High at admission serum Interleukin-6 and cell free DNA as Predictors for Severity and Outcome of Acute pancreatitis Patients
Departments of Internal Medicine, General Surgery* & Clinical Pathology**, Faculty of Medicine, Benha University, Egypt
Abstract
Objectives: To evaluate outcome of therapeutic interventions for cases of acute pancreatitis (AP) and to determined diagnostic yield and predictability of at admission estimation of serum C-reactive protein (CRP), interleukin (IL)-6 and cell free DNA (cfDNA) for such outcome.
Patients & Methods: The study included 67 AP patients. Ranson Criteria scoring (RS) was used to assess AP severity as MAP (RS=<3 points) and SAP (RS=≥3 points). Contrast enhanced CT (CECT) was performed 6–10 days after admission and Balthazar’s CT severity index (CTSI) was used to stratify the severity. Acute Physiology and Chronic Health Evaluation (APACHE II) score was used to assess the impact of AP on general condition and to help surgical decision making. Intevention policies included conservative treatment for patients with APACHE II score of ≥10 and surgical intervention either early (within 2 weeks) or late (after 2 weeks) according to the indications. At admission venous blood samples were obtained for estimation of serum CRP, IL-6 and cfDNA. Survival rate was defined as the primary outcome.
Results: According to Ranson's criteria 40 patients had MAP and 27 patients had SAP. According to CTSI, 41 patients had 0-3 score, 20 patients had 4-6 score and 6 patients had score of >6. According to APACH II scoring, 23 patients had a score of ≥10. In conservative treatment group, mortality rate (MR) was 13%, while was 18.2% in surgical intervention group with non-significant difference. In early surgical intervention group, MR was 17.6% and 15.4% in the late group with non-significant difference. At admission serum levels of the three parameters were significantly higher in SAP than in MAP cases. ROC defined at admission serum level of cfDNA as the most significant early predictor and CTSI as the most significantly specific diagnostic procedure for SAP. High serum IL-6 and cfDNA could predict mortality with high specificity.
Conclusion: High at admission serum cfDNA and IL-6 levels could be used as early predictors for AP disease severity and survival rates. Therapeutic intervention modalities showed non-significant difference in MR. Proper case selection for early surgical intervention improves outcome without deleterious effect on survival rate.
rate Keywords: Acute pancreatitis, severity scoring, serum markers, mortality
Introduction
Acute pancreatitis (AP) remains an unpredictable, potentially lethal disease with significant morbidity and mortality rates. It is a common disease with a wide spectrum of severity, an incidence rate of about 30-113 cases per 100 000 individuals and overall mortality rate of 10%-15%. Most episodes of AP are mild and self-limiting, but up to 10-20% of patients develop severe AP (SAP) with mortality ranging from 29% to 43%. The most common causes of AP are from gallstones and alcohol. Other causes include hyper-triglyceridaemia, hyperparathyroidism, pancreatic malignancy, endoscopic retrograde cholangiopancreatography, trauma, infectious agents, drugs, autoimmunity, and hereditary (1, 2).
The treatment of acute pancreatitis is mainly supportive; Chang et al. (3) documented that suitable crystalloid-colloid ratio should be considered in the early stage of resuscitation in patients with SAP and result in decreased fluid retention in the third space with an improvement of survival rate. Zhao et al. (4) indicated that combination of normal saline; hydroxyethyl starch and glutamine are more efficient in resuscitation of SAP by relieving inflammation and sustaining the intestinal barrier. However, Cui et al. (5) found early enteral feeding with addition of probiotics (bifidobacterium) resulted in significant lowering of the level of pro-inflammatory cytokines, earlier restoration of gastrointestinal function, decrease of complications such as infection, and shortening of hospital day in patients with SAP. Moreover, Pupelis et al. (6) found that early low volume oral feeding provides physiologic stimulation and promotes recovery of bowel function, preparing the gastrointestinal tract for low-fat hospital food in patients with necrotizing SAP.
Pancreatic necrosis and so called peripancreatic “fluid” collections are local complications of AP defined according to the Atlanta Criteria of AP, which categorizes AP as severe if these complications are present. It has been estimated that about 15% (4%-47%) of patients with AP will have necrotizing pancreatitis; and 21%-46% of patients will develop a peripancreatic “fluid” collection, including acute collections (during the first 4-6 wk), pseudocysts (after 4-6 wk of an acute attack with a defined wall with no or little necrotic tissue), necrotic collections and abscesses (collection after 4-6 wk with pus and a defined wall) (7, 8).
Management of SAP especially if complicated by necrotizing pancreatitis (NP) was mainly surgical and because infection of necrosis is considered as an indication for surgery, operations are often performed early. However, early surgical intervention in NP was associated with high mortality and guidelines recommend fine needle aspiration (FNA) in patients with NP and signs of sepsis. Recent advances include postponing intervention as a strategy to facilitate necrosectomy and improve prognosis and the "step-up approach", in case of infected necrosis, which includes percutaneous catheter drainage as the first step, followed by necrosectomy. This highly conservative approach resulted in significantly lower mortality than previous serial FNA and consecutive indication for surgery in case of proven infection. Open surgery in NP should be reserved for concomitant intra-abdominal complications (9, 10, 11).
However, early surgical intervention was revolved once again, so the current study aimed to evaluate the outcome of therapeutic interventions for cases of AP and to determined the diagnostic yield and predictability of early estimation of serum CRP, IL-6 and cell free DNA (cfDNA) for such outcome.
Patients & Methods
The current prospective study was conducted at Departments of General Surgery, Internal Medicine and Clinical pathology at Hospital, KSA. The study protocol was approved by the Local Ethical Committee and all cases of AP admitted to the hospital till June 2013 and signed written fully informed consent were enrolled in the study. The AP diagnosis relied on the presence of at least 2 of the 3 following criteria: a) acute abdomen with variable levels of peritoneal irritation syndrome with characteristic abdominal pain; b) amylase and/or lipase uprising 3 times above the superior normal limit; and c) characteristic findings of AP at the CT imaging. Collected demographic data included gender, age, pancreatitis etiology and preliminary laboratory.
Severity of AP was categorized based on the clinical and laboratory data using Ranson Criteria as shown in table 1 including the 11 early objective signs used to classify the severity of acute pancreatitis. For each positive sign 1-point was given and cases with <3 points were classified as mild AP (MAP) and those with ≥3 points were classified as SAP. The predicted mortality rate (MR) for cases had <3 point is 1%, 3-4 points is 15%, 5-6 points is 40% and for cases had >6 points is 100% (12). Indications for contrast enhanced CT (CECT) included patients with persistent organ failure, signs of sepsis, or clinical deterioration 6–10 days after admission. Balthazar’s CT severity index (CTSI) (Table 2) was used to stratify the severity and to help predicting morbidity and mortality, and equals CT grade + necrosis score (13). The impact imposed by AP on general condition that could help for surgical decision making was evaluated using the Acute Physiology and Chronic Health Evaluation (APACHE II) score, (14).
Table (1): Ranson Criteria used to classify the severity of AP (12)
At admission or diagnosis / During initial 48 hAge over 55 years / Hematocrit fall >10% points
White blood count over 16,000/mm3 / Blood Urea nitrogen rise >5 mg/dl
Blood glucose >200 mg/dl / Arterial Po2 <60 mmHg
Serum Lactic dehydrogenase (LDH)>350 U/l / Serum calcium <8 mg/dl
Serum Aspartate amino-transferase (AST) >250 U/l / Base deficit >4 µEq/l
Estimated fluid sequestration >6000 ml
Table (2): Balthazar' s CT severity index (13)
Scoring of CT grades / Necrosis grading / SignificanceGrade of AP based on non-contrast CT findings / Point / Degree of pancreatic necrosis based on contrast CT findings / Point / CTSI / Morbidity rate / Mortality rate
A= Normal pancreas / 0 / A= No necrosis / 0 / 0-3 / 8% / 3%
B= Focal or diffuse enlargement, including contour irregularities
and inhomogeneous parenchymal attenuation (Edematous pancreas) / 1 / B=Necrosis of up to one third of pancreas / 2 / 4-6 / 35% / 6%
C= Grade B + peripancreatic inflammation / 2 / C=Necrosis of up to half of pancreas / 4 / 7-10 / 92% / 17%
D= Grade C plus a single fluid collection / 3 / D=Necrosis of > half of pancreas / 6
E= Grade C plus two or more fluid collections or retroperitoneal gas / 4
Intevention policies
A. Conservative treatment: Patients with APACHE II score of ≥10 were admitted to ICU to strengthen the functional support for important organs. At ICU conservative treatment consisted of fasting, gastrointestinal decompression, antacid, and somatostatin and its analogues to inhibit the secretion of pancreatic enzyme. Fluid resuscitation, maintenance of water and electrolyte balance, dynamic monitoring of central venous pressure with adjustment of the ratio of crystal and colloid fluid were provided. Intra-abdominal pressure and organ functions were monitored. Antibiotic prophylaxis against Gram-negative bacteria, and anaerobic bacteria that can pass through blood pancreatic barrier was initiated early. Once general condition was stabilized enteral feeding was initiated through the pre-applied nasojejunal tube (7).
B. Surgical intervention: timing of surgical intervention as early (within 2 weeks) or late (after 2 weeks) was determined according to the indications. Early surgical intervention was indicated in cases with biliary pancreatitis secondary to bile duct obstruction, SAP with abdominal compartment syndrome and patients required CT or ultrasound-guided therapeutic percutaneous puncture, catheter drainage, or debridement and decompression of pancreatic necrotic tissue. The indications for late intervention include biliary pancreatitis with no bile duct obstruction, pancreatitis combined with infection and pancreatic or peripancreatic abscess, pancreatic pseudocyst or other local complications (15).
Laboratory investigations
Blood samples were drawn at time of admission under complete aseptic conditions and was put in clean dry tube and allowed to clot and then serum was separated by centrifugation at 3000 rpm for 10 min and was collected in clean dry Eppendorff tube to be stored at -80oC till:
1. Colorimetric estimation of serum C-reactive protein (CRP).
2. ELISA estimation of serum IL-6 (16) using commercial kits (Boehringer GmbH, Mannheim, Germany) for ELISA assay of IL-6.
3. Real-time quantitative reverse transcriptase polymerase chain reaction (PCR) for quantification of serum cell free DNA (cfDNA)
Quantification of serum cell-free DNA
DNA extraction and quantification of serum cell-free DNA were performed as described by Saukkonen et al. (17). Briefly, samples were centrifuged at 16,000 g for 10 minutes before DNA extraction to remove any residual cells (18). DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the “blood and body fluid protocol”. Serum cell-free DNA was measured by real-time quantitative PCR assay for the β-globin gene using the ABI PRISM 7000 sequence detection system (Applied Biosystems). The sequences were as follows:
Forward primer 5'-GCA CCT GAC TCC TGA GGA GAA-3'
Reverse primer 5'- CAC CAA CTT CAT CCA CGT TCA-3'
PCR cycling conditions were two minutes at +50°C, 10 minutes at +95°C, and 46 cycles of 20 seconds at +95°C and one minute at +60°C. Plasma DNA was measured in duplicate samples. A 10-fold serial dilution of human genomic DNA (Roche Diagnostics GmbH, Mannheim, Germany) was used as a standard curve in the PCR assay. Results are expressed as genome equivalents (GE)/ml; 1 GE equals 6.6 pikograms of DNA. Cell-free DNA concentration of 4,000 GE/ml was considered as an upper limit of normal range (19).
Statistical analysis
Obtained data were presented as mean±SD, ranges, numbers and ratios. Results were analyzed using Wilcoxon; ranked test for unrelated data (Z-test) and Chi-square test (X2 test). Sensitivity & specificity of estimated parameters as predictors were evaluated using the receiver operating characteristic (ROC) curve analysis judged by the area under the curve (AUC) compared versus the null hypothesis that AUC=0.05. Statistical analysis was conducted using the SPSS (Version 15, 2006) for Windows statistical package. P value <0.05 was considered statistically significant.
Results
The study included 67 patients; 39 males and 28 females with mean age of 42.5±7.9; range: 28-62 years. Forty patients (59.7%) were younger than 45 years and 27 patients (40.3%) were older than 45 years. Pain is the cardinal presenting symptom and was present in all patients. Nausea and vomiting were reported in 39 patients, jaundice in 20 patients, 13 patients had positive Cullen's sign and 5 patients had positive Gray-Turner's sign. At admission, mean serum amylase level was 759.1±150.4; range: 345-945 U/L and mean serum lipase level was 1066.3±293 U/L. Details of demographic and clinical data and at admission routine laboratory data are shown in table 3 & 4.
Table (3): At admission demographic and clinical data of studied patients
FrequencyAge (years) / <45 / Frequency / 40 (59.7%)
Value / 37.4±4.4 (28-44)
≥45 / Frequency / 27 (40.3%)
Value / 50.1±5.3 (45-62)
Total / 42.5±7.9 (28-62)
Gender / Males / 39 (58.2%)
Females / 28 (41.8%)
Clinical presentation / Pain / 67 (100%)
Nausea and vomiting / 39 (58.2%)
+ Cullen's sign / 20 (29.9%)
+ Gray-Turner's sign / 5 (7.5%)
Etiology of AP / Biliary / 45 (67.2%)
Alcoholism / 7 (10.4%)
Idiopathic / 13 (19.4%)
Post ERCP / 2 (3%)
Data are presented as numbers & mean±SD; percentages & ranges, AP: acute pancreatitis; ERCP: endoscopic retrograde cholangiopancreatography
Table (4): At admission laboratory data of studied patients
Parameter / Strata / Frequency / LevelsHematocrite value (%) / 30-35 / 6 (9%) / 32.5±1.9 (30-35)
>35-40 / 13 (19.4%) / 37.4±1.4 (36-40)
>40-45 / 15 (22.4%) / 42.9±1.6 (40-45)
>45-50 / 18 (26.8%) / 48.4±1.4 (46-50)
>50-55 / 10 (14.9%) / 52.7±1.1 (51-54)
>55 / 5 (7.5%) / 58.7±2.1 (56-61)
Total / 67 (100%) / 45.3±7.5 (30-61)
Total leucocytic count (103/ml) / <15 / 49 (73.1%) / 9.7±2.7 (5-14)
>15 / 18 (26.9%) / 18.7±1.9 (15-23)
Total / 67 (100%) / 12.1±4.8 (5-23)
Serum AST (IU/L) / <250 / 41 (61.2%) / 82.1±16.9 (55-114)
>250 / 26 (38.8%) / 290.1±17.8 (260-320)
Total / 67 (100%) / 163.2±103.9 (55-320)
Serum amylase / <700 / 25 (37.3%) / 595±106.5 (345-697)
>700 / 42 (62.7%) / 858.8±49 (260-320)
Total / 67 (100%) / 759.1±150.4 (345-1240)
Serum lipase / <1000 / 33 (49.3%) / 829.2±120 (598-983)
>1000 / 34 (50.7%) / 1296.4±217 (1050-2100)
Total / 67 (100%) / 1066.3±293 (598-2100)
Data are presented as numbers & mean±SD; percentages & ranges, AST: aspartate transaminase