2023.08.04.32
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]
Available from: http://dx.doi.org/10.21931/RB/2023.08.04.32
ABSTRACT
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
INTRODUCTION
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.
MATERIALS AND METHODS
Subjects
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.
RESULTS AND DISCUSSION
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).