新生儿17α-羟孕酮和总半乳糖测定试剂及妊娠相关蛋白A(PAPP-A)质控品的研制
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摘要
研究背景及目的：
本篇论文主要包括两部分内容。
第一部分为新生儿筛查试剂的研制,包括新生儿17α-羟孕酮定量测定试剂(时间分辨免疫荧光法)和新生儿总半乳糖定量测定试剂(荧光分析法)的研制。
新生儿筛查是指用快速、敏感的实验室方法对新生儿的先天性内分泌异常、遗传代谢病以及某些危害严重的遗传性疾病进行筛查,以期对患病新生儿进行早期诊断、早期治疗,防止其机体组织器官发生不可逆的损伤,从而避免精神发育不可逆损害、智力低下、言语障碍甚至死亡。目前在我国新生儿筛查的基本项目是甲状腺功能低下症和苯丙酮尿症,随着新生儿疾病筛查技术发展,筛查病种越来越多。部分地区相继开展了葡萄糖6-磷酸脱氢酶缺乏症、先天性肾上腺皮质增生症、半乳糖血症、氨基酸代谢病等疾病的筛查工作。
先天性肾上腺皮质增生症(congenital adrenal hyperplasia, CAH)属常染色体隐性遗传病,是一组肾上腺皮质激素合成过程中某种酶先天性缺陷所引起的疾病。受累女性新生儿可有外生殖器男性化体征,男性则出现假性性早熟;并发的醛固酮缺失可引起发育停滞、血容量减少、新生儿肾上腺危象、休克,严重者甚至危及生命。半乳糖血症则是由于代谢过程中酶缺陷,半乳糖及其代谢产物在血液和组织中堆积所致的一种代谢功能紊乱的症候群,亦为常染色体隐性遗传性疾病。主要受损器官为肝肾、脑及晶状体,引起肝硬变、智力发育障碍、白内障等。这两种先天性疾病若能在新生儿早期得到确诊,有助于降低患儿病死率、减少伤残率,对促进我国儿童的正常生长发育非常重要。
临床上多采用检测血中17α-羟孕酮含量进行新生儿CAH筛查。这是因为17α-羟孕酮是皮质醇的前体之一,其水平可反映体内21-羟化酶的活性状态。而CAH中最常见的是21-羟化酶缺乏(约占90%～95%)。因此测定新生儿血中17α-羟孕酮含量可筛查大部分CAH。目前17α-羟孕酮进口或国产试剂,其采用的检测方法多为时间分辨免疫荧光法,这是一种基于以镧系稀土离子作为标记物来标记抗原或抗体的高灵敏度定量免疫检测技术。它具有制备简便、储存时间长、无放射性污染、重复性好、操作流程短、剂量-反应曲线范围宽、不受样品自然荧光干扰等优点,与酶免分析、放射免疫分析等相比,临床应用价值更高。而对于半乳糖血症,国外通常采用荧光分析法测定新生儿血中总半乳糖含量来进行筛查。荧光分析法是利用物质的荧光特性对样品进行定性和定量分析的方法,具有灵敏度高、方法简便、重现性好、剂量-反应曲线范围较宽等特点。
目前我国这两种先天性疾病筛查用试剂主要为进口试剂,如美国Perkin-Elmer公司、芬兰Labsystem公司等,只有一家国产17α-羟孕酮检测试剂,而总半乳糖检测试剂则暂无国产试剂。进口试剂价格昂贵,检测成本较高,临床应用难度大,不利于筛查项目的开展,而国产试剂虽价格较低,但生产厂家较少,远远不能满足我国新生儿CAH和总半乳糖血症的筛查需求,这也是我国这两种新生儿疾病筛查率较低的主要原因之一,因此我们拟研制新生儿17α-羟孕酮定量测定试剂(时间分辨免疫荧光)和新生儿总半乳糖定量测定试剂(荧光分析法)。
本篇论文的第二部分内容为妊娠相关蛋白A (PAPP-A)质控品的研制。
临床实验室要获得可靠的测定结果,实验室的质量控制是一个重要的环节,而质控品是保证质控工作的重要物质基础。随着PAPP-A基础研究的深入,以及作为孕早期筛查重要的血清标志物之一在临床上的广泛应用,PAPP-A的检测得到了越来越多临床工作者的重视,因此准确可靠的检测PAPP-A有着重要的临床价值。
PAPP-A主要由胚胎合体滋养细胞产生,是一种锌离子依赖性金属蛋白酶。在正常妊娠过程中随孕周的增加,母血中PAPP-A水平持续上升,妊娠结束达到高峰。研究表明,测定孕母血清中的PAPP-A水平有助于筛查胎儿染色体异常。唐氏综合征孕妇PAPP-A浓度显著下降。此外,孕早期低水平PAPP-A与其他不良妊娠结局,如流产、死胎和早产有关。最近的研究表明PAPP-A亦是急性冠脉综合征早期识别、进行危险分层以及评价预后的一个新标志物。当前,我国大部分地区逐步开展了产前筛查工作,测定PAPP-A已成为常规检验项目。由于检测方法和仪器不尽相同,各厂家试剂盒测定值之间存在一定差异。为保证PAPP-AI临床测定值的准确可靠,实验室应定期采用质控品开展室内质量控制或者参加室间质量评价,并且PAPP-A试剂在研发过程中,也需要质控品进行性能评价和质量控制。然而目前PAPP-A质控品的提供主要依赖于国外生产商,价格昂贵、购买周期长,给实际应用造成不便。故第二部分的研究拟制备质量可靠、价格低廉、可长期稳定供应的PAPP-A质控品。同时,在不同试剂上测定该质控品和血清样本,以考察各试剂间PAPP-A测定值是否具有可比性。
方法：
新生儿17a-羟孕酮检测试剂(时间分辨免疫荧光法)的反应原理为竞争抑制法。采用羊抗兔IgG的多克隆抗体(二抗)包被反应板,捕获定量加入的17a-羟孕酮抗体(一抗),检测时加入校准品或待测样品和铕标记17a-羟孕酮抗原,样品中的17a-羟孕酮和铕标记17a-羟孕酮抗原竞争结合被二抗捕获的一抗,形成二抗-一抗-铕标记17a-羟孕酮抗原复合物。当铕在增强液中从复合物上解离后发出强的荧光,荧光强度与样品中的17a-羟孕酮浓度成反比。本研究以脱纤维绵羊全血作为校准品、质控品配制用基质。具体过程是：采用17a-羟孕酮抗原配制校准品,以17a-羟孕酮欧洲标准品为对照,在PE公司17a-羟孕酮试剂盒上进行校准品的标定。标定后的校准品浓度分别为Ong/ml、1.3ng/ml、3.2ng/ml、7ng/ml、25ng/ml和100ng/ml。通过本底荧光值及剂量-反应曲线范围、相关系数等指标,确定包被板、包被体积等条件；对二抗和一抗进行两两配对比较；选择抗原偶联物进行铕标记并纯化；对不同稀释比例的标记抗原偶联物与不同浓度的二抗进行配对实验等。
新生儿总半乳糖定量测定试剂(荧光分析法)的反应原理是：滤纸干血片中的总半乳糖在半乳糖脱氢酶的催化作用下,与NAD反应,生成半乳糖内酯和NADH。用荧光检测仪在激发波长355nm和发射波长460nm条件下,测定生成物NADH的荧光强度,其荧光强度与总半乳糖的浓度成正比。以脱纤维绵羊全血作为校准品、质控品配制用基质。采用半乳糖-1-磷酸配制校准品,通过芬兰Labsystem公司新生儿半乳糖检测试剂盒(荧光分析法)进行标定,标定后的校准品浓度依次为：Omg/dl、5mg/dl、10mg/dl、20mg/dl、40mg/dl、60mg/dl。采用本底荧光值及剂量-反应曲线范围、相关系数等指标,选择合适的包被板和主要原材料,选择碱性磷酸酶：半乳糖脱氢酶:NADH混合反应液最佳配比,并对铜试剂的各反应条件进行优化,如硫酸铜溶液的不同稀释比例(1：10000、1：1000、1：100和1：10)、碳酸钠-酒石酸钾钠缓冲液和硫酸铜溶液的不同比例(3：2、1：1、2：1)；对试剂加样体积和反应时间进行研究。
通过上述研究,选定了这两种自制试剂的主要原材料,确定了基本的反应模式和生产工艺流程；然后进行中试生产,并对自制试剂进行分析性能评价,包括最低检测量、准确性、剂量-反应曲线的相关系数、精密度、特异性、抗干扰实验、HOOK效应、稳定性等性能评价。通过健康新生儿血片样本测定值、文献资料及临床现行参考值范围,设定自制试剂的正常参考值范围；采用对照试剂与自制试剂对血清样本同时进行检测,计算两种方法测值的一致性,以考核自制试剂的临床检测性能。
PAPP-A质控品的研制：收集孕晚期孕妇血清为原料,以正常人血清稀释至三水平目标浓度,加入防腐剂,经无菌过滤、充分混匀后,按0.5ml/瓶进行分装,冻干制备得到PAPP-A质控品,于-20℃保存。参考《中国生物制品检定规程》和《中国药典》的相关要求对质控品的物理性状、复溶性和水分含量进行初步评价；符合要求之后再对质控品的均匀性和稳定性进行分析。通过PE公司妊娠相关血浆蛋白A测定试剂盒(时间分辨荧光法),在该公司VICTORTM X4检测系统上对PAPP-A质控品进行定值。
为了解各厂家PAPP-A试剂的反应性能差异以及评价PAPP-A检测结果的可比性,我们选择已取得国家食品药品监督管理局注册证的2家进口与3家国产试剂盒对本研究中制备的PAPP-A质控品和20份新鲜孕妇血清进行分析测定。
结果：
(一)新生儿17a-羟孕酮定量测定试剂的研制
采用竞争抑制法研制了新生儿17a-羟孕酮定量测定试剂。自制试剂准确性高,以欧洲标准品标化的工作校准品为对照,自制试剂校准品的实测浓度与标示浓度的比值在0.94-1.04之间；最低检测量不高于0.4ng/ml;剂量-反应曲线范围宽,可达100ng/ml,三次测试的剂量-反应曲线相关系数绝对值均大于0.9980,分别为-1.000、-0.999和-0.999；分析内和分析间精密度均低于10.0%；特异性强,浓度为100ng/ml的17a-羟孕烯醇酮、雌二醇和雌三醇及浓度为85ng/ml的11-脱氧皮质醇在自制试剂上的测定值均小于0.15ng/ml；对甘油三酯、胆红素的抗干扰能力强。检测浓度高至500ng/ml的17a-羟孕酮,未出现HOOK效应。稳定性检测中的各项指标也均符合要求。该试剂在两家不同公司仪器上的性能比较无显著差异,表明适用性强。自制试剂测定新生儿17a-羟孕酮的正常参考值范围为0-9.9ng/ml。本次研究共检测新生儿滤纸干血片样本1020例,其中阳性61例,阴性959例,以PE公司试剂为对照,自制试剂的阳性符合率为93.44%,阴性符合率为99.37%,总符合率为99.02%,Kappa值为0.914(P=0.000)。两种试剂所测数据具有显著相关性,y=0.989x-0.071,斜率95%CI(0.982～0.997),截距95%CI(-0.177～0.036),相关系数为0.992(P=0.000)。
(二)新生儿总半乳糖定量测定试剂的研制
采用荧光分析法研制了新生儿总半乳糖定量测定试剂。以企业校准品为对照,自制试剂校准品的实测浓度与标示浓度的比值均在0.95-1.00之间,表明自制试剂的准确性好；最低检测量不高于0.4mg/dl；剂量-反应曲线范围宽,可达70mg/dl；相关系数分别为0.999、0.996和0.998；分析内和分析间精密度均低于10.0%。特异性强,浓度为100mg/dl的L-半乳糖、果糖-6-磷酸及浓度为200mg/dl的葡萄糖-1-磷酸、葡萄糖-6-磷酸在自制试剂上的测定值均小于0.5mg/dl。自制试剂测定新生儿总半乳糖的正常参考值范围为0～8.0mg/dl。采用自制试剂和Labsy stems公司试剂对720份滤纸干血片样品平行检测,其中阳性6例,阴性714例,以Labsystem公司试剂为对照,阳性符合率为100.0%,阴性符合率为99.72%,Kappa值为0.856(P=0.000)。两种试剂所测数据具有显著相关性,y=0.899x-0.011,斜率95%CI(0.883-0.914),截距95%CI(-0.046-0.025),相关系数为0.973,(P=0.000)。
(三) PAPP-A质控品的研制
本研究成功制备了PAPP-A质控品。该质控品外观符合要求、复溶性好、三水平质控品的水分含量分别为1.72%、2.01%、2.16%,均低于3.0%。均匀性评价结果显示,同一水平PAPP-A质控品之间的变异系数均小于5%,表明均匀性良好；稳定性试验结果表明,PAPP-A质控品在-20℃条件下至少可保存60w,室温条件(18℃～25℃)放置7天内稳定,在37℃条件下至少可保存3w, PAPP-A质控品复溶后,在2℃～8℃放置3d、室温放置3h、于-20℃放置5w稳定；三水平PAPP-A质控品的测定值及95%可信区间分别为58.34(53.87～62.81) mIU/L、208.40(185.16～231.64) mIU/L和802.82(765.23～840.40)mIU/L。本研究制备的质控品和新鲜血清样本在各试剂上的测定结果差异较大。
结论：
第一部分：
成功研制了新生儿17α-羟孕酮和总半乳糖定量测定试剂,各项指标(准确性、灵敏度、相关系数、精密性、特异性、抗干扰性等)均满足临床检测要求,已达到国外同类产品水平,有望取代进口试剂。其中,新生儿总半乳糖定量测定试剂的研制,属于国内首创,填补了我国该类试剂空白。
随着国内对新生儿先天性肾上腺增生症及半乳糖血症的认识不断提高,开展这两类筛查项目的地区也将不断增加,因此本研究研制的试剂在新生儿筛查领域必将拥有广阔的应用前景,获得巨大的经济效益。
第二部分：
本研究制备的PAPP-A质控品外观、复溶性、水分含量、均匀性和稳定性等性能均令人满意,适合于实验室PAPP-A检测的质量评价及PAPP-A试剂盒精密度等评价。今后若作为商品化的质控品推向市场,必将具有较高应用价值和经济效益。
由于各PAPP-A试剂盒所用的抗原、检测原理和检测仪器不尽相同,导致PAPP-A质控品在各试剂盒上的测定结果较为分散,可比性较差。因此今后通过该质控品的广泛应用,将促使各试剂厂家分析其测定值的不一致性原因,加以改进,最终为促进检测结果的互换性打下基础。
Background and Objective
This dissertation includes two parts.
The first part is the development of newborn screening reagents, including neonatal17a-hydroxyprogesterone diagnostic reagent (Time-resolved fluoroimmunoassay) and neonatal total galactose diagnostic reagent (Fluorescence analysis).
Newborn screening refers to screening congenital endocrine abnormalities, genetic metabolic disease and certain serious genetic disease in newborns with rapid, sensitive laboratory methods. Its purpose is to diagnosis and treat early for sick newborns; prevent irreversible tissue and organ damage, thereby avoid irreversible damage of mental retardation, speech disorders, even death.Currently the basic items of newborn screening are hypothyroidism and phenylketonuria in China. With the development of neonatal screening technology, more and more screening items appear. Some areas carried out screening of glucose6-phosphate dehydrogenase deficiency, congenital adrenal hyperplasia, galactosemia, amino acids metabolic diseases and other diseases.
Congenital adrenal hyperplasia (CAH) belong to an autosomal recessive genetic disorder, which are a group diseases caused by some congenital enzyme defects in the process of adrenal cortical hormone synthesis. Affected female newborns usually have ambiguous genitalia, men appear pseudo-precocious puberty. Concurrent aldosterone lacking can cause retardation of growth, hypovolemia, neonatal adrenal crisis, shock, even life-threatening. Due to enzyme defects of galactose metabolism function, galactose and its metabolites accumulate in the blood and tissues, which cause galactosemia. galactosemia are metabolic dysfunction syndrome and belong to autosomal recessive genetic diseases. The main affected organs are liver, kidney, brain and lens, which can cause cirrhosis, mental retardation and cataracts. If these two congenital disease can be diagnosed in early neonatal stage, it will help to reduce the rate of mortality and disability of children. It is very important to promote normal growth and development of children in China.
Usually, neonatal CAH screening carry out clinically by detecting the content of newborn17a-hydroxyprogesterone in blood. This is due to17a-hydroxyprogesterone as a precursor of cortisol can reflect21-hydroxylase activity state in body. The most common is21-hydroxylase deficiency (about90%to95%). So most of CAH can be screened by detecting17a-hydroxyprogesterone level in neonatal blood. Currently the detection methods of17a-hydroxyprogesterone reagents, imported or domestically, are mostly time-resolved fluoroimmunoassay (TRFIA). Because TRFIA is a sensitive quantitative immunoassay technology using rare earth ion to label antigens or antibodies. It has advantages of preparation simply, long storage time, no radioactive contamination, good reproducibility, short operation process, wide range of standard curve and without disturbance of natural fluorescence from sample. It has wider applications range compared with enzyme immunoassay, radioimmunoassay, etc. For neonatal galactosemia, It is screened by detecting total galactose content in blood usually using fluorescence analysis at home and abroad. Fluorescence analysis is a qualitative and quantitative analysis using fluorescence properties of sample, which possess the advantages of high sensitivity, simple method, good reproducibility and wide range of dose-response curves, etc.
Currently, the reagents screening for both of these two congenital disorders are primarily imported in China, such as PerkinElmer, Inc. USA, Labsystem, Inc. Finland, et al. Only one17a-hydroxyprogesterone diagnostic reagent is domestic-made and total galactose diagnostic reagent is no one domestic-made. Because imported reagent is expensive and testing cost is high, It is very difficult to promote the clinical applications. Although domestic agents is cheaper, manufacturers are too few to meet the need of our nationwide newborn screening for CAH and Galactosemia.lt is also one of main reason for low screening rates of these two neonatal dieases. So, this study intends to develop neonatal17a-hydroxyprogesterone diagnostic reagent using TRFIA and neonatal total galactose diagnostic reagent using fluorescence analysis.
The second part of this dissertation is the preparation of control materials for pregnancy associated plasma protein-A (PAPP-A).
Quality control of laboratory is an important link to obtain reliable results for clinical laboratories, and control material is an important material foundation to ensure quality control work. With the intensive study of basic research and as one of the important serum markers in first trimester screening, the detection of PAPP-A is gaining more and more attention from clinicians. So it has important clinical value to detect PAPP-A accurately and reliably.
PAPP-A is a zinc-dependent metalloproteinases which is mainly produced by embryonic syncytiotrophoblast. With gestational age increasing in the course of a normal pregnancy, maternal PAPP-A level continues to rise and reaches a peak in the last trimester of pregnancy. Measurement of PAPP-A has been reported to improve the performance of screening for fetal aneuplodies. In Down syndrome pregnancies, PAPP-A concentrations are markedly decreased. In addition, low first trimester PAPP-A levels are associated with other adverse pregnancy outcomes, such as stillbirth and preterm delivery. Recent studies show that PAPP-A is also a new prognostic marker to identify acute coronary syndrome in early phage, stratify risk level and evaluate prognosis. Today, with prenatal screening carrying out gradually in most regions of China, the determination of PAPP-A has become a routine test item. Since vary detection methods and instruments, there are differences between the measured values by different diagnostic reagents. To ensure the accuracy and reliability of clinical determination value of PAPP-A, Laboratory should carry out regularly internal quality control or participate in external quality assessment using control materials. PAPP-A reagent in the development process also need control materials to carry out performance evaluation and quality control.However, control materials of PAPP-A is mainly provided by foreign manufacturers, which are expensive, long purchase cycles. These factors cause inconvenience of practical applications for control materials of PAPP-A. Therefore, the purpose of second part of study is to prepare reliable quality, low price, long-term stable supply of PAPP-A quality control materials. Meanwhile, to carry out the determination of controls material and serum samples at different reagents to investigate PAPP-A measured value between the reagents are comparable. Meanwhile, we try to evaluate comparability of results between different reagents by detecting the PAPP-A controls and serum samples.
Methods
According to small molecular weight of17a-hydroxyprogesterone, the reaction principle of diagnostic reagent is competitive inhibition method. First, reaction plate is coated using polyclonal antibody of goat anti-rabbit IgG (secondary antibody), which can capture17a-hydroxyprogesterone antibody (primary antibody) added quantitatively. Second, calibrators or samples and17a-hydroxyprogesterone antigens labeled by europium are added in plate.17a-hydroxyprogesterone in the sample and17a-hydroxyprogesterone antigen labeled by europium are compete to bind primary antibody captured by the second antibody. Then all of above these can form secondary antibody-primary antibody-17a-hydroxyprogesterone antigen complex labeled by europium, when dissociate from the complex in enhancement solution, europium emits strong fluorescence. The17a-hydroxy-progesterone concentration in sample is inversely proportional to fluorescence intensity. In this study, the solution of calibrators and controls were prepared by defibrinated sheep blood as matrix. The detailed process is:calibrators made of17a-hydroxyprogesterone antigen were calibrated in the17a-hydroxyprogesterone reagent of PerkinElmer, Inc.,17a-hydroxyprogesterone European standards as controls. After calibration, calibrators'concentrations were Ong/ml,1.3ng/ml,3.2ng/ml,7ng/ml,25ng/ml and100ng/ml respectively. The coated plates, coated solution volume were confirmed by indicators of background fluorescence, the range of dose-response curve and linear correlation coefficient. We compared paired secondary antibody and primary antibody of17a-hydroxy progesterone. Then selected antigen conjugates to label Eu3+and purify. We also compared different dilutions of labeled antigen conjugate with different concentrations of secondary antibody by paired experiments.
The reaction principle of neonatal total galactose diagnostic reagent is:total galactose in filter paper dried blood spot reacts with NAD by galactose dehydrogenase catalysis, then produce galactose lactone and NADH. The fluorescence intensity of NADH is measured at an excitation wavelength of355nm and emission wavelength of460nm conditions by fluorescent detector, which fluorescence intensity is proportional to the total concentration of galactose. The solution of calibrators and controls were also prepared by defibrinated sheep blood as matrix. The detailed process is:calibrators prepared by Galactose-1-phosphate were calibrated in the neonatal total galactose reagent (Fluorescence analysis) of Finland Labsystem, Inc. After calibration, calibrators'concentrations were Omg/dl、5mg/dl、10mg/dl、20mg/dl、40mg/dl、60mg/dl respectively. Using indicators of background fluorescence, the range of dose-response curve and linear correlation coefficient, we selected appropriate coated plates and main raw material and optimum mixing ratio of reaction mixture of alkaline phosphatase:galactose dehydrogenase:NADH. We optimized various reaction conditions of copper reagent, such as different dilution ratio of copper sulfate solution (1:10000,1:1000,1:100and1:10), different ratios of sodium carbonate-sodium tartrate buffer with copper sulfate solution (3:2,1:1,2:1). We also studied sample volume in reaction and the reaction time.
Through above studies, we selected the main raw material of these two self-made reagents, also determined basic reaction patterns and production technics process. Then we processed pilot production and evaluated the performance of self-detection reagent, including the minimum detectable amount, accuracy, correlation coefficient of dose-response curve, precision, specificity, interference experiments, HOOK effect, stability and so on. We established the normal reference value range of reagent through detecting healthy newborn blood spot samples, literature and current clincal reference range. By using homemade reagents and control reagent to detect filter paper dried blood simultaneously, We assessed the clinical performance of self-made reagents by calculating the measured values' consistency of of two methods.
Development of PAPP-A control materials:Third trimester maternal serum as raw materials were collected and diluted to three target levels by normal serum. Then we added preservatives. PAPP-A control materials were sterile filtrated, thoroughly mixed, and dispensed0.5ml to each bottles, then freeze-dried, stored at-20℃.The general characteristics such as appearance, rehydration quality and moisture content were evaluated according to the protocol of control of Lyophilized Biological Products and Chinese pharmacopoeia. Only meet the requirements, we evaluated the homogeneity and stability of preparations. PAPP-A control materials were calibrated and valued by pregnancy associated plasma protein-A assay kit (TRFIA) of PE, Inc. in the VICTOR TM X4detection system of PE, Inc. In order to understand reactive differences of different reagents and evaluate comparability between PAPP-A reagent for the detection result, We chose2imported and3domestic PAPP-A reagents which have registration certificate approved by State Food and Drug Administration for controls to assay PAPP-A control materials and20fresh maternal sera.
Results
1. Development of neonatal17a-hydroxyprogesterone diagnostic reagent (TRFIA)
Using competitive inhibition assay, we developed a neonatal17a-hydroxy-progesterone diagnostic reagent. The reagent has high accuracy. The ratio of measured and marked value were between0.94-1.04using higher calibrators calibrated by European standards for the reference substance. The minimum detectable amount using this reagent was no more than0.4ng/ml. This reagent had wide dose-response range and the maximum detection range was up to100ng/ml. The correlation coefficients of dose-response curve in three tests were-1.000、-0.999and-0.999. The intra-and inter-assay coefficients of variation were better than10.0%. The results of measuring the concentration of the present assay against17a-hydroxypregnenolone (100ng/ml), estradiol (100ng/ml) and estriol (100ng/ml) and11-deoxycortisol (85ng/ml) were all less than0.15ng/ml, which indicated this assay has a very low cross-reactivity against these materials. The serum concentrations in present kit did not interfered by triglyceride and bilirubin. HOOK effect did not appear when detect high concentrations were up to500ng/ml of17a-hydroxyprogesterone. Stability results were in line with the requirements of the indicators. There was no significant difference in performance on the instrument from two different companies, which means PAPP-A control has good applicability. The17a-hydroxyprogesterone normal reference range of newborn is0-9.9ng/ml by using this reagent.1,020newborn filter paper dried blood samples were detected in this study, which included61positive cases and959negative cases. Compared with PE reagent, the positive rate, the negative rate, total coincidence rate of this reagent were93.44%,99.37%and99.02%respectively. Kappa value was0.914(P=0.000). The data measured by Self-made and control reagent had significant correlation [slope (95%CI),0.989(0.982-0.997); y-intercept (95%CI),-0.071(-0.177-0.036) ng/mL]. The two assays showed a correlation coefficient of0.992(P=0.000).
2. Development of neonatal total galactose diagnostic reagent
Using fluorescence analysis, we developed a neonatal total galactose diagnostic reagent. The ratios of measured and marked concentration were between0.95～1.00using calibrator of higher calibrators calibrated for the reference substance, which means this assay has high accuracy. The minimum detectable amount of this reagent was no more than0.4mg/dl. This reagent had wide range of dose-response curve and the maximum detection range was up to70mg/dl. The correlation coefficients in three tests were0.999,0.996and0.998. The intra-and inter-assay coefficients of variation were better than10.0%. The results of measuring the concentration of the present assay against of L-galactose (100mg/dl), fructose-6-phosphate (100mg/dl) and glucose-1-phosphate (200mg/dl), glucose-6-phosphate (200mg/dl) were all less than0.5mg/dl, which indicated this assay has a very low cross-reactivity against these materials. The total galactose normal reference range of newborn is0～8.0mg/dl by using this reagent.720newborn filter paper dried blood samples were detected in this study, which included61positive cases and959negative cases. Compared with Labsy stems reagent, the positive rate, the negative rate, total coincidence rate of this reagent were100.0%and99.72%respectively. Kappa value was0.856(P=0.000)The data measured by Self-made and control kit had significant correlation [slope (95%CI),0.899(0.883～0.914); y-intercept (95%CI),-0.011(-0.046～0.025) mg/dl]. The two assays showed a correlation coefficient of0.973(P=0.000).
3. Development of PAPP-A control materials
PAPP-A control materials were prepared successfully by this study. The PAPP-A control materials had an acceptable appearance, good rehydration quality. The moisture content of three level controls were all lower than3.0%and were1.72%,2.01%,2.16%respectively. In the homogeneity evaluation, the results showed that the difference were insignificant(P>0.05) within the same levels of PAPP-A control materials. The stability evaluation results showed that the control samples can be stable for at least60w at-20℃conditions,7d at room temperature (18℃-25℃) and3w at37℃.After reconstitution, PAPP-A control materials can be stable for3d at2℃-8℃,3h at room temperature and5w at-20℃. The measured values and95%intervals of three levels of PAPP-A control materials were58.34(53.87-62.81) mIU/L,208.40(185.16-231.64) mIU/L and802.82(765.23-840.40) mIU/L respectively. There were obvious differences in determination results of the control materials prepared in this study and fresh serum samples measured by each reagent.
Conclusions
PartⅠ:
We successfully developed neonatal17a-hydroxyprogesterone and total galactose diagnostic reagent. The indicators (including accuracy, sensitivity, correlation coefficients, precision, specificity, interference, etc.) met the requirements of clinical testing applications and reach the level of the same kind of products abroad. So, these two diagnostic reagent is expected to replace the imported reagent. The development of neonatal total galactose diagnostic reagent is the first one developed in China.
With congenital adrenal hyperplasia and galactosemia increasing awareness at home, the number of regions carrying out these two screening programs will also continue to increase. However, homemade reagent manufacturer is currently only one. The reagent developed by this study will surely have a wonderful application prospects and access to huge economic benefits, get enormous economical benefits in neonatal screening field.
Part II:
The results of the second part demonstrated that the indicators (including appearance, rehydration quality, moisture content, homogeneity and stability) of PAPP-A control materials in this study are satisfactory. PAPP-A control materials are suitable for quality evaluation of laboratory detecting PAPP-A and precision et al. evaluation for PAPP-A reagents. If PAPP-A control materials as commodity are provided in the market in the future, it will have a high application value and economic benefit.
Since antigen, detection principle and instrumentation varied in these five reagents of PAPP-A, the scatter of measurement result of PAPP-A control is big and the comparability is poor. Therefore, through the extensive application of the control samples in the future, it will enable manufacturers to analyze reasons for difference value and improve reagent quality. At last, It will lay the foundation for the promotion of interchangeability of test results.
本篇论文主要包括两部分内容。
第一部分为新生儿筛查试剂的研制,包括新生儿17α-羟孕酮定量测定试剂(时间分辨免疫荧光法)和新生儿总半乳糖定量测定试剂(荧光分析法)的研制。
新生儿筛查是指用快速、敏感的实验室方法对新生儿的先天性内分泌异常、遗传代谢病以及某些危害严重的遗传性疾病进行筛查,以期对患病新生儿进行早期诊断、早期治疗,防止其机体组织器官发生不可逆的损伤,从而避免精神发育不可逆损害、智力低下、言语障碍甚至死亡。目前在我国新生儿筛查的基本项目是甲状腺功能低下症和苯丙酮尿症,随着新生儿疾病筛查技术发展,筛查病种越来越多。部分地区相继开展了葡萄糖6-磷酸脱氢酶缺乏症、先天性肾上腺皮质增生症、半乳糖血症、氨基酸代谢病等疾病的筛查工作。
先天性肾上腺皮质增生症(congenital adrenal hyperplasia, CAH)属常染色体隐性遗传病,是一组肾上腺皮质激素合成过程中某种酶先天性缺陷所引起的疾病。受累女性新生儿可有外生殖器男性化体征,男性则出现假性性早熟;并发的醛固酮缺失可引起发育停滞、血容量减少、新生儿肾上腺危象、休克,严重者甚至危及生命。半乳糖血症则是由于代谢过程中酶缺陷,半乳糖及其代谢产物在血液和组织中堆积所致的一种代谢功能紊乱的症候群,亦为常染色体隐性遗传性疾病。主要受损器官为肝肾、脑及晶状体,引起肝硬变、智力发育障碍、白内障等。这两种先天性疾病若能在新生儿早期得到确诊,有助于降低患儿病死率、减少伤残率,对促进我国儿童的正常生长发育非常重要。
临床上多采用检测血中17α-羟孕酮含量进行新生儿CAH筛查。这是因为17α-羟孕酮是皮质醇的前体之一,其水平可反映体内21-羟化酶的活性状态。而CAH中最常见的是21-羟化酶缺乏(约占90%～95%)。因此测定新生儿血中17α-羟孕酮含量可筛查大部分CAH。目前17α-羟孕酮进口或国产试剂,其采用的检测方法多为时间分辨免疫荧光法,这是一种基于以镧系稀土离子作为标记物来标记抗原或抗体的高灵敏度定量免疫检测技术。它具有制备简便、储存时间长、无放射性污染、重复性好、操作流程短、剂量-反应曲线范围宽、不受样品自然荧光干扰等优点,与酶免分析、放射免疫分析等相比,临床应用价值更高。而对于半乳糖血症,国外通常采用荧光分析法测定新生儿血中总半乳糖含量来进行筛查。荧光分析法是利用物质的荧光特性对样品进行定性和定量分析的方法,具有灵敏度高、方法简便、重现性好、剂量-反应曲线范围较宽等特点。
目前我国这两种先天性疾病筛查用试剂主要为进口试剂,如美国Perkin-Elmer公司、芬兰Labsystem公司等,只有一家国产17α-羟孕酮检测试剂,而总半乳糖检测试剂则暂无国产试剂。进口试剂价格昂贵,检测成本较高,临床应用难度大,不利于筛查项目的开展,而国产试剂虽价格较低,但生产厂家较少,远远不能满足我国新生儿CAH和总半乳糖血症的筛查需求,这也是我国这两种新生儿疾病筛查率较低的主要原因之一,因此我们拟研制新生儿17α-羟孕酮定量测定试剂(时间分辨免疫荧光)和新生儿总半乳糖定量测定试剂(荧光分析法)。
本篇论文的第二部分内容为妊娠相关蛋白A (PAPP-A)质控品的研制。
临床实验室要获得可靠的测定结果,实验室的质量控制是一个重要的环节,而质控品是保证质控工作的重要物质基础。随着PAPP-A基础研究的深入,以及作为孕早期筛查重要的血清标志物之一在临床上的广泛应用,PAPP-A的检测得到了越来越多临床工作者的重视,因此准确可靠的检测PAPP-A有着重要的临床价值。
PAPP-A主要由胚胎合体滋养细胞产生,是一种锌离子依赖性金属蛋白酶。在正常妊娠过程中随孕周的增加,母血中PAPP-A水平持续上升,妊娠结束达到高峰。研究表明,测定孕母血清中的PAPP-A水平有助于筛查胎儿染色体异常。唐氏综合征孕妇PAPP-A浓度显著下降。此外,孕早期低水平PAPP-A与其他不良妊娠结局,如流产、死胎和早产有关。最近的研究表明PAPP-A亦是急性冠脉综合征早期识别、进行危险分层以及评价预后的一个新标志物。当前,我国大部分地区逐步开展了产前筛查工作,测定PAPP-A已成为常规检验项目。由于检测方法和仪器不尽相同,各厂家试剂盒测定值之间存在一定差异。为保证PAPP-AI临床测定值的准确可靠,实验室应定期采用质控品开展室内质量控制或者参加室间质量评价,并且PAPP-A试剂在研发过程中,也需要质控品进行性能评价和质量控制。然而目前PAPP-A质控品的提供主要依赖于国外生产商,价格昂贵、购买周期长,给实际应用造成不便。故第二部分的研究拟制备质量可靠、价格低廉、可长期稳定供应的PAPP-A质控品。同时,在不同试剂上测定该质控品和血清样本,以考察各试剂间PAPP-A测定值是否具有可比性。
方法：
新生儿17a-羟孕酮检测试剂(时间分辨免疫荧光法)的反应原理为竞争抑制法。采用羊抗兔IgG的多克隆抗体(二抗)包被反应板,捕获定量加入的17a-羟孕酮抗体(一抗),检测时加入校准品或待测样品和铕标记17a-羟孕酮抗原,样品中的17a-羟孕酮和铕标记17a-羟孕酮抗原竞争结合被二抗捕获的一抗,形成二抗-一抗-铕标记17a-羟孕酮抗原复合物。当铕在增强液中从复合物上解离后发出强的荧光,荧光强度与样品中的17a-羟孕酮浓度成反比。本研究以脱纤维绵羊全血作为校准品、质控品配制用基质。具体过程是：采用17a-羟孕酮抗原配制校准品,以17a-羟孕酮欧洲标准品为对照,在PE公司17a-羟孕酮试剂盒上进行校准品的标定。标定后的校准品浓度分别为Ong/ml、1.3ng/ml、3.2ng/ml、7ng/ml、25ng/ml和100ng/ml。通过本底荧光值及剂量-反应曲线范围、相关系数等指标,确定包被板、包被体积等条件；对二抗和一抗进行两两配对比较；选择抗原偶联物进行铕标记并纯化；对不同稀释比例的标记抗原偶联物与不同浓度的二抗进行配对实验等。
新生儿总半乳糖定量测定试剂(荧光分析法)的反应原理是：滤纸干血片中的总半乳糖在半乳糖脱氢酶的催化作用下,与NAD反应,生成半乳糖内酯和NADH。用荧光检测仪在激发波长355nm和发射波长460nm条件下,测定生成物NADH的荧光强度,其荧光强度与总半乳糖的浓度成正比。以脱纤维绵羊全血作为校准品、质控品配制用基质。采用半乳糖-1-磷酸配制校准品,通过芬兰Labsystem公司新生儿半乳糖检测试剂盒(荧光分析法)进行标定,标定后的校准品浓度依次为：Omg/dl、5mg/dl、10mg/dl、20mg/dl、40mg/dl、60mg/dl。采用本底荧光值及剂量-反应曲线范围、相关系数等指标,选择合适的包被板和主要原材料,选择碱性磷酸酶：半乳糖脱氢酶:NADH混合反应液最佳配比,并对铜试剂的各反应条件进行优化,如硫酸铜溶液的不同稀释比例(1：10000、1：1000、1：100和1：10)、碳酸钠-酒石酸钾钠缓冲液和硫酸铜溶液的不同比例(3：2、1：1、2：1)；对试剂加样体积和反应时间进行研究。
通过上述研究,选定了这两种自制试剂的主要原材料,确定了基本的反应模式和生产工艺流程；然后进行中试生产,并对自制试剂进行分析性能评价,包括最低检测量、准确性、剂量-反应曲线的相关系数、精密度、特异性、抗干扰实验、HOOK效应、稳定性等性能评价。通过健康新生儿血片样本测定值、文献资料及临床现行参考值范围,设定自制试剂的正常参考值范围；采用对照试剂与自制试剂对血清样本同时进行检测,计算两种方法测值的一致性,以考核自制试剂的临床检测性能。
PAPP-A质控品的研制：收集孕晚期孕妇血清为原料,以正常人血清稀释至三水平目标浓度,加入防腐剂,经无菌过滤、充分混匀后,按0.5ml/瓶进行分装,冻干制备得到PAPP-A质控品,于-20℃保存。参考《中国生物制品检定规程》和《中国药典》的相关要求对质控品的物理性状、复溶性和水分含量进行初步评价；符合要求之后再对质控品的均匀性和稳定性进行分析。通过PE公司妊娠相关血浆蛋白A测定试剂盒(时间分辨荧光法),在该公司VICTORTM X4检测系统上对PAPP-A质控品进行定值。
为了解各厂家PAPP-A试剂的反应性能差异以及评价PAPP-A检测结果的可比性,我们选择已取得国家食品药品监督管理局注册证的2家进口与3家国产试剂盒对本研究中制备的PAPP-A质控品和20份新鲜孕妇血清进行分析测定。
结果：
(一)新生儿17a-羟孕酮定量测定试剂的研制
采用竞争抑制法研制了新生儿17a-羟孕酮定量测定试剂。自制试剂准确性高,以欧洲标准品标化的工作校准品为对照,自制试剂校准品的实测浓度与标示浓度的比值在0.94-1.04之间；最低检测量不高于0.4ng/ml;剂量-反应曲线范围宽,可达100ng/ml,三次测试的剂量-反应曲线相关系数绝对值均大于0.9980,分别为-1.000、-0.999和-0.999；分析内和分析间精密度均低于10.0%；特异性强,浓度为100ng/ml的17a-羟孕烯醇酮、雌二醇和雌三醇及浓度为85ng/ml的11-脱氧皮质醇在自制试剂上的测定值均小于0.15ng/ml；对甘油三酯、胆红素的抗干扰能力强。检测浓度高至500ng/ml的17a-羟孕酮,未出现HOOK效应。稳定性检测中的各项指标也均符合要求。该试剂在两家不同公司仪器上的性能比较无显著差异,表明适用性强。自制试剂测定新生儿17a-羟孕酮的正常参考值范围为0-9.9ng/ml。本次研究共检测新生儿滤纸干血片样本1020例,其中阳性61例,阴性959例,以PE公司试剂为对照,自制试剂的阳性符合率为93.44%,阴性符合率为99.37%,总符合率为99.02%,Kappa值为0.914(P=0.000)。两种试剂所测数据具有显著相关性,y=0.989x-0.071,斜率95%CI(0.982～0.997),截距95%CI(-0.177～0.036),相关系数为0.992(P=0.000)。
(二)新生儿总半乳糖定量测定试剂的研制
采用荧光分析法研制了新生儿总半乳糖定量测定试剂。以企业校准品为对照,自制试剂校准品的实测浓度与标示浓度的比值均在0.95-1.00之间,表明自制试剂的准确性好；最低检测量不高于0.4mg/dl；剂量-反应曲线范围宽,可达70mg/dl；相关系数分别为0.999、0.996和0.998；分析内和分析间精密度均低于10.0%。特异性强,浓度为100mg/dl的L-半乳糖、果糖-6-磷酸及浓度为200mg/dl的葡萄糖-1-磷酸、葡萄糖-6-磷酸在自制试剂上的测定值均小于0.5mg/dl。自制试剂测定新生儿总半乳糖的正常参考值范围为0～8.0mg/dl。采用自制试剂和Labsy stems公司试剂对720份滤纸干血片样品平行检测,其中阳性6例,阴性714例,以Labsystem公司试剂为对照,阳性符合率为100.0%,阴性符合率为99.72%,Kappa值为0.856(P=0.000)。两种试剂所测数据具有显著相关性,y=0.899x-0.011,斜率95%CI(0.883-0.914),截距95%CI(-0.046-0.025),相关系数为0.973,(P=0.000)。
(三) PAPP-A质控品的研制
本研究成功制备了PAPP-A质控品。该质控品外观符合要求、复溶性好、三水平质控品的水分含量分别为1.72%、2.01%、2.16%,均低于3.0%。均匀性评价结果显示,同一水平PAPP-A质控品之间的变异系数均小于5%,表明均匀性良好；稳定性试验结果表明,PAPP-A质控品在-20℃条件下至少可保存60w,室温条件(18℃～25℃)放置7天内稳定,在37℃条件下至少可保存3w, PAPP-A质控品复溶后,在2℃～8℃放置3d、室温放置3h、于-20℃放置5w稳定；三水平PAPP-A质控品的测定值及95%可信区间分别为58.34(53.87～62.81) mIU/L、208.40(185.16～231.64) mIU/L和802.82(765.23～840.40)mIU/L。本研究制备的质控品和新鲜血清样本在各试剂上的测定结果差异较大。
结论：
第一部分：
成功研制了新生儿17α-羟孕酮和总半乳糖定量测定试剂,各项指标(准确性、灵敏度、相关系数、精密性、特异性、抗干扰性等)均满足临床检测要求,已达到国外同类产品水平,有望取代进口试剂。其中,新生儿总半乳糖定量测定试剂的研制,属于国内首创,填补了我国该类试剂空白。
随着国内对新生儿先天性肾上腺增生症及半乳糖血症的认识不断提高,开展这两类筛查项目的地区也将不断增加,因此本研究研制的试剂在新生儿筛查领域必将拥有广阔的应用前景,获得巨大的经济效益。
第二部分：
本研究制备的PAPP-A质控品外观、复溶性、水分含量、均匀性和稳定性等性能均令人满意,适合于实验室PAPP-A检测的质量评价及PAPP-A试剂盒精密度等评价。今后若作为商品化的质控品推向市场,必将具有较高应用价值和经济效益。
由于各PAPP-A试剂盒所用的抗原、检测原理和检测仪器不尽相同,导致PAPP-A质控品在各试剂盒上的测定结果较为分散,可比性较差。因此今后通过该质控品的广泛应用,将促使各试剂厂家分析其测定值的不一致性原因,加以改进,最终为促进检测结果的互换性打下基础。
Background and Objective
This dissertation includes two parts.
The first part is the development of newborn screening reagents, including neonatal17a-hydroxyprogesterone diagnostic reagent (Time-resolved fluoroimmunoassay) and neonatal total galactose diagnostic reagent (Fluorescence analysis).
Newborn screening refers to screening congenital endocrine abnormalities, genetic metabolic disease and certain serious genetic disease in newborns with rapid, sensitive laboratory methods. Its purpose is to diagnosis and treat early for sick newborns; prevent irreversible tissue and organ damage, thereby avoid irreversible damage of mental retardation, speech disorders, even death.Currently the basic items of newborn screening are hypothyroidism and phenylketonuria in China. With the development of neonatal screening technology, more and more screening items appear. Some areas carried out screening of glucose6-phosphate dehydrogenase deficiency, congenital adrenal hyperplasia, galactosemia, amino acids metabolic diseases and other diseases.
Congenital adrenal hyperplasia (CAH) belong to an autosomal recessive genetic disorder, which are a group diseases caused by some congenital enzyme defects in the process of adrenal cortical hormone synthesis. Affected female newborns usually have ambiguous genitalia, men appear pseudo-precocious puberty. Concurrent aldosterone lacking can cause retardation of growth, hypovolemia, neonatal adrenal crisis, shock, even life-threatening. Due to enzyme defects of galactose metabolism function, galactose and its metabolites accumulate in the blood and tissues, which cause galactosemia. galactosemia are metabolic dysfunction syndrome and belong to autosomal recessive genetic diseases. The main affected organs are liver, kidney, brain and lens, which can cause cirrhosis, mental retardation and cataracts. If these two congenital disease can be diagnosed in early neonatal stage, it will help to reduce the rate of mortality and disability of children. It is very important to promote normal growth and development of children in China.
Usually, neonatal CAH screening carry out clinically by detecting the content of newborn17a-hydroxyprogesterone in blood. This is due to17a-hydroxyprogesterone as a precursor of cortisol can reflect21-hydroxylase activity state in body. The most common is21-hydroxylase deficiency (about90%to95%). So most of CAH can be screened by detecting17a-hydroxyprogesterone level in neonatal blood. Currently the detection methods of17a-hydroxyprogesterone reagents, imported or domestically, are mostly time-resolved fluoroimmunoassay (TRFIA). Because TRFIA is a sensitive quantitative immunoassay technology using rare earth ion to label antigens or antibodies. It has advantages of preparation simply, long storage time, no radioactive contamination, good reproducibility, short operation process, wide range of standard curve and without disturbance of natural fluorescence from sample. It has wider applications range compared with enzyme immunoassay, radioimmunoassay, etc. For neonatal galactosemia, It is screened by detecting total galactose content in blood usually using fluorescence analysis at home and abroad. Fluorescence analysis is a qualitative and quantitative analysis using fluorescence properties of sample, which possess the advantages of high sensitivity, simple method, good reproducibility and wide range of dose-response curves, etc.
Currently, the reagents screening for both of these two congenital disorders are primarily imported in China, such as PerkinElmer, Inc. USA, Labsystem, Inc. Finland, et al. Only one17a-hydroxyprogesterone diagnostic reagent is domestic-made and total galactose diagnostic reagent is no one domestic-made. Because imported reagent is expensive and testing cost is high, It is very difficult to promote the clinical applications. Although domestic agents is cheaper, manufacturers are too few to meet the need of our nationwide newborn screening for CAH and Galactosemia.lt is also one of main reason for low screening rates of these two neonatal dieases. So, this study intends to develop neonatal17a-hydroxyprogesterone diagnostic reagent using TRFIA and neonatal total galactose diagnostic reagent using fluorescence analysis.
The second part of this dissertation is the preparation of control materials for pregnancy associated plasma protein-A (PAPP-A).
Quality control of laboratory is an important link to obtain reliable results for clinical laboratories, and control material is an important material foundation to ensure quality control work. With the intensive study of basic research and as one of the important serum markers in first trimester screening, the detection of PAPP-A is gaining more and more attention from clinicians. So it has important clinical value to detect PAPP-A accurately and reliably.
PAPP-A is a zinc-dependent metalloproteinases which is mainly produced by embryonic syncytiotrophoblast. With gestational age increasing in the course of a normal pregnancy, maternal PAPP-A level continues to rise and reaches a peak in the last trimester of pregnancy. Measurement of PAPP-A has been reported to improve the performance of screening for fetal aneuplodies. In Down syndrome pregnancies, PAPP-A concentrations are markedly decreased. In addition, low first trimester PAPP-A levels are associated with other adverse pregnancy outcomes, such as stillbirth and preterm delivery. Recent studies show that PAPP-A is also a new prognostic marker to identify acute coronary syndrome in early phage, stratify risk level and evaluate prognosis. Today, with prenatal screening carrying out gradually in most regions of China, the determination of PAPP-A has become a routine test item. Since vary detection methods and instruments, there are differences between the measured values by different diagnostic reagents. To ensure the accuracy and reliability of clinical determination value of PAPP-A, Laboratory should carry out regularly internal quality control or participate in external quality assessment using control materials. PAPP-A reagent in the development process also need control materials to carry out performance evaluation and quality control.However, control materials of PAPP-A is mainly provided by foreign manufacturers, which are expensive, long purchase cycles. These factors cause inconvenience of practical applications for control materials of PAPP-A. Therefore, the purpose of second part of study is to prepare reliable quality, low price, long-term stable supply of PAPP-A quality control materials. Meanwhile, to carry out the determination of controls material and serum samples at different reagents to investigate PAPP-A measured value between the reagents are comparable. Meanwhile, we try to evaluate comparability of results between different reagents by detecting the PAPP-A controls and serum samples.
Methods
According to small molecular weight of17a-hydroxyprogesterone, the reaction principle of diagnostic reagent is competitive inhibition method. First, reaction plate is coated using polyclonal antibody of goat anti-rabbit IgG (secondary antibody), which can capture17a-hydroxyprogesterone antibody (primary antibody) added quantitatively. Second, calibrators or samples and17a-hydroxyprogesterone antigens labeled by europium are added in plate.17a-hydroxyprogesterone in the sample and17a-hydroxyprogesterone antigen labeled by europium are compete to bind primary antibody captured by the second antibody. Then all of above these can form secondary antibody-primary antibody-17a-hydroxyprogesterone antigen complex labeled by europium, when dissociate from the complex in enhancement solution, europium emits strong fluorescence. The17a-hydroxy-progesterone concentration in sample is inversely proportional to fluorescence intensity. In this study, the solution of calibrators and controls were prepared by defibrinated sheep blood as matrix. The detailed process is:calibrators made of17a-hydroxyprogesterone antigen were calibrated in the17a-hydroxyprogesterone reagent of PerkinElmer, Inc.,17a-hydroxyprogesterone European standards as controls. After calibration, calibrators'concentrations were Ong/ml,1.3ng/ml,3.2ng/ml,7ng/ml,25ng/ml and100ng/ml respectively. The coated plates, coated solution volume were confirmed by indicators of background fluorescence, the range of dose-response curve and linear correlation coefficient. We compared paired secondary antibody and primary antibody of17a-hydroxy progesterone. Then selected antigen conjugates to label Eu3+and purify. We also compared different dilutions of labeled antigen conjugate with different concentrations of secondary antibody by paired experiments.
The reaction principle of neonatal total galactose diagnostic reagent is:total galactose in filter paper dried blood spot reacts with NAD by galactose dehydrogenase catalysis, then produce galactose lactone and NADH. The fluorescence intensity of NADH is measured at an excitation wavelength of355nm and emission wavelength of460nm conditions by fluorescent detector, which fluorescence intensity is proportional to the total concentration of galactose. The solution of calibrators and controls were also prepared by defibrinated sheep blood as matrix. The detailed process is:calibrators prepared by Galactose-1-phosphate were calibrated in the neonatal total galactose reagent (Fluorescence analysis) of Finland Labsystem, Inc. After calibration, calibrators'concentrations were Omg/dl、5mg/dl、10mg/dl、20mg/dl、40mg/dl、60mg/dl respectively. Using indicators of background fluorescence, the range of dose-response curve and linear correlation coefficient, we selected appropriate coated plates and main raw material and optimum mixing ratio of reaction mixture of alkaline phosphatase:galactose dehydrogenase:NADH. We optimized various reaction conditions of copper reagent, such as different dilution ratio of copper sulfate solution (1:10000,1:1000,1:100and1:10), different ratios of sodium carbonate-sodium tartrate buffer with copper sulfate solution (3:2,1:1,2:1). We also studied sample volume in reaction and the reaction time.
Through above studies, we selected the main raw material of these two self-made reagents, also determined basic reaction patterns and production technics process. Then we processed pilot production and evaluated the performance of self-detection reagent, including the minimum detectable amount, accuracy, correlation coefficient of dose-response curve, precision, specificity, interference experiments, HOOK effect, stability and so on. We established the normal reference value range of reagent through detecting healthy newborn blood spot samples, literature and current clincal reference range. By using homemade reagents and control reagent to detect filter paper dried blood simultaneously, We assessed the clinical performance of self-made reagents by calculating the measured values' consistency of of two methods.
Development of PAPP-A control materials:Third trimester maternal serum as raw materials were collected and diluted to three target levels by normal serum. Then we added preservatives. PAPP-A control materials were sterile filtrated, thoroughly mixed, and dispensed0.5ml to each bottles, then freeze-dried, stored at-20℃.The general characteristics such as appearance, rehydration quality and moisture content were evaluated according to the protocol of control of Lyophilized Biological Products and Chinese pharmacopoeia. Only meet the requirements, we evaluated the homogeneity and stability of preparations. PAPP-A control materials were calibrated and valued by pregnancy associated plasma protein-A assay kit (TRFIA) of PE, Inc. in the VICTOR TM X4detection system of PE, Inc. In order to understand reactive differences of different reagents and evaluate comparability between PAPP-A reagent for the detection result, We chose2imported and3domestic PAPP-A reagents which have registration certificate approved by State Food and Drug Administration for controls to assay PAPP-A control materials and20fresh maternal sera.
Results
1. Development of neonatal17a-hydroxyprogesterone diagnostic reagent (TRFIA)
Using competitive inhibition assay, we developed a neonatal17a-hydroxy-progesterone diagnostic reagent. The reagent has high accuracy. The ratio of measured and marked value were between0.94-1.04using higher calibrators calibrated by European standards for the reference substance. The minimum detectable amount using this reagent was no more than0.4ng/ml. This reagent had wide dose-response range and the maximum detection range was up to100ng/ml. The correlation coefficients of dose-response curve in three tests were-1.000、-0.999and-0.999. The intra-and inter-assay coefficients of variation were better than10.0%. The results of measuring the concentration of the present assay against17a-hydroxypregnenolone (100ng/ml), estradiol (100ng/ml) and estriol (100ng/ml) and11-deoxycortisol (85ng/ml) were all less than0.15ng/ml, which indicated this assay has a very low cross-reactivity against these materials. The serum concentrations in present kit did not interfered by triglyceride and bilirubin. HOOK effect did not appear when detect high concentrations were up to500ng/ml of17a-hydroxyprogesterone. Stability results were in line with the requirements of the indicators. There was no significant difference in performance on the instrument from two different companies, which means PAPP-A control has good applicability. The17a-hydroxyprogesterone normal reference range of newborn is0-9.9ng/ml by using this reagent.1,020newborn filter paper dried blood samples were detected in this study, which included61positive cases and959negative cases. Compared with PE reagent, the positive rate, the negative rate, total coincidence rate of this reagent were93.44%,99.37%and99.02%respectively. Kappa value was0.914(P=0.000). The data measured by Self-made and control reagent had significant correlation [slope (95%CI),0.989(0.982-0.997); y-intercept (95%CI),-0.071(-0.177-0.036) ng/mL]. The two assays showed a correlation coefficient of0.992(P=0.000).
2. Development of neonatal total galactose diagnostic reagent
Using fluorescence analysis, we developed a neonatal total galactose diagnostic reagent. The ratios of measured and marked concentration were between0.95～1.00using calibrator of higher calibrators calibrated for the reference substance, which means this assay has high accuracy. The minimum detectable amount of this reagent was no more than0.4mg/dl. This reagent had wide range of dose-response curve and the maximum detection range was up to70mg/dl. The correlation coefficients in three tests were0.999,0.996and0.998. The intra-and inter-assay coefficients of variation were better than10.0%. The results of measuring the concentration of the present assay against of L-galactose (100mg/dl), fructose-6-phosphate (100mg/dl) and glucose-1-phosphate (200mg/dl), glucose-6-phosphate (200mg/dl) were all less than0.5mg/dl, which indicated this assay has a very low cross-reactivity against these materials. The total galactose normal reference range of newborn is0～8.0mg/dl by using this reagent.720newborn filter paper dried blood samples were detected in this study, which included61positive cases and959negative cases. Compared with Labsy stems reagent, the positive rate, the negative rate, total coincidence rate of this reagent were100.0%and99.72%respectively. Kappa value was0.856(P=0.000)The data measured by Self-made and control kit had significant correlation [slope (95%CI),0.899(0.883～0.914); y-intercept (95%CI),-0.011(-0.046～0.025) mg/dl]. The two assays showed a correlation coefficient of0.973(P=0.000).
3. Development of PAPP-A control materials
PAPP-A control materials were prepared successfully by this study. The PAPP-A control materials had an acceptable appearance, good rehydration quality. The moisture content of three level controls were all lower than3.0%and were1.72%,2.01%,2.16%respectively. In the homogeneity evaluation, the results showed that the difference were insignificant(P>0.05) within the same levels of PAPP-A control materials. The stability evaluation results showed that the control samples can be stable for at least60w at-20℃conditions,7d at room temperature (18℃-25℃) and3w at37℃.After reconstitution, PAPP-A control materials can be stable for3d at2℃-8℃,3h at room temperature and5w at-20℃. The measured values and95%intervals of three levels of PAPP-A control materials were58.34(53.87-62.81) mIU/L,208.40(185.16-231.64) mIU/L and802.82(765.23-840.40) mIU/L respectively. There were obvious differences in determination results of the control materials prepared in this study and fresh serum samples measured by each reagent.
Conclusions
PartⅠ:
We successfully developed neonatal17a-hydroxyprogesterone and total galactose diagnostic reagent. The indicators (including accuracy, sensitivity, correlation coefficients, precision, specificity, interference, etc.) met the requirements of clinical testing applications and reach the level of the same kind of products abroad. So, these two diagnostic reagent is expected to replace the imported reagent. The development of neonatal total galactose diagnostic reagent is the first one developed in China.
With congenital adrenal hyperplasia and galactosemia increasing awareness at home, the number of regions carrying out these two screening programs will also continue to increase. However, homemade reagent manufacturer is currently only one. The reagent developed by this study will surely have a wonderful application prospects and access to huge economic benefits, get enormous economical benefits in neonatal screening field.
Part II:
The results of the second part demonstrated that the indicators (including appearance, rehydration quality, moisture content, homogeneity and stability) of PAPP-A control materials in this study are satisfactory. PAPP-A control materials are suitable for quality evaluation of laboratory detecting PAPP-A and precision et al. evaluation for PAPP-A reagents. If PAPP-A control materials as commodity are provided in the market in the future, it will have a high application value and economic benefit.
Since antigen, detection principle and instrumentation varied in these five reagents of PAPP-A, the scatter of measurement result of PAPP-A control is big and the comparability is poor. Therefore, through the extensive application of the control samples in the future, it will enable manufacturers to analyze reasons for difference value and improve reagent quality. At last, It will lay the foundation for the promotion of interchangeability of test results.
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