| Background:Prolonged cholestatic jaundice in the neonatal period is a common pediatric clinical category of disease with complex etiology, and it is still a difficult problem in differential diagnosis. The two main causes of neonatal cholestasis are neonatal hepatitis syndrome and extrahepatic biliary atresia, which constitute 50-70% of cases, it is extremely important to distinguish between the two for appropriate management. Intrahepatic cholestatic jaundice resulting from neonatal hepatitis, infectious disease, metabolic abnormalities or enzymatic defects can be treated medically or managed conservatively. On the other hand, biliary atresia can be a devastating disease in infancy, resulting in cirrhosis, liver failure and death if not corrected surgically in a timely. Because early surgical intervention significantly improves the ouTcome in biliary atresia, it is important to have a reliable noninvasive test to distinguish it from other causes of cholestatic jaundice. Hepatobiliary scintigraphy is one of the oldest and most widely used tests for evaluation of neonatal cholestasis. In 2004, the Cholestasis Guideline Committee of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN) examined the value of diagnostic tests commonly used for the evaluation of cholestatic jaundice and how they can be applied to the clinical scenario. About hepatobiliary scintigraphy, these guidelines stated, "Although the high sensitivity for biliary atresia makes this a fairly good single test for detecting disease, it is time-consuming and expensive and has significant false positive and false-negative results. Hepatobiliary scintigraphy generally adds little to the routine evaluation of the cholestatic infant but may be of value if other means for excluding biliary obstruction are not available".This subject adopted the method of combining prospective and retrospective,63 cases of hospitalized children with persistent jaundice systematic examined from January 2007 to January 2014 in department of nuclear medicine of Nanfang hospital for laboratory examination as well as the history of follow-up in order to understand the prognosis of infantile cholestasis liver disease, to explore the role of radionuclide hepatobiliary scintigraphy in the differential diagnosis of the cause of persistent jaundice, and to save time for early diagnosis and timely treatment.Objection:1. To investigate the value of 99mTc-EHIDA hepatobiliary scintigraphy combined with total bile acid (TBA) and y-glutamyltransferase (y-GT) detection in the differential diagnosis of persistent jaundice induced by infantile hepatitis syndrome (IHS) and congenital extrahepatic biliary atresia (EHBA).2. Because of the limit of 99mTc-EHIDA hepatobiliary scintigraphy in the diagnosis of severe cholestatic infant hepatitis syndrome, to establish extrahepatic cholestasis model by bile duct ligation in rabbits, for testing 99mTc-MIBI used as a new hepatobiliary scintigraphy imaging agent, and evaluate value in diagnosis of severe cholestatic infant hepatitis syndrome.3. To establish models of extrahepatic cholestasis and hepatitis, observe the characteristics of 99mTc-MIBI hepatobiliary scintigraphy, and study P-glycoprotein (P-gp) expression in liver, renal and intestinal tissue of rats, and discuss their correlation.Materials and methods:1 Value of hepatobiliary scintigraphy combined with total bile acid and y-glutamyltransferase detection in etiological diagnosis of the persistent jaundice infants.A total of 63 persistent jaundiced patients were examined in department of nuclear medicine of Nanfang Hospital from January 2007～January 2014, excluding three patients over the age of two years. A retrospective analysis of 60 infants with persistent jaundice undertaking 99mTc-diethylacetanilide iminodiacetic acid (EHIDA) hepatobiliary scintigraphy was done in department of nuclear medicine of Nanfang hospital by single photon emission computed tomography (SPECT). Meanwhile, these infants’sera were collected and measured by AU5431 automatic biochemical assay; the sensitivity, specificity and accuracy of hepatobiliary scintigraphy with TBA and y-GT were evaluated.For hepatobiliary dynamic imaging, the detector of the SPECT were played the surface of the region of liver and intestinal tract, the hepatobiliary dynamic imaging was done by means of 5s/frame (1min), and 1min/frame (4min) with continuous dynamic acquisition. Then the plane static imaging was collected at 5 minutes,30 minutes and 1 h with anterior position, respectively. Supposing the radioactive didn’t emerge in the area of intestinal tract at 1 h, the delayed imaging was done in 3 to 4 hour,6 to 8 hour, and 24 hour. When radioactive was found in intestinal tract, the imaging was stopped. In order to exclude the surface pollution from urine radioactive it is necessary to add lateral imaging.The measurement data were presented as mean and standard deviation(x±s), the data of two independent samples were compared by Mann-Whitney U test, receiver operating characteristic curves were used to compare test performance, and there is statistical significance when probability is less than 0.05.2 Differential diagnostic value of 99mTc-MIBI hepatobiliary scintigraphy in infants with severe cholestasis.2.1 Clinical dataA total of 18 persistent severe cholestasis patients were examined in department of nuclear medicine of Nanfang Hospital from June 2011-January 2014. Criteria of requirement:1) TB<85.1 μmol/L and DB>17.1μmol/L or TB>85.1 μmol/L and DB/TB>20%; 2)99mTc-EHIDA hepatobiliary scintigraphy:radioaction was not found in intestinal tract within 24 hour; 3) unknown etiology or inefficiency after clinical treatment.99mTc-EHIDA hepatobiliary dynamic imaging was done by means of 5s/frame (lmin), and lmin/frame (4min) with continuous dynamic acquisition and the delayed imaging was done in 5 minutes,30 minutes,1 hour,3 to 4 hour,6 to 8 hour, and 24 hour after injection. Supposing the radioactive did not emerge in intestinal tract at 24 h, then 99mTc-MIBI was intravenously injected in the next day, and the imaging was stopped when radioactive was found in intestinal tract.2.2 Experimental animals and methods2.2.1 Experimental animals5 New Zealand rabbits (ordinary level), the average weight 2.5kg, were purchased from the laboratory animal center of Southern Medical University.2.2.2 The cholestasis model induced by extrahepatic bile duct ligationThe rabbit was fasted for 12 hours before operation, and then administered anesthesia with 3% sodium phenobarbital, the rabbit skin was cleaned and disinfected in operation field. The incision in rabbits’ upper abdomen was chosen, the bile duct along the hepatoduodenal ligament was found and then ligated above the duodenum, last layered suture was done and penicillin injection after operation was given.2.2.3 Blood biochemical examinationAt the day before operation and at first, second, third, fifth, seventh day after operation, the rabbit’s serum was taken for testing the serum bilirubin level and liver function index. After 1 week the rabbit was sacrificed and the liver tissue was taken for pathological examination.2.2.4 Experimental animal imaging2.2.4.1 Normal animal imagingThe rabbit was fasted for 4-6 hours before imaging. After=anesthetized successfully,99mTc-EHIDA was intravenously injected into the rabbit for continuous dynamic acquisition of SPECT in 1 hour. Supposing the radioactive did not emerge in intestinal tract at 1 h, the delayed imaging was done at 2 h,3 h, and 4 h, respectively. Then 99mTc-MIBI was injected intravenously in the next day, the imaging was stopped when radioactive was found in intestinal tract.2.2.4.2 Postoperative imaging of bile duct ligationAt the fourth day after bile duct ligation, animal model of biliary atresia was established successfully, and the imaging agents and the imaging method were similar to the preoperative’.3 The P-gp expression of liver, kidney and intestines in rats’model with cholestasis and its clinical significance.3.1 Experimental animals10 SD rats, at age of 6 to 8 weeks-old, weight 180～210g, were bred in the laboratory animal center of Southern Medical University.3.2 Establishing animal models of cholestasis3.2.1 The group of bile duct ligationThe method of bile duct ligation saw the section 2.2.2.Animal model of extrahepatic cholestasis was established successfully in rats by bile duct ligation, the imaging of postoperative was performed at the end of second, third, fourth weeks.3.2.2 The group of CCl4 inducedThe rat was perfused at 3.0 ml per kg with 40% CCL4 of peanut oil solution twice a week by gastric tube, and given a mixed solution of edible alcohol and distilled water as drinking water for drinking.After animal model of cholestasis hepatitis was successfully established, animal imaging was performed at the end of fourth week of the experiment.3.3 Experimental Animal ImagingAfter rats were anesthetized successfully,99mTc-MIBI was intravenously injected for SPECT dynamic imaging. Supposing the radioactive did not emerge in intestinal tract at 1 h, the delayed imaging was done in 2 h,3 h, and 4 h, respectively.3.4 Sample collection and processingAll rats were sacrificed at the end of imaging, some organs and tissues such as liver, kidney, duodenum, jejunum, ileum colon and cecum were taken by opening the abdominal cavity, and placed in 4% paraformaldehyde for fixation.3.4.1 The preparation of paraffin section3.4.2 Hematoxylin-Eosin staining procedure and mounting3.4.3 Expression of P-gp in liver, kidney and intestines tissue of rats detected by immunohistochemistry.Results:1 Diagnostic value of hepatobiliary scintigraphy combined with total bile acid and y-glutamyltransferase detection in etiological identification of the persistent jaundice infants.1.1 Comparison of radionuclide hepatobiliary scintigraphy between EHBA and IHS groups.The sensitivity of 99mTc-EHIDA hepatobiliary scintigraphy in the diagnosis of IHS and EHBA was respectively 100%(17/17) and 67.57%(25/37), the specificity was respectively 67.57%(25/37) and 100%(17/17), and the accuracy was respectively 77.78%(42/54) and 77.78%(42/54).1.2 Comparison of liver function index and bilirubin level between EHBA group and IHS groupThe level of TBA and y-GT were higher in infants with EHBA than that in IHS (all P<0.05, U=209.0,19.5, respectively), and ROC curve analysis indicated that TBA in the IHS group and y-GT in EHBA group had some diagnostic value (AUC=0.736,0.968, respectively).1.3 The diagnostic efficacy of hepatobiliary scintigraphy combined with total bile acid and y-glutamyltransferase analysisIt was suggested that the sensitivity, specificity and accuracy for EHBA diagnosis were 100%(17/17),100%(37/37) and 100%(54/54), respectively, when there was no radioactive appearance in intestines in 99mTc-EHIDA hepatobiliary imaging, and TBA and y-GT were 98.5μmol/L and y-GT was 298U/L, respectively.2 Differential diagnostic value of 99mTc-MIBI hepatobiliary scintigraphy in infants with severe cholestasis.2.1 Results of 99mTc-MIBI hepatobiliary scintigraphyA total of 18 severe cholestasis infants were recruited, of which 6 cases were diagnosed as biliary atresia, including male 5 cases and female 1 case, and another 12 cases were diagnosed as infantile hepatitis syndrome, including male 6 cases and female 6 cases.2.2 Comparison of liver function index and bilirubin level between EHBA group and IHS groupThe level of TBIL、DBIL、IBIL、AST、ALT、ALP、LDH、TBA and γ-GT had no statistical significance between EHBA group and IHS group(P>0.05).2.3 The variation of liver function index and bilirubin level in experimental animalAt 1 week after bile duct ligation, liver function index and bilirubin level were significantly higher than that at preoperation. With the obstruction time prolonged, the level of ALT, AST, TBIL, DBIL and IBIL gradually increased, and reached the peak at fifth day, and then decreased gradually, while the level of ALP and y-GT trended to sustain increasing.2.4 Hepatobiliary scintigraphy of animals2.4.1 Hepatobiliary scintigraphy in normal animalsAfter intravenous injection of 99mTc-EHIDA, gallbladder, bile duct and duodenal clearly visualized at 15～30min and a large amount of radioactivity was seen in the intestinal tract at 30min. While after intravenous injection of 99mTc-MIBI at the next day, slight radioactive can be seen in gall bladder and intestinal tract at 20 ～30 min, and a large amount of radioactive was found in intestinal tract with the extension of time.2.4.2 Postoperative imaging of bile duct ligationAfter intravenous injection of 99mTc-EHIDA at fourth day after bile duct ligation, radioactive did not appear in gallbladder, bile duct and intestine within 4 h. However after intravenous injection of 99mTc-MIBI at the next day, slight radioactive can be visualized in intestinal tract at 60-75min.3 The expression of P-gp in liver, kidney and intestines of Rats’ model with cholestasis and its clinical significance.3.1 Expression of P-gp in liver, kidney and intestines tissue of rats detected by immunohistochemistry.P-gp was expressed in liver, kidney and intestinal tissues.In normal liver, only the apical membrane of the epithelial cells in bile ducts was weakly positive. At 2 week after bile duct ligation, with the obstruction time prolonged, the expression degree of P-gp in liver cell membrane increased gradually, and the expression area also enlarged gradually. However liver cell damage was serious at four weeks after operation and the expression of P-gp decreased in some of the specimens. In the group of CCl4 induced, the positive reaction was found in the membrane of liver cell and hepatic portal vein, the expression level of P-gp was similar to the group of bile duct ligation.In the normal kidney, immunoreactivity was obvious in the proximal and distal tubules. Expression of P-gp was positive in the glomerular and glomerular basal membrane, in which was strong positive with the obstruction time prolonged after bile duct ligation. In the group of CCl4 induced, the expression level of P-gp in the proximal tubules and the glomerular basal membrane was also strongly positive.In the normal intestines, P-gp was localized in the apical membranes of the enterocytes and the epithelial cells of intestinal gland, and showed positive in duodenum, jejunum and ileum and strong positive in colon and cecum in rats. After bile duct ligation, the expression level of P-gp declined gradually in duodenum, jejunum and ileum apart from colon and cecum segments with the obstruction time being longer. In the group of CCl4 induced, the level of expression of P-gp in duodenum, jejunum and ileum had no statistical significance between groups of bile duct ligation at the third week and CC14 induced at the end of fourth week of the experiment, but the positive expression in colon and cecum was weaker than the group of bile duct ligation.Conclusion:1 99mTc-EHIDA hepatobiliary scintigraphy combined with TBA and γ-GT examination can effectively identify EHBA and IHS early, noninvasively and safely, and have important role for further treatment in infants with persistent jaundice.2 For clinical infant hepatitis syndrome caused by severe cholestatic liver disease, the diagnostic sensitivity of 99mTc-MIBI hepatobiliary imaging was higher than that of 99mTc-EHIDA. But the imaging results of bile duct ligation animal model show that the intestinal system seems to have autocrine function; the differential diagnosis value of 99mTc-MIBI imaging agent cannot be applied to the differential diagnosis of the children with serve cholestasis jaundice.3 The extrahepatic cholestasis model and hepatitis model are successfully established, and the research results show that the excretion of 99mTc-MIBI is associated with P-glycoprotein expression. There is a direct excretion pathway from the circulation system to intestinal system; and it is also show that the 99mTc-MIBI imaging agent cannot be applied to the differential diagnosis of the children with serve cholestasis jaundice. |
PDF Full Text Request