Makkus A C, van 't Hof-Grootenboer A E, Pahlplatz M M, de Wilde P C, Gemmink A J, Cuypers V M, Vooijs G P
Department of Pathology, University Hospital Nijmegen, The Netherlands.
Cytometry. 1994 Apr 1;15(4):302-10. doi: 10.1002/cyto.990150405.
Three different methods of fixation (ethanol/Carbowax, formaldehyde, and Carnoy) and four different protocols (without Böhm post-fixation on an uncoated slide, and with Böhm post-fixation on Poly-L-Lysine coated slide, an uncoated slide and a previously Papanicolaou stained slide) were evaluated for their application in high resolution image analysis of Feulgen stained nuclei. The aim of the study was to find a combination with the best reproducibility and the least variance under normal laboratory conditions. Care was taken not to exclude any "normal" laboratory variability. The combinations were evaluated for densitometric, geometric, as well as texture features. Selected features were determined on a CAS-100 using the Cell Measurement Program (Cell Analysis Systems, Inc. Lombard, IL). Diploid and tetraploid rat liver nuclei were used. Ethanol/Carbowax fixation-with Böhm post-fixation proved most stable. This fixation method also gave feature values for previously Papanicolaou stained slides that were comparable to direct Feulgen stained nuclei. Acceptable results were achieved with all three fixatives and the various combinations if one adhered strictly to protocol. In routine practice this usually does not happen. Therefore Ethanol/Carbowax fixation with Böhm post-fixation was considered most suited for routine determination of feature values on the CAS-100.
评估了三种不同的固定方法（乙醇/聚乙二醇、甲醛和卡诺固定液）以及四种不同的方案（在未涂层载玻片上不进行博姆后固定，以及在聚-L-赖氨酸包被的载玻片、未涂层载玻片和先前巴氏染色的载玻片上进行博姆后固定）在福尔根染色细胞核高分辨率图像分析中的应用。该研究的目的是在正常实验室条件下找到重现性最佳且方差最小的组合。注意不排除任何“正常”的实验室变异性。对这些组合进行了光密度、几何以及纹理特征方面的评估。使用细胞测量程序（细胞分析系统公司，伊利诺伊州伦巴德）在CAS - 100上确定选定的特征。使用二倍体和四倍体大鼠肝细胞核。结果表明，乙醇/聚乙二醇固定并进行博姆后固定最为稳定。这种固定方法对于先前巴氏染色的载玻片所给出的特征值与直接福尔根染色的细胞核相当。如果严格遵循方案，所有三种固定剂和各种组合都能取得可接受的结果。但在常规实践中通常不会如此。因此，乙醇/聚乙二醇固定并进行博姆后固定被认为最适合在CAS - 100上常规测定特征值。
