WO2021134505A1 - 一种用于预测早期流产风险的试剂盒及应用 - Google Patents

一种用于预测早期流产风险的试剂盒及应用 Download PDF

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WO2021134505A1
WO2021134505A1 PCT/CN2019/130599 CN2019130599W WO2021134505A1 WO 2021134505 A1 WO2021134505 A1 WO 2021134505A1 CN 2019130599 W CN2019130599 W CN 2019130599W WO 2021134505 A1 WO2021134505 A1 WO 2021134505A1
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platelet
early
risk
miscarriage
kit
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PCT/CN2019/130599
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French (fr)
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项耀祖
王丹丹
徐越
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同济大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

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  • the present invention relates to the field of biomedicine, in particular to a kit and application for predicting the risk of early miscarriage.
  • Missed abortion (Early fetal demise, which shows that there is no heart activity in the embryo, that is, fetal arrest) is the most common pregnancy complication, and its incidence accounts for 10% of all clinically diagnosed pregnancies. 80% of missed abortions occur in the first trimester (8-10 weeks of gestation), but there is no reliable biomarker or index to predict the risk of missed abortion. Although genetic abnormalities are the most common cause of miscarriage in early pregnancy, many other factors (including thrombosis status) may also be the main cause of miscarriage.
  • Platelet count refers to the number of platelets contained in a unit volume of blood. The number of platelets in the blood of normal people is maintained at a certain level, but certain diseases can lead to a decrease in the number of platelets (such as aplastic anemia, acute leukemia, radiation therapy and chemotherapy, thrombocytopenic purpura, hypersplenism, etc.) or Increased (myelodysplastic syndrome, chronic myelogenous leukemia, acute infection, acute blood loss, acute hemolysis, after splenectomy, etc.).
  • Mean platelet volume is a measure of platelet size and a potential biomarker of platelet function, which can usually be used in clinical practice.
  • Higher MPV is associated with traditional cardiovascular risk factors (such as diabetes, obesity, hypertension, and hypercholesterolemia), and is associated with an increased risk of arterial thrombosis and venous thrombosis.
  • Patients with pre-existing coronary artery disease and elevated MPV have a higher risk of myocardial infarction.
  • platelet count or MPV are related to pregnancy or missed miscarriage.
  • the invention patent "Pathology Specimen Retention Device for Missed Abortion" (application number: CN201810402476.6) relates to a publicly missed abortion pathology specimen retention device.
  • the invention is placed in the toilet, and the patient sits on the invention to have a fetus.
  • the stool, vaginal discharge (embryos, villi and blood clots, etc.) and urine are separated by the partition plate, and the discharged vaginal discharge (embryos, villi and Blood clots, etc.) will remain on the filter and separate from urine and blood.
  • the trouble of using tools for medical staff to separate stool, urination, embryos, villi, blood clots, etc. is saved, and the use effect is good.
  • the invention patent application CN201810068147.2 relates to the application of miR-184 in the early warning effect of spontaneous abortion, specifically to the method of intravenous injection of miR-184-3p agomir (miRNA agonist) to establish a spontaneous abortion model and the detection of miR- in the peripheral blood of pregnant women in the early trimester. 184 is used to assess the risk of spontaneous abortion.
  • miR-184-3p agomir miRNA agonist
  • the invention patent application CN201711172943.2 relates to an application for predicting the risk of miscarriage in pregnant women suffering from autoimmune thyroiditis (AIT) by detecting the level of ⁇ -enolase (ENO1)-specific autoantibodies (ENO1Ab) in the serum.
  • ENO1Ab ⁇ -enolase-specific autoantibodies
  • the invention patent application CN201810397317.1 relates to a set of blood DNA methylation markers, primers and their applications for assessing the risk of early miscarriage.
  • the DNA methylation marker of the invention includes 8 DNA methylation sites located near the transcription start region of the PRDM1 gene; cg25608130, cg19577529, cg10573915, cg11072009, cg02667677, cg23778363, cg24793124, and cg07331652.
  • the invention patent application US20040185515 relates to a kit for diagnosing spontaneous abortion, and specifically relates to detecting the content of platelet activating factor PAF autoantibodies in body fluids.
  • the technical problem to be solved by the present invention is to overcome the shortcomings in the prior art such as the lack of effective markers for predicting the risk of early miscarriage, and provides a kit, combination of kits, detection method and application for predicting the risk of early miscarriage.
  • the present invention proposes for the first time that the platelet parameters are applied to the risk prediction of early miscarriage, and the influence of platelet parameters on early miscarriage (especially missed miscarriage) is clarified.
  • the kit, kit combination and detection method of the present invention are used to predict the risk of early miscarriage, sampling is more convenient, detection is faster, and the accuracy and sensitivity of predicting early miscarriage, especially missed miscarriage risk are high, and thus Prenatal and postnatal care provides the basis and guarantee for intervention.
  • platelet parameters are related to the incidence of early miscarriage.
  • High levels of platelet parameters in early pregnancy (such as average platelet volume and platelet count) will significantly increase the risk of early miscarriage, especially missed miscarriage. It is currently in the art This is the first time.
  • the first aspect of the present invention provides a kit for predicting the risk of early miscarriage, the kit including a reagent for detecting platelet parameters.
  • the platelet parameter is platelet count (PLT), mean platelet volume (MPV), platelet volume distribution width (PDW) and/or platelet packed volume (PCT).
  • the early abortion is an early missed abortion.
  • the platelet parameter is the platelet parameter of the early pregnancy, and the early pregnancy is preferably 6-12 weeks of gestation.
  • the kit also includes reagents related to negative control and the like.
  • the reagents for detecting platelet parameters generally include solvents and equipment conventional in the art for detecting platelet parameters.
  • the solvent can be, for example, a calculation method that can calculate blood cells, such as specific reagents or equipment used in single-channel electrical impedance technology: indirect platelet count (PLT) can use fluorescence (or fluorescent antibody) to label platelets, use flow cytometry, according to fluorescence Intensity and scattered light intensity set the threshold at the position where PLT can be distinguished from RBC, detect the ratio of red blood cell (RBC) and PLT, at the same time use a semi-automatic cell counter with single-channel impedance principle to accurately count RBC, divide RBC by RBC and PLT The ratio of PLT is the count value of PLT.
  • PLT indirect platelet count
  • RBC red blood cell
  • PLT red blood cell
  • This method also uses diluent (PBS) and filter membrane (average pore size between 0.20 ⁇ 0.25 ⁇ m).
  • the fluorescent antibodies are CD41 and CD61 antibodies labeled with fluorescein isothiocyanate, and these two antibodies can be combined with platelet membrane glycoprotein IIb/IIIa complex for detecting platelets.
  • the Human Peripheral Blood Platelet Separating Solution Kit is a mixture of Ficoll, hydroxyethyl starch 550 and meglumine diatrizoate.
  • the equipment can be, for example, a hemocytometer (for example, a modified bovine Bowman counter), a platelet counter, a polypropylene or polystyrene container, a microscope (microscopic counting method, phase contrast microscopy), a flow cytometer, and the like.
  • Mean platelet volume is generally a clinical test index obtained after using an automated blood cell analyzer, which is a routine blood test.
  • the second aspect of the present invention provides a kit combination for predicting the risk of early miscarriage, which includes a kit for detecting platelet count, a kit for detecting average platelet volume, and a reagent for detecting platelet volume distribution width. Two, three, or four of the kits and kits for detecting platelet compaction.
  • the early abortion is an early missed abortion.
  • the third aspect of the present invention provides the use of the kit according to the first aspect of the present invention and/or the kit combination according to the second aspect of the present invention in the preparation of a preparation for predicting the risk of early miscarriage .
  • the fourth aspect of the present invention provides the application of the kit according to the first aspect of the present invention and/or the kit combination according to the second aspect of the present invention in predicting the risk of early miscarriage.
  • the fifth aspect of the present invention provides a method for predicting the risk of early miscarriage, the method comprising using the kit as described in the first aspect of the present invention and/or the method as described in the second aspect of the present invention
  • the kit combination makes predictions.
  • the platelet count is more than 237.5 ⁇ 10 9 /L, and/or the average platelet volume is more than 9.9 fL, it can be judged that there is a risk of early miscarriage; more preferably, when the platelet count is 272 ⁇ When 10 9 /L or more, and/or the average platelet volume is 10.6 fL or more, it can be judged that there is a risk of early miscarriage.
  • the sixth aspect of the present invention provides an application of platelet parameters in predicting the risk of early miscarriage.
  • the platelet parameters are platelet count, average platelet volume, platelet volume distribution width, and/or platelet compaction.
  • the platelet count is preferably 237.5 ⁇ 10 9 /L or more, more preferably 272 ⁇ 10 9 /L or more; and/or, the average platelet volume is preferably 9.9 fL or more, more preferably 10.6 fL or more.
  • the early abortion is an early missed abortion.
  • the platelet parameter is the platelet parameter of the early pregnancy, and the early pregnancy is preferably 6-12 weeks of gestation.
  • the seventh aspect of the present invention provides an application of a reagent for detecting platelet parameters in preparing preparations, kits and/or kit combinations for predicting the risk of early miscarriage.
  • the platelet parameters are platelet count, average platelet volume, platelet volume distribution width, and/or platelet compaction.
  • the platelet count is preferably 237.5 ⁇ 10 9 /L or more, more preferably 272 ⁇ 10 9 /L or more; and/or, the average platelet volume is preferably 9.9 fL or more, more preferably 10.6 fL or more.
  • the early abortion is an early missed abortion.
  • the platelet parameter is the platelet parameter of the early pregnancy, and the early pregnancy is preferably 6-12 weeks of gestation.
  • the eighth aspect of the present invention provides the application of platelet parameters as biomarkers for predicting the risk of early miscarriage.
  • the platelet parameters are platelet count, average platelet volume, platelet volume distribution width, and/or platelet compaction.
  • the platelet count is preferably 237.5 ⁇ 10 9 /L or more, more preferably 272 ⁇ 10 9 /L or more; and/or, the average platelet volume is preferably 9.9 fL or more, more preferably 10.6 fL or more.
  • the early abortion is an early missed abortion.
  • the platelet parameter is the platelet parameter of the early pregnancy, and the early pregnancy is preferably 6-12 weeks of gestation.
  • the present invention also provides a biomarker for predicting the risk of early miscarriage, the biomarker including platelet parameters.
  • the platelet parameters are platelet count, average platelet volume, platelet volume distribution width, and/or platelet compaction.
  • the early abortion is an early missed abortion.
  • the platelet parameter is the platelet parameter of the early pregnancy, and the early pregnancy is preferably 6-12 weeks of gestation.
  • missed abortion can be divided into the following types: in the first trimester (the first three months) there has been no fetal heart beat (determined by B-ultrasound); In the first three months), the fetal heart beat (determined by B-ultrasound) and then stopped, and combined with the menopausal time and the size of the gestational sac, the specific time of fetal arrest was comprehensively determined.
  • the early fetal demise usually refers to the type of fetal arrest in early abortion.
  • the sample used for detection by the kit is generally a blood sample.
  • the platelet parameters for example, platelet count, average platelet volume, platelet volume distribution width, and/or platelet hematocrit
  • the platelet parameters can be a necessary option in routine blood tests in hospitals, that is, blood routines.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the positive and progressive effect of the present invention is that the kit, the combination of the kit, and the detection method of the present invention are used to predict the risk of early miscarriage, and sampling is more convenient and detection is faster (generally only 2ml of blood is required, which can be used in 1 hour The detection results are generated within), and the accuracy and sensitivity of predicting the risk of early miscarriage, especially missed miscarriage are high, and thus provide intervention basis and guarantee for prenatal and postnatal care.
  • Figure 1 shows the comparison of platelet related parameters between missed abortion and normal pregnancy.
  • Figure 2 shows the two risk factors of platelet count and average volume and the risk of missed miscarriage.
  • Figure 3 shows the sensitivity of two risk factors, the number of platelets and the average volume, in predicting missed miscarriage.
  • Figure 1 Clinical research uses SPSS and R for all statistical analysis. Since continuous variables (age, parity, miscarriage, MPV, and platelet count) do not follow a normal distribution, they were compared using the Wilcoxon-Mann-Whitney U test (both sides).
  • the table in C of Figure 1 lists the platelet count and volume MPV of women from normal delivery (normal pregnancy group), women diagnosed with missed abortion (missed abortion group), and non-pregnant non-pregnant control women.
  • Statistical comparison found that the platelet number and volume MPV of the normal early pregnancy were significantly reduced, while the platelet number and volume MPV of patients with missed abortion were significantly increased (compared with the normal pregnancy group, p ⁇ 0.001), which was statistically significant (Figure 1).
  • the correlation between MPV and platelet count is calculated by a non-parametric measure of rank correlation.
  • each variable was evaluated through univariate analysis; then, the main risk factors for missed miscarriage were calculated by binary logistic regression model.
  • the lowest quintile in the normal pregnancy group and ⁇ 80% (p80) were used as the reference category.
  • Model 1 (OR1) was not adjusted, and model (OR2) included adjustments for potential confounding factors.
  • Quantile also known as Quantile, refers to the numerical points that divide the probability distribution range of a random variable into several equal parts. Commonly used are the median (ie, the quartile) and the quartile. Digits, quintiles, percentiles, etc. Quintile is a kind of quantile in statistics, that is, all values are arranged from small to large and divided into quintiles. The value at the position of three division points is the quintile.
  • the first quintile (Q1 or p20), also known as the "smaller quintile", is equal to the 20th percentile of all values in the sample in descending order; the fourth quintile (Q4 or p80), also known as the “larger quintile”, is equal to the 80th percentile of all values in the sample in descending order.
  • OR value (odds ratio), also known as odds ratio, odds ratio mainly refers to the ratio of the exposed people to the non-exposed people in the case group divided by the ratio of the exposed people to the non-exposed people in the control group. It is a case-control study in epidemiological research. A commonly used indicator in. An OR value equal to 1, indicates that the factor has no effect on the occurrence of the disease; an OR value greater than 1, indicates that the factor is a risk factor; an OR value less than 1, indicates that the factor is a protective factor.
  • Figure 2 compares the two risk factors of platelet count and average volume with the risk of missed miscarriage. The results show that pregnant women with MPV ⁇ p80 and platelet count have the highest risk of missed miscarriage, with an OR1 value of 30.498 (95% CI, 4.032-230.672) (figure 2). Since age, miscarriage history and other factors will affect the risk of missed miscarriage, it is necessary to eliminate the interference of this factor in statistics.
  • OR1 represents the statistical result of unadjusted age, miscarriage history and other influencing factors
  • OR2 is the adjustment of the above factors
  • the statistics of platelet parameter variables For the risk of missed abortion the sample size corresponding to the MPV and platelet count ⁇ p80 group is small, which is in line with the actual situation, because the incidence of missed abortion in the normal population is only about 15%, and the present invention can just identify such a population earlier And risk warning.
  • Example 3 The sensitivity of the two risk factors of platelet count and average volume in predicting missed abortion
  • the sensitivity is quantified by the combined effect of MPV p80 and platelet count and the area under the receiver operating characteristic curve (AUC) of only one risk factor. All reported values are two-sided tests, and P ⁇ 0.05 is considered statistically significant.
  • Figure 3 quantified by AUC on the combined effect of MPV and platelet count p80, the discrimination reached 0.724 (95% CI, 0.689-0.759), which is different from the combined effect of AUC on MPV and platelet count p75 and p80.
  • the combined effect of MPV higher than the fourth quintile of p80 and platelet count increases the risk of missed miscarriage compared with only one risk factor (high MPV or high platelet count), and p80 can be used for both The best critical value of risk assessment brought about by the combination of factors.
  • Example 4 Prospective verification of the sensitivity of the product of the two risk factors of platelet number and average volume in predicting missed miscarriage

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Abstract

一种用于预测早期流产风险的试剂盒，所述试剂盒包括检测血小板参数的试剂。将所述血小板参数应用于早期流产的风险预测，明确了血小板参数在早期流产(特别是稽留流产)中的影响。将所述试剂盒、试剂盒组合、检测方法用于预测早期流产风险中，采样更方便、检测更加快捷，预测早期流产、特别是稽留流产风险的准确性更高、敏感性更好，从而为优生优育提供干预依据和保障。

Description

一种用于预测早期流产风险的试剂盒及应用 技术领域
本发明涉及生物医药领域，具体涉及一种用于预测早期流产风险的试剂盒及应用。
背景技术
稽留流产(Early fetal demise，可见胚胎中没有心脏活动，即胎停)是最常见的妊娠并发症，其发生率在所有临床确诊的妊娠中占10％。80％的稽留流产发生在孕早期(孕期8-10周)，但是尚无可靠的生物标志物或指数来预测稽留流产的风险。尽管遗传异常是导致早孕流产的最常见原因，但许多其他因素(包括血栓形成状态)也可能是导致流产的主要原因。
血小板在止血、血栓形成和动脉粥样硬化形成中均起关键作用。血小板计数(PLT，platelet count)是指单位体积血液中所含的血小板数目。正常人血液中的血小板数量维持在一定的水平，但某些疾病原因可导致血小板数量的减少(如再生障碍性贫血、急性白血病、放射治疗和化疗、血小板减少性紫癜、脾功能亢进等)或增多(骨髓增生异常综合征、慢粒白血病、急性感染、急性失血、急性溶血、脾切除术后等)。平均血小板体积(MPV，mean platelet volume)是血小板大小的度量，是血小板功能的潜在生物学标记，在临床实践中通常可以使用。较高的MPV与传统的心血管危险因素(如糖尿病，肥胖，高血压和高胆固醇血症)有关，并且与动脉血栓和静脉血栓形成的风险增加有关。既有冠状动脉疾病和MPV升高的患者发生心肌梗塞的风险较高。但是，目前尚无将血小板计数或MPV与妊娠或稽留流产相关的报道。
发明专利“稽留流产病理标本留取装置”(申请号：CN201810402476.6)涉及一种公开稽留流产病理标本留取装置。该发明置于马桶，患者坐在该发 明上排胎，其排出的大便、阴道排出物(胚胎、绒毛及血块等)及尿液通过分隔板分离，排出的阴道排出物(胚胎、绒毛及血块等)会残留于过滤网上与尿液、血液分离。由此，即使得可自动分离出胚胎、绒毛及血块等，省去医护人员取用工具进行大便、小便、胚胎、绒毛及血块等分离的麻烦，使用效果好。
发明专利申请CN201810068147.2涉及miR-184在自然流产预警作用中的应用，具体涉及静脉注射miR-184-3p agomir(miRNA激动剂)建立自然流产模型的方法和孕早期检测孕妇外周血的miR-184用于评估自然流产风险。
发明专利申请CN201711172943.2涉及一种通过检测血清中α烯醇化酶(ENO1)特异性自身抗体(ENO1Ab)水平来预测患有自身免疫性甲状腺炎(AIT)的孕妇流产风险的应用。其中发现了ENO1Ab的存在与流产的发生和胎盘功能障碍的发病之间密切相关，可为及时筛选出具有高流产风险的AIT孕妇，提供一种新的临床应用。
发明专利申请CN201810397317.1涉及一组用于评估早期流产风险的血液DNA甲基化标记物、引物及其应用。该发明的DNA甲基化标志物包括位于PRDM1基因转录起始区附近的8个DNA甲基化位点；cg25608130,cg19577529，cg10573915，cg11072009，cg02667677，cg23778363，cg24793124，cg07331652。
发明专利申请US20040185515涉及一种用于诊断自发性流产的试剂盒，具体涉及检测体液中血小板活化因子PAF自身抗体的含量。
上述文献和专利各自具有一定的优点，但仍然存在不足：(1)上述专利申请主要围绕早期流产风险的标记物包括抗体、DNA甲基化标志物、外周血miRNA，并没有指出血小板指标与早期流产风险的关联；(2)少数专利或文献仅仅涉及稽留流产的病理标本留取装置的发明，或者仅涉及诊断自发性流产(即自然流产)的试剂盒。上述文献均未提示指出血小板数量或平均 体积或与稽留流产风险有直接相关性。
引起早期复发性流产包括稽留流产的因素有很多，包括解剖因素、夫妇染色体异常、内分泌异常、感染因素、血栓前状态、免疫功能异常和不良环境及生活习惯等。尽管腹部彩超能有效地诊断稽留流产，但目前没有报道哪一种因素与发生稽留流产的具体相关系数和风险强弱。因此急需一种临床实践可行的预测早期流产、特别是稽留流产风险的因子或标记物，以提高当前生殖健康和效率。
发明内容
本发明所要解决的技术问题是为克服现有技术中缺乏有效预测早期流产风险的标记物等缺陷，提供了一种用于预测早期流产风险的试剂盒、试剂盒组合、检测方法及其应用。本发明首次提出将所述血小板参数应用于早期流产的风险预测，明确了血小板参数在早期流产(特别是稽留流产)中的影响。将本发明的试剂盒、试剂盒组合、检测方法用于预测早期流产风险中，采样更方便、检测更加快捷，预测早期流产、特别是稽留流产风险的准确性高、敏感性更好，从而为优生优育提供干预依据和保障。
本发明人意外发现，血小板参数与早期流产的发生率相关，妊娠早期高水平的血小板参数(例如血小板平均体积和血小板计数)将显著增加早期流产、特别是稽留流产的风险，目前在本领域内尚属首次。
为了解决上述技术问题，本发明第一方面提供了一种用于预测早期流产风险的试剂盒，所述试剂盒包括检测血小板参数的试剂。
较佳地，所述血小板参数为血小板计数(PLT)、血小板平均体积(MPV)、血小板体积分布宽度(PDW)和/或血小板压积(PCT)。
较佳地，所述早期流产为早期稽留流产。
较佳地，所述血小板参数为妊娠早期的血小板参数，所述妊娠早期优选妊娠6-12周。
较佳地，所述试剂盒还包括阴性对照相关的试剂等。
本发明中，所述检测血小板参数的试剂一般包括本领域常规的用于检测血小板参数的溶剂和设备。溶剂可以是例如可以计算血细胞的计算方法，例如单通道电阻抗技术用到的具体试剂或设备：间接法血小板计数(PLT)可用荧光(或荧光抗体)标记血小板，用流式细胞仪，根据荧光强度和散射光强度将阈值设在可从RBC中区分PLT的位置，检测红细胞(RBC)和PLT的比值，同时用单通道阻抗原理的半自动细胞计数仪准确计数RBC，用RBC除以RBC和PLT的比值得出PLT的计数值。该方法还用到了稀释液(PBS)、滤膜(平均孔径在0.20～0.25μm之间)。所述荧光抗体是异硫氰酸荧光素标记的CD41和CD61抗体，这两种抗体可以与血小板膜糖蛋白Ⅱb/Ⅲa复合物结合，用于检测血小板。此外还有鞘流电阻抗法、草酸铵(草酸铵1g，EDTA钠盐14mg，加蒸馏水至100ml)手工计数法，但国标是用单克隆CD41/CD61抗体标记法。人外周血血小板分离液试剂盒为Ficoll、羟乙基淀粉550与泛影酸葡甲胺的混合液。设备可以是例如血球计数板(例如改良牛鲍氏计数板)、血小板计数器、聚丙烯或聚苯乙烯容器、显微镜(显微镜计数法、相差显微镜法)、流式细胞仪等。
平均血小板体积(MPV)一般是采用自动化血细胞分析仪后得到的一项临床检测指标，属于血常规检查。
为了解决上述技术问题，本发明第二方面提供了一种用于预测早期流产风险的试剂盒组合，其包括检测血小板计数的试剂盒、检测血小板平均体积的试剂盒、检测血小板体积分布宽度的试剂盒和检测血小板压积的试剂盒中的两种、三种或者四种。
较佳地，所述早期流产为早期稽留流产。
为了解决上述技术问题，本发明第三方面提供了如本发明第一方面所述的试剂盒和/或如本发明第二方面所述的试剂盒组合在制备预测早期流产风险的制剂中的应用。
为了解决上述技术问题，本发明第四方面提供了如本发明第一方面所述的试剂盒和/或如本发明第二方面所述的试剂盒组合在预测早期流产风险中的应用。
为了解决上述技术问题，本发明第五方面提供了一种预测早期流产风险的方法，所述方法包括利用如本发明第一方面所述的试剂盒和/或如本发明第二方面所述的试剂盒组合进行预测。
较佳地，当血小板计数为237.5×10 9/L以上，和/或所述血小板平均体积为9.9fL以上时，可以判断为有早期流产的风险；更佳地，当所述血小板计数272×10 9/L以上，和/或所述血小板平均体积10.6fL以上时，可以判断为有早期流产的风险。
为了解决上述技术问题，本发明第六方面提供了一种血小板参数在预测早期流产风险中的应用。
较佳地，所述血小板参数为血小板计数、血小板平均体积、血小板体积分布宽度和/或血小板压积。其中，所述血小板计数优选为237.5×10 9/L以上，更优选272×10 9/L以上；和/或，所述血小板平均体积优选为9.9fL以上，更优选10.6fL以上。
较佳地，所述早期流产为早期稽留流产。
较佳地，所述血小板参数为妊娠早期的血小板参数，所述妊娠早期优选妊娠6-12周。
为了解决上述技术问题，本发明第七方面提供了一种检测血小板参数的试剂在制备预测早期流产风险的制剂、试剂盒和/或试剂盒组合中的应用。
较佳地，所述血小板参数为血小板计数、血小板平均体积、血小板体积分布宽度和/或血小板压积。其中，所述血小板计数优选为237.5×10 9/L以上，更优选272×10 9/L以上；和/或，所述血小板平均体积优选为9.9fL以上，更优选10.6fL以上。
较佳地，所述早期流产为早期稽留流产。
较佳地，所述血小板参数为妊娠早期的血小板参数，所述妊娠早期优选妊娠6-12周。
为了解决上述技术问题，本发明第八方面提供了血小板参数在作为预测早期流产风险的生物标记物中的应用。
较佳地，所述血小板参数为血小板计数、血小板平均体积、血小板体积分布宽度和/或血小板压积。其中，所述血小板计数优选为237.5×10 9/L以上，更优选272×10 9/L以上；和/或，所述血小板平均体积优选为9.9fL以上，更优选10.6fL以上。
较佳地，所述早期流产为早期稽留流产。
较佳地，所述血小板参数为妊娠早期的血小板参数，所述妊娠早期优选妊娠6-12周。
为了解决上述技术问题，本发明还提供了一种用于预测早期流产风险的生物标志物，所述生物标志物包括血小板参数。
较佳地，所述血小板参数为血小板计数、血小板平均体积、血小板体积分布宽度和/或血小板压积。
较佳地，所述早期流产为早期稽留流产。
较佳地，所述血小板参数为妊娠早期的血小板参数，所述妊娠早期优选妊娠6-12周。
按照世界卫生组织的发表的“稽留流产”的命名和诊断标准，可将“稽留流产”分为以下类型：孕早期(前三个月)一直无胎心搏动(B超确定)；孕早期(前三个月)先有胎心搏动(B超确定)后停跳，并结合停经时间和孕囊大小，综合判断胎停具体时间。本发明中，所述稽留流产(Early fetal demise)通常是指早期流产中发生胎停的这一类型。
本发明中，所述试剂盒用于检测的样品一般为血液样品。
本发明中，所述的血小板参数(例如血小板计数、血小板平均体积、血小板体积分布宽度和/或血小板压积)可为医院常规血液学检查即血常规中的 必选项。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：将本发明的试剂盒、试剂盒组合、检测方法用于预测早期流产风险中，采样更方便、检测更加快捷(一般仅需取血2ml，即可在1个小时内出检测结果)，预测早期流产、特别是稽留流产风险的准确性高、敏感性更好，从而为优生优育提供干预依据和保障。
附图说明
图1为稽留流产与正常妊娠血小板相关参数的比较。
图2为血小板数量和平均体积两种风险因素与稽留流产的风险。
图3为血小板数量和平均体积两种风险因素预测稽留流产的敏感性。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1 稽留流产与正常妊娠血小板相关参数的比较
收集在2017年1月1日至2018年12月31日期间在同济大学附属医院分娩的588名妇女中，最初纳入研究，其中68名患有妊娠相关并发症，40名患有血小板减少症。其余467名妇女无妊娠并发症，被纳入研究。我们从467例未患有妊娠并发症的妇女的前三个月(不超过妊娠12周)获得的数据(正常妊娠组)与被诊断为稽留流产、394例妊娠6-12周获得的数据(稽留流产组)进行了比较。此外，同时纳入了1485名非孕妇作为未孕对照。
图1临床研究应用SPSS和R进行所有统计分析。由于连续变量(年龄， 胎次，流产，MPV和血小板计数)不服从正态分布，因此通过Wilcoxon-Mann-Whitney U检验(两侧)进行比较。图1的C中的表格中列出从正常分娩(正常妊娠组)的妇女，被诊断稽留流产的妇女(稽留流产组)和未怀孕的未孕对照妇女的血小板数量和体积MPV。统计比较发现，正常妊娠早期血小板数量和体积MPV都显著降低，而稽留流产患者的血小板数量和体积MPV都显著升高(与正常妊娠组比较p＜0.001)，具有统计学意义(图1)。
实施例2 血小板数量和平均体积两种风险因素与稽留流产的风险
MPV和血小板计数之间的相关性是通过秩相关性的非参数度量来计算。为评估稽留流产风险与两个因素(MPV和血小板计数)的存在之间的关联，首先，通过单变量分析评估每个变量；随后，通过二元逻辑回归模型计算了稽留流产的主要危险因素的风险比(OR)，其可信度为95％置信区间(95％CI)。以正常妊娠组中最低的五分位数和<80％(p80)被用作参考类别。模型1(OR1)未进行调整，模型(OR2)中包括了对潜在混杂因素的调整。
其中，分位数(Quantile)，亦称分位点，是指将一个随机变量的概率分布范围分为几个等份的数值点，常用的有中位数(即二分位数)、四分位数、五分位数、百分位数等。五分位数(Quintile)是统计学中分位数的一种，即把所有数值由小到大排列并分成五等份，处于三个分割点位置的数值就是五分位数。第一五分位数(Q1或p20)，又称“较小五分位数”，等于该样本中所有数值由小到大排列后第20％的数字；第四五分位数(Q4或p80)，又称“较大五分位数”，等于该样本中所有数值由小到大排列后第80％的数字。OR值(odds ratio)又称比值比、优势比，主要指病例组中暴露人数与非暴露人数的比值除以对照组中暴露人数与非暴露人数的比值，是流行病学研究中病例对照研究中的一个常用指标。OR值等于1，表示该因素对疾病的发生不起作用；OR值大于1，表示该因素是危险因素；OR值小于1，表示该因素是 保护因素。
图2比较血小板数量和平均体积两种风险因素与稽留流产的风险，结果表明：≥p80的MPV和血小板计数孕妇发生稽留流产的风险最高，其OR1值为30.498(95％CI，4.032-230.672)(图2)。由于年龄、流产史等因素会影响稽留流产的风险，因此统计时需要消除此因素的干扰，OR1表示未调整年龄、流产史等影响因素的统计结果，OR2是调整了上述因素，统计血小板参数变量对稽留流产的风险；MPV和血小板计数≥p80组对应的样本量少，符合实际情况，因为正常人群中稽留流产的发生率也就15％左右，本发明正好可以较早的识别出这样的人群和风险预警。
实施例3 血小板数量和平均体积两种风险因素预测稽留流产的敏感性
通过MPV p80与血小板计数的联合作用和仅一个危险因素的受试者操作特征曲线(AUC)下的面积来量化敏感性。所有报告值均为双侧检验，P<0.05被认为具有统计学意义。
图3由AUC量化的对MPV和血小板计数的p80联合作用的判断，其区分度达0.724(95％CI，0.689–0.759)，与对AUC对MPV和血小板计数p75和p80的联合作用有区别。显然，MPV高于p80第四五分位数和血小板计数的联合作用与仅具有一个危险因素(高MPV或高血小板数量)相比，增加了稽留流产的风险，p80可以被用作这两个因素共同带来的风险评估的最佳临界值。
实施例4 前瞻性验证血小板数量和平均体积两种风险因素乘积预测稽留流产的敏感性
收集在2019年5月1日至2019年12月1日期间在同济大学附属医院确诊的114名孕妇，其中16名患有妊娠并发症，81例数据不全，失访17例。其余17名妇女无妊娠并发症，被纳入研究。
从纳入研究的妇女孕7周开始，采集病人血小板数量和平均体积，随访至稽留流产或孕10周，以前期回顾性研究推算的模型确定的阈值2469.70(血小板数量和平均体积乘积)。以大于阈值预测稽留流产，预测成功为1，预测失败0。经卡方分析，结果发现两组具有统计学差异(pearson卡方p值＝0.022)，即，用本发明中的血小板数量和平均体积这些风险因素对孕妇进行预测后，之后回访的结果与预测的结果具有一致性。说明用该技术方法预测稽留流产的敏感性较好、准确性高。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (10)

  1. 一种用于预测早期流产风险的试剂盒，其特征在于，所述试剂盒包括检测血小板参数的试剂。
  2. 如权利要求1所述的试剂盒，其特征在于，所述血小板参数为血小板计数、血小板平均体积、血小板体积分布宽度和/或血小板压积；
    和/或，所述早期流产为早期稽留流产；
    和/或，所述血小板参数为妊娠早期的血小板参数，所述妊娠早期优选妊娠6-12周。
  3. 如权利要求1或2所述的试剂盒，其特征在于，所述试剂盒还包括阴性对照。
  4. 一种用于预测早期流产风险的试剂盒组合，其特征在于，其包括检测血小板计数的试剂盒、检测血小板平均体积的试剂盒、检测血小板体积分布宽度的试剂盒和检测血小板压积的试剂盒中的两种、三种或者四种；
    较佳地，所述早期流产为早期稽留流产。
  5. 如权利要求1～3任一项所述的试剂盒和/或权利要求4所述的试剂盒组合在制备预测早期流产风险的制剂中的应用。
  6. 如权利要求1～3任一项所述的试剂盒和/或如权利要求4所述的试剂盒组合在预测早期流产风险中的应用。
  7. 一种预测早期流产风险的方法，其特征在于，所述方法包括利用如权利要求1～3任一项所述的试剂盒和/或如权利要求4所述的试剂盒组合进行预测；
    较佳地，当血小板计数为237.5×10 9/L以上，和/或所述血小板平均体积为9.9fL以上时，可以判断为有早期流产的风险；更佳地，当所述血小板计数272×10 9/L以上，和/或所述血小板平均体积10.6fL以上时，可以判断为有早期流产的风险。
  8. 一种血小板参数在预测早期流产风险中的应用；
    较佳地，所述血小板参数为血小板计数、血小板平均体积、血小板体积分布宽度和/或血小板压积；所述血小板计数优选为237.5×10 9/L以上，更优选272×10 9/L以上；所述血小板平均体积优选为9.9fL以上，更优选10.6fL以上；
    和/或，所述早期流产为早期稽留流产；
    和/或，所述血小板参数为妊娠早期的血小板参数，所述妊娠早期优选妊娠6-12周。
  9. 一种检测血小板参数的试剂在制备预测早期流产风险的制剂、试剂盒和/或试剂盒组合中的应用；
    较佳地，所述血小板参数为血小板计数、血小板平均体积、血小板体积分布宽度和/或血小板压积；所述血小板计数优选为237.5×10 9/L以上，更优选272×10 9/L以上；所述血小板平均体积优选为9.9fL以上，更优选10.6fL以上；
    和/或，所述早期流产为早期稽留流产；
    和/或，所述血小板参数为妊娠早期的血小板参数，所述妊娠早期优选妊娠6-12周。
  10. 一种血小板参数在作为预测早期流产风险的生物标记物中的应用；
    较佳地，所述血小板参数为血小板计数、血小板平均体积、血小板体积分布宽度和/或血小板压积；所述血小板计数优选为237.5×10 9/L以上，更优选272×10 9/L以上；所述血小板平均体积优选为9.9fL以上，更优选10.6fL以上；
    和/或，所述早期流产为早期稽留流产；
    和/或，所述血小板参数为妊娠早期的血小板参数，所述妊娠早期优选妊娠6-12周。
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