Preservation solution for pregnant woman peripheral blood sample and preservation method for pregnant woman peripheral blood sampleTechnical FieldThe application relates to the field of blood sample preservation, in particular to a preservation solution for preserving a peripheral blood sample of a pregnant woman and a preservation method for the peripheral blood sample of the pregnant woman.
BackgroundThere are several methods currently available for prenatal diagnosis of genetic diseases. The effects on the fetus can be classified into invasive prenatal diagnosis and non-invasive prenatal diagnosis according to whether the sampling route or the diagnosis method constitutes the influence. Invasive prenatal diagnosis includes amniocentesis, villus cell aspiration, and fetal cord blood puncture; although the diagnosis accuracy is high, the method can cause complications such as abortion and the like and can be only implemented in high-risk pregnant women. The non-invasive prenatal diagnosis mainly aims at prenatal diagnosis of fetal cells entering maternal blood at present, and the diagnosis mode has the advantages of capability of prenatal diagnosis and no danger, is suitable for screening large groups of low-risk pregnant women, finds and stops abnormal fetuses as early as possible, and achieves the purpose of prenatal care.
According to the current research, the amount of fetal blood entering maternal blood circulation in normal pregnancy of human is estimated to be 0.1-0.3 ml, the main types of fetal cells in the maternal blood include trophoblast cells, lymphocytes, granulocytes, monocytes, Nucleated Red Blood Cells (NRBC), etc., wherein the fetal cell type most suitable for prenatal diagnosis is first derived from fetal nucleated red blood cells because (1) fetal nucleated red blood cells are mononuclear and abundant in fetal blood at early pregnancy but rare in maternal peripheral blood, (2) it expresses several specific antigens such as transferrin receptor and specific fetal hemoglobin peptide chains such as α chain, β chain and gamma chain, as cell markers to facilitate cell classification, identification and aggregation, and (3) the life cycle of nucleated red blood cells in maternal peripheral blood is short, and is continued to be delivered for 2 months after 3 weeks from the start of fertilization, and is not continued to the next pregnancy, and the fetal nucleated red blood cells are a characteristic of the blood circulation system.
The fetal blood circulation is established 3 weeks after fertilization and its red blood cells are mainly from the yolk sac. In the 10 th week of gestation, the liver becomes the main organ for erythropoiesis, and the bone marrow and spleen gradually have hematopoietic function. At term of gestation, 90% of the erythrocytes are derived from the bone marrow. With the progressive development and maturation of the fetal hematopoietic system, there is a progressive decrease in nucleated red blood cells in the blood circulation. Simpson et al found that fetal nucleated red blood cells accounted for 10% of the total number of red blood cells in the fetal circulation at 11 weeks gestation, decreased to 0.5% at 19 weeks gestation, and even lower at term. The amount of fetal nucleated red blood cells detected in maternal blood at different stages varies. Analysis of fetal cells in peripheral blood of 61 pregnant women of 21-35 years old at 11-34 weeks of gestation has been reported, and the results show that NRBC exists in maternal blood in early, middle and late gestation, the NRBC content in the early gestation period is in an increasing trend along with the progress of pregnancy, and reaches a peak around 17 and 18 weeks and then becomes a descending trend. And proposes that about 17 and 18 weeks of pregnancy is the best time for prenatal diagnosis. It was not detected 2 months after delivery, most of which were not detected 2 hours after delivery, indicating that the detection process was less affected by false positives of fetal DNA remaining from the last pregnancy.
However, the peripheral blood of pregnant women contains very little fetal cells, about 5 to 20 fetal cells per 1ml maternal blood, and less NRBC 2 to 6 fetal cells per 1 ml. Therefore, the pregnant woman peripheral blood sample must be separated and purified to facilitate further analysis and prenatal diagnosis. The transfer of the blood sample drawn from the pregnant woman to the laboratory from the hospital takes a lot of time, and the time for reaching the laboratory cannot be fixed, so that the time for the next experiment cannot be reasonably arranged. The research on the preservation of blood in a refrigerator can affect cells in the sample blood, and the result of the research on the preservation of blood by doctor of Dopithei, university of the fourth military region, shows that the sample blood is preserved at a low temperature of 4-6 ℃ in a weak acid state of pH 5-6, the metabolism of red blood cells is reduced along with the reduction of the temperature in the weak acid environment, but the preservation period of a cell system is weakened continuously because the blood-like preservation temperature is reduced to 0 ℃ and even 0 ℃, the metabolism of the blood is still carried out, namely, the preservation damage effect is continuous, and along with the prolonging, the preservation period is not supplemented because damage-resistant substances are continuously consumed, and the accumulation of cell cracking products and various metabolites can induce stronger damage reactions, such as peroxidation, acceleration of cell aging process, starting of apoptosis process and the like, so as to change a series of structures and functions. Among these changes, the first damage is that the respective damage factors of biological membranes, such as basal and apoptotic signals, on one hand, activate MAPK and other pathways to induce apoptosis through CM, PS and other phospholipid messenger molecules, and on the other hand, the damage factors can accelerate the cell aging process by changing or destroying membrane structures, such as phospholipid, protein and the like, and finally, the cell aging death and the loss of effective blood functions are caused. And a large amount of manpower and material resources are consumed, so that expected experimental results cannot be obtained.
Ashwage's solution is a red blood cell preservation solution, and mainly comprises glucose, sodium citrate, citric acid and sodium chloride. Wherein the glucose provides a nutrient for the cells; sodium citrate, also known as trisodium citrate, is used for anticoagulation of blood by forming soluble complex with calcium ion, thus coagulation factor-Ca necessary for blood coagulation2＋Can not participate in blood coagulation to achieve anticoagulation. The aldrin liquid is mainly used for preserving red blood cells, and the red blood cells can be preserved for 1-2 weeks under the environment of 4 ℃ without changing the activity and the characteristics, thereby having great benefits for subsequent gradient centrifugation and flow sorting. However, since the content of NRBC in the peripheral blood of pregnant women is very rare, when the peripheral blood sample of pregnant women is preserved by using the Ashi's solution, the cells are still active, the metabolism of blood is still carried out, the originally rare NRBC is irreversibly preserved and damaged, and the subsequent non-invasive prenatal diagnosis cannot be met even seriouslyRequired for the test.
Disclosure of InventionThe application aims to provide an improved preservation solution capable of well preserving rare cells such as nucleated red blood cells in a pregnant woman peripheral blood sample, and provides two preservation methods of the pregnant woman peripheral blood sample.
In order to achieve the purpose, the following technical scheme is adopted in the application:
the application discloses a preservation solution for pregnant woman peripheral blood samples, which consists of aldrin and an apoptosis inhibitor, wherein the aldrin: the volume ratio of the apoptosis inhibitor is 5-15: 0.03-0.1.
The pregnant woman peripheral blood sample is prepared by adding an apoptosis inhibitor into a preserving fluid based on an Ashi fluid, so that the preserving fluid can better preserve the pregnant woman peripheral blood sample, wherein the apoptosis inhibitor mainly plays a role in delaying apoptosis of cells in the sample, maintaining various indexes of the cells and keeping the sample fresh as much as possible; especially has good protection effect on rare cells in the peripheral blood of pregnant women, such as nucleated red blood cells and the like. It will be appreciated that conventional apoptosis inhibitors may be used in the present application, but in a preferred embodiment of the present application, it is preferred to use a Caspase inhibitor, which is a pan-Caspase inhibitor that penetrates the cell membrane, has an apoptosis inhibiting effect on most cells, and when acting in the peripheral blood of a pregnant woman, has a very good protective effect on rare cells in addition to nucleated red blood cells.
The application also discloses a preservation method of a pregnant woman peripheral blood sample, which comprises the steps of adding the preservation solution into freshly extracted pregnant woman peripheral blood, conveying the pregnant woman peripheral blood to a laboratory through a low-temperature sample conveying box, and preserving at 4 ℃ for later use.
It should be noted that the low temperature sample transport box, i.e., the sample transport box for transporting blood samples, which is conventionally used, has a temperature of about 2 to 8 ℃.
Preferably, the volume ratio of the pregnant woman peripheral blood to the preservation solution is that the pregnant woman peripheral blood: preservation solution =1-5: 5-15.
The preservation solution has the functions of protecting the peripheral blood of the pregnant woman and rare cells in the peripheral blood, particularly nucleated red blood cells; in consideration of the protection effect, cost and subsequent treatment, the preferable dosage of the controlled preservation solution is that the volume ratio of the peripheral blood of the pregnant woman: preservation solution =1-5: 5-15; too little preservation solution can influence the preservation effect, and too much preservation solution is extravagant reagent, increases the cost firstly, dilutes too much to be unfavorable for sample processing of low reaches to blood, like density gradient centrifugation etc..
The application further discloses a second preservation method of a pregnant woman peripheral blood sample, which comprises the steps of extracting the peripheral blood of a pregnant woman in a pregnancy period of 10-20 weeks, adding an apoptosis inhibitor into the freshly extracted peripheral blood of the pregnant woman, conveying the blood to a laboratory through a low-temperature sample conveying box, adding an aldrin solution into the peripheral blood of the pregnant woman, uniformly mixing, and preserving at 4 ℃ for later use.
The basic idea is to store the peripheral blood of the pregnant woman by using the preservation solution of the application, but the dosage of the aldrin is larger, the apoptosis inhibitor is only added in advance under the condition of more sample collection quantity, so that the transportation is more convenient, and the field operation is more convenient due to the small addition amount of the apoptosis inhibitor.
Preferably, the volume ratio of the pregnant woman peripheral blood to the apoptosis inhibitor is that the pregnant woman peripheral blood: apoptosis inhibitor =1-5: 0.03-0.1.
Preferably, the volume ratio of the liquid A to the liquid A is as follows: ashi solution =1-5: 5-15.
It should be noted that the volume ratio of the peripheral blood of the pregnant woman to the apoptosis inhibitor and the volume ratio of the peripheral blood of the pregnant woman to the aphrodisiac are calculated according to the dosage of the preservation solution for preserving the peripheral blood of the pregnant woman.
Similarly, in the second method for preserving a peripheral blood sample from a pregnant woman, it is preferred that the inhibitor of apoptosis is a Caspase inhibitor.
One key role of the preservation solution and preservation method of the application is to preserve fetal cells in the peripheral blood of a pregnant woman, including trophoblast cells, lymphocytes, granulocytes, monocytes, nucleated red blood cells; especially has good protection effect on nucleated erythrocyte for subsequent non-invasive prenatal diagnosis. Thus, a further aspect of the present application provides the use of a preservation solution of the present application for the preservation of foetal cells in a peripheral blood sample from a pregnant woman. The application further comprises the preservation of fetal cells in the peripheral blood of a pregnant woman, in particular the preservation of nucleated red blood cells, with the preservation solution and the preservation method of the present application.
The preservation solution and the preservation method for the peripheral blood of the pregnant woman have the final aim of subsequent non-invasive prenatal diagnosis, so that the key function is to preserve the nucleated red blood cells in the peripheral blood of the pregnant woman, namely, preserve the nucleated red blood cells; however, the preservation solution and the preservation method of the present application can effectively preserve nucleated red blood cells and still protect other fetal cells.
Due to the adoption of the technical scheme, the beneficial effects of the application are as follows:
the application rate firstly applies the combination of the Arundina solution and the apoptosis inhibitor to the preservation of the peripheral blood of the pregnant women, and provides the preservation solution consisting of the Arundina solution and the apoptosis inhibitor. The preservation solution can effectively delay apoptosis of cells in peripheral blood of pregnant women, maintain various indexes of the cells and keep samples as fresh as possible; and can maintain the cell morphology, provide nutrients for the cells and maintain the physicochemical properties of the cells; lays a foundation for subsequent gradient centrifugation and flow sorting of nucleated erythrocytes and other rare cells, preserves a large amount of fresh nucleated erythrocytes, and provides guarantee for the accuracy of non-invasive prenatal diagnosis.
DrawingsFIG. 1: is a microscope observation result graph of a fresh sample in the second embodiment of the application, wherein a is a bright field observation result, and b is a fluorescence channel observation result;
FIG. 2: is a microscope observation result graph after being stored for 1 day in the second embodiment of the application, wherein a is a bright field observation result, and b is a fluorescence channel observation result;
FIG. 3: is a microscope observation result graph after being stored for 2 days in the second embodiment of the application, wherein a is a bright field observation result, and b is a fluorescence channel observation result;
FIG. 4: is a microscope observation result graph after being stored for 3 days in the second embodiment of the application, wherein a is a bright field observation result, and b is a fluorescence channel observation result;
FIG. 5: is a microscope observation result graph after being stored for 5 days in the second embodiment of the application, wherein a is a bright field observation result, and b is a fluorescence channel observation result;
FIG. 6: is a microscope observation result chart after being stored for 7 days in the second embodiment of the application, and the chart is a bright field observation result;
FIG. 7: is a microscope observation result graph after being stored for 7 days in the second embodiment of the present application, and the graph shows the observation result of the fluorescence channel;
FIG. 8: the cell capture staining fluorescence detection result is obtained after Caspase inhibitor and alpha liquid are added and stored for 3 days in the second embodiment of the application, wherein a, b and c are respectively three different fluorescence channel observation results;
FIG. 9: the cell capture staining fluorescence detection result is obtained after the sample directly stored in the second embodiment of the application is stored for 3 days, and a, b and c are respectively three different fluorescence channel observation results;
FIG. 10: is the sequencing analysis result after 3 days of adding Caspase inhibitor and solution of Arabic in the second embodiment of the application;
FIG. 11: is the result of sequencing analysis after 3 days of storage of the sample directly stored in the second application example.
Detailed DescriptionThe ashwagen solution is a blood sample preservation solution which is relatively conventionally used at present, but when used for preserving a pregnant woman peripheral blood sample, the ashwagen solution does not have ideal effect on preserving rare cells in the pregnant woman peripheral blood, particularly nucleated red blood cells for non-invasive prenatal diagnosis. Therefore, the application is improved on the basis of the Ashi solution, and the novel preservation solution consisting of the Ashi solution and the apoptosis inhibitor is provided, so that the preservation solution can provide nutrients for cells, the cells can keep the shapes and various physical and chemical properties of the cells as much as possible, the apoptosis can be effectively inhibited, the samples can be kept fresh, and the damage to rare cells in the peripheral blood of pregnant women, particularly nucleated red blood cells, is reduced.
The storage temperatures of the ashwagen's solution and the apoptosis inhibitor, particularly the Caspase inhibitor preferred in the present application, are completely different from each other, and the physical forms of the ashwagen's solution and the apoptosis inhibitor are also different from each other at the respective storage temperatures; the pregnant woman peripheral blood preservation solution and the preparation method creatively combine the two to form the preservation solution for the pregnant woman peripheral blood, have a good preservation effect on rare cells in the pregnant woman peripheral blood, particularly nucleated red blood cells, and provide guarantee for non-invasive prenatal diagnosis.
On the basis of the preservation solution, the preservation method for the peripheral blood sample of the pregnant woman is provided, the preservation method for the peripheral blood sample of the pregnant woman is firstly to directly add the preservation solution into the freshly extracted peripheral blood of the pregnant woman, and the second method is to add the apoptosis inhibitor into the freshly extracted peripheral blood of the pregnant woman and then add the aldrin solution. In any preservation method, the basic principle is to preserve the peripheral blood of the pregnant woman by using the preservation solution. Only the second method is more convenient for transportation and field treatment; it should be noted that, although the apoptosis inhibitor has a protective effect during the period of time after the pregnant woman peripheral blood is transported to a laboratory and then the ashlar solution is added, the period of time is still not too long, the apoptosis inhibitor only temporarily keeps the sample fresh, and finally needs to act together with the ashlar solution, so that rare cells in the pregnant woman peripheral blood can be effectively protected; therefore, under the condition that the conditions allow, the preservation solution is directly added into the peripheral blood of the pregnant woman, and the effect is the best.
The present application is described in further detail below with reference to specific embodiments and the attached drawings. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.
Example one
This example was a comparative experiment on the added components of a fresh blood sample. A fresh tube of pregnant woman peripheral blood is taken firstly to directly carry out density gradient centrifugation and observe the centrifugation effect and the cell state as reference. Then set, test group a: fresh peripheral blood of pregnant women without any addition. Test group b: fresh pregnant woman peripheral blood is added with the Ashi solution, the volume ratio of the pregnant woman peripheral blood to the Ashi solution is that the pregnant woman peripheral blood: aldrin liquid =1: 15. Test group c: adding a Caspase inhibitor into fresh peripheral blood of the pregnant woman, wherein the volume ratio of the peripheral blood of the pregnant woman to the Caspase inhibitor is as follows: caspase inhibitor =1: 0.1. test group d: fresh pregnant woman peripheral blood is added with preservation solution consisting of alpha liquid and Caspase inhibitor, and the volume ratio of the pregnant woman peripheral blood to the preservation solution is that the pregnant woman peripheral blood: preservation solution =1:5, aldrin solution in preservation solution: the volume ratio of the apoptosis inhibitor is 15: 0.1. The four experimental groups were mixed and left to stand overnight in a refrigerator at 4 c, centrifuged with density gradient the next day and the centrifugation effect and cell status were observed and compared with the results of the fresh samples observed on the first day as a reference. The Caspase inhibitor in this example is a commercially available Z-VAD-FMKCcaspase inhibitor, and the other reagents or devices are conventional reagents and devices in the laboratory. In this example, two dyes, FDA + Hochest, were used to observe the surface and nucleus of cells.
The results showed that hemolysis occurred in experimental group a and the cells were coagulated into clumps. The experiment group b is normal in centrifugation, and cells after FDA staining have no fluorescence, which indicates that the cells are apoptotic. Experimental group c was slightly hemolyzed with clumping. Experimental group d was centrifuged normally and stained normally with essentially no difference from the fresh sample observed on the first day as a reference. Moreover, in test group a without any protective agent added, only 2 to 3 nucleated erythrocytes could be found; in test group d with the addition of aphaki and the inhibitor, an equivalent amount of the blood sample found about 30 nucleated red blood cells, as in the fresh blood; the other two groups found nucleated red blood cells were between test group a and test group d.
In addition, in this example, the amounts of a Caspase inhibitor and a D-alpha inhibitor were optimized on the basis of the test group, and the results showed that the ratio of peripheral blood of pregnant women: the volume ratio of the apoptosis inhibitor is 1-5:0.03-0.1, the ratio of the peripheral blood of the pregnant woman: the volume ratio of the Ashi solution is 1-5:5-15, the blood sample can be well protected, and the observation effect is equivalent to that of the test group d. In addition, different commercially available Caspase inhibitors were tested and the results were comparable to test group d using the Z-VAD-FMKCaspase inhibitor.
Example two
Two tubes of fresh pregnant woman peripheral blood are extracted from a hospital, a Caspase inhibitor is added into one tube and evenly mixed, and then the mixture is stably transported to a laboratory at a low temperature, wherein the volume ratio of the pregnant woman peripheral blood to the Caspase inhibitor is as follows: caspase inhibitors = 1-0.1. After being transported to a laboratory, the blood is measured according to the proportion of the peripheral blood of the pregnant woman: adding peripheral blood of pregnant woman into the Ashi solution at volume ratio of Ashi solution =1:3, and storing in a refrigerator at 4 deg.C. And observing the morphological integrity and the FDA + Hochest fluorescence detection cell viability of the cells in the blood sample after the blood sample is stored for 1 day, 2 days, 3 days, 5 days and 7 days, respectively, performing gradient centrifugation, and observing the centrifugation effect.
And directly storing the peripheral blood of the other tube of pregnant woman in a refrigerator at 4 ℃ for 3 days, comparing the sample with a sample which is stored for 3 days after adding the alpha liquid and the Caspase inhibitor, and respectively performing microfluidic capture and whole genome sequencing.
The results of microscopic observation and fluorescence detection are shown in fig. 1-7, and the results prove that the morphology integrity and cell viability of the samples after being stored for 1 day, 2 days, 3 days, 5 days and 7 days are more than 95% of the morphology integrity and cell viability of the cells in the fresh samples. As shown, the results of the samples stored for 1 day and 2 days are shown in fig. 2 and 3, and no cellular abnormality was found in fig. 1 compared with the fresh samples; and the sample stored for 3 days was found to have a cell abnormality, the portion shown by the circle in FIG. 4; samples were stored for 5 days and two cellular abnormalities were found, the circled portion in FIG. 5; the samples stored for 7 days were counted on a counting plate 4X4 grid and showed that 50 cells were observed in the bright field and 48 FDA stained cells, which is a cell viability of 96%.
The fluorescence effect of microfluidic capture staining is shown in fig. 8 and 9, and the results of sequencing analysis are shown in fig. 10 and 11. The results show that the cell morphology of the cells with the inhibitor and the aldrin solution is intact and the fluorescence is bright, fig. 8, while the blood samples stored directly without any preservation solution have rough cell morphology and dull fluorescence, fig. 9. The sequencing result shows that the chromosome sequencing result of the sample cell is uniform after the aldrin liquid and the apoptosis inhibitor are added, and the pregnant woman is successfully judged to be pregnant with a T18 male fetus, and the result is consistent with the DNA sequencing result of the aborted tissue, as shown in FIG. 10; the samples without the addition of the aldrin and the apoptosis inhibitor, i.e., the directly stored blood samples, had heterogeneous cell chromosome sequencing results, and many chromosome abnormalities failed to diagnose the chromosomal disease, as shown in fig. 11.
The results prove that: no matter the number, the form and the dyeing effect of the captured target cells, the sample added with the aldrin liquid and the apoptosis inhibitor is superior to the blood sample which is directly stored without treatment, and the data uniformity obtained by adding the aldrin liquid and the apoptosis inhibitor is better from the whole genome sequencing data, and the good uniformity is the premise of diagnosing chromosome and gene diseases, so the method for storing the peripheral blood of the pregnant woman effectively stores the characteristics of the cells to ensure that the pregnant woman obtains an ideal data result, and the sample added with the two reagents ensures that the sick children of T18 trisomy be successfully detected, and the blood sample directly stored cannot be judged without adding any storage liquid.
EXAMPLE III
In addition to example two, the solution of Arundina and Caspase inhibitor were prepared into a preservative solution for testing. Specifically, 1, preparing a preservative solution in a laboratory: 100ul Caspase apoptosis inhibitor is added into 15ml of the Alzheimer's disease solution, the final concentration is 0.67 percent, the mixture is evenly mixed, a 50ml centrifuge tube is stored at the temperature of 80 ℃, and the mixture is taken out and melted to the room temperature before sampling. 2. Carrying a 50ml storage liquid tube to a hospital at normal temperature, extracting a peripheral blood sample of the pregnant woman, using a 5ml heparin sodium tube after 10-20 weeks of pregnancy, shaking up, and immediately transferring to the 50ml storage liquid tube. 3. The samples were quickly transported from the hospital to the laboratory in a cold specimen presentation box and stored in a refrigerator at 4 ℃. The rest of the arrangement is the same as the embodiment.
The results show that the morphological integrity and the cell viability of the sample added with the preservation solution after being preserved for 7 days are 98% of the cell viability and the detection result is not shown.
In the contrast design, a sample added with preservation solution is preserved for 3 days, and the microfluidic capture staining fluorescence detection result shows that the cell shape is complete and the fluorescence is bright; and the sample without the preservation solution is directly preserved for 3 days, the cell morphology is rough, the fluorescence is dark, and the detection result is slightly shown. Similarly, the sequencing results were the same as in the examples.
The results confirmed that the peripheral blood sample of the pregnant woman could be well preserved both in the method of adding the inhibitor and the aphakic acid in example two and in the method of preparing the inhibitor and the aphakic acid into the preservation solution in example three. In addition, although the method of example two, in which the inhibitor and the aldrin solution are added separately, may be disadvantageous for sample storage, the method of example two and example three achieve almost the same test results, and we analyzed that this is probably because the time taken for the sample to be transported to the laboratory is relatively short, that is, although only the inhibitor is added after the sample is taken in example two, blood cells can be effectively protected in a short time, and the effect of both is equivalent to that of example three in which the inhibitor and aldrin solution are directly added.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. For those skilled in the art to which the present application pertains, several simple deductions or substitutions may be made without departing from the concept of the present application, and all should be considered as belonging to the protection scope of the present application.