| BackgroundHirschsprung disease (HSCR) is a congenital intestinal disease. The pathologic change is that a portion of the intestinal wall lacks ganglion cells. Its incidence rate is 1:5000; the proportion between male and female is 4:1. Its main cause is that ganglion cells cannot cluster and locate in the intestinal wall. Genetic predisposition and intestinal microenvironment changes may contribute to development of HSCR.Diagnosis of HSCR mainly relies on clinical manifestations noted by an observant caregiver and subsequent diagnostic testing using variously invasive techniques, including barium enema reduction, rectal mucus biopsy, and anorectal pressure measurement. Some newborns with HSCR are missed because their clinical signs are not typical when they are born. In newborn infants, the medical literature reports that 20% of the rectal contrasts of barium enema reduction are mistakenly diagnosed. This is most likely because in newborns, the dividing lines among the spasm section, transfer section, and extension section are not clear; that is, the expression of HSCR X line is not typical in this period. In addition, when carrying out barium enema reduction, because of the improper operation in the preparation of purifying intestine and injecting bubbles into the anus, "false megacolon" may be found. The barium enema is also not without risk. If the barium is not diluted properly, then water intoxication may develop; if before examination the child has developed enterocolitis to the contrast, then enema may result in intestinal perforation and consequently lead to barium peritonitis. Another frequently used diagnostic test, rectal mucus biopsy, is a traumatic and imperfect examination. If the scope of biopsy is extensive, then the trauma is relatively large; if the scope of biopsy is narrow, then the probability of missed diagnosis increases, especially for patients with short-segment and super short-segment HSCR. Besides, transfer mucus receiving histochemical examination is at the risks of hemorrhage and perforation, and the pathologic technology requirements are relatively high. The positive rate is 83%. The diagnostic accuracy rate for anorectal pressure measurement in newborn infants with HSCR is approximately 64.3% to 71.43%; false positives that lead to missed diagnosis may occur as a result of improper operation of the measuring device.SELDI-TOF-MS technology is a new proteome technology that was developed in 2002. It uses the principles of gene chip, reasonably combining chromatography and mass spectrum technology with protein chip, and it is able to detect proteins and peptides that are hard to verify with the traditional methods, although it does not specifically identify the proteins other than by size. It has the characteristics of rapidity, sensitivity, and high throughput, and it has made major breakthroughs in the marker screening and early diagnosis of ovarian cancer, prostate cancer, breast cancer, lung cancer, liver cancer, and colon cancer.Objects: The objective of this study was to identify a specific fingerprint chromatogram model of serum proteins for early screening and diagnosis of Hirschsprung disease.Subjects and materials:1. Subjects Seventy-eight serum specimens were obtained from the Pediatric Surgery Department of the First Affiliated Hospital of Zhengzhou University, among those, 42 case specimens were of patients with HSCR (including long-segment type [7], short-segment type [30], super short-segment type [4], and leap type [1]), 16 specimens were of adhesive ileus including appendicitis (8) and Meckel diverticulum (3) after operation and inflammatory bowel disease (5), and the 20 controls are from healthy children who had a physical checkup in Pediatric Surgery Department in our hospital. All of the specimens with HSCR were previously confirmed by pathologic results. Among the children with HSCR, 33 were male and 9 were female; their ages were 3 days to 18 years.2. Main reagents and devices CHAPS, Urea, DTT, NaAC, SPA (Sinapinic acid), they are all purchased from the Pomega Company of USA. Ciphergen PBS II ~+SELDI -TOF-MS and WCX2 protein chips are purchased from Ciphergen Company of USA.3. Protein chip technical route To unfreeze the serum specimens in ice bath, turn them centrifugally at 10000 rpm under 4℃for 2 minutes. Place a 96-hole plate on the ice box, add 10 ul of U9 (9MUrea, 2%CHAPS, 1%DTT) and 5 ul of serum to each hole, vibrate at 600 rpm under 4℃for 30 minutes in cold lab chamber. Before 15 minutes of finishing the vibration, pre-process the chip. The chip is placed in the Bioprocessor, note down the chip number, add 200 ul of NaNC (100 Mm, pH4) to each hole, vibrate at 600 rpm for 2 minutes in cold lab chamber, and repeat the above-mentioned operation once. Place the 96-hole plate being processed by U9 on the ice, use a medical gun to add 185 ul of NaNC, vibrate at 600 rpm under 4℃for 2 minutes in cold lab chamber. Add 100 ul of processed specimens to the chip, place them in the cold lab chamber under 4℃combining 600 rpm for 60 minutes, swing off the remaining liquid and dry out rapidly. Add 200 ul of NaAC, after vibrating at 600 rpm for 5 minutes, swing off and dry out, repeat this operation for three times. Wash each hole twice using 200 ul of deionized water, swing off the excessive water. After the chip is air dried, each hole is added 50% saturated SPA1 ul in two stages, after drying, place them on the device for testing.4. Data collection and processing Use a protein chip whose molecular weight is known to adjust the SELDI-TOF-MS system, until the tolerance of molecular weight is less than 0.1%. Use mass spectrum reader to analyze the WCX2 protein chip combined with protein. Analysis parameters: laser strength is 170, sensitivity is 6, and the total number of each specimen collection is 140 times. The scope of data collection is 1000-30000 daltons; the optimized scope is 2000-20000 daltons. Use quality control serum to make repeated tests, the coefficients of variation of the peak value and the strength are 0.05% and 19.7% respectively.5. Support vector machine (SVM) Use linear SVM classifier. The selection of feature vector uses the method of statistical filtration combining with model dependent screening, to build discrimination model, use the method of leave-one-out crossing verification to assess the discrimination result of the model.This experiment also uses discrimination analysis method to process mass spectrum data, to verify the result being processed by SVM.6. Statistical analysis After the original data of mass spectrum have been filtered out the noise and after clustering analysis, the mass-charge-ratio peaks data being preliminary screened out is carried out Wilcoxon rank sum test, the testing standard is set at a=0.01.Results:1. HSCR Group and Normal Control GroupAfter the mass spectrum data of the HSCR group and the normal control group were filtered and screened, 213 m/z peaks were attained. After carrying out Wilcoxon rank sum tests to test relative signal strength, 13 m/z peaks with P<0. 01 were obtained. From the random combination of protein peaks with remarkable variation, SVM screened out the combination model with the maximum Youden index of the predicted value, identifying 3 markers positioned at 3221.7, 5639.2, and 6884.2. In the HSCR group, the proteins were not significantly expressed, whereas in normal control group, they were noted to have high expression. Combining 3 potential markers, using the method of leave-1-out to make crossing detection, in the test collection of 62 patients, the specificity of discrimination model was 100%, and its sensitivity was 100%. 2. Adhesive Ileus Group and Normal Control GroupThrough deleting 16 specimens of adhesive ileus including appendicitis and Meckel diverticulum after operation and inflammatory bowel disease and normal control group, there is no significant difference between the 2 groups positioned at the 3 markers.Conclusions: The fingerprint chromatogram model of serum protein using surfaceenhanced laser desorption/ionization time of flight mass spectrometry technology combining support vector machine is a new method of early screening anddiagnosis of Hirschsprung disease that is worthy of additional research and application. |
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