| Isolation and purification of leukocytes from whole blood is a basic method in many biological and medical applications.It has been widely used in many aspects,including the early diagnosis of cytomegalovirus infection in bone marrow transplantation(BMT)and the diagnosis of leukemia immunotyping.For example,by sorting and enriching Leukemia and normal people’s peripheral blood leukocytes,real-time quantitative PCR(RT-q PCR)was used to analyze lnc.The expression level of RNAs and the correlation between RNAs and clinical indexes are very important in the immunophenotyping of leukemia.In the study of acute lymphocytic leukemia,it is indicated that the blood transfusion scheme with leukocyte removal can reduce the non-hemolytic fever reaction,improve the immune function of patients,and improve the therapeutic effect of acute lymphocytic leukemia.In the clinical treatment of autoimmune hemolytic anemia(AHA),the most effective way to treat the patients with severe anemia is to separate white blood cells from the whole blood,and 90%white blood cells from the whole blood is conducive to reduce the allergic reaction and other transfusion related adverse reactions.Studies have shown that cells are commonly known as leukocytes.The number of leukocyte cells in normal human body(4.0～10.0×10~9/L),but the number of leukocyte cells in human peripheral blood cells is one thousandth of the number of red blood cells.At present,the methods of leukocyte separation and enrichment reported at home and abroad are:flow cytometry,Percoll continuous density gradient centrifugation,filtration,immunomagnetic beads adsorption,physical centrifugation,immunomagnetic beads and red blood cell lysis buffer,etc.The feasibility of flow cytometry in leukocyte classification and counting has been widely confirmed,especially in immature cells and new cell subsets.It has been widely used in many diseases,but flow cytometry also has its limitations.For example,if there are some differences between different antibody combinations in leukocyte recognition and disease judgment,it is also the key to quality management to realize the automation and standardization of this method.In addition,commercial multi antibody reagents are gradually coming out,and there are still some constraints such as expensive reagents,complex operation,large cell damage,design of analysis software and automatic door setting.Percoll density gradient centrifugation is a simple and fast method with low separation cost and good collection effect of target cells.At present,this method is widely used in experimental research.It uses hydroxyethyl starch,gelatin,methylcellulose and other precipitated red blood cells,and then uses density gradient centrifugation to separate mononuclear cells.Good results have been obtained,but the cells obtained by this method have poor growth status Disadvantages,affecting cell activity.The method of immunomagnetic beads is based on the combination of cell surface antigen and specific monoclonal antibody connected with magnetic beads.In the external magnetic field,the cells connected with magnetic beads by antibody are adsorbed and detained in the magnetic field,so that the cells can be separated.However,the price of immunomagnetic beads is relatively expensive,which limits its clinical application.Leukocyte filtration is the most simple cell analysis method at present.The principle of microstructure filtration method is simple.It can design different scale microstructure in the channel,realize the rapid separation of red blood cells and white blood cells,and separate platelets and red blood cells with different sizes of plasma or particles from other blood cell components such as white blood cells.This separation effect is easily blocked by a large number of red blood cells,and the separation efficiency is low.Red blood cell lysis buffer is one of the most convenient methods to remove red blood cells,that is,to use lysate to lyse red blood cells,it can’t damage leukocytes and remove red blood cells fully.Lysis buffer is a relatively mild method of red blood cell removal.NH4Cl in lysate will cause red blood cell deformation,biological channel expansion,expansion and cracking until it causes red blood cell rupture without attacking other cells,so as to achieve the separation and purification of white blood cells in blood cells.However,it will lead to unnecessary activation of white blood cell surface proteins and cracking of a small number of target cells.Centrifugal blood cell separation technology is the most widely used in clinical and blood.The basic principle of centrifugal blood cell separation technology is based on the different size and density of blood cells.Under certain centrifugal separation conditions,the sedimentation rate of cells is also different.From the top to the bottom are plasma layer,buffy coat layer and erythrocyte layer.The main components of buffy coat layer are platelets lymphocyte,monocyte and granulocyte can be isolated by this method,but cell purity and cell concentration are low.Although many of the reported methods can achieve a high standard of cell isolation,they only aim at the isolation of a single type of leukocyte in the whole blood.The emergence of 3D printing technology provides a new way to solve this problem.In recent years,3D printing technology,as one of the representative technologies of the third industrial revolution,is widely developed in the world due to its advantages of integrated rapid prototyping,high-precision entity replication,complex structure and relatively low cost.Biomedical micro-nano devices processed by 3D printing technology are also widely used in cell biology,medical detection,gene diagnosis,etc.Biomedical micro-nano devices based on 3D printing have the incomparable flexibility of traditional micromachining technology.Generally,the whole design and processing process can be completed in a very short time.In addition,many kinds of 3D printing raw materials have biocompatibility to meet the research and application needs of life science and medicine.At the same time,in order to prevent cross contamination,biomedical micro-nano devices used in medical diagnosis and other fields are all disposable,and the manufacturing of biomedical micro-nano devices through 3D printing technology significantly reduces the cost,which has a very positive significance for the application and promotion of medical diagnosis technology based on biomedical micro-nano devices.In addition,3D printing technology has been widely used in various fields of medicine,such as medical props,models,supplies,etc.,such as copying human bones,making artificial limbs,medical equipment design,etc.Shallan et al.used a concentration gradient generator made of stereolithography.After many times of separation and mixing,the concentration gradient was evenly distributed along the vertical direction of the main channel.In this study,the stereolithography technology of 3D printing realized one-time forming,with very low cost,high precision and the whole processing only needed 4 hours.Although3D printing technology has been widely used in the field of medicine,it has not been reported that 3D printing technology is used to process biomedical micro-nano devices for sorting and enriching human peripheral blood cells.Objective:The purpose of this study is to independently develop a biomedical micro-nano device that can effectively separate white blood cells by 3D printing technology,and constantly optimize the experimental conditions of white blood cell separation and enrichment,so as to achieve high recovery rate of white blood cells,no need to label target cells,no chemical reagent pollution samples,short time,simple operation,no impact on the shape,function and activity of target cells.In order to be able to diagnose,predict the curative effect and judge the prognosis in the early stage of clinical research,and design the treatment plan based on it,so as to improve the clinical curative effect,which has great influence and clinical value for the credibility of the follow-up research results.Method:1.To screen the special internal structure of three kinds of leukocyte sorting accumulator(LSA)devices,and to establish a new idea of leukocyte sorting and enriching methodology to preliminarily determine the best structure of LSA device;2.On the basis of the above research,we studied the influence of the experimental operation and other factors in the pushing style and pulling style methods on the whole leukocytes enrichment effect for the LSA-3.The main factors include the location of the buffy coat layer in the upper funnel chamber(0～1/3 and 1/3～2/3)of the LSA-3and the second centrifugation time(1min,2min,3min,4min,5min,10 min,respectively),and finally establish the methodology system of leukocyte separation and enrichment3.According to the established methodology system of leukocyte enrichment,using LSA-3,using pushing style and pulling style methods to enrich leukocytes in 51clinical blood samples,further verifying the stability and applicability of the methodology system of leukocyte enrichment.Results:1.Three kinds of LSA devices with different internal structures,LSA～1,LSA-2and LSA～3,were independently developed.The average recovery of leukocytes was44.8±15.1%(P=0.034),64.7±13.5%(P=0.029),84.7±5.3%(P=0.019),respectively.The results showed that the efficiency of the three kinds of leukocyte sorting and enrichment devices was significantly different from high to low,which was LSA～3>LSA～2>LSA～1;2.On the basis of the above research,when the buffy coat layer is located in the0～1/3 position of the upper funnel cavity of the LSA～3,the average recovery rate of leukocytes is 85±4.9%(P=0.009)and 86.5±2.89%(P=0.007)respectively by using pushing style and pulling style methods,Similarly,when the buffy coat layer is located in the 1/3～2/3 position of the upper funnel chamber of LSA～3,the average recovery of leukocyte is 96.20±2.38%(P=0.005)and 94.40±0.8%(P=0.003),respectively.The results showed that the average recovery rate of leukocytes in the1/3～2/3 position of the upper funnel chamber of LSA-3 was significantly higher than that in the 0～1/3 position;3.Based on the above research,we continue to explore the influence of the second centrifugation time on the efficiency of leukocyte sorting.The results show that the second centrifugation time gradient(1 min,2 min,3 min,4 min,5 min,10 min)using LSA-3,using pushing style and pulling style methods,The results showed that the average recovery rate of leukocytes was 94.4±1.1%,96.2±2.4%,95±0.02%,95.5±0.01%,93.5±0.02%,96.7±1%(P=0.175,P=0.415,P=0.267,P=0.258,P=0.163,respectively),.There was no significant difference.The average recovery of leukocytes in the pulling style method was 89.2±1.3%,94.4±0.8%,91.1±3.14%,94.9±1.2%,92.3±2.9%,95.0±3.5%(P=0.061,P=0.276,P=0.377,P=0.167,P=0.423),respectively,and there was no significant difference.The results show that the second centrifugation time has no effect on the enrichment of leukocytes by the pulling style method.Considering the whole time of leukocyte enrichment,the second centrifugation time is set as 2 minutes;4.There was no significant difference(P>0.05)in the recovery rate of leukocytes and the removal rate of erythrocytes between the two methods(pushing style and pulling style method).The results showed that the two methods did not affect the efficiency of leukocyte separation and enrichment;5.The target cells obtained by red blood cell lysis buffer and LSA-3 pushing style method were separated and enriched respectively,and the morphological changes of the target cells obtained by the two methods were compared with each other by staining with Wright-Giemsa solution,The results showed that the morphology and structure of the leukocytes collected by pushing style method were complete,and no fragments of erythrocytes and broken leukocytes were found.The cytoplasm of the leukocytes collected by the method of red blood cell lysis shrinks,which is rich in red blood cell fragments and broken leukocytes cells fragments.The results showed that the shape and structure of leukocytes collected by LSA-3 pushing style method was better than that of lysis buffer method.6.The method system of leukocyte separation and enrichment was established.The methods of pushing style and pulling style were used to separate and enrich leukocytes in 51 cases of clinical blood samples.The results showed that the average recovery rate of leukocytes and the average removal rate of erythrocyte were 95±2.8%and 93±2.7%,respectively.The results showed that the average recovery rate of leukocytes and the average removal rate of erythrocytes were 94±2.5%and 91±2.9%,respectively.The results show that it is stable and applicable to separate and enrich leukocytes by using the leukocyte sorting and enriching device 3.0 and using pushing style and pulling style methods.In this study,the LSA device independently developed and designed by 3D printing technology,from a new point of view,to find another way to solve the shortcomings of the current clinical sorting of leukocytes.The LSA-3 realizes the unmarked sorting and enrichment of leukocytes in peripheral blood,and obtains the characteristics of high recovery rate,no chemical reagent pollution samples,short time,simple operation,good target cell morphology and cell activity after separation.Moreover,it effectively solves the problem that red blood cells and granulocytes are not easy to separate in the whole leukocytes extraction process.With the advantages of one-time molding,low cost,high precision and short processing cycle,the LSA device can provide a better treatment plan for the early diagnosis of cytomegalovirus infection in bone marrow transplant patients and the diagnosis of leukopenia immune typing,and has a wide range of applications. |
PDF Full Text Request