WO2016045022A1 - 用于孕妇外周血样的保存液和孕妇外周血样的保存方法 - Google Patents

用于孕妇外周血样的保存液和孕妇外周血样的保存方法 Download PDF

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WO2016045022A1
WO2016045022A1 PCT/CN2014/087371 CN2014087371W WO2016045022A1 WO 2016045022 A1 WO2016045022 A1 WO 2016045022A1 CN 2014087371 W CN2014087371 W CN 2014087371W WO 2016045022 A1 WO2016045022 A1 WO 2016045022A1
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peripheral blood
solution
cells
pregnant woman
preservation
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PCT/CN2014/087371
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English (en)
French (fr)
Inventor
邓乐
潘健昌
刘萍
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深圳华大基因科技有限公司
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Priority to CN201480081717.1A priority Critical patent/CN106793771B/zh
Priority to PCT/CN2014/087371 priority patent/WO2016045022A1/zh
Publication of WO2016045022A1 publication Critical patent/WO2016045022A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/18—Erythrocytes

Definitions

  • the present application relates to the field of blood sample preservation, and in particular to a preservation solution for preservation of a peripheral blood sample of a pregnant woman, and a method for preserving a peripheral blood sample of a pregnant woman.
  • prenatal diagnosis includes amniocentesis, villous cell aspiration, and fetal cord blood puncture; although the diagnostic accuracy is high, it can lead to complications such as abortion, and can only be implemented in high-risk pregnant women.
  • Non-invasive prenatal diagnosis currently mainly for prenatal diagnosis of fetal cells entering the maternal blood, the advantage of this diagnostic method is that it can be prenatal diagnosis and non-risk, suitable for screening large groups of low-risk pregnant women. Discover and eliminate abnormal fetuses as soon as possible to achieve eugenic purposes.
  • Normal placenta is not a perfect, cell-permeable barrier. According to current research, in normal human pregnancy, the amount of fetal blood entering the maternal blood circulation is estimated to be 0.1 to 0.3 ml.
  • the main types of fetal cells in the blood of pregnant women include trophoblast cells, lymphocytes, granulocytes, monocytes, and nucleated red blood cells (NRBC). Among them, the most suitable fetal cell type for prenatal diagnosis is the primordial fetal nucleated red blood cells.
  • fetal nucleated red blood cells are mononuclear, and the fetal blood in the early pregnancy is abundant but rare in the peripheral blood of pregnant women; It expresses several specific antigens, such as transferrin receptors and specific fetal hemoglobin peptide chains, such as ⁇ -chain, ⁇ -chain and ⁇ -chain, as cell markers for cell sorting, identification and aggregation; (3) The survival time of nucleated red blood cells in the peripheral blood of pregnant women is short, from 2 weeks after the start of fertilization for 3 weeks, and will not last until the next pregnancy. Fetal nucleated red blood cells are a characteristic marker of fetal hematopoietic system and fetal blood circulation.
  • the fetal blood circulation is established at the end of the third week after fertilization, and its red blood cells are mainly derived from the yolk sac.
  • the liver becomes the main organ of erythropoiesis, and the bone marrow and spleen gradually have hematopoietic function.
  • 90% of the red blood cells are derived from the bone marrow.
  • nucleated red blood cells in the blood circulation gradually decrease.
  • Simpson et al found that at 11 weeks of gestation, fetal nucleated red blood cells accounted for 10% of the total number of red blood cells in the fetal circulation, down to 0.5% at 19 weeks of pregnancy, and lower at full term.
  • the amount of fetal nucleated red blood cells that can be detected in maternal blood is also different. It has been reported that 61 cases of pregnant women aged 11 to 34 weeks, 21 to 35 years old pregnant women in the peripheral blood analysis of fetal cells, the results show that in the early, middle and late pregnancy, maternal blood in the presence of NRBC, early pregnancy NRBC content with pregnancy Progress has shown an upward trend, reaching a peak around 17 and 18 weeks, and has since declined. And put the best time for prenatal diagnosis around 17 and 18 weeks of pregnancy. It could not be detected 2 months after delivery, most of which could not be detected 2 hours after delivery, indicating that the detection process was less affected by false positives of fetal DNA left over from the last pregnancy.
  • the amount of fetal cells in the peripheral blood of pregnant women is extremely small, with only about 5 to 20 fetal cells per 1 ml of maternal blood, and NRBC is less, containing 2 to 6 per 1 ml. Therefore, it is necessary to separate and purify the peripheral blood samples of pregnant women in order to facilitate further analysis and to make prenatal diagnosis. It takes a lot of time for the blood sample taken from the pregnant woman to be transferred from the hospital to the laboratory, and the time to arrive at the laboratory cannot be fixed, which may result in the subsequent experiment being unable to arrange the time reasonably. The preservation of the sample blood in the refrigerator will affect the cells in it. Dr.
  • the first damage is the damage factors such as biofilm free radicals and apoptosis signals.
  • the phospholipid messenger molecules such as CM and PS activate MAPK and other pathways to induce apoptosis, and on the other hand, the membrane can be altered or destroyed. Structures, such as phospholipids, proteins, etc., accelerate the process of cell aging, eventually leading to aging death of cells and loss of effective blood function. It takes a lot of manpower and material resources to get the expected experimental results.
  • Alsever's Solution is a red blood cell preservation solution.
  • the main components are glucose, sodium citrate, citric acid, and sodium chloride. Among them, glucose provides nutrients for cells; sodium citrate, also known as trisodium citrate, is used for anticoagulation of blood.
  • the principle is to form a soluble complex with calcium ions, thereby making the coagulation factor-Ca 2+ necessary for blood coagulation. Can not participate in blood coagulation to achieve anticoagulant effect.
  • Aristotle is mainly used to preserve red blood cells. At 4 ° C, red blood cells can be stored for 1-2 weeks without changing the activity and characteristics, which is of great benefit for subsequent gradient centrifugation and flow sorting.
  • the purpose of the present application is to provide an improved preservation solution for preserving rare cells such as nucleated red blood cells in peripheral blood samples of pregnant women, and to provide a method for preserving peripheral blood samples of two pregnant women.
  • the present application discloses a preservation solution for a peripheral blood sample of a pregnant woman, the preservation solution consisting of a fluid and an apoptosis inhibitor, and the volume ratio of the albino solution: the apoptosis inhibitor is 5-15:0.03. -0.1.
  • the peripheral blood sample of the pregnant woman of the present application is the peripheral blood sample of the pregnant woman.
  • This application firstly adds an apoptosis inhibitor to the preservation solution based on the A's solution, so that the preservation solution can better preserve the peripheral blood of the pregnant woman.
  • the sample, wherein the apoptosis inhibitor mainly serves to delay the apoptosis of the cells in the sample, maintain the various indexes of the cells, and keep the sample fresh as much as possible; especially for rare cells in the peripheral blood of pregnant women, such as nucleated red blood cells, Has a good protection.
  • apoptosis inhibitors can be used in the present application, however, in a preferred embodiment of the present application, it is preferred to use a Caspase inhibitor which is a pan-Caspase inhibitor which can penetrate the cell membrane. It has an inhibitory effect on apoptosis in most cells. When it is applied to the peripheral blood of pregnant women, it has a good protective effect on other rare cells except for nucleated red blood cells.
  • the other side of the application discloses a method for preserving a peripheral blood sample of a pregnant woman, comprising adding the preservation solution of the present application to the peripheral blood of the freshly drawn pregnant woman, and then transporting it to the laboratory through the cryosampled sample box, and storing at 4 ° C for use.
  • cryosampled sample box is a sample delivery box for transporting blood samples which is conventionally used, and the temperature thereof is about 2-8 °C.
  • the role of the preservation solution is to protect the peripheral blood of the pregnant woman and the rare cells therein, especially the nucleated red blood cells; in view of the protective effect, cost and subsequent treatment, the preferred amount of the control preservation liquid used in the present application is the volume.
  • Peripheral blood than pregnant women: preservation solution 1-5:5-15; too little preservation solution will affect the preservation effect, while too much preservation solution is a waste of reagents, increase costs, and second, the dilution of blood is too large to be conducive to downstream samples Treatment, such as density gradient centrifugation.
  • a further aspect of the present application discloses a method for preserving a peripheral blood sample of a second pregnant woman, comprising extracting peripheral blood of a pregnant woman for 10-20 weeks of pregnancy, adding an apoptosis inhibitor to the peripheral blood of the freshly drawn pregnant woman, and then delivering the sample by cryolysis.
  • the box is transported to the laboratory, and then the aristotle liquid is added to the peripheral blood of the pregnant woman, mixed, and stored at 4 ° C for use.
  • the basic idea is to use the preservation solution of the present application to preserve the peripheral blood of pregnant women, but the amount of albis solution is relatively large, In the case of a large number of sample collections, it is convenient to transport the apoptosis inhibitor in advance, and it is more convenient for on-site operation due to the small amount of apoptosis inhibitor added.
  • volume ratio of peripheral blood to apoptosis inhibitor in pregnant women is calculated according to the dosage of the preservation solution of the present application for peripheral blood storage of pregnant women. of.
  • the apoptosis inhibitor is a Caspase inhibitor.
  • a key role of the preservation solution and preservation method of the present application is to preserve fetal cells in the peripheral blood of pregnant women, including trophoblast cells, lymphocytes, granulocytes, monocytes, and nucleated red blood cells; The subsequent non-invasive prenatal diagnosis of nucleated red blood cells has a good protective effect.
  • a further aspect of the present application provides the use of a preservation solution of the present application in the preservation of fetal cells in a peripheral blood sample of a pregnant woman.
  • the application further includes using the preservation solution of the present application and using the preservation method of the present application to preserve fetal cells in the peripheral blood of pregnant women, especially for nucleated red blood cells.
  • the preservation solution and preservation method for the peripheral blood of pregnant women of the present application are ultimately intended for subsequent non-invasive prenatal diagnosis, and therefore, the key function is to preserve the nucleated red blood cells in the peripheral blood of pregnant women. That is, the key is to preserve the nucleated red blood cells; however, the preservation solution and the preservation method of the present application can not only effectively preserve the nucleated red blood cells, but also have protection effects on other fetal cells.
  • This application is the first to combine the application of albenol and apoptosis inhibitors in the preservation of peripheral blood of pregnant women, and proposed a preservation solution consisting of albenol and apoptosis inhibitors.
  • the preservation solution of the present application can effectively delay the apoptosis of cells in the peripheral blood of pregnant women, maintain various indexes of the cells, keep the samples as fresh as possible, and maintain the cell morphology, provide nutrients for the cells, and maintain the physical and chemical properties of the cells; Subsequent gradient centrifugation and flow sorting of nucleated red blood cells and other rare cells lay the foundation, and the preservation of a large number of fresh nucleated red blood cells provides a guarantee for the accuracy of non-invasive prenatal diagnosis.
  • Figure 1 is a microscopic observation result of a fresh sample in the second embodiment of the present application, wherein a is a bright field observation result, and b is a fluorescence channel observation result;
  • a is a bright field observation result
  • b is a fluorescence channel observation result
  • a is a bright field observation result
  • b is a fluorescence channel observation result
  • Figure 4 is a microscopic observation result after storage for 3 days in the second embodiment of the present application, wherein a is a bright field observation result, and b is a fluorescence channel observation result;
  • Figure 5 is a microscopic observation result after 5 days of storage in the second embodiment of the present application, wherein a is a bright field observation result, and b is a fluorescence channel observation result;
  • Figure 6 is a microscopic observation result after 7 days of storage in the second embodiment of the present application, and the figure is a bright field observation result;
  • Figure 7 is a diagram showing the results of microscopic observation after 7 days of storage in the second embodiment of the present application, which is shown as a result of observation of a fluorescent channel;
  • Figure 8 is a fluorescence detection result of cell capture staining after adding Caspase inhibitor and A. sinensis solution for 3 days in the second embodiment of the present application, and a, b, and c are respectively observed results of three different fluorescent channels;
  • Figure 9 is a result of fluorescence detection of cell capture staining after 3 days of preservation of the sample directly stored in Example 2 of the present application, and a, b, and c are respectively observed results of three different fluorescent channels;
  • Figure 10 is a sequencing analysis result after adding Caspase inhibitor and A. sinensis solution for 3 days in the second embodiment of the present application;
  • Figure 11 is a result of sequencing analysis after 3 days of preservation of the sample directly stored in Example 2 of the present application.
  • Aristotle is a blood sample preservation solution that is currently used routinely, but it is used for the storage of peripheral blood samples of pregnant women, and it is rare cells in the peripheral blood of pregnant women, especially nucleated red blood cells for non-invasive prenatal diagnosis.
  • the preservation effect is not ideal. Therefore, the present application is based on the improvement of aristoleine, and proposes a new preservation solution consisting of albenol and an apoptosis inhibitor, which not only can provide nutrients to the cells, but also keep the cells as much as possible. Its morphology and physical and chemical properties, and can effectively inhibit apoptosis, keep the sample fresh, reduce the damage of rare cells in the peripheral blood of pregnant women, especially nucleated red blood cells.
  • the storage temperature of the albens and apoptosis inhibitors, particularly the preferred Caspase inhibitors of the present application are completely different, and the physical forms of the two are at their respective storage temperatures.
  • the present invention also creatively combines the two to form a preservation solution for the peripheral blood of pregnant women, which has a good preservation effect on rare cells in the peripheral blood of pregnant women, especially nucleated red blood cells, and is a non-invasive prenatal period. Diagnosis provides protection.
  • the present application provides two methods for preserving peripheral blood samples of pregnant women.
  • the first method is to directly add the preservation solution of the present application to the peripheral blood of freshly drawn pregnant women, and the second is to first A freshly drawn pregnant woman is given an apoptosis inhibitor in the peripheral blood and then added to the fluid.
  • the basic principle is to preserve the peripheral blood of pregnant women by using the preservation solution of the present application. It is only the second method, which is more convenient for transportation and on-site treatment.
  • the apoptosis inhibitor is added to the peripheral blood of the pregnant woman until it is transported to the laboratory, and then added to the albino solution, although there are cells.
  • apoptosis inhibitors only temporarily keeps the sample fresh, and finally needs to work with the albino solution to be effective in the peripheral blood of pregnant women.
  • the rare cells are protected; therefore, if the conditions permit, the preservation solution of the present application is directly added to the peripheral blood of the pregnant woman, and the effect is the best.
  • test group a fresh pregnant women's peripheral blood, without any addition.
  • the four experimental groups were mixed and allowed to stand overnight in a refrigerator at 4 ° C. The density gradient was centrifuged on the next day and the centrifugation effect and cell state were observed and compared with the results of the fresh samples as the reference observed on the first day.
  • the Caspase inhibitor of this example is commercially available as Z-VAD-FMK. Caspase inhibitors, other reagents or equipment are routine laboratory reagents and equipment. In this case, the FDA+Hochest dyes were used to observe the cell surface and nucleus.
  • the experimental group b was centrifuged normally, and the cells showed no fluorescence after FDA staining, indicating that the cells had apoptosis.
  • Experimental group c was slightly hemolyzed with agglomeration.
  • the experimental group d was normalized to centrifugation, and the staining was normal, and there was substantially no difference from the fresh sample as the reference observed on the first day.
  • the test group a without adding any protective agent only 2-3 nucleated red blood cells could be found; and in the test group d to which the agar solution and the inhibitor were added, an equivalent amount of blood samples could be found about 30.
  • the number of erythrocytes is the same as in fresh blood; the nucleated red blood cells found in the other two groups are between test group a and test group d.
  • the amount of albino solution and Caspase inhibitor was optimized.
  • the results showed that the volume ratio of peripheral blood: apoptosis inhibitor in pregnant women was 1-5: 0.03-0.1.
  • the peripheral blood of pregnant women the volume ratio of the arsenic solution is between 1-5:5-15, and the blood sample can be well protected, and the observation effect is equivalent to that of the test group d.
  • this example also tested different market-purchased Caspase inhibitors, and the results were compared with Z-VAD-FMK.
  • the test group d of the caspase inhibitor was equivalent.
  • the morphological integrity of the cells in the blood samples and the FDA+Hochest fluorescence detection cell viability were observed after storage for 1 day, 2 days, 3 days, 5 days, and 7 days, respectively, and gradient centrifugation was performed to observe the centrifugation effect.
  • the other peripheral blood of the pregnant woman was directly placed in a refrigerator at 4 ° C for 3 days.
  • the sample was compared with the sample stored with the Caspase inhibitor for 3 days, and subjected to microfluidic capture and whole genome sequencing.
  • FIG. 1-7 The results of microscopic observation and fluorescence detection are shown in Figure 1-7.
  • the results showed that the peripheral blood of pregnant women who were added with Caspase inhibitor and A's solution was preserved for 1 day, 2 days, 3 days, 5 days, 7 days after the sample and fresh.
  • the morphological integrity of the cells in the sample is more than 95% of the fresh sample compared to the cell viability.
  • the samples were stored for 1 day and 2 days.
  • the results are shown in Fig. 2 and Fig. 3. Compared with the fresh samples, no abnormalities were observed in Fig. 1. However, after storage for 3 days, a cell abnormality was found.
  • the sample stored for 5 days was found to have two cell abnormalities, the part shown by the circle in Fig. 5; the sample stored for 7 days was counted by the 4X4 square of the counting plate, and the result showed that in the bright field 50 cells were observed, while the FDA stained cells were 48, ie the cell survival rate was 96%.
  • the microfluidic capture staining fluorescence effect is shown in Figures 8 and 9, and the sequencing analysis results are shown in Figures 10 and 11.
  • the results showed that the cells with the addition of inhibitor and albenol were intact and the fluorescence was bright.
  • Figure 8 without adding any preservation solution, directly preserved blood samples, the cell morphology was rough and the fluorescence was dim, Figure 9.
  • the sequencing results showed that the chromosome sequence of the sample was uniform after adding the albenol solution and the apoptosis inhibitor, and the pregnant woman was successfully judged to have the T18 male fetus.
  • the result is consistent with the DNA sequencing result of the abortion tissue, Fig. 10; Samples of the liquid and apoptosis inhibitors, ie, directly preserved blood samples, have inconsistent results in cell chromosome sequencing, and multiple chromosomal abnormalities cannot diagnose chromosomal diseases, Figure 11.
  • the results showed that no matter the number of target cells captured, the morphology or the staining effect, the samples with the addition of albenol and apoptosis inhibitors were superior to the blood samples that were not directly preserved, and from the whole genome sequencing data,
  • the data obtained by the Aristotle liquid and the apoptosis inhibitor are more uniform, and the good uniformity is the premise for the diagnosis of chromosomal and genetic diseases. Therefore, the peripheral blood preservation method of the pregnant woman in this example effectively preserves the characteristics of the cells.
  • the sample with the addition of two reagents ensured that we successfully detected the T18 trisomy, without adding any preservation solution, the blood sample directly saved can not be judged.
  • this example also prepares a preservation solution of the albenic solution and the Caspase inhibitor for the test. Specifically, 1. Prepare a preservation solution in the laboratory: 100 ul of Caspase cell apoptosis inhibitor is added to 15 ml of albenol solution, the final concentration is 0.67%, mix, 50 ml centrifuge tube -80 ° C, take out and melt before sampling. Room temperature. 2. Carry a 50ml preservation solution tube to the hospital at room temperature, and take the peripheral blood sample of pregnant women. The gestational age is about 10-20 weeks, use 5ml heparin sodium tube, shake it and transfer it to the 50ml preservation solution tube immediately. 3. The sample was quickly transported from the hospital to the laboratory in a cryosampled sample box and stored in a refrigerator at 4 °C. The rest of the settings are the same as in the second embodiment.
  • the sample of the preservation solution was added, and after 3 days of storage, the microfluidic capture staining fluorescence detection showed that the cell morphology was complete and the fluorescence was bright; while the sample without the preservation solution was directly preserved for 3 days, the cell morphology was rough and fluorescent. Dim, the test results are slightly. Similarly, the sequencing results are the same as in the second embodiment.
  • the method of adding the inhibitor and the aristole solution in the second embodiment, or the method of preparing the inhibitor and the agar solution in the third embodiment can well preserve the peripheral blood sample of the pregnant woman.
  • the method of the second embodiment separately adds the inhibitor and the arsenic solution, it may be unfavorable for the preservation of the sample, but the second and the third examples obtain almost the same test results, and we analyze that It may be because the time we sampled and transported to the laboratory is relatively short. That is to say, although the inhibitor is added after sampling in the second embodiment, the blood cells can be effectively protected in a short time. Compared with the direct addition inhibitor and the arsenic solution of the third embodiment, the effects were similar in a short transportation time.

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Abstract

本申请公开了一种用于孕妇外周血样的保存液和孕妇外周血样的保存方法。本申请的保存液由阿氏液和细胞凋亡抑制剂组成，阿氏液：细胞凋亡抑制剂的体积比为5-15:0.03-0.1。本申请的保存液率先将阿氏液和细胞凋亡抑制剂联合应用于孕妇外周血液的保存，能有效的延缓孕妇外周血液中细胞的凋亡、维持细胞的各项指标，使样品尽量保持新鲜；并且可以维持细胞形态、为细胞提供养分、保持细胞各项理化性；为后续的有核红细胞和其它稀有细胞的梯度离心和流式分选奠定基础，大量新鲜的有核红细胞的保有，为非侵入性产前诊断的准确性提供了保障。

Description

用于孕妇外周血样的保存液和孕妇外周血样的保存方法 技术领域
本申请涉及血液样品保存领域，特别是涉及一种用于孕妇外周血液样品保存的保存液，以及孕妇外周血液样品的保存方法。
 背景技术
目前对遗传性疾病的产前诊断有数种方法。根据采样途径或诊断方法是否构成对胎儿的影响可分为侵入性产前诊断和非侵入性产前诊断。侵入性产前诊断包括羊膜腔穿刺术、绒毛细胞吸取术及胎儿脐血穿刺等；虽然诊断精确度较高，但可导致流产等并发症，只能在高危孕妇中实施。非侵入性产前诊断，目前主要是对进入母体血液的胎儿细胞进行产前诊断，这种诊断方式的优点是既能行产前诊断又无危险性，适用于低风险的孕妇大群体筛选，尽早发现和杜绝异常胎儿，达到优生目的。
正常胎盘并非是完善的、细胞透不过的屏障。根据目前的研究，在人类正常妊娠当中，进入母体血液循环中的胎儿血量估计是0.1～0.3ml。孕妇血液中胎儿细胞的主要类型包括滋养叶细胞、淋巴细胞、粒细胞、单核细胞、有核红细胞（NRBC）等。其中最合适做产前诊断的胎儿细胞类型当首推胎儿有核红细胞，理由是：（1）胎儿有核红细胞是单核的，孕早期胎儿血中含量丰富但孕妇外周血中罕见；（2）其表达几种特异的抗原，如运铁蛋白受体和特异的胎儿血红蛋白肽链，如α链、β链和γ链，作为细胞标记而利于细胞的分类、鉴定和聚集；（3）有核红细胞在孕妇外周血中的生存期短，从开始受精3周持续分娩后2月，不会持续到下一次妊娠，胎儿有核红细胞是胎儿造血系统及胎儿血液循环的一个特征性标志。
胎儿血循环于受精后3周末建立，其红细胞主要来源于卵黄囊。妊娠第10周，肝脏成为红细胞生成的主要器官，以后骨髓、脾脏逐渐具有造血功能。于妊娠足月时，90％的红细胞来源于骨髓。随着胎儿造血系统的逐渐发育成熟，血液循环中有核红细胞逐渐减少。Simpson等研究发现，孕11周时，胎儿循环中胎儿有核红细胞占红细胞总数的10％，孕19周时降至0.5％，足月时更低。在不同阶段，在母体血中所能检出胎儿有核红细胞的量亦不同。有报道对61例孕11～34周，21～35岁的孕妇外周血中的胎儿细胞进行分析，结果显示在早、中、晚期妊娠时母血中都存在NRBC，孕早期NRBC含量随妊娠的进展呈上升趋势，17、18周左右达高峰，此后成下降趋势。并提出孕17、18周左右为产前诊断的最佳时间。在分娩后2个月即不能检出，其中大部分在产后2小时就不能测出，这表明检测过程较少受到上次妊娠遗留的胎儿DNA的假阳性的影响。
但是，孕妇外周血液中胎儿细胞含量极少，大约每1ml母体血液中只含5～20个胎儿细胞，NRBC则更少，为每1ml含2～6个。因此，必须对孕妇外周血样进行分离纯化才能利于进一步分析，做产前诊断。而从孕妇体内抽取的血样从医院转移至实验室需要花费大量时间，而且到达实验室的时间无法固定，从而导致接下来的实验无法合理安排时间。把样品血液保存在冰箱中又会对其中的细胞造成影响，第四军区大学杜静平博士对血液保存进行了研究，结果显示在pH5～6的弱酸性状态下于4～6℃的低温保存，在弱酸性环境中，随着温度的下降，红细胞的代谢也下降，但由于血液状保存温度降至0℃已达极限，即便是0℃，血液的代谢仍在进行，即保存损伤作用仍在持续，随着保存期延长，由于抗损伤物质不断消耗而没有后援补充，使得细胞系统不断削弱，而细胞的裂解产物及各种代谢产物的堆积又可以诱发更为强烈的损伤反应，如过氧化反应、加速细胞老化进程、启动凋亡程序等，引起一系列结构和功能的改变。在这些变化中，首先受损的是生物膜各自由基及凋亡信号等损伤因子一方面通过CM、PS等磷脂信使分子激活MAPK等途径诱发凋亡反应，另一方面可通过改变或破坏膜结构，如磷脂、蛋白质等，加速细胞老化进程，最终导致细胞的衰老死亡，血液有效功能丧失。耗费大量人力物力而得不到预期的实验结果。
阿氏液（Alsever's Solution）是一种红细胞保存液，主要成分为葡萄糖、构檬酸钠、枸檬酸、氯化钠。其中葡萄糖为细胞提供营养成分；构檬酸钠，又称为柠檬酸三钠，用于血液抗凝，其原理是与钙离子形成可溶性的复合物，从而使血液凝固必须的凝血因子-Ca2＋不能参与血液凝固而达到抗凝作用。阿氏液主要用于保存红细胞，在4℃的环境下，红细胞可以保存1-2周而不改变活性和特性，对于后续的梯度离心和流式分选有很大的好处。但是，由于孕妇外周血液中的NRBC含量非常稀少，采用阿氏液对孕妇外周血液样品进行保存时，由于细胞仍然是具有活性的，血液的代谢仍在进行，对原本稀少的NRBC造成不可逆的保存损伤，严重的甚至无法满足后续的非侵入性产前诊断试验需要。
 发明内容
本申请的目的是提供一种改进的能够很好的保存孕妇外周血液样品中的有核红细胞等稀有细胞的保存液，并提供了两种孕妇外周血液样品的保存方法。
为了实现上述目的，本申请采用了以下技术方案：
本申请一方面公开了一种用于孕妇外周血样的保存液，该保存液由阿氏液和细胞凋亡抑制剂组成，阿氏液：细胞凋亡抑制剂的体积比为5-15:0.03-0.1。
需要说明的是，本申请的孕妇外周血样即孕妇外周血液样品，本申请率先在以阿氏液为基础的保存液中添加了细胞凋亡抑制剂，使得保存液能够更好的保存孕妇外周血液样品，其中细胞凋亡抑制剂主要起到延缓样品中细胞的凋亡、维持细胞各项指标，尽量使样品保持新鲜的作用；特别是对孕妇外周血液中的稀有细胞，如有核红细胞等，有很好的保护作用。可以理解，常规的细胞凋亡抑制剂都可以用于本申请，但是，本申请的一个优选方案中，优选使用Caspase抑制剂，该细胞凋亡抑制剂是一种可以穿透细胞膜的泛Caspase抑制剂，对大多数细胞都有凋亡抑制作用，作用于孕妇外周血液中时，除对有核红细胞外，对其它的稀有细胞也都有很好的保护作用。
本申请的另一面公开了一种孕妇外周血样的保存方法，包括在新鲜抽取的孕妇外周血液中加入本申请的保存液，然后通过低温送样箱运输至实验室，4℃保存备用。
需要说明的是，低温送样箱即常规使用的运输血液样品的送样箱，其温度约为2-8℃。
优选的，孕妇外周血液与保存液的体积比为，孕妇外周血液：保存液=1-5:5-15。
需要说明的是，保存液的作用是保护孕妇外周血液及其中的稀有细胞，特别是有核红细胞；考虑到其保护效果、成本以及后续的处理，本申请优选的控制保存液的用量为，体积比孕妇外周血液：保存液=1-5:5-15；保存液太少会影响保存效果，而保存液太多一是浪费试剂，增加成本，二是对血液稀释过大不利于下游的样本处理，如密度梯度离心等。
本申请的再一面公开了第二种孕妇外周血样的保存方法，包括抽取孕期10-20周的孕妇的外周血液，向新鲜抽取的孕妇外周血液中加入细胞凋亡抑制剂，然后通过低温送样箱运输至实验室，再向孕妇外周血液中加入阿氏液，混匀，4℃保存备用。
需要说明的是，在孕妇外周血液中加入细胞凋亡抑制剂然后在加入阿氏液，其基本思路还是采用本申请的保存液对孕妇外周血液进行保存，但是阿氏液的用量比较大，在样品采集数量比较多的情况下，只是预先加入细胞凋亡抑制剂会比较方便运输，并且由于细胞凋亡抑制剂的加入量少，也更方便现场操作。
优选的，孕妇外周血液与细胞凋亡抑制剂的体积比为，孕妇外周血液：细胞凋亡抑制剂=1-5:0.03-0.1。
优选的，阿氏液按体积比，孕妇外周血液：阿氏液=1-5:5-15的量添加。
需要说明的是，无论是孕妇外周血液与细胞凋亡抑制剂的体积比，还是孕妇外周血液与阿氏液的体积比，都是按照本申请的保存液用于孕妇外周血液保存时的用量计算的。
同样的，在第二种孕妇外周血样的保存方法中，优选的，细胞凋亡抑制剂为Caspase抑制剂。
本申请的保存液和保存方法的一个关键作用是用于保存孕妇外周血液中的胎儿细胞，这些胎儿细胞包括滋养叶细胞、淋巴细胞、粒细胞、单核细胞、有核红细胞；尤其是对用于后续的非侵入性产前诊断的有核红细胞具有很好的保护作用。因此，本申请的再一面提供了一种本申请的保存液在孕妇外周血样中胎儿细胞的保存中的应用。该应用进一步的包括，采用本申请的保存液，并采用本申请的保存方法，对孕妇外周血液中的胎儿细胞进行保存，尤其是对有核红细胞进行保存。
需要说明的是，本申请的用于孕妇外周血液的保存液和保存方法，其最终的目的是为了后续的非侵入性产前诊断，因此，其关键作用在于保存孕妇外周血液中的有核红细胞，即关键是对有核红细胞进行保存；但是，本申请的保存液和保存方法不仅可以有效的保存有核红细胞，对其它胎儿细胞仍然具有保护作用。
由于采用以上技术方案，本申请的有益效果在于：
本申请率先将阿氏液和细胞凋亡抑制剂联合应用于孕妇外周血液的保存，并提出了由阿氏液和细胞凋亡抑制剂组成的保存液。本申请的保存液可以有效的延缓孕妇外周血液中细胞的凋亡、维持细胞的各项指标，使样品尽量保持新鲜；并且可以维持细胞形态、为细胞提供养分、保持细胞各项理化性；为后续的有核红细胞和其它稀有细胞的梯度离心和流式分选奠定基础，大量新鲜的有核红细胞的保有，为非侵入性产前诊断的准确性提供了保障。
附图说明
图1：是本申请实施例二中新鲜样品的显微镜观察结果图，a为明场观察结果，b为荧光通道观察结果；
图2：是本申请实施例二中保存1天后的显微镜观察结果图，a为明场观察结果，b为荧光通道观察结果；
图3：是本申请实施例二中保存2天后的显微镜观察结果图，a为明场观察结果，b为荧光通道观察结果；
图4：是本申请实施例二中保存3天后的显微镜观察结果图，a为明场观察结果，b为荧光通道观察结果；
图5：是本申请实施例二中保存5天后的显微镜观察结果图，a为明场观察结果，b为荧光通道观察结果；
图6：是本申请实施例二中保存7天后的显微镜观察结果图，图示为明场观察结果；
图7：是本申请实施例二中保存7天后的显微镜观察结果图，图示为荧光通道观察结果；
图8：是本申请实施例二中添加Caspase抑制剂和阿氏液保存3天后的细胞捕获染色荧光检测结果，a、b、c分别为三个不同的荧光通道观察结果；
图9：是本申请实施例二中直接保存的样品保存3天后的细胞捕获染色荧光检测结果，a、b、c分别为三个不同的荧光通道观察结果；
图10：是本申请实施例二中添加Caspase抑制剂和阿氏液保存3天后的测序分析结果；
图11：是本申请实施例二中直接保存的样品保存3天后的测序分析结果。
具体实施方式
阿氏液是目前比较常规使用的血液样品保存液，但是，用于孕妇外周血液样品保存时，其对孕妇外周血液中的稀有细胞，特别是对用于非侵入性产前诊断的有核红细胞的保存效果并不理想。因此，本申请在阿氏液的基础上进行改进，提出了一种新的由阿氏液和细胞凋亡抑制剂组成的保存液，这种保存液不仅可以给细胞提供养分，使细胞尽量保持其形态和各项理化性能，而且可以有效的抑制细胞凋亡，使样品保持新鲜，减小了孕妇外周血液中稀有细胞，特别是有核红细胞的损伤。
需要说明的是，阿氏液和细胞凋亡抑制剂，特别是本申请优选的Caspase抑制剂，两者的保存温度是完全不同的，并且，在其各自的保存温度下，两者的物理形态也不同；本申请创造性的将两者组合在一起，形成用于孕妇外周血液的保存液，对孕妇外周血液中的稀有细胞，特别是有核红细胞具有良好的保存作用，为非侵入性产前诊断提供了保障。
在本申请的保存液的基础上，本申请提供了两种孕妇外周血样的保存方法，第一种是直接将本申请的保存液加入到新鲜抽取的孕妇外周血液中，第二种是先在新鲜抽取的孕妇外周血液中加入细胞凋亡抑制剂，然后再加入阿氏液。需要说明的是，无论是哪种保存方法，最终其基本原理都是采用本申请的保存液对孕妇外周血液进行保存。只是第二种方法，更方便运输和现场处理；需要说明的是，在孕妇外周血液中加入细胞凋亡抑制剂，直至运输到实验室，再加入阿氏液的这段时间中，虽然有细胞凋亡抑制剂的保护作用，但是，这段时间仍然不宜太长，细胞凋亡抑制剂只是暂时的保持样品的新鲜，最终还是需要和阿氏液一起作用，才能够有效的对孕妇外周血液中的稀有细胞进行保护；因此，在条件允许的情况下，还是直接向孕妇外周血液中加入本申请的保存液，效果是最好的。
下面通过具体实施例和附图对本申请作进一步详细说明。以下实施例仅对本申请进行进一步说明，不应理解为对本申请的限制。
实施例一
本例对新鲜血样中添加的成分进行了对比试验。先取一管新鲜的孕妇外周血液直接进行密度梯度离心并观察离心效果及细胞状态作为参考。然后设置，试验组a：新鲜的孕妇外周血液，无任何添加。试验组b：新鲜的孕妇外周血液，添加阿氏液，孕妇外周血液与阿氏液的体积比为，孕妇外周血液：阿氏液=1:15。试验组c：新鲜的孕妇外周血液，添加Caspase抑制剂，孕妇外周血液与Caspase抑制剂的体积比为，孕妇外周血液：Caspase抑制剂=1：0.1。试验组d：新鲜的孕妇外周血液，添加由阿氏液和Caspase抑制剂组成的保存液，孕妇外周血液与保存液的体积比为，孕妇外周血液：保存液=1:5，保存液中阿氏液：细胞凋亡抑制剂的体积比为15:0.1。四组实验组混匀后静置在4℃冰箱内过夜，第二天密度梯度离心并观察离心效果及细胞状态并与第一天观察到的作为参考的新鲜样品的结果对比。本例的Caspase抑制剂为市场购买的Z-VAD-FMK Caspase抑制剂，其它试剂或器材都是实验室常规的试剂和器材。本例采用FDA+Hochest两种染料，分别对细胞表面和细胞核进行染色观察。
结果显示，实验组a出现溶血现象，细胞凝结成块状。实验组b离心正常，FDA染色后细胞出现无荧光，说明细胞已凋亡。实验组c轻微溶血，带有结块。实验组d离心正常，染色正常，与第一天观察到的作为参考的新鲜样品基本无差别。并且，在不添加任何保护剂的试验组a中，仅能找到2-3颗有核红细胞；而添加阿氏液和抑制剂的试验组d中，等量的血液样品可以找到约30颗有核红细胞，与新鲜血液中的数量一样；其它两组所找到的有核红细胞介于试验组a和试验组d之间。
此外，本例在试验组d的基础上，对阿氏液和Caspase抑制剂的用量进行了优化，结果显示，孕妇外周血液：细胞凋亡抑制剂的体积比在1-5:0.03-0.1之间，孕妇外周血液：阿氏液的体积比在1-5:5-15之间，都能够对血样进行很好的保护，观察效果与试验组d相当。此外，本例还对不同的市场购买的Caspase抑制剂进行了试验，结果与采用Z-VAD-FMK Caspase抑制剂的试验组d相当。
实施例二
从医院抽取新鲜的孕妇外周血液两管，其中一管加入Caspase抑制剂混匀后低温平稳运输至实验室，其中孕妇外周血液与Caspase抑制剂的体积比为，孕妇外周血液：Caspase抑制剂=1-0.1。运输至实验室后，按孕妇外周血液：阿氏液=1:3的体积比，将孕妇外周血液加入阿氏液中，放入4℃冰箱保存。分别在保存1天、2天、3天、5天、7天后观察血样中的细胞的形态完整度和FDA+Hochest荧光检测细胞活率，并做梯度离心，观察离心效果。
另一管孕妇外周血液直接放入4℃冰箱保存3天，该样品与添加阿氏液与Caspase抑制剂后保存3天的样品进行对比，分别进行微流控捕获及全基因组测序。
显微镜观察和荧光检测结果如图1-图7所示，结果证明，加入Caspase抑制剂和阿氏液的孕妇外周血液，其保存1天、2天、3天、5天、7天后样品与新鲜样品中细胞的形态完整度和细胞活率相比，均在新鲜样品的95%以上。如图所示，保存1天和2天的样品，其结果图2和图3，与新鲜样品相比，图1，都没有发现细胞异常；而保存3天的样品，发现有一个细胞异常，图4中圆圈所示部分；保存5天的样品，发现有两个细胞异常，图5中圆圈所示部分；保存7天的样品，采用计数板4X4方格进行统计，结果显示，明场中观察到50个细胞，而FDA染色细胞为48个，即细胞存活率96%。
微流控捕获染色荧光效果如图8和图9所示，测序分析结果如图10和11所示。结果显示，添加抑制剂和阿氏液的细胞形态完整，荧光明亮，图8，而没有添加任何保存液，直接保存的血样，其细胞形态粗糙，荧光暗淡，图9。测序结果显示，添加阿氏液与细胞凋亡抑制剂后样品细胞染色体测序结果均一，且成功判断该孕妇怀有T18男胎，该结果与流产组织DNA测序结果一致，图10；而不添加阿氏液和细胞凋亡抑制剂的样品，即直接保存的血样，其细胞染色体测序结果不均一，多条染色体异常无法进行染色体疾病的诊断，图11。
结果证明：无论是捕获到的目标细胞数量，形态还是染色效果，添加了阿氏液和凋亡抑制剂的样品都优于不处理直接保存的血液样品，且从全基因组测序数据来看，加入阿氏液和凋亡抑制剂后得到的数据均一性更佳，而好的均一性是我们进行染色体及基因疾病诊断的前提，所以本例的孕妇外周血液保存方法有效的保存了细胞的特性进而保证我们得到理想的数据结果，添加了两种试剂后的样本保证了我们成功检出了T18三体的患儿，而没有添加任何保存液，直接进行保存的血样则无法判断。
实施例三
在实施例二的基础上，本例还将阿氏液与Caspase抑制剂制成保存液，用于试验。具体的，1.实验室内配制保存液：100ul的Caspase细胞凋亡抑制剂加入15ml的阿氏液中，终浓度为0.67%，混匀，50ml离心管-80℃保存，取样前取出融化至室温。2.常温携带50ml保存液管至医院，抽取孕妇外周血液样品，孕周在10-20周左右，使用5ml肝素钠管，摇匀后立即转移到50ml保存液管中。3.样品放置在低温送样箱中从医院快速运输至实验室，并保存在4℃的冰箱中。其余设置与实施例二相同。
结果显示，添加保存液的样品，保存7天后其形态完整度和细胞活率，与新鲜样品相比，细胞存活率98%，检测结果图略。
在对比设计中，添加保存液的样品，保存3天后，微流控捕获染色荧光检测结果显示，细胞形态完整，荧光明亮；而没有添加保存液的样品，直接保存3天后，细胞形态粗糙，荧光暗淡，检测结果图略。同样的，测序结果也与实施例二相同。
结果证实，无论是实施例二中分别添加抑制剂和阿氏液的方法还是实施例三中将抑制剂和阿氏液制成保存液的方法，都能够很好的保存孕妇外周血液样品。另外，虽然我们认为，实施例二的方法将抑制剂和阿氏液分开来添加，可能对样品的保存不利，但是，实施例二和实施例三获得了几乎相同的试验结果，我们分析，这有可能是因为，我们采样到运输至实验室的时间比较短，也就是说，虽然实施例二中采样后仅仅添加抑制剂，但是，在短时间内，还是可以有效的对血液细胞进行保护，与实施例三的直接添加抑制剂和阿氏液相比，在短的运输时间内，两者效果相当。
以上内容是结合具体的实施方式对本申请所作的进一步详细说明，不能认定本申请的具体实施只局限于这些说明。对于本申请所属技术领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本申请的保护范围。

Claims (10)

  1. 一种用于孕妇外周血样的保存液，其特征在于：由阿氏液和细胞凋亡抑制剂组成，所述阿氏液：细胞凋亡抑制剂的体积比为5-15:0.03-0.1。
  2. 根据权利要求1所述的保存液，其特征在于：所述细胞凋亡抑制剂为Caspase抑制剂。
  3. 一种孕妇外周血样的保存方法，其特征在于：包括在新鲜抽取的孕妇外周血液中加入权利要求1或2所述的保存液，然后通过低温送样箱运输至实验室，4℃保存备用。
  4. 根据权利要求3所述的保存方法，其特征在于：所述孕妇外周血液与所述保存液的体积比为，孕妇外周血液：保存液=1-5:5-15。
  5. 一种孕妇外周血样的保存方法，其特征在于：包括抽取孕期10-20周的孕妇的外周血液，向新鲜抽取的孕妇外周血液中加入细胞凋亡抑制剂，然后通过低温送样箱运输至实验室，再向孕妇外周血液中加入阿氏液，混匀，4℃保存备用。
  6. 根据权利要求5所述的保存方法，其特征在于：所述孕妇外周血液与所述细胞凋亡抑制剂的体积比为，孕妇外周血液：细胞凋亡抑制剂=1-5:0.03-0.1。
  7. 根据权利要求5所述的保存方法，其特征在于：所述阿氏液按体积比，孕妇外周血液：阿氏液=1-5:5-15的量添加。
  8. 根据权利要求5-7任一项所述的保存方法，其特征在于：所述细胞凋亡抑制剂为Caspase抑制剂。
  9. 根据权利要求1或2所述的保存液在孕妇外周血样中胎儿细胞的保存中的应用，所述胎儿细胞包括滋养叶细胞、淋巴细胞、粒细胞、单核细胞和有核红细胞中的至少一种，尤其包括有核红细胞。
  10. 根据权利要求9所述的应用，包括采用权利要求3-8任一项所述的保存方法对孕妇外周血样中的胎儿细胞进行保存，尤其是对孕妇外周血样中的有核红细胞进行保存。
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