This protocol describes the process of immunostaining rectal suction biopsies for calretinin, S100 protein, and protein gene product 9.5. This novel adjuvant diagnostic method for Hirschsprung's disease has preferable sensitivity and specificity rates.
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.
Hirschsprung's disease (HD) is a common congenital gut intestinal disorder characterized by a lack of ganglion cells in different segments of the distal intestinal tract1. The human enteric nervous system is formed when the invasion of the embryonic neural cells is completed. If there is a disturbance of the process and the invasion fails to complete, the distal intestine of the newborn becomes aganglionic2. This potentially fatal condition is called Hirschsprung's disease. Proliferation, motility, and gut growth are the three main components of successful colonization.
Traditional hematoxylin and eosin (H&E) staining of a limited submucosal biopsy cannot attain as satisfactory of a result as H&E staining of a full-thickness tissue obtained from surgery. Additionally, acetylcholinesterase (AChE) staining of rectal suction tissue is theoretically challenging due to its inadequate sensitivity, which is 91%, and complex processing of frozen sections3,4. Several other immunohistochemical markers of ganglion cells and nerve fibers that can be stained in formalin-fixed and paraffin-embedded specimens are gradually becoming the mainstream HD diagnostics. Calretinin is a vitamin D-dependent calcium-binding protein that is not expressed in the myenteric and submucosal plexus of HD-affected segments5. S100 protein is expressed in cells derived from the neural crest, such as nerve fibers and glial cells, which often present neural hypertrophy in the submucosal tissue of HD-affected segments6. Protein gene product 9.5 (PGP9.5) reliably stains nerve fibers and ganglion cells; PGP9.5 staining acts as a supplement to calretinin staining, especially in cases of isolated hypoganglionosis. Double staining with S100 and PGP9.5 can decrease the false-negative rate and increase the sensitivity. As a prerequisite, the current study aims to ensure adequate specificity and high sensitivity of this novel diagnostic method. Our novel protocol used all three markers for the discrimination of aganglionic intestine and hypertrophic nerve fibers. A prospective study of 318 children was performed by our lab and previously published without a detailed protocol7. The detailed protocol and precautions are discussed in this article. Any neonates who suffered from a severe defecation problem since birth or children with chronic constipation excluding other common diseases are potential candidates for rectal suction biopsy (RSB). Our novel protocol is suitable for staining not only RSBs but also full-thickness biopsies or surgical specimens to make a final diagnosis.
This protocol was approved by the Research Ethics Board of the Union Hospital of Huazhong University of Science and Technology.
1. Rectal Suction Biopsy
2. Preparation of the RSB Sections
3. Antigen Retrieval
4. Blocking
5. Antigen-antibody Reaction
6. 3,3-Diaminobenzidine and Hematoxylin Staining
In total, 318 patients were enrolled in our study. All of the patients underwent RSB, and the tissues were stained for calretinin, S100, and PGP9.5. The diagnosis based on our novel protocol was HD in 97 cases, non-HD in 213 cases, and suspected HD in 8 cases. Among the 132 surgical patients, 99 patients were diagnosed with HD by immunostaining of full-thickness specimens after surgery. S100 and PGP9.5 staining showed that 92% and 93% of the 99 patients, respectively, had hypertrophied nerve trunks. The other 83 of the 186 patients underwent full-thickness biopsies and showed ganglion cells. A total of 103 patients were clinically cured by conservative therapies, and HD was excluded. Of the 8 patients with suspected HD, 3 patients were categorized as short-segment HD, and the other 5 patients were categorized as non-HD.
According to our results, 97 patients were diagnosed by RSB initially. Detailed information is shown in Table 1. The protocol of immunohistochemical staining for calretinin, S100, and PGP9.5, which we conducted on RSBs, has high sensitivity and specificity rates of 96.49% (95% confidence interval [CI], 0.88-0.99) and 100% (95% CI, 0.97-1.00), respectively. The positive predictive value is 94.3% (95% CI, 0.87-0.99), and the negative predictive value is 99.1% (95% CI, 0.95-1.00).
Figure 1A and 1B represent typical results after immunostaining for calretinin. Many ganglion cells, which express calretinin, can be found in the normal tissues. However, no ganglion cells were detected in any area of the tissue from the HD segment. As shown in Figure 1C-F, S100 and PGP9.5 immunostaining shows hypertrophied nerve trunks in the submucosa, while only granular staining of some small nerve twigs was observed in the normally innervated intestine. In our study, hypertrophic nerve trunks are nerve fibers with a diameter of ≥ 40 μm.
Figure 1: Immunostaining of rectal suction biopsies for calretinin, PGP9.5, and S100. Immunostaining for calretinin in HD-affected tissue (A) and in ganglion cells of myenteric plexuses in normally innervated tissue (B). No calretinin staining was detected in the myenteric plexuses of the HD-affected segment. PGP9.5 staining in HD-affected tissue showed dense hypertrophic nerve fibers in the submucosa (C) in contrast to nerve fibers in the normal tissues (D).(E) Dense and prominent S100 immunostaining showed hypertrophic nerve trunks in the submucosa of HD-affected tissue. (F) Normal nerve fibers in the submucosa. Please click here to view a larger version of this figure.
protein markers | HD(99) | Non-HD(219) | ||
ganglion cells (+) | Hypertrophic nerve trunks (+) | ganglion cells (+) | Hypertrophic nerve trunks (+) | |
calretinin | 2 | — | 214 | — |
S100 | — | 91 | — | 3 |
PGP9.5 | — | 92 | — | 4 |
Table 1: Immunohistochemical staining scores for the three protein markers in rectal suction biopsies.
Here we described a procedure using three different immunohistochemical antibodies to stain RBS sections for the diagnosis of HD. The sensitivity of our diagnostic protocol was 96.49% (95% CI, 0.88-0.99), and the specificity was 100% (95% CI, 0.97-1.00).
The most critical steps of the protocol are RSB and antigen-antibody reaction. The size of the biopsy determines the accuracy of the staining. A small biopsy will not provide enough tissue to make a precise diagnosis, while a large biopsy leads to a higher risk of bleeding. As a matter of experience, biopsies with a diameter of 2 mm are the optimal size. For the antigen-antibody reaction, proper preservation of the antibody cannot be ignored. Following the protocol step by step and gently working are needed. Reverse osmosis-purified water is recommended in all steps, yielding a clearer field for observation and making it easier for pathologists to make an accurate diagnosis.
Contrast enema, anorectal manometry, and biopsy with histology are three preoperative diagnostic methods that have been used in recent years. According to Takawira et al.'s research, biopsy with histology has the highest sensitivity and specificity8. Meier-Ruge et al. first reported the use of acetylcholinesterase (AChE) staining on frozen rectal suction tissue to help diagnose HD in 19729. This staining method detects hypertrophic nerve trunks in the submucosa. After that report, many institutions around the world started to use this method as an adjunct for the diagnosis of HD. Due to its diagnostic limitations, researchers started to concentrate on other markers that can also stain the ganglion cells and nerve fibers in formalin-fixed and paraffin-embedded specimens. In 2004, Barshack et al. found that the loss of calretinin expression may help diagnose aganglionic segments in HD10. Kapur et al. and Guinard-Samuel et al. repeated the protocol in 2009 and concluded that immunohistochemical staining for calretinin was superior to staining for acetylcholinesterase11,12. In 1988, S100 immunostaining for the diagnosis of HD was first introduced by Robey et al.13. Monforte-Muñoz et al. repeated the method in 1998 and found that 90% of their 20 Hirschsprung cases had nerve fibers wider than 40 μm14. Therefore, S100 immunostaining may serve as an ancillary diagnostic method in cases without ganglion cells. Furthermore, Bachmann et al. noticed that immunohistochemical staining for MAP2, calretinin, GLUT1, and S100 could obtain a reliable result as accurate as frozen section techniques15. However, in 1992, Sams et al. evaluated the value of PGP9.5 in the diagnosis of HD16. Although S100 can detect more lamina propria nerve fibers, it has a higher false-negative rate. Therefore, we chose to use PGP9.5 and S100 together for the immunohistochemical diagnosis of HD, as previous studies recommended. Many cases of staining for neuron-specific enolase in transitional zones also showed positive ganglion cells in the myenteric and submucosal plexus, but these cells were sparse and small12. Similarly, calretinin-positive ganglion cells are small and have a clustered formation. A previous study indicated that some slightly thickened nerve trunks could also be detected in the transitional segments of HD, but with total colonic aganglionosis, the nerve trunks may be within normal limits (< 40 μm)17. We should analyze the results case by case and combine these results with clinical manifestations to avoid misdiagnosis.
This protocol could not represent the final pathological diagnosis, so we carried out a minimum of one year of thorough clinical follow-up for each child. Additionally, this protocol requires more samples to prove its reliability.
In conclusion, the diagnosis of HD by RSB is a convenient method that provides an ideal histological diagnosis with acceptable minimal injury. The sensitivity is closely related to whether the site of sampling is correct. Immunohistochemical staining of RSB for calretinin, S100, and PGP9.5 is sensitive and specific in the detection of ganglion cells and hypertrophic nerve trunks. The combination of these three markers offers a more reliable diagnostic method for HD.
The authors have nothing to disclose.
The authors thank Weibing Tang for his great help in providing the filming lab. This article is supported by the Public Welfare Research, and special funds were received from the National Health and Family Planning of China (Grant No.201402007).
calretinin antibody | MXB Biotechnologies | MAB-0716 170416405c | antibody: primary antibody |
S-100 antibody | MXB Biotechnologies | Kit-0007 | antibody: primary antibody |
PGP9.5 antibody | Shanghai long island antibody Co. Ltd | R-0457-03 | antibody: primary antibody |
enhancer reagent | MXB Biotechnologies | KIT-9902-A 170416405a | antibody: secondary antibody A |
Goat anti-Rabbit/Mouse IgG Secondary Antibody | MXB Biotechnologies | KIT-9902-B | antibody: secondary antibody B |
DAB staining kit (containing reagent A B and C) | MXB Biotechnologies | DAB-0031 | staining kit |
Heat incubator | Shanghai yiheng instrument Co. Ltd | DHP-9082 | instrument |
Rbi2 suction rectal biopsy system | Aus Systems Pty Ltd, South Australia, Australia | CP1200 HP1000 SS1000 | instrument |
microtome | Leica | leica RM2016 | instrument |
citric acid sodium citrate buffer(100X) | MXB Biotechnologies | MVS-0101 | antigen retrieval buffer |
pathological tissue dehydrator | wuhan junjie electronic Co. Ltd | JT-12F | instrument |