Preliminary Exploration Of Dual-color Fluorescence Staining Of Glioma Based On Laser Confocal Microscopy

Objective:To explore a dual-color fluorescence staining technique based on laser scanning confocal microscopy,which is expected to achieve a similar diagnostic effect to traditional HE staining,and lay a preliminary foundation for the application of this technique in guiding the development of surgical resection strategy for glioma and to find the cut edges of intraoperative glioma and normal brain tissue.Methods:Fresh human brain tissue specimens of glioma were collected and divided into two samples of the same size for dual-color fluorescence staining and HE tissue staining.Then the lipid and DNA of brain tissue were stained with BODIPY and DRAQ5 respectively in one sample and fluorescence imaging was performed under laser scanning confocal microscope to collect fluorescence images.The fluorescence images were randomly evaluated by two pathologists and then compared with the HE images.After 90 days,the two pathologists evaluated the dual-color fluorescence images again.The consistency test was performed on the evaluation results of the two pathologists and κvalue was calculated to judge the diagnostic consistency of dual-color fluorescence staining compared with HE staining.The diagnostic accuracy,sensitivity and specificity of dual-color fluorescence staining were calculated.Results:The dual-color fluorescence staining technique based on laser scanning confocal microscopy had histological characteristics very similar to HE staining and lipid retention was intact.The κ value of the first dual-color fluorescence stain diagnosis and HE staining diagnosis of Pathologist A was 0.48,which had a generally satisfactory consistency and the second κ value was 0.76,which had a fairly satisfactory consistency;the κ value of the first fluorescence stain diagnosis of Pathologist B was 0.64,which was generally consistent with the HE staining diagnosis,and the second κ value was 0.82,which had a fairly satisfactory consistency.The diagnostic accuracy of pathologists A and B increased from 76% and 83% in the first time to 89% and 92% in the second time respectively,which has a high accuracy.In addition to the high accuracy,Tables 2-3 also show an encouraging result in terms of sensitivity and specificity.Of the 28 low-grade gliomas,16(57% specificity)and 23(71% specificity)were identified by pathologist A,respectively.Pathologist B identified 20 and 25 low-grade gliomas in the first and second sessions,with specificity of 82% and 89%,respectively.Among the 43 high-grade gliomas,Pathologist A correctly identified 38 cases for the first time with a sensitivity of88%,Pathologist B correctly identified 39 high-grade gliomas with a sensitivity of 90%for the first evaluation,and the second evaluation correctly identified 40 high-grade gliomas,and the sensitivity increased to 93%.Semi-quantitative analysis of the fluorescence intensity of the dual-color fluorescence-stained images showed that the green fluorescence intensity,red fluorescence intensity and total fluorescence intensity were stronger in high-grade gliomas than in low-grade gliomas(P<0.05).Conclusion:1.The dual-color fluorescence staining images have histological characteristics similar to that of HE staining,and compared with HE staining,the "gold standard",the diagnostic consistency is basically satisfactory,and the diagnostic accuracy,sensitivity and specificity are high.It provides a preliminary foundation for us to apply two-color fluorescence image in the intraoperative diagnosis of glioma,guide the formulation of surgical strategy,achieve higher tumor total resection rate and sub-total resection rate,and improve the prognosis of patients.2.Pathologists can initially master the evaluation of dual-color fluorescence staining images with the help of HE staining reading experience,saving huge learning and time costs.3.The fluorescence intensity of dual-color fluorescence staining of high-grade gliomas is stronger than that of low-grade gliomas.Lipid metabolism may affect the green fluorescence intensity of different grades of gliomas and the dual-color fluorescence staining technique preserves lipids completely,making it possible to study the important process of lipid metabolism in fresh human glioma cells.