Abstract
Minimally invasive techniques allow tissue sampling while limiting procedural invasiveness. This study compared the quality of biopsy samples obtained through laparoscopically guided core needle (16 G or 18 G), laparoscopic pancreatic forceps or laparoscopic cup forceps with teeth for sampling non-grossly enlarged cecocolic lymph nodes in dog cadavers. Three techniques were performed in randomized order across 17 fresh cadavers, collecting three specimens per technique (153 total specimens, yielding 51 technique-level histological assessments). Sample collection time, observer-reported perceived difficulty, and a composite histological sample-adequacy score (0–14) were evaluated. Core needle biopsy required longer collection time than both forceps techniques (adjusted p = 0.002 for both comparisons). Perceived difficulty was lowest for pancreatic forceps, followed by cup forceps with teeth, and highest for core needle biopsy (all adjusted p ≤ 0.004). Both pancreatic forceps and cup forceps with teeth yielded higher composite adequacy scores than core needle biopsy (adjusted p = 0.019 and 0.006, respectively), whereas the two forceps did not differ (p = 0.504). Core needle biopsy was associated with greater fragmentation, lower structural preservation, and more histopathological changes limiting interpretation. Exploratory comparisons showed no differences between 16 G and 18 G needles. In this cadaveric model, both laparoscopic forceps techniques provided shorter collection times, lower perceived difficulty, and superior histological sample adequacy than laparoscopically guided core needle biopsy. Clinical safety and disease-specific diagnostic performance warrant further evaluation.
Keywords: Minimally invasive surgery, Diagnostic techniques, Surgical instruments, Histological techniques, Feasibility studies
Introduction
The advancement of minimally invasive diagnostic techniques in veterinary medicine has expanded the options for tissue sampling. Videolaparoscopy and core needle biopsy are widely used to obtain histopathological samples, particularly from abdominal organs (Grimes, 2018; Nowicki et al., 2010). These methods permit tissue acquisition through limited surgical access or under direct image guidance, thereby reducing tissue trauma compared to open surgical biopsy (Grimes, 2018; Willard et al., 2019).
Mesenteric lymph nodes are diagnostically significant due to their involvement in inflammatory, neoplastic, and systemic diseases. Anatomically, they are located deep within the abdominal cavity and are often adjacent to major vascular and intestinal structures, making their identification and sampling technically challenging (Nyman & O’Brien, 2007). These characteristics make them relevant targets for evaluating minimally invasive sampling techniques, particularly when precision and tissue preservation are essential.
Among the mesenteric nodes, the cecocolic lymph nodes are associated with the cecocolic ligament, which serves as a consistent laparoscopic landmark that facilitates standardized identification and surgical access. This landmark is particularly advantageous in a model lacking gross lymph node enlargement, where the visibility of normal abdominal lymph nodes can be variable. Indeed, colic lymph nodes were not consistently identified by computed tomography in a series of dogs without lymphadenopathy (Beukers et al., 2013).
Core needle biopsy is a relatively accessible technique; however, the smaller specimen size obtained may incompletely represent the lesion and is susceptible to fragmentation, crushing, or structural distortion during handling and fixation (Murgia, 2014; Rawlings et al., 2003). In contrast, laparoscopic techniques provide direct visualization and targeted tissue manipulation, which may facilitate the collection of larger, more intact specimens (Willard et al., 2019; Buote et al., 2022).
Despite the increasing use of minimally invasive lymph node biopsies in dogs, comparative studies evaluating different sampling instruments remain scarce, particularly in controlled experimental models. This gap limits the standardization of surgical protocols and the comprehensive evaluation of operative and histopathological parameters. Therefore, this study aimed to compare laparoscopically guided core needle biopsy with two laparoscopic forceps biopsy techniques in dog cadavers with non-grossly enlarged cecocolic lymph nodes, focusing on sample collection time, observer-reported perceived difficulty, and histological sample adequacy.
Material and methods
Specimens
Seventeen fresh canine cadavers (10 males and seven females) were obtained from the institutional veterinary teaching hospital where the study was conducted. The dogs had either died naturally or were euthanized for clinical reasons unrelated to the study; no animal was euthanized for research purposes. All cadavers were voluntarily donated by owners, with written informed consent obtained for post-mortem research use. Cadaver donation, collection, handling, storage, and disposal adhered to institutional animal care guidelines and applicable national legislation.
Inclusion criteria comprised dog cadavers weighing between 4 and 25 kg, across various ages and breeds, with body condition scores ranging from 4/9 to 6/9 (WSAVA, 2011). Seventeen cadavers without anatomical or traumatic abnormalities, abdominal wall hernias, or intra-abdominal neoplasms were selected. To minimize post-mortem tissue degradation, all cadavers were stored under refrigeration (2 °C to 8 °C) and used within a maximum of 7 h post-mortem.
Experimental design
To isolate the effect of the sampling instrument, all biopsy techniques were performed under direct laparoscopic visualization in cadavers lacking gross enlargement of the cecocolic lymph nodes. Each cadaver underwent three laparoscopically guided lymph node biopsy techniques: core needle biopsy (GCN), laparoscopic pancreatic forceps (GPF), and laparoscopic toothed cup forceps (GCF).
For GCN, a 16 G or 18 G core needle was used. Initial needle gauge selection was stratified by body weight: an 18 G needle was assigned to cadavers weighing ≤12.5 kg, and a 16 G needle to those weighing >12.5 kg. This criterion was followed in 15 of the 17 cadavers. In two cadavers weighing ≤12.5 kg, a 16 G needle was used due to a temporary shortage of cadavers weighing >12.5 kg. Overall, 11 cadavers were sampled using an 18 G needle and six using a 16 G needle; the assigned gauge remained constant for all three needle samples within each cadaver.
Each technique was repeated three times per cadaver, yielding nine biopsy specimens per animal and 153 specimens in total. Specimens were taken from different regions of the same cecocolic lymph node or from an adjacent node to minimize structural damage from prior sampling. The three specimens obtained per technique within a cadaver were evaluated jointly as a single sample set and assigned a single histological adequacy score. Consequently, the histological dataset comprised 51 technique-level assessments (three assessments per cadaver).
The sequence of the three biopsy techniques was randomized using a draw-of-lots system based on six predefined sequences: A (GPF, GCN and GCF); B (GPF, GCF and GCN); C (GCF, GCN and GPF); D (GCF, GPF and GCN); E (GCN, GCF and GPF); and, F (GCN, GPF and GCF).
Laparoscopic procedures
Surgical procedures were conducted by a standardized laparoscopic team consisting of a primary surgeon, a camera assistant, and a surgical assistant. Cadavers were clipped, and tissues were handled gently to minimize crush artifacts.
Cadavers were initially placed in dorsal recumbency. A 10 mm portal was established caudal to the umbilicus using the modified Hasson technique. The abdominal cavity was insufflated with CO₂ to a pressure of 10 mmHg at a flow rate of 1 L/min. Two additional 5 mm portals were placed under direct vision in the prepubic and left flank regions, creating a triangular portal layout (Fig. 1). Portals were secured to the skin using nylon sutures. Cadavers were then repositioned into left lateral recumbency.
Fig. 1.
- (Left) Triangular portal positioning on a canine cadaver abdomen for cecocolic lymph node biopsy: 10-mm umbilical port (A), 5-mm prepubic port (B), and 5-mm left flank port (C). (Right) Sampling instruments evaluated: laparoscopic pancreatic forceps (D), laparoscopic toothed cup forceps (E), 18 G core needle (F), and 16 G core needle (G).
The cecocolic ligament was identified as an anatomical landmark to expose the corresponding lymph node. A pair of Kelly forceps introduced through the prepubic cannula was used to elevate the intestine by grasping the cecocolic ligament. Concurrently, the surgeon used atraumatic forceps through the left flank port to dissect and isolate the lymph node. For GCN, the designated 16 G or 18 G needle was introduced transabdominally and advanced into the node under direct visual monitoring. For GPF, a 4-mm laparoscopic pancreatic forceps was applied to the tissue; samples were harvested by clamping followed by gentle traction. For GCF, a 4-mm laparoscopic toothed cup forceps was used to grasp the tissue, and specimens were excised using a clamping and twisting motion.
Following sample acquisition, pneumoperitoneum was evacuated. The abdominal fascia and muscle layers were closed with interrupted cruciate sutures using 2–0 polyglactin 910. Subcutaneous tissue was apposed with horizontal mattress sutures using the same material, and the skin was closed with simple interrupted nylon sutures.
The execution time for each surgical step was recorded in minutes: portal placement, pneumoperitoneum establishment, secondary portal insertion, repositioning, node exposure, sample collection, site inspection, and port closure. Total procedural time was calculated as the sum of these phases. Sample collection time was recorded separately for each specific biopsy technique.
Surgical difficulty
Perceived procedural difficulty was evaluated independently by the primary surgeon and surgical assistant immediately after completing each procedure. Evaluated domains included instrument and port manipulation, operative field/ working space, lymph node exposure, mesenteric stabilization, technical execution of sample collection, and physical/ mental fatigue. Technical execution was scored separately for each biopsy technique, whereas the other domains were scored for the global procedure.
A study-specific 5-point Likert-type rating scale, prospectively adapted from previous laparoscopic literature (Tapia-Araya et al., 2015) was implemented. The scale was used to systematically quantify the evaluators’ perceived difficulty using ordered response categories. For the procedural-difficulty domains, scores ranged from 1 (no perceived difficulty), 2 (low perceived difficulty), 3 (moderate perceived difficulty), 4 (high perceived difficulty) to 5 (very high perceived difficulty). For the fatigue domains, scores ranged from 1 (no perceived fatigue) to 5 (very high perceived fatigue). Evaluators recorded their scores independently without prior domain-specific behavioral anchors, rendering the scores a measure of subjective perception rather than objective performance metrics. For descriptive and comparative analyses, the mean of the surgeon's and assistant's ratings was calculated for each cadaver.
Sample fixation, processing and histological evaluation
Immediately following collection, the three specimens obtained per technique were placed together in a coded plastic cassette and immersed in 10% neutral buffered formalin Fig. 2. To prevent loss of core needle samples through cassette mesh openings, needle specimens were placed between histological biopsy sponges prior to cassette insertion. Each cadaver yielded nine specimens (153 total). The three specimens per technique were evaluated jointly as a single pooled sample set, yielding 51 technique-level assessments.
Fig. 2.
Specimen processing prior to formalin fixation: core needle samples (18 G) placed on biopsy sponges (01), laparoscopic pancreatic forceps samples (02), and laparoscopic toothed cup forceps samples (03).
Tissues were processed and stained with hematoxylin and eosin (H&E) according to standard histopathological protocols (Nunes & Cinsa, 2016). Assessments were performed by a veterinary pathologist blinded to the sampling technique. Cassette codes were unblinded only after all histological scoring was completed.
Sample adequacy was scored using light microscopy based on five semiquantitative criteria adapted from Park et al. (2017): fragmentation, crush artifact, adipose tissue content, structural preservation, and interpretation-limiting histopathological changes. Lesions such as necrosis, fibrosis, and hemorrhage were categorized as underlying changes rather than sampling artifacts and were scored according to the proportion of parenchymal replacement. Detailed scoring criteria are outlined in Table 1.
Table 1.
Semiquantitative scoring system for histological adequacy of cecocolic lymph node biopsy samples in dogs.
| Criterion | Score | Operational definition |
|---|---|---|
| Fragmentation | 0 | Severe fragmentation affecting >50% of the tissue section |
| 1 | Moderate fragmentation affecting >25% to ≤50% of the tissue section | |
| 2 | Mild fragmentation affecting >0% to ≤25% of the tissue section | |
| 3 | No fragmentation (0%) | |
| Crush artifact | 0 | Severe crush artifact affecting >50% of the tissue section |
| 1 | Moderate crush artifact affecting >25% to ≤50% of the tissue section | |
| 2 | Mild crush artifact affecting >0% to ≤25% of the tissue section | |
| 3 | No crush artifact (0%) | |
| Adipose tissue content | 0 | Adipose tissue occupying >50% of the tissue section |
| 1 | Adipose tissue occupying >25% to ≤50% of the tissue section | |
| 2 | Adipose tissue occupying >0% to ≤25% of the tissue section | |
| 3 | No adipose tissue identified (0%) | |
| Structural preservation | 0 | Absence of both lymphoid follicles and corticomedullary differentiation |
| 1 | Presence of either lymphoid follicles or corticomedullary differentiation | |
| 2 | Presence of both lymphoid follicles and corticomedullary differentiation | |
| Histopathological changes limiting interpretation | 0 | Extensive necrosis, hemorrhage, and/or fibrosis affecting >50% of the tissue section |
| 1 | Moderate necrosis, hemorrhage, and/or fibrosis affecting >25% to ≤50% of the tissue section | |
| 2 | Mild necrosis, hemorrhage, and/or fibrosis affecting >0% to ≤25% of the tissue section | |
| 3 | No necrosis, hemorrhage, or fibrosis identified (0%) |
Note: Percentages were estimated visually across the combined tissue section area of the three pooled specimens per technique. For histopathological changes, the percentage represented the combined area occupied by necrosis, hemorrhage, and/or fibrosis that replaced or obscured diagnostically interpretable lymphoid parenchyma. Overlapping alterations within the same area were counted only once. The composite score ranges from 0 to 14 points.
Structural preservation was scored from 0 to 2 points based on the presence of lymphoid follicles and corticomedullary distinction. The remaining four criteria were scored from 0 to 3 points each, resulting in a composite histological adequacy score ranging from 0 to 14 points. Higher scores indicated greater tissue integrity, architectural preservation, and availability of diagnostically interpretable lymphoid parenchyma. A total score of 0 represented the lowest possible adequacy across all five criteria, whereas a score of 14 represented the highest possible adequacy, characterized by the absence of fragmentation, crush artifact, adipose tissue, and interpretation-limiting histopathological changes, together with preservation of both lymphoid follicles and corticomedullary differentiation.
Statistical analysis
Data were analysed using Python 3.8 (Pandas, SciPy, statsmodels) and figures generated using Matplotlib. Continuous variables are reported as mean ± standard deviation, median (interquartile range, IQR), and range. Observer-reported perceived-difficulty scores and composite histological sample-adequacy scores were summarized using the same descriptive measures, whereas individual histological criterion scores were summarized as median and interquartile range.
Because GPF, GCF, and GCN were evaluated in the same cadavers, collection time, perceived difficulty, and composite adequacy scores were compared using the Friedman test, with cadaver treated as the blocking factor. Post-hoc pairwise comparisons were conducted using two-sided Wilcoxon signed-rank tests with Holm adjustment for multiple comparisons. Total procedural time was reported descriptively.
Exploratory analysis of individual histological criteria among GPF, GCF, and GCN was conducted using the Friedman test, applying Holm adjustment across the five criterion-level omnibus p-values. Significant criteria underwent pairwise Wilcoxon signed-rank tests with Holm correction.
Comparisons between 16 G and 18 G core needles were treated as exploratory and evaluated using two-sided Mann-Whitney U tests for independent groups.
Inter-observer reliability between the surgeon and assistant for difficulty scores was evaluated using linearly weighted Cohen's kappa (k) with 95% confidence intervals, interpreted according to Landis and Koch (1977) as poor (κ <0.00), slight (κ from 0.00 to 0.20), fair (κ from 0.21 to 0.40), moderate (κ from 0.41 to 0.60), substantial (κ from 0.61 to 0.80), and almost perfect (κ from 0.81 to 1.00). Significance was set at p < 0.05.
Results
The demographic characteristics of the 17 canine cadavers included in this study are summarized in Table 2. The sample included both males (n = 10, 58.8%) and females (n = 7, 41.2%). Age was available in hospital records for nine cadavers and ranged from 1 to 20 years (mean 8.33 ± 6.87 years; median 8.0 years, IQR 2.0–10.0). Body weight ranged from 4.7 to 23.3 kg (mean 9.42 ± 4.82 kg), and body condition scores ranged from 4/9 to 6/9 (mean 4.88 ± 0.78) (WSAVA, 2011).
Table 2.
Mean, median and range of distribution of 17 dog cadavers included in the study.
| Dog cadavers characteristic | Available data, n | Mean ± SD | Median (IQR) | Range/distribution |
|---|---|---|---|---|
| Age (years) | 9 | 8.33 ± 6.87 | 8.0 (2.0–10.0) | 1–20 |
| Body weight (kg) | 17 | 9.42 ± 4.82 | 8.0 (6.0–12.0) | 4.7–23.3 |
| Body condition score | 17 | 4.88 ± 0.78 | 5 (4–5) | 4/9: n = 6 5/9: n = 7 6/9: n = 4 |
| Sex | 17 | — | — | Male: n = 10 (58.8%); female: n = 7 (41.2%) |
Abbreviations: SD, standard deviation; IQR, interquartile range.
Mean total procedural execution time was 37.06 ± 6.85 min. Mean sample collection times were 2.41 ± 1.42 min for GPF, 3.18 ± 1.63 min for GCF, and 6.18 ± 2.60 min for GCN. In the exploratory gauge-specific description, mean collection times were 6.67 ± 3.39 min for GCN 16 G (n = 6) and 5.91 ± 2.21 min for GCN 18 G (n = 11).
Sample collection time differed significantly among the three primary biopsy techniques (Friedman test, p < 0.001). GCN required significantly longer collection times than GPF and GCF (Holm-adjusted p = 0.002 for both comparisons), whereas GPF and GCF did not differ significantly from each other (adjusted p = 0.144). In exploratory gauge comparison, collection time did not differ between 16 G and 18 G (p = 0.839).
Perceived difficulty in the technical execution of sample collection was lowest for GPF (median 1.0; IQR, 1.0–1.5; range, 1.0–2.5), followed by GCF (median 1.5; IQR, 1.5–2.0; range, 1.0–3.0), and highest for GCN (median 4.0; IQR, 3.5–4.0; range, 2.5–4.5) (Fig. 3). For needle gauges, median scores were 3.75 (IQR, 3.5–4.0; range, 2.5–4.5) for 16 G GCN (n = 6) and 4.0 (IQR, 4.0–4.0; range, 3.0–4.5) for 18 G GCN (n = 11).
Fig. 3.
- Observer-reported perceived-difficulty scores for the technical execution of cecocolic lymph node sampling using laparoscopic pancreatic forceps (GPF), laparoscopic toothed cup forceps (GCF), and laparoscopically guided core needle biopsy (GCN). Horizontal lines represent medians, boxes represent interquartile ranges, and points display individual cadaver values. Scores range from 1 (no difficulty) to 5 (very high difficulty).
Technical execution difficulty differed significantly among techniques (Friedman test, p < 0.001). All pairwise comparisons were statistically significant: GPF versus GCF (Holm-adjusted p = 0.004), GPF versus GCN (adjusted p < 0.001), and GCF versus GCN (adjusted p < 0.001). Technical execution difficulty did not differ between 16 G and 18 G needles (p = 0.429).
Difficulty in overall procedural domains was evaluated for the laparoscopic procedure as a whole. Median perceived-difficulty scores were 2.0 for lymph node localization, 2.0 for mesenteric stabilization, 1.5 for lymph node access, 1.5 for working space, and 1.5 for instrument and portal handling. Median physical and mental fatigue scores were both 1.0. Complete descriptive and comparative results are detailed in Table 3.
Table 3.
Procedural time and observer-reported perceived-difficulty scores during laparoscopic cecocolic lymph node biopsy in canine cadavers.
| Outcome | Technique or domain | n | Mean ± SD | Median (IQR) | Range | p-value |
|---|---|---|---|---|---|---|
| Procedural time (min) | Total procedure | 17 | 37.06 ± 6.85 | 37 (32 to 40) | 26 to 56 | - |
|
Sample collection time (min) |
GPF | 17 | 2.41 ± 1.42 | 2 (1 to 3) | 1 to 6 |
<0.001ᵃ |
| GCF | 17 | 3.18 ± 1.63 | 3 (2 to 4) | 1 to 7 | ||
| GCN | 17 | 6.18 ± 2.60 | 6 (4 to 8) | 2 to 12 | ||
|
Gauge-specific collection time (min) |
GCN 16G | 6 | 6.67 ± 3.39 | 6 (4.25 to 8.50) | 3 to 12 |
0.839ᵇ |
| GCN 18G | 11 | 5.91 ± 2.21 | 6 (4.50 to 7.50) | 2 to 9 | ||
|
Technical execution difficulty |
GPF | 17 | 1.26 ± 0.44 | 1.0 (1.0 to 1.5) | 1.0 to 2.5 |
<0.001ᵃ |
| GCF | 17 | 1.68 ± 0.64 | 1.5 (1.5 to 2.0) | 1.0 to 3.0 | ||
| GCN | 17 | 3.82 ± 0.50 | 4.0 (3.5 to 4.0) | 2.5 to 4.5 | ||
| Gauge-specific technical execution difficulty | GCN 16G | 6 | 3.67 ± 0.68 | 3.75 (3.5 to 4.0) | 2.5 to 4.5 |
0.429ᵇ |
| GCN 18G | 11 | 3.91 ± 0.38 | 4.0 (4.0 to 4.0) | 3.0 to 4.5 | ||
| Overall procedural domains | Lymph node localization | 17 | 2.26 ± 1.03 | 2.0 (1.5 to 2.5) | 1.0 to 4.5 | — |
| Mesenteric stabilization | 17 | 1.85 ± 0.63 | 2.0 (1.5 to 2.0) | 1.0 to 3.0 | — | |
| Lymph node access | 17 | 1.82 ± 0.81 | 1.5 (1.0 to 2.5) | 1.0 to 3.5 | — | |
| Working space | 17 | 1.47 ± 0.65 | 1.5 (1.0 to 1.5) | 1.0 to 3.5 | — | |
| Instrument and port handling | 17 | 1.62 ± 0.70 | 1.5 (1.5 to 1.5) | 1.0 to 4.0 | — | |
| Physical fatigue | 17 | 1.38 ± 0.45 | 1.0 (1.0–2.0) | 1.0 to 2.0 | — | |
| Mental fatigue | 17 | 1.32 ± 0.39 | 1.0 (1.0–1.5) | 1.0 to 2.0 | — |
Note: Values are presented as mean ± SD and median (IQR). Perceived difficulty values represent the mean rating of the primary surgeon and surgical assistant per cadaver (1 = no difficulty; 5 = very high difficulty). GPF, laparoscopic pancreatic forceps; GCF, laparoscopic toothed cup forceps; GCN, laparoscopically guided core needle biopsy. ᵃ Friedman test, followed by Wilcoxon signed-rank tests with Holm adjustment. ᵇ Mann-Whitney U test. —, not applicable. Difficulty scale: 1, no perceived difficulty; 5, very high perceived difficulty.
Inter-rater agreement between the primary surgeon and surgical assistant varied across evaluated domains, with linearly weighted Cohen’ κ values ranging from −0.05 to 0.64. The agreement estimates and corresponding 95% confidence intervals are presented in Table 4.
Table 4.
Inter-rater agreement between the surgeon and surgical assistant for observer-reported perceived-difficulty domains.
| Domain | Linearly weighted Cohen’s κ (95% CI) | Interpretation of point estimate |
|---|---|---|
| Instrument and port handling | 0.28 (95% CI, −0.27 to 0.84) | Fair |
| Working space | 0.46 (95% CI, 0.13 to 0.80) | Moderate |
| Lymph node access | 0.49 (95% CI, 0.24 to 0.74) | Moderate |
| Lymph node localization | 0.41 (95% CI, 0.16 to 0.66) | Moderate |
| Mesenteric stabilization | 0.29 (95% CI, −0.05 to 0.64) | Fair |
| Technical execution GPF | 0.11 (95% CI, −0.25 to 0.46) | Slight |
| Technical execution GCF | 0.15 (95% CI, −0.23 to 0.54) | Slight |
| Technical execution GCN | −0.05 (95% CI, −0.29 to 0.19) | Poor |
| Physical fatigue | 0.64 (95% CI, 0.29 to 0.98) | Substantial |
| Mental fatigue | 0.13 (95% CI, −0.31 to 0.57) | Slight |
Note: Qualitative interpretations follow Landis and Koch (1977) benchmark criteria based on point estimates.
Sample sets obtained using laparoscopic forceps had higher composite histological sample-adequacy scores than those obtained using core needles (Fig. 4). Mean scores were 10.47 ± 2.53 for GCF, 10.00 ± 2.26 for GPF, and 5.53 ± 5.14 for GCN on a scale ranging from 0 to 14 (Fig. 5). In the exploratory gauge-specific description, mean scores were 6.50 ± 5.32 for GCN 16 G (n = 6) and 5.00 ± 5.22 for GCN 18 G (n = 11).
Fig. 4.
- Photomicrographs of H&E-stained cecocolic lymph node biopsy specimens. (A) Specimen obtained via laparoscopic toothed cup forceps (GCF), demonstrating preserved tissue architecture and intact lymphoid follicles. (B) Specimen obtained via core needle biopsy (GCN), displaying marked tissue fragmentation and architectural disruption. (C) and (D) Specific artifacts: intra-sample adipose tissue deposition ($*$) and crushing artifacts (arrows). Hematoxylin and eosin stain; $10\times$ objective lens; eyepiece field number, FN 20.
Fig. 5.
Composite histological sample-adequacy scores (scale 0–14) for cecocolic lymph node samples collected using laparoscopic pancreatic forceps (GPF), laparoscopic toothed cup forceps (GCF), and core needle biopsy (GCN). Horizontal lines represent medians, boxes represent interquartile ranges, and points display individual cadaver values.
The frequency distributions of categorical histological findings are presented in Table 5.
Table 5.
Distribution of selected histological features among technique-level sample sets (n = 17 per technique).
| Findings | Laparoscopic Pancreatic forceps (GPF) | Laparoscopic cup forceps (GCF) | Core needle (GCN) | Total |
|---|---|---|---|---|
| No fragmentation | 9/17 | 13/17 | 5/17 | 27/51 |
| No crush artifact | 3/17 | 0/17 | 4/17 | 7/51 |
| Lymphoid follicles or corticomedullary differentiation | 3/17 | 2/17 | 1/17 | 6/51 |
| Lymphoid follicles and corticomedullary differentiation | 7/17 | 10/17 | 0/17 | 17/51 |
| Histopathological changes limiting interpretation* | 0/17 | 1/17 | 7/17 | 8/51 |
| Adipose tissue | 8/17 | 7/17 | 11/17 | 26/51 |
Includes extensive necrosis, fibrosis, or hemorrhage replacing lymphoid parenchyma. Values are presented as n/N.
Exploratory criterion-level analyses revealed significant differences among techniques for tissue fragmentation (Holm-adjusted omnibus p = 0.039), structural preservation (adjusted p < 0.001), and interpretation-limiting histopathological changes (adjusted p = 0.019). Fragmentation scores were significantly lower (more fragmented) for GCN compared to GPF (Holm-adjusted pairwise p = 0.031) and GCF (p = 0.011), with no difference between GPF and GCF (p = 0.357). Structural preservation scores were also lower for GCN than for GPF (p = 0.008) and GCF (p = 0.004), with no difference between the two forceps techniques (p = 0.366). Interpretation-limiting histopathological changes were more severe/frequent in GCN samples compared to both GPF and GC (p = 0.024 for both comparisons), while GPF and GCF did not differ (p = 0.317). No technique-related differences were detected for crush artifact severity (adjusted omnibus p = 0.423) or adipose tissue content (adjusted omnibus p = 0.064). Complete criterion-level descriptive and comparative results are presented in Table 6.
Table 6.
Median and interquartile interval of individual histological sample-adequacy criterion scores according to biopsy technique.
| Criterion | GPF | GCF | GCN | Friedman p | Holm- adjusted p |
|---|---|---|---|---|---|
| Fragmentation | 3.0 (2.0–3.0) | 3.0 (3.0–3.0) | 1.0 (0.0–3.0) | 0.013 | 0.039 |
| Crush artifact | 1.0 (0.0–2.0) | 1.0 (1.0–2.0) | 0.0 (0.0–2.0) | 0.423 | 0.423 |
| Adipose tissue content | 3.0 (2.0–3.0) | 3.0 (2.0–3.0) | 2.0 (0.0–3.0) | 0.032 | 0.064 |
| Structural preservation | 1.0 (0.0–2.0) | 2.0 (0.0–2.0) | 0.0 (0.0–0.0) | <0.001 | <0.001 |
| Histopathological changes limiting interpretation* | 3.0 (3.0–3.0) | 3.0 (3.0–3.0) | 3.0 (0.0–3.0) | 0.005 | 0.019 |
Note: Fragmentation, crush artifact, adipose content, and interpretation-limiting changes scored from 0 to 3; structural preservation scored from 0 to 2. Higher scores indicate superior sample quality. Omnibus comparisons performed via Friedman test with Holm adjustment across the 5 criteria. Significant omnibus tests were followed by pairwise Wilcoxon signed-rank tests with Holm correction within each criterion. GPF, laparoscopic pancreatic forceps; GCF, laparoscopic toothed cup forceps; GCN, laparoscopically guided core needle biopsy. *Includes necrosis, fibrosis, and hemorrhage.
Composite histological sample-adequacy scores differed significantly among the three primary biopsy technique (Friedman test, p = 0.014). Post-hoc pairwise comparisons revealed significantly lower composite scores for GCN compared to GPF (Holm-adjusted p = 0.019) and GCF (adjusted p = 0.006), whereas GPF and GCF did not differ significantly from each other (adjusted p = 0.504). In the exploratory gauge-specific comparison, composite adequacy scores did not differ between 16 G and 18 G core needles (p = 0.566).
Discussion
The experimental design of the present study incorporated direct laparoscopic visualization to standardize target identification and instrument positioning across two distinct core needle gauges and two distinct laparoscopic forceps. Although percutaneous ultrasound-guided biopsy is widely utilized in clinical practice, its feasibility relies heavily on adequate acoustic windows, clear identification of the target lymph node, and a safe needle trajectory that avoids adjacent vascular structures (Mattoon et al., 2021; Nyman & O’Brien, 2007). Mattoon et al. (2021) recommend ultrasound-guided biopsy of sublumbar lymph nodes primarily when nodes are sufficiently enlarged to permit accurate identification and safe needle advancement. Because the cecocolic lymph nodes evaluated in the present study were not grossly enlarged, direct laparoscopic visualization provided a reliable method for identifying the target tissue and standardizing instrument placement. Consequently, the present findings should not be directly extrapolated to percutaneous ultrasound-guided core needle procedures.
Sample collection time differed significantly among the three biopsy techniques. Core needle biopsies required longer collection times than either laparoscopic forceps technique, whereas execution times did not differ between the two forceps types or between the two core needles gauges. Because access creation, pneumoperitoneum maintenance, visualization, and port closure were standardized across procedures, these temporal differences were restricted to tissue sampling itself and likely reflect instrument-specific handling demands. Surgical time in laparoscopic procedures is inherently operator-dependent and influenced by surgeon experience, technical proficiency, and progression along the learning curve (Runge et al., 2014).
In the current study, cadavers were initially positioned in dorsal recumbency before being transitioned to left lateral recumbency to elevate the target cecocolic lymph node uppermost. A similar positioning strategy was previously described for laparoscopic retrieval of medial iliac lymph nodes in dogs (Gibson, Fransson & Dupre, 2022).
To enable standardized comparison across techniques, perceived technical difficulty was evaluated using a 5-point Likert-type rating scale prospectively adapted from established veterinary laparoscopic literature (Tapia-Araya et al., 2015). Likert-type scales provide a structured means of translating subjective perceptions into ordered response categories, allowing the perceived intensity of a construct to be compared systematically (Likert, 1932). Accordingly, the scores in the present study represent standardized ratings of perceived technical difficulty rather than objective measurements of technical performance. Observer-reported difficulty differed among techniques: pancreatic forceps received the lowest scores, followed by cup forceps, while core needles were rated as the most technically demanding. The exploratory gauge-specific comparison revealed no difference between 16 G and 18 G core needles. Although the study-specific adaptation of the Likert-type scale was not formally validated and inter-rater agreement varied across domains, its prospective application provided a standardized framework for recording perceived difficulty. Therefore, these findings should be interpreted as subjective ratings of perceived difficulty rather than objective measures of technical performance.
Loss of depth perception, altered tactile feedback, and the fulcrum effect are well-established ergonomic challenges in laparoscopic surgery (Dejescu et al., 2023). Because these constraints were inherent to all procedures in the present study, they do not account for the differences observed among instruments. Given that all procedures were performed through the same access and surgical team, the higher difficulty scores for core needles likely reflected instrument-specific demands, particularly the requirement to align the needle, stabilize the lymph node within the surrounding mesentery, and control needle advancement under direct visualization. Technical challenges related to tissue stabilization and accurate instrument alignment during laparoscopic manipulation of mesenteric lymphatic structures have been reported previously (Brisson et al., 2006). Instrument design, caliber, and activation mechanisms are known to influence core needle handling (Murgia, 2014). Conversely, laparoscopic forceps allowed direct grasping of the target tissue prior to separation, simplifying tissue capture.
Regarding specific difficulty domains, lymph node localization and mesenteric stabilization yielded the highest median difficulty scores for the overall procedure. This was particularly evident in cadavers with substantial abdominal fat, where instrument slippage occurred during cecocolic ligament suspension, prolonging the time required to dissect and isolate the target nodes. Previous imaging studies have shown that overall body fat and the distribution of visceral versus subcutaneous abdominal fat are not equivalent measurements in dogs (Turner et al., 2020), which aligns with our observations. Furthermore, excessive adipose tissue within the falciform ligament and ventral abdominal cavity can severely compromise the surgical field and working space during laparoscopy (Van Goethem and Katic, 2022), a constraint that similarly affected node visualization in the present study.
In contrast, physical and mental fatigue scores were consistently low. Operator perception of fatigue and difficulty is inherently subjective and heavily influenced by surgical experience and training. For instance, veterinary surgery residents trained on minimally invasive simulators reported greater comfort during basic laparoscopic tasks (Balsa et al., 2020). Similarly, simulation training has been shown to improve movement efficiency and procedural time in veterinarians without prior laparoscopic experience (Hincapié-Gutiérrez et al., 2023). These factors highlight how operator familiarity and training modalities can modulate perceived procedural difficulty.
Histopathological evaluation demonstrated significantly higher composite sample-adequacy scores for specimens obtained using the laparoscopic forceps compared to those collected via core needle biopsy. Although cup forceps achieved the highest numerical score, performance did not differ significantly from pancreatic forceps. Buote et al. (2022) demonstrated that the choice of laparoscopic cup forceps technique significantly influenced specimen size and artifact formation during canine liver biopsies, with rotational/ twisting technique producing larger samples and fewer tearing artifacts than other laparoscopic forceps, as observed in this study. Although that study evaluated hepatic tissue rather than lymphoid tissue, its findings underscore the critical impact of tissue-separation mechanics on specimen integrity.
The lower adequacy scores observed for core needle samples align with previously reported limitations of core biopsy techniques, including smaller total tissue volume, potential underrepresentation of the target lesion, and increased susceptibility of small specimens to handling and fixation artifacts (Murgia, 2014). Although our exploratory comparison showed no difference between 16 G and 18 G calibers, previous research in canine renal biopsies demonstrated that 18 G core needles caused more frequent crushing and fragmentation than 14 G needles (Rawlings et al., 2003). However, the wider gauge disparity in that study, differences in target organ architecture, and the small, non-randomized subgroup comparisons in our study preclude direct comparison.
Tearing and fragmentation can also occur with laparoscopic forceps, as sample adequacy depends not only on the physical instrument but also on tissue acquisition mechanisms and post-collection handling. Nakashima et al. (2025) reported improved histological adequacy in small intestinal endoscopic biopsies mounted on filter paper prior to fixation compared to specimens placed freely in formalin. In the present study, histological sponges were utilized exclusively for core needle samples to prevent tissue loss during processing. Consequently, it cannot be determined whether the use of sponges independently influenced tissue preservation or adequacy scores for the core needle group.
Detailed microscopic evaluation provided insight into specific qualitative features dictating diagnostic utility. While crush artifact frequencies did not differ among techniques, both forceps demonstrated superior structural preservation (preservation of lymphoid follicles and corticomedullary architecture) and lower fragmentation rates compared to core needles. Intact nodal architecture is essential for definitive histopathological diagnosis, particularly in lymphoproliferative disorders where architectural pattern recognition is paramount (Seelig et al., 2016).
Our findings mirror those of Park et al. (2017) in a laparoscopic renal biopsy model in Beagles, where 18 G core needles yielded significantly higher rates of fragmentation and loss of tissue architecture than laparoscopic forceps. These parallel findings indicate that the primary limitation of laparoscopic core needle biopsy stems from structural fragmentation during acquisition rather than simple mechanical compression or crushing.
Regarding histopathological changes that limit diagnostic interpretation (such as necrosis, fibrosis, and hemorrhage), core needle samples exhibited a significantly higher frequency of interpretation-limiting histopathological changes. In human diagnostic oncology, core needle lymph node biopsies frequently yield inconclusive diagnoses due to crush artifacts, tissue distortion, fragmentation, and specimen shrinkage (Singh et al., 2025). Furthermore, Singh et al. (2025) observed higher rates of non-diagnostic samples with core needles compared to excisional biopsy due to high proportions of surrounding perinodal adipose tissue. In the present study, however, adipose tissue content did not differ significantly among biopsy techniques after adjustment for multiple comparisons. Instead, the lower histological adequacy observed in the core needle group was primarily associated with greater fragmentation, reduced structural preservation, and more interpretation-limiting histopathological changes. It should also be noted that samples in the present study were acquired under direct laparoscopic visualization, whereas core needle biopsies in Singh et al. (2025) were performed percutaneously in a clinical patient population.
This study has several limitations. The use of a cadaveric model precluded the evaluation of tissue perfusion, active hemorrhage, intestinal peristalsis, pain, healing, and postoperative morbidity. Consequently, clinical safety and diagnostic accuracy in diseased tissue could not be assessed. Because the evaluated lymph nodes were not grossly enlarged or diseased, findings reflect technical sample adequacy rather than disease-specific diagnostic sensitivity. The modest sample size of 17 cadavers may also have limited the ability to detect smaller differences, particularly in exploratory subgroup analyses. Additionally, although biopsy order was randomized and samples were taken from distinct regions or adjacent nodes, sequential sampling effects on tissue integrity cannot be entirely ruled out. Finally, all procedures were performed by a single operator, and subjective difficulty domain assessments exhibited variable inter-rater reliability. Although the Likert-type scale provided a standardized framework for recording perceived difficulty, the study-specific adaptation was not formally validated. Further studies could evaluate and validate procedure-specific rating instruments with predefined domain-specific anchors and assess their reproducibility across different surgeons and surgical teams.
In summary, within the context of this cadaveric model, laparoscopic forceps required shorter sample collection times, were perceived as technically easier to execute, and yielded superior composite histological adequacy scores compared to core needle biopsy. These findings support the use of laparoscopic forceps for obtaining histologically adequate samples from non-grossly enlarged cecocolic lymph nodes in this cadaveric model. However, prospective clinical studies in living patients with lymphadenopathy are necessary to confirm procedural safety, diagnostic accuracy, and clinical utility. Such studies should also investigate the potential influence of patient-related and procedural variables, including body weight, comorbidities, lymph node disease characteristics, and differences among surgeons and surgical teams.
Conclusion
Laparoscopically guided pancreatic and cup forceps required shorter sample collection times, were perceived as less technically difficult, and yielded significantly higher composite histological sample-adequacy scores than core needle biopsy. Although cup forceps achieved the highest numerical adequacy score, performance did not differ significantly from pancreatic forceps. Both laparoscopic forceps techniques yielded higher histological sample-adequacy scores than core needle biopsy in this canine cadaver model.
Informed Consent Statement
Informed consent of cadaver donation was obtained from all dog owners. Consonant with the Brazilian Law (n° 11.794/2008, article 3°, section III) and the Normative n° 30/2016/CONCEA, cadaveric studies are not included as animal experimentation.
Ethics in Publishing Statement
I testify on behalf of all co-authors that our article submitted followed ethical principles in publishing.
Title: LAPAROSCOPIC FORCEPS YIELD HIGHER QUALITY BIOPSY SAMPLES THAN CORE NEEDLE IN CECOCOLIC LYMPH NODES OF CADAVERS OF DOGS.
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This research presents an accurate account of the work performed, all data presented are accurate and methodologies detailed enough to permit others to replicate the work.
This manuscript represents entirely original works and or if work and/or words of others have been used, that this has been appropriately cited or quoted and permission has been obtained where necessary.
This material has not been published in whole or in part elsewhere.
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That generative AI and AI-assisted technologies have not been utilized in the writing process or if used, disclosed in the manuscript the use of AI and AI-assisted technologies and a statement will appear in the published work.
That generative AI and AI-assisted technologies have not been used to create or alter images unless specifically used as part of the research design where such use must be described in a reproducible manner in the methods section.
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During the preparation of this work the author(s) used ChatGPT in order to create the graphics presented in the article. After using this tool, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.
Funding
This research was supported by “Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Estado do Paraná (FA)”, Paraná, Brazil (Announcement N° 89/GR/UFFS/2022).
CRediT authorship contribution statement
Jane Karlla de Oliveira Matos: Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation, Conceptualization. Vitor Ângelo Musial: Investigation, Data curation. Pauline Silva dos Santos: Investigation. Bernardo Nascimento Antunes: Methodology, Conceptualization. Leonardo Gruchowskei: Investigation. Fabiana Elias: Investigation. Camila Dalmolin: Formal analysis, Data curation. Maurício Veloso Brun: Resources, Methodology, Conceptualization. Fabíola Dalmolin: Writing – review & editing, Writing – original draft, Supervision, Project administration, Investigation, Funding acquisition, Conceptualization.
Declaration of competing interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Fabiola Dalmolin reports financial support was provided by Fundação de Amparo à Pesquisa do Estado do Paraná - Fundação Araucária. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
Victor Angelo Musial was granted a Scientific Initiation Scholarship of “Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Estado do Paraná (FA)”, Paraná, Brazil (Announcement N° 89/GR/UFFS/2022).
Contributor Information
Jane Karlla de Oliveira Matos, Email: jane.karlla@gmail.com.
Vitor Ângelo Musial, Email: vitor.musial94@gmail.com.
Pauline Silva dos Santos, Email: paulinesilvadossantos@gmail.com.
Bernardo Nascimento Antunes, Email: bernardonascimentoantunes@gmail.com.
Leonardo Gruchowskei, Email: leonardo.gruchouskei@ufsc.br.
Fabiana Elias, Email: fabiana.elias@uffs.edu.br.
Camila Dalmolin, Email: camidal@gmail.com.
Maurício Veloso Brun, Email: mauriciovelosobrun@hotmail.com.
Fabíola Dalmolin, Email: fabiola.dalmolin@uffs.edu.br.
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