Article, Surgery

A pilot study on potential new plasma markers for diagnosis of acute appendicitis

Original Contribution

A pilot study on potential new plasma markers for diagnosis of acute appendicitis?

Geertje Thuijls MD 1, Joep P.M. Derikx MD 1, Fred J. Prakken MD 2, Bregje Huisman MD, Annemarie A. van Bijnen Ing, Ernest L.W.E. van Heurn MD, PhD,

Wim A. Buurman PhD?, Erik Heineman MD, PhD 3

NUTRIM School for Nutrition, Toxicology and Metabolism, Department of Surgery, Maastricht University Medical Centre, 6229 ER, Maastricht, the Netherlands

Received 23 June 2009; revised 25 September 2009; accepted 26 September 2009

Abstract

Background: Diagnosis of Acute appendicitis remains a surgical dilemma, with Negative appendectomy rates of 5% to 40% and perforation suggestive for late operative intervention in 5% to 30%. The aim of this study is to evaluate new plasma markers, representing early neutrophil activation, to improve diagnostic accuracy in patients suspected for AA.

Materials and Methods: Fifty-one patients who underwent surgery for AA were included (male-female = 28:23), and blood was sampled. Plasma concentrations of 2 neutrophil proteins were measured: lactoferrin (LF) and calprotectin (CP). Controls consisted of 27 healthy volunteers. C-reactive protein (CRP) and white blood cell count concentrations were measured for routine patient care.

Results: Median plasma concentrations for LF and CP were significantly higher in 51 patients with proven AA (665 and 766 ng/mL, respectively) than in 27 healthy volunteers (198 and 239 ng/mL, respectively, P b .001).

No clinically relevant correlation exists between the plasma levels of LF and CP and the conventional laboratory tests for CRP and WBC.

Conclusions: Circulating LF and CP levels are significantly elevated in patients with appendicitis and are detectable in plasma using relatively simple and low-cost enzyme-linked immunosorbent assays. Furthermore, plasma levels of LF and CP give additional information to conventional markers WBC and CRP, making them potential new markers for AA diagnosis.

(C) 2011

? Grant support:This research was supported by AGIKO-stipendium 920-03-438 (to JPM Derikx) from the Netherlands Organisation for Health research and development. This work was partially funded by a “Profileringsfonds” grant from the University Hospital Maastricht.

* Corresponding author. Tel.: +31 43 3881499; fax: +31 43 3884154.

E-mail address: [email protected] (W.A. Buurman).

1 Equally contributed to this work.

2 Currently at Rijnland Hospital, Leiderdorp, the Netherlands.

3 Currently at Maxima Medisch Centrum, Veldhoven, the Netherlands.

Introduction

acute appendicitis is a common clinical entity with significant morbidity and mortality, particularly at the extremes of age, mainly because of the Atypical clinical presentation leading to a low diagnostic and therapeutic accuracy [1-3]. In general, the risk of 2 primary adverse outcomes must be balanced in diagnosis and consequent management of suspected AA. Firstly, misdiagnosis leads to removal of a normal appendix; and secondly, delayed diagnosis may lead to

0735-6757/$ - see front matter (C) 2011 doi:10.1016/j.ajem.2009.09.029

perforation and peritonitis. Because Delayed diagnosis and treatment of AA are associated with an increased rate of perforation, with resulting increased Morbidity and mortality rates, Timely intervention is crucial [1]. To reduce the risk of appendiceal perforation, surgeons have traditionally favored early appendectomy. As a consequence, the appendix is normal in approximately 5% to 40% of patients who undergo explorative laparotomy for suspected AA [4]. However, perforation of an inflamed appendix still occurs in 5% to 30% of patients treated surgically for suspected AA [4].

To improve the diagnostic accuracy of patients suspected for AA, the value of diagnostic imaging modalities and conventional inflammatory parameters has been studied. Ultrasound and/or computed tomographic scanning can improve diagnostic accuracy of appendicitis [5-7]. However, ultrasonography cannot be used for early diagnosis because only significant distension of the appendix can be detected using this imaging modality. Furthermore, diagnosis in adults using ultrasonography is hampered by fat and bowel gas that make visualization of the appendix difficult. Despite these limitations, ultrasonography can improve diagnostic accuracy in experienced hands. Computed tomographic scanning has a very high sensitivity and specificity for diagnosis of appendicitis and therefore reduces negative appendectomy rates. However, limited use of computed tomographic scanning for diagnosis of appendicitis is mandatory because of radiation exposure of patients.

Furthermore, no major improvement of diagnostic accuracy was found using white blood cell count and C-reactive protein (CRP) [8,9]. Therefore, diagnosis of appendicitis remains based on clinical symptoms and physical examination in most cases.

The pathophysiology of AA is characterized by defects in or increased permeability of the mucosal barrier, leading to inflammation and concomitant sequestration of large numbers of neutrophils into the gut wall [1]. Therefore, we sought tests to identify gut wall inflammation in patients with appendicitis to improve diagnosis of appendicitis after clinical suspicion. The neutrophil proteins lactoferrin (LF) and calprotectin (CP) are released upon neutrophil activation and are readily detectable in feces, as observed in patients with inflammatory bowel disease [10-13]. Appendicitis is an acute inflammatory disease, which makes a fecal test too time consuming and not suitable for diagnosis of Acute disease onset.

This pilot study is the first to investigate the potential usefulness of circulating neutrophil activation products LF and CP in diagnosing AA.

Materials and methods

Patients

From May 2004 to July 2005, 51 patients with acute AA who underwent appendectomy were consecutively included

at the emergency department (ED) of the University Hospital of Maastricht (28 males, 23 females; mean age, 34 years; range, 6-77 years). In addition, 27 healthy volunteers were included in this study to obtain reference values for plasma concentrations of LF and CP (20 males, 7 females; mean age, 24 years; age range, 3-61 years). Informed consent was obtained from each participant, and the study was approved by the local ethics committee. All patients included had the Dutch nationality and were white.

On admission to the ED, EDTA plasma samples were collected and stored at -20?C until analyses. Diagnosis was based on findings during surgery and histopathologic examination of the appendix. Out of the 51 appendicitis

Fig. 1 Lactoferrin (A) and CP (B) plasma concentrations for patients with appendicitis and healthy volunteers (outcome is reported as median +- range).

specimens, 4 (8%) showed acute mucosal inflammation indicating catarrhal appendicitis. Thirty-seven (72.5%) showed signs of a suppurative (phlegmonous) appendix; and in 10 cases (19.5%), the final diagnosis of a gangrenous (necrotizing) appendix was defined.

Methods

Lactoferrin and CP concentrations in plasma samples were determined using standard enzyme-linked immunosor- bent assays (ELISAs) kindly provided by HyCult Biotech- nology (Hbt, Uden, the Netherlands). The detection limits for LF and CP were 50 and 10 ng/mL, respectively. The human LF and CP ELISA kits are solid-phase ELISAs based on the sandwich principle with a working time of 3 1/2 hours. C- reactive protein and WBC concentrations were measured for routine patient care and retrospectively collected.

Statistics

Statistical analysis was performed with GraphPad Prism 4 for Windows (GraphPad Software Inc, San Diego, CA). Normality was tested using Kolmogorov-Smirnov test, and normal distribution was found for plasma CP. The distribution of plasma LF did not pass the normality test. Because of the relatively small group sizes and to keep the analyzed data comparable, Mann-Whitney U test was used

for between-group comparison for both markers tested. Data were presented as median +- range.

To test if LF and CP may have additional value to conventional laboratory tests (CRP and WBC), correlation between these new markers and WBC and CRP was measured in patients with proven appendicitis using Pearson correlation after normalizing the data by computing the natural logarithm of all data.

P b .05 is considered statistically significant.

Results

Fifty-one patients with histopathologically confirmed AA were included. Median plasma concentrations for LF and CP were significantly higher in the 51 patients with proven AA (665 [162-7008] and 766 [130-2424] ng/mL,

respectively) than in the 27 healthy volunteers (198 [100-

543] and 239 [100-585] ng/mL, P b .001) (Fig. 1).

Additional value of the plasma markers LF and CP to conventional laboratory tests (CRP and WBC) was tested using Pearson correlation. No correlation was found between LF and CRP (r2 = 0.010, P = .488). Lactoferrin and WBC correlated significantly (r2 = 0.083, P = .040). Significant correlation was found for CP and CRP and for CP and WBC (r2 = 0.114, P = .015 and r2 = 0.099, P = .024, respectively). Scatter plots are shown in Fig. 2.

Fig. 2 Scatter plots for LF and CRP (A), LF and WBC (B), CP and CRP (C), and CP and WBC (D).

Discussion

Early and correct diagnosis for appendicitis is crucial at the one hand to prevent perforation with resultant morbidity and at the other hand to avoid unnecessary surgery. Surgeons rely on disease history and clinical examination for diagnosing AA. laboratory investigations (CRP, WBC) and expensive advanced Imaging techniques can help improve diagnostic accuracy in some cases. As promising as these tests might seem, diagnostic accuracy improved only marginally in recent decades; and as a consequence, both negative appendectomy rates and Perforation rates remain high [4,14].

In this pilot study, we evaluated the potential usefulness of circulating neutrophil activation products LF and CP in diagnosing AA. The tests are relatively simple and cheap ELISAs that can be performed in clinical laboratories within 4 hours. Median plasma concentrations for LF and CP were significantly higher in the 51 patients with proven AA than in the 27 healthy volunteers. Next, we investigated if plasma levels of LF and CP give additional information to the conventional markers CRP and WBC. A correlation exists between LF and WBC and between CP and both CRP and WBC. However, Pearson correlation coefficient is low for these 3 correlations, with 0.114 as highest squared correlation coefficient (for CP and CRP), indicating that 89% of the CP values cannot be explained by CRP. Therefore, plasma levels of LF and CP can all give additional information in patients with appendicitis.

Elevated WBC levels measured in patients with appen- dicitis are mainly caused by increased neutrophil numbers. During appendicitis, the transformation of neutrophils from passively circulating cells to potent effector cells is mediated by exocytosis of granules and cytosolic proteins leading to the release of LF and CP. Therefore, we consider neutrophil activation products more specific for detection of appendi- citis than WBC. Neutrophil granules contain antimicrobial and potentially cytoToxic substances that are delivered to the exterior of the cell after degranulation. Lactoferrin is present in polymorphonuclear neutrophil granules. Lactoferrin (78 kd) is a glycoprotein mainly stored in specific granules. Upon activation, it is called lactoferricin and possesses antimicrobial activity against a broad spectrum of gram- positive and Gram-negative bacteria. The antimicrobial activity is exerted by impairment of bacterial growth by sequestration of iron, and by causing bacterial cell lysis by binding to the cell membrane [15]. Calprotectin (36 kd) is a heterodimer of 2 calcium binding cytosolic proteins, MRP14 and MRP8, constituting 60% of soluble proteins in neutrophils. Calprotectin is released upon neutrophil activa- tion and has bacteriostatic and cytokine-like effects in the local environment [11]. Calprotectin is also present in monocytes and mucosal epithelial cells [16]. Therefore, leakage of epithelial cells can also contribute to elevated plasma levels of CP. Recently, Sudan et al [16] showed that fecal CP is a useful marker for intestinal allograft monitoring.

Fecal CP levels were significantly higher in patients with allograft rejection than in patients with viral or nonspecific enteritis. Research on inflammatory bowel disease showed elevation of fecal CP in patients with active disease, with good correlation between amount of CP and the degree of mucosal damage measured endoscopically and between amount of CP and the extensiveness of inflammation assessed by leukocyte scan [12,17,18].

Although the markers are elevated in other intestinal Inflammatory conditions, they will allow discrimination of serious intestinal inflammatory pathologies, among others, AA, from simple gastrointestinal problems. Therefore, they can be of value in diagnosing inflammatory bowel conditions by reducing the differential diagnosis. This pilot study is an early step toward determining the utility of LF and CP in diagnosing appendicitis. Next step is to study the accuracy of these markers for diagnosing appendicitis in a large patient population admitted to the hospital with a clinical suspicion of AA.

In conclusion; circulating LF and CP levels are significantly elevated in patients with appendicitis and are detectable in plasma using relatively simple and low-cost ELISAs. Furthermore, plasma levels of LF and CP give additional information to the conventional markers WBC and CRP, making them potential new markers for AA diagnosis.

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