Investigation of Keyes Skin Biopsy Instrument for Intestinal Biopsy Versus a Standard Biopsy Technique

Introduction

Intestinal biopsy is a commonly performed procedure in small animal practice. Intestinal biopsies can be obtained in a variety of ways. Biopsies may be obtained via percutaneous methods, peroral suction catheter, endoscopy, laparoscopy, and celiotomy.^1–5 The choice of biopsy method used depends on the type of disease suspected, location of the disease (both anatomical and histopathological), anesthetic and surgical risks, and clinician preference or experience.³⁶

Open (i.e., celiotomy) biopsy is often selected for numerous reasons. If the disease is thought to involve bowel beyond the proximal small intestine, endoscopic biopsy is not usually possible. If other abdominal organs (e.g., lymph nodes, liver, spleen, kidneys) require examination or biopsy, these procedures can also be accomplished at the time of celiotomy. In addition, if partial-thickness biopsy (i.e., endoscopic) has not yielded a diagnosis, full-thickness biopsy may be needed.⁶ Celiotomy also allows the placement of an enterostomy tube and the excision of discrete intestinal masses at the time of biopsy.

Indications for intestinal biopsy are diarrhea (usually chronic), vomiting, weight loss, protein-losing enteropathy, or the presence of an intestinal mass.²³⁶ Typical differential diagnoses for dogs undergoing surgical intestinal biopsy are inflammatory bowel disease (IBD), lymphosarcoma (LSA), lymphangiectasia, histiocytosis, phycomycosis, intestinal masses, and foreign bodies.⁷

Many different techniques for obtaining a surgical intestinal biopsy exist.² Most involve sharp excision of a small portion of antimesenteric bowel wall and subsequent closure of the defect. The purpose of this study was to investigate the use of a Keyes skin biopsy punch for obtaining intestinal biopsies and to compare those biopsies with tissue specimens obtained by scissor excision, an accepted standard technique. Variables assessed in making the comparison included biopsy collection time, diagnostic value of the specimens, histological quality of the specimens, and complication rates.

Materials and Methods

Twelve dogs requiring abdominal exploration for diseases of the small intestine were included in the study. Laboratory data obtained from all animals included a complete blood count, serum electrolytes, and biochemistries. A urinalysis was performed in five dogs. All dogs underwent abdominal imaging (e.g., plain radiography, ultrasonography, or both) prior to surgery. If significant proteinuria or hypoalbuminemia was present, a urine protein:creatinine ratio was also submitted to quantify renal protein loss.⁸⁹

For this study, paired jejunal biopsy samples were obtained via standard ventral midline celiotomy. Biopsies of the intestines were taken if there were no gross abnormalities found during abdominal exploration or if diffuse intestinal disease was present. Additional biopsies (e.g., lymph nodes, liver, etc.) were taken as indicated. Animals with focal intestinal masses were not included in the study.

During surgery, one biopsy sample was collected using a Keyes biopsy punch, and this method was given the designation “P” for punch biopsy. A segment of jejunum was isolated, and a new no. 6 Keyes biopsy punch^a was used to obtain a full-thickness sample from the antimesenteric surface of the jejunum. The defect was then closed in a transverse direction using 4-0 polydioxanone^b with a swaged-on taper needle in a simple interrupted appositional pattern. The time from initiation of the biopsy to closure was recorded in seconds.

A second, separate biopsy sample was collected by creating a small stab incision with a new no. 11 scalpel blade directed in a longitudinal direction along the antimesenteric surface. In the study, this method was given the designation “S” for standard biopsy. A full-thickness sample was excised from the cut edge using Metzenbaum scissors. The defect was closed in a transverse direction in the same manner as for the “P” technique. The time to completion of the biopsy was also recorded in seconds. The biopsy times were compared using a paired t-test, with significance set at P<0.05.

After collection, all biopsy specimens were atraumatically removed from the surgical field and placed directly into formalin. All the specimens were evaluated histologically at the end of the study by a single pathologist (Greenlee), blinded to the study’s protocols and hypotheses. In case no. 8, the samples lost their identifying labels, making them indistinguishable, so they were excluded from the analysis of the histopathological data.

A scoring system was used by the pathologist to grade the severity of the intestinal disease based on the tissue layers involved, the types of cellular infiltrates identified, and the presence of fibrosis, edema, lymphangiectasia, abscessation, or goblet cell hyperplasia. This scoring system used a scale of 1 to 5, with 1 indicating mild disease and 5 denoting severe disease. Results of the histopathological examinations are summarized in Table 1. A second scoring system was used to rate the overall quality of the tissue submitted. The quality was rated from 1 to 5, with 1 indicating a poor sample quality and 5 denoting a high sample quality. The scores for quality were considered to be substantially different if there was >1 gradation between the samples. Analysis of biopsy method on specimen quality was performed using a Fisher’s exact probability test, with significance set at P<0.05.

After surgery and after suture removal, each surgeon completed a questionnaire pertaining to the biopsy techniques. Any intra- or postoperative complications identified with either technique were noted. The likelihood of dogs developing major life-threatening complications (e.g., biopsy wound dehiscence) was evaluated using a Fisher’s exact probability test, with significance set at P<0.05.

Results

Twelve dogs met the inclusion criteria for the study [Table 2]. The average age of the dogs was 7.9 years (range, 4 months to 13 years). Seven dogs were spayed females, four were castrated males, and one was an intact male. Overall, there were nine breeds represented in the study, including beagles (n=2), mixed-breed dogs (n=2), the German shepherd dog, Portuguese water dog, curly-coated retriever, West Highland white terrier, Yorkshire terrier, miniature schnauzer, border collie, and Shetland sheepdog (n=1 each).

Most clinical complaints upon presentation were related to the gastrointestinal system. Four dogs were presented for vomiting alone. Vomiting and diarrhea were noted in six dogs. In three of these dogs, weight loss was also present. One dog was presented for only weight loss, and another dog was presented for only chronic small-bowel diarrhea.

In all cases, the same clinical diagnoses were made with both biopsy methods. Diagnoses included IBD (n=11) and LSA (n=1). In two dogs with IBD, lymphangiectasia was also noted. The IBD was further differentiated into lymphocytic plasmacytic enteritis (n=5); lymphocytic, plasmacytic, and eosinophilic enteritis (n=3); eosinophilic enteritis (n=1); lymphocytic and eosinophilic enteritis (n=1); and plasmacytic and eosinophilic enteritis (n=1) [Table 2].

In three sample pairs, individual tissue layers were scored differently [Table 1]. In case no. 3, significant infiltrates were noted in three tissue layers, and fibrosis was noted in the “S” sample; however, these findings were not reported in the “P” sample. In case nos. 2 and 9, infiltrates were noted in one tissue layer with the “P” specimen and were not seen with the “S” specimen. In case no. 2, edema was identified in the “P” sample and was not noted in the “S” specimen. The opposite was true for case no. 10. The “S” sample had no serosa present in the tissue sample from case no. 2.

The qualities of the jejunal samples were different in six cases. In four dogs (case nos. 2, 4, 7, 9), the “P” sample was of higher quality, and in case nos. 10 and 11, the opposite was true. Analysis of biopsy technique on specimen quality showed no significant difference between the two collection methods (P=0.32).

There were technical problems encountered in four dogs with the punch method. These occurred primarily when the surgeons had not previously used the punch for obtaining this device.¹² In the authors’ practice, intestinal biopsies. In three dogs (case nos. 2, 5, 10), the punch did not cut through the intestinal wall for 360° around the edge of the blade of the punch. The resulting tissue tag was then cut with Metzenbaum scissors in order to release the sample. In case no. 4, the punch crushed and tore the sides of the intestine as it cut. This event did not result in any noticeable complications, other than to make closure of the defect more difficult.

In two dogs, gross abnormalities of the intestines were noted by the surgeons; however, only one of the dogs developed a major postoperative complication. In case no. 3, generalized thickening and lymphangiectasia were noted, and in this dog dehiscence occurred at the site where the “S” method was used. Septic peritonitis was diagnosed 2 days after surgery based on abdominal distension and pain, hypotension, tachycardia, and a leukocytosis with a left shift. The diagnosis was confirmed during emergency celiotomy. The site of the leakage could not be closed effectively, and an intestinal resection and anastomosis were performed. Despite recovery from the second surgery, vomiting, diarrhea, and lethargy persisted, resulting in euthanasia 7 days after the second surgery. The diagnosis in this dog was IBD characterized as lymphocytic plasmacytic enteritis. Statistical analysis of the effects of biopsy method on the occurrence of complications (P=0.5), however, showed that the complication was not statistically related to the biopsy method.

Mild intestinal edema was noted in case no. 7, but no clinical problems were encountered in this case with either biopsy technique. In case no. 1, reinforcement techniques were used after closure of the enterotomy sites. The serosa was noted to be friable during closure and, therefore, the surgeon elected to reinforce both biopsy sites using an omental patch without subsequent complications.

The average time for biopsy collection in the “P” group was 278 seconds (range, 144 to 447 seconds). The average time for biopsy collection using the “S” technique was 333 seconds (range, 94 to 540 seconds). On average, the “P” method was 55 seconds faster; however, this was not statistically significant (P=0.18).

Discussion

The Keyes biopsy instrument was initially designed for obtaining full-thickness skin biopsies, but it has also been used for obtaining liver and muscle biopsies.¹⁰¹¹ Recently, cartilage biopsies were also obtained experimentally with intestinal biopsies are often obtained with this instrument, as well as with the “S” method. The study reported here was designed to evaluate whether one technique had an advantage over the other technique with regard to diagnostic value of samples, time for collection of samples, and post-biopsy complication rate.

A wide range of biopsy times occurred between surgeons. Statistically, however, the time for specimen collection was not different between the two techniques. For the most part, it was felt that the Keyes punch device was easy to use, with minimal preparatory learning required.

The crushing of the sides of the “P” biopsy site that occurred in case no. 4 was an isolated incident and appeared to be related to the manner in which the bowel was isolated and stabilized while the specimen was harvested. This was the first time that particular surgeon had used the Keyes punch for intestinal biopsy purposes. In retrospect, the surface of the bowel that is in contact with the instrument should be held as flat as possible so that the blade of the instrument is perpendicular to the tissue to be cut. The depth of the cut on the Keyes biopsy instrument is limited to the blade length (approximately 8 mm), but, theoretically, injury could occur to the opposite side of the bowel if too much force is applied to the punch or if the bowel wall is extremely thin. Additionally, in smaller dogs (and maybe all cases), a smaller sized punch (i.e., 4 mm instead of the 6-mm size) may be more appropriate. The 4-mm punch is routinely used by the authors to obtain intestinal biopsies in cats.

General complications reported with intestinal biopsies are dehiscence, strictures, and adhesions.^13–19 Only one dog in this study developed a major complication associated with intestinal biopsy. In case no. 3, septic peritonitis developed 2 days after surgery from leakage at the “S” biopsy site. This dog’s bowel was thickened and visibly abnormal at the time of biopsy, and the intestinal tissue was friable and thickened, with lymphangiectasia. At the time of biopsy, the surgeon tested both biopsy sites for leaks, and no leakage was detected. If leakage had been detected, serosal or omental patching could have been used to reinforce the site. In this study, only one (8.3%) of 12 dogs developed a dehiscence, which is below the previously reported level of 15.7% dehiscences for all types of small intestinal surgery.²⁰

The biopsy samples were dissimilar in overall quality in 54% (6/11) of the samples, but this difference was not statistically different. One of the “S” biopsy samples did not contain a serosal layer when evaluated. It was not known whether loss of serosa occurred from damage during collection, handling, or processing of the sample, or if it was related to the disease present. Samples in three cases had substantially different histological tissue scoring results. Despite these differences, all samples yielded the same diagnosis regardless of the method of collection. Because marked variation in interpretation of intestinal biopsy samples can occur between pathologists, only one pathologist examined all the samples retrieved in this study.²¹

Conclusion

Based upon the results of this study, intestinal biopsy with a Keyes biopsy punch was safe, rapid, and provided accurate diagnostic information. There were no statistical differences in sample collection times, histopathological grade and diagnosis, complication rates, or sample quality between use of a Keyes biopsy punch and intestinal biopsy using a standard scissors technique.