WO2020052294A1 - 汞、镉混合暴露检测在非梗阻性无精子症辅助诊断中的应用 - Google Patents
汞、镉混合暴露检测在非梗阻性无精子症辅助诊断中的应用 Download PDFInfo
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- WO2020052294A1 WO2020052294A1 PCT/CN2019/090512 CN2019090512W WO2020052294A1 WO 2020052294 A1 WO2020052294 A1 WO 2020052294A1 CN 2019090512 W CN2019090512 W CN 2019090512W WO 2020052294 A1 WO2020052294 A1 WO 2020052294A1
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- obstructive azoospermia
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- the invention belongs to the fields of analytical chemistry and clinical medicine, and relates to an exposure biomarker related to non-obstructive azoospermia and a detection method and application thereof.
- the exposure biomarker is a mixed exposure of mercury and cadmium, and its detection method is based on ICP -MS detection method.
- Exposure omics as a complement to genomics refers to environmental exposures (including lifestyle factors) throughout life from the beginning of pregnancy. Exposure sources include external sources (pollution, radiation, diet, etc.) and endogenous sources (inflammation, infection, microorganisms, etc.)
- EWAS Full Exposure Association Study
- GWAS Whole Genome Association Study
- Biomarkers and in turn use these biomarkers to elucidate exposure-effect relationships (biochemical epidemiology), sources of exposure and human kinetics (exposure biology), and mechanisms of action (systems biology).
- the research on the relationship between environmental exposure and health can start by measuring pollutants in the air, water, and food in the environment outside the case group and the control group to test whether there is a difference between the two groups, and then through the intake of major substances, Metabolism and other considerations are used to estimate the exposure dose. This is called a bottom-up method.
- Another method is to measure the substances in the blood or other body fluids of the case group and the control group and test the various substances.
- the statistical connection with the disease which ultimately determines the substance that causes the disease and the source of its exposure, is called a top-down approach.
- the “bottom-up” and “top-down” methods are of scientific value in identifying individual exposures.
- the “top-down” method is used to reveal unknown sources of exposure to human diseases, while the “bottom-up” method It is a method for analyzing external exposures and establishing interventions and prevention.
- Biomarkers can be used not only to study external exposures, but also to study internal exposures.
- Internal exposure omics is studied by omics methods, such as genomics, proteomics, epigenomics, metabolomics, transcriptomics, adductomics, etc. Exposure omics involves cross-disciplinary research (epidemiology, biostatistics, bioinformatics, analytical chemistry, etc.).
- Mercury (Hg) and cadmium (Cd) are important toxic metals and widely exist in environments of natural and man-made sources [5] . Because of its extensive exposure, heavy metals can be detected in biological samples from most people, including blood, seminal plasma and urine [6-8] . Previous studies have shown that exposure to either mercury or cadmium in men can cause sperm production disorders [9-12] . Studies have shown that mercury exposure can reduce the level of 17 ⁇ -estradiol, which can make testicular tissue more susceptible to oxidative damage, leading to functional inactivation and sperm disturbance [13] . Exposure to cadmium can target the disruption of the blood-testis barrier, leading to impaired spermatogenesis [14] .
- mercury and cadmium may be an important cause of non-obstructive azoospermia and should be given more attention and attention in the clinic.
- men are exposed to a variety of heavy metals and other chemicals, which work together to cause health effects or increase sensitivity to toxic effects [15] .
- synergy and antagonism between metals which affects the absorption, distribution and excretion of each other [15] .
- environmental toxicants are in a mixed exposure state, the effects of multiple heavy metals on sperm quality should be considered simultaneously [16] . Therefore, the combined exposure of mercury and cadmium may be an important cause of spermatogenesis disorders, which should cause more attention and attention in the clinic. Therefore, whether the single exposure of mercury and cadmium at safe concentrations can cause non-obstructive azoospermia when mixed exposure remains to be further studied.
- GFAAS Graphite furnace atomic absorption spectrometry
- ICP-MS Inductively coupled plasma mass spectrometry
- ICP-MS In past practice, ICP-MS was originally used for the analysis of heavy metal pollution levels in environmental samples such as water samples and soil. It has since been used to detect levels of lead, cadmium, mercury and other heavy metals in bodily fluid samples of workers at risk of occupational exposure. In recent years, more and more research and practice use ICP-MS as an early marker for the identification and detection of various human diseases, such as diabetes, neurodevelopmental disorders in children, cardiovascular disease, adverse birth outcomes, etc., related papers Published in academic journals "Environ Health Perspective”, “Epidemiology”, “Environ Pollut”, “Occup Environ Med”, reflecting its great potential and value in the diagnosis of human diseases.
- Exposure biomarkers related to non-obstructive azoospermia are a mixed exposure of mercury and cadmium.
- a method for detecting the exposed biomarker is to measure the concentration of mercury and cadmium in male whole blood by ICP-MS, especially to detect the concentration of male whole blood by ICP-MS with internal standard and standard curve mixed. Concentrations of mercury and cadmium.
- the detection method, the specific steps and parameters of the detection method are:
- ICP-MS is stable for about half an hour after the ignition is started; pump 1ppb tuning solution (provided by the instrument manufacturer, Ba, Bi, Ce, Co, Ln, Li, U, each concentration 1 ⁇ g / L, the matrix is 2.5 % HNO 3 , 0.5% HCl), according to the sensitivity of Li, In and U, the level of oxide (CeO) and double charge (Ba 2+ ), the whole mass range (4 ⁇ 290amu) is tuned to reach the optimal state ; Tuning requirements meet the following standards: standard mode sensitivity 7Li ⁇ 4000cps / ppb, 115In ⁇ 15000cps / ppb; 238U ⁇ 20000cps / ppb; standard mode double charge Ba 2+ /Ba ⁇ 3.0%; standard mode oxide 156CeO / 140Ce ⁇ 2.0 %; See Table 1.1 for optimized operating parameters;
- the instrument collection parameters are shown in Table 1.2.
- the instrument operates in the single collision cell mode of Kinetic Energy Discrimination (KED) when testing samples. Pure helium is used as the collision gas and the system is flushed with a cleaning solution.
- KED Kinetic Energy Discrimination
- kits for detecting non-obstructive azoospermia contains a reagent for detecting the concentration of mercury and cadmium in male whole blood by ICP-MS.
- the auxiliary diagnostic kit contains the following reagents:
- Reagent A Diluent (containing 0.1% v / v triton + 1% v / v nitric acid + 10 ⁇ g / L rhodium + 10 ⁇ g / L bismuth, the rest is deionized water);
- Reagent B Mercury standard curve: the concentration is 0, 0.1, 0.5, 1, 5, 10, 50 ⁇ g / L;
- Reagent C cadmium standard curve: the concentration is 0, 0.01, 0.05, 0.1, 0.5, 1, 5 ⁇ g / L;
- Reagent D cleaning solution: 2% v / v nitric acid.
- the standard curve uses a diluent configuration.
- the concentration of the standard curve 0 is the diluent.
- the content of the internal standard element is diluted by the standard solution of the element.
- the inventors collected standard male whole blood samples using standard operating procedures (SOPs), systematically collected complete population basic information and clinical data, and used ICP-MS-based detection methods for analysis.
- SOPs standard operating procedures
- ICP-MS-based detection methods for analysis.
- the experimental methods of research mainly include the following parts:
- Group A Healthy control group (100 people, normal semen examination and fertility):
- Group B Case group (100 people, azoospermia by semen examination, non-obstructive azoospermia diagnosed by testicular puncture):
- ICP-MS is stable for about half an hour after ignition is started; pump 1ppb tuning solution (provided by the instrument manufacturer, Ba, Bi, Ce, Co, Ln, Li, U, each concentration 1 ⁇ g / L, the matrix is 2.5 % HNO 3 , 0.5% HCl), according to the sensitivity of Li, In and U, the level of oxide (CeO) and double charge (Ba 2+ ), the whole mass range (4 ⁇ 290amu) is tuned to reach the optimal state ; Tuning requirements meet the following standards: standard mode sensitivity 7Li ⁇ 4000cps / ppb, 115In ⁇ 15000cps / ppb; 238U ⁇ 20000cps / ppb; standard mode double charge Ba 2+ /Ba ⁇ 3.0%; standard mode oxide 156CeO / 140Ce ⁇ 2.0 %; See Table 1.1 for optimized operating parameters;
- the instrument collection parameters are shown in Table 1.2.
- the instrument operates in the single collision cell mode of Kinetic Energy Discrimination (KED) when testing samples. Pure helium is used as the collision gas and the system is flushed with a cleaning solution.
- KED Kinetic Energy Discrimination
- Injection method automatic injection, injection and stabilization time 30s, cleaning time 30s.
- Exposure levels above 75% of the total sample are considered high exposure (+), otherwise they are low exposure (-).
- the constituent ratios of individuals with different exposure levels in the healthy control group and the case group were compared using the chi-square test.
- the inventor also prepared an auxiliary diagnostic kit that can be used for non-obstructive azoospermia.
- the auxiliary diagnostic kit includes a set of reagents and consumables for detecting whole blood mercury and cadmium.
- the present inventors compared the exposure levels of mercury and cadmium in whole blood of normal control and non-obstructive azoospermia cases by using ICP-MS, and found that there is a marker with non-obstructive azoospermia diagnosis value in male whole blood In combination with the application of ICP-MS for the detection of this marker, an auxiliary diagnostic kit that can facilitate the clinical application of non-obstructive azoospermia was developed.
- the invention uses the mixed exposure level of mercury and cadmium in male whole blood as a marker for non-obstructive azoospermia evaluation.
- the advantages are:
- the mixed exposure detection of mercury and cadmium provided by the present invention can be used for the auxiliary diagnosis of non-obstructive azoospermia, and the early diagnosis of non-obstructive azoospermia through a minimally invasive method can provide a basis for further in-depth examination by clinicians.
- the present invention uses non-obstructive azoospermia cases and whole blood samples from healthy control groups for verification, and proves that non-obstructive azoospermia cases have higher exposure levels of mercury and cadmium and can be used as markers.
- the ICP-MS technology has simple sample processing, rapid and accurate instrument analysis, and results can be issued within 30 minutes after sampling, which has high clinical diagnostic value.
- the sample operation steps of the present invention are simple, can prevent sample contamination, and improve sensitivity.
- a 100 ⁇ l whole blood sample can be analyzed by adding it to the prepared diluent, which reduces the requirements on the environment and operators.
- the present invention uses ICP-MS technology to optimize the pre-treatment steps for detection of combined exposure of mercury and cadmium, which can better reduce the cost and time of detection.
- the top and bottom of the box plot represent the 75th and 25th percentiles, respectively, and the top to bottom of the box plot represent the 90th and 10th percentiles.
- FIG 2 Cadmium exposure levels in healthy control and case groups. Refer to Figure 1 for legends.
- the present inventors collected whole blood samples of non-obstructive azoospermia cases and normal control men from the Nanjing Maternal and Child Health Hospital of Nanjing Medical University. Semen examination and normal fertility were the controls, and non-obstructive azoospermia was the case. A total of 100 cases were randomly selected, and a total of 200 matched males were matched according to age, BMI, and abstinence time. A total of 200 people were used as subjects for the assessment of non-obstructive azoospermia mercury and cadmium mixed exposure.
- the specific sample classification criteria are as follows:
- Group A Healthy control group (100 people, normal semen examination and fertility):
- Group B case group (100 people, non-obstructive azoospermia):
- Example 2 Analysis of ICP-MS detection of mixed exposure of mercury and cadmium and occurrence of non-obstructive azoospermia
- ICP-MS is stable for about half an hour after ignition is started; pump 1ppb tuning solution (provided by the instrument manufacturer, Ba, Bi, Ce, Co, Ln, Li, U, each concentration 1 ⁇ g / L, the matrix is 2.5 % HNO 3 , 0.5% HCl), according to the sensitivity of Li, In and U, the level of oxide (CeO) and double charge (Ba 2+ ), the whole mass range (4 ⁇ 290amu) is tuned to reach the optimal state ; Tuning requirements meet the following standards: standard mode sensitivity 7Li ⁇ 4000cps / ppb, 115In ⁇ 15000cps / ppb; 238U ⁇ 20000cps / ppb; standard mode double charge Ba 2+ /Ba ⁇ 3.0%; standard mode oxide 156CeO / 140Ce ⁇ 2.0 %; See Table 1.1 for optimized operating parameters;
- the instrument collection parameters are shown in Table 1.2.
- the instrument operates in the single collision cell mode of Kinetic Energy Discrimination (KED) when testing samples. Pure helium is used as the collision gas and the system is flushed with a cleaning solution.
- KED Kinetic Energy Discrimination
- Injection method automatic injection, injection and stabilization time 30s, cleaning time 30s.
- the data was quantitatively evaluated online using Qtegra (ISDS) software (Thermo Fisher Scientific, USA), and the internal standards rhodium and bismuth were used to correct the concentrations of cadmium and mercury, respectively.
- ISOS Quality of Service
- rhodium and bismuth were used to correct the concentrations of cadmium and mercury, respectively.
- Exposure levels above 75% of the total sample are considered high exposure (+), otherwise they are low exposure (-).
- the constituent ratios of individuals with different exposure levels in the healthy control group and the case group were compared using the chi-square test.
- the ICP-MS method was used to determine the high levels of mercury and cadmium in the whole blood of the case group, and then the chi-square test was used to find that the composition ratios of individuals in the healthy control group and the case group at different exposure levels were significantly different.
- the number of high-exposure was higher than that of the control group.
- the high exposure of mercury and cadmium significantly increased the incidence of non-obstructive azoospermia.
- Detection of mixed exposure of mercury and cadmium can be used for the auxiliary diagnosis of non-obstructive azoospermia.
- This kit includes a batch of reagents and consumables for the detection of mercury and cadmium in whole blood.
- the diluent is used for sample dilution, and it contains a stable and detectable internal standard for experimental quality control. Each concentration gradient of mercury and cadmium standard curve It is used to draw a standard curve for quantitative analysis.
- the cleaning solution is used for container cleaning and blank control. The value of this kit is that only 100 ⁇ l of male whole blood can be used to detect the mercury and cadmium content in the whole blood at the same time, and then the comparison of the analysis results with the reference value is used to assist the diagnosis of non-obstructive azoospermia, and it is easy to perform dynamic Monitor and observe the effect of treatment.
- the specific kit composition is as follows:
- Reagent A Diluent (containing 0.1% v / v triton + 1% v / v nitric acid + 10 ⁇ g / L rhodium + 10 ⁇ g / L bismuth, the rest is deionized water);
- Reagent B Mercury standard curve: the concentration is 0, 0.1, 0.5, 1, 5, 10, 50 ⁇ g / L;
- Reagent C cadmium standard curve: the concentration is 0, 0.01, 0.05, 0.1, 0.5, 1, 5 ⁇ g / L;
- Reagent D cleaning solution: 2% v / v nitric acid.
- TAYLOR CM, EMOND, M, LINGAM, R et al. Prenatal, Lead, Cadmium, Mercury, Exposure, and Associations with motors, skills, 7years, ina, UK, Observational, birth, cohort, etc. -7.
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Abstract
本发明属于分析化学及临床医学领域,公开了汞、镉混合暴露检测在非梗阻性无精子症辅助诊断中的应用。与非梗阻性无精子症相关的暴露生物标志物为汞和镉的混合暴露。采用ICP-MS检测全血中的汞和镉的浓度,可用于辅助诊断非梗阻性无精子症,用血量少、操作简单、快速、准确,具有较好的临床推广价值。
Description
本发明属于分析化学及临床医学领域,涉及一种与非梗阻性无精子症相关的暴露生物标志物及其检测方法和应用,该暴露生物标志物为汞镉混合暴露,其检测方法为基于ICP-MS的检测方法。
大约30%-55%的人类不孕不育与男性因素有关
[1],男性不育症最常见的原因是精子发生障碍,临床上称为弱精子症或无精子症
[2]。其中10-15%归因于无精子症
[1]。近年来,随着人们生殖健康需求的不断提高,男性不育越来越成为社会关注的焦点。WHO不育症防治专题组的研究表明生殖内分泌功能紊乱,特别是精液质量低下是男性不育的重要病因。在过去50年中,男性精液质量,尤其是精子生成数量有了相当程度的下降
[3],我国的研究也呈现出相类似的情况
[4]。男性不育受到多方面因素的影响,包括环境因素,遗传因素以及表观遗传因素。由于短期内遗传变异相对有限,因此我们有理由相信,非梗阻性无精子症更可能是受到了某些环境危险因素的影响。
基因组学进行了大量广泛的研究,相对于投入的大量资金及人员,其研究结果对健康的影响并不明显。暴露组学作为基因组学的补充是指从妊娠开始贯穿整个人生的环境暴露(包括生活方式因素).暴露源包括外源(污染、辐射、饮食等)和内源(炎症、感染、微生物等),继全基因组关联研究(GWAS)之后发展的全暴露组关联研究(EWAS)的目的是对在未知方式下暴露的评估,EWAS方法通过比较患者和健康受试者暴露组的分析结果,确定有效的生物标志物,进而利用这些生物标志物来阐明暴露-效应关系(生化流行病学)、暴露和人体动力学来源(暴露生物学)、以及作用机制(系统生物学)。环境暴露与健康之间的关系研究可从测定病例组和对照组外环境中的空气、水、食物内的污染物入手,检验两组间是否存在差异,进而通过对主要物质的摄入量、代谢等进行考量,估计暴露剂量,这称之为自下而上的方法(bottom-up);另一种方法是从测量病例组和对照组血液或其他体液内的物质入手,检验各类物质与疾病之间的统计学联系,最终确定导致疾病的物质及其暴露来源,称之自上而下的方法(top-town)。 “自下而上”和“自上而下”的方法在识别个体暴露上都具有科学价值,“自上而下”法用于揭示人类疾病的未知暴露源,而“自下而上”法是用于分析外暴露以及建立干预与预防的方法。生物标志物不仅可以用于研究外暴露,也可以用于研究内暴露。内暴露组学采用组学的方法进行研究,如基因组学、蛋白质组学、表观基因组学、代谢组学、转录组学、加合物组学等。暴露组学涉及多学科交叉(流行病学、生物统计学、生物信息学、分析化学等),目前研究方向如发展更先进的分析技术(提高通量、降低样本量及费用等)、高效寻找合适的暴露标志物、混合暴露与疾病的关系、在群体水平找到与疾病相关的暴露因素、暴露组关联研究,等等。
环境暴露与健康关系研究中存在一些问题,比如,针对单一环境暴露因素研究(忽略混合暴露的存在及各个暴露间的联合作用),环境暴露因素种类局限(集中在已知的和法规重点控制的暴露),环境暴露采样时机与疾病的自然发生进程分离(较少考虑慢性病存在潜伏期)、环境暴露采样与检测不准确,等等。对暴露内部标志物的测定要与疾病发生的“时间窗”相对应,否则,研究结果将可能因为暴露和疾病发生时间上的先后关系不清而无法得出可靠结论。
汞(Hg)和镉(Cd)是重要的有毒金属,广泛存在于自然和人为来源的环境中
[5]。因其广泛暴露,大多数人群的生物样本中都能检测到重金属,包括血液、精浆和尿液
[6-8]。以往的研究表明男性的汞或镉暴露均会造成精子生成障碍
[9-12]。有研究表明汞暴露后可导致17β-雌二醇的水平降低,可使睾丸组织更容易受到氧化损伤,从而导致功能失活,导致精子发生障碍
[13]。镉的暴露可以靶向破坏血睾屏障,从而导致精子生成障碍
[14]。因此,汞、镉暴露可能是非梗阻性无精子症的重要病因,应当在临床上引起更多的关注和重视。通常情况下,男性会接触多种重金属和其他化学物,它们会共同作用导致健康效应或增加毒作用的敏感性
[15]。金属之间因存在相加作用,协同作用,拮抗作用,从而影响彼此的吸收,分配和排泄
[15]。因环境毒物均呈混合暴露的状态,应该同时考虑多种重金属对精子质量的影响
[16]。所以汞、镉混合暴露可能是精子生成障碍的重要病因,应当在临床上引起更多的关注和重视。因此单独暴露均处于安全浓度的汞、镉,是否在混合暴露时能够导致非梗阻性无精子症尚有待进一步研究。
石墨炉原子吸收光谱法(GFAAS)是目前全血中铅和镉常用的检测方法,可以做到较快速的准确定量分析
[17]。然而这一方法标准工作曲线的线性范围窄,样品前处理较复杂,特别是其无法进行多元素同时分析,因此局限了其在混合暴露评估中的 应用。其他常用方法诸如原子荧光光谱(AFS)、电化学分析法等也具有特定的不足。电感耦合等离子体质谱(ICP-MS)是用于痕量及超痕量多元素分析的质谱仪,具有灵敏度高、检出限低、选择性好、分析速度快、动态范围宽等优点,由于其多元素高通量检测的特点,因而被广泛应用于混合暴露特别是多种重金属混合暴露的检测当中
[18-20]。
在既往的实践中,ICP-MS最初被用于环境样本如水样、土壤的重金属污染水平分析。此后又被用于具有职业暴露风险的工人体液样本中铅、镉、汞等重金属水平检测。近年来,越来越多的研究和实践中使用ICP-MS作为人类各类疾病的早期标志物的识别和检测应用,如糖尿病,儿童神经发育障碍,心血管疾病,不良出生结局等,相关论文发表在学术期刊《Environ Health Perspect》、《Epidemiology》、《Environ Pollut》、《Occup Environ Med》,体现出其在人类疾病诊断中的巨大潜力与价值。然而目前采用ICP-MS分析男性全血中汞、镉混合暴露水平在非梗阻性无精子症辅助诊断中的应用还未得到相应的关注。若能发现汞、镉混合暴露作为辅助诊断标志物与非梗阻性无精子症的明确关联,并研发相应疾病的ICP-MS检测方法,提高检测结果的准确性与灵敏度,不仅可以在该领域获得国际领先的研究成果,还能创造可观的经济价值,并且为促进和提高我国男性生殖健康水平做出贡献。
发明内容
本发明的目的是:
1.将汞、镉混合暴露作为非梗阻性无精子症辅助诊断标志物;
2.建立基于ICP-MS的男性全血中汞、镉混合暴露的检测方法;
3.开发用于非梗阻性无精子症辅助诊断的汞、镉混合暴露检测和辅助诊断试剂盒。
本发明的目的通过下列技术措施实现的:
与非梗阻性无精子症相关的暴露生物标志物,该暴露生物标志物为汞和镉的混合暴露。
用于检测所述暴露生物标志物的方法,该方法为采用ICP-MS检测男性全血中的汞和镉的浓度,特别是采用内标与标准曲线混合的ICP-MS检测男性全血中的汞和镉的浓度。
所述的检测方法,该检测方法的具体步骤及参数为:
a.ICP-MS点火启动后,稳定约半小时;泵入1ppb调谐液(由仪器厂家配套提供, Ba、Bi、Ce、Co、Ln、Li、U,每种浓度1μg/L,基质为2.5%HNO
3、0.5%HCl),根据Li、In和U的灵敏度,氧化物(CeO)以及双电荷(Ba
2+)的水平,对全部质量范围(4~290amu)进行调谐并达到最佳状态;调谐要求达到以下标准:标准模式灵敏度7Li≥4000cps/ppb、115In≥15000cps/ppb;238U≥20000cps/ppb;标准模式双电荷Ba
2+/Ba<3.0%;标准模式氧化物156CeO/140Ce<2.0%;优化的操作参数见表1.1;
表1.1 ICP-MS优化的操作参数
b.仪器采集参数见表1.2,仪器在检测样本时在动能歧视(Kinetic Energy Discrimination,KED)的单一碰撞池模式下运行,采用纯氦气作为碰撞气体,使用清洗液冲洗系统。
表1.2 ICP-MS采集参数
其他步骤详见厂商说明书。
所述的暴露生物标志物在制备检测非梗阻性无精子症的辅助诊断试剂盒中的应用。
一种用于检测非梗阻性无精子症的辅助诊断试剂盒,该试剂盒含有采用ICP-MS检测男性全血中的汞和镉的浓度的试剂。
所述的辅助诊断试剂盒,该试剂盒含有下列试剂:
试剂A:稀释剂(含0.1%v/v曲拉通+1%v/v硝酸+10μg/L铑+10μg/L铋,其余为去离子水);
试剂B:汞标准曲线:浓度分别为0、0.1、0.5、1、5、10、50μg/L;
试剂C:镉标准曲线:浓度分别为0、0.01、0.05、0.1、0.5、1、5μg/L;
试剂D:清洗液:2%v/v硝酸。
(注:标准曲线采用稀释剂配置,标准曲线0浓度就是稀释剂。内标元素含量通过该元素的标准液稀释而来。)
本发明详细描述如下:
本发明人以标准操作程序(SOP)采集符合标准的男性全血样本,系统收集完整的人群基础信息和临床资料,并采用了基于ICP-MS检测方法进行分析。
具体来说研究的实验方法主要包括以下几个部分:
一、研究对象选择和分组依据
纳入病例男性100人,根据年龄、BMI、禁欲时间匹配对照100人,共200人。
A组:健康对照组(100人,精液检查和生育力正常):
1.年龄在22到37岁间;
2.无高血压、糖尿病等慢性疾病;
3.无吸烟、饮酒史;
4.无泌尿生殖道炎症、附睾炎、睾丸损伤、隐睾等疾病史;
5.无性功能异常或排精异常;
6.无其他与男性生殖障碍有关的疾病史。
B组:病例组(100人,精液检查无精子,睾丸穿刺确诊非梗阻性无精子症):
1.年龄在25到38岁间;
2.无高血压、糖尿病等慢性疾病;
3.无吸烟、饮酒史;
4.无泌尿生殖道炎症、附睾炎、睾丸损伤、隐睾等疾病史;
5.无性功能异常或排精异常;
6.无其他与男性生殖障碍有关的疾病史。
二、ICP-MS检测汞、镉混合暴露与非梗阻性无精子症发生的分析
1.样本前处理
取100μl全血,加入4.9ml试剂A(即稀释50倍),充分混匀。
2.仪器检测
分析仪器:ICAP RQ ICP-MS(Thermo)
a.ICP-MS点火启动后,稳定约半小时;泵入1ppb调谐液(由仪器厂家配套提供,Ba、Bi、Ce、Co、Ln、Li、U,每种浓度1μg/L,基质为2.5%HNO
3、0.5%HCl),根据Li、In和U的灵敏度,氧化物(CeO)以及双电荷(Ba
2+)的水平,对全部质量范围(4~290amu)进行调谐并达到最佳状态;调谐要求达到以下标准:标准模式灵敏度7Li≥4000cps/ppb、115In≥15000cps/ppb;238U≥20000cps/ppb;标准模式双电荷Ba
2+/Ba<3.0%;标准模式氧化物156CeO/140Ce<2.0%;优化的操作参数见表1.1;
表1.1 ICP-MS优化的操作参数
b.仪器采集参数见表1.2,仪器在检测样本时在动能歧视(Kinetic Energy Discrimination,KED)的单一碰撞池模式下运行,采用纯氦气作为碰撞气体,使用清洗液冲洗系统。
表1.2 ICP-MS采集参数
其他步骤详见厂商说明书。
进样方式:自动进样,进样及稳定时间30s,清洗时间为30s。
3.数据处理:
使用Qtegra(ISDS)软件(赛默飞世尔科技,美国)在线对数据进行定量评估,以内标铑(Rh)、铋(Bi)分别用于校正镉和汞的浓度。将样品名称、待测元素名称、浓 度或信号强度(Counts Per Second,CPS)值等导出至excel表格中。
4.数据分析:
暴露水平在总样本75%分位数以上时被认为是高暴露(+),否则为低暴露(-)。健康对照组与病例组中不同暴露水平个体的构成比采用卡方检验进行比较。
5.健康对照组、病例组全血样本中汞、镉混合暴露的差异和诊断意义。
经Welch's t-test发现病例组汞和镉暴露水平均高于对照组。卡方检验发现健康对照组与病例组中不同暴露水平个体的构成比具有显著差异,病例组的高暴露人数多于对照组,汞、镉高暴露显著增加了非梗阻性无精子症发病率。
三、诊断试剂盒制备方法
根据上述一系列实验结果,本发明人还制备了一种能用于非梗阻性无精子症的辅助诊断试剂盒,所述辅助诊断试剂盒包含一套全血汞、镉检测的试剂及耗材。
本发明的有益效果:
本发明人通过采用ICP-MS比较正常对照和非梗阻性无精子症病例全血中的汞、镉混合暴露水平,发现了男性全血中存在具有非梗阻性无精子症辅助诊断价值的标志物组合,以及该标志物检测的ICP-MS的应用,研制出可便于临床应用非梗阻性无精子症的辅助诊断试剂盒。
本发明采用男性全血中汞、镉混合暴露水平作为非梗阻性无精子症评价的标志物的优越性在于:
(1)汞、镉混合暴露是一组新型生物标志物,其与疾病结局关联强,不仅稳定、微创、易于检测,且定量精确,将大大提高非梗阻性无精子症辅助诊断的敏感性和特异性,该类生物标志物的成功开发将为男性不育的防治开创全新局面,为其他疾病生物标志物的研制提供借鉴。
(2)本发明提供的汞、镉混合暴露检测可用于非梗阻性无精子症的辅助诊断,可在早期通过微创方式早期诊断非梗阻性无精子症,从而为临床医生进一步深入检查提供依据,为快速准确掌握患者的疾病状态和病情严重程度、及时采取更具个性化的防治方案提供支持,延缓和阻止疾病进展。
(3)本发明采用非梗阻性无精子症病例和健康对照人群的全血样本进行验证,证明了非梗阻性无精子症病例具有较高的汞、镉暴露水平,可作为标志物使用。
(4)ICP-MS技术样本处理简单,仪器分析迅速准确,取样后,30分钟内可出具 结果,具有较高的临床诊断实用价值。
(5)本发明样本操作步骤简单,且能防止样品污染,提高灵敏度,100μl全血样本加入配备好的稀释剂中即可上机进行分析,对环境及操作人员的要求降低。
(6)本发明采用ICP-MS技术对汞、镉联合暴露进行检测的前处理步骤进行了优化,能够更好地减少检测的成本和时间。
图1健康对照组和病例组的汞暴露水平。
箱式图的顶部和底部分别代表第七十五和第二十五百分位,箱式图的上端到下端代表第九十和第十百分位。
图2健康对照组和病例组的镉暴露水平,图注参考图1。
图3卡方检验发现健康对照组与病例组中不同暴露水平个体的构成比具有显著差异,病例组的高暴露人数多于对照组,汞、镉高暴露显著增加了非梗阻性无精子症发病率。
以下通过实施例对本发明作进一步的阐述。
实施例1研究对象选择和分组依据
本发明人从南京医科大学附属南京妇幼保健院采集符合要求的非梗阻性无精子症病例及正常对照男性的全血样本。精液检查和生育力正常的为对照,非梗阻性无精子症者为病例。随机纳入100例病例,根据年龄、BMI、禁欲时间匹配对照男性100人,共200人作为非梗阻性无精子症汞、镉混合暴露评估的实验对象。具体的样本归类标准如下:
第一阶段关联探索阶段
纳入病例男性100人,根据年龄、BMI、禁欲时间匹配对照100人,共200人。
A组:健康对照组(100人,精液检查和生育力正常):
1.年龄在22到37岁间;
2.无高血压、糖尿病等慢性疾病;
3.无吸烟、饮酒史;
4.无泌尿生殖道炎症、附睾炎、睾丸损伤、隐睾等疾病史;
5.无性功能异常或排精异常;
6.无其他与男性生殖障碍有关的疾病史。
B组:病例组(100人,非梗阻性无精子症者):
1.年龄在25到38岁间;
2.无高血压、糖尿病等慢性疾病;
3.无吸烟、饮酒史;
4.无泌尿生殖道炎症、附睾炎、睾丸损伤、隐睾等疾病史;
5.无性功能异常或排精异常;
6.无其他与男性生殖障碍有关的疾病史。
实施例2 ICP-MS检测汞、镉混合暴露与非梗阻性无精子症发生的分析
1.样本前处理
取100μl全血,加入4.9ml试剂A(即稀释50倍),充分混匀。
2.仪器检测
分析仪器:ICAP RQ ICP-MS(Thermo)
a.ICP-MS点火启动后,稳定约半小时;泵入1ppb调谐液(由仪器厂家配套提供,Ba、Bi、Ce、Co、Ln、Li、U,每种浓度1μg/L,基质为2.5%HNO
3、0.5%HCl),根据Li、In和U的灵敏度,氧化物(CeO)以及双电荷(Ba
2+)的水平,对全部质量范围(4~290amu)进行调谐并达到最佳状态;调谐要求达到以下标准:标准模式灵敏度7Li≥4000cps/ppb、115In≥15000cps/ppb;238U≥20000cps/ppb;标准模式双电荷Ba
2+/Ba<3.0%;标准模式氧化物156CeO/140Ce<2.0%;优化的操作参数见表1.1;
表1.1 ICP-MS优化的操作参数
b.仪器采集参数见表1.2,仪器在检测样本时在动能歧视(Kinetic Energy Discrimination,KED)的单一碰撞池模式下运行,采用纯氦气作为碰撞气体,使用清洗液冲洗系统。
表1.2 ICP-MS采集参数
其他步骤详见厂商说明书。
进样方式:自动进样,进样及稳定时间30s,清洗时间为30s。
3.数据处理:
使用Qtegra(ISDS)软件(赛默飞世尔科技,美国)在线对数据进行定量评估,以内标铑、铋分别用于校正镉和汞的浓度。将样品名称、待测元素名称、浓度或信号强度(Counts Per Second,CPS)值等导出至excel表格中。
4.数据分析:
暴露水平在总样本75%分位数以上时被认为是高暴露(+),否则为低暴露(-)。健康对照组与病例组中不同暴露水平个体的构成比采用卡方检验进行比较。
5.健康对照组、病例组全血样本中汞、镉混合暴露的差异和诊断意义。
经Welch's t-test发现病例组汞和镉暴露水平均高于对照组。卡方检验发现健康对照组与病例组中不同暴露水平个体的构成比具有显著差异(结果见图1、图2、图3),病例组高暴露人数与对照组高暴露人数具体比例为汞高暴露2.55:1,镉高暴露:2.00:1,汞、镉均高暴露2.00:1,病例组的高暴露人数多于对照组,显著增加了非梗阻性无精子症发病率。
实施例3用于检测男性全血汞、镉混合暴露辅助诊断非梗阻性无精子症的试剂盒制作
首先通过ICP-MS的方法确定病例组全血中具有较高水平的汞、镉浓度,然后通过卡方检验发现健康对照组与病例组中不同暴露水平个体的构成比具有显著差异,病例组的高暴露人数多于对照组,汞、镉高暴露显著增加了非梗阻性无精子症发病率。 检测汞、镉混合暴露可以用于非梗阻性无精子症的辅助诊断。此试剂盒包括一批全血中汞、镉检测用试剂和耗材,其中稀释剂用于样品稀释,其中含有稳定且可检测的内标用于实验质量控制,汞和镉标准曲线浓度梯度各一条用于绘制标准曲线进行定量分析,清洗液用于容器清洗和空白对照。此试剂盒的价值在于只需要100μl男性全血,即可同时检测全血中的汞、镉含量,再通过分析结果与参考值的比较用于辅助诊断非梗阻性无精子症,并易于进行动态监测和观察治疗效果。
具体试剂盒组成如下:
试剂A:稀释剂(含0.1%v/v曲拉通+1%v/v硝酸+10μg/L铑+10μg/L铋,其余为去离子水);
试剂B:汞标准曲线:浓度分别为0、0.1、0.5、1、5、10、50μg/L;
试剂C:镉标准曲线:浓度分别为0、0.01、0.05、0.1、0.5、1、5μg/L;
试剂D:清洗液:2%v/v硝酸。
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Claims (7)
- 与非梗阻性无精子症相关的暴露生物标志物,该暴露生物标志物为汞和镉的混合暴露。
- 用于检测权利要求1所述暴露生物标志物的方法,其特征在于该方法为采用ICP-MS检测男性全血中的汞和镉的浓度。
- 根据权利要求2所述的检测方法,其特征在于该方法为采用内标与标准曲线混合的ICP-MS检测男性全血中的汞和镉的浓度。
- 根据权利要求3所述的检测方法,其特征在于该检测方法的具体步骤及参数为:a.ICP-MS点火启动后,稳定约半小时;泵入1ppb调谐液,根据Li、In和U的灵敏度,氧化物(CeO)以及双电荷(Ba 2+)的水平,对全部质量范围(4~290amu)进行调谐并达到最佳状态;调谐要求达到以下标准:标准模式灵敏度7Li≥4000cps/ppb、115In≥15000cps/ppb;238U≥20000cps/ppb;标准模式双电荷Ba 2+/Ba<3.0%;标准模式氧化物156CeO/140Ce<2.0%;优化的操作参数见表1.1;表1.1 ICP-MS优化的操作参数b.仪器采集参数见表1.2,仪器在检测样本时在动能歧视(Kinetic Energy Discrimination,KED)的单一碰撞池模式下运行,采用纯氦气作为碰撞气体,使用清洗液冲洗系统。表1.2 ICP-MS采集参数
- 权利要求1所述的暴露生物标志物在制备检测非梗阻性无精子症的辅助诊断试剂盒中的应用。
- 一种用于检测非梗阻性无精子症的辅助诊断试剂盒,其特征在于该试剂盒含有采用ICP-MS检测男性全血中的汞和镉的浓度的试剂。
- 根据权利要求6所述的辅助诊断试剂盒,其特征在于该试剂盒含有下列试剂:试剂A:稀释剂(含0.1%v/v曲拉通+1%v/v硝酸+10μg/L铑+10μg/L铋,其余为去离子水);试剂B:汞标准曲线:浓度分别为0、0.1、0.5、1、5、10、50μg/L;试剂C:镉标准曲线:浓度分别为0、0.01、0.05、0.1、0.5、1、5μg/L;试剂D:清洗液:2%v/v硝酸。
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