目的:

室内质量控制是实验室质量控制的关键环节，目前国内尚未见全国性血细胞分析室内质量控制的相关研究。本研究通过对血细胞分析项目全国质评参加实验室的室内质量控制数据进行统计分析，发现存在的问题，提出针对性的质量改进建议，以促进血细胞分析室内质控的规范开展；同时，在前期精密度研究结果和对生物学变异数据进行科学评估的基础上，根据制订分析质量要求的模式按照浓度水平对血细胞分析精密度分析质量要求进行模拟研究，为相关指南、标准的制修订提供参考依据。

方法：

    1. 血细胞分析室内质量控制数据分析：收集2012至2017年参加全国血细胞分析室间质量评价实验室5项参数的室内质量控制数据，包括白细胞计数（WBC）、红细胞计数（RBC）、血红蛋白（Hb）、红细胞比容（Hct）和血小板计数（PLT），共计12次。经数据有效性确认后，对以下内容进行分析：①统计实验室一般信息，包括：实验室等级/类型、不同品牌仪器使用情况、不同浓度水平质控物和质控规则的使用情况。②对室内质控CV进行统计分析，包括：分析12次实验室回报CV的P₂₅、P₅₀、P₇₅、P₉₀随时间的变化趋势；比较同时使用3个浓度水平质控物的实验室检测CV的差异；不同浓度水平、不同等级/类型实验室和不同地理区域实验室CV满足行业标准的比例；将2017年实验室回报CV与其它来源的精密度要求（德国）进行比较。

2. 精密度分析质量要求的模拟研究：①基于生物学变异推导允许精密度：检索2017年4月以前发表的血细胞分析生物学变异研究文献，按照BIVAC标准排除质量较差的文献，对剩余文献进行评分，给予不同权重；筛选适用于评估成人血细胞分析生物学变异的研究；按照权重推导出可靠的生物学变异数据。②基于西格玛度量推导允许精密度：采用西格玛度量理论，取行业标准允许总误差和以基于生物学变异推导的允许总误差中较大者作为允许精密度的制订标准之一，分别计算达到3σ、4σ、5σ和6σ水平的允许精密度。③获取各种来源的精密度数据：计算2017年质评参加实验室不同浓度水平80%和90%实验室所能达到的精密度；收集专业机构（如国外协会）推荐意见；收集不同仪器品牌的精密度数据；检索国内外性能验证报道。④精密度分析质量要求的拟订：根据以上信息在制订分析质量要求的参考价值和重要程度，确定制订血细胞分析精密度质量要求的原则和方法，拟订血细胞分析精密度分析质量要求。⑤分析质量要求的适用性验证：以2018年第1次全国质评活动回报的室内质控变异系数及格率是否大于80%评估其适用性。

结果：

1. 血细胞分析室内质量控制数据分析：剔除无效数据后，剩余1431~2332家（占96.0%~99.2%）实验室的数据用于以下分析。所有等级/类型实验室数量均逐年增加，三级医院占总体比例最高（62.0%~73.9%）；使用比例最高的仪器品牌为Sysmex（占61.8%~66.8%）；分别有61.9%~66.1%、18.2%~ 23.6%和14.3%~17.3%的实验室使用1个、2个和3个浓度水平的质控物，使用2个以上浓度水平质控物的实验室由33.9%增加至38.1%。使用含1_3s/2_2s质控规则的实验室比例由59.2%上升至76.0%。不同浓度水平变异系数随时间有下降趋势，每个参数P₇₅和P₉₀下降趋势较为显著；RBC、Hb和Hct不同浓度水平室内质控CV无明显差异，PLT和WBC中值与高值室内质控CV无明显差异，低值与中值、低值与高值室内质控CV有明显差异；近6年WBC 3个浓度水平、PLT高值的及格率始终在90%以上，PLT、Hb不同浓度的及格率差异较大。三级医院、独立实验室各参数及格率均大于80%，而二级、一级医院部分年份的部分参数及格率小于80%；华东、华南地区CV及格率相对较高（及格率均大于85%），东北地区及格率多次低于80%；与德国医学协会的标准相比，80%实验室的CV能够达到其要求（PLT低值质控物的CV除外）。

2. 精密度分析质量要求的模拟研究：①共检索到19篇血细胞分析生物学变异研究，分别排除10篇方法废除、3篇研究期限≤1周、1篇研究对象为老年妇女、1篇标本类型为指血、1篇研究对象为慢性疾病人群的研究后，剩余3篇，其文献质量评分相同，赋予相同权重，其中WBC、PLT生物学变异研究文献各2篇，取其均值作为可靠的BV数据，剩余参数文献各1篇，取其数值作为可靠的BV数据。本次推导的WBC、PLT两个参数的生物学变异数据较Westgard网站生物学变异数据库生物学变异数值低，其余参数无明显差异。②WBC和Hct 3个浓度水平、Hb高值和PLT中、高值以基于行业标准规定的TEa，剩余参数采用基于生物学变异的TEa为评价标准，计算其3σ、4σ、5σ、6σ水平的允许精密度。③计算我国2017年80%、90%实验室达到的精密度，汇总德国医学会标准和检索到的文献精密度验证结果，列表表示。④以基于生物学变异推导的精密度、西格玛度量推导的精密度和我国80%、90%实验室达到的精密度水平为制订标准，若基于生物学变异推导的任一等级允许精密度（最佳、适当、最低）或基于西格玛度量计算的任一σ水平（3σ、4σ、5σ、6σ）对应的允许精密度在我国80%和90%实验室达到的精密度之间，则以较大者作为推荐的精密度分析质量要求，若以上允许精密度不在我国80%和90%实验室达到的精密度之间，则以我国90%实验室达到的精密度为推荐的精密度分析质量要求。WBC低、中、高值精密度分析质量要求的模拟结果分别为：3.8%、3.8%和3.1%， RBC均为2.1%，Hb分别为2.1%、2.1%和1.5%，Hct分别为3.0%、3.0%和2.3%、PLT分别为8.4%、6.7%和4.0%。⑤拟订的精密度分析质量要求适用性验证结果：各参数2018年第1次回报的室内质控CV满足模拟分析质量要求的比例均大于80%。

结论：

    1. 6年来，实验室室内质量控制CV随时间变化总体呈下降趋势，使用符合《医疗机构临床实验室管理办法》实施细则及CNAS-CL43质控规则的实验室数量和比例增加，但部分实验室使用的质控物浓度水平和质控规则未满足管理要求，一些实验室的室内质控CV未达到我国行业标准规定的最低要求，需进一步实施质量改进。对于未规范开展室内质控、室内质控变异系数未满足行业标准要求的实验室，应查找原因，加强质量控制关键环节的技术培训并实施改进。

    2. 本研究首先对其BV研究文献质量进行科学的评估，保证了基于生物学变异精密度要求的可靠性，然后按照EFLM制订分析质量要求的模式，并引入基于σ度量模拟了精密度分析质量要求，最后对其适用性进行初步验证，为相关标准的制修订提供参考依据。

第二部分：抗缪勒氏管激素和常规性激素检测性能验证

及相关临床意义探讨实验方案的研究

目的：

抗缪勒氏管激素和常规性激素检测是反映卵巢储备功能的指标。为验证抗缪勒氏管激素和常规性激素检测系统的分析性能，保证检测结果的准确性和可靠性，本研究参考相关指南文件和厂商说明书要求对AMH和常规性激素检测系统进行性能验证。

子宫切除术对卵巢储备的影响以及子宫切除合并双侧输卵管切除术术否加重对卵巢储备的影响目前尚无定论。本研究拟设计使用特定手术方式切除子宫患者手术前后AMH和血清性激素5项（FSH、LH、PRL、E2、T）浓度水平变化的实验方案，用于探讨子宫切除术对卵巢储备功能的影响，并进一步研究合并双侧输卵管切除是否会加重对卵巢功能的影响。

方法：

    1. 检测系统性能验证：参照CLSI EP15-A3、EP6-A、EP17-A等指南文件和厂家的要求，明确性能验证的方法和指标，对Beckman Access 2 enhance全自动免疫仪AMH和性激素5项检测的精密度、正确度、线性以及AMH检测限进行验证。

    2. 相关临床意义探讨实验方案的研究：根据研究目的，通过大量查阅文献、咨询临床专家，选择用于研究的手术术式，明确研究影响因素，拟订研究对象纳入和排除标准、观察对象个数，设置观察期限，制订标准化标本采集流程，设计研究路线图，起草满足伦理要求的知情同意书，并选择合适的数据分析方式。

结果：

    1. 性能验证结果：明确了Beckman Access 2 enhance全自动免疫仪AMH、 FSH、LH、PRL、E2、T检测性能验证的方法和指标，各检测项目验证结果如下：①精密度小于厂家声称的总不精密度要求。②正确度小于可接受允许范围。③AMH、E2、FSH、LH、PRL、T分别在0.0~21.23ng/mL、3~4856pg/mL、0.0~172.75 mIU/mL、0.23~248.51 mIU/mL、0.0~177.96ng/mL、0.0~14.37ng/mL范围内线性验证通过。④AMH检测限验证结果符合厂商说明书要求。

2. 相关临床意义探讨实验方案的研究结果：选择特定方式子宫切除术为研究的手术术式，明确内分泌疾病、年龄、附件手术史等为影响卵巢储备的因素；确定患某些子宫良性病变行子宫切除术（合并或不合并双侧输卵管切除）患者为研究对象，同时排除内分泌疾病、附件手术史等人群；拟订子宫切除术及合并双侧输卵管切除数患者观察例数分别为100例；拟订每个研究对象的临床观察期限为术后6个月；根据分析前影响因素制订标准化标本采集流程；根据以上信息设计了技术路线路；制订了知情同意告知等材料；拟采用配对t检验进行患者术前和术后的比较，采用独立样本t检验比较不进行预防性输卵管切除术和合并双侧输卵管切除术对卵巢储备的影响。

结论：

1. 本研究以Beckman Access 2 enhance检测系统为代表，对AMH及常规性激素检测性能进行验证，结果显示其性能满足厂商声明的要求和基于生物学变异的允许范围，保证了后续临床实验检测患者标本的准确性和可靠性，同时其验证方案可为其它实验室开展性能验证提供参考。

2. 本研究拟订了特定子宫切除术对卵巢储备的影响，以及合并双侧输卵管切除术是否加重对卵巢储备影响的实验方案，该方案已通过伦理委员会的审批，为后续实验的推进奠定了基础。

Part1. Analysis of internal quality control data for complete blood count and study on precision analytical quality requirement

Objective:

    Internal quality control (IQC) is a key part of laboratory quality control. At present, there is no country-wide study on the analysis of internal quality control data of complete blood count. This study aims to investigate the current status and problems of IQC of complete blood count in China so as to perform IQC normally， to help clinical laboratories find problems and to propose quality improvement suggestions. At the same time, we conducted a simulation study on the precision requirements of the complete blood count, and provided a basis for formulating and improving related policies and standards based on the model of the quality requirements.

Method:

1.Analysis of the complete blood count IQC data: The IQC data of complete blood count(CBC) for five parameters were collected from laboratories participating in national external quality assessment during 2012-2017 (totally 12 times)，including WBC，RBC，Hb，Hct and PLT. After confirmation of all data，relative analysis are as follows ①collection of general laboratory information including the laboratory level/type, instrument brand, the proportion of use of quality control materials at different concentrations, and the use of quality control rules. ②Statistical analysis of CV of IQC, including analysis of the change trend of P25, P50, P75, and P90 of CV in the lab for 12 times; compare the difference of CV in the laboratory using 3 concentration control materials at the same time; Laboratories with different concentration levels, different grades/types, and different geographical regions have coefficients of variation that meet industry standards; the 2017 laboratory CV is compared with the precision requirements of other criteria（Germany）.

2. study on precision quality requirement:  ①Derivation of allowable precision based on biological variation: Search for literature on biological variation in blood cell analysis published before April 1, 2017, exclude poor quality literature according to the BIVAC criteria, and score the remaining literature.We assign different weights to the remaining literature, screening studies that are suitable for assessing biological variation in adult blood cell analysis, and generalizing biological variation data based on weights. ② Derivation of allowable precision based on Sigma standards: the allowable total error allowed by the healthy standard and the above allowable total error derived based on the biological variation were calculated using the Sigma theory. The allowable precision reaching the levels of 3σ, 4σ, 5σ, and 6σ were calculated respectively. ③Acquisition of precision data from various sources: professional institutions; 2017 quality assessment participating in the laboratory with different concentrations of 80%, 90% laboratory precision can be achieved; performance verification reports; precision data from different instrument brands; Academic experience. ④Determine the blood cell analysis precision quality requirements based on the above information in the development of the reference value and importance of the analysis of quality requirements. ⑤Develop quality requirements for the analysis of blood cell precision. Whether or not the coefficient of variation of  IQC used in the first national quality assessment program in 2018 is greater than 80% is used to assess its applicability.

Results:

1. Analysis of the IQC data of CBC : After eliminating invalid data, the number of laboratories remaining for analysis was 1431 to 2332 (96.0% to 99.2%). Three-level medical institutions accounted for the highest proportion (62.0%~73.9%); the highest-use instrument brand was Sysmex (61.8%~66.8%); 61.9%~66.1%, 18.2%~23.6%, and 14.3%~ respectively. 17.3% of laboratories used 1, 2, and 3 concentration controls, and laboratories using more than 2 concentration controls increased from 33.9% to 38.1%. The proportion of laboratories using 1_3s/2_2s quality control rules increased from 59.2% to 76.0%. The coefficient of variation at different levels had a decreasing trend with time, with the lowest values ​​of PLT and Hct being the most significant; the pass rates of the three WBC and PLT high values ​​were always above 90%. The pass rates of different concentrations of PLT and Hb varied greatly. The passing rate of all parameters of the Three-level medical institutions and independent laboratories was greater than 80%, while the pass rates of some parameters of the secondary and primary medical institutions were less than 80%; the pass rates of CV in North China, East China, and South China were relatively high (pass rate (More than 85%). The pass rate in the northeast region was below 80% for many times. Compared with the German Medical Association's standards, 80% of the other laboratory parameters of the CV reached the standards of the German Medical Association (except CVs with lower PLT quality controls).

2. study on precision analysis quality requirements: ①A total of 19 blood cell BV studies were searched. Eleven methods were abolished, three studies were completed for one or less weeks, one was for elderly women, and one for fingertip blood. The remaining 3 study quality scores were the same. Two of the literatures on biological variation in WBC and PLT were taken as the reliable BV data, and each of the remaining parameters was taken as reliable CV_I. The remaining parameters were only 1 and were taken as the reliable BV data. ②Take the maximum allowable precision calculated based on the σ metric as the evaluation criteria. ③Summarize China's precision data, German standards, and list the retrieved literature results. ④Based on biological variability, sigma metrics, and the level of precision achieved in 80% and 90% laboratories in China. The simulation results for the low, medium, and high value analytical quality requirements of the WBC were: 3.8%, 3.8%, and 3.1, respectively. RBC were 2.1%, 2.1%, 2.1%, Hb was 2.1%, 2.1%, 1.5%, Hct was 3.0%, 3.0%, 2.3%, and PLT was 8.4%, 6.7%, respectively. 4.0%. ⑤Each parameter takes the simulated analytical quality requirement as the evaluation standard, and the passing rate in 2018 is more than 80%.

Conclusion:

1. The laboratory quality control CV has shown a decreasing trend over the past 6 years. However, the control level and quality control rules used by some laboratories have not met the management requirements. Some laboratory CVs have not reached the minimum requirements of the China's quality control industry standards. Further implementation of quality improvement is needed. For those laboratories that have not standardized the IQC and CV of IQC that do not meet the requirements of the industry standards, they should find out the reasons. Labs should strengthen technical training in key steps of quality control and take quality improvement measures.

2. This study first scientifically evaluated the quality of its BV research literature. And then ensured the transferability of precision requirements based on BV, and then formulated the model for analyzing quality requirements according to EFLM, and introduced precision quality requirements based on σ measures. Finally, preliminary appraisal of its applicability provides data and basis for improving relevant standards and criteria.

Part2. Performance Verification of Anti-Mullerian Hormone and Routine Sex Hormone              

      Tests and Study Protocol to Explore Their Clinical Significance in

Ovarian Reserve Evaluation

Objective:

Anti-Müllerian hormone (AMH) and routine sex hormone tests are good indicators of ovarian reserve function. In order to confirm the analytical performance of AMH and routine sex hormone tests and to ensure the accuracy and reliability of the above test results, this study performes performance verification of AMH and routine hormone testing accroding to relative guidelines and manufacturer's instructions.

The impact of hysterectomy and hysterectomy with bilateral salpingectomy on ovarian reserve are inconclusive. This study plans to design an experimental protocol through the changes of AMH and serum sex hormone levels before and after surgery in patients undergoing hysterectomy to investigate the effect of hysterectomy on ovarian reserve, and further explore whether bilateral salpingectomy will aggravate the effects on overian reserve.

Method:

1. Performance verification: According to the requirements of the CLSI EP15-A3, EP-6, EP17 guide documents and the manufacturer's requirements, the performance verification methods and indicators are clearly defined, and five items (including precision, accuracy, linearity,AMH detection limit) of the Beckman Access 2 enhanced automatic immunoassay AMH and sex hormones are identified for verification.

2. Design clinical trial program: According to the purpose of the study, through searching a large number of professional literature, consulting clinical experts, to understand the influencing factors of this study, draw up the inclusion and exclusion criteria for the study object, the number of objects to be observed, set the observation period, formulate a standardized sample collection process, design the research road Lines, and select the appropriate way to analyze the data.

Results:

1. Performance verification: The methods and indicators for verifying the detection performance of the Beckman Access 2 enhanced automatic immunoassay unit (FSH, LH, PRL, E2, T) were defined. The result were as follows:① The precision is less than the manufacturer's stated total imprecision requirement. ②The accuracy is less than the acceptable range. ③Linear verification results AMH, E2, FSH, LH, PRL, and T were 0.0 ~21.23 ng/mL, 3~ 4856 pg/mL, 0.0 ~ 172.75 mIU/mL, 0.23 ~ 248.51 mIU/mL, 0.0 ~177.96 ng/mL, and 0.0~14.37ng/mL, respectively. ④AMH detection limit meet the manufacturer's instruction requirments.

2. Design clinical trial program: Choose specific hysterectomy as target surgical procedure. Endocrine diseases, age, history of ovarian surgery were identified as factors affecting ovarian reserve; patients with particular benign lesions were determined as research objects, and patients with endocrine diseases, history of ovarian surgery were excluded; the proposed number of patients with hysterectomy and bilateral salpingectomy were 100 cases, separately; the clinical follow-up period for each observation object was setted as 6 months. the standardized specimen collection process was established  according to the influencing factors before analysis; the paired t test was intended to use for preoperative and postoperative comparison, independent t test was use to compare no preventive salpingectomy combined with bilateral salpingectomy on the impact of ovarian reserve.

Conclusion:

1. This study is based on the Beckman Access 2 enhance detection system and verified the performance of AMH and routine sex hormone tests. The results show that the performance meets the manufacturer's stated requirements and allowable requirements based on biological variation. The verification scheme can be used for performance in other laboratories.

2. This study developed a study programe of the impact of  hysterectomy on ovarian reserve, and whether the combination of bilateral salpingoectomy aggravated the impact on overian reserve, which has passed the approval of the ethics committee and laid the foundation for the follow-up experiment.