Research On Isolation Of Peripheral Blood Leukocytes Applying To Immunophenotyping For Leukemia

ObjectiveIsolation and purification of human leukocytes subpopulations from whole blood are fundamental in many biological and medical applications. It is widely applied to diagnosing human cytomegalovirus infection in bone marrow transplant patients, Immunophenotyping for Leukemia and many other areas. As known, white blood cell separation is the first and critical step in many biological experiments, because credibility of following research results to a large extent depends on the cell viability and purity. Therefore, the research of separating WBC becomes hot topic.It has been reported at home and abroad that there are many ways to separate human leukocytes now, including density gradient centrifugation, physical adsorption, cell electrophoresis and flow cytometry methods. Some methods of WBC separation can achieve at a very high level of cell purity and concentration. However, these methods were only applied to separation of single subsets of leukocytes. The main purpose of this experiment is to find a new cell separation method which is Applied to Immunophenotyping for leukemia. The final result is that all of leukocyte should be extracted from the peripheral blood.To isolate morphological and functionally intact human leukocyte subpopulations, this study had improved the conventional separation methods. The whole WBC separation liquid prepared by changing osmotic pressure and specific gravity of separation liquid, According to the principle of equal density gradient centrifugation. After repeated experiment to explore the conditions of the whole WBC separation liquid, we can separate all of the leukocytes at one time.Methods1. Preparation of Dextran-hypaque and Ficoll-hypaque Dextran and Hypaque or Ficoll and Hypaque were mixed in different proportions to the gravity of 1.080g/ml,1.110g/ml,1.112g/ml,1.113g/ml .Various amount of sodium chloride was added into separation liquid to regulate the osmotic pressure.2. Groping for the best osmotic pressure and specific. gravity of separation liquidTake 50 normal peripheral blood samples. By comparing cell isolation effects of separation liquid select the best gravity and osmotic pressure of separation liquid. Parameters for effects of cell separation are cell concentration, purity and viability.3. Comparison of two separating liquidTake 20 normal peripheral blood specimens. By comparing cell separation effects of two separation liquid (Dextran-hypaque and Ficoll-hypaque) determine the best separation liquid. Parameters for effects of cell separation are cell concentration, purity and viability.4. Determination of antibody concentration which be fixed on the chipTo the concentration of 1.0mg/ml monoclonal antibodies diluted in proportion 1:2; 1:4; 1:8; 1:16, which were fixed on the slide to form cytochip. Then the whole WBC which were dyed with fluorescence were incubated on cytochip. After detected by MicroGrid II and observed by Microscope, we choose the best concentration of antibodies.5. The whole WBC separation liquid applying to Immunophenotyping for LeukemiaTake 50 normal peripheral blood specimens. First, the blood specimens were separated by the whole WBC separation liquid, and then the WBC were incubated on the chip. Finally by observing cell morphology and statistics cell number, we detected isolation effects of the WBC separation liquid.6. Leukemia blood cell separationTake 10 leukemia peripheral blood specimens, through separating leukemia cells by the whole WBC separation liquid and capturing leukemia cell on the chip, we detected the effects of the separation of leukemia cells.Results1. The whole WBC separation liquid with density of 1.112g/ml and osmolarity of 520m0sm/l was the best results. The cell concentration was the highest. The whole cell components were fully consistent with the proportion of the normal peripheral blood .2. By comparing the Ficoll-Hypaque and Dextran-Hypaque,there was no significant difference in the concentration of cells, but in cell purity Ficoll-Hypaque was better than Dextran-Hypaque.3. The Best antibody concentration was 50μg/ml, Not only the cell density but also the specific of cell which were captured by chip were good.4.The chip test results : 50 cases of isolated peripheral blood specimens statistics, it showed 90% cells were captured on the CD3, CD4, CD7, CD8, CD19, CD20, CD21, CD22 were lymphocytes, while most of cells were captured on the CD13, CD14, CD15 were granulocytes, The cells were captured on the CD4, CD15, CD19 were positive by using immunohistochemical staining.5. After separated by the whole WBC separation liquid. Leukemia cells were successful capture on the Cytochip which applied to Immunophenotyping for Leukemia. T-ALL in four cases, the naive lymphocytes were capture on the CD4, CD7 antibody points, B-ALL in three cases., the naive lymphocytes can be seen on the CD19, CD22 point, AML in three cases, naive granulocyte were captured on the point of CD13, CD33, CD15. The results for Immunophenotyping of leukemia by cytochip are consistent with the result of clinical diagnosis.ConclusionThe whole WBC separation liquid which we had prepared could isolate the whole leukocytes successfully, the cells concentration and purity were very high, the cell shape and viability maintained well, The whole cell components were fully consistent with the proportion of the peripheral blood .we had not only solved the problem that it was difficult to isolate erythrocytes and granulocytes, but also minimized the contamination of RBC. The whole WBC separation liquid corresponded fully with chip demand. And the whole WBC separation liquid could also be used for isolation of leukemia cells. For many applications in biology and medicine, It is an effective method of cell separation.