529273

research-article2014

SJS104010.1177/1457496914529273W. Farooqui, H-C. Pommergaard, J. Burcharth, J. R. EriksenScandinavian Journal of Surgery

ORIGINAL ARTICLE

Scandinavian Journal of Surgery 104: 7278, 2014

The Diagnostic Value of a Panel of Serological

Markers in Acute Appendicitis
W. Farooqui, H-C. Pommergaard, J. Burcharth, J. R. Eriksen
Gastroenheden, Kirurgisk Sektion, Herlev Hospital, Herlev, Denmark

Abstract

Background: Appendicitis is a frequent reason for hospital admissions. Elevated C-reactive

protein, white blood cell count, and serum bilirubin have been suggested as individual
markers for appendicitis and appendiceal perforation. The aim of this study was to analyze
if a combination of serologic markers could increase the prognostic accuracy of diagnosing
non-perforated and perforated appendicitis.
Material and Methods: Demographic data, histological findings, blood tests, and clinical
symptoms were collected on all patients who underwent a diagnostic laparoscopy, a
laparoscopic appendectomy, or conventional (open) appendectomy between May 2009 and
May 2012 from a surgical department. The patients were grouped into those with either
perforated appendicitis, non-perforated appendicitis, or differential diagnosis. Univariate
and multivariate models were used to identify which markers were useful in predicting
acute and perforated appendicitis, and receiving operating characteristics curves were
used to find the specificity, sensitivity, and the negative and positive predictive values.
Results: A total of 1008 patients were operated under suspicion of appendicitis.
From these, 700 patients had a pathologically verified inflamed appendix and 190 had
a perforated appendix. Patients with acute appendicitis had significantly higher blood
levels of white blood cell, bilirubin, C-reactive protein, and alanine transaminase than
patients without appendicitis. Patients with perforated appendicitis had significantly
higher levels of white blood cell, bilirubin, and C-reactive protein than patients with nonperforated appendicitis. The highest positive predictive value to discriminate between
acute appendicitis and non-appendicitis was of a linear regression model combining
white blood cell count, bilirubin, and alanine transaminase. C-reactive protein levels
and a linear regression model, including white blood cell count, bilirubin, and C-reactive
protein levels as variables, had the highest negative predictive values when discriminating
between perforated and non-perforated appendicitis.

Correspondence:
Waqas Farooqui
Lundtoftegrdsvej 27, st th
2800 Lyngby
Denmark
Email: waqas.farooqui@regionh.dk

Diagnostic value of serological markers in appendicitis

73

Conclusion: Combining blood markers was useful in predicting appendicitis and

perforated appendicitis. In addition to C-reactive protein and white cell count, blood
levels of bilirubin, and alanine transaminase may be useful.
Key words: Appendicitis; prognosis; serology; accuracy; perforation; combination

Introduction

Statistical Analysis

Appendicitis is a result of a bacterial infection in the

appendix (13) and is a frequent cause of abdominal
pain and of hospital admissions (4). Usually, clinical
symptoms are enough to hint the diagnosis (5); however, a diagnosis can only be confirmed upon surgery
with subsequent pathological evaluation. Several blood
markers, including bilirubin, C-reactive protein (CRP),
and white blood cell (WBC) count may be increased in
patients with appendicitis and even more in patients
suffering from perforated appendicitis (5, 6).
Numerous scoring systems evaluating clinical
symptoms and blood tests to increase the prognostic
accuracy of appendicitis have been designed (79).
However, none of the scoring systems evaluate the
risk of appendiceal perforation, and none of the scoring systems use a combination of blood markers. The
aim of this study was to analyze whether multiple
serological markers, including CRP, bilirubin, and
WBC count, in combination, could increase the prognostic accuracy of the diagnoses of appendicitis and
perforated appendicitis.

For the statistical analysis, SPSS version 20 (SPSS Inc.,

Chicago, IL, USA) for Windows (Microsoft
Coorporation, Redmond, WA, USA) was used.
Univariate analysis and receiving operating characteristics (ROC) curves were used to obtain the specificity
and sensitivity of WBC, bilirubin, CRP, alanine
transaminase (ALAT), and aspartate transaminase
(ASAT). From these, the significant (p < 0.05) variables
were then inserted into a multivariate logistic regression model using forced entry mode. The significant
variables from the multivariate model were used as
variables in a multiple linear regression equation
(y = a x1 + b x2 + c x3 ...+ z xn ) with letters representing the logistic regression coefficients (B) for each significant variable found in the multivariate logistic
regression, and the x-values representing the level of
the serologic marker. A ROC curve was then performed using the equation to yield a sensitivity and
specificity for the variables combined. The specificity
and sensitivity of each value were used to calculate
the negative and positive predictive values (PPVs).
This study was approved by the Danish Data
Protection Agency (No. HEH-2013-051).

Method
During a 3-year study period from May 2009 to May
2012, we retrospectively included all patients suspected of or suffering from appendicitis, who underwent an acute diagnostic laparoscopy, laparoscopic
appendectomy, or conventional (open) appendectomy in a surgical department. Data was extracted
from the electronic patient journal system and
included patient demographics, histological findings, results of blood tests (liver function test, WBC,
bilirubin, and CRP), and clinical symptoms on admission. Patients were excluded if one of the following
criteria were present: appendectomy or laparotomy
for other reasons than appendicitis; known liver, biliary, or hematologic diseases; recent severe illness
(defined as any illness resulting in an increased CRP
and WBC count); pregnancy at the time of admission;
missing results from relevant blood tests; or an age
below 16 years.
Patients were divided into two groups consisting of
patients with histologically verified appendicitis and
patients with other differential diagnoses. Patients
with a histologically verified appendicitis were further divided into two subgroups depending on
whether they had a perforated or non-perforated
appendicitis. Perforation was defined as the presence
of one of the following criteria: visible perforation at
operation, documented presence of a periappendicular abscess, or histologically verified perforation (a
break of the serosa layer).

Results
A total of 1656 patients were initially included. Out of
those, 39% were excluded due to the presence of one
or more of the exclusion criteria. A total of 1008
patients were operated with a preoperative suspicion
of appendicitis. Of these, 700 patients had a pathologically verified inflamed appendix. Of the patients with
an inflamed appendix, 27% had a perforated appendix
(Fig. 1). A total of 698 patients had their appendix
removed through laparoscopy, 24 patients through
open surgery, and 286 patients underwent a diagnostic laparoscopy. Patient demographics are shown in
Table 1.
In the univariate analysis, patients with acute
appendicitis had significantly higher blood levels of
WBC (p < 0.001), bilirubin (p < 0.001), CRP (p < 0.001),
and ALAT (p = 0.001) than patients without appendicitis. Levels of ASAT were not significantly different
between the two groups (p = 0.818). Patients with perforated appendicitis had significantly higher levels of
WBC count (p = 0.004), bilirubin (p < 0.001), and CRP
(p < 0.001) than patients with a non-perforated appendicitis. No differences in levels of ASAT (p = 0.331)
and ALAT (p = 0.178) were seen between the two
groups.
In the multivariate analysis for detecting markers
that predicted appendicitis versus non-appendicitis,
we found that the WBC count, bilirubin, and ALAT

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W. Farooqui, et al.

Fig.1. Flowcharts of patients included/excluded from the study.

Table 1
Patient demographics and efficiency data.
Parameter

Age (years)
Gender
Male
Female
WBC
Bilirubin
CRP
ALAT
ASAT

Non
appendicitis
(n = 308)

Appendicitis
(n = 700)

33 (1691)

40 (1697)

84 (27.3%)
224 (72.7%)
10.4 (3.2027.6)
9 (163)
25.50 (3427)
17 (3455)
26 (13607)

343 (49%)
357 (51%)
13.5 (3.7033.40)
12 (291)
39.50 (3486)
19 (3828)
27 (9326)

Statistical
significance
p < 0.001

p < 0.001
p < 0.001
p = .002
p = 0.001
p = 0.818

Non-perforated
appendicitis
(n = 510)
37 (1697)
246 (48.2%)
264 (51.8%)
13.2 (3.730.3)
12 (278)
26.5 (3486)
19 20.82 (3203)
27 (9166)

Perforated
appendicitis (n = 190)
51 (1695)
97 (51.1%)
93 (49.9%)
13.85 (5.433.4)
15.5 (291)
86 (3379)
17.5 75.43 (3828)
27 (12326)

Statistical
significance
p < 0.001

p = 0.004
p < 0.001
p < 0.001
p = 0.331
p = 0.178

WBC: white blood count; CRP: C-reactive protein; ALAT: alanine transaminase; ASAT: aspartate transaminase.
Data are presented as median (range).

levels were all significant factors (Table 2). In the multivariate analysis, discriminating perforated from
non-perforated appendicitis, we found that blood levels of WBC, bilirubin, and CRP were significant
parameters for predicting appendiceal perforation
(Table 2).
A linear regression model, including the WBC
count, bilirubin, and ALAT (y = 4.624 logWBC +
1.378 log Bilirubin + 0.684 log ALAT) had the highest
PPV to discriminate between acute appendicitis and
non-appendicitis (Table 3). In order to discriminate
between perforated and non-perforated appendicitis
level of CRP, a linear regression model, including
WBC count and level of bilirubin and CRP as variables (y = 1.842logWBC + 0.815log Bilirubin + 1.091log
CRP), had the highest negative predictive value
(NPV) (Table 4). A comparison of the ROC curves is
shown in Figs 2 and 3.
Discussion
This study found that the WBC count, levels of bilirubin, CRP, and ALAT levels were all significantly

increased in patients with appendicitis compared to

patients without. Furthermore, it was shown that
WBC count, bilirubin levels, and CRP levels were significantly increased in patients with perforated appendicitis compared to patients suffering from
non-perforated appendicitis.
We found that increased WBC count and bilirubin
levels were useful in predicting appendicitis, and furthermore, combining the markers with ALAT
increased the predictive value in our model. In the
group of patients with perforated appendicitis, we
found that bilirubin levels and CRP levels were useful
markers alone and in combination with WBC count.
Combining the markers even increased the predictive
value slightly. Our results affirm the findings of other
studies that the level of bilirubin is increased in
patients with acute and perforated appendicitis. High
levels of bilirubin have previously been shown among
patients with perforated appendicitis (1012), and
one study has shown that an elevated bilirubin level
may be a good predictor for acute appendicitis (13).
A recent meta-analysis (14) concluded that bilirubin alone as a marker for identifying patients with

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Diagnostic value of serological markers in appendicitis

Table 2
Multivariate analysis showing factors discriminating appendicitis from non-appendicitis and perforated from non-perforated appendicitis.
Parameters

Logistic regression
coefficient (B)

Appendicitis and non-appendicitis

WBC count
4.624
Bilirubin
1.378
CRP
0.154
ALAT
0.684
Perforated and non-perforated appendicitis
WBC count
1.842
Bilirubin
0.815
CRP
1.091

SE

Statistical
significancea

Exp (B)

0.522
0.263
0.118
0.257

p < 0.001
p < 0.001
p = 0.191
p = 0.008

101.930
3.966
1.166
1.981

0.673
0.342
0.162

p = 0.006
p = 0.017
p < 0.001

6.311
2.158
2.977

95% confidence
interval
36.7283.3
2.46.6
0.91.5
1.23.3
1.68823.602
1.1554.417
1.1674.090

WBC: white blood count; CRP, C-reactive protein; ALAT: alanine transaminase; ASAT: aspartate transaminase.
Parameters were analyzed using their logarithmic value.
aLogistic regression analysis.
Table 3
Sensitivities, specificities, positive predictive values, and negative predictive values of parameters used in the early diagnosis of acute appendicitis.
Parameters

Sensitivity

Specificity

Positive
predictive value

Negative
predictive value

WBC count
Bilirubin
CRP
ALAT
ASAT
Linear model

0.68
0.69
0.81
0.73
0.52
0.64

0.64
0.56
0.32
0.39
0.51
0.75

0.81
0.78
0.73
0.73
0.71
0.86

0.46
0.45
0.42
0.39
0.32
0.49

WBC: white blood cell; CRP: C-reactive protein; ALAT: alanine transaminase; ASAT: aspartate transaminase.
Parameters significant in the multivariate analysisWBC, bilirubin, and ALAT. Linear model is a combination of the significant
parameters in the multivariate analysis.

Table 4
Sensitivities, specificities, positive predictive values, and negative predictive values of parameters used as indicators for identifying patients with a
perforated appendix among patients with acute appendicitis.
Parameters

Sensitivity

Specificity

Positive
predictive value

Negative
predictive value

WBC
Bilirubin
CRP
ALAT
ASAT
Linear model

0.34
0.50
0.54
0.03
0.61
0.60

0.77
0.71
0.79
0.99
0.43
0.76

0.35
0.39
0.49
0.46
0.28
0.48

0.76
0.79
0.82
0.73
0.75
0.83

WBC: white blood cell; CRP: C-reactive protein; ALAT: alanine transaminase; ASAT: aspartate transaminase.
Parameters significant in the multivariate analysisWBC, bilirubin, and CRP. Linear model is a combination of the significant parameters
in the multivariate analysis.

appendicitis was not sufficiently useful, but had to be

included in a scoring system comprising other factors
such as clinical symptoms and other blood markers.
Our results showed bilirubin having a sufficient significance alone, and increased when combined with
other markers.
The level of CRP was not significantly increased
among patients suffering from acute appendicitis compared to patients suffering a differential diagnosis.

An explanation could be that CRP reacts slower compared to, for example, WBC (15). Thus, in the patient
with appendicitis, who normally has a short and
acute symptomatic history, the CRP may not react
until later. In contrast, the patients with perforated
appendicitis may have a more severe disease and a
longer duration of inflammation. This may result in
the significant elevation of CRP levels for perforated
disease.

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W. Farooqui, et al.

Fig.2. ROC curve analysis for patients with appendicitis versus patients with a differential diagnosis. Top left: WBC count (AUC = 0.707);
top right: bilirubin (AUC = 0.661); bottom left: ALAT (AUC = 0.569); and bottom right: linear model (AUC = 0.745).
ROC: receiving operating characteristics; WBC: white blood cell; AUC: area under curve; ALAT: alanine transaminase.

Acute appendicitis causes an inflammatory

response. Literature has shown that WBC count was
significantly increased during an inflammatory
response, which was caused by a bacterial infection
in the appendix (5). Other studies have shown that
levels of bilirubin and CRP were significantly
increased in the early diagnosis of acute appendicitis. The mechanisms behind this sepsis-related
hyperbilirubinemia may be explained through
increased hemolysis, and a decrease in bile uptake
and excretion (16). Other studies have shown that
bacterial endotoxins, including toxins produced by
the bacteria Escherichia coli, decreased the hepatic
bile secretion contributing to intrahepatic cholestasis (1719) and sinusoidal damage (20). In rodent
models, endotoxins reduced bile-salt uptake in
hepatocytes (21). Our study showed that levels of
ALAT were significantly increased, especially among
patients suffering from appendicitis. This could be a

result of an inflammatory reaction in the hepatocytes

or sinusoidal damage.
Being retrospective, this study has inherent limitations. Nonetheless, since the parameters recorded in
this study were collected prospectively, information
bias may be limited. However, due to missing data
among blood tests some patients with appendicitis
were excluded. Furthermore, this study did not
include patients under the age of 16, many of who
were operated for appendicitis. They could not be
included since blood tests are not performed on children under the age of 16, if admitted under the suspicion of appendicitis. Compared to the other studies in
this field, our study is strong because we had a large
patient material. Furthermore, we have attempted to
limit selection bias by including all patients operated
on the suspicion of appendicitis in the cohort.
Diagnosing appendicitis is mainly done through
symptomatic history and clinical evaluation. Over

Diagnostic value of serological markers in appendicitis

77

Fig.3. ROC curve analysis for patients with non-perforated appendicitis versus patients with perforated appendicitis. Top left: WBC count
(AUC = 0.565); top right: bilirubin (AUC = 0.617); bottom left: CRP (AUC = 0.700); and bottom right: linear model (AUC = 0.715).
ROC: receiving operating characteristics; WBC: white blood cell; AUC: area under curve; CRP: C-reactive protein.

past few years, many studies have shown that blood

tests can be useful in strengthening the prognostic
accuracy of the appendicitis diagnosis and in differentiating between perforated and non-perforated appendicitis. Our study has shown that there is a greater
chance of diagnosing acute appendicitis when using a
combination of multiple blood markers compared to
using only one.
In a clinical setting, many patients admitted with
the suspicion of appendicitis are kept under close
observation rather than being operated immediately
since their symptomatic history or clinical picture is
not well defined. In these cases, a scoring system could
be useful in strengthening the suspicion. In cases of
prioritizing the patient in greater risk of perforated
appendicitis a scoring system could be useful as well.
Numerous studies have been conducted to identify factors or biomarkers, which can be used

diagnostically to differentiate between patients suffering from appendicitis and differential diagnoses
and thereby reduce the number of patients undergoing surgical treatment. The similar has been
attempted for patients suffering from perforated
and non-perforated appendicitis. As stated above,
many biomarkers have indeed been identified as
being related to appendicitis and appendiceal perforation, the most important and well documented
being WBC, CRP, and bilirubin. These biomarkers
have generally had a high specificity, but low
sensitivity (22, 23). Combining biomarkers increases
the specificity without a great change in sensitivity.
Therefore, relevant biomarkers, alone or in
combination, cannot be used as a differential tool
but rather as a supportive tool along side
the patients clinical appearance and symptomatic
history.

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W. Farooqui, et al.

Conclusion
WBC count and bilirubin, CRP, and ALAT levels are
useful biomarkers in predicting appendicitis and
appendiceal perforation. Combining the biomarkers
increases the predictive values. Therefore, blood levels
of bilirubin, CRP, and ALAT should be taken in consideration when predicting appendicitis.
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Received: September 15, 2013

Accepted: March 2, 2014