The Circulating Fibrocyte - a Novel and Accurate Biomarker in Diagnosing Acute Appendicitis in Adults
The Circulating Fibrocyte - a Novel and Accurate Biomarker in Diagnosing Acute Appendicitis in Adults
Accurate diagnostic biomarkers for acute appendicitis (AA) are lacking. Circulating
 fibrocytes (CFs) are increased in inflammatory states. It is not known whether they are
 altered in appendicitis. This study aimed to assess if CF altered in AA, and compared their
 diagnostic accuracy with white cell count (WCC), C-reactive protein (CRP), neutrophils,
 lymphocytes, neutrophils-lymphocytes ratio (NLR), monocytes, basophils and eosinophils in
 diagnosis of AA in patients presenting with right iliac fossa pain (RIFP).

 95 consecutive adults were recruited in a cohort based prospective study at University
 Hospital Limerick, 15 were healthy individuals and 80 were patients with RIFP. Peripheral
 venous samples were obtained at presentation. Clinical, biochemical and histo-pathological
 parameters were recorded. CF levels were determined by dual-staining for CD45 and Col-1 using
 FACS and correlated with histopathological diagnoses. P<0.05 was taken as significant.

 The study hypothesis is that in patients with suspected appendicitis, CF levels may improve
 the diagnostics, reduce unnecessary radiation and negative appendicectomy rates.
Acute appendicitis (AA) is the commonest abdominal emergency. Despite its high prevalence,
 the diagnosis of appendicitis remains a challenge. The clinical presentation is frequently
 atypical and symptoms often overlap with many other conditions. Coupled with this, failure to
 reach a prompt diagnosis can result in adverse outcomes.

 Circulating fibrocytes (CFs) were first described in 1994 by Richard Bucala. They are
 haematopoietic cells derived from the bone marrow. They circulate in the monocyte fraction
 and can differentiate into myelofibrocytes, fibroblasts and adipocytes (among other cell
 types). They have a prominent role in inflammatory and healing processes, and in the
 development of fibrosis.

 No studies to date have examined the role of CFs in acute appendicitis. Thus, the aims of
 this clinical trial were (1) to determine if CFs are altered in acute appendicitis, (2) to
 assess and compare their diagnostic accuracy with CRP, WCC, neutrophils, lymphocytes,
 neutrophils-lymphocytes ratio (NLR), monocytes, basophils and eosinophils, and (3) to
 determine the most reliable predictors in diagnosing AA.

 A prospective cohort study was undertaken to (1) determine circulating fibrocytes levels (CF)
 in patients with histologically confirmed acute appendicitis (AA), and (2) to determine the
 sensitivity and specificity of circulating CF levels in the diagnosis of AA, in adults (age ≥
 16 years) presenting acutely with right iliac fossa pain, at the University hospital
 Limerick. The diagnosis of appendicitis was confirmed by histopathological assessment
 following appendicectomy.

 Patients were divided in three cohorts following their final diagnosis on discharge and based
 on the histopathology results. (1) patients with RIFP who underwent an appendicectomy and had
 a normal appendix at histological assessment i.e histologically proven normal appendix group
 (HPNG). (2) patients with RIFP who underwent an appendicectomy and had an inflamed appendix
 at histological assessment i.e histologically proven appendicitis group (HPAG). (3) patients
 with RIFP who had an alternate diagnosis on discharge other than appendicitis i.e alternative
 diagnosis group (ADG). Another cohort was added as (4) healthy controls.

 Data collected included date of admission, patient's gender, age, presenting symptoms,
 duration of symptoms, CF levels, preoperative WCC and its differentials (neutrophil,
 lymphocyte, neutrophils-lymphocytes ratio (NLR), monocyte, basophil and eosinophil counts),
 CRP, final clinical diagnosis, operation and post-operative diagnosis based at histological
 assessment.

 Peripheral venous samples were obtained from patients in cohort 1, 2 and 3 (n= 80) and
 healthy controls (n=15). A single 10 mL sample of heparinized venous blood was collected via
 peripheral, upper extremity venipuncture. Samples were collected in sodium heparin (EDTA)
 vacutainer tubes and transferred to the laboratory within 3 hours. Samples were then
 processed to isolate the buffy coat layer using density gradient centrifugation (Histopaque,
 Sigma-Aldrich, Wicklow, Ireland). The resulting peripheral blood mononuclear cells were
 subsequently washed in Phosphate-buffered saline (PBS) and re-suspended in freezing medium
 (50% foetal bovine serum, 40% RPMI medium and 10% dimethyl sulfoxide) prior to transfer to
 cryogenic vials in 1-ml aliquots. Finally, samples were cooled in a cryogenic temperature
 control rate container to -80 °C until processing for flow cytometry. Following white blood
 cell isolation using density gradient centrifugation, 1×106cells were re-suspended in flow
 cytometry buffer (RPMI medium supplemented with 10% horse serum, 0.1% sodium azide and 25 mM
 HEPES). Cells were fixed and permeabilised using BD Cytofix/Cytoperm solution (BD
 Biosciences, Oxford, England) and blocked prior to intracellular staining of Collagen-I with
 mouse anti-human Collagen-I antibody (Millipore, Cork, Ireland, Product code MAB3391) which
 was subsequently stained with Alexa-Fluor 488 goat anti-mouse secondary antibody (Jackson
 ImmunoResearch Europe, Suffolk, England; Product code 115-545-146). Cells were then stained
 for cell surface antigen CD45 using PerCP anti-human CD45 (Biolegend, London, England). Cells
 were then re-suspended in PBS before subsequent analysis on the flow cytometer (BD
 FACSVerse). All analysis was done on a BD FACSVerse (BD Biosciences) using BD FACSuite v1.0.5
 (BD Biosciences). Fibrocyte levels were displayed as a percentage of the total white blood
 cell population.

 Data analyses were performed using IBM SPSS for Mac OSX version 25.0. Data were presented as
 means and standard deviations, or medians and interquartile ranges, as appropriate.
 Distribution of variables was assessed by histograms, Q-Q plots, and box plots. Analysis of
 Variance (One-Way ANOVA) was used to compare between different independent groups.
 Kruskal-Wallis test was used to compare between the biomarkers and the different groups (HPNG
 vs. HPAG vs. ADG), and for subgroup analysis pairwise comparisons were performed, a P - value
 of less than 0.05 was considered statistically significant. Receiver operating characteristic
 (ROC) curve was used to characterize and compare between the diagnostic accuracy of CFs, WCC,
 CRP, neutrophils, NLR and monocytes. Multinomial logistic regression analysis was used to
 assess for independent predictors for AA.
Inclusion Criteria:

 -  Any patient presents with right iliac fossa pain

 -  Age 16 years old or more

 Exclusion Criteria:

 -  Previous appendicectomy