Picrosirius red (PSR) staining is generally used to evaluate liver fibrosis; however, PSR sometimes causes nonspecific nuclear staining. In this study, we evaluated the ability of phosphomolybdic acid (PMA) pretreatment to prevent nonspecific nuclear staining by PSR. In a manual evaluation of 27 non-tumor samples from patients with hepatocellular carcinoma, nonspecific nuclear staining was observed in 3.7% of PMA-treated specimens, compared with 85.2% of untreated specimens. Conversely, computer-assisted image analysis (CAIA) identified nonspecific nuclear staining in 0% of PMA-treated samples, vs 44.4% of untreated samples. Surprisingly, after mounting, PMA-treated specimens exhibited a blue tinge because of molybdenum blue (MB) production following sunlight exposure or virtual slide scanning. Using UV cut film, MB production induced by sunlight exposure was prevented; however, the film did not prevent MB production during virtual slide scanning. Moreover, only blue light–emitting diode exposure resulted in a blue tinge in PMA solution. Our data indicated that PMA pretreatment is effective for evaluating liver fibrosis using CAIA. Meanwhile, improvements in virtual slide scanning protocols would directly improve the quality of PMA-pretreated specimens subjected to CAIA:

Picrosirius红（PSR）染色通常用于评估肝纤维化。但是，PSR有时会引起非特异性核染色。在这项研究中，我们评估了磷钼酸（PMA）预处理通过PSR防止非特异性核染色的能力。在对来自肝细胞癌患者的27个非肿瘤样本进行的人工评估中，在3.7％的PMA处理的样本中观察到非特异性核染色，而未处理的样本为85.2％。相反，计算机辅助图像分析（CAIA）在0％的PMA处理的样品中发现了非特异性核染色，而未处理的样品为44.4％。出人意料的是，安装后，由于暴露在阳光下或进行虚拟载玻片扫描后会产生钼蓝（MB），因此经过PMA处理的样品呈现出蓝色。使用紫外线切割膜 防止了由于阳光照射而导致的MB产生；但是，该胶卷并没有阻止在虚拟幻灯片扫描过程中产生MB。而且，只有蓝色发光二极管曝光会在PMA解决方案中产生蓝色调。我们的数据表明PMA预处理可有效评估使用CAIA的肝纤维化。同时，虚拟载玻片扫描协议的改进将直接提高经受CAIA的PMA预处理样品的质量：