Greil R, Fasching B, Weger A, Loidl P
Department of Internal Medicine, University of Innsbruck, Austria.
Lab Invest. 1992 Feb;66(2):251-60.
The biologic functions mediated by the nuclear protooncogene, c-MYC are correlated to gene dosage. Since automated quantification programs are expensive, time-consuming and not easily available, and since analysis by flow cytometry is difficult in the case of nuclear antigens, we examined the suitability and reproducibility of a semiquantitative in situ evaluation system. This system was based on the percentage of nuclear area staining positively, and comprised the following categories: 0: negative, 1+: single scattered grains of the immunocytochemical staining product, 2+: confluence of grains to patches but less than 50% nuclear area positive, and 3+: greater than 50% positive nuclear area. In addition, sensitivity and specificity of two anti-c-MYC antibodies were investigated. Although both antibodies differed slightly in staining pattern and sensitivity, the four quantification categories were applicable for immunostainings of both antisera and highly reproducible when re-evaluated by the same observer (r = 0.98; p = 0.0001) or a second investigator (rAb155 = 0.98, rAb DCPm = 0.96; p = 0.0001), both reading blindly and independently. Comparing our semiquantitative evaluation categories and results of computer-assisted image analysis, the percentage of positive nuclear area (p less than 0.0001), the median staining intensity (p less than 0.0001), and the product of both (p less than 0.0001) differed significantly in the four evaluation categories. This result still held true after correction for nuclear size, which differed appreciably in various cell types (p less than 0.0001). The product of positive nuclear area, staining intensity and nuclear size (microns 2), which best approximates the absolute amount of c-MYC within a certain cell, was clearly different within the four staining categories (p less than 0.0001) and did not depend on cellular morphology within the staining categories 0 to 2. Also, the immunocytochemical technique proved highly reproducible (median day/day variance 0.65% (0-13); r = 0.995). The practicability of this system for semiquantification was demonstrated by (a) correlation of H score values of immunocytochemical stainings with densitometric scans of Western blots and (b) by the fact that peripheral blood lymphocytes, Phytohemagglutinin stimulated blasts, 13 cases of multiple myeloma and HL-60 cells differed concerning their estimated c-MYC amounts (p = 0.0125). This confirms on the effector molecule level results previously reported from mRNA in situ and Northern blotting analyses. We conclude that a simple and highly reproducible evaluation system can be used for in situ comparison of nuclear oncogene dosage.
由核原癌基因c-MYC介导的生物学功能与基因剂量相关。由于自动化定量程序昂贵、耗时且不易获得，并且由于在核抗原的情况下通过流式细胞术进行分析很困难，我们研究了一种半定量原位评估系统的适用性和可重复性。该系统基于阳性染色核面积的百分比，包括以下类别：0：阴性；1+：免疫细胞化学染色产物的单个散在颗粒；2+：颗粒融合成片但阳性核面积小于50%；3+：阳性核面积大于50%。此外，还研究了两种抗c-MYC抗体的敏感性和特异性。尽管两种抗体在染色模式和敏感性上略有不同，但这四个定量类别适用于两种抗血清的免疫染色，并且当由同一位观察者（r = 0.98；p = 0.0001）或另一位研究者（rAb155 = 0.98，rAb DCPm = 0.96；p = 0.0001）重新评估时具有高度可重复性，两者均盲目且独立地进行读取。将我们的半定量评估类别与计算机辅助图像分析的结果进行比较，阳性核面积百分比（p < 0.0001）、中位染色强度（p < 0.0001）以及两者的乘积（p < 0.0001）在四个评估类别中存在显著差异。在校正核大小后该结果仍然成立，核大小在不同细胞类型中差异明显（p < 0.0001）。阳性核面积、染色强度和核大小（微米²）的乘积最接近特定细胞内c-MYC的绝对量，在四个染色类别中明显不同（p < 0.0001），并且在0至2类染色中不依赖于细胞形态。此外，免疫细胞化学技术证明具有高度可重复性（中位日/日方差0.65%（0 - 13）；r = 0.995）。该系统用于半定量的实用性通过以下两点得以证明：（a）免疫细胞化学染色的H评分值与蛋白质印迹的光密度扫描结果的相关性；（b）外周血淋巴细胞、植物血凝素刺激的母细胞、13例多发性骨髓瘤和HL-60细胞在估计的c-MYC量方面存在差异（p = 0.0125）。这在效应分子水平上证实了先前从mRNA原位和Northern印迹分析中报道的结果。我们得出结论，一种简单且高度可重复的评估系统可用于原位比较核癌基因剂量。
