Raskin Rose E, Vickers Julie, Ward Jennifer G, Toland Angus, Torrance Andrew G
Purdue University, West Lafayette, Indiana.
VGP, formerly TDDS, a member of SYNLAB, Exeter, Devon, UK.
Vet Clin Pathol. 2019 Oct;48 Suppl 1:88-97. doi: 10.1111/vcp.12759. Epub 2019 Jul 25.
Romanowsky staining is often the initial method used to stain hematologic and cytologic materials. While immunocytochemistry (ICC) is a well-established method on air-dried smears, there are rare veterinary reports of ICC involving Romanowsky-stained slides.
This study aimed to compare immunoreactivity of unstained vs Romanowsky-stained specimens, evaluate reactions over time, and assess ICC associations with confirmatory tests of 50 lymphoma cases. Another goal aimed to optimize manual ICC protocols with cellular and tissue immunomarkers to detect CD3ε, CD20, Pax5, MHCII, lysozyme, MUM1, vimentin, cytokeratin, and Melan-A antigens on Romanowsky-stained specimens.
Cytologic specimens from cases of lymphoid and nonlymphoid neoplasms were stained with a methanolic Romanowsky method. Additional unstained slides from these cases were used for comparison with the stained materials. Antigen retrieval involved a citrate buffer pH6 or Tris/EDTA pH9 at 95°C for 25 minutes in a decloaking chamber. Immunocytochemistry used known positive and secondary antibody-only negative cytologic controls. Immunoreactivity of unstained and prestained lymphoma slides was graded by the intensity and percent of stained cells. Signal grading was monitored over time for diagnostic differences.
Unstained and Romanowsky-stained slides had similar membrane/cytoplasm graded reactions, but unstained slides produced stronger signals. Romanowsky-stained blood films from B-cell and T-cell leukemias showed minimal loss of signal when monitored over 20 weeks. Signal differences did not change the diagnosis. There was a significant association between ICC and confirmatory tests. Optimization involved antibody dilution and antigen retrieval methodology for each antibody tested.
Immunocytochemistry of Romanowsky-stained material can be successfully performed using antibodies against CD3ε, CD20, cytokeratin, lysozyme, Melan-A, MHCII, MUM1, Pax5, and vimentin.
罗曼诺夫斯基染色法通常是用于对血液学和细胞学材料进行染色的初始方法。虽然免疫细胞化学（ICC）是一种在空气干燥涂片上成熟的方法，但涉及罗曼诺夫斯基染色玻片的兽医ICC报告很少。
本研究旨在比较未染色与罗曼诺夫斯基染色标本的免疫反应性，评估随时间的反应，并评估50例淋巴瘤病例的ICC与确诊试验的相关性。另一个目标是优化使用细胞和组织免疫标志物的手动ICC方案，以检测罗曼诺夫斯基染色标本上的CD3ε、CD20、Pax5、MHCII、溶菌酶、MUM1、波形蛋白、细胞角蛋白和Melan-A抗原。
用甲醇罗曼诺夫斯基法对淋巴样和非淋巴样肿瘤病例的细胞学标本进行染色。这些病例的其他未染色玻片用于与染色材料进行比较。抗原修复在脱盖室中于95°C用pH6柠檬酸盐缓冲液或pH9 Tris/EDTA缓冲液处理25分钟。免疫细胞化学使用已知阳性和仅含二抗的阴性细胞学对照。未染色和预染色淋巴瘤玻片的免疫反应性通过染色细胞的强度和百分比进行分级。随时间监测信号分级以观察诊断差异。
未染色和罗曼诺夫斯基染色玻片具有相似的膜/细胞质分级反应，但未染色玻片产生更强的信号。对B细胞和T细胞白血病的罗曼诺夫斯基染色血涂片在20周内进行监测时，信号损失最小。信号差异未改变诊断。ICC与确诊试验之间存在显著相关性。优化涉及针对每种测试抗体的抗体稀释和抗原修复方法。
使用针对CD3ε、CD20、细胞角蛋白、溶菌酶、Melan-A、MHCII、MUM1、Pax5和波形蛋白的抗体，可以成功地对罗曼诺夫斯基染色材料进行免疫细胞化学检测。
