Post endoscopic pancreatitis (PEP) has different initial immunologic response to primary injury compared to acute pancreatitis of other etiology (non-PEP AP).

The purpose of this study is to compare initial immunologic response, 24 h after primary injury, in patients with PEP and patients with acute pancreatitis of other etiology.

PROTOCOL:

All patients with indication for endoscopic retrograde cholangiopancreatography (ERCP) will be prepared for ERCP on the following protocol: they will be instructed not to eat 4 to 6 hours before the procedure. Immediately before the procedure they will be administrated with Diclophenac Sodium 100 mg suppositories. One hour before the procedure 10 mL of blood sample and urine sample will be taken and analyzed in laboratory premises incorporated in University Hospital Centre Rijeka for following parameters: values of amylase in serum and urine and lipase in serum, liver enzyme tests and kidney function. 4-6 hours after ERCP is performed, 10 mL of blood sample will be taken and analyzed in laboratory premises incorporated in University Hospital Centre Rijeka for following parameters: values of amylase in serum and urine and lipase in serum, liver enzyme tests and kidney function.

24 hours after the procedure all patients will be taken 30 ml of heparinized peripheral venous blood and urine sample. 10 mL will be examined in the Department of Laboratory Medicine, Clinical Hospital Centre for following parameters: values of amylase in serum and urine and lipase in serum, liver enzyme tests and kidney function. Urine sample collected after the procedure will be used to evaluate amylase levels. Other 20 mL of blood samples will be sent to Department of Physiology and Immunology, School of Medicine where immunologic analysis will be performed. All patients who underwent ERCP will be divided into two groups. First group (PEP group) will be made of patients who developed acute pancreatitis following ERCP. Upon clinical and laboratory confirmation of acute pancreatitis according to European Society of Gastrointestinal Endoscopy (ESGE) guidelines for post-ERCP pancreatitis, will be further monitored during hospitalization for evaluation of the severity of the disease. Severity of the disease will be assessed according to the Cotton criteria for the severity of post-ERCP pancreatitis. Group of patients who underwent ERCP but didn't develop acute pancreatitis within 24 hours after the procedure, according to ESGE guidelines will be used as a control group (non-PEP group).

All patients with acute pancreatitis according to Atlanta criteria admitted through Emergency Department will be taken 30 ml of heparinized peripheral venous blood and urine sample upon 24 hours after the clinical symptoms have started (upper abdominal pain often radiating through to the back). 10 mL of collected blood samples will be examined in the Department of Laboratory Medicine, Clinical Hospital Centre for following parameters: values of amylase in serum and urine and lipase in serum, liver enzyme tests and kidney function. Other 20 mL of blood samples will be sent to Department of Physiology and Immunology, School of Medicine where immunologic analysis will be performed. This group of patients will be further monitored during hospitalization for evaluation of the severity of the disease. Severity of the disease will be assessed according to the Atlanta criteria.

METHODS AND MATERIALS

Laboratory parameters

For each patient included in the study blood and urine analysis will be performed in the Department of Laboratory Medicine, Clinical Hospital Centre Rijeka. Basic hematology tests will include: total blood count - a count of the total number of red blood cells, white blood cells and platelets present in blood, values of hemoglobin and hematocrit. Biochemical analysis of blood serum will include: values of amylase and lipase in serum, sodium, potassium, glucose, liver enzymes (aspartate aminotransferase (AST), alanine aminotransferase (ALT), Gama-glutamyl transferase (GGT), alkaline phosphatase (ALP)) direct and indirect bilirubin, kidney function (serum urea and creatinine, estimated glomerular filtration (eGFR)), C-reactive protein and acid-base status of arterial blood.

Immunological methods

For each patient included in the study immunologic analysis of blood samples (24 hours after the procedure or onset of the symptoms) will be performed in Department of Physiology and Immunology, School of Medicine in Rijeka, Croatia.

Isolation of peripheral blood mononuclear cells Twenty milliliters of peripheral blood will be acquired in Vacutainer (Becton Dickinson, Franklin Lakes, NY), overlaid onto Lymphoprep (Nycomed Pharma AS, Oslo, Norway) and centrifuged (20 min at 600 g). After the centrifugation, the cells from the interface will be collected and washed twice in Roswell Park Memorial Institute (RPMI) 1640 medium (Auckland, NZ) and used immediately for further experimental procedure. The viability of the cells was >95% will be assessed with propidium iodide 0.5 mg/ml/106 cells (Sigma-Aldrich Chemie) and a flow cytometer (FACSCalibur, Becton Dickinson, San Jose, USA)

Detection of cell surface Peripheral blood mononuclear cells (PBMC) will be stained for 30 min at 40 degrees Celsius with different combinations of Phycoerythrin (PE) - Cyanine (Cy) anti-cluster of differentiation (CD) 3 monoclonal antibody (mouse Antibody (mAb), Immunoglobulin G 1 (IgG1)), PE-conjugated anti-cluster of differentiation (CD)56 mAb (mouse B159, IgG1) and fluorescein isothiocyanate (FITC)-conjugated anti-natural-killer group 2, member D (NKG2D) mAb (5C6). FITC-, PE- and PE-Cy conjugated mouse isotype match antibodies will be used to set negative controls for each class of antibody used.

Stained cell samples will be analyzed by flow cytometry using FACSCalibur flow cytometer (Becton Dickinson & Co, San Jose, USA).

Detection of cytokine Interleukin (IL)-1 beta (β) and heat shock protein (HSP) 70, 27.

For the quantitative determination of human IL-1 β concentrations in plasma,Human IL-1 ELISA (Enzyme-Linked Immunosorbent Assay) Kit will be used.

For the quantitative determination of heat shock protein (HSP) 70 and 27 in plasma, HSP 70 High sensitivity ELISA Kit and HSP 27 ELISA kit will be used.

Detection of pentraxin 3 (PTX 3) and procalcitonin For the quantitative determination of human pentraxin 3 (PTX) and procalcitonin concentrations in plasma specific ELISA Kit will be used.

Inclusion Criteria:

• all patients underwent to ERCP irrespectively about the diagnosis
• all patients with diagnosed acute pancreatitis according to Atlanta criteria admitted through Emergency Department within 24 hours of onset of symptoms.

Exclusion Criteria:

• unwillingness or inability to consent for the study
• anticipated inability to follow protocol, previous ERCP, acute cholecystitis and/or cholangitis
• active or recent (within 4 weeks) gastrointestinal hemorrhage
• existing acute pancreatitis (lipase peak) within 72 hours prior to ERCP
• intrauterine pregnancy, breast feeding mother
• patients with chronic inflammatory diseases (e.g. IBD) systemic inflammatory and autoimmune disorders (e.g. systemic lupus erythematosus, ) or acute inflammatory diseases (e.g. pneumonia, pyelonephritis or sepsis of any cause)
• patients on immunomodulatory or immunosuppressive therapy.

All patients with indication for ERCP and all patients with acute pancreatitis admitted through Emergency Department