Study Of Two-Dimensional Light Scattering Static Cytometry And Its Application For Leukemia Detection

Particles are related to environmental inspection,mechanical manufacture,food testing,pharmaceutical screening and so on.Size measurement of particles not only affects the quality of product,but also is closely associated with performance testing of instrument.Leukemia is one of the top ten malignant cancers with an increasing incidence and mortality rate.Early diagnosis and personalized therapies are crucial for significant reductions in leukemia mortality.Currently,the methods for the detection of leukemia in clinics are mostly based on cytochemical,immunophenotypic and cytogenetic features,and clinical characteristics of the leukemic cells.Moreover,these methods which depend on fluorochromes for labeling the cells and require intensive laboratory studies are complex-to-use,time-consuming.With the virtues of simplicity,high speed,excellent detection sensitivity,good repeatability and non-destruction,light scattering technique offers advantages when be applied to particle size measurement and biological cell detection.This dissertation aimed to probe deeply into the significance of 2D light scattering static cytometry in the particle size measurement and leukemic cell detection.After introduction of the 2D light scattering static cytometer,its performance was tested via measurements on standard microspheres.This static cytometer was used for size measurement of yeast cells and classification of normal and leukemic cells.Then,coupled with machine-learning technique,the improved wide-angle 2D light scattering static cytometer was applied to the automatic classification of acute and chronic leukemic cells.The main research achievements are listed as follows.(1)The factors influenced measurement precision of the 2D light scattering static cytometer were discussed.Scattered light in the polar angle range of 79° ≤ θ ≤ 101°and azimuthal angle range of 79°≤φ≤101° can be obtained by using this static cytometer,which contains structural information of individual scatterer.Modular design of this static cytometer was convenient for function expansion and maintenance of device.(2)Performance indexes of the 2D light scattering static cytometer were evaluated via experiment.The experimentally observed 2D patterns from 4.19 μm microspheres(mean diameter)agreed well with the simulated ones based on Mie theory.This validated the consistency and repeatability of the static cytometer.The resolution of the static cytometer was validated via size measurement on standard microspheres with mean diameters of 3.87 and 4,19 μm.The estimated sizes agreed well with the given size ranges.These results demonstrated that the 2D light scattering static cytometric technique may provide an easy-to-use,low-cost and label-free method for particle size measurement and biological cell analysis with submicron resolution.(3)Application of the 2D light scattering static cytometer to particle size measurement and leukemia screening was studied.The static cytometer collected the scattered light in the angular range from 79 to 101 degrees.Size measurement of yeast cells demonstrated that the 2D light scattering static cytometer has great potential in size measurement of biological particles.Two parameters,namely the number of speckles and their average area in each 2D pattern,were extracted from the 2D patterns of both normal and leukemic cells.Statistical analysis of these two parameters were used for the classification of different cells.The accuracy rate for the differentiation of acute myeloid leukemic cells HL-60 from normal granulocytes was 92.5%,and was 100%for the differentiation of chronic myeloid leukemic cells K562 from normal granulocytes by using the static cytometer.The results showed that the 2D light scattering static cytometer may provide a label-free method for leukemia screening in clinics.(4)An improved wide-angle 2D light scattering static cytometer was developed based on the improvement of both static liquid-based chip and 2D light scattering pattern recording module in the 79°-101° static cytometer.Scattered light in the polar angle range of 26° ≤θ≤154° and azimuthal angle range of 26° ≤φ≤154° can be obtained by using this wide-angle static cytometer.Good agreement between experimental and simulated 2D light scattering patterns from standard microspheres with mean diameters of 3.87 and 4.19 μm,validated the capability of the wide-angle cytometer accurately collecting 2D light scattering signals in the angular range both from 40 to 100 degrees and from 26 to 154 degrees.The wider scattering angular range was detected by the cytometer,the much richer information of the cells was obtained.The wide-angle static cytometer may provide a better resolution for cell analysis as compared with the 79°-101° static cytometer.(5)Application of the wide-angle 2D light scattering static cytometer to the classification of leukemia subtypes was studied.The 2D light scattering patterns from acute myeloid leukemic cells HL-60 and chronic myeloid leukemic cells K562 were obtained by the wide-angle static cytometer.The 2D patterns were obtained in the angular range from 26 to 154 degrees.Texture features such as contrast(CON),angular second moment(ASM)and entropy(ENT)were extracted from the wide-angle 2D patterns with gray level differential statistics(GLDS)method.Coupled with support vector machine(SVM),HL-60 cells and K562 cells were automatically identified with a sensitivity of 92%and a specificity of 95%by the wide-angle static cytometer.The method can be universally established to classification the other leukemia subtypes and detection the other cancer cells.The results of the research indicate that the label-free 2D light scattering static cytometric technique features low-cost,easy-to-use,excellent-specificity and high-resolution in the particle size measurement and leukemic cell detection.In addition,the label-free and non-invasive analysis may provide unperturbed cellular information that is important for the study of live cells.Most of all,this works serve as a new method for leukemia screening and the classification of leukemia subtypes in clinics,and laid a certain foundation for the detection of leukemic cells from whole blood.