the diagnosis and extent of resection in the management of hd depend on the sensitive and specific identification of ganglion cells.[13 ] however , documenting aganglionosis is often difficult and tedious on routine hematoxylin - eosin ( h and e ) stained sections .
acetylcholinesterase ( ache ) has evolved as the gold standard in diagnosing hd ; however , this histochemical analysis is technically challenging , and to date , has not gained worldwide utilization and applicability .
the aim of this study was to evaluate the efficacy of calretinin immunostaining in the ganglionic and aganglionic hd colon biopsy specimens and correlate with the h and e , thereby exploring its utility in suspicious cases of hd .
our standard protocol of care for a child presenting with hd is barium enema study at presentation followed by a laparotomy where multiple biopsies are taken from the spastic aganglionic segment , transition zone , and the normal colon .
a colostomy is sited at the level of the junction between the normal colon and the transition zone .
the definitive surgery of choice is duhamel 's pull - through after 6 months of age .
thirty - six were full - thickness rectal biopsies ( for suspected hd ) and 24 were bowel segments resected during the definitive pull - through surgery .
calretinin ( monoclonal mouse antihuman antibody ( dako ) , ( clone : dak  calret 1 , code : ir627 ) immunohistochemistry ( ihc ) staining was done on all paraffin - embedded blocks after routine h and e examination .
the age of patients ranged from 1 day to 14 years ( mean 8.2 months ) , and there were 46 boys and 14 girls ( m : f = 3.2 - 3.3:1 ; normal ratio 3:1 - 4:1 ) .
twenty - three patients ( 63.8% ) presented at less than 1 month of age .
five ( 8.3% ) had long segment disease , 6 ( 10% ) had total colonic aganglionosis . during the analysis of 36 initial full - thickness colon biopsy specimens , h and e staining revealed absence of ganglion cells ( negative ) in 19 cases ( 52.7% ) , presence of ganglion cells ( positive ) in 2 cases ( 0.05% ) , and suspected presence of ganglion cells in 15 cases ( 41.6% ) .
of the 19 cases reported negative through h and e staining , 17 ( 47.22% ) were reported negative , and 2 ( 0.05% ) positive for calretinin histochemistry for the ganglion cells and nerve fibers [ table 1 ] .
hematoxylin and eosin staining versus calretinin in rectal biopsies in 15 patients , the h and e sections were suspicious of a presence of ganglion cell .
calretinin ihc showed immunopositivity in 3 slides , whereas 5 slides showed immunopositivity in nerve fibers . among the 5 slides
positive for nerve fibers , in 2 of the cases ( 2/60 ) , calretinin gave a slight positive staining of nerve fibers , but with no staining of other areas .
the slight calretinin positivity in the 2 specimens was observed in some large bundles with no staining in other areas , and thus indicating the beginning of transition zone . in the 24 patients where resected specimens from the definitive surgery were sent , serial biopsies were taken from the aganglionic segment , transition zone , and the ganglionic segment [ table 2 ] .
calretinin was not expressed in the ganglion cells and nerve fibers of submucosal and myenteric plexus of 24 aganglionic ( spastic ) segments . in the transition zone ,
calretinin staining was positive in the ganglion cells in 20 cases ( 83.3% ) and was focally positive in the nerve fibers of 22 cases ( 91.6% ) , both in the submucosal and myenteric plexus .
in ganglionic bowel segments of hd , calretinin showed immunopositivity in > 90% of ganglion cells and nerve fibers of submucosal and myenteric plexus .
the histological diagnosis of hd is challenging , requiring the expertise of a senior pathologist and access to specialized techniques for handling frozen specimens for ache staining .
this makes the diagnosis of hd difficult in centers where cases are infrequent , causing a delay in the treatment of the child .
calretinin is proven to be highly sensitive for the presence of ganglion cells and nerve fibers .
this protein is involved in calcium transport ; and its absence allows accumulation of cytoplasmic calcium , causing excess neuroexcitability and ultimate neurodegeneration .
barshack , et al . , procured the colons of 10 patients with proven hd .
calretinin ihc was performed on sections from the aganglionic zone , ganglionic zone , and transition zone of these colons .
they identified calretinin staining in interstitial nerve fibres ( inf ) and ganglion cells in normal colon [ figures 1 and 2 ] and focal inf staining in 92% of transition zones .
in contrast , there was no inf or ganglion cell staining in the aganglionic zone .
it can be interpreted as positive or negative for immune reactivity reducing the ambiguity in diagnosis . in our study
, in the rectal biopsy specimens , diagnosis was ambiguous in 15 patients on h and e examination . by using calretinin ,
immune positivity was demonstrated in 3 patients ( 3 for ganglion cells and 5 for nerve fibers ) ; thereby , ruling out hd in these patients .
hematoxylin and eosin section with ganglion cell ( arrow ) ( h and e , 10 ) calretinin staining of ganglion cells ( h and e , 40 ) guinard - samuel , et al . , have studied the largest number of suction biopsies using calretinin .
they have concluded that calretinin is accurate in proving the absence of ganglion cells , it is easy to interpret , and can replace ache to diagnose hd .
they , however , recommend that it should be used as an aid along with h and e examination , especially in ultrashort segment disease and transition zone specimens . in our study
, we found that calretinin correlated with h and e examination in both rectal biopsies and the resected bowel specimens . in the rectal biopsy specimens , calretinin also aided in the diagnosis of 15 patients with ambiguous findings .
calretinin ihc is accurate in detecting the presence or absence of ganglion cells and holds several advantages such as follows : ( 1 ) it can be carried out on paraffin - embedded tissue sections ; ( 2 ) staining pattern is simple ; ( 3 ) binary pattern of interpretation ( negative or positive ) ; ( 4 ) it is cost effective . in the present study
it can serve as a valuable cost - effective diagnostic aid in the centers where ache enzyme histochemistry is not available .
 aim : to evaluate the efficacy of calretinin immunostaining in diagnosing hirschsprung 's disease ( hd).materials and methods : sixty cases were studied over a period of 1 year ( july 2010-june 2011 ) . 
 there were 36 full - thickness biopsies and 24 resected specimens . 
 calretinin processing was done on the paraffin - embedded blocks after routine histopathological examination.results:of the 36 biopsy specimens , in 19 cases hd was diagnosed by hematoxylin and eosin ( h and e ) staining earlier . in 2 patients , 
 ganglion cells were seen and hd was ruled out . in 15 cases , there was a diagnostic dilemma and calretinin was used . 
 ganglion cells were found in 3 specimens and nerve fibers in 5 . in all 24 resected specimens , calretinin correlated with the findings on h and 
 e staining.conclusions:calretinin was extremely useful in solving the suspicious and indeterminate cases of hd . 
 it can serve as a valuable cost - effective diagnostic aid in the centers where acetylcholinesterase enzyme histochemistry is not available . 
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS