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J Vet Clin 2024; 41(3): 143-149

https://doi.org/10.17555/jvc.2024.41.3.143

Published online June 30, 2024

Investigation of Circulating Cell-Free DNA Concentration in Dogs with Pancreatitis

Jae-Hun Kim1 , Soo-Yeon Jeong1,2 , Chul Park1,*

1Department of Veterinary Internal Medicine, College of Veterinary Medicine, Jeonbuk National University, Iksan 54596, Korea
2Department of Companion Animal Health Care, Kyung-in Women’s University, Incheon 21041, Korea

Correspondence to:*chulpark0409@jbnu.ac.kr

Received: February 29, 2024; Revised: April 19, 2024; Accepted: May 16, 2024

Copyright © The Korean Society of Veterinary Clinics.

Circulating cell-free DNA (cfDNA) constitutes a fragment of DNA released into the blood through cellular apoptosis or necrosis. In human medicine, cfDNA has been studied as a disease severity biomarker. Recent studies have shown that concentrations of cfDNA in dogs with immune-mediated and tumor-related diseases are increased. Pancreatitis is known to be caused by excessive release of trypsin, which leads to edema, inflammation, necrosis, and apoptosis in the pancreas. Based on the results of research showing an increase of cfDNA due to apoptosis and necrosis of cells, we hypothesized that cfDNA concentration would increase in the presence of pancreatitis. A total of 35 dogs were studied, including 21 with pancreatitis and 14 without any inflammatory diseases (normal group). The results showed that the concentration of cfDNA in dogs with pancreatitis was approximately twice that of normal dogs (median 0.0912 ng/μL. p-value 0.028). This result suggests that cfDNA can serve as a new biomarker for estimating pancreatitis severity.

Keywords: dog, circulating cell free DNA, apoptosis, pancreatitis, inflammation

Circulating cell-free DNA (cfDNA) consisting of small double-stranded DNA fragments found in blood was first discovered in 1948 by Mandel and Metais (23). cfDNA that consists of degraded DNA fragments can be released to body fluids and blood. It has been reported that both healthy and ill human have cfDNAs (6).

cfDNA flows into the bloodstream via apoptosis and necrosis. It is directly released from viable cells after lysis of circulating cells (23). Consequently cfDNA levels can increase in various physiological and pathological states such as inflammation, smoking, sepsis, trauma, and cancer that can lead to increased apoptotic cell death (16). One study has found that cfDNA fragment’s length in circulation has a laddering pattern of 170-180 base pairs, the size of DNA produced by apoptosis (4). Therefore, major sources of cfDNA have been thought to be apoptotic and necrotic cells nowdays (23).

In human, increased concentrations of cfDNA have been reported in serum, plasma, and urine samples of patients with various conditions such as stroke, trauma, myocardial infarction, cancer, and immune mediated disease. This suggests an association between cfDNA and several disorders (15). Many researchers have endeavored to utilize cfDNA as a marker in liquid biopsy to assist diagnosis of prenatal, tumor, stroke, autoimmune disorders, and myocardial infarction or in transplant medicine (13,15). In veterinary medicine, several investigations have been conducted to find association between cfDNA and various diseases. Reports have indicated that cfDNA concentrations are increased in parasite disorders, immune-mediated hemolytic anemia, neurologic disorders, and various tumors and other inflammatory disease of dogs (1,15,18,20). In human medicine, cfDNA has been studies in the context of inflammation. An association between an inflammatory state (acute or chronic inflammation, autoimmune diseases, and infections) and cfDNA has been observed (3). This correlation may also exist in veterinary medicine (7,8).

Pancreatitis refers to inflammation in the pancreas with the presence of neutrophilic infiltrate, edema, and necrosis. This disease can be a localized inflammation. But, it can also lead to a systemic inflammatory response syndrome. The exact cause of pancreatitis in dogs and cats remains unclear. However, it has been discovered that hereditary mutations of trypsin may predispose people to pancreatitis in human medicine. This might be similar in dogs and cats (11).

Trypsin is a protease secreted in the pancreas. It is typically stored in zymogen granules in an inactive state under normal conditions. However, if excessive activation of trypsin occurs, this can lead to autodigestion and severe inflammation. This process can cause peripancreatic fat necrosis, sterile peritonitis, and systemic inflammatory response (2). So as pancreatitis is a kind of inflammatory disease with necrosis and apoptosis process, it may be possible to increase in serum cfDNA concentration.

C-reactive protein (CRP) is a kind of plasma protein that increase in inflammatory, infectious, neoplastic states and traumatic, infectious state (9,22). CRP is often used as an indirect indicator to assess the severity of a disease. And while increases in cfDNA have been seen in a variety of conditions where CRP is elevated, the correlation is not yet clear (10,17).

Based on the previous studies, we hypothesized that pancreatitis might also be associated with cfDNA in dogs. And we also hypothesized that if cfDNA was increased, it would also be associated with CRP. Therefore, this study aimed to determine the association between cfDNA and pancreatitis and to discover difference in cfDNA level between healthy dogs and dogs with pancreatitis. And to find the association between CRP and cfDNA in pancreatitis state. To the best of our knowledge, this is the first study that investigates concentrations of cfDNA in dogs with pancreatitis in veterinary medicine.

Animals

This was a prospective study of 21 dogs diagnosed with pancreatitis. The pancreatitis group consisted of dogs at the Jeonbuk Animal Medical Center (JAMC) between November 2020 and July 2021, all accompanied by their owners, that exhibited mild to severe digestive symptoms like vomiting, diarrhea, anorexia. Dogs with negative results from canine pancreatic lipase test (SNAP cPL kit, IDEXX Laboratories Ltd, Maine, USA) were excluded. They were also diagnosed with pancreatitis with abdominal ultrasonography. The CRP level was also measured. However, it was not used as a marker to differentiate between the pancreatitis and normal groups. A normal group consisted of dogs without digestive clinical signs among patients visiting the animal hospital. Normal dogs had no problems associated with pancreatitis based on x-ray, ultrasound, blood, and physical examinations and had no any inflammatory disease and tumor based on CRP concentration, radiography, ultrasonography (Table 1). There were 21 dogs with pancreatitis in the pancreatitis group and 14 healthy neutered dogs in the normal group. In the pancreatitis group, there were 11 females and 10 males. In the normal group, there were 5 females and 9 males. In this study, we devided the pancreatitis groups into acute and chronic pancreatitis groups based on ultrasonographic findings, CRP, and cPL. The dogs with other inflammatory lesions that were thought to be secondary to severe pancreatitis were categorized as acute pancreatitis. Patients were included in the chronic pancreatitis group if there were no other inflammatory lesions and the ultrasound showed features of chronic pancreatitis, even if there was an increase in CRP levels. There were 8 dogs in the acute pancreatitis group and 13 dogs in the chronic pancreatitis group (Table 2).

Table 1 Concentrations of cell free DNA in normal group

NumberBreedSex/AgeALT (U/L)ALP (U/L)WBC (K/ul)cfDNA (ng/μL)cPLCRP (mg/L)Ultrasond
Normal value--17-7847-2546-17-Negative10-20Normal
1BeagleM/64715415.10.0565Negative12Normal
2MalteseM/8775613.60.0512Negative10Normal
3BeagleM/64313411.40.0626Negative13Normal
4MixF/8331609.40.0777Negative12Normal
5BeagleM/64121014.80.151Negative10Normal
6MalteseF/13411229.40.0279Negative15Normal
7BeagleF/4471318.20.0866Negative10Normal
8BeagleM/645267170.0669Negative14Normal
9MixF/72929515.50.0464Negative14Normal
10BeagleM/63523712.40.0572Negative10Normal
11BeagleF/43523611.60.0442Negative12Normal
12PomeranianM/62979120.0677Negative11Normal
13BeagleM/41831111.10.0795Negative10Normal
14MalteseM/9651157.80.0321Negative10Normal

The normal group consisted of 14 dogs. All dogs were neutered. Concentration of cell free DNA was determined as the average of three measurements. M, male; F, Female; Normal, dog without pancreatitis; Ultrasound, ultrasonogrphic finding of pancreas.



Table 2 Concentrations of cell free DNA in patient group

NumberBreedSex/AgeALT (U/L)ALP (U/L)WBC (103/μL)cfDNA (ng/μL)cPLCRP (mg/L)Ultrasound
Reference interval17-7847-2546-17-Negative10-20Normal
1PoodleM/15601009.30.1726Positive10Abnormal
2Yorkshire terrierF/17437812.90.1176Positive33.6Abnormal
3ChihuahuaM/69119880.05385Positive13.9Abnormal
4MalteseF/1550848.10.0637Positive10Abnormal
5DachshundM/1212681680.0504Positive14.8Abnormal
6MalteseM/155175,5697.70.1575Positive36.4Abnormal
7Miniature pinscherF/12352,14350.80.451Positive138.8Abnormal
8Jack russell terrierM/787896.90.0605Positive35.5Abnormal
9Miniature pinscherF/12502,00029.10.134Positive117.1Abnormal
10Yorkshire terrierF/172913213.80.186Positive33.9Abnormal
11Yorkshire terrierM/13681787.40.231Positive22.7Abnormal
12Yorkshire terrierF/17287812.90.0465Positive33.6Abnormal
13PoodleM/10399310.30.0307Positive17Abnormal
14Golden retrieverM/5646199.20.0691Positive86.6Abnormal
15Shih-tzuF/151972,00014.90.027Positive64.7Abnormal
16Yorkshire terrierM/132717012.90.246Positive78.8Abnormal
17MixF/152821218.90.0912Positive33.8Abnormal
18SchnauzerF/4454310.50.0685Positive10Abnormal
19JindoM/1120086112.10.152Positive22.5Abnormal
20PoodleF/826698.70.1Positive26.4Abnormal
21Golden retrieverF/92899100.0867Positive95Abnormal

The patient group was 21 dogs. All dogs were neutered. Concentration of cell free DNA was determined as the average of three measurements. M, male; F, Female; Patient, dog with pancreatitis. Ultrasound, ultrasonogrphic finding of pancreas. cPL, canine pancreas-specific lipase; CRP, c-reactive protein



Radiographic examination

Abdominal radiographs were obtained from both ventrodorsal view and right lateral view. Evaluations included serosal detail, intestinal opacity, intestinal size, and liver size. All dogs with radiographs suggestive of foreign bodies were excluded.

Abdominal ultrasonography

Ultrasonographic examinations were performed using a Toshiba Aplio 300 (Aplio 300, Canon Medical Systems, Otawara, Japan). Ultrasound assessments involved measuring the shape and thickness of pancreas and evaluating echogenicity. We considered the normal thickness range for the pancreas up to 6.5 mm for the left lobe, 6.3 mm for the body, and 8.1 mm for the right lobe (12). If the ultrasonographic finding showed increased thickness of the pancreas and hypoechoic regions, it was classified as acute pancreatitis group. If the thickness of the pancreas was normal but showed hyperechoic non-shadowing region, it was classified as chronic pancreatitis group (Fig. 1).

Figure 1.Ultrasonographic findings in dogs with pancreatitis group. (A) Acute pancreatitis. Hypoechoic region and hyperechoic peripheral region was observed in pancreatic body. The thickness of pancreas body was 13.3 mm and this is abnormal. (B) Chronic pancreatitis. Hyperechoic non-shadowing region was observed in right lobe pancreas. The thickness was normal range (4.5 mm).

Blood sample collection

Approximately 2 mL of blood was collected into ethylenediaminetetraacetic acid (EDTA) tubes from cephalic and external jugular veins using a 3 mL syringe. Plasma separation was performed after centrifuging blood samples at 2,000 g for 10 minutes. And we were careful not mix white blood cells and plasma. All samples were tested within one week of collection. All samples were stored at or below minus 78 degrees celsius as plasma (14).

Blood analysis

Blood analysis was conducted using a bench-top dry chemistry analyzer (Vettest 8008, IDEXX Laboratories Ltd, Maine, USA) and CRP analyzer (Vcheck V200, Bionote, Hwasung, Republic of Korea). Blood tests were performed to assess amylase, lipase, alanine transaminase (ALT), alkaline phosphatase (ALP), C-reactive protein (CRP), and aspartate transaminase (AST) levels. Additionally, canine pancreatic lipase was tested using SNAP cPL kit (IDEXX Laboratories Ltd, Maine, USA).

Quantitative analysis

Storage and quantitative analysis of samples were performed according to the promega manual. Extraction of cfDNA from plasma sample (0.5 mL) was performed using a Maxwell RSC cfDNA plasma kit (Promega, Madison, Wisconsin, USA). Calibration was performed using blank samples (1X TE Buffer) and standard samples (200 ng cfDNA) to determine the reference point. Concentration of extracted cfDNA was measured using a Quantifluor dsDNA system (Promega, Madison, Wisconsin, USA). Measurements were taken three times for each sample and the average of the three values was set to be the result value.

Statistical analysis

All statistical analyses were conducted using IBM SPSS 26.0 statistical software (SPSS Inc., USA). Group data are presented as medians and interquartile range (IQR). A value of p < 0.05 was considered significant for the analyses, unless otherwise indicated.

The normality test was conducted using the Shapiro-Wilk method, which resulted in no normality. Because of the lack of normality, Mann-Whitney tests were conducted to compare normal and pancreatitis groups. Statistical analysis was also attempted to compare the three groups of normal, acute pancreatitis, chronic pancreatitis. Because three of the groups did not satisfy normality, the Kruskal-Wallis test was used to compare the three groups. p-values below 0.05 were considered statistically significant. Statistical analysis was attempted to find a correlation between CRP and cfDNA. Because the sample size was small and normality was not satisfied, statistical analysis was performed using the Spearman and Kendall methods. A correlation coefficient of 0.5 or higher was considered a significant correlation, and a significance probability of less than 0.05 was considered statistically significant.

A total of 21 dogs were diagnosed with pancreatitis based on physical examination, blood analysis, cPL kit test, radiographic evaluation, and ultrasonographic evaluation. The average age of pancreatitis group was 11.8 years. In the pancreatitis group, the average concentration of cfDNA was 0.1236 ng/μL and the median was 0.0912 ng/μL (IQR 0.1650-0.0571). There were 14 dogs in the normal group based on physical examination, blood analysis, cPL kit test, and ultrasonography. The average age of normal group was 6.6 years. In the normal group, the average concentration of cfDNA was 0.0648 ng/μL and the median was 0.0599 ng/μL (IQR 0.0781-0.0458) (Fig. 2).

Figure 2.The graph about comparison of average cfDNA concentration. Patient group: median 0.0912 ng/μL (IQR 0.1650-0.0571), Normal group: median 0.0599 ng/μL, p-value = 0.028 (IQR 0.0781-0.0458). cfDNA; cell-free DNA.

When comparing mean cfDNA concentrations between the two groups, the pancreatitis group had about two-fold higher concentration than the normal group (Fig. 2). Statistical results using Mann-Whitney tests showed that there was a significant difference in cfDNA concentration between the two groups (Fig. 2) (p-value = 0.028).

Based on CRP levels, clinical symptoms, and ultrasound imaging findings, dogs in the pancreatitis group were classified into those with acute and chronic pancreatitis. In the acute pancreatitis group, a definite inflammatory response was seen on ultrasound and CRP levels. Their data were then analyzed. When comparing 8 patients with acute pancreatitis to 13 patients with chronic pancreatitis, the average cfDNA concentration was 0.1495 ng/μL for those with acute pancreatitis and 0.1076 ng/μL for those with chronic pancreatitis. The cfDNA concentration in the acute pancreatitis group was roughly 1.4 times higher than that in the chronic pancreatitis group and 2.3 times higher than that in normal group (Fig. 3). However, statistical analysis using the Kruskal-Wallis test showed low statistical significance (p-value = 0.082 > 0.05).

Figure 3.The graph about comparison of average cfDNA concentration in normal dogs and chronic pancreatitis patients dogs and acute pancreatitis patients dogs groups (Normal group: median 0.0599 ng/μL, Chronic group: median 0.0867 ng/μL, Acute group: median 0.1044 ng/μL, p-value = 0.082 > 0.05). cfDNA; cell-free DNA.

Statistical analysis to find the association between cfDNA and CRP showed a correlation coefficient of 0.18 (<0.5) with a significance of 0.269 (p-value > 0.05) by the Kendall method, and a correlation coefficient of 0.289 (<0.5) with a significance of 0.217 (p-value > 0.05) by the Spearman method. In conclusion, the correlation between the two indicator seems to be weak and the statistical significance is low.

The aim of this research was to quantify concentrations of cfDNA in dogs with pancreatitis and to find association between cfDNA and pancreatitis. In human medicine, there have been recent attempts to use cfDNA as a new liquid biomarker for estimating severity both mild and severe acute pancreatitis (19). However, to the best of our knowledge, this is the first study in veterinary medicine to measure and evaluate cfDNA concentrations in dogs with pancreatitis based on pathogenesis of pancreatitis.

A recent study in human medicine has shown a significant difference in cfDNA when comparing mild acute pancreatitis to severe acute pancreatitis (19). Based on these findings and the mechanism of cfDNA, we hypothesized that there could be a significant difference in cfDNA level between dogs with pancreatitis and normal dogs.

The analysis showed a significant difference between the pancreatitis group and the normal group (p-value = 0.028). Dogs with pancreatitis had higher average concentrations of cfDNA than normal dogs. These results indicate that the concentration of cfDNA is significantly increased in dogs affected by pancreatitis (p-value = 0.028).

We classified the pancreatitis groups into two groups, acute pancreatitis, and chronic pancreatitis. And then we compared these two group with normal group. When comparing the three groups, we found differences in the mean values of cfDNA concentrations, but not statistically significant (p-value = 0.082).

We also attempted to find an association between CRP, one of the most clinically used inflammatory markers, and cfDNA. We supposed that as the inflammation in the pancreas becomes more severe, pancreatic cell necrosis and apoptosis will increase, so we expected to see a clear correlation between cfDNA and CRP. However, statistical analysis showed that the correlation was very weak and the statistical significance was very low (p-value = 0.269/ p-value = 0.217).

This study has some limitations. First, the source of cfDNA in dogs could not be clearly classified. This is because if inflammatory reactions caused by pancreatitis progress throughout the body, the concentration of cfDNA might increase due to apoptosis or necrosis occurring in the liver, digestive system, heart, and muscles. Second. The lack of biopsy or fine-needle aspiration tests makes it difficult to clearly separate acute and chronic pancreatitis groups. We used cPl, CRP, and ultrasound to differentiate between the two groups, but this is not enough to clearly differentiate between the two groups. Further studies should use histologic examination or fine needle aspiration to clearly distinguish between chronic pancreatitis and acute pancreatitis. Third, the limitation is that we did not consider the age of the pancreatitis and normal groups. The concentration of cfDNA is thought to be affected by the aging of the body (21). Further studies should include the age of the subjects as a factor.

Finally, the sample size was too small to meet normality and this may have affected the statistical analysis. A larger study with a larger sample size is needed.

In this study, dogs with pancreatitis had significantly higher cfDNA levels than normal dogs. This result suggests that apoptosis and necrosis occur in the pancreas when pancreatitis occurs.

In conclusion, the results of this study suggest the possibility of using cfDNA as one of the markers to assess the severity of pancreatitis in dogs. However, further studies are needed.

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through a Companion Animal Life Cycle Industry Technology Development Program funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (322094).

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Article

Original Article

J Vet Clin 2024; 41(3): 143-149

Published online June 30, 2024 https://doi.org/10.17555/jvc.2024.41.3.143

Copyright © The Korean Society of Veterinary Clinics.

Investigation of Circulating Cell-Free DNA Concentration in Dogs with Pancreatitis

Jae-Hun Kim1 , Soo-Yeon Jeong1,2 , Chul Park1,*

1Department of Veterinary Internal Medicine, College of Veterinary Medicine, Jeonbuk National University, Iksan 54596, Korea
2Department of Companion Animal Health Care, Kyung-in Women’s University, Incheon 21041, Korea

Correspondence to:*chulpark0409@jbnu.ac.kr

Received: February 29, 2024; Revised: April 19, 2024; Accepted: May 16, 2024

This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Circulating cell-free DNA (cfDNA) constitutes a fragment of DNA released into the blood through cellular apoptosis or necrosis. In human medicine, cfDNA has been studied as a disease severity biomarker. Recent studies have shown that concentrations of cfDNA in dogs with immune-mediated and tumor-related diseases are increased. Pancreatitis is known to be caused by excessive release of trypsin, which leads to edema, inflammation, necrosis, and apoptosis in the pancreas. Based on the results of research showing an increase of cfDNA due to apoptosis and necrosis of cells, we hypothesized that cfDNA concentration would increase in the presence of pancreatitis. A total of 35 dogs were studied, including 21 with pancreatitis and 14 without any inflammatory diseases (normal group). The results showed that the concentration of cfDNA in dogs with pancreatitis was approximately twice that of normal dogs (median 0.0912 ng/μL. p-value 0.028). This result suggests that cfDNA can serve as a new biomarker for estimating pancreatitis severity.

Keywords: dog, circulating cell free DNA, apoptosis, pancreatitis, inflammation

Introduction

Circulating cell-free DNA (cfDNA) consisting of small double-stranded DNA fragments found in blood was first discovered in 1948 by Mandel and Metais (23). cfDNA that consists of degraded DNA fragments can be released to body fluids and blood. It has been reported that both healthy and ill human have cfDNAs (6).

cfDNA flows into the bloodstream via apoptosis and necrosis. It is directly released from viable cells after lysis of circulating cells (23). Consequently cfDNA levels can increase in various physiological and pathological states such as inflammation, smoking, sepsis, trauma, and cancer that can lead to increased apoptotic cell death (16). One study has found that cfDNA fragment’s length in circulation has a laddering pattern of 170-180 base pairs, the size of DNA produced by apoptosis (4). Therefore, major sources of cfDNA have been thought to be apoptotic and necrotic cells nowdays (23).

In human, increased concentrations of cfDNA have been reported in serum, plasma, and urine samples of patients with various conditions such as stroke, trauma, myocardial infarction, cancer, and immune mediated disease. This suggests an association between cfDNA and several disorders (15). Many researchers have endeavored to utilize cfDNA as a marker in liquid biopsy to assist diagnosis of prenatal, tumor, stroke, autoimmune disorders, and myocardial infarction or in transplant medicine (13,15). In veterinary medicine, several investigations have been conducted to find association between cfDNA and various diseases. Reports have indicated that cfDNA concentrations are increased in parasite disorders, immune-mediated hemolytic anemia, neurologic disorders, and various tumors and other inflammatory disease of dogs (1,15,18,20). In human medicine, cfDNA has been studies in the context of inflammation. An association between an inflammatory state (acute or chronic inflammation, autoimmune diseases, and infections) and cfDNA has been observed (3). This correlation may also exist in veterinary medicine (7,8).

Pancreatitis refers to inflammation in the pancreas with the presence of neutrophilic infiltrate, edema, and necrosis. This disease can be a localized inflammation. But, it can also lead to a systemic inflammatory response syndrome. The exact cause of pancreatitis in dogs and cats remains unclear. However, it has been discovered that hereditary mutations of trypsin may predispose people to pancreatitis in human medicine. This might be similar in dogs and cats (11).

Trypsin is a protease secreted in the pancreas. It is typically stored in zymogen granules in an inactive state under normal conditions. However, if excessive activation of trypsin occurs, this can lead to autodigestion and severe inflammation. This process can cause peripancreatic fat necrosis, sterile peritonitis, and systemic inflammatory response (2). So as pancreatitis is a kind of inflammatory disease with necrosis and apoptosis process, it may be possible to increase in serum cfDNA concentration.

C-reactive protein (CRP) is a kind of plasma protein that increase in inflammatory, infectious, neoplastic states and traumatic, infectious state (9,22). CRP is often used as an indirect indicator to assess the severity of a disease. And while increases in cfDNA have been seen in a variety of conditions where CRP is elevated, the correlation is not yet clear (10,17).

Based on the previous studies, we hypothesized that pancreatitis might also be associated with cfDNA in dogs. And we also hypothesized that if cfDNA was increased, it would also be associated with CRP. Therefore, this study aimed to determine the association between cfDNA and pancreatitis and to discover difference in cfDNA level between healthy dogs and dogs with pancreatitis. And to find the association between CRP and cfDNA in pancreatitis state. To the best of our knowledge, this is the first study that investigates concentrations of cfDNA in dogs with pancreatitis in veterinary medicine.

Materials|Methods

Animals

This was a prospective study of 21 dogs diagnosed with pancreatitis. The pancreatitis group consisted of dogs at the Jeonbuk Animal Medical Center (JAMC) between November 2020 and July 2021, all accompanied by their owners, that exhibited mild to severe digestive symptoms like vomiting, diarrhea, anorexia. Dogs with negative results from canine pancreatic lipase test (SNAP cPL kit, IDEXX Laboratories Ltd, Maine, USA) were excluded. They were also diagnosed with pancreatitis with abdominal ultrasonography. The CRP level was also measured. However, it was not used as a marker to differentiate between the pancreatitis and normal groups. A normal group consisted of dogs without digestive clinical signs among patients visiting the animal hospital. Normal dogs had no problems associated with pancreatitis based on x-ray, ultrasound, blood, and physical examinations and had no any inflammatory disease and tumor based on CRP concentration, radiography, ultrasonography (Table 1). There were 21 dogs with pancreatitis in the pancreatitis group and 14 healthy neutered dogs in the normal group. In the pancreatitis group, there were 11 females and 10 males. In the normal group, there were 5 females and 9 males. In this study, we devided the pancreatitis groups into acute and chronic pancreatitis groups based on ultrasonographic findings, CRP, and cPL. The dogs with other inflammatory lesions that were thought to be secondary to severe pancreatitis were categorized as acute pancreatitis. Patients were included in the chronic pancreatitis group if there were no other inflammatory lesions and the ultrasound showed features of chronic pancreatitis, even if there was an increase in CRP levels. There were 8 dogs in the acute pancreatitis group and 13 dogs in the chronic pancreatitis group (Table 2).

Table 1 . Concentrations of cell free DNA in normal group.

NumberBreedSex/AgeALT (U/L)ALP (U/L)WBC (K/ul)cfDNA (ng/μL)cPLCRP (mg/L)Ultrasond
Normal value--17-7847-2546-17-Negative10-20Normal
1BeagleM/64715415.10.0565Negative12Normal
2MalteseM/8775613.60.0512Negative10Normal
3BeagleM/64313411.40.0626Negative13Normal
4MixF/8331609.40.0777Negative12Normal
5BeagleM/64121014.80.151Negative10Normal
6MalteseF/13411229.40.0279Negative15Normal
7BeagleF/4471318.20.0866Negative10Normal
8BeagleM/645267170.0669Negative14Normal
9MixF/72929515.50.0464Negative14Normal
10BeagleM/63523712.40.0572Negative10Normal
11BeagleF/43523611.60.0442Negative12Normal
12PomeranianM/62979120.0677Negative11Normal
13BeagleM/41831111.10.0795Negative10Normal
14MalteseM/9651157.80.0321Negative10Normal

The normal group consisted of 14 dogs. All dogs were neutered. Concentration of cell free DNA was determined as the average of three measurements. M, male; F, Female; Normal, dog without pancreatitis; Ultrasound, ultrasonogrphic finding of pancreas..



Table 2 . Concentrations of cell free DNA in patient group.

NumberBreedSex/AgeALT (U/L)ALP (U/L)WBC (103/μL)cfDNA (ng/μL)cPLCRP (mg/L)Ultrasound
Reference interval17-7847-2546-17-Negative10-20Normal
1PoodleM/15601009.30.1726Positive10Abnormal
2Yorkshire terrierF/17437812.90.1176Positive33.6Abnormal
3ChihuahuaM/69119880.05385Positive13.9Abnormal
4MalteseF/1550848.10.0637Positive10Abnormal
5DachshundM/1212681680.0504Positive14.8Abnormal
6MalteseM/155175,5697.70.1575Positive36.4Abnormal
7Miniature pinscherF/12352,14350.80.451Positive138.8Abnormal
8Jack russell terrierM/787896.90.0605Positive35.5Abnormal
9Miniature pinscherF/12502,00029.10.134Positive117.1Abnormal
10Yorkshire terrierF/172913213.80.186Positive33.9Abnormal
11Yorkshire terrierM/13681787.40.231Positive22.7Abnormal
12Yorkshire terrierF/17287812.90.0465Positive33.6Abnormal
13PoodleM/10399310.30.0307Positive17Abnormal
14Golden retrieverM/5646199.20.0691Positive86.6Abnormal
15Shih-tzuF/151972,00014.90.027Positive64.7Abnormal
16Yorkshire terrierM/132717012.90.246Positive78.8Abnormal
17MixF/152821218.90.0912Positive33.8Abnormal
18SchnauzerF/4454310.50.0685Positive10Abnormal
19JindoM/1120086112.10.152Positive22.5Abnormal
20PoodleF/826698.70.1Positive26.4Abnormal
21Golden retrieverF/92899100.0867Positive95Abnormal

The patient group was 21 dogs. All dogs were neutered. Concentration of cell free DNA was determined as the average of three measurements. M, male; F, Female; Patient, dog with pancreatitis. Ultrasound, ultrasonogrphic finding of pancreas. cPL, canine pancreas-specific lipase; CRP, c-reactive protein.



Radiographic examination

Abdominal radiographs were obtained from both ventrodorsal view and right lateral view. Evaluations included serosal detail, intestinal opacity, intestinal size, and liver size. All dogs with radiographs suggestive of foreign bodies were excluded.

Abdominal ultrasonography

Ultrasonographic examinations were performed using a Toshiba Aplio 300 (Aplio 300, Canon Medical Systems, Otawara, Japan). Ultrasound assessments involved measuring the shape and thickness of pancreas and evaluating echogenicity. We considered the normal thickness range for the pancreas up to 6.5 mm for the left lobe, 6.3 mm for the body, and 8.1 mm for the right lobe (12). If the ultrasonographic finding showed increased thickness of the pancreas and hypoechoic regions, it was classified as acute pancreatitis group. If the thickness of the pancreas was normal but showed hyperechoic non-shadowing region, it was classified as chronic pancreatitis group (Fig. 1).

Figure 1. Ultrasonographic findings in dogs with pancreatitis group. (A) Acute pancreatitis. Hypoechoic region and hyperechoic peripheral region was observed in pancreatic body. The thickness of pancreas body was 13.3 mm and this is abnormal. (B) Chronic pancreatitis. Hyperechoic non-shadowing region was observed in right lobe pancreas. The thickness was normal range (4.5 mm).

Blood sample collection

Approximately 2 mL of blood was collected into ethylenediaminetetraacetic acid (EDTA) tubes from cephalic and external jugular veins using a 3 mL syringe. Plasma separation was performed after centrifuging blood samples at 2,000 g for 10 minutes. And we were careful not mix white blood cells and plasma. All samples were tested within one week of collection. All samples were stored at or below minus 78 degrees celsius as plasma (14).

Blood analysis

Blood analysis was conducted using a bench-top dry chemistry analyzer (Vettest 8008, IDEXX Laboratories Ltd, Maine, USA) and CRP analyzer (Vcheck V200, Bionote, Hwasung, Republic of Korea). Blood tests were performed to assess amylase, lipase, alanine transaminase (ALT), alkaline phosphatase (ALP), C-reactive protein (CRP), and aspartate transaminase (AST) levels. Additionally, canine pancreatic lipase was tested using SNAP cPL kit (IDEXX Laboratories Ltd, Maine, USA).

Quantitative analysis

Storage and quantitative analysis of samples were performed according to the promega manual. Extraction of cfDNA from plasma sample (0.5 mL) was performed using a Maxwell RSC cfDNA plasma kit (Promega, Madison, Wisconsin, USA). Calibration was performed using blank samples (1X TE Buffer) and standard samples (200 ng cfDNA) to determine the reference point. Concentration of extracted cfDNA was measured using a Quantifluor dsDNA system (Promega, Madison, Wisconsin, USA). Measurements were taken three times for each sample and the average of the three values was set to be the result value.

Statistical analysis

All statistical analyses were conducted using IBM SPSS 26.0 statistical software (SPSS Inc., USA). Group data are presented as medians and interquartile range (IQR). A value of p < 0.05 was considered significant for the analyses, unless otherwise indicated.

The normality test was conducted using the Shapiro-Wilk method, which resulted in no normality. Because of the lack of normality, Mann-Whitney tests were conducted to compare normal and pancreatitis groups. Statistical analysis was also attempted to compare the three groups of normal, acute pancreatitis, chronic pancreatitis. Because three of the groups did not satisfy normality, the Kruskal-Wallis test was used to compare the three groups. p-values below 0.05 were considered statistically significant. Statistical analysis was attempted to find a correlation between CRP and cfDNA. Because the sample size was small and normality was not satisfied, statistical analysis was performed using the Spearman and Kendall methods. A correlation coefficient of 0.5 or higher was considered a significant correlation, and a significance probability of less than 0.05 was considered statistically significant.

Results

A total of 21 dogs were diagnosed with pancreatitis based on physical examination, blood analysis, cPL kit test, radiographic evaluation, and ultrasonographic evaluation. The average age of pancreatitis group was 11.8 years. In the pancreatitis group, the average concentration of cfDNA was 0.1236 ng/μL and the median was 0.0912 ng/μL (IQR 0.1650-0.0571). There were 14 dogs in the normal group based on physical examination, blood analysis, cPL kit test, and ultrasonography. The average age of normal group was 6.6 years. In the normal group, the average concentration of cfDNA was 0.0648 ng/μL and the median was 0.0599 ng/μL (IQR 0.0781-0.0458) (Fig. 2).

Figure 2. The graph about comparison of average cfDNA concentration. Patient group: median 0.0912 ng/μL (IQR 0.1650-0.0571), Normal group: median 0.0599 ng/μL, p-value = 0.028 (IQR 0.0781-0.0458). cfDNA; cell-free DNA.

When comparing mean cfDNA concentrations between the two groups, the pancreatitis group had about two-fold higher concentration than the normal group (Fig. 2). Statistical results using Mann-Whitney tests showed that there was a significant difference in cfDNA concentration between the two groups (Fig. 2) (p-value = 0.028).

Based on CRP levels, clinical symptoms, and ultrasound imaging findings, dogs in the pancreatitis group were classified into those with acute and chronic pancreatitis. In the acute pancreatitis group, a definite inflammatory response was seen on ultrasound and CRP levels. Their data were then analyzed. When comparing 8 patients with acute pancreatitis to 13 patients with chronic pancreatitis, the average cfDNA concentration was 0.1495 ng/μL for those with acute pancreatitis and 0.1076 ng/μL for those with chronic pancreatitis. The cfDNA concentration in the acute pancreatitis group was roughly 1.4 times higher than that in the chronic pancreatitis group and 2.3 times higher than that in normal group (Fig. 3). However, statistical analysis using the Kruskal-Wallis test showed low statistical significance (p-value = 0.082 > 0.05).

Figure 3. The graph about comparison of average cfDNA concentration in normal dogs and chronic pancreatitis patients dogs and acute pancreatitis patients dogs groups (Normal group: median 0.0599 ng/μL, Chronic group: median 0.0867 ng/μL, Acute group: median 0.1044 ng/μL, p-value = 0.082 > 0.05). cfDNA; cell-free DNA.

Statistical analysis to find the association between cfDNA and CRP showed a correlation coefficient of 0.18 (<0.5) with a significance of 0.269 (p-value > 0.05) by the Kendall method, and a correlation coefficient of 0.289 (<0.5) with a significance of 0.217 (p-value > 0.05) by the Spearman method. In conclusion, the correlation between the two indicator seems to be weak and the statistical significance is low.

Discussion

The aim of this research was to quantify concentrations of cfDNA in dogs with pancreatitis and to find association between cfDNA and pancreatitis. In human medicine, there have been recent attempts to use cfDNA as a new liquid biomarker for estimating severity both mild and severe acute pancreatitis (19). However, to the best of our knowledge, this is the first study in veterinary medicine to measure and evaluate cfDNA concentrations in dogs with pancreatitis based on pathogenesis of pancreatitis.

A recent study in human medicine has shown a significant difference in cfDNA when comparing mild acute pancreatitis to severe acute pancreatitis (19). Based on these findings and the mechanism of cfDNA, we hypothesized that there could be a significant difference in cfDNA level between dogs with pancreatitis and normal dogs.

The analysis showed a significant difference between the pancreatitis group and the normal group (p-value = 0.028). Dogs with pancreatitis had higher average concentrations of cfDNA than normal dogs. These results indicate that the concentration of cfDNA is significantly increased in dogs affected by pancreatitis (p-value = 0.028).

We classified the pancreatitis groups into two groups, acute pancreatitis, and chronic pancreatitis. And then we compared these two group with normal group. When comparing the three groups, we found differences in the mean values of cfDNA concentrations, but not statistically significant (p-value = 0.082).

We also attempted to find an association between CRP, one of the most clinically used inflammatory markers, and cfDNA. We supposed that as the inflammation in the pancreas becomes more severe, pancreatic cell necrosis and apoptosis will increase, so we expected to see a clear correlation between cfDNA and CRP. However, statistical analysis showed that the correlation was very weak and the statistical significance was very low (p-value = 0.269/ p-value = 0.217).

This study has some limitations. First, the source of cfDNA in dogs could not be clearly classified. This is because if inflammatory reactions caused by pancreatitis progress throughout the body, the concentration of cfDNA might increase due to apoptosis or necrosis occurring in the liver, digestive system, heart, and muscles. Second. The lack of biopsy or fine-needle aspiration tests makes it difficult to clearly separate acute and chronic pancreatitis groups. We used cPl, CRP, and ultrasound to differentiate between the two groups, but this is not enough to clearly differentiate between the two groups. Further studies should use histologic examination or fine needle aspiration to clearly distinguish between chronic pancreatitis and acute pancreatitis. Third, the limitation is that we did not consider the age of the pancreatitis and normal groups. The concentration of cfDNA is thought to be affected by the aging of the body (21). Further studies should include the age of the subjects as a factor.

Finally, the sample size was too small to meet normality and this may have affected the statistical analysis. A larger study with a larger sample size is needed.

Conclusions

In this study, dogs with pancreatitis had significantly higher cfDNA levels than normal dogs. This result suggests that apoptosis and necrosis occur in the pancreas when pancreatitis occurs.

In conclusion, the results of this study suggest the possibility of using cfDNA as one of the markers to assess the severity of pancreatitis in dogs. However, further studies are needed.

Acknowledgements

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through a Companion Animal Life Cycle Industry Technology Development Program funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (322094).

Conflicts of Interest

The authors have no conflicting interests.

Fig 1.

Figure 1.Ultrasonographic findings in dogs with pancreatitis group. (A) Acute pancreatitis. Hypoechoic region and hyperechoic peripheral region was observed in pancreatic body. The thickness of pancreas body was 13.3 mm and this is abnormal. (B) Chronic pancreatitis. Hyperechoic non-shadowing region was observed in right lobe pancreas. The thickness was normal range (4.5 mm).
Journal of Veterinary Clinics 2024; 41: 143-149https://doi.org/10.17555/jvc.2024.41.3.143

Fig 2.

Figure 2.The graph about comparison of average cfDNA concentration. Patient group: median 0.0912 ng/μL (IQR 0.1650-0.0571), Normal group: median 0.0599 ng/μL, p-value = 0.028 (IQR 0.0781-0.0458). cfDNA; cell-free DNA.
Journal of Veterinary Clinics 2024; 41: 143-149https://doi.org/10.17555/jvc.2024.41.3.143

Fig 3.

Figure 3.The graph about comparison of average cfDNA concentration in normal dogs and chronic pancreatitis patients dogs and acute pancreatitis patients dogs groups (Normal group: median 0.0599 ng/μL, Chronic group: median 0.0867 ng/μL, Acute group: median 0.1044 ng/μL, p-value = 0.082 > 0.05). cfDNA; cell-free DNA.
Journal of Veterinary Clinics 2024; 41: 143-149https://doi.org/10.17555/jvc.2024.41.3.143

Table 1 Concentrations of cell free DNA in normal group

NumberBreedSex/AgeALT (U/L)ALP (U/L)WBC (K/ul)cfDNA (ng/μL)cPLCRP (mg/L)Ultrasond
Normal value--17-7847-2546-17-Negative10-20Normal
1BeagleM/64715415.10.0565Negative12Normal
2MalteseM/8775613.60.0512Negative10Normal
3BeagleM/64313411.40.0626Negative13Normal
4MixF/8331609.40.0777Negative12Normal
5BeagleM/64121014.80.151Negative10Normal
6MalteseF/13411229.40.0279Negative15Normal
7BeagleF/4471318.20.0866Negative10Normal
8BeagleM/645267170.0669Negative14Normal
9MixF/72929515.50.0464Negative14Normal
10BeagleM/63523712.40.0572Negative10Normal
11BeagleF/43523611.60.0442Negative12Normal
12PomeranianM/62979120.0677Negative11Normal
13BeagleM/41831111.10.0795Negative10Normal
14MalteseM/9651157.80.0321Negative10Normal

The normal group consisted of 14 dogs. All dogs were neutered. Concentration of cell free DNA was determined as the average of three measurements. M, male; F, Female; Normal, dog without pancreatitis; Ultrasound, ultrasonogrphic finding of pancreas.


Table 2 Concentrations of cell free DNA in patient group

NumberBreedSex/AgeALT (U/L)ALP (U/L)WBC (103/μL)cfDNA (ng/μL)cPLCRP (mg/L)Ultrasound
Reference interval17-7847-2546-17-Negative10-20Normal
1PoodleM/15601009.30.1726Positive10Abnormal
2Yorkshire terrierF/17437812.90.1176Positive33.6Abnormal
3ChihuahuaM/69119880.05385Positive13.9Abnormal
4MalteseF/1550848.10.0637Positive10Abnormal
5DachshundM/1212681680.0504Positive14.8Abnormal
6MalteseM/155175,5697.70.1575Positive36.4Abnormal
7Miniature pinscherF/12352,14350.80.451Positive138.8Abnormal
8Jack russell terrierM/787896.90.0605Positive35.5Abnormal
9Miniature pinscherF/12502,00029.10.134Positive117.1Abnormal
10Yorkshire terrierF/172913213.80.186Positive33.9Abnormal
11Yorkshire terrierM/13681787.40.231Positive22.7Abnormal
12Yorkshire terrierF/17287812.90.0465Positive33.6Abnormal
13PoodleM/10399310.30.0307Positive17Abnormal
14Golden retrieverM/5646199.20.0691Positive86.6Abnormal
15Shih-tzuF/151972,00014.90.027Positive64.7Abnormal
16Yorkshire terrierM/132717012.90.246Positive78.8Abnormal
17MixF/152821218.90.0912Positive33.8Abnormal
18SchnauzerF/4454310.50.0685Positive10Abnormal
19JindoM/1120086112.10.152Positive22.5Abnormal
20PoodleF/826698.70.1Positive26.4Abnormal
21Golden retrieverF/92899100.0867Positive95Abnormal

The patient group was 21 dogs. All dogs were neutered. Concentration of cell free DNA was determined as the average of three measurements. M, male; F, Female; Patient, dog with pancreatitis. Ultrasound, ultrasonogrphic finding of pancreas. cPL, canine pancreas-specific lipase; CRP, c-reactive protein


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Vol.41 No.3 June 2024

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The Korean Society of Veterinary Clinics

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eISSN 2384-0749

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