Immunohistochemical Diagnosis of Hirschsprung’s Disease and Allied Disorders

Abstract

Summary Introduction. Hirschsprung’s disease accounts for approximately 20% of neonatal bowel obstruction and has a mortality rate of 20 to 25%. As the pathology severely affects a child’s quality of life, quick and precise diagnosis is mandatory. For years, diagnosis relied completely on histopathological analysis of rectal biopsies using haematoxylin-eosin and acetylcholinesterase stains. However, there have been many attempts to find an immunohistochemical marker that would simplify diagnosis of Hirschsprung’s disease. Acceptable markers should be highly sensitive and specific, easy to use and reliable. Aim of the study. The aim is to disclose the full morphological scope of Hirschsprung’s disease and allied disorders by using data available at the Children’s Clinical University Hospital (Riga, Latvia) and to identify the most sensitive and specific immunohistochemical marker for the diagnosis of Hirschsprung’s disease and allied disorders. Material and methods. In a retrospective study, all patients diagnosed with Hirschsprung’s disease and allied disorders at the Pathology Bureau of the Children’s Clinical University Hospital between April 2010 and October 2014 were identified. Immunohistochemical visualisation of calretinin, chromogranin A and synaptophysin was carried out. A conjugated polymer system EnVision was used to detect the bound primary antibodies. The study was controlled by findings in the bowel wall of adults and children lacking aganglionosis. The intensity and pattern of ganglion cell and nerve fibre staining were evaluated semi-quantitatively on a scale of one to three in both submucosal and myenteric plexuses of bowel. The specificity and sensitivity of each immunohistochemical marker were determined with a MedCalc online calculator. Results. During the relevant period, Hirschsprung’s disease (23) and allied disorders (12) were diagnosed in 35 patients. Among these children, 45.7% were diagnosed during the first year of life. Three types of Hirschsprung’s disease were present - short segment disease (73.9%), long segment disease (21.8%) and total colonic aganglionosis (4.3%). Allied Hirschsprung’s disorders were of three different types - hypoganglionosis (75%), zonal aganglionosis (16.7%) and immaturity of ganglion cells (8.3%). After evaluation of the reactivity of immunohistochemical markers, calretinin, synaptophysin and chromogranin showed staining intensity of ganglion cells of 2.47, 0.58 and 0.63, respectively, while nerve plexus staining intensity was 1.84 for calretinin, 2.68 for synaptophysin and 1.79 for chromogranin. Calretinin was characterised by sensitivity/ specificity as high as 90.5%/ 92.9% while these parameters were only 52.4%/ 100.0% for chromogranin A and 33.3%/ 14.3% for synaptophysin. Conclusions. In Latvia, the diagnosis of Hirschsprung’s disease or allied disorder is made later in life than it is in other countries. Hirschsprung’s disease was more frequent than the allied disorders. It can present as short segment disease, long segment disease and total colonic aganglionosis. Calretinin showed the highest reactivity to ganglion cells and proved to be the most specific and sensitive marker for diagnosis of Hirschsprung’s disease and allied disorders. Synaptophysin showed strong staining of myenteric plexuses and can be used for assessment of extrinsic nerve fibres despite its low reactivity to ganglion cells.

Comparative study between use of calretinin and synaptophysin immunostaining in diagnosis of Hirschsprung disease

“…Hirschsprung Disease (HD) is a developmental disorder of the enteric nervous system with incidence of 1: 5000, it is characterized by absence of ganglion cells in the myenteric and submucosal plexuses and hypertrophied nerve terminal along a variable portion of the distal intestine. 1 The rectum is always affected and in 90% patients the disease extends up to rectosigmoid region. 2 The most widely accepted etiopathogenic hypothesis is based on a defect of craniocaudal migration of neuroblasts originating from the neural crest.…”

Diagnostic roles of calretinin in hirschsprung disease: A comparison to neuron-specific enolase

“…They also mentioned that the application of combined one positive (AChE) and one negative (calretinin) could take full advantage of the precision to diagnose HD. [ 14 ]…”

Comparison of Haematoxylin and Eosin Staining and Calretinin Immunohistochemistry in Clinically Suspected Hirschsprung Disease to Evaluate the Diagnostic Utility of Calretinin in Hirschsprung Disease

“…Although, most of these have been proved to be diagnostically insignificant, some, like calretinin are shown to be of diagnostic value. 13 Calretinin is a vitamin D-dependent calcium-binding protein which is involved in the physiological buffering of excess cytosolic calcium ions; calcium transport and protection against calcium-ion overload. 14 It is normally expressed in enteric nervous system, and when stained, can be seen as a distinct granular network of fibres in lamina propria between the crypts, submucosa and muscularis propria which is an indirect evidence of ganglionosis.…”