Hirschsprung ’ s disease diagnosis : Comparison of immunohistochemical , hematoxilin and eosin staining

Background: The diagnosis of Hirschsprung’s disease (HD) is based on the absence of ganglion cells. In hemotoxilin and eosin (H and E) as well as acetylcholine esterase staining there are limitations in the diagnosis of immature ganglion cells in neonates. Methods: In this prospective study, 54 biopsies taken from suspected HD patients (five mucosal specimens and 49 full thickness specimens) were studied. In the laboratory, after preparing sections of paraffin embedded tissues, H and E staining slides were compared with immunohistochemical (IHC) staining including: S100, NSE, CD117, CD56, Cathepsin D, Vimentin, BCL2, GFAP, Synaptophysin and chromogranin. Results: The study revealed 30 negative (absence of ganglion cells) cases (55.5%), 17 positive cases (31.04%) and seven suspected cases (12.9%) of ganglion cells on the H and E staining. On IHC staining with CD56 and Cathepsin D, all of the 17 positive cases detected through H and E, were confirmed for having ganglion cells and out of 30 cases reported negative on H and E staining, 28(93.3%) were reported negative and two (6.7%) positive by IHC staining. Of the seven suspected cases H and E staining), IHC staining detectedganglion cells only in five slides; two remained negative. Conclusions: IHC staining using CD56 and Cathepsin D improved the accuracy of diagnosis in HD when used in addition to H and E staining technique, especially for negative or suspicious slides.

that increase dramatically in the lamina propria mucosa and sub mucous layer, but is not a specific marker for ganglion cell. [3]AChE staining requires the experience of pathologists and in some instances interpretation is difficult. [4]There are reports of false positive and false negative results using this technique. [5][8] In this study, we compare IHC staining using neural markers with H and E staining to find out the best diagnostic panel for HD.

matErial anD mEthoDs
This is a prospective study conducted in the period introDUction H e m a t ox i l i n a n d E o s i n ( H a n d E ) s t a i n i n g , Acetylcholinesterase staining (AChE) are commonly used in the diagnosis of Hirschprung's disease (HD).However, diagnosis is not possible with H and E every times, because staining has limitations in the diagnosis of immature ganglion cells in neonates and the sub mucosal area in which the ganglion cells are small (three to five cells per ganglion) and irregularly distributed and so their identification is difficult and requires high expertise. [1,2]n t h e o t h e r h a n d a l t h o u g h AC h E a c t i v i t y is diagnostically the most useful set of enzymehistochemical reactions, it is not sufficient.AChE stains the parasympathetic nerve fibers and trunks of fibers Memarzadeh, et al.: Application of immunohistochemical staining 2001 to 2004.Rectal biopsy specimens from 54 infants suspected to be having HD constituted the material for the study.There were five mucosal and 49 full thickness biopsies.The specimens were kept in 10% formalin solution.
In the laboratory, after preparing sections of paraffin embedded tissues, H and E staining slides were compared with IHC staining including S 100 , NSE, CD 117, CD 56, Cathepsin D, Vimentin, BCL2, GFAP, Synaptophysin, chromogranin.
The slides for IHC were processed as follows: • First sections of four µm were obtained and fixed on the slides with polyelizine.This was followed by antigen retrieval for 10 minutes using heat and citrate buffer (pH is equal to six).Then 3% H 2 O 2 and pure methanol were added for five minutes and sections were washed with distilled water.Next, primary antibody (with negative control) was added for 10 minutes and washing was performedSecondary antibody (biotinylated link) was added for 10 minutes and washed Streptavidin -HRP was added for 10 minutes and washed with P.B.S.After adding substrate chromogen (D.A.B) for 10 minutes, counter was stained.All the antibodies were from DAKO.-Co and the slides were scanned for ganglion cells.
The best staining method was appreciated based on the degree of staining of ganglion cell versus its background and clear detection of ganglion cells.

rEsUlts
In the microscopic study of 54 specimens, H and E staining revealed absence of ganglion cells (negative) in 30 cases (55.5%), presence of cells (positive) in 17(31.04%)and suspected presence in seven cases (12.9%).In the study of specimens through IHC staining with CD56 and Cathepsin D [Figure 1 and 2], all 17 cases detected positive through H and E, were confirmed for having ganglion cells and of the 30 cases reported negative through H and E staining, 28 (93.3%) were reportednegative and two (6.7%) positive by IHC.Out of seven cases suspected of having ganglion cells on H and E staining, we could find ganglion cells in five slides while two remained negative [Table 1].

DiscUssion
Detection of ganglion cells in H and E sections can be a difficult process for the pathologist. [10]The maturation of ganglion cells is incomplete at the time of birth, especially in the sub mucosal area. [7]Immature ganglion cells may be unipolar or bipolar and can be mistaken for stromal cells. [7]Sub mucosal ganglion cells are smaller than myenteric plexus ganglion cells, [2] and pathologists have to prepare between 50 to 400 sections of H and E stained slides to find ganglion cells. [11]On the other hand, although AChE staining is the chosen technique for some pathologists [5] its diagnosis needs experience and its interpretation is difficult in some instances. [4]ne of the problems is the interference of red blood cell (RBC) acetyl cholinesterase due to hemorrhage in lamina propria. [10]Also, false positive [11] and false negative [10] reactions were reported using this staining technique.18] Park et al. [10] found that the main diagnostic pitfall was the interpretation of the enteric nervous plexuses in the transitional zone and the detection of the indistinct or immature neurons indistinguishable from enteric glial cells or satellite cells.They showed immunohistochemical study was a very helpful diagnostic adjunct to delineating the immature neurons (BCL2), and the size of the enteric ganglia and neuromuscular innervation (S-100 protein, Synaptophysin, and CD56).Another study [12][13][14] found that Synaptophysin-positive synapses distribution in circular and longitudinal colonic muscles and intermuscular ganglions can reflect functional disturbances of large bowel motility and could be helpful in the description of the innervation status of colonic specimens in HD patients.
Facing a wide diversity of opinions, we decided to compare IHC markers with H and E staining to find out the best diagnostic panel for detection of ganglion cells.As shown in figures 1 to 3, ganglion cell detection and its staining with CD56 and Cathepsin D were better than BCL2 and Synaptophysin.
To conclude, our study shows that IHC markers, including both Cathepsin D and CD56, especially for negative or suspicious slides are the best diagnostic panel for detection of ganglion cells.In Cathepsin D staining, the ganglion cells are stainable, but the background is not.On the contrary, in CD56 staining the background is stainable, but ganglion cells are not,