Morphology Analysis Methods Of Unlabeled Red Blood Cells And Applications In The Evaluation Of Storage Lesion

Storage of blood in a liquid suspension medium for blood transfusion has practiced for more than 90 years.In clinical surgery,transfusion of suspended red blood cells（RBCs）is an effective treatment for patients with severe symptoms or massive bleeding.However,during storage,Red Blood Cells（RBCs）undergo several biochemical and biophysical changes,and disc-sphere changes known as storage lesions are induced in their morphology.Clinical studies demonstrated that,in patients who had been transfused with older RBCs,poorly deformable RBCs such like spherocytes can be trapped in microcirculation,which may lead to tissue hypoxia.Therefore,it is important to identify the morphological changes of RBCs throughout the storage process.In this thesis,the non-invasive detection of RBCs morphology is studied,and the abilities of qualitative imaging techniques and quantitative phase imaging techniques are demonstrated,respectively.The research content of this thesis includes:（1）The relationship among blood storage lesion,environmental factors affecting the changes of RBCs morphology,RBCs deformation and disease was investigated.In addition,the detection methods of RBCs morphology,especially the standard methods in clinical practice,are briefly introduced.Their advantages and problems are also analyzed.（2）In this thesis,the literature of microscopic imaging technology is reviewed,which mainly includes intensity-based microscopic imaging,qualitative phase imaging and quantitative phase imaging.The advantages and limits of these imaging techniques are discussed.The basic principles of four imaging techniques in this thesis are summarized and the core ideas of qualitative phase imaging and quantitative phase microscopic imaging are briefly introduced.The methods of quantitative differential phase contrast phase imaging and digital holographic imaging are also introduced emphatically.（3）In this thesis,the methods of defocus imaging based on bright field,differential phase contrast imaging based on oblique illumination and quantitative phase imaging based on differential phase contrast are used to detect the morphology of red blood cells.The main technologies are described as follows: based on a commercial microscope（Olympus,IX73）,different additional modules are added to carry out experimental design from the aspects of optical design,mechanical production and microscopic imaging,which realized the multi-mode imaging of RBCs.The application of these imaging methods in the analysis of erythrocyte morphology was further studied.（4）Using the LED array as lighting source,multi-mode imaging is realized.Furthermore,difference phase contrast imaging technique is applied to suspended red blood cells and blood smears samples,and different forms of red blood cells in bright field,oblique illumination,differential phase contrast and quantitative phase imaging are observed and compared demonstrating that our quantitative phase imaging method is able to improve the image contrast of blood cells.（5）An automatic classification method of red blood cell morphology by binary decision tree was established.To evaluate the morphology of RBCs,four morphological indexes were extracted from the micrographs of individual RBC.By manually adjusting the appropriate threshold and establishing automatic classification standard,higher automatic classification accuracy can be achieved.The morphological indexes of bright-field imaging and quantitative phase imaging were analyzed statistically.The results indicated that compared with bright-field imaging,phase imaging can significantly improve the accuracy of RBCs morphology analysis.In this study,a non-destructive,unlabeled method for analyzing the morphology of RBCs is developed.The q DPC microscopy provides a valuable tool for studying the behavior of a single cell in real time.This technique does not require the introduction of dyes or probes to stain cells,and cell samples can be continuously imaged for phase diagram calculations without damaging their function or structure.The q DPC method described in thesis have more extensive research and application prospects in the evaluation and detection of RBC storage lesions and blood diseases.