Rectal Biopsy for Hirschsprung’s Disease 
Catherine Jackson-Cole, Marlene Ishimwe, Jason Axt 
 
OPEN MANUAL OF SURGERY IN RESOURCE-LIMITED SETTINGS 
www.vumc.org/global-surgical-atlas 
This work is licensed under a Creative Commons Attribution-ShareAlike 3.0 Unported License  
 
Introduction:  
 
Surgeons in resource-limited settings are 
often asked to evaluate children with chronic 
constipation for Hirschsprung’s Disease. The triad 
of abdominal distention, vomiting, and passage of 
first meconium stool later than 24 hours raise 
suspicion of Hirschsprung’s. Constipation can be 
caused by a multitude of dietary, environmental, 
and disease processes, and the clinician should 
maintain a broad differential. If the history is highly 
suggestive, or if the patient has remained refractory 
to medical treatment of constipation the patient 
should be evaluated for Hirschsprung’s Disease.  
 
A definitive diagnosis of is confirmed by 
pathological evaluation of full thickness rectal 
tissue taken at least 2 cm above the dentate line.  
This tissue will not have ganglion cells in the 
submucosal plexus.  In addition, nerve hypertrophy 
can be demonstrated in the submucosal plexus and 
myenteric 
plexus. 
Adjuncts 
such 
as 
acetylcholinesterase and calretinin stains can also be 
used for greater diagnostic certainty, but the cost of 
specialized stains may be prohibitive. 
Tissue can be obtained using a device for 
suction rectal biopsy, by endoscopic methods, or by 
an operative rectal biopsy. Often in resource-limited 
settings, the device for suction rectal biopsy is not 
available, and suction rectal biopsies may be of 
small size. In our experience, pathologists who 
evaluate for Hirschsprung’s infrequently may not 
have a high degree of certainty. Therefore, it has 
been our practice to confirm presumptive diagnoses 
of Hirschsprung’s with a full thickness rectal biopsy.   
 
Steps: 
1. The patient should not have active enterocolitis.  
If rectal washouts are being performed for 
presumed Hirschsprung’s, a washout should be 
performed shortly before operating.  
2. General anesthesia is induced, and the patient is 
placed in frog-leg lithotomy position at the end 
of the operating table.  
 
The proper position for rectal biopsy. Note that the child’s 
buttocks are right next to the edge of the bed, so the bed itself 
does not block the surgeon’s ability to view inside the anus 
from any angle necessary. Note also that due to the short 
duration of the procedure, general anesthesia is maintained 
with a mask held against the child’s face by an expert 
anesthetist rather than through an endotracheal tube.  
 
3. Prophylactic antibiotics are not necessary. 
4. The operation may be done using sterile towels 
and sterile gloves with a protecting apron 
(“Mackintosh”). Full gowning and draping are 
not necessary. 
5. The operating surgeon wears a surgical 
headlight.  
6. A digital rectal exam is performed, and any 
abnormalities or strictures noted. 
7. A lubricated nasal or anal speculum is 
introduced into the anus with the handle at the 
vertical position.  
8. A suture is placed into a small betadine-soaked 
gauze covered with lubricating gel and it is 
placed into the rectum using a forceps.  The 
suture is allowed to hang out of the anus and is 
directed above the speculum, so it is out of the 
way.  
Rectal Biopsy for Hirschsprung’s Disease 
Catherine Jackson-Cole, Marlene Ishimwe, Jason Axt 
 
OPEN MANUAL OF SURGERY IN RESOURCE-LIMITED SETTINGS 
www.vumc.org/global-surgical-atlas 
This work is licensed under a Creative Commons Attribution-ShareAlike 3.0 Unported License  
 
 
A nasal or small anal speculum is inserted with the handle 
facing upwards. A sutured betadine-soaked gauze is inserted  
through the anus into the rectum. The suture is directed 
upwards out of the way. It can be pulled to remove the gauze 
once the biopsy is complete.  
 
9. The dentate line is identified. Place a traction 
suture about 1cm above this structure. This will 
help pull the tissue downwards.  
 
With insertion of the speculum, excellent positioning and 
relaxation under anesthesia, the dentate line as well as rectum 
proximal to it can easily be seen. Note also the suture attached 
to the gauze, out of the way behind the speculum.  
 
10. While holding tension on the traction suture, a 
second full thickness biopsy suture is placed 1 
cm above it. Angle the needle 45 degrees 
forward and rotate the wrist. This will be the 
biopsy site. The suture is cut and held with a 
hemostat. 
11. A third full thickness suture is placed 1 cm 
above the biopsy site and tied, without cutting 
off the needle. This will be used to close the 
biopsy site. A 3-0 polyglycolic acid (Vicryl) 
suture is often used, but any absorbable suture 
will suffice.  
 
All three sutures have been placed demonstrating their 
importance in elevating the tissue, underneath the Biopsy 
Suture, which will now be cut. 
 
12. Holding all three sutures under tension a tissue 
scissors is used to make a vertical full thickness 
cut between the biopsy stitch and the closing 
stitch. 
 
The first cut is made above the biopsy suture. The second will 
be made below it, yielding a full thickness piece of tissue 
about 2mm wide.  
 
Rectal Biopsy for Hirschsprung’s Disease 
Catherine Jackson-Cole, Marlene Ishimwe, Jason Axt 
 
OPEN MANUAL OF SURGERY IN RESOURCE-LIMITED SETTINGS 
www.vumc.org/global-surgical-atlas 
This work is licensed under a Creative Commons Attribution-ShareAlike 3.0 Unported License  
 
13. Use the tissue scissors to cut tissue from beneath 
the biopsy stitch to connect the cuts and 
obtaining a ~2 mm piece of rectal tissue. The 
biopsy site will bleed briskly, but this will be 
controlled by closure. 
14. Use the already placed closing suture to close 
both sides of the biopsy site in running locking 
fashion. Once the wound is closed, tie the suture 
to itself.  
 
The completed closure, with the traction suture removed. The 
remaining suture is attached to the gauze.  
 
15. Check for hemostasis, remove the speculum and 
the gauze.  
16. All incisions and sutures placed should be above 
the dentate line and therefore painless. No 
postoperative analgesics are necessary.  
 
Pitfalls 
• Ensure that the biopsy is at minimum 1 cm 
above the dentate line, or the biopsy may be 
falsely read as no ganglion cells.  
• Do not stricture the anus by being excessively 
“generous” with the closure- close only the 
mucosa adjacent to the wound.  
• Avoid cutting the closure suture while taking 
the biopsy. It is difficult to replace that suture 
when there is bleeding, and one has lost the 
traction of the other suture. 
• Preterm infants may not have fully developed 
nerves yet. Rectal biopsies should not be trusted 
until the infant has reached full term gestational 
age.  
 
Catherine Jackson-Cole, MBChB 
Marlene Ishimwe, MD 
Jason Axt, MD MPH FAAP FCS(ECSA) 
AIC Kijabe Hospital  
Kenya 
 
May 2024 
