Introduction
Acute pancreatitis is a reversible inflammatory disease1–4 with clinical signs that range from mild and transient to severe with life-threatening complications including inflammatory response syndrome and multiorgan dysfunction.2–6 Cardiovascular and hemostatic derangements, as well as renal and pulmonary complications, account for the high fatality rates in dogs (23% to 58%) and people with acute pancreatitis.2,4,7,8
Histones are evolutionally highly conserved alkaline nuclear proteins that constitute the basic structure DNA interacts with to form nucleosomes.9 Nuclear histones are released into the extracellular space from injured cells or activated neutrophils (eg, so-called neutrophil extracellular traps).9–12 Extracellular his-tones bind to toll-like receptors, leading to increased production of proinflammatory cytokines (eg, TNF-α and IL-6).11–13 Moreover, experimental administration of histones to mice and monkeys induces neutrophil migration and activation, cytotoxic effects, endothelial damage, platelet activation, and thrombosis.9,12 Administration of heparins, albumin, or activated protein C mitigates histone-induced toxic effects and platelet activation in vitro and in vivo.9,13–16 Serum H3 and serum H4 are implicated in the pathogenesis of sepsis-induced tissue injury, and neutralization of these factors by specific anti-histone antibodies or nonanticoagulant heparin significantly decreases their deleterious effects.9,16,17 Histones present in serum have recently emerged as disease severity markers in several autoimmune, inflammatory, infectious, and malignant diseases in people.9 Serum histone concentrations strongly correlate with histologic scores of pancreatic necrosis in mice with experimentally induced acute pancreatitis10 and are predictive for persistent (≥ 48-hour duration) organ failure and death in people with acute pancreatitis.17–19 Although serum histone concentrations are significantly increased in dogs with heatstroke, compared with results for healthy dogs,20 the role of extracellular histones in other diseases in dogs, particularly in acute pancreatitis, is unknown.
The primary purpose of the study reported here was to compare serum concentrations of histones and inflammatory markers in dogs affected by acute pancreatitis with those in healthy control dogs and investigate associations of these variables with coagulation test results and survival (vs nonsurvival) to hospital discharge in affected dogs. Our first hypothesis was that serum total histone, histone H3, and histone H4 concentrations would be greater in dogs with acute pancreatitis than in control dogs. We also hypothesized that concentrations of these analytes would be associated with other hematologic values (serum CRP, IL-6, and TNF-α concentrations; CHT results; and thromboelastometry results) and with disease severity and outcome in affected dogs.
Owing to the paucity of information regarding hemostatic perturbations and changes in inflammatory mediators in dogs with acute pancreatitis, our secondary objectives were to characterize hemostatic findings in dogs with acute pancreatitis by means of CHTs and thromboelastometry and investigate the potential prognostic utility of physical examination and ultrasonographic findings; CBC, serum biochemical analysis, CHT, and thromboelastometry results; and serum CRP, IL-6, and TNF-α concentrations in affected dogs.
Materials and Methods
Dogs and study design
The prospective observational study was conducted at a university veterinary referral hospital that provided primary emergency and referral patient care. Client-owned dogs with a diagnosis of acute pancreatitis and staff-owned healthy control dogs evaluated at the facility between September 3, 2017, and August 9, 2018, were eligible for enrollment. Staff-owned control dogs were recruited for the study and were deemed healthy on the basis of a lack of history of disease and unremarkable physical examination, CBC, and serum biochemical analysis findings. Dogs with acute pancreatitis were enrolled when owners consented to medical treatment and hospitalization; dogs that were not hospitalized or were discharged from the hospital against medical recommendations and those euthanized for financial reasons were removed from the study. The study was approved by the institutional ethics committee. All owners were informed of the study and agreed to have their dog included.
Diagnosis of acute pancreatitis was made on the basis of a compatible history, physical examination and ultrasonographic findings consistent with acute pancreatitis,21 and DGGR lipase activity greater than the hospital laboratory's upper reference limit (125 U/L) or an abnormal result for a point-of-care canine pancreas–specific lipase test.a Ultrasonographic diagnosis of pancreatitis was made when ≥ 2 of the following findings were present: pancreatomegaly, pancreatic echogenicity and echotexture abnormalities, irregular pancreatic contours, surrounding hyperechoic mesentery, peripancreatic fluid accumulation, and irregular or abnormal pancreatic duct dilatation.21 Acute kidney injury was diagnosed according to the International Renal Interest Society grading system.22
Relevant history, signalment (age, sex and neuter status, and purebred vs mixed-breed dog), clinical signs, body weight, body condition score (scale of 1 to 9), rectal temperature, heart rate, comorbidities, results of ultrasonographic examination (including evaluation of PAPF if applicable) and hematologic tests (CBC, serum biochemical analysis, CHTs, thromboelastometry, and measurement of serum histones, IL-6, and TNF-α), and outcomes were recorded for dogs with acute pancreatitis. Dogs that survived to hospital discharge were classified as survivors, and dogs that died or were euthanized during hospitalization because of clinical deterioration despite ongoing treatment were classified as nonsurvivors. Signalment and serum concentrations of histones, IL-6, and TNF-α were recorded for control dogs.
Sample collection and laboratory methods
Blood samples (standardized volumes for all dogs) were collected at the time of initial evaluation at the hospital. Blood samples for routine serum biochemical analysis (2 mL) and measurement of CRP, IL-6, TNF-α, total histones, histone H3, and histone H4 (an additional 2 mL) were collected in plain tubes with gel separators and allowed to clot, and serum was harvested ≤ 45 minutes later. Routine biochemical variables and CRP were analyzedb in samples at 37 °C ≤ 60 minutes after collection. Serum aliquots were stored at –80 °C for up to 8 months prior to measurement of histone and cytokine concentrations. Samples for CBCs (1 mL) were collected in potassium EDTA–containing tubes and analyzedc ≤ 60 minutes after collection. Blood samples for CHTs (1.8 mL), including PT, aPTT, fibrinogen concentration (determined with the Clauss method), and antithrombin activity, were collected in tubes containing 3.2% tri-sodium citrate solution and centrifuged ≤ 30 minutes after collection. The plasma was immediately harvested and analyzedd; citrated plasma was also used to measure D-dimer concentratione,f at 37 °C. Blood samples for thromboelastometryg (1.8 mL) were collected in the same type of citrate-containing tubes, and 3-hour-duration tests were initiated ≤ 30 minutes after sample collection, with recombinant tissue factor and kaolin used as accelerators for the extrinsic and intrinsic clotting pathway assays, respectively.
Stored serum samples were frozen and thawed only once. Commercially available ELISA immunoas-says were used to measure total histone (all histone family members regardless of type),h histone H3,i his-tone H4,j IL-6,k and TNF-αl concentrations according to the manufacturers’ instructions. Intraassay coefficients of variance were determined from the means of simple duplicates. The optical density of the solutions was measured at 450 nm with a spectrophotometer.m All measurements were performed in duplicate, and the mean of the 2 measurements was used for statistical analyses. Measured values below the detection limit of the assays were entered as the value of the detection limit of the assay divided by the square root of 2.23
Statistical analysis
Distribution of the data was assessed with the Shapiro-Wilk test and Q-Q plots. Because most variables had nonnormal distributions, intergroup comparisons were performed with nonparametric Mann-Whitney U tests, and the Kruskal-Wallis test was used to compare serum concentrations of total histones, histone H3, histone H4, IL-6, and TNF-α among survivors, nonsurvivors, and control dogs. The results are presented as median and range, and Spearman correlation analysis was used to examine relationships between 2 continuous variables of interest, including duration of hospitalization (as a surrogate marker of disease severity), laboratory test results, and serum histone concentrations for dogs with pancreatitis. Associations between 2 categorical variables (eg, the presence of jaundice, hypercoagulability, or azotemia and outcome of survival) were evaluated with the Fisher exact test. All tests were 2-tailed, and values of P < 0.05 were considered significant for all comparisons. Statistical analyses were performed with a software package.n
Results
Dogs
The study included 29 dogs with acute pancreatitis (23 survivors and 6 nonsurvivors); 11 were spayed females (10 survivors and 1 nonsurvivor), 9 were castrated males (7 survivors and 2 nonsurvivors), 5 were sexually intact males (3 survivors and 2 non-survivors), and 4 were sexually intact females (3 survivors and 1 nonsurvivor). Nineteen of the 29 (66%) patients with pancreatitis were mixed-breed dogs. The control group comprised 7 mixed-breed dogs (4 spayed females and 3 castrated males). Age was not significantly different between survivors (median, 120 months; range, 24 to 192 months) and nonsurvivors (median, 104 months; range, 14 to 129 months) in the acute pancreatitis group or between the acute pancreatitis (median, 120 months; range, 14 to 192 months) and control (median, 72 months; range, 36 to 132 months) groups.
History, clinical, and clinicopathologic findings for dogs with acute pancreatitis
Presumptive causes of acute pancreatitis and outcomes—Presumptive causes of acute pancreatitis included dietary indiscretion (n = 7) and previous general anesthesia (3 to 7 days prior to presentation to our hospital; 3). No presumptive cause was determined for the remaining 19 dogs. In addition, 2 dogs with acute pancreatitis had concurrent diabetes mellitus.
Overall, 23 of 29 (79%) dogs in the acute pancreatitis group survived to hospital discharge, and all survivors were alive 1 month after discharge from the hospital. The median duration of hospitalization was 4 days (range, 1 to 8 days). Four of the 6 nonsurvivors died < 24 hours after presentation, 1 was euthanized during this same interval (owing to systemic complications including shock, persistently prolonged CHT times despite treatment, severe obtundation, and seizures), and 1 died 4 days after presentation. Among nonsurvivors, additional disorders that developed during hospitalization included DIC (n = 2), acute respiratory distress syndrome (1), seizures of unknown cause (1) and anuric acute kidney injury (1).
Additional history, physical examination, and ultra-sonographic findings—The median duration of clinical signs prior to presentation was 3 days (range, 1 to 10 days) for survivors and 5 days (range, 1 to 43 days) for nonsurvivors. There were no significant differences between the survivor and nonsurvivor groups with regard to body weight (median, 10.5 kg [range, 2.5 to 63.3 kg] and 20.5 kg [range, 3.3 to 40.0 kg], respectively), body condition score (median, 4 [range, 3 to 8] and 4.5 [range, 3 to 8], respectively), rectal temperature (median, 39.1 °C [range, 33.8 to 40.0 °C] and 38.9 °C [range, 36.3 to 39.8 °C], respectively), or heart rate (median, 120 beats/min [range, 88 to 180 beats/min] and 116 beats/min [range, 80 to 164 beats/min], respectively). Additional history and physical examination findings were summarized (Table 1). Obtundation and jaundice were significantly more common in nonsurvivors than in survivors.
Comparison of selected history and physical examination findings at the time of presentation to the hospital for 29 dogs with acute pancreatitis grouped by outcome (survival vs nonsurvival to hospital discharge).
| Finding | Survivors (n = 23) | Nonsurvivors (n = 6) | P value |
|---|---|---|---|
| First episode of acute pancreatitis | 21 (91) | 5 (83) | 0.51 |
| Dietary indiscretion* | 5 (22) | 2 (33) | 0.61 |
| Home-cooked diet† | 12 (52) | 3 (50) | 0.99 |
| Anorexia | 22 (96) | 6 (100) | 0.99 |
| Vomiting | 14 (61) | 4 (67) | 0.99 |
| Diarrhea | 13 (57) | 1 (17) | 0.17 |
| Melena | 4 (17) | 0 (0) | 0.55 |
| Hematochezia | 6 (26) | 0 (0) | 0.29 |
| Hematemesis | 1 (4) | 1 (17) | 0.37 |
| Obtundation | 3 (13) | 4 (67) | 0.006 |
| Signs of abdominal pain | 15 (65) | 4 (67) | 0.99 |
| Jaundice | 2 (9) | 3 (6) | 0.017 |
Data are shown as number (%) of dogs. The Fisher exact test was used to compare frequencies between outcome groups. Values of P < 0.05 were considered significant.
Consumption of table scraps, raw or cooked meat, items in the trash, or other items not part of the routine diet ≤ 3 days prior to presentation.
Consumption of a home-cooked diet on a regular basis.
All affected dogs had evidence of pancreatomegaly and pancreatic echotexture abnormalities on ultrasonographic examination. Additional ultra-sonographic findings in the pancreas and surrounding structures included increased echogenicity of the peripancreatic mesentery (survivors, 10/23; nonsurvivors, 3/6), ascites (survivors, 5/23; nonsurvivors, 5/6), irregular pancreatic contours (survivors, 7/23; nonsurvivors, 3/6), and presence of an echogenic nodule or cyst (1/23 survivors for each). The proportion of dogs with ultrasonographically confirmed as-cites was significantly (P = 0.005) higher among non-survivors (5/6) than among survivors (5/23). For all dogs with PAPF, a sample of the fluid was analyzed and identified as a nonseptic exudate.
CBC and serum biochemical data—There were no significant differences in the RBC, WBC, or platelet concentrations or the frequencies of deviations from the respective reference intervals for these variables between outcome groups (Table 2). Most other CBC variables did not differ between the 2 groups. Serum total bilirubin concentration (P = 0.009) and ALT (P = 0.016) and AST (P = 0.033) activities were higher, whereas serum cholesterol concentration was lower (P = 0.014) in nonsurvivors, compared with survivors (Table 3). There were no significant differences between nonsurvivors and survivors in the frequencies of hyperbilirubinemia (6/6 and 13/23, respectively) or abnormally high ALT activity (6/6 and 12/23, respectively), but abnormally high AST activity was significantly (P = 0.028) more frequent among non-survivors than among survivors (6/6 and 11/23, respectively). The remaining serum biochemical analytes did not differ significantly between the outcome groups when treated categorically as abnormally high vs within the respective reference intervals. Within the survivor group, serum concentrations of total bili-rubin (rS = 0.553; P = 0.012), creatinine (rS = 0.485; P = 0.030), and urea (rS = 0.485; P = 0.042) and DGGR lipase activity (rS = 0.520; P = 0.019) were positively correlated with the duration of hospitalization. Serum CRP concentrations were higher (P = 0.036) in survivors than in nonsurvivors. However, within the survivor group, CRP concentrations were positively correlated with the duration of hospitalization (rS = 0.535; P = 0.022).
Comparison of CBC and CHT results at the time of presentation to the hospital for the 29 dogs in Table 1.
| Analyte | Reference interval | Survivors (n = 23) | Nonsurvivors (n = 6) | P value |
|---|---|---|---|---|
| WBCs (× 103/mL) | 5.2–13.9 | 18.6 (5.5–58.5) | 15.3 (5.3–19.9) | 0.17 |
| RBCs (× 106/mL) | 5.7–8.8 | 6.5 (4.8–10.3) | 5.9 (4.2–9.2) | 0.60 |
| Hemoglobin (g/dL) | 12.8–18.4 | 15.5 (10.1–23.6) | 14.3 (9.1–20.5) | 0.94 |
| Hct (%) | 37.1–57.0 | 45.0 (29.5–66.4) | 42.4 (28.8–62.2) | 0.94 |
| MCV (fL) | 58.8–71.2 | 66.4 (54.3–71.9) | 69.0 (64.0–75.3) | 0.045 |
| MCHC (g/dL) | 31.0–36.2 | 34.9 (32.7–37.9) | 33.5 (31.6–36.3) | 0.033 |
| RDW (%) | 11.9–14.5 | 14.3 (12.0–22.8) | 14.7 (13.7–19.9) | 0.59 |
| Platelets (103/μL) | 143–400 | 276 (67–884) | 312 (127–506) | 1.00 |
| MPV (fL) | 7–11 | 13.4 (9.4–27.0) | 16.5 (10.5–27.7) | 0.57 |
| Neutrophils (× 103/μL) | 3.9–8.0 | 14.3 (3.7–51.8) | 13.0 (4.4–16.9) | 0.41 |
| Lymphocytes (× 103/μL) | 1.3–4.1 | 1.9 (0.3–5.0) | 1.2 (0.4–1.6) | 0.07 |
| Monocytes (× 103/μL) | 0.2–1.1 | 1.0 (0.2–7.7) | 0.9 (0.3–1.8) | 0.59 |
| Eosinophils (× 103/μL) | 0.0–0.6 | 0.12 (0.0–0.7) | 0.1 (0.0–0.3) | 0.26 |
| Basophils (× 103/μL) | 0.0–0.1 | 0.1 (0.0–0.6) | 0.1 (0.0–0.1) | 0.48 |
| PT (sec) | 6.0–8.4 | 7.6 (6.2–17.1) | 10.9 (7.0–28.8) | 0.008 |
| aPTT (sec) | 11.5–17.4 | 13.0 (9.9–67.0) | 23.5 (12.4–100.0) | 0.011 |
| Fibrinogen (mg/dL) | 200–400 | 477 (29–961) | 250 (43–598) | 0.20 |
| Antithrombin activity (%) | 87–140 | 111 (58–161) | 75 (35–121) | 0.047 |
| D-dimers (ng/dL) | 0–250 | 136 (0–8,232) | 510 (31–22,426) | 0.30 |
Data are shown as median (range). The Mann-Whitney U test was used to compare variables between outcome groups. Values of P < 0.05 were considered significant.
MCHC = Mean corpuscular hemoglobin concentration. MCV = Mean corpuscular volume. MPV = Mean platelet volume. RDW = RBC distribution width.
Comparison of serum biochemical analysis results at the time of presentation to the hospital for the 29 dogs in Table 1.
| Analyte | Reference interval | Survivors (n = 23) | Nonsurvivors (n = 6) | P value |
|---|---|---|---|---|
| Albumin (g/dL) | 3.0–4.4 | 3.2 (2.1–4.4) | 2.8 (2.3–3.4) | 0.37 |
| Total protein (g/dL) | 5.4–7.6 | 6.3 (3.7–8.5) | 5.4 (4.2–6.9) | 0.061 |
| Total bilirubin (mg/dL) | 0.0–0.2 | 0.25 (0.0–17.3) | 1.2 (0.4–11.3) | 0.009 |
| Cholesterol (mg/dL) | 135–361 | 275 (108–408) | 169 (151–259) | 0.014 |
| Creatinine (mg/dL) | 0.3–1.2 | 1.0 (0.4–3.2) | 1.0 (0.5–3) | 0.73 |
| Glucose (mg/dL) | 64–123 | 91 (20–740) | 75 (40–160) | 0.28 |
| Triglycerides (mg/dL) | 19–133 | 82 (33–554) | 168 (35–400) | 0.53 |
| Total calcium (mg/dL) | 9.7–11.5 | 9.2 (6.5–11.1) | 9.0 (8.4–11.3) | 0.98 |
| Urea (mg/dL) | 10.7–53.5 | 47.1 (11.1–295.0) | 33.8 (11.4–174.8) | 0.68 |
| Phosphorus (mg/dL) | 3.0–6.2 | 4.1 (1.5–12.8) | 4.3 (3.6–9.1) | 0.44 |
| ALP (U/L) | 21–170 | 239 (59–3,099) | 382 (128–735) | 0.45 |
| ALT (U/L) | 19–67 | 75 (16–4,218) | 198 (76–2,736) | 0.016 |
| Amylase (U/L) | 103–1,510 | 1,708 (107–12,729) | 1,237 (379–2,577) | 0.32 |
| DGGR lipase (U/L) | 0–125 | 1,077 (217–9,612) | 846 (75–2,572)* | 0.48 |
| AST (U/L) | 19–42 | 54 (27–2,345) | 184 (60–1,321) | 0.033 |
| Creatine kinase (U/L) | 51–399 | 270 (59–7,430) | 320 (224–7,679) | 0.30 |
| GGT (U/L) | 0.0–6.0 | 3.5 (0.0–189.0) | 10.0 (0.0–58.0) | 0.075 |
| Chloride (mmol/L) | 108–118 | 102 (61–117) | 102 (85–112) | 0.88 |
| Potassium (mmol/L) | 3.6–5.3 | 4.1 (3.2–5.8) | 4.1 (3.8–5.0) | 1.00 |
| Sodium (mmol/L) | 145–154 | 140 (125–156) | 143 (132–152) | 0.90 |
| CRP (mg/L) | 0.0–6.0 | 121.7 (5.1–280.0) | 40.1 (0.0–129.2) | 0.036 |
Data are shown as median (range).
One dog had DGGR lipase activity within the reference interval but had an abnormal point-of-care test result for canine specific lipase activity. ALP = Alkaline phosphatase. GGT = g-Glutamyltransferase.
See Table 2 for remainder of key.
CHT and thromboelastometry findings—The PT and aPTT were longer (P = 0.008 and P = 0.011, respectively), and antithrombin activity was lower (P = 0.047) in nonsurvivors, compared with survivors (Table 2). The proportion of dogs with abnormally prolonged PT was higher among nonsurvivors than survivors (5/6 and 6/23, respectively; P = 0.010), but similar results were not observed for abnormally prolonged aPTT (4/6 and 6/23, respectively; P = 0.06). The proportions of dogs with abnormally shortened aPTT did not differ between groups (0/6 nonsurvivors vs 7/23 survivors; P = 0.29); abnormally shortened PT was not recorded for any dog. The aPTT was positively correlated with the duration of hospitalization for survivors (rS = 0.480; P = 0.032).
Numerous thromboelastometric changes consistent with hypercoagulability (median values at or exceeding the upper reference limits) were noted among survivors, including extrinsic and intrinsic pathway assay maximal clot firmness, maximal clot elasticity, and shear elastic modulus strength (Table 4). However, the proportions of dogs with hypercoagulability, as judged by the extrinsic and intrinsic pathway assay shear elastic modulus strength values, were not significantly different between the 2 outcome groups (12/23 survivors vs 1/6 nonsurvivors [P = 0.11] and 9/23 survivors vs 1/6 nonsurvivors [P = 0.30], respectively). Hypocoagulability was detected in only 2 dogs, and both were nonsurvivors (1/assay type).
Comparison of thromboelastometry results at the time of presentation to the hospital for the 29 dogs in Table 1.
| Variable | Assay | Reference interval | Survivors (n = 29) | Nonsurvivors (n = 6) | P value |
|---|---|---|---|---|---|
| CT (s) | EPT | 31–97 | 50 (18–130) | 69 (15–135) | 0.41 |
| IPT | 97–203 | 153 (50–253) | 187 (107–299) | 0.30 | |
| CFT (s) | EPT | 58–282 | 76 (31–152) | 151 (54–306) | 0.06 |
| IPT | 50–287 | 67 (34–144) | 101 (36–226) | 0.19 | |
| MCF (mm) | EPT | 39–72 | 74 (59–82) | 53 (5–80) | 0.024 |
| IPT | 42–70 | 70 (56–77) | 59 (44–78) | 0.041 | |
| α Angle (°) | EPT | 48–78 | 76 (61–84) | 70 (44–83) | 0.11 |
| IPT | 46–78 | 76 (63–83) | 70 (59–83) | 0.16 | |
| A10 (mm) | EPT | 32–66 | 65 (46–79) | 46 (30–74) | 0.036 |
| IPT | 28–63 | 61 (45–72) | 47 (30–72) | 0.041 | |
| LI60 (%) | EPT | 54–99 | 99 (85–100) | 100 (36–100) | 0.33 |
| IPT | 93–100 | 99 (94–100) | 100 (98–100) | 0.14 | |
| MV (mm/min) | EPT | 6–22 | 23 (9–44) | 13.5 (5–31) | 0.31 |
| IPT | 4–25 | 19 (9–44) | 13 (9–36) | 0.24 | |
| AUC (mm × 100) | EPT | 3,912–7,149 | 7,263 (5,868–8,135) | 5,267 (472–7,896) | 0.026 |
| IPT | 4,273–6,972 | 6,995 (5,562–7,675) | 6,034 (4,432–7,817) | 0.07 | |
| MCF-t (s) | EPT | 1,269–2,878 | 2,069 (995–2,868) | 2,041 (11–3,129) | 1.00 |
| IPT | 1,726–2,822 | 1,954 (1,063–3,378) | 2,735 (1,805–3,024) | 0.036 | |
| MCE (mm) | EPT | 64–274 | 278 (141–449) | 118 (5–398) | 0.022 |
| IPT | 91–265 | 230 (127–332) | 143 (79–357) | 0.036 | |
| G (dynes/cm2) | EPT | 3,205–13,689 | 14,230 (7,195–22,777) | 5,879 (263–20,000) | 0.019 |
| IPT | 4,273–5,073 | 11,666 (6,363–16,739) | 7,202 (3,928–17,727) | 0.041 |
Data are shown as median (range).
A10 = Clot firmness at 10 minutes. AUC = Area under the first derivative curve. CFT = Clot formation time. CT = Clotting time. EPT = Extrinsic pathway thromboelastometry. G = Shear elastic modulus strength. IPT = Intrinsic pathway thromboelastometry. LI 60 = Lysis index at 60 minutes. MCE = Maximal clot elasticity. MCF = Maximal clot firmness. MCF-t = Time to maximal clot firmness. MV = Maximal velocity.
See Table 2 for remainder of key.
Serum concentrations of histones, IL-6, and TNF-α in dogs with acute pancreatitis and control dogs
The intraassay coefficients of variance were 5.9%, 7.7%, 6.9% and 8.6% for serum concentrations of total histones, histone H3, histone H4, and IL-6, respectively. Calculation of the coefficient of variance was infeasible for serum TNF-α concentrations, as the analyte was detectable in only 2 samples (both from dogs with acute pancreatitis) and calculation was based on the means of simple duplicates. Concentrations of total his-tones, histone H3, and histone H4 did not differ among survivors, nonsurvivors, and control dogs (Table 5). The IL-6 concentration was below the lower limit of detection for all control dogs. Concentrations of total histones, histone H3, and histone H4 were not associated with TNF-α, IL-6, or CRP concentrations or with CHT or thromboelastometry results in affected dogs.
Comparison of serum total histone, histone 3, histone 4, and IL-6 concentrations at the time of presentation to the hospital for the 29 dogs with acute pancreatitis in Table 1 and 7 healthy control dogs.
| Analyte | Dogs with pancreatitis | P value | ||
|---|---|---|---|---|
| Survivors (n = 23) | Nonsurvivors (n = 6) | Controls (n = 7) | ||
| Total histones (ng/mL) | 25.3 (5.5–103.2) | 51.2 (1.1–78.7) | 27.8 (6.7–56.5) | 0.75 |
| Histone H3 (ng/mL) | 4.6 (1.4–27.4) | 3.1 (1.3–22.8) | 2.0 (0.8–7.2) | 0.25 |
| Histone H4 (ng/mL) | 1.2 (0.4–8.4) | 1.1 (0.4–5.4) | 0.9 (0.2–2.3) | 0.56 |
| IL-6 (pg/mL) | 16.3 (10.7–247.0) | 32.5 (10.7–1,940.0) | NA | 0.97 |
Data are reported as median (range). The Kruskal-Wallis test was used to compare variables among the survivor, nonsurvivor, and control groups.
NA = Not applicable (IL-6 was undetectable in all controls).
Among affected dogs, serum IL-6 concentrations were positively correlated with serum CRP concentrations (rS = 0.556; P = 0.003) and DGGR lipase activities (rS = 0.436; P = 0.018) and negatively correlated with antithrombin activities (rS = –0.405; P = 0.029). Additionally, serum IL-6 concentrations were positively correlated with the duration of hospitalization among survivors (rS = 0.528; P = 0.017).
Post hoc power calculation on the basis of the difference in serum total histone concentrations between survivors and nonsurvivors in the present study revealed that the power to detect this difference was approximately 13%. For a statistical power of 80% and α of 5%, with a mortality rate of 21% as reported here, the calculations indicated that enrollment of 254 survivors and 66 nonsurvivors would have been required to detect a significant difference in this variable between groups.
Discussion
In the present study of dogs with naturally occurring acute pancreatitis, serum total histone, histone H3, and histone H4 concentrations at the time of hospital presentation were not significantly associated with outcome (survival to discharge vs nonsurvival [death or euthanasia because of deteriorating condition while hospitalized]), serum concentrations of markers of inflammation, or CHT or thromboelastometry results, contrary to studies of rodents with experimentally induced acute pancreatitis10 and of human patients with naturally occurring acute pancreatitis.18,19 In the present study, interleukin-6 was detectable in the serum of dogs with pancreatitis but not in the serum of healthy control dogs, and the IL-6 concentration was positively correlated with hospitalization time among survivors. Clotting times in CHTs were significantly greater for nonsurvivors, and results for many thromboelastometry parameters differed between survivors and nonsurvivors. Last, the associations of nonsurvival with the presence of PAPF and indicators of hepatic injury and jaundice in the present study might have clinical implications, as suggested by previous findings for human patients24,25 and for rodents with experimentally induced acute pancreatitis.26,27
Results of multiple studies10,12,14 support that extracellular histones have proinflammatory, procoagulant, and cytotoxic effects. Furthermore, serum histone concentrations are associated with outcomes in people with various inflammatory diseases, including acute pancreatitis,17–19 and circulating histones induce pancreatic acinar necrosis in mice.19 In mice, circulating histone concentrations were strongly correlated with the magnitude of pancreatic necrosis but, conversely, were barely detected in mice with edematous pancreatitis.10 This observation might partly account for our findings regarding the lack of correlation between serum histone concentrations and outcome in dogs. Additionally, the present study might have been underpowered to detect significant differences, warranting larger future studies to corroborate or refute the lack of clinical utility of total histone concentrations for purposes of prognostication in dogs with acute pancreatitis.
Complications of acute pancreatitis can be devastating, as pancreatic zymogens are activated, and an inflammatory response of variable intensity and extent (ie localized vs systemic) ensues.3,4 The deleterious effects of active pancreatic enzymes on cellular membranes, endothelium, adipose tissue, and circulating clotting factors engender tissue damage, necrosis, edema formation, and hypoxia.3 Serum concentrations of several cytokines and chemokines, including IL-1β, IL-2, IL-6, IL-18, TNF-α, and monocyte chemoattractant protein-1, are increased in dogs with acute pancreatitis.1,3 Interleukin-6 is an extensively investigated proinflammatory cytokine involved in a plethora of inflammatory, autoimmune, and malignant diseases.28 In support of its role, serum IL-6 concentration was positively correlated with concentrations of serum CRP, a positive acute-phase protein, in the present study. Previous studies have demonstrated an increase in serum IL-6 concentration in dogs with experimentally induced29 and naturally occurring30 acute pancreatitis. Interestingly, and despite its many reported deleterious effects on tissues,28 IL-6 administration to dogs with experimentally induced acute pancreatitis significantly reduces bacterial translocation to mesenteric lymph nodes, alluding to a beneficial role of this cytokine in regulating the immune response and protecting against invading pathogens.31 In the present study, serum IL-6 was detectable only in dogs with acute pancreatitis and was positively correlated with duration of hospitalization among the survivors. Nonetheless, serum IL-6 concentration failed to differentiate survivors from nonsurvivors in our study, similar to previous findings in dogs30 and contrary to a study of people3 with acute pancreatitis.
Tumor necrosis factor-α, produced and released by immune system cells as well as pancreatic acinar cells,32 is also implicated in acute pancreatitis in people4 and dogs,29,30,33 but was undetectable in most dogs regardless of group in the present study. Previously, TNF-α was measurable in small proportions of dogs with acute pancreatitis, and plasma concentrations of the cytokine failed to differentiate among various disease severity scores.33 A similar discrepancy between its apparent role in the pathogenesis of disease and lack of clinical utility has been observed in human medical studies, and likely stems from its short half-life and phasic release as well as the presence of circulating inhibitors, and most importantly, its primarily paracrine activity.4,34
A plethora of potential causes have been described, but in many cases, acute pancreatitis remains idiopathic in dogs.1 Dietary indiscretion and table scrap consumption (prior to acute pancreatitis or throughout life) are known risk factors for pancreatitis.35 Specifically, dietary fat composition affects exocrine pancreatic secretion and the risk for pancreatitis.36,37 Correspondingly, the most commonly reported potential cause of acute pancreatitis in the present study was diet related, with a recent history of dietary indiscretion in 7 of 29 (24%) dogs and roughly half of the dogs that regularly consumed a home-cooked diet. In the remaining cases, possible pancreatic hypoperfusion during anesthesia and diabetes mellitus were noted, and both factors have been previously associated with pancreatitis in dogs and people.1,2,38
In the present study, obtundation was significantly more frequent among nonsurvivors than among survivors. Impaired mental status imparts a worse prognosis in human patients with acute pancreatitis,39 and presumably, this condition results from electrolyte and metabolic imbalances, as well as direct complications of pancreatitis (eg, coagulopathy, edema, and acute kidney injury). Another potential prognostic marker in our study was the presence of ascites. In human patients and dogs with acute pancreatitis, PAPF is occasionally present24,40 as a result of increased vascular permeability, pancreatic and duct-ular enzyme leakage, hypoalbuminemia, and portal vein thrombosis.40,41 In human patients, PAPF increases intraperitoneal pressure, which might culminate in abdominal compartment syndrome, decreased abdominal perfusion, and organ dysfunction.24,25 Furthermore, PAPF collected from rats with experimentally induced pancreatitis upregulates proinflammatory cytokine expression in human vascular endothelium42 and murine macrophages26 in vitro and in the lung tissue of mice26; it also induces lung injury in rats,27 whereas peritoneal lavage in rats shortly after the onset of acute peritonitis has systemic antiinflammatory effects.43 In human patients, PAPF drainage has been associated with decreased serum CRP, IL-1, IL-6, and TNF-α concentrations.24 To our knowledge, the effects of PAPF have not been investigated in dogs, but the effects described in other species might have accounted for the association between presence of PAPF and the outcome of nonsurvival in the study reported here.
Acute pancreatitis, in its more severe forms, can cause a systemic inflammatory state and disseminated coagulopathy, which together with gastrointestinal fluid loss due to vomiting and diarrhea contributes to renal hypoperfusion, ischemia, and, consequently, acute kidney injury.1,2,44 Azotemia has inconsistently been associated with outcome in dogs,7,8,44,45 people,4 and cats46 with acute pancreatitis. In agreement with most previous studies, the present study found that serum creatinine concentration was positively associated with a longer duration of hospitalization for survivors. Nevertheless, azotemia was not associated with survival, although the study might have been underpowered to detect such an association.
The prognostic importance of concurrent hepatic injury in patients with acute pancreatitis is similarly unclear, and previous reports are contradictory. In 1 study47 of dogs with acute pancreatitis, high serum ALT activity was associated with an extended duration of hospitalization, whereas in 2 other studies,7,8 evidence of hepatic injury was unassociated with patient outcomes. Pancreatitis-associated liver injury is a multifactorial process caused by hepatic hypoperfusion, biliary obstruction, impaired metabolic activity secondary to pancreatic enzyme release, and direct hepatotoxic effects of PAPF.48,49 Hepatic injury in cases of acute pancreatitis independently promotes and exacerbates hemostatic abnormalities and organ dysfunction, including lung injury.49–51 In the present study, although median serum bilirubin concentration and serum ALT and AST activities were higher in nonsurvivors than in survivors, comparison of the proportions of dogs with abnormal results revealed that only the frequency of abnormally high AST activity differed between the 2 outcome groups. Future studies are warranted to investigate the pathophysiology of hepatic injury in dogs with acute pancreatitis and its clinical and therapeutic implications.
Leakage of pancreatic enzymes into the circulation and the presence of systemic inflammation can activate the coagulation cascade and result in thromboembolic events, bleeding, and DIC.1,2,4,52 In dogs with experimentally induced acute pancreatitis, evidence of consumptive coagulopathy included prolonged PT and aPTT, decreased plasma fibrinogen concentration in face of increased fibrinogen degradation products, and thrombocytopenia.53 In the clinical setting, hemostatic abnormalities (ie, thrombocytopenia and prolonged PT or aPTT) are associated with poorer short-term outcomes in dogs with acute pancreatitis,7 and in human patients with acute pancreatitis, development of DIC is an independent negative prognostic factor.4 Circulating antithrombin activity also decreases in patients with acute pancreatitis, likely owing to consumption of the protein during the clotting process and systemic inflammation, and decreased antithrombin activity imparts a worse prognosis in people54 and dogs.55 Hemostatic derangements were frequently observed in the present study. Compared with results for survivors, the nonsurvivors had significantly greater PT and aPTT. Nevertheless, corresponding thromboelastometry changes were lacking, and hypocoagulability was detected in only 2 dogs (both nonsurvivors) by thromboelastometry. Survivors in the study reported here had several significant differences in thromboelastometry parameters, compared with nonsurvivors, that collectively suggested a tendency toward hypercoagulability among survivors. In addition, shortened aPTT, possibly reflective of hypercoagulability,56 was only observed among the survivors, although the apparent difference in frequencies between groups was not statistically significant. Two recent publications57,o described similar tendencies on thromboelastography for dogs with naturally occurring acute pancreatitis, but limited sample sizes precluded comparisons between survivors and nonsurvivors. The possible clinical ramifications of these variables should be prospectively investigated. In rats, antithrombin injections have been shown to preclude development of experimentally induced acute pancreatitis58 and attenuate acute kidney injury following experimental induction of the disease.59 Similar studies have not been conducted in dogs, and 1 retrospective study60 found no clinical benefit of fresh frozen plasma administration to dogs with pancreatitis. However, fresh frozen plasma is sometimes administered regardless of the hemostatic status in dogs with acute pancreatitis, in an attempt to replenish exhausted blood antiprotease stores. Considering those findings60 and results of the present study, a different, CHT-driven approach to fresh frozen plasma administration might prove more beneficial and should be prospectively evaluated.
The magnitude of increase in circulating canine pancreatic-lipase immunoreactivity,61 but not serum amylase or total serum lipase activities,6 is associated with outcome in dogs with acute pancreatitis. In the present study, DGGR lipase activity at the time of presentation was positively correlated with the duration of hospitalization and with serum IL-6 concentrations, alluding to a possible association of inflammation severity with the magnitude of DGGR lipase activity and ultimately with morbidity and clinical course of the disease.
The present study had several limitations. The fairly small sample size (for both the acute pancreatitis and control groups) was likely statistically under-powered to detect differences in histone concentrations and probably other variables among the 3 groups as well. The heterogeneity of breeds and sex in the study sample might have affected some results, notwithstanding a previously described lack of association between circulating histone concentration and sex in a study of people18 with naturally occurring acute pancreatitis. Additionally, our study included dogs that might have had underlying chronic pancreatitis, and some dogs had concurrent diseases (eg, diabetes mellitus). Clinical signs of acute pancreatitis are nonspecific,62 and because differentiation between acute pancreatitis and chronic pancreatitis, with or without an acute flareup, is infeasible in the clinical setting,1,62 we opted to include all dogs that had acute signs of pancreatitis and supportive results for appropriate tests. Serial measurements of serum cytokines and histones, CHTs, and thromboelastometry were not performed during hospitalization, and the present study relied on measurements obtained from dogs with various disease severities at the time of presentation. The concentrations of cytokines and histones as well as coagulation variables can fluctuate as the disease progresses; changes in serum CRP concentration have been described over time in hospitalized dogs with acute pancreatitis,8,61 suggesting that serial measurements of some variables may provide added information. We did not perform independent analytical validation of the commercial assays used in the study, and to our knowledge, they have not been independently validated by other groups. Finally, correction for multiple comparisons was not carried out in this study because the secondary objectives were to examine several additional potential prognostic markers of disease severity and outcome without prespecified hypotheses. The use of multiplicity adjustment in such cases is controversial, as it engenders loss of power and a substantial increase in the rate of type II errors while reducing the chance of type I errors. In other words, it is possible that potentially significant, clinically relevant factors may be overlooked. Consequently, many prefer to avoid such adjustments and instead present exploratory results that must be subsequently tested in confirma-tory, hypothesis-driven studies.63–65
Although the number of dogs in the present study was small and no associations were detected between serum histone concentrations and the presence of pancreatitis or outcome in affected dogs, we identified several potential prognostic indicators for outcome in dogs with acute pancreatitis: PAPF, obtundation, and jaundice were more common among nonsurvivors than survivors. The serum values of several analytes associated with hepatic injury were also greater for nonsurvivors than for survivors, and several CHT and thromboelastometry results differed significantly between these 2 groups, with nonsurvivors having slower clotting times and lower values for maximal clot firmness, elasticity, and strength. Larger studies of dogs with acute pancreatitis are needed to further evaluate whether serum histone concentrations have prognostic value in affected dogs and to prospectively investigate the clinical utility of specific medical interventions in patients with hypocoagulability, presence of PAPF, and evidence of hepatic injury, given the present findings.
Acknowledgments
Supported by the American Kennel Club Canine Health Foundation. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the views of the Foundation.
The authors declare that there were no conflicts of interest.
Footnotes
SNAP cPL test, Idexx, Westbrook, Me.
Cobas 6000, Roche Diagnostics, Mannheim, Germany.
Advia 2120i, Siemens Healthcare, Erfurt, Germany.
ACL-9000, Instrumentation Laboratory SpA, Milano, Italy.
Tina-quant D-dimer assay, Roche Diagnostics, Mannheim, Germany.
Cobas 6000, RocheDiagnostics, Mannheim, Germany.
ROTEM delta analyzer, TEM International GmbH, Munich, Germany.
Canine histone ELISA, Mybiosource, San Diego, Calif.
Canine histone H3 ELISA, Mybiosource, San Diego, Calif.
Canine serum histone H4 ELISA, Mybiosource, San Diego, Calif.
Canine IL-6 ELISA, R&D Systems Inc, Minneapolis, Minn.
Canine TNF-α ELISA, R&D Systems Inc, Minneapolis, Minn.
Infinite F50 Absorbance, Tecan Trading AG, Männedorf, Switzerland.
SPSS, version 25.0 for Windows, IBM Corp, Armonk, NY.
Park C, Lee J, Rye J, Chung H. Thromboelastographic evaluation in dogs with acute pancreatitis (abstr), in Proceedings. Am Coll Vet Intern Med Forum 2019;575.
Abbreviations
| ALT | Alanine aminotransferase |
| aPTT | Activated partial thromboplastin time |
| AST | Aspartate aminotransferase |
| CHT | Conventional citrated plasma-based hemostatic test |
| CRP | C-reactive protein |
| DGGR | 1,2-o-dilauryl-rac-glycero glutaric acid-(6′-methylresorufin) ester |
| DIC | Disseminated intravascular coagulopathy |
| IL | Interleukin |
| PAPF | Pancreatitis-associated peritoneal fluid |
| PT | Prothrombin time |
| TNF | Tumor necrosis factor |
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