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Files > Volume 8 > Vol 8 no 4 2023

Evaluation of Calretinin and enumeration of mast cells in rectum tissue biopsies of Hirschsprung and non-Hirschsprung disease in neonate and infant
Rusul A. Abdul Hussein 1*, Sahar A. H. AL-Sharqi 2, Nada K. Mehdi3 and Ali E. Joda 4,5
1,2 Department of Biology, College of Science, Mustansiriyah University, Baghdad/Iraq
3 Histopathology Specialist, Central Child Teaching Hospital, Baghdad, Iraq.
4 Pediatrics department, College of Medicine, Mustansiriyah University, Baghdad/Iraq
5 Consultant pediatric surgeon, Central Child Teaching Hospital, Baghdad, Iraq
* Corresponding Author: Rusul A. Abdul Hussein, Email: [email protected]
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The Hirschsprung disease (HD) is a complex genetic congenital condition characterized by the absence of ganglion cells in the myenteric and submucosal plexuses of the colon and rectum, leading to functional intestinal obstruction. A study was conducted from July 2022 to December 2022. The Toluidine blue stain and calretinin immunohistochemistry were applied to 36 cases of neonates and infants who clinically presented with symptoms suspicious of having HD, And the hematological study of cell blood counts test and compared the result of the HD group with the non-HD group and control group. The study showed an increase in mast cell numbers in the rectal biopsy tissue of HD patients compared with non-HD patients using Toluidine blue stain. The Immunohistochemistry for calretinin result displayed 27 (75%) cases as HD, while the remaining 9 (25%) cases were confirmed as non-HD and showed hypertrophied nerve fiber in HD cases. at the same time, the complete blood count result was unrelated to HD. Some worrying maternal risk factors were highlighted during pregnancy were the age of the mother at conception, maternal illness, intake of drugs, type of Childbirth, and number of previous maternal abortions; all of them show a non-significant difference between the HD group and non-HD group, also consanguineous marriage was detected and shows a significant difference between the HD group and non-HD group.
Keywords: Hirschsprung, Calretinin, Toluidine blue, CBC count

The Hirschsprung disease (HD) is a complex genetic congenital condition. The Latin name for HD is megacolon congenitum 1,2. According to reports, HD has a 4:1 male-to-female ratio and impacts 1 case out of every 5,000 live births globally 3,4. The gold standard for diagnosing HD is a rectal biopsy 5. If a patient's clinical symptoms (failure to pass meconium, abdominal distension, constipation)  and radiological findings raise suspicion of HD, the Rectal suction biopsy is obtained 6. Calretinin is calcium signaling that involves a binding protein 7, found in enteric neurons that project into the mucosal and submucosal layers of the gut. This protein might be used as a marker for aganglionosis in HD; it is a crucial component of how cells function and is expressed by the CALB2 gene 8. Mast cells (MCs) are immune cells that migrate from the bone marrow and perform their primary functions in various peripheral tissues 9. The proximity of MCs to nerve fibers, the possibility that they play a physiological role in nerve fiber growth and repair, and the fact that they produce, store, and release the nerve growth factor necessary for the growth and repair of nerve fibers all point suggest that MCs are to blame for the hyperplasia and hypertrophy of adrenergic and cholinergic nerve fibers, which are typical HD symptoms. However, the precise function of MCs in HD is still unknown. MCs can be seen thanks to the Toluidine Blue (TB) stain. The part mast cells play in HD has recently attracted much attention 10. Because most specialist doctors find it challenging to diagnose HD, and the different methods of diagnosis depend on clinical signs and radiological examinations.
The study aims to establish a scientific basis for diagnosing the disease based on the Evaluation of Histopathological and Immunohistochemical (IHC) changes that occur in the layers of the colon, with detecting changes that occur in blood cells and collecting, analyzing, and studying data, to specify diagnosis fundamentals.

The study included the histopathological, hematological, and IHC examination of 36 cases suspected of having HD. The ages of cases ranged between (1day-1year) for both sexes, 9 females and 27 males; the sample was obtained from the Central Teaching Hospital for Children and teaching laboratories at the Medical City Hospital in Baghdad from July 2022 to December 2022. All participants agreed to provide the investigator with the specimens. The College of Science, Mustansiriyah University's ethics committee approved this work. According to the Declaration of Helsinki, informed consent was obtained from all participants (Ref. No.: BCSMU/0622/0012Z Appendix-1).
Collection and preparation of the rectum tissue specimens
The tissue specimens were obtained from the rectum of 36 neonates and infants (27 male and 9 female) who clinically presented with symptoms suspicious of having HD after being diagnosed by specialized doctors, the cases were rectal punch biopsies as shown in Figure (1), the tissue samples were maintained in the fixative solution (formalin 10%) for histopathological study.

Figure 1. Image of cases in which patients showed stages of obtaining a biopsy from the rectum.
Tissue samples from the rectal biopsy were prepared for histological study using Suvara 11. Each tissue sample was cut into small fragments about 2- 3 cm long before fixation in a buffered isotonic solution of 10% formaldehyde for 24 hours. Each biopsy was processed for the dehydration process, which was by passing them through progressive concentrations of ethanol alcohol. They were cleared by passing them through two steps of xylene. Then, tissues were infiltrated with paraffin wax and were embedded in a metal template, and after that, the paraffin blocks were sectioned by rotary microtome into sections 5 μ in thickness. After staining with TB for MC examination, the slides were examined using a light microscope.
Enumeration of Mast Cells in the Tissue   
After sections were stained with TB, the grading of MCs in rectal tissue was done using the following method of Amerada et al. 12: - /+: No cells or few; +: 10 cells seen per 10 high power fields; ++: Clusters of more than 10 cells seen per 10 high power fields; +++: > 10 clusters seen in 10 per high power fields. The results were analyzed after grading.  
Immunohistochemical study of Calretinin
The IHC stain for Calretinin was conducted in cases that included 36 formalin-fixed, paraffin-embedded rectal incisional biopsies from neonate and infant patients. IHC of Calretinin was graded as A total absence of staining (negative) or presence of brown staining (positive) according to the method of Leica Company from Germany.
Collection of Blood Samples
From the 36 patients and 20 controls, about 3 ml of venous blood was collected from a suitable vein and withdrawn from the cases using 3 ml disposable syringes.
The blood (3 ml) was then collected in a tube containing ethylene diamine tetra acidic acid (EDTA) as an anticoagulant with a slow mix for a hematological investigation.
Some Complete Blood Count (CBC)  
In this test, 3 ml of the non-hemolyzed blood is anti-coagulated with EDTA at the collection and was examined by using an automated system Sysmex XP300 hematology analyzer, which is a computerized, highly specialized machine that counts the number of total WBCs and different types of cells such as neutrophil, lymphocyte, RBCs, HCT, and platelets in a blood sample.
Statistical analysis  Statistical Package for Social Sciences (SPSS) version 21 is used to interpret the data. The information is given as a mean, standard deviation, and ranges. Frequencies and percentages are used to display categorical data. ANOVA was used to compare the tested mean Data expressed as mean± SD. Values of p>0.05 were considered statically non-significant, while p≤0.05 considered significant results.

Enumeration of mast cells in the rectal biopsy tissue
           Enumeration of MCs in the rectal biopsies tissue of non-HD and HD cases by using a TB special stain, table (1)  represents the MCs for each HD case and non-HD case, which is divided into four categories that are (Occasional cells, Few natural cells, Moderate number cells, and clumps). In the HD case, the highest grade registered for a Moderate number of cells, while the lowest was for Occasional cells. In contrast, in the non-HD case, the highest grade was reported for Occasional cells, while the lowest was for a Moderate number of cells. That could be because MC is observed in significant amounts in the digestive tract.

Table 1. Enumeration of mast cells in the rectal biopsies tissue of non-HD and HD cases
Results are expressed as percentage - /+: No cells or few; +: 10 cells seen per 10 high power fields; ++: Clusters of more than 10 cells seen per 10 high power fields; +++: > 10 clusters seen in 10 per high power fields In this study mast cells were seen in the submucosa and showed clump of mast cell in the muscular layer (Figure 2).

Figure 2. A cross-section of the patient's rectal biopsy tissue showed (A) a mast cell in the submucosa. (B) a clump of mast cells in the muscular layer (black arrows) (TB staining, X40).

Recently, there has been a lot of interest in the role of MCs in HD, where TB stain highlights MCs, according to 13.. Their description of the transmural distribution of these cells in HD cases, particularly around nerve fibers and perivascular, is consistent with the findings of our study. This may be because MCs secrete a wide range of biologically active substances. MC synthesizes, stores, and releases nerve growth factor, essential for nerve fiber growth and repair 14. A similar finding was mentioned by 15. This could result from the MCs potentially having a significant impact on the regeneration and differentiation of the intestinal neural system. MCs are more prevalent in HD patients 16. However, Hermanowicz  17 carried out a study that found no statistically significant difference existed between the number of MCs in the submucosa of the HD group compared to the number of MCs in the other investigations and when comparing the mean number of MCs in the submucosa of the non-HD group in their study with a mean number of MCs in earlier 16. This study proved that the number of MCs in the submucosa of HD and non-HD groups did not differ statistically significantly. It may be caused by the MC's reaction to allergens or pathogenic pathogens rather than by their relationship with aganglionosis 15.18. Other gastrointestinal conditions like acute appendicitis, ulcerative colitis, celiac disease, and gluten enteropathy have also been linked to an increase in MCs. In the case of suspected HD, this poses a problem for interpreting a rectal biopsy 19. However, one study done by 20 reported an increase in MCs in the mucosa of HD but no statistically significant change in the number of MCs in the submucosa, muscularis propria, and serosa; it has been discovered that numerous inflammatory illnesses at this site are connected with an increase in MCs, which are near arteries and peripheral nerves, which may be because the MCs are primarily present in the gastrointestinal tract 21, Given that many of our patients have several comorbid conditions, this may be explained by the rising number of Mcs, which may not necessarily be related to HD but may instead be caused by these conditions.22
Immunohistochemistry of Calretinin
Calretinin IHC was applied to all 36 studied cases, and 27 (75%) of the cases were identified as HD, while the remaining 9 (25%) cases were identified as non-HD Table (2). Figure (3) showed positive expression in the testis as (positive control) of IHC staining of Calretinin.

Figure 3. The immunohistochemical staining method detected Calretinin in the testis as (positive control), showing positive expression (X10).

Table 2. Expression of Calretinin in the non-HD and HD cases.
The IHC for Calretinin was applied to all 36 cases after the application of Calretinin IHC; out of 36 cases, 27 (75%) cases were confirmed as HD while the remaining 9 (25%) cases were confirmed as non-HD; hence, all suspicious HD cases had been confirmed and categorized in HD and non-HD. In our study, strong calretinin immunoreactivity was observed in all ganglionic segments (non-HD cases), figure (4), showing positive expression between the two muscularis layers and positive expression in the submucosa layer.
Figure 4. Immunohistochemical staining method detection of Calretinin in the rectal biopsies of non-HD case (A) showing positive expression between the two layers of muscularis (B) showing positive expression in the submucosa (Large figure: X10, small figure: X40)
Whereas any immunoreactivity was not observed in almost all aganglionic segments (HD cases), (Figure 5) shows a negative expression of Calretinin in the two layers of the muscularis layer while showing a complete absence of staining expression of Calretinin in the mucosa and submucosa layers.

Figure 5. Immunohistochemical staining method detection of Calretinin in the rectal biopsies of HD case (A) showing negative expression of Calretinin in the two layers of muscularis layer (B) showing negative expression of Calretinin in the mucosa and submucosa layers (X4)
For HD to be pathologically diagnosed, the colonic neural plexus must be devoid of ganglion cells. Identifying tiny immature ganglion cells is made more accessible by IHC labeling of Calretinin, which causes strong ganglia staining 23. IHC expression in this study found that the calretinin IHC approach is less complicated to use, easier to interpret, and requires fewer serial sections of the microscopic rectal biopsy to detect and identify small immature ganglion cells 24. Aganglionic and ganglionic regions differed significantly from one another. Using Calretinin it was successful in detecting the presence of ganglions. The current results showed that Calretinin IHC has good diagnostic value and that Calretinin is an extremely valuable, sensitive, and specific marker for detecting aganglionosis in patients who are believed to have HD 25. This outcome is consistent with the research by 26. Barshack et al. were the first authors to report that expression of Calretinin was not observed in aganglionic areas in HD, but it was kept in ganglionic areas. They also concluded that aganglionic segments showed negative calretinin expression while positive in all rectal biopsies with ganglionic cells. Various research has reported that Calretinin is a good marker in displaying ganglia in HD, as Musa ZA et al. in Iraq revealed in 2017 23, Calretinin is a perfect and trustworthy diagnostic aid to histological examination of HD, where claimed sensitivity and specificity are 100%.
Various research have reported that Calretinin is a good marker for displaying HD ganglia. The presence of hypertrophic submucosal nerve bundles is a beneficial positive finding because HD is diagnosed based on the absence of a histological characteristic, namely the ganglion cells 24. many large nerves are usually present in Submucosal nerve hypertrophy, shown in the aganglionic rectal submucosa of a patient with HD cases in (Figure 6).