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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2021  |  Volume : 64  |  Issue : 2  |  Page : 266-276
Estimation of plasma and RBC acetylcholinesterase in children: An evaluation tool for Hirschsprung's disease?


1 Department of Chemical Engineering, Indian Institute of Technology, Bombay, Powai, Maharashtra, India
2 Department of Pathology, St. John's Medical College, Bengaluru, Karnataka, India
3 Department of Paediatric Surgery, Indira Gandhi Institute of Child Health Hospital, Bengaluru, Karnataka, India

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Date of Submission18-Jul-2019
Date of Decision13-Oct-2019
Date of Acceptance12-Apr-2020
Date of Web Publication9-Apr-2021
 

   Abstract 


Background: Increased acetylcholinesterase (AChE) activity on frozen sections of rectal mucosal biopsies accurately diagnoses Hirschsprung disease (HD). But the quest for a biomarker in blood as a screening test prompts one to look for AChE in blood and study its role in HD diagnosis. Aim: To develop a low-cost reliable method to estimate the AChE activity in plasma and red blood cells (RBCs) in normal children (control) and study its role in HD (test). Materials and Methods: Optimized method derived after modifying and standardizing known AChE assay protocols for blood were employed on 30 controls to define the AChE cut-off range, on 40 suspected HD cases to categorize them as HD/non-HD based on cut-off values and later compared with gold standard tissue AChE histochemistry of rectal mucosal biopsies. Results: An optimal in-house modified methods of Ellman's was found best suited to analyze plasma AChE activity, method by Wilson and Henderson was optimal for extraction and AChE estimation in RBCs. AChE levels (controls) obtained were 1.03 ± 0.31 U/mL and 5.17 ± 1.52 U/mL in plasma and RBCs, respectively while the plasma AChE was 1.35 ± 0.84 U/mL (HD) and 1.62 ± 0.85 U/mL (non-HD) while RBC AChE was 4.29 ± 3.2 U/mL (HD) and 6.48 ± 4.31 U/mL (non-HD). Sensitivity was 66.67% and 55.56%, specificity was 22.73% and 45.45%, and an accuracy rate of 42.5% and 50% for plasma and RBC, respectively. Conclusions: Mutually exclusive AChE activity range identified for test blood samples overlapped with the normal and hence, not considered a diagnostic tool for HD.

Keywords: Acetylcholinesterase enzyme assay, Ellman's method, Hirschsprung's disease, plasma, red blood cells

How to cite this article:
Kuvelkar N, DSouza S, Vidhyashree K, Shankar G, Bukel MF, Noronha S, Kini U. Estimation of plasma and RBC acetylcholinesterase in children: An evaluation tool for Hirschsprung's disease?. Indian J Pathol Microbiol 2021;64:266-76

How to cite this URL:
Kuvelkar N, DSouza S, Vidhyashree K, Shankar G, Bukel MF, Noronha S, Kini U. Estimation of plasma and RBC acetylcholinesterase in children: An evaluation tool for Hirschsprung's disease?. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 May 16];64:266-76. Available from: https://www.ijpmonline.org/text.asp?2021/64/2/266/313259





   Introduction Top


Acetylcholinesterase (AChE) histochemistry on rectal mucosal biopsy is considered as the gold standard and hence a very useful ancillary technique in the routine and intraoperative diagnosis of Hirschsprung's disease (HD).[1] Classical HD characterized by aganglionosis of the rectosigmoid region is treated surgically. The absence of ganglion cells is characterized by increased AChE activity in the neuronal plexuses well noted in the submucosa with varying patterns of enzyme localization in the mucosa. The enzyme changes seen as definite patterns of enzyme localization on frozen sections of rectal mucosal biopsy at microscopy is used to assess AChE activity in the mucosa for rapid diagnosis of HD. This requires the expertise of histopathologist who in turn needs to be equipped with high-end infrastructure settings for enzyme histochemistry on frozen sections of the mucosal biopsy to confirm the same. Due to these constraints in the existing diagnostic tools, this study aims to device a technique that is minimally invasive (blood-based) and interpretable by a clinician in a primary healthcare center to make a definitive diagnosis in patients with lower gut motility disorders such as HD.

We hypothesize that increased AChE activity seen in the rectal biopsy due to aganglionosis, may reflect in increase in AChE activity in the peripheral blood too. To this effect, there have been few earlier studies,[2],[3],[4] which have made an attempt at assessing AChE activity in blood but have not been correlated with the AChE activity of the rectal biopsies studied and therefore have no concrete conclusive data for its implementation in the diagnostic workup. At our translational laboratory for gut motility disorders, which exclusively studies biopsies for HD, we have undertaken this study as phase 1, attempting to initially assess AChE activity in red blood cells (RBCs)[5],[6],[7] and plasma samples. The results of this study will probably help in (a) developing an economically viable assay protocol for estimating AChE levels in the blood (plasma and RBC) with minimal laboratory infrastructure and (b) assess the credibility of using AChE activity in plasma and RBC samples as a diagnostic tool for HD.


   Materials and Methods Top


This prospective double-blind study was conducted on Indian children after obtaining institutional ethics approval (IEC Study Ref. No. 23/2017) at the exclusive Translational Research Laboratory for Gut Motility Disorders and Department of Pathology, St. John's Medical College, Bangaluru, India in the tertiary referral healthcare center that serves as a referral center for the diagnosis of HD.

Recruitment of control patients and AChE activity estimation

Infants and children attending the outpatient department at the tertiary children's hospital from April 2018 to July 2018 and subjected for routine hematology investigations for symptoms not related to gastrointestinal/liver pathology or hematological conditions were recruited as controls. With informed consent, 1 ml of whole blood collected in ethylenediaminetetraacetic acid (EDTA) vacutainers (BD Biosciences) was centrifuged in a four-degree cold centrifuge (Thermo Scientific, Roskilde, Denmark) to separate the plasma from RBCs under strict sterile conditions. Blood samples that were hemolyzed/inadequate or not maintained in a cold chain during transport to the lab were excluded from the study. The algorithmic approach for the work pattern for these samples is depicted in [Figure 1]
Figure 1: Flowchart to explain methodology employed for AChE assay optimization. Method A * In-house Modified Ellman's, Method B ** Wilson and Henderson 2007[11], Method C *** Linhares et al.2013[13], Method 1 x Wilson and Henderson 2007[11], Method 2 y Gupta et al.2015[12]

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Recruitment of test patients and AChE activity estimation

Blood samples of those children suspected of HD (Test patients) referred to this center during the period October 2018 till February 2020 were received along with fresh rectal mucosal biopsies, in a cold chain.

Children were suspected of 'the latter, for diagnostic purposes HD if any of them had one or more of the following symptoms:

  1. Failure to pass meconium within 24 hrs of birth
  2. Abdominal distension/vomiting, mostly non-bilious and constipation dating back to early infancy
  3. Symptoms mentioned above with associated dysmorphology, trisomy 21 and stigmata of neurocristopathies or syndromic
  4. Child with constipation and having a sibling with total colonic aganglionosis or long segment HD.


The AChE activity on blood was estimated using the optimized protocol as shown in [Figure 1], and samples were further classified as HD or non-HD (NHD) based on the AChE activity range obtained from the controls. These results were later compared with the final diagnosis report issued based on AChE histochemistry[8],[9] performed on rectal mucosal biopsies. The detailed algorithmic approach for a given sample is depicted in [Figure 2].
Figure 2: Optimized methodology employed for estimating AChE activity in blood samples. Method A * In-house Modified Ellman's, Method 1 x Wilson and Henderson 2007[11], Method B ** Wilson and Henderson 2007[11]

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   Control Samples Top


AChE assay in plasma

Fresh plasma obtained after centrifuging the blood samples collected in an EDTA vacutainer was employed for the AChE assay. Ellman's method[10] was employed to measure the AChE activity. The latter method was performed using 50 μl of plasma sample, mandating higher concentrations of the reagents required for the assay in a cuvette original protocol. In the given scenario of a neonate, the required plasma volume may not be available. Hence, the method was modified to perform the assay with a smaller volume in this study; 10 μl of the plasma with a proportionate reduction in the reagent concentration best suited for a microtiter plate. This method is being addressed as in-house modified Ellman's method in this study.

The plasma samples were diluted 1:10 with 20 mM phosphate buffer (pH 7.4) and enzyme activity (U/mL) was measured as absorbance change at 412 nm for 5 minutes with a 1-minute interval. Further quantification was carried out using the formula



Where,

Δ Absorbance = Change in absorbance per minute

AChE assay in RBC

The AChE activity in RBCs was assayed as a two-step process. The first step involved lysis of RBCs to extract the AChE followed by the second step of assaying the enzyme for its activity. The AChE enzyme was extracted using the two known protocols namely, Wilson and Henderson[11] (referred to as Method 1) and Gupta et al.[12] (referred to as Method 2) which after lysis, the lysate was centrifuged to obtain the supernatant containing the enzyme for assay. The AChE activity was estimated using three known protocols namely, in-house Modified Ellman's, Wilson and Henderson,[11] and Linhares et al.[13] referred to as Methods A, B, and C, respectively in this study. For the assay, the supernatant was diluted 1:200-fold with 20 mM phosphate buffer (pH 7.4). The enzyme activity (U/mL) was measured for absorbance change at 412 nm using the formula as described earlier for plasma. The initial runs were carried out to select the most efficient protocol for AChE extraction and activity measurement in terms of time required for extraction and ease of performance by a laboratory technician. The optimal protocol confirmed in this study was employed thereafter to assess the range of AChE enzyme activity in the supernatant after RBC lysis on test samples collected for the study.


   Test Samples Top


AChE assay in plasma

Fresh plasma obtained after centrifuging the blood samples collected in EDTA vacutainer was used for the AChE assay. The method optimized for the estimation of AChE activity in the control samples was used to quantify the enzyme activity in test samples. The calculation of enzyme activity was as mentioned for the plasma control.

AChE assay in RBC

The method optimized for the extraction and estimation of AChE in the control samples was used for AChE estimation in test samples. Enzyme activity was calculated as per the specifications of the optimized method for RBC AChE activity estimation.

AChE histochemistry on rectal mucosal biopsies

Frozen sections were cut from fresh rectal biopsies at 10 μm thickness using Leica CM 1950 cryostat at different levels. Two sections were stained with rapid hematoxylin and eosin (H and E) and two with AChE by using protocols standardized in our laboratory for rapid final diagnosis of HD.[8],[9] A frozen section positive for HD was run as a positive control. The frozen tissue remains were preserved at -25°C for further use if necessary. The results were kept blinded until the AChE assay was executed on the plasma and RBC samples.

Performance parameters of the in-house modified AChE assay

Sensitivity, specificity, and prediction accuracy of the in-house modified AChE assay was calculated for plasma and RBC samples using the below-mentioned formula keeping the frozen diagnosis on rectal mucosal biopsies using AChE histochemistry as the gold standard.[1]

Sensitivity = Number of correctly predicted HD cases/Number of True HD cases*100

Sensitivity = Number of correctly predicted NHD cases/Number of True NHD cases*100

Prediction accuracy = Number of correctly predicted cases/Number of total cases *100

The data for the initial RBC standardization was statistically analyzed by performing paired t-test.


   Results Top


In this prospective double-blind study, 40 blood samples in the control group were obtained from neonates and infants of Indian origin in the mean age group of 7.1 ± 4.48 years (M:F = 3:1); 10 samples were excluded as eight were hemolyzed and two were inadequate. In-house modified methods by Ellman and Wilson and Henderson[11] were found to be the most optimal methods for AChE estimation in plasma and RBC, respectively.

The details of the methods used for assessing the activity of AChE in plasma and RBC of the 30 control samples included in the study have been summarized in [Table 1]. The volume of blood sample required for the assay have been scaled down and AChE levels in plasma were studied.
Table 1: Methods employed to estimate AChE activity in plasma and RBC samples

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   Control Samples Top


AChE activity in plasma

The AChE activity assessed initially in the 10 control plasma samples using Method A (in-house Modified Ellman's method) was most optimal and then used further to analyze the rest of the 20 samples. We observed the AChE activity in the plasma of 30 samples of the pediatric cohort to be 1.03 ± 0.31 U/mL [Table 2] and [Figure 3] and was considered as the “reference range for plasma” for further analysis. The following formula was used to calculate the activity:
Table 2: AChE assay values in Plasma and RBC of the 30 control samples

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Figure 3: Plasma AChE assay values in control, HD and non-HD samples, Control indicates those patients who were not referred on suspicion of HD, Test samples (HD/Non-HD) indicates those patients who were referred on suspicion of HD

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Where,

Δ Absorbance = Change in absorbance per minute

Volume of reaction = 200 μL

Dilution factor = 1:10

Volume of sample = 10 μL

AChE activity in RBC

The initial run with 10 RBC samples enabled us in selecting the optimal protocol for both AChE enzyme extraction and estimation of its activity. We observed a combination of Method 1 (Wilson and Henderson, 2007)[11] and Method B (Wilson and Henderson, 2007)[11] to be the best protocol (P < 0.0001) as shown in [Figure 1]. The AChE enzyme activity was found to be 5.75 ± 1.35 U/mL when estimated with the above mentioned combination. [Table 3.1] with statistical significance calculated in [Table 3.2] and shown in [Figure 4]. Using this protocol, the AChE activity was analyzed for the other 20 samples of the cohort. The AChE activity in RBC samples was found to be 5.17 ± 1.52 U/mL [Table 2] and [Figure 5] and was considered as the “reference range for RBC” for further analysis. The following formula was used to calculate the activity:


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Figure 4: Comparison of methods for AChE estimation. AChE enzyme activity estimation method: Method A* Inhouse Modified Ellman's, Method B† Wilson and Henderson 2007[11], Method C Linhares et al. 2013[13] Method 1§ Wilson and Henderson 2007[11], Method 2 || Gupta et al. 2015[12]. Method A1: Method A Method 1; Method B1: Method B Method 1; Method C1: Method C Method 1

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Figure 5: RBC AChE assay values in control, HD and non-HD samples, Control indicates those patients who were not referred on suspicion of HD. Test samples (HD/Non-HD) indicates those patients referred on suspicion of HD

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Where,

Δ Absorbance = Change in absorbance per minute

Volume of reaction = 320 μL

Dilution factor = 1:200

Volume of sample = 30 μL


   Test Samples Top


The test samples comprising 40 infants and children in the mean age group of 2.19 ± 2.84 years (M:F = 3.3:1) were assayed for AChE with the protocol optimized with the control samples.

AChE activity in plasma

The AChE activity was assessed in the 40 test plasma samples using the most optimal Method A (in – house Modified Ellman's method). We observed the AChE activity in the test plasma of 40 samples of the cohort to be 1.35 ± 0.84 U/mL and 1.62 ± 0.85 U/mL in HD and non-HD cases, respectively [Table 4] and [Figure 3].
Table 4: AChE assay values in Plasma and RBC samples from the 40 cases suspected of HD

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AChE activity in RBC

The AChE activity was assessed in the 40 test RBC samples using the optimized protocol namely a combination of Method 1 (Wilson and Henderson, 2007)[11] and Method B (Wilson and Henderson, 2007).[11] The test RBC AChE activity was 4.29 ± 3.2 U/mL and 6.48 ± 4.31 U/mL in HD and non-HD cases respectively [Table 4] and [Figure 4].

AChE histochemistry on rectal mucosal samples

The rectal mucosal biopsies of the test 40 cases were read based on the following criteria:

A rectal mucosal biopsy was considered as HD [Figure 6] if
Figure 6: Shows frozen section of rectal mucosal biopsy in Hirschsprung's disease (HD). (a and b) Stained with H and E show hypertrophic nerve bundles (1.25x, 10x), (c and d) Stained for AChE histochemistry showing increased AChE activity of pattern A as evident by increased AChE positive fibers in the submucosa and muscularis mucosa, diagnostic of Hirschsprung's disease. (4x, 10x)

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  • Hypertrophic nerve bundles were identified in the submucosa with no ganglion cells
  • Increased AChE activity with positive staining of hypertrophic nerve fibers as dark green-black staining in specific patterns of either (1) Pattern A where nerve fibers in the submucosa extending through the muscularis mucosa into the lamina propria akin to an arborizing tree trunk, or (2) Pattern B where nerve fibers extending only up to the base of the crypts, (3) Equivocal pattern where hypertrophic nerve bundles in the submucosa alone with no specific pattern in the lamina propria.


  • A rectal mucosal biopsy was considered as non-HD [Figure 7] if
    Figure 7: Shows frozen section of rectal mucosal biopsy in Non-Hirschsprung's (non-HD) cases. (a and b) Show neither ganglion cells nor hypertrophic nerve bundles (1.25x, 4x), (c and d) AChE histochemistry showing equivocal pattern with no increase in AChE activity evident by the presence of an occasional AChE positive fiber in the submucosa and muscularis indicative of normal innervation (Non-Hirschsprung's disease) (4x, 10x)

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  • At least one ganglion cell was identified in one/both the H and E sections
  • The AChE stained an occasional nerve twig in the submucosa and highlighted a ganglion cell if included in the biopsy. However, no stainable AChE fibers were identifiable (negative staining) in the muscularis mucosa and lamina propria.


Eighteen showed increased AChE activity in the submucosa and/or mucosa with no ganglion cells and were diagnosed as HD mandating surgical intervention. Twenty-two of the 40 cases were ganglionic and showed no increase in AChE activity, thus, diagnosing them as non-HD cases. The results, thus, obtained were correlated with the results of plasma and RBC AChE values as shown in [Table 5].
Table 5: Shows concordance between diagnoses made on AChE assay of plasma and RBCs with diagnosis based on frozen AChE histochemistry

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Performance parameters of the in-house modified AChE assay for plasma [Table 6]
Table 6: Performance parameters of the in-house modified AChE assay

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The in-house modified AChE assay was, thus, found to have a sensitivity of 66.67%, specificity of 22.73% and the prediction accuracy rate of 42.5%.

Performance parameters of the Wilson and Henderson Method for RBC [Table 6]

The Wilson and Henderson method was found to have a sensitivity of 55.56% and specificity to be 45.45%. The prediction accuracy was calculated to be 50%.


   Discussion Top


The AChE enzyme in the enteric nervous system plays a role in terminating neuronal impulses by the catalytic hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid in the synaptic cleft. Absence of ganglion cells as seen in HD results in an increased concentration of acetylcholine[4] and in effect increased AChE activity expressed in the neural plexus and in blood. Blood is considered as the liquid biopsy and easily accessible, its AChE estimation may reflect the tissue AChE levels in HD in the absence of hemolysis and hemolytic disorders. However, AChE enzyme activity in blood samples has not yet been employed as a diagnostic modality in the evaluation of patients with suspected HD. This could probably be due to the lack of evidence of correlation of AChE activity between the two types of samples or probably requires modification of methodology.

The AChE enzyme distribution in whole blood is concentrated in the RBC as a transmembrane protein[5],[6] with the enzyme partitioned in the ratio of 4:1 in the RBC and plasma fraction, respectively. This 4:1 ratio may get altered due to lysis of RBC when the enzyme gains entry into the plasma.[7] Given the partitioning, it could be possible to obtain an exclusive classifying range for AChE activity in plasma and RBC fraction, which could be used for screening of neonates with HD.

Commercially available kits in the market for AChE activity quantification, which are validated to their respective population, are exclusively employed in the management of patients with organophosphorus poisoning and are available only at certain diagnostic centers with a turnaround time of a minimum of 3 days. Additionally, they are expensive, restricting their use in under-developed and developing countries like India. The same kits, however, are not suitable for diagnostic purposes such as for use in infants and children with HD and they may not have been validated in the pediatric population. Keeping this in mind, known AChE assay protocols available for plasma and RBC were tested on Indian children, were further modified and employed in this study to make it economically sustainable for use in any healthcare center. Further, the AChE assays in both plasma and RBCs have been carried out from a single blood sample and the methodology for both have been designed for a smaller volume of blood, i.e., 1 ml, keeping in mind the difficulty of getting blood from neonates and children.

AChE assay in control plasma

Plasma as a sample is easier to work with as it does not require pre-processing steps for the AChE assay. The modifications carried out in the Ellman's protocol included reduction of plasma volume to 10 μL, raised concentration of acetylthiocholine iodide, and 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB) to 1 mM and 0.45 mM from 0.48 mM and 0.32 mM, respectively as noted in [Table 1]. This modified protocol was found to be a one-step simple assay protocol. This study concludes the normal range of AChE activity in the plasma in Indian children to be 1.03 ± 0.31 [Table 2].

AChE assay in control RBC

Assessing the activity of AChE was found to be a relatively complex process given that it is a two-step process (extraction and estimation). Although the principle used by the known methods is the same, the methods differ from each other in certain aspects such as the reaction time, the concentration of the reagents, and the volume of the sample used. We, therefore, tested the efficiency of these methods referred to in [Figure 1] for both the steps, namely extraction and estimation of the AChE. From the initial run of experiments, we found Method 1 (Wilson and Henderson[11]) and Method B (Wilson and Henderson[11]) to be best suited for the extraction of the enzyme and estimation of the AChE activity in RBC fraction, respectively. Similar AChE enzyme activity levels [Table 3.1] were obtained when the RBCs were processed for extraction using Method 1 (Wilson and Henderson[11]) and Method 2 (Gupta et al.[12]) and estimation as per Method B (Wilson and Henderson[11]). However, the shortened extraction step of 1 hour achieved by using 0.5% Triton X -100 in Method 1 as opposed to 0.2% Triton X -100 used in the 20- hour extraction step of Method 2, prompted us to employ Method 1 for the rest of the samples. In addition to the extraction procedure, the assay procedure plays an important role in estimating the AChE activity. We observed that optimal results for AChE enzyme activity were obtained when we employed Method B (Wilson and Henderson[11]) [Table 2].

An AChE activity of 5.17 ± 1.52 U/mL was obtained when a combination of Method 1 (extraction of enzyme) and Method B (estimation of activity) was used to assess the AChE activity in the RBC fraction of Indian children [Table 3.1].

On performing statistical analysis by paired t-test [Table 3.2], we found only Method C Method 1 combination Vs Method C Method C2 to be significant (P < 0.03) when 95 % confidence interval was used. If the confidence interval were to be changed to 99 %, the method would no longer be statistically significant. This indicates that Method 1 or Method 2 can be used for extraction of the AChE from RBC. We preferred Method 1 due to its shorter extraction time as already mentioned. Also of the tested combinations, Method B Method 1 was found to have a wide range of AChE activity value that as compared to others [Figure 5] indicating a relatively sensitivity over the other methods for the same AChE activity level.

AChE assay in test plasma and RBC

On the estimation of AChE activity in the plasma and RBC samples of test patients with the optimized protocol, we observed the test AChE activity range to overlap with the respective control AChE activity range. On calculating the performance parameters, we found that the prediction accuracy for both the in-house modified assay for plasma and RBC was 42.5% and 50%, respectively [Table 6]. Thus, indicating that AChE levels in plasma and RBC were not elevated in HD positive cases to serve as a diagnostic tool, which is against the hypothesis of this study.

Literature review has shown conflicting reports on the utility of AChE activity for diagnosing HD. The first study by Boston et al. in 1978[14] demonstrates that there are systemic effects of HD and are seen as a significantly higher concentration of enzyme in both serum and erythrocytes. On the other hand, the study by Okasora et al. in 1983[15] showed that study of acetylcholine esterase in serum and erythrocytes carried out in nine patients with HD and 16 normal controls has shown significantly elevated enzyme levels in serum, while no significant difference was observed in erythrocytes which is similar to that noted in this study. However, this study is different in many aspects: it is a double-blind study carried out with much larger sample size and age-matched controls with the application of modified improvised rapid techniques of enzyme estimation on plasma and RBCs obtained from a 1 ml of EDTA blood (a better and cheaper anticoagulant) against 3 ml of heparinized blood (relatively more expensive).

Subsequent studies by Bamforth et al.[16] and Atias et al.[17] had concluded that AChE activity in RBC do not differentiate HD from non-HD cases. On the other hand, three studies [Yanagihara,[18] Ya-xiong,[19] and Chalkoo[20]] reported the ability of erythrocyte AChE activity to differentiate between HD and non-HD cases. We further analyzed the above mentioned papers to decipher how distinctly the control AChE activity levels differed from the test AChE activity [Table 7]. In two studies (Yanagihara[18], and Ya-xiong[19]), the AChE activity range in the test overlapped with that of the control. Yet these studies have credited the result to have diagnostic value. The third study (Chalkoo[20]) has considered only those patients suspected of HD in their study without providing data for control patients and hence cannot be discussed upon. Their studies[18],[19],[20] were not blinded and modifications of the study not elaborated upon with respect to procedure.[19] They, thus, failed to differentiate between the test and control groups with a mutually exclusive AChE range for each group.
Table 7: Comparison of the design studies reported in the literature

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Further, the relationship between the increased AChE of the aganglionic colon in HD and that of serum and erythrocytes is not clear and requires further studies. The cause of this elevation will remain speculative as very little is known of the physiological role of the enzymes. Is the cholinesterase present locally in the affected segment of the colon the source of the elevated blood levels in HD? Okasora et al.[15] have suggested the level of serum enzyme level to be closely related to the extent of the aganglionic bowel segment. However, it has not been clarified, whether AChE excessively present in the aganglionic bowel segment is directly released into the bloodstream or not. In two cases, they proved that the AChE levels in inferior mesenteric vein blood samples obtained from pre and post-surgical intervention for HD were the same and state that the aganglionic bowel segment itself is not the site of AChE production which may influence the elevation of AChE in serum. Is there a relative failure locally of the complete hydrolysis of acetylcholine in the affected aganglionic bowel resulting in elevated enzyme activity as a compensatory mechanism for its degradation systemically or is there something in the aganglionic bowel segment that stimulate the production of AChE in the liver via some unknown mechanism. These questions remain open for debate.

In summary, this study documents a mutually exclusive AChE activity range in plasma and RBC samples of normal Indian children without gut motility/liver/hematological disorders and describes simple modified improvised rapid assay protocols which are economically viable (INR 10). This AChE enzyme activity range may help in assessing children with neurological and gastrointestinal disorders. However, the AChE enzyme levels in plasma and RBC, obtained in patients with HD, were not statistically significant to employ it as a screening/diagnostic test for screening and diagnosing HD.

Acknowledgements

We thank Dr. Hemalatha Lokanatha, MD, Professor and Head, Department of Pediatric Hematology, Indira Gandhi Institute of Child Health, Bangalore and her team for helping in sample collection; Dr. Rita Christopher, MD, Professor of Neurochemistry, National Institute of Mental Health and Neurosciences, Bangalore for sharing the assay protocol of AChE estimation in cerebrospinal fluid. We wholeheartedly thank all the paediatric surgeons and their staff who supported this study by providing the blood samples of their patients.


   Ethical Standards Top


Ethical clearance

This study was conducted after the ethical approval in the following three centers - St John's Medical College, Bangalore (IEC Study Ref. No. 23/2017), Indira Gandhi Institute for Child Health, Bangalore (No IGICH/ACA/EC/1/2017-18), and Indian Institute of Technology-Bombay, Mumbai (IITB-IEC/2017/018). All procedures performed in the study involving human participants were in accordance with the ethical standards of the concerned institutes and with the 1964 Helsinki declaration and its amendments. The study did not involve animals.

Informed consent

Informed consent was obtained from the parents of the participants enrolled in the study. Participants' information has not been disclosed in any format in the manuscript or abstract or in the form of images or tables.

Financial support and sponsorship

Dr. Usha Kini was supported by the research grant from Rajiv Gandhi University of Health Sciences, Bangalore, Karnataka, India (Project No 17 MO 12) for this study.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Correspondence Address:
Usha Kini
Translational Research Laboratory for Gut Motility Disorders, Department of Pathology, St. John's Medical College, Bengaluru - 560 034, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_567_19

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