Discussion
In this study, we aimed to measure the adequacy of the SRB method applied to take the rectal biopsy sample, which is the gold standard diagnostic method for HD diagnosis, and in addition to report the results of the test. We found that the SRB results obtained for HD were sufficient for diagnosis. 62.5% of samples receiving SRB were sufficient, and 37.5% of samples were insufficient or suspicious. Insufficient sampling rates have been reported at different rates in several studies in the literature. Although debates on this issue continue, studies reported insufficient sampling rates at the range between 11-50% (16-22). The main difference between these studies is the design of the biopsy procedure that has taken with different procedures. When we investigate the studies in the literature, we observed that most of the studies have low insufficient material rate, 3 or 4 samples were taken in each biopsy procedure, and also biopsy was repeated if the submucosal tissue was small. Mostly, studies reported that 1-4 samples must be taken for each biopsy procedure. In many studies, at least three samples were taken (17-22). Although we took two samples from each patient in our study, we consider that our insufficient sampling rate was acceptable. However, we considered that the reason why this rate was higher when compared to other studies in the literature was that we only obtained two samples in each procedure, did not have SRB for the second time, and that we have just started to apply this protocol in our clinic. In addition, the biopsy of our two patients who were reported suspicious but considered insufficient may have increased this rate.
In our study, when samples containing sufficient tissue and insufficient samples were examined to confirm or exclude the disease in the pathological examinations of biopsies; the average volume of sufficient samples was 8.4 mm3 and 4.7 mm3 for insufficient samples. Although these values are not statistically significant, it is noteworthy that the volume of sufficient samples is high. There are limited studies in the literature regarding the volume measurement of samples. In the study of Muise, the average volume obtained from SRB samples was reported as 14.8±7.8 mm3 (14). Several studies reported that an adequate biopsy sample should be at least 3.5 mm in diameter (17, 23, 24). The increase in the volume of the samples contributes to make a decision of diagnosis, but the depth of the biopsy also is another important criterion for the diagnosis. In the histopathological examinations of the samples in our study, the average percentage of submucosal tissue of sufficient samples was found to be 52%, and the average of the insufficient samples was 40%. The difference was statistically significant (p=0.033). Although there are few prospective studies in this direction in the literature, it is remarkable that retrospective studies reported that the rate of submucosa in the biopsy tissue should not be less than 35-50% among the inclusion criteria for the samples (14, 16, 17). In our study, similar rates were reported with these studies in the literature in terms of both the volume of SRB samples and the percentage of submucosal tissue.
In our study, we found that 73.3% of the biopsy tissues obtained from the newborn age group were sufficient for histopathological examination, while 44.4% of the patients aged between one month and three years were considered sufficient. Although this difference is not statistically significant, tissue adequacy in SRB was found to be higher in newborns than in older babies in our study. When the studies in the literature are examined, different results draw attention when the diagnostic feature of SRB according to age groups is evaluated. While some studies reported that there was no difference in the diagnostic yield of SRB between age groups, several studies reported that insufficiency rate was higher in infants younger than 1.5 months compared to infants older than 1.5 months (5, 22). In another study, it was reported that the tissue adequacy rate was higher in babies older than one year compared to babies older than one year old (25). In some studies, it has been reported that the tissue adequacy ratio of SRB decreases after the age of 3 (20, 26, 27). Since different results have been reported in the literature on this issue, there is no consensus yet. Because in older children, obtaining biopsy under sedation and the presence of hypertrophic nerve fibres in the pathological evaluation of the tissue obtained is a positive factor in diagnosis, previous enterocolitis attacks and thicker intestinal mucosa could be considered as a negative factor and may play a role in obtaining different results in each study. Although the immaturity of the ganglion cells in the neonatal period and the lack of hypertrophic nerve fibres are factors that challenge the pathologist, an experienced pathologist can make a definitive diagnosis with a sample that has reached a sufficient amount of submucosal tissue. It is well-known that full-thickness rectal biopsy procedure is more difficult, especially in the newborn period compared to older ages. In our study, a higher material adequacy rate in newborn period could be achieved with the SRB method, and lower complication rates were observed in all-aged patients. These results show that the SRB technique is an easily applicable and safe method in newborn periods.
In patients with a pre-diagnosis of HD, often anal dilatation and rectal irrigation are recommended methods in terms of both diagnosis and treatment during the decision of biopsy procedure (4). We planned a prospective study by eliminating this suspicious procedure for the first time in the literature, with the suspicion that both procedures may have an oedema-forming effect on the rectum wall and may prevent sufficient submucosal tissue obtain during the SRB procedure. We concluded that our insufficient material rates were parallel to similar studies in the literature. As a result, we revealed that giving patients a 48-hour period without anal intervention before SRB does not provide any benefit, and it is possible to perform biopsy whenever desired. When 9 of the biopsies obtained from 24 patients included in the study, which were considered as insufficient material, were not included in the evaluation, the specificity and sensitivity of SRB from 15 patients was found to be 100%. It has been reported that the sensitivity of SRB in the diagnosis of HD is 88-93% and its specificity is 95-99% (22, 23, 28-30). In the study of Nicole, no false negative or false positive results were reported, similar to our study (28).
The limitations of our study were; when compared to previous studies, the reason why our study was 100% accurate, and there was no margin of error caused by the low number of cases. However, we consider that excluding suspicious SRB results from the evaluation and working with an experienced pathologist for the diagnosis of HD increased the accuracy of the study results. Further prospective-randomized double-blind studies including large-patient population, it may be expected that the sensitivity and specificity rate, which is 100%, will decrease slightly. Therefore, this high rate supports the opinions that the SRB technique should be preferred over FTRB.