Hirschsprung disease is a disorder resulting from a congenital absence of ganglion cells in the bowel wall extending from the anus over a variable distance (1). It may present in the newborn as intestinal obstruction and accounts for almost 33% of neonatal intestinal obstructions (1). Although usually diagnosed in infants, Hirschsprung disease may rarely be diagnosed in the older child presenting with chronic, relentless constipation (2). In older children with chronic constipation unresponsive to treatment, it is necessary to distinguish between those with Hirschsprung disease and other related neuropathies and those with the more common “functional” constipation. A rectal biopsy demonstrating normal ganglion cells in the submucosal layer excludes Hirschsprung disease and related conditions such as neuronal intestinal dysplasia. Therefore, obtaining an adequate specimen is 1 of the requirements for a successful diagnosis of Hirschsprung disease. The gross specimen should ideally be 3.5 mm in diameter with equal amounts of mucosa and submucosa (3,4).
Most rectal biopsies are performed with a suction-and-cut instrument modified and popularized by Noblett (3) in the late 1960s. Although it is relatively easy to use, the disadvantage of this instrument is its inability to consistently obtain tissue of adequate depth and size. In a retrospective study of 258 children 1 day to 14 years of age undergoing rectal biopsies to exclude Hirschsprung disease, Alizai et al. (5) observed a trend for suction biopsies to be inadequate in children older than age 6 months compared with a “cup” biopsy. Similar to Alizai et al., it had been our clinical impression for several years that the suction biopsy often obtains inadequate samples in children over the age of 12 months. We have not found any prospective studies seeking to determine the age at which the suction biopsy can no longer be relied on to provide a specimen of adequate size. Therefore, the aim of this study was to determine at what age the suction rectal biopsy is less likely to provide an adequate tissue sample to detect submucosal ganglion cells.
PATIENTS AND METHODS
This was a prospective study of children ≥1 year old with chronic unrelenting constipation undergoing a rectal biopsy to exclude Hirschsprung disease at our institution from February 1998 through November 2003. Children undergoing rectal biopsies for other indications, such as to exclude graft-versus-host disease after stem cell transplant or to investigate rectal bleeding, were excluded, as were children with a known diagnosis of Hirschsprung disease. As part of the evaluation of these patients, proctosigmoidoscopy was performed to exclude any obstructing or mucosal lesions of the distal rectum that would account for the unrelenting nature of their symptoms. No enema or other bowel preparation was given before the procedure. For 92 patients (60%), a manual disimpaction was needed to clear the rectum of a large fecal mass. For many others, an examination under anesthesia was necessary because a thorough rectal examination was resisted in the clinic. Because of the need for these additional procedures, all of the biopsies were performed under general anesthesia. Endotracheal intubation was at the discretion of the anesthesiologist.
Each child had 1 biopsy obtained by the grasp technique and a second by the suction technique. A Kevorkian-Younge uterine biopsy forceps (V-Mueller Instrument Division of Baxter International, Deerfield, IL) with a cup size of 8.5 × 3.5 mm was used for the grasp biopsy. The forceps was introduced into the rectum with the cutting edge oriented sideways and advanced 3 cm from the dentate line; it was then rotated 90° to align the cutting edge perpendicular to the lateral wall of the rectum. The jaws of the forceps were then opened to grasp the mucosa. A gentle tug obtained the biopsy. The model SBT-100 rectal suction biopsy tool (Medical Measurements, Inc, Hackensack, NJ) was used for the suction biopsy (Fig. 1). The biopsy knife was sharpened after ≈5 uses and changed if sharpness could no longer be maintained. To obtain the biopsy, a standard suction pressure of 20 to 25 inches of mercury was applied with a Luer-Loc syringe assembly. The suction was applied with rapid withdrawal of the plunger, and the knife was pulled when the pressure began to fall from 25 inches of mercury. There were no restrictions on the number of passes necessary to obtain a biopsy, but rarely was more than 1 pass needed. All of the biopsies were taken at 3 cm above the dentate line to avoid the physiological hypoganglionic region. The biopsies were performed by 6 of the authors, all pediatric gastroenterologists, with the patient in the left lateral decubitus position. All of the biopsies were obtained from the left lateral wall of the rectum. Hemostasis was maintained by applying pressure to the biopsy site with gauze. The 2 specimens were immediately oriented on pieces of paper towel and placed in separate, appropriately labeled formalin containers. Paraffin-embedded biopsies were sectioned, stained with hematoxylin and eosin, and examined for adequacy of submucosa and for the presence or absence of ganglion cells in the submucosa. Adequacy of the submucosa was graded on a scale of 0 to 3, with 0 = absence of submucosa, 1 = scant submucosa (submucosa less than one third the mucosa), 2 = adequate submucosa (submucosa more than one third and up to same as mucosa), and 3 = ample submucosa (submucosa more than mucosa). At least 50 but up to 340 serial sections of each biopsy were examined independently by 2 pathologists. The pathologists were not blinded to the type of biopsy specimen. Both signed off on the final report, which was used for analysis.
For ease of analysis, the subjects were grouped into 4 age categories: 1 to 3, 4 to 6, 7 to 9, and ≥10 years. Analysis was performed with Sigmastat Statistical Software version 2 (SPSS, Chicago, IL). The χ2 test was used to compare adequacy of biopsies and the yield in submucosal ganglion cell detection between the 2 biopsy techniques in the different age groups.
The study was approved by the Indiana University-Purdue University Indianapolis/Clarian Health Partners Institutional Review Board. Written informed consent was obtained from a parent or legal guardian of each participant before enrollment in the study.
One hundred fifty-two patients were recruited for the study; 99 were male. The mean age was 6.6 years; the median age was 6 years; and the age range was 1 to 17 years. There were 41 patients in the 1- to 3-year-old group, 37 in the 4- to 6-year-old group, 36 in the 7-to 9-year-old group, and 38 in the ≥10-year-old group.
Hirschsprung disease was diagnosed in a 5-year-old boy who had an adequate biopsy sample from both techniques but no identifiable ganglion cells and easily identifiable abnormal nerve trunks in the submucosa. Three patients with ≥1 abnormalities of ganglion cells and nerves, such as multiple giant ganglia, neuronal hypertrophy or ectopic ganglion cells, were diagnosed with intestinal neuronal dysplasia on grasp biopsies but not the corresponding suction biopsies. Two patients, a 9- and an 11-year-old, had inadequate submucosa and no ganglion cells detected on either specimen. These patients needed repeat rectal biopsies to exclude Hirschsprung disease. The repeat biopsies were done surgically and were normal.
One patient, on aspirin therapy for postsurgical management of congenital heart disease, experienced prolonged bleeding from the biopsy site, which was managed conservatively. There were no other complications. For age groups 1 to 3 years, 4 to 6 years, 7 to 9 years, and ≥10 years, the suction biopsy produced no submucosa in 12%, 30%, 29% and 55% and yielded submucosal ganglion cells in 73%, 51%, 53% and 50%, respectively. The grasp biopsy produced no submucosa in 2%, 5%, 3% and 8% and yielded submucosal ganglion cells in 98%, 97%, 92% and 92%, respectively (Table 1).
For all age groups combined, there was a statistically significant difference between the 2 techniques with respect to adequacy of submucosa and yield in ganglion cell detection (P < 0.001 and P < 0.001, respectively). In the 1- to 3-year-old group, however, there was no significant difference between the grasp biopsy and suction biopsy with regard to adequacy of the sample (P = 0.19), although more ganglion cells were detected from the grasp sample (P < 0.05). There was a statistically significant difference between the 1- to 3-year-old group and the 3 older age groups with respect to the adequacy of the specimen and the yield in submucosal ganglion cells on biopsies obtained with the suction device (P < 0.001 and P < 0.05, respectively). There was no significant difference within the 3 older age groups with respect to adequacy and yield in submucosal ganglion cells on biopsies obtained with the suction device (P = 0.305 and P = 0.997, respectively). There was no significant difference between all 4 age groups with respect to the adequacy of specimen and the yield in submucosal ganglion cells on biopsies obtained with the grasp forceps (P = 0.835 and P = 0.995, respectively).
For all age groups individually, there was a significant difference between the grasp biopsy and the suction biopsy with respect to the yield in submucosal ganglion cells (P < 0.05, P < 0.001, P < 0.001, and P < 0.001, respectively). Significant differences also were observed in the 3 older age groups in the ability to obtain some submucosa (P < 0.05, P < 0.05, and P < 0.001, respectively) (Table 1).
For the physicians performing the biopsies, the number of biopsies with insufficient submucosa to identify ganglion cells correlated with the total number of biopsies performed. Thus, the physicians performing the most biopsies had the most insufficient samples. There were no significant differences in the percentages of biopsies inadequate for ganglion cell identification between physicians (Table 2).
Constipation accounts for 3% of visits to a general pediatrician's office and 25% of visits to a pediatric gastroenterologist (6). Ninety-five percent of children with chronic constipation have functional constipation, and only ≈5% have an organic cause for their symptoms (7). If medical management fails to relieve symptoms, then a rectal biopsy, the gold standard for the diagnosis of Hirschsprung disease, is necessary to exclude this condition (8). An adequate specimen, one that includes submucosa, is necessary for reliable interpretation by a pathologist. In 1955, Swenson and colleagues (9) described a method of biopsy that included the muscle layers of the rectal wall. The aim of their technique was to obtain a specimen with an adequate sample of Auerbach plexus. The technique required an open procedure and was prone to serious complications, including bleeding, infection, and scarring (10). After the discovery that the ganglionic-aganglionic junction was at the same level for both the submucosal (Meissner) plexus and the intermyenteric (Auerbach) plexus in patients with Hirschsprung disease (11), it was suggested that a large superficial biopsy of mucosa and submucosa alone would be sufficient to diagnose Hirschsprung disease (10). This led to the development of instruments for both suction and grasp biopsies (5). The first grasp biopsy technique was introduced by Shandling (12) in 1961; the first suction biopsy technique was introduced by Dobbins and Bill (13) in 1965. In 1969, Noblett (3) introduced a suction biopsy tube with a side aperture through which rectal mucosa and submucosa could be suctioned and cut with a cylindrical knife. Most rectal biopsies are performed using the Noblett technique. Although Campbell and Noblett (14) did not obtain any inadequate biopsies in 116 biopsies obtained from 45 patients of all ages, many subsequent investigators reported inadequate biopsies with the suction device (5,15–17). None, however, reported the age at which this device is more likely to produce an inadequate sample.
Our aim in this study was to determine the age above which the suction device is less likely to provide adequate tissue to identify submucosal ganglion cells when present. We compared its performance with that of a grasp biopsy device. The 2 biopsies from each patient were obtained at the same time under general anesthesia. Thus, there were no confounding factors to favor 1 technique over the other. We found that the suction biopsy was less likely to produce a sample with some submucosa after the ages of 1 to 3 years, and submucosal ganglion cells were identified in only 50% of biopsies obtained with the suction device beyond this age group (Fig. 2). The grasp biopsy provided a sample with some submucosa and yielded submucosal ganglion cells in >90% of patients in all age groups. Thus, the single grasp biopsy excluded Hirschsprung disease in at least 92% of all patients, whereas the single suction biopsy, obtained at the same time, excluded Hirschsprung disease in 73% of patients 1 to 3 years old and only 50% of patients older than 3. Viewed another way, 50% of our patients older than 3 years undergoing a suction biopsy of the rectum would have had to have a repeat biopsy compared with at most 8% of patients in the same age group undergoing a grasp biopsy. We must mention, however, that no patient with Hirschsprung disease was missed by either technique in this group of patients. A possible explanation for the inadequacy of the suction biopsy with increasing age may be that as they got older, patients with chronic constipation acquired a megarectum, which made it difficult to approximate the suction chamber of the biopsy gun with the rectal mucosa.
There are a few limitations to this study. Because of restrictions imposed by our institutional review board, we were able to obtain only 1 biopsy each with the 2 devices. One biopsy is obviously not standard of care, and certainly, the probability of obtaining an adequate sample or identifying ganglion cells may increase with more biopsies. Thus, it is possible that we could have improved the yield from the suction biopsy if more biopsies had been done with this equipment. In addition, at the Fourth International Symposium on Hirschsprung Disease and Related Disorders held in Genoa, Italy, in April 2004, participants concluded that acetylcholinesterase staining is “the best diagnostic technique to demonstrate hypertrophic nerve trunks in lamina propria mucosae,” while acknowledging that many pathologists from different centers use hematoxylin-and-eosin staining effectively (18).
Although our pathologists do not use acetylcholinesterase staining routinely and did not in the patients described here, failure to perform this stain could potentially affect the ability to obtain the most information from a rectal suction biopsy, including identifying ganglion cells or diagnosing neuronal intestinal dysplasia in inexperienced hands. Finally, in her original paper, Noblett (3) cautioned against the use of bowel washouts and enemas before suction biopsy because they may cause mucosal edema and reduce the depth of submucosa obtained. Although none of our patients received any washouts or enemas, we do not know how manipulations such as manual disimpaction and proctosigmoidoscopy may have affected the suction biopsy. In most patients, however, we would not have been able to biopsy without disimpaction, and we cannot envision that proctosigmoidoscopy affected the biopsies in any way.
There is widespread experience with performing a suction rectal biopsy in the outpatient clinic but little experience with performing a grasp biopsy in the same setting. Although all of our biopsies were done under anesthesia, we have no reason to believe that a grasp biopsy with the Kervokian-Younge uterine biopsy forceps cannot be done safely in the outpatient clinic with or without mild sedation, depending on the level of cooperation of the patient.
In conclusion, we found the suction biopsy to be less likely to provide adequate tissue for identification of submucosal ganglion cells in patients older than age 3 years. Although the grasp biopsy was superior to the suction biopsy at all ages in providing an adequate sample size, for patients 3 years of age or younger, the suction biopsy was still reliable in producing an adequate sample even if it did not produce as many ganglion cells as the grasp biopsy.
The authors would like to thank Vicki Haviland-Wilhite for her expert secretarial assistance and Isaac Hammond, MD, PhD, and Steven Dadzie for their statistical advice.
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