应用于白血病免疫分型芯片的外周血白细胞分离方法的研究

发布时间：2018-05-22 14:14

本文选题：白细胞 + 全白细胞分离液　；参考：《中国医科大学》2007年硕士论文

【摘要】： 目的从全血中分离和纯化白细胞是许多生物学和医学应用中的基本方法，它广泛被应用于包括骨髓移植患者巨细胞病毒感染的早期诊断和白血病的免疫分型的诊断等许多方面。众所周知，白细胞分离是许多生物学实验的第一步，也是至关重要的一步，因为后续的研究成果的可信度很大程度上取决于分离的白细胞活性与纯度。因此对白细胞分离方法的研究成为当血液学一个颇为热点的研究课题。目前国内外报道的分离白细胞的方法很多，包括密度梯度离心法、物理吸附法、细胞电泳法、免疫磁珠法、流式细胞法等，其中有的分离方法所提取的细胞纯度和细胞浓度已经可以达到很高的标准。但这些方法只适用于单种白细胞或单个白细胞亚群的分离，而本实验研究的主要目的是为了寻找一种适用于白血病免疫分型芯片的细胞分离方法，它的最终结果是要将外周血中的全白细胞分离提取出来。为了分离、提取形态及功能完整的全白细胞，本实验在既往细胞分离方法的基础上作了改进，根据等密度梯度离心原理，通过改变细胞分离液比重及渗透压自制了一种全白细胞分离液，经过我们反复对全白细胞分离液条件探索以期达到一次性分离全白细胞的目的。方法 1、全白细胞分离液的配制右旋糖苷(或聚蔗糖)和泛影葡胺按不同比例混合配制成1.108g/ml、1.110g/ml、1.112g/ml、1.113g/ml四种比重分离液，并且在不同比重的分离液中分别加入五种不同浓度的氯化钠来调节分离液渗透压。 2、全白细胞分离液最佳比重及最佳渗透压探索取50例正常外周血标本，采用四种比重五种渗透压全白细胞分离液，比较分离效果，筛选分离液的最佳渗透压和最佳比重。细胞分离效果测定参数为自细胞回收率、红细胞去除率和各种白细胞比例三方面。 3、两种全白细胞分离液比较取20例正常外周血标本，，通过我们配制的两种全白细胞分离液(Dextran-全白细胞分离液和Ficoll-全白细胞分离液)对白细胞分离效果进行比较，筛选最佳全白细胞分离液。细胞分离效果比较参数为细胞浓度，细胞纯度，细胞活性。 4、全白细胞分离液适用的芯片上固定抗体浓度确定将原始浓度为0.2mg/ml的单克隆抗体按比例1：2；1：4；1：8；1：16稀释，用MicroGridⅡ芯片点样仪将其点在氨基硅烷—醛基修饰的玻片上，制备单克隆微阵列；然后用全白细胞分离液分离提取白细胞，制备自细胞悬液；经过吖叮橙染色后孵育芯片；通过扫描仪扫描及显微镜观察结果比较筛选芯片上固定抗体浓度。 5、全白细胞分离液对正常血细胞的分离及芯片对细胞分离结果的检测取临床50例正常外周血标本，首先用全白细胞分离液进行白细胞分离，芯片孵育及细胞染色，然后通过对显微镜下各抗体点捕获细胞形态观察和数量统计，检测全白细胞分离液分离白细胞效果。6、全白细胞分离液对白血病细胞的分离及其在白血病免疫分型芯片的应用通过全白细胞分离液对10例白血病标本的分离及芯片对白血病细胞捕获，检测全白细胞分离液对肿瘤细胞分离效果。结果 1、全白细胞分离液中以比重为1.112g/ml、渗透压为520m0sm/l左右分离液分离细胞效果最好。白细胞回收率及红细胞去除率最高，分离所获得的白细胞组分全，各种白细胞的比例正常。 2、Ficoll全白细胞分离液分离提取细胞的效果好于Dextran全白细胞分离液：两种分离液分离的细胞浓度及细胞活性无明显差异，但Ficoll全白细胞分离液分离的细胞纯度高于Dextran全白细胞分离液。 3、全白细胞分离液适用的芯片上固定最佳抗体稀释比例为1：4，即固定的抗体浓度为50μg/ml。此点样条件的确定不仅使芯片捕获细胞的密度大，捕获细胞的特异性好，而且保证了芯片对全白细胞分离液检测结果的准确性。 4、芯片对全白细胞分离液分离外周血白细胞检测结果：取50例正常外周血标本，用全白细胞分离液分离，再用白血病免疫分型细胞芯片捕获细胞后，统计分析。在芯片上可见CD3、CD4、CD7、CD8、CD19、CD20、CD21、CD22八个抗体点上捕获的细胞90％以上为淋巴细胞，CD13、CD14、CD15、CD33四个抗体点上捕获的细胞90％以上为粒细胞，细胞的形态正常。CD4、CD15、CD19三个抗体点上捕获的细胞做免疫细胞化学染色，可见CD4、CD15、CD19阳性。 5、利用全白细胞分离液对10例白血病标本的分离，白血病免疫分型芯片成功地捕获到白血病细胞。急性T淋巴细胞白血病(T-ALL)4例，在CD4、CD7抗体点上捕获到幼稚的淋巴细胞，急性B淋巴细胞白血病(B-ALL)3例，在CD19、CD22点上可见幼稚的淋巴细胞，急性髓系白血病(AML)3例，在CD13、CD33、CD15抗体点上捕获到幼稚的粒细胞。通过白血病免疫分型芯片对白血病诊断的结果与临床相符合。结论本实验自制的全白细胞分离液成功地分离全白细胞。所获得的细胞浓度及细胞纯度高，细胞形态及细胞活性保持良好，细胞组分全，细胞比例正常，不仅解决了白细胞提取过程中红细胞和粒细胞不易分离的难题，而且也最大限度的减少了红细胞污染，完全符合白血病免疫分型芯片对细胞标本的要求。为许多生物学和医学的应用提供了一种有效的细胞分离方法。
[Abstract]:objective
The separation and purification of white blood cells from the whole blood is a basic method in many biological and medical applications. It is widely used in many aspects, including early diagnosis of cytomegalovirus infection in bone marrow transplant patients and diagnosis of immunophenotyping of leukemia. It is well known that leukocyte separation is the first step of many biological experiments. An important step, because the reliability of subsequent research results depends largely on the separation of leukocyte activity and purity. Therefore, the study of leukocyte isolation has become a hot research topic in hematology.
There are many methods to separate white cells at home and abroad, including density gradient centrifugation, physical adsorption, cell electrophoresis, immunomagnetic beads and flow cytometry, among which the purity and cell concentration of some separation methods can reach very high standard. But these methods are only suitable for single white cells or single cells. The main purpose of this study is to find a cell separation method suitable for leukemic immunophenotyping chips. The final result is to isolate and extract all white blood cells from peripheral blood.
In order to isolate and extract complete white cells with complete morphology and function, this experiment was improved on the basis of previous cell separation methods. According to the principle of ISO density gradient centrifugation, a kind of full white cell separation liquid was made by changing the proportion of cell separation fluid and osmotic pressure. After repeated exploration of the conditions of the whole white cell separation solution, we could reach a period of time. The purpose of one-off separation of all leukocytes.
Method
1, the preparation of the total leukocyte separation solution
Dextran (or polysucrose) and meglumine are mixed into four kinds of specific gravity separation liquid, 1.108g/ml, 1.110g/ml, 1.112g/ml, 1.113g/ml, and five different concentrations of sodium chloride are added to the separation liquid of different specific gravity to regulate the osmotic pressure of the separation liquid.
2, the best proportion and optimal osmotic pressure of total leukocyte separation fluid.
50 normal peripheral blood samples were taken, and four specific gravity and five osmotic total leukocyte separation fluid were used to compare the separation effect. The optimum osmotic pressure and optimum specific gravity were screened. The parameters of cell separation effect were AutoCyte recovery rate, red cell removal rate and all kinds of white blood cell ratio in three aspects.
Comparison of 3, two total white blood cell separation fluid
In 20 normal peripheral blood samples, the white cell separation effect was compared through the two total leukocyte separation liquid (Dextran- total leukocyte separation solution and Ficoll- total white cell separation solution), and the optimum total white cell separation solution was screened. The cell separation effect was compared with the cell concentration, cell purity and cell activity.
4, the concentration of antibody immobilized on the whole white cell separation solution is determined.
The monoclonal antibodies with the original concentration of 0.2mg/ml were proportionately 1:2; 1:4; 1:8; diluted at 1:16. The monoclonal microarray was prepared on the glass slides modified by the amino silane aldehyde group by the MicroGrid II chip. Then the white cell suspension was separated and extracted with the total white cell separation solution to prepare the self cell suspension. After staining with acridine orange, it was stained with acridine orange. The chip was incubated, and the concentration of immobilized antibody on the chip was screened by scanning and microscopic observation.
5, separation of normal blood cells by whole leukocyte separation fluid and detection of cell separation results by chips.
In 50 normal peripheral blood samples, white cell separation was first used for leukocyte separation, microchip incubation and cell staining. Then, by observing and counting the cell morphology of the antibody points under the microscope, the leukocyte effect was detected by the total white cell separation solution.6, and the leukocyte separation solution was separated to the leukemia cells. Its application in leukemic immunophenotyping
Isolation of 10 leukemia samples and capture of leukemic cells by leukocyte isolation fluid were used to detect the effect of whole leukocyte separation fluid on tumor cells.
Result
1, the proportion of the total white cell separation liquid was 1.112g/ml and the osmotic pressure of 520m0sm/l separation liquid was the best. The rate of leucocyte recovery and the rate of red cell removal was the highest, the white cell components obtained by separation were all, and the proportion of all kinds of white blood cells was normal.
2, the effect of Ficoll total leucocyte separation solution was better than that of Dextran total white cell separation solution. There was no significant difference in cell concentration and cell activity separated by two kinds of separation solution, but the purity of cell separated by Ficoll total white cell separation solution was higher than that of Dextran total white cell separation solution.
3, the optimal antibody dilution ratio of the full white cell separation solution is 1:4, that is, the fixed antibody concentration is 50 mu g/ml., which not only makes the chip capture cell density, the specificity of the capture cell is good, but also ensures the accuracy of the detection results of the whole white cell separation solution.
4, the microchip was used to separate the peripheral blood white blood cells from the total leukocyte separation solution. 50 cases of normal peripheral blood samples were collected with full white cell separation solution, and then the leukemia immunophenotyping cell chip was used to capture cells. On the chip, CD3, CD4, CD7, CD8, CD19, CD20, CD21, CD22 were found to capture more than 90% cells on the eight antibody points. More than 90% of the cells captured on the four antibody points of the lymphocyte, CD13, CD14, CD15, and CD33 were granulocytes. The cell morphology was normal.CD4, CD15, and CD19 three antibody points were stained by immunocytochemical staining, and CD4, CD15, CD19 positive.
5, the leukemic cells were isolated from 10 leukemic specimens with full leucocyte separation solution. Leukemia cells were successfully captured by leukemic immunophenotyping chips. 4 cases of acute T lymphocytic leukemia (T-ALL) were captured at CD4, CD7 antibody points and 3 cases of acute B lymphocyte leukaemia (B-ALL). The immature lymphoid lymphatic cells were observed at the CD19 and CD22 points. In 3 cases of acute myeloid leukemia (AML), naive granulocytes were captured at the CD13, CD33, and CD15 antibody points. The results of leukemia diagnosis by the leukemia immunophenotyping chip were in accordance with the clinic.
conclusion
The total white cell separation liquid was successfully separated. The cell concentration and cell purity were high, the cell morphology and cell activity remained well, the cell components were complete, and the proportion of cells was normal. It not only solved the difficult problem of the separation of red cells and granulocytes in the process of leukocyte extraction, but also the maximum decrease. The red cell pollution is fully in line with the requirements of the leukemic immunophenotyping chip for the cell specimens. It provides an effective method of cell separation for many biological and medical applications.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

【参考文献】