Diagnostic utility of D-dimer and peritoneal fluid cytology in traumatic reticuloperitonitis in bovines

Introduction

Traumatic reticuloperitonitis (TRP) remains one of the most frequently encountered and economically significant thoraco-abdominal disorders in bovines, attributed largely to indiscriminate feeding behavior and increasing environmental exposure to metallic foreign bodies. Ingestion of sharp metallic objects may result in reticular perforation, localized or diffuse peritonitis, and in severe cases, extension to adjacent structures such as the diaphragm or pericardium. Given the overlapping clinical presentation of TRP with other digestive disorders such as vagal indigestion, tympany, ruminal impaction, or intestinal obstruction, the diagnostic process remains complex. Traditional diagnostic approaches encompass clinical evaluation, imaging, hematology, and biochemical profiling. However, peritoneal fluid analysis has emerged as a rapid, minimally invasive, and highly informative diagnostic tool to evaluate peritoneal inflammation and infection. Normal bovine peritoneal fluid was straw-colored, watery, and low in cellularity and protein (Radostits et al., 2007) [8] . In contrast, inflammatory or septic peritonitis causes alterations such as increased turbidity, neutrophilic infiltration, elevated total protein, and the presence of fibrin, RBCs, and bacteria (Hussain & Uppal, 2014; Wittek et al., 2010) [4,11] . The diagnostic accuracy of cytological and biochemical analysis-especially D-dimer estimation-has been established in numerous studies, showing high sensitivity and specificity for septic peritonitis International Journal of Advanced Biochemistry Research 2025; SP-9(7): 505-510 (Wittek et al. 2010) [11] . Hussain et al. (2021) [6] further emphasized that cytology can detect degenerated neutrophils, engulfed bacteria, and fibrinous debris, which are essential for staging peritonitis. In bovines, where systemic indicators may be less pronounced than in species like horses or dogs, peritoneal fluid analysis holds particular diagnostic value (Hussain et al., 2021) [6] . Recent studies have expanded its application to diagnose complications such as perforated abomasal ulcers, intestinal obstruction, uterine torsion, and even reticular abscesses (Bhutia et al., 2019; Brar et al., 2006) [2,3] . This study was designed to evaluate the cytological and ddimer changes in peritoneal fluid in bovines affected with TRP and to establish a semi-quantitative scoring system for cytological evaluation to aid in diagnosis and clinical staging.

Materials and Methods

A total of six clinically diagnosed cases of traumatic reticuloperitonitis (TRP) in bovines were enrolled during the study period along with six healthy bovines during the period of March 2022 to November 2023 at Department of Veterinary Medicine, DGCN College of Veterinary and Animal Sciences, CSK Himachal Pradesh Agriculture University, Palampur, Himachal Pradesh, India.

Peritoneal Fluid Collection and Gross Evaluation

Peritoneal fluid was obtained via abdominocentesis from predetermined anatomical landmarks (Vermunt J, 2011) [10] or under ultrasonographic guidance in recumbent animals. Gross evaluation of the fluid included assessment of color, odor, turbidity, and volume.

Peritoneal Fluid Analysis

Peritoneal fluid samples collected in EDTA vials were analyzed using the Mindray BC-5000 Vet Hematology Analyzer for total leukocyte count (TLC), red blood cell (RBC) count, and neutrophil count (both absolute and differential). For D-dimer estimation, fluid was collected in 3.2% sodium citrate tubes and processed using the ICHROMA™ II analyzer. A 10 µl aliquot of the sample was mixed with the detection buffer, and 75 µl of the mixture was loaded into the cartridge for automated quantification. Results were generated within 12 minutes. Peritoneal fluid total protein concentration was estimated using a handheld refractometer.

Cytological Examination and Scoring

Cytological evaluation was performed on direct smears prepared from centrifuged peritoneal sediment. Smears were air-dried, methanol-fixed, and stained with Giemsa. Wet mount and stained preparations were examined under low (10×) and high-power (40× and 100× oil immersion) objectives. The following cytological parameters were evaluated: polymorphonuclear cells (PMNC), mononuclear cells (MNC), fibrin strands, bacteria, and RBCs. Each parameter was semi-quantitatively scored based on visual estimation, averaging findings across 10 high-power (40×) microscopic fields. Scoring criteria were defined as follows:

PMNC and MNC (percentage of total nucleated cells/ average of 10 high-power (40×) microscopic fields):0 = <10%, + = 10-30% (Mild), ++ = 31-60% (Moderate), +++ = >60% (Severe) Bacteria and RBCs (relative abundance): 0 = Absent, + = Mild = ++, +++ = Marked For statistical analysis, these categorical scores were converted to numerical values: 0 = 0, + = 1, ++ = 2, +++ = 3.

Tube Clot Test: Approximately 2-3 ml of peritoneal fluid was placed in a clean, dry glass test tube and left undisturbed at room temperature for 30 minutes. Clot formation indicated fibrin presence and was graded semiquantitatively as + (thin film), ++ (moderate clot), or +++ (dense clot) (Radostits et al., 2007 and Athar et al., 2010) [1,8] .

Statistical Analysis

Quantitative data were expressed in Mean ± S.E. and analyzed using Welch's t-test. All statistical analyses were performed using GraphPad Prism version 9. Cytology scores and Tube clot scores from TRP and healthy control groups were analyzed using the Mann-Whitney U test due to the ordinal nature of the data and small sample size (n = 6 per group). Results were presented as median (range), and differences were considered statistically significant at p<0.05.

Results

Gross appearance

Peritoneal fluid analysis in six bovines diagnosed with TRP (Table 1) revealed distinct deviations from normal findings. In terms of color, three animals (50%) exhibited yellow fluid, while the remaining three (50%) had brownish-red fluid (Fig. 1), indicating inflammatory and hemorrhagic reactions respectively. No cases showed straw-colored fluid, which was typical in healthy animals (Fig. 2). Odour was absent in three animals (50%), while the other three showed a putrid smell, suggesting bacterial infection and tissue necrosis. The volume of peritoneal fluid was >50 ml in four animals (66.67%), consistent with inflammatory effusion, while two animals (33.33%) had fluid volumes <50 ml. Turbidity was present in four animals (66.67%), further indicating active inflammation and exudate accumulation, whereas it was absent in the remaining two (33.33%).

Peritoneal Fluid Parameters

Various recorded peritoneal fluid parameters (Table 2) revealed significant alterations were observed in the peritoneal fluid profiles of TRP-affected bovines compared to healthy controls. Total red blood cell count was significantly higher in the diseased group (p<0.05), indicating possible hemorrhagic effusion. Total leukocyte count and neutrophil count were markedly elevated in TRP cases (p<0.05), with neutrophils comprising a significantly greater percentage of the leukocyte population (p<0.05), consistent with a purulent inflammatory response. Total protein concentration was significantly elevated in TRP animals (p<0.01), indicating an exudative nature of the effusion. D-dimer levels were also significantly higher in the diseased group compared to healthy controls (p<0.05), reflecting ongoing fibrinolysis and inflammation.

Cytological Evaluation of Peritoneal Fluid

Cytological evaluation of bovines (Table 3) revealaled normal peritoneal cytology characterized by mononuclear cell predominance (PMNC score: 0 to +) and absence of bacteria and red blood cells in healthy animals, establishing the baseline for comparison. Among TRP cases, the mildest changes were observed in TRP 5, which showed mild neutrophilic infiltration without detectable bacteria or erythrocytes. This pattern was suggestive of early, or a sterile exudative response (Fig. 3). Moderate cytological changes (Fig. 4) were noted in TRP 2 and TRP 4, marked by intracellular bacteria and mild to moderate hemorrhage, indicating mid-stage or localized peritonitis (Fig. 4). Severe cytological alterations were present in TRP 1, TRP 3, and TRP 6, which showed marked (+++) polymorphonuclear cell infiltration, presence of intra-and extracellular bacteria, and a hemorrhagic background (Fig. 5 and Fig. 6). These findings were consistent with advanced septic TRP and exudative effusion (Fig. 5 and6). These stage-wise cytological patterns, when matched with reference Fig. images, enabled a clearer understanding of TRP progression from early sterile inflammation to advanced septic peritonitis.

Tube Clot Test Results

Peritoneal fluid samples subjected to the tube clot test (Table 3) demonstrated variable fibrin content. One sample showed a thin fibrin film (+), two samples exhibited moderate clot formation (++) (Fig. 7), and three samples formed dense gelatinous clots (+++), indicating substantial fibrin presence suggestive of ongoing or advanced inflammatory processes.

Cytological Score Comparison between Groups

Peritoneal cytology scores (Table 4) differed significantly between TRP-affected and healthy bovines. The PMNC score was significantly elevated in the TRP group compared to controls (p = 0.0022), indicating neutrophilic inflammation. Conversely, the MNC score was significantly reduced in affected animals (p = 0.0022), reflecting a shift from mononuclear to polymorphonuclear predominance. Bacterial presence, although absent in healthy bovines, was significantly increased in TRP animals (p = 0.0152), with both intracellular and extracellular organisms noted on smears. RBC scores were also significantly higher in the diseased group (p = 0.0152), suggesting hemorrhagic contamination or vascular leakage. These findings confirm the presence of septic, exudative peritonitis in TRP cases. Fibrin presence (Tube clot test) was also markedly higher in TRP cases (p = 0.0022), consistent with exudative inflammation.

A significant positive Spearman rank correlation was observed between tube clot scores and D-dimer concentrations in TRP-affected bovines (ρ = 0.926, p = 0.033), suggesting that greater fibrin deposition on cytology was associated with increased fibrinolytic activity in the peritoneal cavity.

Discussion

Peritoneal fluid analysis revealed significant alterations in bovines with TRP. Color changes from straw (healthy) to yellow and brownish-red in affected animals reflected the inflammatory and hemorrhagic nature of the effusion. The presence of turbidity and putrid odor in a majority of TRP cases suggests ongoing bacterial infection and necrosis, which aligns with the findings of Athar et al. (2010) [1] and Hussain et al. (2021) [6] , who reported elevated neutrophils, degenerated PMNCs, and engulfed bacteria in peritonitis. Significant increase in total protein (p<0.05) confirmed the exudative character of peritoneal effusions in TRP, consistent with the classical differentiation of transudate and exudate described by Kopcha and Schultze (1991) [7] and Radostits et al. (2007) [8] . The markedly elevated leukocyte and neutrophil counts in diseased animals (p<0.05) were suggestive of inflammation. These findings were further corroborated by elevated D-dimer levels, indicating active fibrinolysis, as previously validated by Wittek et al. (2010) [11] . Cytological scoring provided critical insights into disease severity. TRP animals showed significantly higher PMNC scores and reduced MNC scores (p = 0.0022), reflecting acute inflammatory progression (Fig. 3). Fibrin deposition, RBC infiltration, and bacterial presence were significantly higher in TRP animals (p<0.05), supporting the findings of Hussain and Uppal (2014) [4] and Constable et al. (2017) [5] , who observed degenerated neutrophils and engulfed bacteria in septic peritonitis. These findings were also in agreement with Radostits et al. (2007) [8] and Constable et al. (2017) [5] , who described severe peritoneal inflammation in TRP as being characterized by high protein, neutrophilic dominance, fibrin deposition, and bacterial presence. In the current study, gross fluid characteristics such as turbid appearance, putrid smell, and red-yellow discoloration corroborated the cytological and biochemical findings. Peritoneal fluid analysis also enabled differential diagnosis from other causes of abdominal distress. While intestinal obstruction may present with high neutrophils and protein, bacterial presence and D-dimer elevations were more suggestive of septic TRP than ischemic or non-septic obstruction (Bhutia et al., 2019) [2] .

Acknowledgement

AcknowledgmentsThe authors would like to express their sincere gratitude to the Department of Veterinary Medicine, CSKHPKV, Palampur, and especially to the Head of Department and Dr. Ajay Katoch, for providing the necessary facilities and support for this research. We also extend our heartfelt thanks to Dr. Ankur Sharma, Dr. R.D. Patil, and Dr. Nainika for their valuable guidance and cooperation throughout the study. The contributions of co-workers, field assistants, and local stakeholders are also gratefully acknowledged. The authors declare no conflict of interest related to this work.

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