Rectal Suction Biopsy Research Articles

Gastrointestinal Amyloidosis in Children with Familial Mediterranean Fever Disease and Anorectal Manometry Results

Objectives: The aim of this study was to evaluate the effect of amyloid deposition in colon mucosa on the results of anorectal manometry test and the use of ROME III questionnaire in patients with Familial Mediterranean Fever (FMF). Methods: The files of patients diagnosed with FMF were scanned to evaluate the patients at risk for amyloid deposition. Predisposing factors were identified. The patients were sampled using anorectal manometry test, Rome III questionnaire and the rectal mucosa suction biopsy and the test results were compared. Results: 17 (63%) of the patients were female and 10 (37%) were male. The mean age was 12.15 ± 2.40 years. The number of patients with amyloid deposition in the rectal mucosa sample was 2 (7.4%). Both of these patients had regularly used colchicine. Amyloid deposition was found to be high in patients with M694V homozygous mutation (P = 0.05). According to the Rome III questionnaire, findings suggestive of irritable bowel syndrome were found in 5 patients and abdominal migraine was found in 3 patients. However, no statistical difference was found when the results of the Rome III questionnaire were compared with the results obtained from the anorectal manometry test (P > 0.05). Conclusions: Amyloidosis can be seen due to various environmental factors, regardless of age, even when colchicine is regularly used. Anorectal manometry is an easy to perform test, helping diagnosis in this patient group. Although it has been shown that in patients with predisposing factors for amyloid deposition RAIR can be detected at lower pressures in anorectal manometry, the clinical significance of this finding is unclear. Since the Rome III questionnaire showed findings consistent with functional abdominal pain disease in one third of the patients, independent of the presence of FMF, it was found that the application of this questionnaire in patients with FMF could be misleading.

To assess calretinin immunohistochemistry in Hirschsprung’s disease and allied disorders

Hirschsprung’s disease (HD) is the congenital aganglionosis of the human colon due to disruption of migration, imbalanced proliferation and differentiation of neural crest cell. The diagnosis and extent of resection for the management of HD depend on the sensitive and specific identification of ganglion cells. Negative immunohistochemical expression of calretinin, shown its superiority as diagnostic method for full thickness as well as partial thickness rectal suction biopsies. Total 86 colonic and rectal biopsies of 43 patients attending Sassoon hospital, Pune (between 2015-2017) with surgically resected specimen proven Hirschsprung’s disease cases and follow up cases of non HD cases were evaluated with both Haematoxylin and Eosin stain and calretinin IHC. Age group was 2 days to 7 year with having Male: Female ratio 2.91:1. Out of 43 cases, 26 were diagnosed as Hirschsprung’s Disease, 16 were not having Hirschsprung’s disease, 1 case of hypogangliosis. 13 out of 86 biopsy and 9 out of 43 cases show false positive result with use of Haematoxylin and Eosin stain only. Specificity and negative predictive value of H&E stain was 72% and 78.12% respectively with accuracy of 86%. Use of calretinin IHC gave 100% accuracy in all 86 biopsies studied including 17 partial thickness biopsies. Only use of Haematoxylin and Eosin stain will lead to significant false positive result leading to misdiagnosis and in turn wrong treatment. Loss of calretinin expression in aganglionic segment help in the interpretation of even partial biopsies and easy to interpret with improvement in diagnosis upto 100%.

Hirschsprung's disease

Hirschsprung's disease (HD) is a congenital functional disorder characterized by the absence of ganglion cells in the enteric nervous system. The estimated incidence of HD is 1:5000 with up to 60% of patients having associated anomalies. Short-segment rectosigmoid disease is seen in 75% of cases with infants presenting with abdominal distension, bilious vomiting and delayed passage of meconium. A bedside rectal suction biopsy is the gold-standard in confirming the diagnosis of HD. The absence of ganglion cells in the myenteric and submucosal plexi in the presence of thickened hypertrophic nerves (more than 40 microns diameter) is diagnostic of HD. There are no medical options to treat HD, however rectal washouts can be used to bridge the gap to surgery. Contemporary management of this condition involves a single-stage pull-through that can be performed using various techniques including minimal access surgery. Long-term problems in with HD are common and these include obstructive symptoms (10–30%), constipation (10–15%), enterocolitis (10–15%) and true faecal incontinence (1–2%). Furthermore 20–30% of adults report symptoms of incontinence and constipation as well as poor sexual function. Future research is being focussed on novel gene therapies including stem-cell therapy and the use of CRISPR/CA9 to edit parts of the genome.

PHOX2B Immunostaining: A Simple and Helpful Tool for the Recognition of Ganglionic Cells and Diagnosis of Hirschsprung Disease.

Hirschsprung disease (HD) is a congenital disorder of the enteric nervous system that occurs in ∼1 in 5000 live births. It is characterized by the absence of ganglionic cells (GCs) in the distal intestine. The diagnosis relies on the thorough analysis of a rectal suction biopsy (RSB), which must show a complete absence of GCs after careful examination of at least 100 serial sections. Such a negative characteristic explains the difficulty of this diagnosis. Moreover, GCs may be immature in very young or preterm born children, making them hard to recognize. Therefore, ancillary techniques have been developed as diagnostic help, such as acetylcholinesterase histochemistry and calretinin immunostaining. These techniques reveal only indirect clues, focusing mainly on the changes in nerve fibers, but not on GCs themselves. As PHOX2B has been shown to be a very specific transcription factor in GCs and in progenitor enteric nerve cells, we have assessed (i) PHOX2B immunostaining in immature enteric ganglia and (ii) the use of PHOX2B immunostaining for the recognition of GCs on RSBs for suspicion of HD. We have observed PHOX2B expression in all GCs, both mature and immature, and its complete absence in Hirschsprung cases. We suggest that the use of PHOX2B immunostaining is of great help (i) in the recognition of GCs on RSBs regardless of their differentiation and therefore (ii) in the diagnosis of HD.

Calretinin versus Hematoxylin and Eosin Stain for Diagnosis of Hirschsprung’s Disease; Comparison in Ganglionic, Transitional, and Aganglionic Zones

Objective: To compare the results of calretinin stain with hematoxylin and eosin (H&E) stain for diagnosis of Hirschsprung’s disease. Materials and Methods: A prospective double-blind diagnostic study was done. Hirschsprung’s patients who underwent transanal endorectal pull through (TERPT) surgery between February 2015 and February 2017 were included. The pulled-through specimens were sampled at the ganglionic, transitional, and aganglionic zones. Each specimen was stained with calretinin and H&E. Agreement and kappa analysis were done. Results: Fifty-one Hirschsprung’s patients (153 specimens) were included for analysis. There were 37 males (72.5%) and 14 females (27.5%) with a median age at surgery of four months. Thirty-one specimens showed a negative stain for calretinin (aganglionic bowel) and 33 specimens showed no ganglion cells in the H&E stain. One hundred twenty-two specimens in calretinin stain were consistently positive with ganglionic bowel and 120 specimens in the H&E stain showed ganglion cells. Agreement and Cohen’s kappa coefficient were 97.4% and 0.921 (95% confidence interval 0.845 to 0.997), respectively. Disconcordance was found in four specimens. Three out of four were in the transitional zone. One was in the aganglionic zone, which has no muscular layer attached. (Thirty-six specimens had no muscular layer.) Conclusion: Calretinin stain was found to be comparable with the H&E stain and could be used for diagnosis of Hirschsprung’s disease. In rectal suction biopsy specimen in which the muscular layer was not included, both calretinin and H&E can be used. Keywords: Megacolon, Hematoxylin and eosin (H&E), Calretinin, Rectal suction biopsy (RSB), Full thickness biopsy

Functional constipation or redundancy of the colon?

Constipation is a common problem in children, and most of the time, the cause is defined as functional. Our hypothesis is that children with functional refractory constipation had anatomic alterations of the colon. All children with chronic refractory constipation who visited our centre underwent accurate clinical examination, contrast enema (CE), anorectal manometry (ARM) and rectal suction biopsies (RSB). In case of functional constipation, three operators measured the size of the colon using radiograms and calculated the ratio based on the width of the second lumbar vertebra. The measurements carried out were compared with those reported in the literature on patients of the same age without constipation. Over a period of 24months, 69 patients with chronic refractory constipation, aged between 1 and 14years, visited our department. A CE was performed on 67, and 2 were excluded because of anal stenosis. Sixty-five underwent anorectal manometry. Rectal suction biopsies were needed in 14 children, and 2 of them were found to have colonic aganglionosis. After a complete evaluation, 57 (82.61%) patients were diagnosed having functional constipation. By comparing the data of the patients with those of normal children reported by the other authors, we found that none of the measurements was statistically significant except for the rectosigmoid length: the mean value in one-year-old patients was 19.03 vs. 9.75, and in older children, it was 19.46 vs. 9.59. Recognizing an anatomic anomaly in patients suffering from functional constipation is important for specific treatment, especially when the ratio (rectosigmoid length/L2) is higher than 15.

Comparison of inconclusive rates between suction rectal biopsy and open strip rectal biopsy in children of different age groups: a single-center retrospective study

BackgroundRectal biopsy for the diagnosis for Hirschsprung's disease (HD) can be performed in several ways. Suction rectal biopsy (SRB) is the most widely used method for neonates and younger infants...

Suction Rectal Biopsy is Accurate in Late Preterm Infants with Suspected Hirschsprung Disease

BackgroundFew data are available to substantiate the dogma commonly held by pediatric surgeons and pediatric pathologists that rectal biopsies may be inaccurate or risky in preterm neonates. We aimed to address these concerns. MethodsWe performed a single-institution retrospective review of patients who underwent suction rectal biopsies at <37 weeks corrected age from 2009 to 2018. The primary outcomes were accuracy of biopsy results and procedural complications. ResultsForty-nine patients underwent suction rectal biopsy at <37 weeks corrected age. Mean gestational age at biopsy was 35.2 weeks (range 32.1–36.9) and mean weight was 2126 g (range 1590–3100). Five (10%) infants had biopsies positive for Hirschsprung Disease. All 5 later underwent pull-through operations and had pathologic confirmation of an aganglionic segment. The remaining 44 (90%) had biopsies showing ganglion cells. None were later found to have HD during the follow up period. Those who underwent biopsy at <2000 g (16/44) had 100% sensitivity [95% CI 48, 100] and specificity [95% CI 92, 100] (2 true positives, 14 true negatives). There were no complications identified. ConclusionSuction rectal biopsy can be performed safely in preterm infants as small as 1590–2000 g with high accuracy. Clinicians should not hesitate to perform a biopsy for a premature infant when clinically appropriate. Type of studyStudy of a diagnostic test. Level of evidenceLevel IV.

Total colonic tubular duplication causing severe constipation

To report the case of a patient with total colonic tubular duplication pointing out the diagnostic and therapeutic difficulties and reviewing the Literature concerning this rare malformation. The patient was a female girl born at term with vertebral and spinal anomalies. At 6 months she underwent emergent left upper lobectomy for a symptomatic, previously undiagnosed left congenital lobar emphysema. She also had intractable coprostasis and abdominal distension. The clinical picture and the results of the contrast enema made us think of congenital megacolon but suction rectal biopsies were inconclusive. Therefore we obtained full thickness colonic and terminal ileum biopsies and we performed an ileostomy for faecal diversion and bowel management. A few months later, the anterograde contrast study from the distal ileostomy showed two colons with proximal communication and a distal pouch. On laparotomy, we confirmed the diagnosis of complete colonic tubular duplication. We divided the common wall between the two colons, we performed a mucosectomy and we removed the duplicated segment. Post-operative course was uneventful. Duplications of the colon are rare and the diagnosis is not easy. Two main problems occurred during surgery: differentiating between normal and duplicated colon and removing the duplication avoiding extensive colonic resections. Resection of the common wall, mucosectomy and removal of the duplicated colon represented a feasible and safe procedure.

Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5.

Hirschsprung's disease (HD) is a congenital intestinal disease that is clinically manifested as an inability to pass meconium in infants or as long-term constipation in children. Rectal suction biopsy (RSB) to determine the absence of ganglion cells and neural hypertrophy is the most accurate test for the diagnosis of HD at present. Traditional hematoxylin-eosin staining lacks sensitivity and specificity. Acetylcholinesterase staining cannot be widely used due to its complex process. Our novel protocol of immunostaining for calretinin, S100 protein, and protein gene product 9.5 (PGP9.5), which we conducted on RSBs, exhibits high sensitivity and specificity rates of 96.49% (95% confidence interval, 0.88-0.99) and 100% (95% confidence interval, 0.97-1.00), respectively. The HD-affected segments often present as the absence of the expression of calretinin, S100 protein, and PGP9.5, which are markers of neural hypertrophy in the submucosal tissue. This protocol describes the detailed operating process of this new diagnostic method.

Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5

Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5

Management of Hirschsprung disease in Australia and New Zealand: a survey of the Australian and New Zealand Association of Paediatric Surgeons (ANZAPS).

To define the practice of management for Hirschsprung disease (HD) in Australia and New Zealand. Online survey of Australian and New Zealand Association of Paediatric Surgeons (ANZAPS) members. 56/80 (70%) members from 17 centres responded. 100% perform suction rectal biopsies; 40% perform a contrast enema. H&E (94%), ACHE (70%) and calretinin (75%). Primary pull-through (PT) is performed by 88% (100% by < 6/12months). The Soave-Boley PT is the preferred approach (85%), with laparoscopic assistance (77%) and muscle cuff division (93%). Routine post-operative dilatations are performed by 63% of respondents. If symptoms persist following PT, majority adopt a conservative approach (enemas/laxatives 90%; Botox 74%). If a long-segment is identified at PT, 60% fashion a stoma and delay definitive surgery. If total colonic aganglionosis is identified at PT, 76% fashion a stoma and delay definitive surgery. A dedicated bowel management program is available in 45% of centres with transition to adult services in 29%. A laparoscopic-assisted Soave-Boley PT is the most common technique for recto-sigmoid HD. Differences are noted in both the management of long-segment/total aganglionosis HD and post-operative management/follow-up.

Too Many Biopsies Performed to Rule Out Hirschsprung's Disease: But It is Worth Doing Them.

The U.K. National Institute for Health and Clinical Excellence (NICE) specify the following indications for performing rectal biopsy to rule out Hirschsprung's disease (HSD): (1) passage of meconium > 48 hours; (2) constipation since first few weeks of life; (3) chronic abdominal distension with vomiting; (4) family history of HSD; and (5) faltering growth in addition to any other indication. The aim of this study was to assess the compliance of a tertiary referral center with the current U.K. NICE guidelines for performing rectal biopsies to rule out HSD. Secondary aims included assessing alternative indications and complication rates. Retrospective analysis of patients undergoing open or suction rectal biopsies to investigate HSD, from 2009 to 2014. A total of 188 patients underwent 214 biopsies (suction: n = 154, open: n = 60). Note that 128 patients (68.1%) had biopsies indicated by NICE. A total of 84.4% of indicated biopsies were ganglionic versus 91.7% of unindicated biopsies (p = 0.17). Twenty-five patients were diagnosed with HSD (mean age: 13.2 weeks, range: 3 days-3.2 years). Only 80% of HSD patients had NICE-indicated biopsies. Presentations for patients with biopsies not indicated by NICE included constipation, distension, and vomiting that did not strictly meet the guidelines. The majority of biopsies were uncomplicated (suction 82.5% vs. open 95.0%)-inadequate/indeterminate histology was the most common complication (16.2% suction vs. 5% open). A significant number of unindicated biopsies were performed, the majority of which were ganglionic. Rectal biopsies are generally safe and strict adherence to the NICE guidelines could have led to missed HSD diagnoses with potential significant morbidity and mortality.

Optimal time for single-stage pull-through colectomy in infants with short-segment Hirschsprung disease.

Short-segment Hirschsprung disease (HSCR) is the predominant type of HSCR that affects approximately 75% of patients. Whether single-stage endorectal pull-through (ERPT) surgery is appropriate for neonatal patients with HSCR has not been definitively determined. This retrospective cohort study concerning infants with short-segment HSCR investigated the optimal age for single-stage ERPT surgery, regardless of the operative approach. The 198 patients were stratified by operative age ≤ 3 or > 3months (groups A or B, respectively, n = 62 and 136, respectively). Diagnoses of short-segment HSCR were conducted by preoperative contrast enema and rectal suction biopsy with acetylcholinesterase immunohistochemical staining. The perioperative clinical course for all patients was reviewed and the accuracy rate of the preoperative diagnoses and postoperative short- and midterm outcomes were assessed. The rates of diagnostic accuracy, according to the results of the preoperative contrast enema or rectal suction biopsy, were lower in group A (67.2 and 93.5%, respectively) than in group B (81.4 and 94.9%, respectively). In groups A and B, 49 (79.1%) and 108 (79.4%) infants, respectively, completed follow-up examinations. The short-term outcomes were postoperative HSCR-associated enterocolitis, adhesive bowel obstruction, anastomosis leakage, and anal stenosis during the first 12months after surgery. The midterm outcomes were incontinence and constipation at ~24months after surgery. Compared with group B, group A experienced more incidences of anastomotic leakage in the short-term and more soiling in the midterm. In groups A and B, the rates of constipation recurrence were nil and 1.9%, respectively. Infants with HSCR ≤3months old at the time of single-stage ERPT surgery showed lower rates of accurate and conclusive diagnostic results and poorer postoperative outcomes. Waiting to perform this surgery until infants are older might be more beneficial.

Air test as a simple method of screening for Hirschsprung's disease

To present the technique and the diagnostic accuracy of the air test to diagnose Hirschsprung's disease (HD). Children who attended hospital for chronic constipation (CC) between January 2012 and December 2016 for whom the air test was performed were enrolled. The test was conducted during contrast enema under fluoroscopic observation using 20-50 ml injections of air into the rectum through a 10 F Nelaton catheter. The demographics, results of the air test, and additional examinations, as well as the outcomes of subsequent treatments were analysed retrospectively. The air test was conducted in 179 patients (median: 3 years, range: 0-14 years), and was positive in 150 and negative in 29 cases. Of the 29 patients with negative results, four were diagnosed with HD by rectal suction biopsy (RSB). Of the remaining 25 patients, RSB was conducted in seven and HD was excluded in all cases. In all 150 patients with positive air test results, CC was adequately controlled with conservative treatment. The sensitivity and specificity of the air test were 100% (4/4) and 85.7% (150/175), respectively. The air test can be used as a new non-invasive screening method for HD, performed simultaneously with contrast enema.

Anorectal Manometry for the Diagnosis of Hirschsprung Disease: New Heights for the Balloon or Just Hot Air?

Anorectal Manometry for the Diagnosis of Hirschsprung Disease: New Heights for the Balloon or Just Hot Air?

Anorectal Manometry May Reduce the Number of Rectal Suction Biopsy Procedures Needed to Diagnose Hirschsprung Disease.

The aim of the study was to evaluate whether anorectal manometry (ARM), which is used to test the rectoanal inhibitory reflex (RAIR), is a safe alternative for reducing the number of invasive rectal suction biopsy (RSB) procedures needed to diagnose Hirschsprung disease (HD). Between 2010 and 2017, we prospectively collected the ARM results of 105 patients suspected of having HD. Following the outcome, the patients either underwent additional tests to confirm HD or they were treated conservatively. Primary ARM-based diagnoses were compared with the definitive diagnoses based on the pathology reports and/or clinical follow-ups. Additionally, we analyzed whether modifications to our ARM protocol improved diagnostic accuracy. The sensitivity of ARM and RSB was comparable (97% vs 97%). The specificity of ARM, performed according to our initial protocol, was significantly lower than that of RSB. After we modified the protocol the difference between the specificity of ARM and RSB was no longer statistically significant (74% vs 84%, respectively, P = 0.260). The negative predictive value of ARM was 100%, while their positive predictive value was significantly lower than that of RSB (56% vs 97%, P < 0.001). ARM is a viable screening tool for HD and, provided it is performed properly, it can be used to exclude HD with absolute certainty. By contrast, an absent rectoanal inhibitory reflex on ARM should always be followed by an RSB to confirm the diagnosis of HD. Using ARM as the diagnostic of first choice could reduce the number of invasive biopsies.

Accuracy of Suction Rectal Biopsy for Diagnosis of Hirschsprung's Disease in Neonates.

Recent publications have questioned the sensitivity of suction rectal biopsy (SRB) for diagnosis of Hirschsprung's disease (HD) in newborns. A recent European survey reported that 39% of pediatric surgeons performed full-thickness transanal biopsies due to concerns about the accuracy of SRB. We sought to examine our contemporary SRB experience in infants. A review was performed (2007-2016) of patients under 6 months of age who had a SRB at our children's hospital. The cohort was subdivided by postmenstrual age at time of SRB: preterm (< 40 weeks, A), term neonate (40-44 weeks, B), and infant (> 44 weeks, C). The pathology reports from endorectal pull-through were used as gold standard confirmation. One-year follow-up of patients with negative SRB was used to confirm accurate diagnosis. A total of 153 patients met the criteria and a total of 159 SRBs (< 2,500 g; n = 26) were performed (A = 60, B = 58, C = 35). Forty-three patients were diagnosed with HD (A = 25, B = 15, C = 3). A second SRB was performed in 6 (3.9%) patients due to inadequate tissue (A = 2, B = 2, C = 2) with HD diagnosed in 5. No complications occurred. Sensitivity and specificity of SRB was 100% in all age groups. Half of the patients with a negative SRB had at least 1 year follow-up, with none subsequently diagnosed with HD. SRB results in adequate tissue for evaluation of HD in nearly all patients less than 6 months of age on the first attempt and is highly accurate in the preterm and newborn infants. No complications occurred, even among infants less than 2,500 g.

Calretinin, S100 and protein gene product 9.5 immunostaining of rectal suction biopsies in the diagnosis of Hirschsprung’ disease

Objective To evaluate the sensitivity and specificity of calretinin (CR) combined with S100 proteins (S100) and protein gene product 9.5 (PGP9.5) immunostaining of rectal suction biopsies for the diagnosis of Hirschsprung’s disease (HD). Methods We conducted a prospective study and collected 318 patients with suspicious HD in Wuhan Union Hospital. All children included underwent the rectal suction biopsy after careful history and physical examinations to confirm or exclude the HD. We applied a protocol using calretinin combined with S100 and PGP9.5 immunostaining for the identification of ganglion cells and hypertrophic nerve trunks. Finally, we evaluated the usefulness of this method for diagnosis of HD. Results Between February 2014 to January 2017, a total of 318 patients were enrolled in our study. Among them, 219 patients (69%) had ganglion cells on the initial diagnostic protocol, and 99 cases were devoid of ganglion cells. 92% and 93% showed submucosal neural hypertrophy on S100 staining and PGP9.5 staining, respectively. 132 patients underwent a colonic resection, and 99 had confirmed aganglionic segment. The sensitivity and speciflcity rates of our diagnostic protocol were 97.0% (95%CI: 88.0%-99.0%) and 100.0% (95%CI: 97.0%-100.0%) respectively, with positive predictive value and negative predictive value of 94.3% (95%CI: 87.0%-99.0%) and 99.1% (95%CI: 95.0%-100.0%), respectively. Conclusion Calretinin coupled with S100 and PGP9.5 immunostaining on suction rectal biopsies is sensitive and specific for diagnosing HD. Each of them offers speciflc advantages, and these complimentary aspects could contribute to an accurate diagnosis. Key words: Calretinin; S100 protein; Protein gene product 9.5; Hirschsprung’s disease

Diagnostic Accuracy of Combined Acetylcholinesterase Histochemistry and Calretinin Immunohistochemistry of Rectal Biopsy Specimens in Hirschsprung's Disease.

Acetylcholinesterase (AchE) histochemistry has been established as an accurate diagnostic tool for Hirschsprung's disease (HD). In addition, calretinin immunohistochemistry is also reported as a reliable and adjunctive method to diagnose HD. We investigated the diagnostic value of combined AchE histochemistry and calretinin immunohistochemistry in rectal suction biopsies from HD and non-HD patients. We retrospectively reviewed 99 rectal suction biopsy specimens including 4 repeat biopsies from 95 patients (34 HD and 61 non-HD). Each specimen was evaluated with hematoxylin-eosin, AchE histochemistry, and calretinin immunohistochemistry. Of 95 patients, only 21 (22.1%) showed some ganglion cells. All 61 non-HD cases revealed no abnormal AchE-positive fibers. Of 34 HD patients, 32 exhibited abnormal AchE fibers, but 2 showed no stained fibers. None of the tissues from the HD patients exhibited calretinin immunoreactivity. Test sensitivity and specificity of AchE histochemistry alone were 93.5% and 100.0%, respectively, while calretinin immunohistochemistry were 100.0% and 85.2%, respectively. AchE histochemistry is a good diagnostic method for HD, if feasible, and a combination of AchE histochemistry and calretinin immunohistochemistry will help increase the accuracy of the diagnosis of HD.