 0.017). Moderate correlation between UP and MM (r = 0.51, P &lt; 0.0001) was shown. ROC analysis predicted eosinophilic CRS with an overall low sensitivity. Once allergic patients were excluded from the analysis, the sensitivity decreased for sampling on IT and increased for MM sampling.
Conclusions. This study suggests that MM cytology gives more accurate information on the degree of tissue eosinophilia. Replication in wide and unbiased cohorts is necessary to verify these results and define accurate thresholds.
application/xml
1827-675X
711
5
en
Paolo Castelnuovo
https://creativecommons.org/licenses/by-nc-nd/4.0
ACTA Otorhinolaryngologica Italica
cytology
Exploring the role of nasal cytology in chronic rhinosinusitis
Text.Serial.Journal
https://www.actaitalica.it/
40
Rhinology
2020-12-03
2020-02-25
2020-12-04
2020-12-04
368-376
1
Paolo Castelnuovo
2020/12/03
10.14639/0392-100X-N0711
368
Department of Otorhinolaryngology, University of Insubria and ASST Sette Laghi, Varese, Italy; Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy
1827-675X
5
cytology
376
Exploring the role of nasal cytology in chronic rhinosinusitis
https://www.actaitalica.it/article/download/711/465
40
ACTA OTORHINOLARYNGOLOGICA ITALICA 2020;40:368-376; doi: 10.14639/0392-100X-N0711




Rhinology


Exploring the role of nasal cytology in chronic
rhinosinusitis
Il ruolo della citologia nasale nella rinosinusite cronica
Stefania Gallo1,2, Francesco Bandi1, Andrea Preti3,4, Carla Facco5, Giorgia Ottini5, Federica Di Candia2,
Francesco Mozzanica3,6, Laura Saderi7, Fausto Sessa5, Marcella Reguzzoni4, Giovanni Sotgiu7, Paolo Castelnuovo1,2
1
  Department of Otorhinolaryngology, University of Insubria and ASST Sette Laghi, Varese, Italy; 2 Department of Biotechnology
and Life Sciences, University of Insubria, Varese, Italy; 3 Department of Otorhinolaryngology, IRCCS Multimedica, Milano, Italy;
4
  Department of Medicine and Surgery, University of Insubria, Varese, Italy; 5 Department of Pathology, University of Insubria and
ASST Sette Laghi, Varese, Italy; 6 Department of Biomedical and Clinical Sciences, University of Milan, Italy; 7 Clinical Epidemiology
and Medical Statistics Unit, Department of Biomedical Sciences, University of Sassari, Italy


SUMMARY
Objective. Characterising the eosinophilic profile represents the main step in chronic rhi-
nosinusitis (CRS) endotyping. The aim of the study is to verify the correlation between
different methods for tissue eosinophilia quantification.
Methods. 33 CRS patients undergoing endoscopic sinus surgery and 30 controls undergo-
ing non-CRS surgeries were enrolled. Blood venous sampling, nasal biopsy on uncinate
process (UP), nasal cytology on inferior turbinate (IT) and middle meatus (MM) were per-
formed.
Results. Differences in eosinophil count in blood (P=0.0001), UP (P<0.0001), IT (P = 0.01)
and MM (P = 0.0006) were significant between CRS cases and controls. A weak correlation         Received: February 25, 2020
was found between UP and blood eosinophil count (r = 0.34, P = 0.006) and between UP            Accepted: May 11, 2020
and IT eosinophil count (r = 0.30, P = 0.017). Moderate correlation between UP and MM
(r = 0.51, P < 0.0001) was shown. ROC analysis predicted eosinophilic CRS with an over-         Correspondence
all low sensitivity. Once allergic patients were excluded from the analysis, the sensitivity    Stefania Gallo
decreased for sampling on IT and increased for MM sampling.                                     Clinica Otorinolaringoiatrica, ASST Sette Laghi e
                                                                                                Università degli Studi dell’Insubria, via Guicciardi-
Conclusions. This study suggests that MM cytology gives more accurate information on
                                                                                                ni 9, 21100 Varese, Italy
the degree of tissue eosinophilia. Replication in wide and unbiased cohorts is necessary to     Tel. +39 0332 278426. Fax +39 0332 278945
verify these results and define accurate thresholds.                                            E-mail: stefania.gallo@me.com
KEY WORDS: rhinosinusitis, nasal polyps, rhinitis, eosinophilia, biomarker, cytology
                                                                                                Funding
                                                                                                None.
RIASSUNTO
Obiettivi. L’identificazione del profilo eosinofilico è uno step fondamentale nell’endoti-      Conflict of interest
pizzazione della rinosinusite cronica (RSC). Lo scopo dello studio è verificare il grado di     The Authors declare no conflict of interest.
correlazione tra le diverse metodiche di quantificazione dell’eosinofilia.
Metodi. Sono analizzati, per 33 pazienti RSC candidati a chirurgia endoscopica nasosinu-          How to cite this article: Gallo S, Bandi F,
sale e 30 controlli sottoposti a chirurgia non RSC-correlata, un campione di sangue venoso        Preti A, et al. Exploring the role of nasal cy-
periferico, una biopsia del processo uncinato (PU), un citologico sul turbinato inferiore         tology in chronic rhinosinusitis.Acta Otorhi-
(TI) e un citologico nel meato medio (MM).                                                        nolaryngol Ital 2020;40:368-376. https://doi.
Risultati. Differenze tra RSC e controlli negli eosinofili su sangue periferico (P = 0,0001),     org/10.14639/0392-100X-N0711
PU (P < 0,0001), TI (P = 0,01) e MM (P = 0,0006) sono risultate statisticamente signifi-
cative. È stata dimostrata una correlazione debole tra sangue periferico e PU (r = 0.34,        © Società Italiana di Otorinolaringoiatria
                                                                                                e Chirurgia Cervico-Facciale
P = 0,006) e tra PU e IT (r = 0,30, P = 0,017), e una correlazione moderata tra PU e MM
(r = 0,51, P < 0,0001). Le curve ROC hanno predetto forme di RSC eosinofila con una
                                                                                                               OPEN ACCESS
sensibilità globalmente bassa. Escludendo i pazienti allergici la sensibilità si riduce ulte-
riormente per TI mentre aumenta per MM.                                                         This is an open access article distributed in accordance with
                                                                                                the CC-BY-NC-ND (Creative Commons Attribution-Non-
Conclusioni. Il presente studio suggerisce di eseguire il prelievo citologico nel MM al fine
                                                                                                Commercial-NoDerivatives 4.0 International) license. The
di identificare le RSC eosinofile. Sono necessari studi più ampi per verificare i risultati e   article can be used by giving appropriate credit and mentio-
definire valori limite adeguati.                                                                ning the license, but only for non-commercial purposes and
                                                                                                only in the original version. For further information: https://
PAROLE CHIAVE: rinosinusite, polipi nasali, rinite, eosinofilia, biomarker, citologia           creativecommons.org/licenses/by-nc-nd/4.0/deed.en




368
                                                                                             A correlational study of tissue eosinophilia




Introduction                                                     ECRS in presence of differently matched clinical criteria 7.
                                                                 Much less widespread among rhinologists is assessment
Chronic rhinosinusitis (CRS) is a generic term for different
                                                                 of the degree of eosinophilia through nasal cytology. This
disease entities, each representing the downstream conse-        technique has been reported as an efficient method to dif-
quence of a specific immune-mediated inflammatory mech-          ferentiate among various forms of non-allergic rhinitis 8.
anism. This is why a blanket treatment approach has been         However, it is still debated if it might be of interest in defin-
proven unsuccessful in some cases 1. The phenotypic di-          ing CRS inflammatory profiles. Controversies are related
chotomy of CRS with and without nasal polyps (CRSwNP             to both sampling site and method of analysis, and only few
and CRSsNP, respectively) is progressively being replaced        reports have examined the cellular inflammatory pattern of
by a more complex biomolecular classification of subtypes        different endonasal subsites in CRS.
(or endotypes) 2.                                                In light of these premises, we wished to verify in a sample
Although the current therapeutic strategy is focused on im-      population (including CRS patients and controls) the exist-
munomodulation (i.e., monoclonal antibodies), more sci-          ence of a correlation among the degree of tissue, blood and
entific evidence is needed to find accurate predictive mo-       cytological eosinophilia. Moreover, standard cytological
lecular markers of CRS endotypes to better tailor effective      data was integrated with analysis of a cytological sample
regimens.                                                        obtained from the middle meatus region. Lastly, by sort-
To date, biological agents tested or in use for moderate/se-     ing the study population into cases (patients with CRS) and
vere inflammatory disorders of the airways and skin target       controls, we investigated the existence of significant differ-
the T helper 2 (Th2) pathway. Conversely, few treatments         ences in the degree of eosinophilia and association with the
are available for non-Th2 and non-eosinophilic cascades 3.       most typical clinical features related to CRS.
However, based on the rate of non-responders to biological
therapies, clinical translation of the endotyping process is
urgently needed 3. Currently, the eligibility for these treat-   Materials and methods
ments depends on the demonstration of an eosinophilic in-        An observational prospective study was conducted accord-
flammatory profile (i.e., blood eosinophil count and serum       ing to the declaration of Helsinki and was previously ap-
IgE). However, cut-off values are not clearly defined, with      proved by the Institutional Review Board of the hospital.
the only exception being thresholds applied in clinical tri-
als 4.                                                           Study population
All these premises are even more vague when applied to           Clinical data were obtained from patients affected by CRS
CRS. A basic attempt of CRS endotyping is based on the           who underwent endoscopic sinus surgery (ESS) at the same
identification of the predominant immune cells in the in-        tertiary care center in the period between January 2018 and
flamed sinonasal mucosa; in particular, it is key the distinc-   July 2018.
tion between eosinophilic and non-eosinophilic mediated          CRS was diagnosed according to the latest European guide-
CRS (ECRS and non-ECRS, respectively) 2. The clinical            lines 9. Each CRS case was assessed by SNOT-22 question-
interest in ECRS arises as it generally shows a poor re-         naire for symptoms, Lund Kennedy (LK) and Lund Mackay
sponse to medical and surgical therapies 5. The therapeutic      (LM) scores to assess the endonasal status and the degree
impact would be significant, as cases with intense eosino-       of opacification of the sinuses, respectively, and skin prick
philia would justify higher dosage of steroids and, theoreti-    test to investigate allergic sensitisation to common inhal-
cally, selected biological antagonists of type 2 inflamma-       ants. Data on asthma, aspirin sensitivity and smoking hab-
tion 6.                                                          its were self-reported by patients. Exclusion criteria were
Although no unanimous histopathological criteria exist for       genetic syndromes, congenital or acquired immunodefi-
discriminating between ECRS and non-ECRS, it is current          ciency, malignancy or history of the head and neck cancers,
practice to define western ECRS when tissue eosinophil           systemic autoimmune diseases, and drug abuse.
count > 5 cells/HPF 5. Moreover, a tissue eosinophil count       Patients scheduled for other non-CRS surgeries (septo-
> 10 cells/HPF correlates with poorer outcomes 5. Diagno-        plasty and dacryocystorhinostomy) in the same time-lapse
sis requires obtaining tissue for histopathological analysis.    served as control group. Each control was assessed by
As sinus mucosa needs to be collected, biopsies may not be       SNOT-22 questionnaire as well as LK and LM scores to
straightforward or performed under local anaesthesia. This       exclude CRS. Control patients affected by asthma and as-
is why different, less invasive, surrogates have been tested     pirin sensitivity were excluded a priori. Lastly, skin prick
to improve their reliability to predict tissue eosinophilia.     tests were performed to investigate allergic sensitisation to
It is worth mentioning the JESREC score, which defines           common inhalants.



                                                                                                                                   369
S. Gallo et al.




All cases and controls were considered eligible for enrol-        absolute and relative frequencies. Mean and standard de-
ment only after a washout period of 15 days from oral and         viation (SD) or median and interquartile range (IQR) were
topical steroids and 1 month from oral antibiotics.               used for quantitative variables with a parametric and non-
All collected data were entered in a specific CRS database        parametric distribution, respectively. Chi-squared or Fish-
as previously reported 10.                                        er’s exact test were used to detect significant differences
                                                                  for qualitative variables. Student’s t and Mann-Whitney
Sampling steps                                                    tests were used for quantitative variables following their
At the beginning of the surgical procedure under general          parametric or non-parametric distribution. Spearman’s cor-
anesthesia, the following sampling steps were taken.              relation was used to assess the relationship between the dif-
A peripheral blood venous sample from antebrachial vein           ferent measurements of eosinophils. P values < 0.05 was
was collected for blood and leukocyte formula counts.             considered statistically significant. Stata 15 statistical soft-
White blood cells (WBC) were expressed both as absolute           ware was used for each statistical computation.
count (cell x 109/L) and percentage of the total WBC count.
A nasal cytological sampling was performed under en-              Results
doscopy along the inferior turbinate (IT) and the middle
meatus – lateral nasal wall (MM) mucosa. The procedure            A total of 33 CRS patients and 30 controls were recruited.
consisted in swiping gently a disposable plastic nasal cu-        Baseline characteristics are shown in Table I. The CRS
rette (Rhinoprobe®), equipped with a small distal collection      group included 21 (63.6%) CRSwNP and 12 (36.4%)
chamber, on the mucosal surface. Samples were swiped on           CRSsNP. Allergic sensitisation was diagnosed in 13 cases
the central area of a slide and smeared with May Grun-            (39.4%), asthma in 14 (42.4%), and aspirin intolerance in 3
wald-Giemsa as described by Gelardi et al. 11. Slides were        (9.1%). Sixteen (48.5%) CRS patients had underwent pre-
observed through an optical microscope (Nikon Eclipse             vious surgery elsewhere. Median baseline SNOT-22 score
600®) at different magnifications (100x, 200x, and 400x).         was 30. Mean baseline LK and LM scores were 6.1 and
Observed cells included intact respiratory epithelial, flak-      13.5, respectively.
ing, and immune cells (i.e., eosinophils, neutrophils, mast-      Only 5 (16.7%) controls showed allergic sensitisation to
cells, macrophages, and plasma cells) and were counted in         inhalants.
10 consecutive fields at 400x. Eosinophils were expressed         The median blood eosinophil count was 0.3x109/L in CRS
both as mean of eosinophil cells per high-power field (HPF)       group (min 0.03, max 1.14) and 0.2x109/L in control group
400x and percentage of eosinophils on total immune cells.         (min 0.01, max 0.36) [P = 0.0001]. The median percentage
This latter parameter was intended to incorporate also the        of blood eosinophils was 3.9% (min 0.4, max 13.3) and 2%
effect of the neutrophilic degree of infiltration of the speci-   (min 0.2, max 6) in CRS and control group, respectively
men.                                                              [P = 0.0008].
A mucosal biopsy was collected on the uncinate process            Increased overall degree of inflammation was found in UP
(UP) at the same side of the cytological sampling. All sam-       CRS samples [P = 0.003]. Eosinophil counts in UP sam-
ples, sized > 0.4 mm, were fixed in 10% buffered formalin,        ples were significantly different between cases and controls
dehydrated by alcohol passages with increasing concentra-         [P < 0.0001]. In detail, among CRS group, the eosinophil
tions of ethanol, clarified in BioClear® and embedded in          count was <5 cells/HPF in 18 (54.5%) cases, 5-10 cells/
paraffin. Histological sections, with a thickness of 3 mm,        HPF in 3 (9.1%), and > 10 cells/HPF in 12 (36.4%); in con-
were stained with haematoxylin-eosin. A conventional              trol samples, the eosinophil count was < 5 cells/HPF in 29
morphological evaluation was carried out according to the         (96.7%) cases and 5-10 cells/HPF in 1 (3.3%).
2017 WHO classification criteria. Additional histopatho-          Similarly, cytological analysis showed higher overall in-
logic features were taken into consideration as reported by       flammatory infiltration in CRS cases, confirmed at both IT
Snidvongs et al. 12. Moreover, an immune cell count was           (P = 0.01) and MM scraping (P = 0.0006). Median IT eo-
performed in 5 HPF using a 400x objective corresponding           sinophil count was 0.5 cells/HPF in CRS group and 0 cells/
to an area of 1 mm2. Tissue eosinophil count was graded in        HPF in control group [P = 0.0002]. Median IT eosinophil
three classes: < 5 cells/HPF, 5-10 cells/HPF and > 10 cells/      percentage was 4.2% and 0% in CRS and control group,
HPF 12.                                                           respectively [P = 0.002]. Median MM eosinophil count
                                                                  was 0.3 cells/HPF in CRS group and 0 cells/HPF in control
Statistical analysis                                              group [P = 0.006]. Median MM eosinophil percentage on
An ad hoc electronic database was created to collect all          total immune cells was 1.9% and 0% in CRS and control
study variables. Qualitative data were summarised with            group, respectively [P = 0.01]. On the whole, these data



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Table I. Demographic data of control and CRS groups.

                                                                                              Control group                  CRS group                    P value
                                                                                                 N = 30                       N = 33
 Males, n (%)                                                                                    17 (56.7)                     8 (24.2)                    0.009
 Mean (SD) age, years                                                                           52.1 (16.8)                  52.7 (15.5)                   0.88
 CRS with nasal polyps, n (%)                                                                        -                        21 (63.6)                      -
 Previous surgery for CRS, n (%)                                                                     -                        16 (48.5)                      -
 Allergy, n (%)                                                                                   5 (16.7)                    13 (39.4)                    0.05
 Asthma, n (%)                                                                                    0 (0.0)                     14 (42.4)                  < 0.0001
 Aspirin intolerance n (%)                                                                        0 (0.0)                       3 (9.1)                    0.24
 Smoker, n (%)
 Nonsmoker                                                                                       29 (96.7)                    24 (72.7)
 Smoker                                                                                           1 (3.3)                      4 (12.1)                    0.03
 Former                                                                                           0 (0.0)                      5 (15.2)
 Median (IQR) SNOT-22 score                                                                          -                       30 (25-42)                      -
 Mean (SD) LK score                                                                                  -                        6.1 (2.8)                      -
 Mean (SD) LM score                                                                                  -                        13.5 (5.7)                     -
SD: standard deviation; CRS: chronic rhinosinusitis; IQR: inter-quartile range; LK: Lund-Kennedy; LM: Lund-Mackay; SNOT-22: Sino-nasal outcome test 22



showed a significant difference in terms of eosinophilic in-                              phil count (MM eosinophils/HPF 400x, P = 0.003; MM
filtrate between CRS cases and controls (Tab. II).                                        eosinophil percentage, P = 0.005) (Tab. IV). The absence
A weak correlation was seen between UP eosinophil count                                   of a significant difference for asthma, aspirin intolerance
and blood eosinophil count (r = 0.34, P = 0.006), and be-                                 and polyp phenotype might be justified by the small size
tween UP and IT eosinophil count (r = 0.30, P = 0.017),                                   of the sample.
whereas a moderate correlation was observed between UP                                    ROC curve analysis on IT eosinophil count predicted ERCS
and MM eosinophil count (r = 0.51, P < 0.0001). Subgroup                                  with a sensitivity of 51.5% and specificity of 90% [posi-
analysis showed that in the control group only the correla-                               tive predictive value (PPV) 85%; negative predictive value
tion between UP and IT cytology was confirmed, whereas                                    (NPV) 62.8%; area under the curve (AUC) 0.76, range
the opposite was seen in the CRS group (loss of correlation                               0.65-0.87], on IT eosinophil percentage on total immune
between UP eosinophil count and IT cytology and con-                                      cells with a sensitivity of 48.5% and specificity of 80%
firmed correlation between UP eosinophil count and MM                                     (PPV 72.7%; NPV 58.5%; AUC 0.72, range 0.59-0.84),
cytology) (Tab. III).                                                                     on MM eosinophil count with a sensitivity of 42.4% and
No significant differences were observed in terms of tissue                               specificity of 90% (PPV 82.4%; NPV 58.7%; AUC 0.69,
eosinophilia (blood, UP, IT, MM eosinophil count) across                                  range 0.57-0.81), on MM eosinophil percentage on total
different clinical parameters, including sex, age, presence                               immune cells with a sensitivity of 42.4% and specificity of
of nasal polyps, previous surgery, allergy, asthma and                                    87.7% (PPV 77.8%; NPV 57.8%; AUC 0.67, range 0.55-
smoking habit. Similarly, no significant differences were                                 0.80). Once allergic patients were excluded from the CRS
evident comparing UP, IT, MM eosinophil count and clini-                                  population, ROC curve analysis on IT eosinophil count
cal staging scores (SNOT-22, LK score, LM score). Con-                                    predicted ERCS with a sensitivity of 11.1% and specificity
versely, higher levels of blood eosinophilia were associated                              of 90.9% (PPV 50%; NPV 55.6%; AUC 0.53, range 0.27-
with an increase in endoscopic and radiological scores (LK                                0.80), on IT eosinophil percentage on total immune cells
score, P = 0.03; LM score, P = 0.01).                                                     with a sensitivity of 11.1% and specificity of 81.8% (PPV
The CRS group was then classified in ECRS and non-                                        33.3%; NPV 52.9%; AUC 0.51, range 0.24-0.77), on MM
ECRS on the basis of the histopathological threshold                                      eosinophil count with a sensitivity of 33.3% and specificity
(ECRS, eosinophil count ≥ 5 cells/HPF; non-ECRS, eo-                                      of 90.9% (PPV 75%; NPV 62.5%; AUC 0.81, range 0.61-
sinophil count < 5 cells/HPF). The analysis of different                                  1), on MM eosinophil percentage on total immune cells
clinical and biological parameters showed only a signifi-                                 with a sensitivity of 55.6% and specificity of 81.8% (PPV
cant difference between the two groups for MM eosino-                                     71.4%; NPV 69.2%; AUC 0.85, range 0.67-1).



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S. Gallo et al.




Table II. Blood, histological and cytological features of control and CRS groups. Statistical difference is expressed as p value; significant results (P < 0.05) are
highlighted in bold.
                                                                                                  Control group               CRS group               P value
 Peripheral blood eosinophilia
 Median (IQR) blood eosinophil count, 109/L                                                                   0.2 (0.1-0.2)    0.3 (0.2-0.4)         0.0001
 Median (IQR) blood eosinophils, %                                                                            2.0 (1.1-3.2)    3.9 (2.4-5.8)         0.0008
 Uncinate process (UP) histological features
 Overall degree of inflammation, n (%)                                               Absent                        14 (46.7)     5 (15.2)
                                                                                       Mild                        16 (53.3)     22 (66.7)           0.003
                                                                                  Moderate                          0 (0.0)       6 (18.2)
 Inflammatory predominance, n (%)                                         Lymphoplasmacytic                        16 (53.3)     27 (81.8)
                                                                                     Absent                        14 (46.7)      5 (15.2)            0.01
                                                                                Eosinophilic                         0 (0.0)       1 (3.0)
 Neutrophilic infiltrate, n (%)                                                                                     4 (13.3)      4 (12.1)             1.0
 Eosinophil count, n (%)                                                              < 5/HPF                      29 (96.7)     18 (54.5)
                                                                                     5-10/HPF                        1 (3.3)       3 (9.1)         < 0.0001
                                                                                       10/HPF                        0 (0.0)     12 (36.4)
 Inferior turbinate (IT) cytological features
 Median (IQR) eosinophils/HPF 400x                                                                             0 (0.0-0.2)      0.5 (0.0-1.3)        0.0002
 Median (IQR) eosinophil percentage on total immune cells                                                      0 (0.0-0.6)     4.2 (0.0-12.5)        0.002
 Eosinophil grading, n (%)                                                               < 5%,                  24 (80.0)         20 (60.6)
                                                                                        5-19%                    3 (10.0)          9 (27.3)
                                                                                                                                                      0.05
                                                                                       20-50%                    3 (10.0)          1 (3.0)
                                                                                          50%                     0 (0.0)          3 (9.1)
 Median (IQR) mast cell count                                                                                    0 (0-1)           3 (1-8)          < 0.001
 Median (IQR) neutrophil count                                                                                   8 (2-43)        46 (8-300)          0.06
 Median (IQR) macrophage count                                                                                    2 (1-3)          3 (1-4)           0.25
 Median (IQR) plasma cell count                                                                                   0 (0-0)          0 (0-0)           0.17
 Median (IQR) total immune cells                                                                                12 (4-50)       80 (20-409)          0.01
 Middle meatus (MM) cytological features
 Median (IQR) eosinophils/HPF 400x                                                                             0 (0.0-0.2)     0.3 (0.0-3.5)         0.006
 Median (IQR) eosinophil percentage on total immune cells                                                        0 (0-4)        1.9 (0-30)            0.01
 Eosinophil grading, n (%)                                                               < 5%,                  24 (80.0)        18 (54.6)
                                                                                        5-19%                    4 (13.3)         4 (12.1)
                                                                                                                                                      0.04
                                                                                       20-50%                     0 (0.0)         6 (18.2)
                                                                                          50%                    2 (6.7)          5 (15.2)
 Median (IQR) mast cell count                                                                                    1 (0-1)         3 (1-12)          < 0.0001
 Median (IQR) neutrophil count                                                                                  3 (2-13)        19 (4-200)           0.04
 Median (IQR) macrophage count                                                                                   1 (1-2)          3 (2-6)            0.001
 Median (IQR) plasma cell count                                                                                  0 (0-0)          0 (0-0)             0.33
 Median (IQR) total immune cells                                                                                7 (4-18)       95 (13-253)          0.0006
IQR: interquartile range; UP: uncinate process; IT: inferior turbinate; HPF: high power field; MM: middle meatus




372
                                                                                                                                    A correlational study of tissue eosinophilia




Table III. Spearman’s rank-order correlation between histological samples, blood tests and cytology. Statistical difference is expressed as p value; significant
results (P < 0.05) are highlighted in bold.
                                                                                                Uncinate process (UP) eosinophil count
 Spearman’s rho                                                                                                   rho                                  P value
 Total population (n = 63)
 Blood eosinophil count, 109/L                                                                                   0.34                                   0.006
 Blood eosinophils, %                                                                                            0.26                                   0.038
 IT eosinophils/HPF 400x                                                                                         0.30                                   0.017
 IT eosinophil percentage on total immune cells                                                                  0.20                                   0.111
 MM eosinophils/HPF 400x                                                                                         0.51                                 < 0.0001
 MM eosinophil percentage on total immune cells                                                                  0.48                                 < 0.0001
 Control group (n = 30)
 Blood eosinophil count, 109/L                                                                                  -0.17                                    0.36
 Blood eosinophils, %                                                                                           -0.07                                    0.69
 IT eosinophils/HPF 400x                                                                                         0.40                                    0.03
 IT eosinophil percentage on total immune cells                                                                  0.26                                    0.16
 MM eosinophils/HPF 400x                                                                                         0.19                                    0.32
 MM eosinophil percentage on total immune cells                                                                  0.11                                    0.56
 CRS group (n = 33)
 Blood eosinophil count, 109/L                                                                                   0.20                                    0.26
 Blood eosinophils, %                                                                                            0.11                                    0.56
 IT eosinophils/HPF 400x                                                                                         0.04                                    0.83
 IT eosinophil percentage on total immune cells                                                                 -0.06                                    0.74
 MM eosinophils/HPF 400x                                                                                         0.53                                   0.002
 MM eosinophil percentage on total immune cells                                                                  0.51                                   0.002
UP: uncinate process; IT: inferior turbinate; HPF: high power field; MM: middle meatus; CRS: chronic rhinosinusitis



Table IV. Blood, histological, cytological and clinical differences between non-ECRS and ECRS group. Statistical difference is expressed as P value; significant
results (P < 0.005) are highlighted in bold.
                            CRS group                                        Non-ECRS                        ECRS                          P value
                                                                               N = 18                      N = 15
 Median (IQR) blood eosinophil count, 109/L                                            0.3 (0.2-0.4)                       0.4 (0.2-0.6)                     0.19
 Median (IQR) blood eosinophils, %                                                     3.7 (2.3-5.4)                       3.9 (2.4-8.0)                     0.42
 Median (IQR) IT eosinophils/HPF 400x                                                  0.4 (0.1-0.8)                        0.7 (0-1.8)                       0.66
 Median (IQR) IT eosinophil percentage on total immune cells                            4.6 (3-9.1)                         3.4 (0-15)                        0.94
 Median (IQR) MM eosinophils/HPF 400x                                                  0.05 (0-0.2)                       3.3 (0.4-10.3)                     0.003
 Median (IQR) MM eosinophil percentage on total immune cells                            0.2 (0-7.7)                       23.3 (1.9-68.8)                    0.005
 Asthma, n (%)                                                                           8 (44.4)                            6 (40.0)                         0.80
 Allergy, n (%)                                                                          7 (38.9)                            6 (40.0)                         0.95
 Aspirin intolerance, n (%)                                                              2 (11.1)                             1 (6.7)                          1.0
 CRSwNP, n (%)                                                                           12 (66.7)                           9 (60.0)                         0.69
IQR: interquartile range; IT: inferior turbinate; HPF: high power field; MM: middle meatus; CRS: chronic rhinosinusitis




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A cut-off of ≥ 1.9% of MM eosinophil percentage provided                                         eosinophil count is > 5 cells/HPF. Moreover, a tissue eosin-
the best sensitivity and specificity (88.9% and 81.8%, re-                                       ophil count > 10 cells/HPF was demonstrated to correlate
spectively) (Tab. V).                                                                            with poorer outcomes and overall prognosis 5.
                                                                                                 To overcome the aforementioned disadvantages of biopsy,
Discussion                                                                                       other types of biological samples have been considered
                                                                                                 as possible indirect assessments of tissue eosinophilia. A
The term ECRS was introduced to identify a subgroup of                                           number of studies demonstrated that there is an association
patients with CRS and eosinophilic infiltration of nasal                                         between peripheral eosinophilia and tissue eosinophilia in
polyps, likely to occur consequent to eosinophil dysregula-                                      paranasal sinuses. Our study confirmed this correlation, al-
tion 13. The aetiology of ECRS encompasses a wide variety                                        beit weak (r = 0.341). The cut-point of > 0.3 x 109/L or
of stimuli and overlapping pathogenic mechanisms 13. There                                       4.4% of WBC is that adopted for administration of biologi-
is evidence that ECRS is associated with greater symptom                                         cal agents in asthma, though still within the normal range.
severity 14, extensive sinus disease and comorbidities (asth-                                    Other thresholds have been proposed to gain better diag-
ma), with intermittent acute exacerbation of secondary bac-                                      nostic reliability. However, their broad variability prevents
terial infections 5. Moreover, ECRS patients seem to have                                        drawing firm conclusions 13. Blood eosinophil count shows
a poorer response to medical and surgical treatments with                                        low specificity depending on other comorbidities (para-
high polyp recurrence rate and severely impaired quality                                         sitic infections, allergy, autoimmune disorders, adverse
of life 5. Therefore, early detection of ECRS, preferably in                                     drug events, etc.); moreover, local eosinophilic activation
outpatient settings, is key to guide overall long-term man-                                      is often independent on blood eosinophils 14. It is reason-
agement and improve prognosis.                                                                   able that on-site biomarkers might provide a more specific
In daily practice, diagnostic criteria for ECRS are based                                        overview on cellular inflammatory pattern. In some studies,
on clinical features. Traditional traits include asthma (late-                                   indeed, asthma subtypes are defined on induced sputum, a
onset), nasal polyps, aspirin intolerance, high serum eosin-                                     non-invasive well standardised procedure of bronchial cy-
ophilia and IgE. Although the presence of polyps predicts                                        tological assessment, able to sort asthma into eosinophilic,
high tissue eosinophilia, a remarkable number of CRSs-                                           neutrophilic, mixed-granulocytic or pauci-granulocytic
NP show the same degree of eosinophilic inflammation                                             subtypes 16. Similarly, the degree of nasal eosinophilia,
(19%) 12. For this reason, the most reliable way to diagnose                                     together with other inflammatory cells, can be measured
ECRS remains histopathological assessment. However, re-                                          by cytological analysis. Numerous techniques have been
lying on biopsy as the main diagnostic tool of ECRS opens                                        described to obtain nasal specimen for cytological assess-
several issues. 1) Unless adequately aware, the pathologist’s                                    ment. Among them, nasal scraping, performed along the
report often concludes generically with “chronic inflamma-                                       medial aspect of the inferior turbinate, has shown several
tion” 12. 2) Diagnosis requires obtaining sufficient tissue                                      advantages 8. Although the technique has been validated as
for histopathological analysis. As sinus mucosa needs to be                                      a semi-quantitative analysis for diagnosis of cellular rhini-
collected, and not just nasal polyp samples, biopsies may                                        tis and correlations have been demonstrated between nasal
not be straightforward or performed under local anaesthe-                                        and bronchial inflammatory cytological patterns, its role in
sia. Moreover, biopsy – due to its intrinsic invasiveness –                                      CRS has not been clarified 17. One controversial issue is
is not an early step in the CRS diagnostic workup. 3) To                                         linked to the sampling site. Some studies debate its use-
date, the definition of eosinophilia in CRS has not reached                                      fulness when performed along the inferior turbinate. For
consensus among researchers 5. This controversy concerns                                         example, De Corso et al. reported that inferior turbinate
both the method and interpretation of the results. Actually,                                     eosinophilic inflammation represents an early marker for
it is accepted practice to define western ECRS when tissue                                       severe CRSwNP 18. Similarly, Gelardi et al. showed that the


Table V. Sensitivity, specificity, positive likelihood ratio and negative likelihood ratio of cytology performed on the inferior turbinate and middle meatus in the non-
allergic CRS population.
 N = 20                                                        Best cut-off Sensitivity, %           Specificity, %      LR+         LR-       Correctly classified, %
 IT eosinophil count in 10 consecutive HPF 400x                          ≥7                  66.7                   54.6           1.5   0.6          60.0
 IT eosinophil percentage on total immune cells                         ≥ 1.3                66.7                   45.5           1.2   0.7          55.0
 MM eosinophil count in 10 consecutive HPF 400x                          ≥5                  88.9                   72.7           3.3   0.2          80.0
 MM eosinophil percentage on total immune cells                         ≥ 1.9                88.9                   81.8           4.9   0.1          85.0
IT: inferior turbinate; HPF: high power field; MM: middle meatus; LR+: positive likelihood ratio; LR-: negative likelihood ratio




374
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association of eosinophilic-mast cell inferior turbinate in-     plied to patients clinically suspect for ECRS, this test might
filtration and the presence of asthma and aspirin sensitivity    confirm diagnosis and drive treatment selection. Lastly, it
is correlated with an increased risk of polyp relapse 19. Our    is reasonable to think that the degree of neutrophilic infil-
analysis confirmed the existence of a significant degree of      tration also produces an effect in terms of CRS classifica-
correlation between tissue eosinophilia and IT cytological       tion. Thus, a more comprehensive grouping should account
eosinophilic count (r = 0.30, P = 0.017). To make matters        for mixed-granulocytic and pauci-granulocytic CRS cases,
complicated, She et al. demonstrated a lack of significant       apart from the classical ECRS and non-ECRS subtypes.
correlation between the total and individual inflammatory        Of course, the study is somewhat limited. It represents a
cell counts in inferior turbinate versus paranasal sinus mu-     preliminary exploration of the role of nasal cytology in
cosa, questioning the diagnostic value of nasal cytology         CRS in a relatively small population. The technical choice
for CRS 20. If it is true that the term CRS has been coined      has fallen upon nasal scraping because it was the only
precisely to express that every sinus inflammation trans-        available in our center. Nonetheless, the literature concern-
lates contextually into an inflammation of the nasal mucosa      ing this topic is limited and extremely variable in terms of
(and therefore also of the inferior turbinate) and that tran-    sampling site and processing techniques, which makes it
scriptomic studies showed a substantially overlapping gene       difficult to carry out comparisons and draw solid conclu-
expression profile of various nasal subsites 21,22, it is also   sions.
true that clinical practice teaches that the phenotypic mani-
festations of CRS usually spare the mucosa of the inferior       Conclusions
turbinates 23. This aspect is the inspiring concept underly-
ing the recent “reboot approach” 24. Moreover, this region,      In summary, assuming that a re-classification of CRS is a
in addition to a different embryological origin, shows a         pressing clinical need, as well as the identification of re-
morpho-histological structure that is not identical to that of   liable biomarkers, nasal cytology conceptually represents
the middle meatus 22,25.                                         an interesting tool. In the same way as bronchial cytology
She et al. demonstrated that 66% of CRS patients with CRS        for asthma, nasal cytology can allow for cellular profiling
show marked inflammation in the inferior turbinate, but that     of CRS which, albeit in its initial stages, is a step forward
the inflammation is much more intense in maxillary sinus         the endotyping process and the thoughtful application of
mucosa 20. Furthermore, the inflammatory response in the         innovative biological therapies. Additionally, it shows sev-
ethmoid sinus seems even more severe than in maxillary           eral practical advantages, such as good tolerability and
sinus or inferior turbinate in other series of patients with     compliance, limited costs and an easy-to use approach. It
chronic sinusitis 26. Taken together, these findings suggest     is reasonable to think that nasal cytology in the MM might
that the paranasal sinuses, especially ethmoid, possibly         provide more accurate information on the degree of tissue
play a pivotal role in CRS. It follows that sampling a typi-     eosinophilia in CRS. The next steps would be to verify
cal site of CRS manifestation might be more representative       these results across other wide and unbiased cohorts (even-
of CRS-related inflammatory profile; moreover, the cel-          tually comparing different sampling methods) and to define
lular inflammatory pattern of the inferior turbinate can be      thresholds values with the best accuracy. However, at pre-
clearly influenced by the coexistence of allergic and non-       sent, its semi-quantitative nature, the lack of standard cut-
allergic rhinitis. Furthermore, as reported by Armengot et       offs and the discrepancy of reported results limit its sys-
al., a significant correlation exists between ethmoid tissue     tematic use in CRS workup, while remaining undisputed its
eosinophilia and MM cytological eosinophilia 27; the same        role in chronic rhinitis.
moderate correlation emerged from our data (r = 0.51,
P < 0.0001). However, the estimated accuracy of nasal            Acknowledgements
cytology seems limited because overall sensitivity values
                                                                 Andrea Preti is a PhD student of the “Sperimental and
are low. Interestingly, once allergic patients were excluded
                                                                 translational medicine” course at University of Insubria.
from the analysis, the sensitivity further decreased for cy-
                                                                 A special thanks goes to Daniele Sabatino, technician of
tological sampling on IT and slightly increased for cyto-
                                                                 the Pathology Department, for his precious collaboration in
logical MM sampling. This fact suggests that the allergy
                                                                 the preparation of the histopathological samples analysed
comorbidity can act as a confounding factor and should be
                                                                 in the present study.
taken into account when interpreting nasal cytology find-
ings. These results, moreover, lead to further reflection. Ap-
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