Calretinin immunohistochemistry: A new cost-effective and easy method for diagnosis of Hirschsprung's disease.

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

The diagnosis and extent of resection in the management of HD depend on the sensitive and specific identification of ganglion cells.[1-3] 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.[4]

The aim of this study was to evaluate the efficacy of calretinin immunostaining[57] 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.

MATERIALS AND METHODS

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.

This was a prospective study from June 2010 to June 2011. Specimens from 60 patients with HD were evaluated. 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.

RESULTS

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. Seven (19.5%) patients presented between 1 month and 1 year of age. Six (16.7%) patients presented after the age of 1. Forty-nine patients had classical segment disease (81.7%). 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].

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. Later, surgery revealed an ultrashort segment HD with only 1 cm of aganglionosis. 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.

Table 2

Calretinin staining in aganglionic, transition and ganglionic zone of resected specimens

DISCUSSION

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.[458] 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.[67] This protein is involved in calcium transport; and its absence allows accumulation of cytoplasmic calcium, causing excess neuroexcitability and ultimate neurodegeneration. Barshack, et al.,[7] 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 is easily applied to paraffin-embedded specimens. 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.

Figure 1

Hematoxylin and eosin section with ganglion cell (arrow) (H and E, 10×)

Figure 2

Calretinin staining of ganglion cells (H and E, 40×)

Guinard-Samuel, et al.,[5] 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.

CONCLUSIONS

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, we found calretinin extremely useful in solving the doubtful cases of HD. It can serve as a valuable cost-effective diagnostic aid in the centers where AChE enzyme histochemistry is not available.