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Tissue Eosinophilia in Chronic Sinusitis
Quantification Techniques
Neil Bhattacharyya, MD;
Darshan K. Vyas, MD;
Frank P. Fechner, MD;
Richard E. Gliklich, MD;
Ralph Metson, MD
Arch Otolaryngol Head Neck Surg. 2001;127:1102-1105.
ABSTRACT
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Objective To ascertain the reliability of a proposed method for quantifying tissue
eosinophilia in sinus mucosa.
Design Prospective cohort study.
Interventions and Outcome Measures Pathology slides from patients undergoing endoscopic sinus surgery for
chronic rhinosinusitis were independently assessed by 2 reviewers. Using a
proposed systematic counting method, the degree of tissue eosinophilia was
quantified. Disease severity was assessed by computed tomographic (CT) staging.
Intrarater, interrater, and intrapatient reliability was determined using
correlational reliability analysis. The degree of correlation between tissue
eosinophilia and CT stage was determined.
Results One hundred thirty-two slides from 65 patients were reviewed. The mean
(SD) eosinophil density was 23.4 (37.2) eosinophils per high-power field.
Only 12 patients (18%) had no eosinophils on histopathologic analysis. Strong
intrarater (r 0.91 for each rater, P<.001) and interrater reliability (r0.82
between raters, P<.001) was noted for the quantification
method. A moderate degree of correlation was found between CT scan stage and
degree of tissue eosinophilia (Spearman  = 0.62, P<.001).
Conclusions The proposed method for quantifying tissue eosinophilia in sinus mucosa
is reliable and valid. A relatively strong correlation exists between CT scan
stage and tissue eosinophilia in chronic rhinosinusitis.
INTRODUCTION
CHRONIC rhinosinusitis is an extremely common clinical problem, affecting
as many as 30 million Americans.1 Despite recent
progress in formalizing clinical diagnostic criteria, radiographic staging,
and management protocols, much remains to be learned about the pathogenesis
of this entity.2 Recent studies have identified
a potential link between chronic rhinosinusitis and the inflammatory cells
and mediators present in sinonasal mucosa. Several authors3-4
have identified hypereosinophilia within the sinonasal mucosa in chronic rhinosinusitis
and linked this finding with the production of inflammatory mediators and
subsequent tissue damage within the respiratory mucosa. In addition, the degree
of tissue eosinophilia has been found to predict extensive disease as measured
by computed tomographic (CT) scan stage.5 Other
data suggest that eosinophilia may be predictive of success or failure with
endoscopic sinus surgery.6
Despite mounting evidence that the eosinophil has a significant role
in the pathogenesis of chronic rhinosinusitis, conflicting data have emerged
in the literature regarding its impact on the disease. Variability in the
definition of tissue hypereosinophilia and quantification of eosinophilia
may account for some of these contradictory findings. To better understand
the role of the eosinophilia in chronic rhinosinusitis, an accurate and reproducible
method for quantifying tissue eosinophilia is required. Therefore, we sought
to establish a statistically reliable and valid method for quantifying tissue
eosinophilia in sinus mucosal specimens. Furthermore, we explored the relationship
between tissue eosinophilia and extent of disease as measured by the CT scan
stage of the chronic rhinosinusitis.
MATERIALS AND METHODS
An inception cohort of 71 consecutive patients undergoing surgical therapy
for medically refractory chronic rhinosinusitis constituted the study population.
All patients met clinical criteria established by the American Academy of
Otolaryngology for the diagnosis of chronic rhinosinusitis; thorough medical
management had failed for all.2 Preoperative
CT scans were performed and staged according to a previously described system.7 A second group of 5 patients without a history of
chronic rhinosinusitis undergoing endoscopic orbital decompression comprised
a control or reference group.
The mucosal specimens obtained at the time of endoscopic sinus surgery
were fixed in formalin embedded in paraffin. Standard 5-µm sections
were stained with hematoxylin-eosin. Light microscopic examination of each
slide was then conducted according to a structured protocol.
Initially, a low-power survey of the entire slide was performed at x20
and x100 magnification. In this survey, sinus mucosa (excluding nasal
mucosa) was identified by its lack of glandular elements and the presence
of goblet cells (when identifiable). Subsequently, the segment of sinus mucosa
containing the greatest degree of eosinophilic inflammation was identified
and then examined under a total magnification of x450, with a 10 x
10-mm reticulate present in the eyepiece. The total number of eosinophils
present within this grid was determined as the eosinophil count per high-power
field (HPF). Second and third eosinophil counts within this same area of diseased
mucosa were performed by the same observer after removing the eyes from the
microscope for 30 seconds and then recounting. The slide was then placed under
low-power magnification and in a random position for a second observer to
repeat the low-power survey, identification of the most diseased area, and
eosinophil count and recounts procedure. Values were recorded for eosinophil
counts, with each observer blinded to the counts of the other observer. For
patients with more than one slide of sinus mucosa (ie, left and right sinus
contents), all slides were processed in the same manner to look for variability
within patients.
Statistical analysis was then performed to determine intrarater reliability,
interrater reliability, and intrapatient consistency. Standard techniques
for determination of Pearson correlation coefficients and statistical significance
of these coefficients were used. Intrarater reliability was determined for
counts 1, 2, and 3 for each slide of each patient, thus determining the counting
error. Interrater agreement was determined by averaging the 3 eosinophil counts
per slide for each observer and then statistically comparing these counts
between observers. Finally, intrapatient variability was assessed by comparing
the mean eosinophil count for all 6 counts (3 counts per observer for 2 observers)
between slides for a given patient. These 3 reliability/variability assessments
are diagrammatically represented in Figure
1. An average eosinophil density for each patient was computed by
averaging the counts from both observers for all slides for that patient,
resulting in a mean eosinophil count per HPF for a given patient.
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Figure 1. Diagrammatic representation of
eosinophil counts and recounts for a single patient. Small bidirectional arrows
indicate comparisons for intrarater reliability; medium bidirectional arrows,
comparisons for interrater reliability; and long bidirectional arrow, intrapatient
variability.
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RESULTS
Of the initial 71 patients in the inception cohort, 6 patients had missing
or unavailable pathology slides and were therefore excluded from the subsequent
analysis. Twenty-three (35%) of the remaining 65 patients had clinical evidence
of sinonasal polyposis. The distribution of patients by CT scan stage is displayed
in Figure 2. Each patient had between
1 and 3 pathology slides of sinus mucosa prepared based on the sidedness of
the surgical procedure (bilateral procedures resulted in at least 2 slides)
and the amount of material present in the paraffin block. A total of 132 pathology
slides were reviewed according to this protocol, resulting in a total of 396
data points per observer and 792 total data points.
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Figure 2. Distribution of computed tomographic
(CT) scan stage.
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For patients in the chronic rhinosinusitis group, the mean (SD) eosinophil
density was 23.4 (37.2) eosinophils per HPF. Three hundred nine (39.0%) data
points had eosinophil counts per HPF of 0. Twelve patients (18%) had no eosinophils
on histopathologic analysis. For the control group, the mean eosinophil density
in the sinus mucosa was 0.0 eosinophils per HPF.
Correlation coefficients and corresponding P
values for the 3 reliability measurements are displayed in Table 1. Strong intrarater and interrater reliabilities were noted
for the counting method. Regarding intrapatient consistency of tissue eosinophilia,
some variability was noted between slide 1 and slide 2, but the correlation
between an individual patient's slides was still strongly significant (r = 0.64, P<.001). A moderately
strong degree of correlation was noted between the CT scan stage and the degree
of tissue eosinophilia (Spearman = 0.62, P<.001; Figure 3).
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Statistics for Reliability Analysis of the Eosinophil Counting Method*
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Figure 3. Mean eosinophil density vs computed
tomographic (CT) scan stage. HPF indicates high-power field. Error bars indicate
95% confidence limits.
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COMMENT
Increasing evidence has emerged that the eosinophil may have a central
role in the pathogenesis of chronic rhinosinusitis. Several authors8-9 have found that tissue eosinophilia
is more prevalent in the mucosa of patients with chronic rhinosinusitis than
controls. Other studies have implicated the eosinophil as a source of immunomodulators
and cellular by-products that influence the mucosal response to allergens
and infection. Therefore, the eosinophil may not only have a role in the pathogenesis
of chronic rhinosinusitis, but may also be a useful indicator of disease severity
and prognosis for various forms of therapy. In an important study by Newman
et al,5 tissue eosinophilia was found to strongly
correlate with chronic rhinosinusitis disease severity on CT scan. Bhattacharyya
and Fried10 found a statistically higher rate
of peripheral blood eosinophilia in patients with chronic rhinosinusitis compared
with controls. Other investigators have also found that tissue eosinophilia
may predict surgical treatment outcome in patients with chronic rhinosinusitis.6
To more fully explore the effect of tissue eosinophilia on chronic rhinosinusitis,
a statistically valid and reliable method of eosinophil quantification within
sinus mucosa is required. Histomorphometric counting methods have been used
in other inflammatory disease states and in chronic rhinosinusitis. However,
different authors have used varying methods for quantifying tissue eosinophilia,
making comparisons among studies somewhat difficult. For example, several
studies have sought to explore the relationship between mucosal eosinophilia
and the severity of chronic rhinosinusitis as measured by CT scan stage. Goldwyn
et al11 found poor correlation between tissue
eosinophilia and CT scan severity (Pearson r = 0.131),
whereas Newman et al5 found a statistically
significant correlation between them. Neither study provided statistical measures
of reliability for their histologic eosinophil quantification method. Baroody
et al9 also found poor correlation between
CT severity and tissue eosinophilia (r = 0.25) and
noted that their eosinophil quantification method had strong interrater reliability
(r = 0.84). In that study, however, a marking pen
was used to identify the area on the pathology slide to be counted or recounted
by the second observer. Therefore, this measure of interrater reliability
is biased by the pen marking, potentially inflating the reliability score.
In fact, the lack of correlation between tissue eosinophilia and chronic rhinosinusitis
severity may be due to variability in the counting method rather than a true
lack of association.
For a histologic quantification method to be clinically useful, it must
be relatively simple to perform and statistically reliable. We sought to establish
a method that could be used in a standardized fashion to further explore the
relationship between the eosinophil and chronic rhinosinusitis. It is clear
from the data that the proposed method of tissue eosinophilia quantification
is statistically reliable and sound. High intrarater and interrater correlation
coefficients were noted for the proposed method with appropriate statistical
significance. This method is simple, uses readily available histologic and
microscopic techniques, and has proven reliability.
Using a statistically reliable eosinophil counting method, we found
a relatively strong correlation between severity of chronic rhinosinusitis
as measured by CT scan stage and degree of tissue eosinophilia (Figure 2). This relationship has come into question based on several
studies.5, 9, 11 In
our study, the relationship found between the CT scan stage and the degree
of tissue eosinophilia was independent of the anatomic side of the patient
that was sampled for tissue measurements.
Although we found our method to be statistically valid and reliable,
problems may still arise with its use in studies of sinus eosinophilia. Variability
in the thickness of the tissue fixed in the pathology slide requires that
the observer "focus through" the depth of the histologic specimen using the
fine-tuning focus control of the microscope. Some inherent variability may
arise between observers based on this operator-dependent portion of the counting
system. Our pathology laboratory consistently uses a 5-µm-thick slice
for processing hematoxylin-eosin specimens. Consistency in the cut thickness
of the pathology specimens is important for the reliability of this method.
Other methods of quantifying tissue eosinophilia have been developed
that use immunologic markers for the eosinophil or its by-products.12-13 These methods have been primarily
used in the study of the pathogenic impact of the eosinophil and chronic rhinosinusitis,
but may also be used to quantify tissue eosinophilia. Although these counting
techniques are considered reliable, they require additional processing, time,
and expense. In addition, they usually require a suitable control to ensure
that the staining or immunofluorescence technique is accurate. Our method
requires no additional processing time or expense and can be applied in a
retrospective fashion to previously obtained sinus tissue specimens. The use
of this reliable technique may allow for better study of the potential impact
of the eosinophil on the pathogenesis, staging, and treatment outcomes of
chronic rhinosinusitis.
AUTHOR INFORMATION
Accepted for publication February 7, 2001.
Corresponding author and reprints: Neil Bhattacharyya, MD, Division
of Otolaryngology, 333 Longwood Ave, Boston, MA 02114.
From the Division of Otolaryngology, Brigham and Women's Hospital (Dr
Bhattacharyya), Department of Otolaryngology, Massachusetts Eye and Ear Infirmary
(Drs Vyas, Fechner, Gliklich, and Metson), and Department of Otology and Laryngology,
Harvard Medical School (Drs Bhattacharyya, Vyas, Fechner, Gliklich, and Metson),
Boston, Mass.
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