| Background and ObjectiveThis 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.MethodsAccording 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.Results1. 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 reagentUsing 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 materialsPAPP-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.ConclusionsPartⅠ: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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