胆道闭锁血清蛋白质标记物的检测

目的 寻找特异性诊断胆道闭锁的血清蛋白标记物.方法 应用表面增强激光解吸电离飞行时间质谱(SELDI-TOF-MS)技术检测28例血清标本(胆道闭锁7例,先天性胆总管囊肿7例,婴肝综合征3例,正常对照11例)的蛋白质质谱表达,结合生物信息学方法(SVM)分析数据.结果 筛选出m/z位于3403、2108、2111、2131、2823的5个蛋白质标记物区分肝胆疾病和正常对照小儿的血清蛋白指纹图谱模型,敏感性94.1%,特异性81.8%;筛选出m/z位于3403蛋白质标记物区分胆道闭锁和正常对照小儿的血清蛋白指纹图谱模型,敏感性100%,特异性100%;筛选出m/z位于3403、4796的2个蛋白质标记物区分胆道闭锁和其他肝胆疾病的血清蛋白指纹图谱模型,敏感性71.4%,特异性80.0%.结论 应用SELDI-TOF-MS结合SVM构建的胆道闭锁血清蛋白指纹图谱模型为胆道闭锁的早期诊断方法开拓了新方向,值得进一步研究.     

血清蛋白质标记物在先天性巨结肠患儿中的应用

目的 通过检测先天性巨结肠(HD)患儿血清蛋白质筛选特异的蛋白质标记物,构建用于HD早期筛选及诊断的血清蛋白质指纹图谱模型.方法 应用表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术检测82例血清标本(HD组42例,其他类型肠梗阻组、健康对照组各20例)的蛋白质质谱,并结合生物信息学方法 (支持向量机)分析数据.结果 1.HD组与健康对照组比较:筛选出3个M/Z位于3 221.7、5 639.2、6 884.2的蛋白质标记物,构建HD早期筛选及诊断模型,3个标记物在HD组低表达,其在HD组和健康对照组的表达强度分别为378.29±273.34、295.65±159.38、444.13±254.06和1 428.18±1 192.61、1 039.60±785.64、1 115.72±680.48,2组比较均有显著性差异(Pa<0.01).经留一法交叉验证,区分HD和健康儿童的血清蛋白质指纹图谱模型的特异性和敏感性均为100%.2.其他类型肠梗阻组与健康对照组比较:在M/Z位于2 694.2、3 520.2位点上均无显著性差异(Pa>0.05).结论 SELDI-TOF-MS结合支持向量机建立HD血清蛋白质指纹图谱模型是早期筛选及诊断HD的一种特异性强、敏感性高的新方法 .     

儿童节细胞神经母细胞瘤血清特异性蛋白的筛选与鉴定

目的构建更为完善的儿童节细胞神经母细胞瘤(GNB)的早期诊断血清蛋白质指纹图谱模型。方法入选30例GNB术前患儿及30例正常对照儿童,采集血清标本;以弱阳离子交换磁珠芯片(MB-WCX)处理血清,用表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)筛选方法检测和分析蛋白m/z峰值,筛选特异性蛋白,通过基质辅助激光解析电离飞行时间质谱(MALDI-TOF/TOF)方法对特异性蛋白进行鉴定。结果 SELDI-TOF-MS质谱筛选GNB术前患儿与正常对照儿童得到m/z峰位于5 920的蛋白标记物,该标记物在GNB术前患儿中高表达(6 180.6±2 328.0),相比正常对照儿童(419.1±493.3),差异有统计学意义(P0.05);经MALDI-TOF/TOF鉴定,该蛋白标记物为类载脂蛋白C-Ⅲ(Apo C-Ⅲ)。结论 m/z峰位于5 920的蛋白标记物考虑为儿童GNB特异性标记物,其有助于儿童GNB的早期诊断。     

急性淋巴细胞白血病患儿血清蛋白质指纹图谱模型建立及应用评价

目的 应用SELDI-TOF-MS技术构建ALL患儿、对照组AML患儿及正常儿童血清蛋白质指纹图谱,寻找血清差异表达蛋白并建立初步模型,分析其在ALL诊断中作用.方法 应用表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术检测98例血清标本(ALL 46例,AML 32例,正常儿童20例)的蛋白质质谱,并...     

基于支持向量机神经母细胞瘤血清蛋白质标记物的检测及临床应用

目的 寻找神经母细胞瘤特异血清蛋白标记物,构建初步诊断模型,并探讨其临床应用价值.方法 收集47例神经母细胞瘤患儿血清标本,30例其它恶性实体肿瘤患儿血清标本以及健康儿童血清标本10例;用ZUCI-Protein Chip Data Analyze System分析软件进行数据处理;经留一法交叉验证,分类器评价模型的预测效果.结果 构建3个模型并筛选出10个蛋白标记物,能够成功区分神经母细胞瘤和健康儿童蛋白质谱的差异,表达模型的敏感性为100%,特异性为100%,区分神经母细胞瘤术前和术后蛋白质潜差异表达模型的敏感性为100%,特异性为100%,区分神经母细胞瘤与其它小儿恶性实体肿瘤血清蛋白质指纹图谱模型的敏感性为88.89%.特异性为100%.结论 用SELDI-TOF-MS及生物信息学技术并结合支持向量机(SVM)初步建立的模型可作为神经母细胞瘤的另一种特异性强、敏感性高的辅助检查手段.     

基于人工神经网络的血清蛋白质指纹图谱模型在先天性巨结肠患儿诊断中的应用

目的建立蛋白质芯片技术检测血清蛋白质指纹图谱的方法，探讨基于人工神经网络（ANN）的血清蛋白质指纹图谱模型在先天性巨结肠（HD）患儿诊断中的应用价值。方法应用蛋白质指纹图谱分析仪测定HD患儿64例、巨结肠类缘病患儿25例和健康儿童23例血清标本的蛋白质指纹图谱，并结合ANN方法进行数据分析。112例标本随机分成训练组66例（HD40例，巨结肠类缘病14例，健康儿童12例）和盲法测试组46例（HD24例，巨结肠类缘病11例，健康儿童11例）。利用从训练组得出的基于ANN的血清蛋白质指纹图谱模型，对46例未知血清进行检测，并与X线影像学检查结果进行比较。结果筛选出质荷比（m／z）位于7211．6和2864．8的蛋白质标志物2个。在HD组表达强度分别为6．15&#177;2．21和2．78&#177;1．21，巨结肠类缘病组表达强度分别为12．82&#177;7．56和4．86&#177;0．91（Pa〈0．01）。筛选出m／z位于6884．2和5639．2的蛋白质标志物2个。HD组表达强度分别为4．09&#177;1．78和15．57&#177;8．87，健康对照组表达强度分别为8．31&#177;3．07和30．31&#177;6．18（P〈0．01）。应用该方法对HD患儿进行诊断的准确率、敏感度和特异度分别为89．13％（41／46例）、87．50％（21／24例）和90．91％（20／22例），明显高于x线影像学检查68．8％（77／112例）、82．8％（53／64例）和50．0％（24／48例）。结论在HD患儿的诊断中，利用从训练组得出的基于ANN的血清蛋白质指纹图谱模型较传统方法有更高的敏感性和特异性。     

儿童肝母细胞瘤潜在标志物载脂蛋白A-I的筛选及鉴定

目的筛选并鉴定出小儿肝母细胞瘤(HB)血清生物学标记物,以助HB的早期诊断。方法使用弱阳离子磁珠技术处理30例HB患儿、20例全身炎症反应综合征(SIRS)患儿和20例正常儿童的血清样品,利用表面增强激光解吸电离飞行时间质谱(SELDI-TOF-MS)平台筛选出肿瘤组和正常组的差异性蛋白。SDS-PAGE纯化分离出目标蛋白质后,应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)鉴定出目标蛋白质的氨基酸序列,并通过Swiss Prot数据库查找与之匹配的蛋白质。最后使用Real-time PCR和ELISA技术验证该蛋白质的表达情况。结果 SELDI-TOF-MS筛选并排除炎症因子干扰后,发现HB组和正常组存在质合比为9 348 Da的差异性蛋白质,在HB组的表达较低(P0.05)。经鉴定此蛋白为载脂蛋白A-I(Apo A-I)。Real-time PCR和ELISA验证Apo A-I基因和蛋白在HB组均低表达,在正常组高表达。结论 Apo A-I可作为HB非炎症性蛋白质标记物,对其早期诊断有意义。     

应用蛋白质芯片技术筛选急性淋巴细胞白血病患儿血清标志物的研究

目的探讨儿童急性淋巴细胞白血病(ALL)急性期、缓解期(CR)患儿血清蛋白质谱的变化,旨在筛选可用于ALL早期诊断、病情变化监测及预后判断的蛋白质标志物。方法应用蛋白质芯片CM10及表面增强激光解吸/离子化飞行时间质谱(SELDI-TOF-MS)技术获得ALL、正常健康儿童血清蛋白表达指纹图谱,并用Biomarker Wizard和Biomarker Patterns System5.0软件对数据进行分析,建立诊断模型。结果通过检测、分析发现,ALL组与正常对照组比较有显著差异(P<0.05)的蛋白质峰有14个,并获得质荷比(M/Z)为2770.43、7576.2和5288.68的蛋白质在ALL患儿血清蛋白质谱中波峰强度明显升高;进一步利用这3个蛋白质建成的诊断模型,可将ALL与正常儿童正确分组,其正确分组率分别为93%(25/27)和90%(19/21)。ALL完全缓解后血清蛋白质谱中,原高表达的蛋白质明显下调。结论SELDI-TOF-MS蛋白质芯片技术是一种快速、简单易行、用量少和高通量分析方法,能够直接检测出ALL患者血清中特异的蛋白标志物,其对于ALL的早期诊断具有一定的临床意义。     

肾母细胞瘤血清蛋白质标记物检测与分期模型构建研究

目的筛选出肾母细胞瘤患儿特异性血清蛋白质标记物，建立肾母细胞瘤临床分期模型与CT分期进行对照分析，并评价其临床应用价值。方法应用SELDI-TOF-MS技术检测80例血清标本（术前肾母细胞瘤30例，其他恶性肿瘤30例，正常儿童20例），用ZUCI-Protein Chip Data Analyze System分析软件进行数据处理，结合支持向量机（support vector machine，SVM）建立肾母细胞瘤临床分期模型。结果筛选出2个m／z位于4153．9和3257．6的蛋白质标记物，区分肾母细胞瘤Ⅲ和Ⅳ期与肾母细胞瘤Ⅰ和Ⅱ期蛋白质谱差异表达模型的敏感性为100％，特异性为93。8％；区分肾母细胞瘤与正常儿童、腹腔实体肿瘤及肾脏其他恶性肿瘤的特异性是100％，敏感性是100％、80．0％、100％；临床分期模型可以特异性地将各期区分开来，其特异性及敏感性均为100％；通过肾母细胞瘤早期诊断模型中的2个m／z（6984．4，6455．5）血清标记物进行分析得出肾母细胞瘤各期情况如下：Ⅳ期相对于Ⅲ期低表达；Ⅲ期相对于Ⅱ期低表达；Ⅱ期相对于Ⅰ期低表达；Ⅰ期相对于正常儿童低表达；后者相对高表达；临床分期越晚，m／z强度就越低表达。蛋白芯片分期准确性与病理一致，达到100％，在分期定性问题上优于CT（Ⅰ期100％，Ⅱ期85．0％，Ⅲ期85．0％，Ⅳ期75．0％）。结论用SELDI-TOF-MS结合SVM建立的肾母细胞瘤临床分期模型可弥补CT在肾母细胞瘤分期定性问题匕的不足。     

胆道闭锁和淤胆型肝炎患儿血清蛋白质指纹图谱比较

目的 比较胆道闭锁与淤胆型肝炎患儿血清蛋白质指纹图谱,以发现二者的差异蛋白质分子.方法 收集60日龄前就诊、经手术证实为胆道闭锁和经过治疗、随访排除胆道闭锁的其他淤胆型婴儿肝炎患儿各21例的血清,采用弱阳离子交换蛋白质芯片(WCX2)为检测介质,经表面加强激光解离-飞行时间-质谱(SELDI-TOF-MS)测定,得到蛋白质指纹图谱,并运用BioMarker Wizard 软件分析比较.结果 在质荷比(m/z)1 500～30 000,检测出的25个蛋白峰中,强度变异系数15%;比较二者血清蛋白质指纹图谱,发现质荷比为8 677的蛋白质峰在胆道闭锁患儿组下调(P＜0.05).该蛋白质的减少可能在胆道闭锁的形成中起到一定作用,可能是胆道闭锁的蛋白质分子标志.根据发现的差异蛋白的分子质量及等电点,在Swiss蛋白质数据库中检索,发现与载脂蛋白AⅡ非常接近.结论 SELDI-TOF-MS是一种快速、简便和高通量的分析方法,重复性好,与主要针对大分子质量的双相电泳研究在技术上形成互补,能直接筛选出胆道闭锁与淤胆型肝炎患儿的血清蛋白质指纹图谱中的差异蛋白质分子,可望用于胆道闭锁的早期鉴别诊断.     

新生儿先天性巨结肠直肠肛管压力监测及其临床意义

目的　探讨直肠肛管测压检查在新生儿先天性巨结肠 (HD)早期诊断中的应用价值。方法　对 4 2例经手术病理或直肠粘膜活检确诊的新生儿HD患儿的术前直肠肛管压力监测结果进行评价分析 ,并与X线钡灌肠进行比较。结果　直肠肛管测压 18例未出现直肠肛管抑制反射(RAIR) ,12例RAIR反射异常 ,确诊HD30例 ,诊断符合率 71 4 3%。X线钡灌肠确诊HD 5例 ,可疑HD14例 ,诊断符合率 4 5 2 4 %。直肠肛管测压阳性而钡灌肠阴性者 16例 ,钡灌肠阳性而直肠肛管测压阴性者 5例。检查结果的不同部分进行统计学处理 χm2 =4 76 ,P <0 0 5 ,差异有显著意义。结论直肠肛管压力监测在新生儿HD诊断中优于X线钡灌肠。该检查方法简单、安全、有效、无创伤性 ,可作为新生儿期怀疑HD患儿的首选检查 ,但必须结合其他检查手段才能作出确切诊断     

Analysis of renal biopsies performed in children with abnormal findings in urinary mass screening

Background: School urinary mass screening tests are performed to make early diagnosis and provide proper treatment for chronic renal diseases. However, very few systemic analyses or studies have been reported regarding final diagnosis made on children with abnormal urinary screening results. Aim: To study the cases of renal biopsy in children detected in urinary screening. Methods: We retrospectively analysed 461 cases of renal biopsy performed on children referred to us with abnormal school urinary mass screening results who satisfied indications for renal biopsy. Results: Pathologically abnormal findings were observed in 285 (61.8%) patients. Thin glomerular basement membrane disease was detected in 127 (27.5%) cases and IgA nephropathy in 121 (26.2%) cases. Among those 461 children, microscopic haematuria was observed in 289 (62.7%) patients, proteinuria in nine (2.0%), and both in 163 (35.4%). In addition, a statistically higher rate of pathological abnormalities on renal biopsy was noted in the group with microscopic haematuria combined with proteinuria and also in cases with more severe haematuria.

Conclusion: School urinary mass screening has greatly contributed to diagnosing chronic renal diseases. Continuous medical observation is required when abnormal urinalysis is observed, and a more aggressive medical approach such as renal biopsy should also be performed if necessary.     

Serum levels and differential expression of CD44 in childhood leukemia and malignant lymphoma: Correlation with prognostic criteria and survival

BACKGROUND: The CD44, a cell surface proteoglycan, participates in a variety of function including tumor dissemination and metastasis. However, there are no available data on the prognostic significance of CD44 expression of tumor tissue correlated with serum sCD44 level in childhood leukemias and lymphomas. METHODS: Serum levels and leukemic cell tumor tissue expression of CD44 were detected in 54 children with acute leukemia and malignant lymphoma. Serum samples were obtained from all patients before treatment and during remission. Twelve age-matched healthy children were included as a control group. RESULTS: The serum CD44 levels were significantly higher in patients with Hodgkin's disease (HD), non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL) and acute lymphoblastic leukemia (ALL) than those in the control group. The median values were 1627.0, 1336.0, 1318.5, 1730.4, 902.7 ng/mL, respectively, and P<0.001, P<0.01, P<0.01, P<0.05 in comparisons, respectively. However, there was no significant difference between acute myeloid leukemia (AML) and the control group (median values: 900.3 and 902.7 ng/mL, respectively, P>0.05). Serum sCD44 levels significantly declined in HD, NHL and ALL patients who were in complete remission (median values: 684.0, 573.8 and 1101.1 ng/mL, respectively, P<0.05 in each comparison). Patients with HD had higher levels of serum sCD44 and correlated well with higher erythrocyte sedimentation rate (ESR), B-symptoms and advanced-stage disease (P<0.05, P<0.05 and P<0.01, respectively). Expression of CD44 was significantly high in patients with HD and NHL who were in advanced stages of disease. High serum CD44 level was also associated with high tumor tissue expression of CD44 in patients with HD and BL. In addition, patients with higher levels of serum sCD44, had a poorer outcome and survival than those with lower sCD44 levels in HD and NHL groups. CONCLUSIONS: A high serum sCD44 level and/or tumor tissue expression at diagnosis is associated with poor prognostic criteria and/or unfavorable outcome in childhood leukemias and lymphomas.     

Effectiveness of the screening programme for galactosemia. New strategy in Poland

Galactosemia is an autosomal recessive disease related to deficiency of one of three different enzymes involved in the metabolism of galactose: galactokinase (GALK), galactoso-J-phosphate uridyltransferase (GALT) or UDP-galactose-4-epimerase (GALE). Classic galactosemia is due to GALT deficiency and is the most common. Longitudinal studies have shown that in spite of early diagnosis and early treatment of children with galactosemia detected in the mass screening programme, the results are poor and mental retardation as well as other complications are of similar severity as in children diagnosed clinically without screening. In many investigations it was also proved that some impairments developed already in the prenatal period. Therefore, many countries among them also Poland, stopped mass screening for galactosemia. At present, in Poland the procedure strategy in galactosemic children and their families include: diagnosis of new cases on the basis of clinical symptoms, selective screening in high-risk families, prophylactic lactose-free diet for mothers during pregnancy. Such management can help to prevent clinical manifestations in newborns and prevent death in the early period of life.     

Guidelines for Mass Screening of Congenital Hypothyroidism (2014 revision)

Purpose of developing the guidelines: Mass screening for congenital hypothyroidism started in 1979 in Japan, and the prognosis for intelligence has been improved by early diagnosis and treatment. The incidence was about 1/4000 of the birth population, but it has increased due to diagnosis of subclinical congenital hypothyroidism. The disease requires continuous treatment, and specialized medical facilities should make a differential diagnosis and treat subjects who are positive in mass screening to avoid unnecessary treatment. The Guidelines for Mass Screening of Congenital Hypothyroidism (1998 version) were developed by the Mass Screening Committee of the Japanese Society for Pediatric Endocrinology in 1998. Subsequently, new findings on prognosis and problems in the adult phase have emerged. Based on these new findings, the 1998 guidelines were revised in the current document (hereinafter referred to as the Guidelines). Target disease/conditions: Primary congenital hypothyroidism. Users of the Guidelines: Physician specialists in pediatric endocrinology, pediatric specialists, physicians referring patients to pediatric practitioners, general physicians, laboratory technicians in charge of mass screening, and patients.     

C-reactive protein (CRP) as tumor marker in pediatric and adolescent patients with Hodgkin disease

BACKGROUND: C-reactive protein (CRP) is an acute phase protein produced in the liver. An elevated CRP is a nonspecific marker of inflammation. Additionally, it also appears to be a prognostic marker in several malignancies. Elevated CRP levels in adult patients with Hodgkin disease (HD) were reported previously. However, levels of CRP have not been evaluated in pediatric and adolescent HD patients. PROCEDURE: We analyzed CRP serum levels in 95 consecutive pediatric and adolescent patients with Hodgkin disease. CRP levels were correlated with stage, absence or presence of B symptoms, and prognosis. RESULTS: At the time of diagnosis increased serum CRP levels were found in 64 % (61/95) of the patients with a median of 21 mg/L (range: <5-211). Serum C-reactive protein levels correlated with stage and were higher in patients with B symptoms. Higher CRP levels were associated with an increased risk of relapse. CONCLUSION: In addition to soluble interleukin 2 receptor (sIL-2R) levels, CRP holds promise as a diagnostic and prognostic index and follow-up monitor in pediatric and adolescent patients with Hodgkin disease, and merits further investigation.     

Impact of antenatal screening on the presentation of infants with congenital heart disease to a cardiology unit

AIM: Antenatal diagnosis of congenital heart disease (CHD) facilitates prenatal treatment and optimal perinatal care. This has been demonstrated to improve perinatal mortality and morbidity in neonates with CHD. Thus, antenatal diagnosis of CHD is most likely to benefit patients who require surgery in early infancy. We aimed to examine the frequency of antenatal diagnosis in neonates presenting to The Royal Children's Hospital severe CHD. METHODS: Main outcome measures were antenatal diagnosis and whether the individual lesion would have been expected to be detected on a four-chamber view or four-chamber and outflow tract view during a routine obstetric anomaly ultrasound. Poisson regression was used to estimate the average trend over the study period. RESULTS: A total of 610 patients met the inclusion criteria, of whom 164 had an antenatal diagnosis (26.8%). If routine ultrasound screening was ideal, we would have expected 63.9% of cases to be detected on four-chamber view and 83.6% on four-chamber and outflow tract view. Trend analysis demonstrated an annual rate of improvement of 9% in actual versus expected antenatal diagnosis of CHD. Malformation-specific analysis showed that antenatal detection was the highest for double inlet/outlet ventricle (51.3%, 95% confidence interval 34.8-67.6%) and the lowest for simple transposition of the great arteries (15.6%, 95% confidence interval 9.0-24.5). CONCLUSION: Despite mass screening for congenital malformations in Victoria with routine antenatal ultrasounds, a large proportion of neonates with severe congenital heart disease still present without an antenatal diagnosis.