圆偏振光与线偏振光对心肌细胞外基质组织学分析的一致性研究:Bland-Altman分析

Analysis on the agreement of linearly polarized light in the histological analysis of myocardial extracellular matrix:A Bland-Altman analysis

【目的】研究圆偏振光和线偏振光结合苦味酸天狼猩红染色,对心肌细胞外基质组织学分析结果的一致性。【方法】对心肌梗死大鼠的心脏组织进行苦味酸天狼猩红染色后,固定其它光学参数,在同一视野分别使用圆偏振光和线偏振光获取图像(共随机获取40个视野)并进行图像分析,计算红色、绿色胶原纤维的面积、胶原容积分数以及胶原平均灰度等反映梗死心脏组织修复过程的组织学指标;采用Bland-Altman分析以及Pearson相关分析,对两种光学体系下测得的上述指标的一致性进行分析。【结果】通过Pearson相关分析显示两种光学系统中测得的红色纤维面积(r2=0.9684,P<0.01)、绿色纤维面积(r2=0.8183,P<0.01)、胶原容积分数(r2=0.9595,P<0.01)和胶原平均灰度(r2=0.6188,P<0.01)均有很高的相关性。然而Bland-Altman分析显示,至少30%以上的红色胶原纤维、50%绿色胶原纤维成分和30%的总细胞外基质成分在线偏振光中无法显示,并且线偏振光系统检测的偏倚随着均数的增加而增大。【结论】线偏振光结合天狼猩红染色在检测细胞外基质的沉积量、平均灰度方面,敏感性显著低于圆偏振光。在细胞外基质的相关研究中,应该尽量避免使用线偏振光。

[ Objective] To investigate the agreement of histological analysis of extracelluar matrix （ECM） by circularly polarized light and linearly polarized light in picrosirius red stained myocardial sections. [Methods] Infarcted rat heart tissue, prepared by ligation of coronary artery was fixed in paraffin and sectioned for picrosirius red staining. In the same field of myocardial tissue, linear polarizer and circular polarizer were used to capture images, respectively. A total of 40 fields were randomly selected for analysis. The area of red collagen fiber, green collagen fiber, collagen volume fraction and average intensity of collagen scar were calculated by quantitative image analysis. Bland-Altman analysis and Pearson＇ s correlation were performed to evaluate the relationships between the above parameters resulted from circularly and linearly polarized light. [ Results] By Pearson＇s correlation analysis, the area of red collagen fiber （ r^2= 0. 9684, P 〈 0.01 ）, green collagen fiber （ r^2=0.8183, P 〈 0.01）, collagen volume fraction （ r^2= 0. 9595, P 〈 0.01 ） and average intensity of collagen scar （ r^2= 0.6188, P 〈 0.01） in myocardial tissue derived from circularly and linearly polarized microscope setups, presented good correlations. However, when Bland- Altman analysis was performed, at least 30% of red collagen fibers, 50% of green fibers and 30% of total collagen fibers were severely underestimated by linearly polarized light; and there was an increasing trend of bias with the increase of average. [Conclusions] Linearly polarized light will significantly underestimate collagen deposition in myocardial tissue stained with picrosirius red, which is not an idea method to detect ECM amount by means of image analysis.