，英文的我看不明白 
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- 请教宫颈癌的DNA检查
Feulgen stain
Feulgen stain is a staining technique discovered by Robert Feulgen and used in histology to identify chromosomal material or DNA in cell specimens. It depends on acid hydrolysis of DNA, therefore fixating agents using strong acids should be avoided.
The specimen is subjected to warm (60 °C) hydrochloric acid, then to Schiff reagent. In the past, a sulfite rinse followed, but this is now considered unnecessary. Optionally, the sample can be counterstained with Light Green SF yellowish. Finally, it is dehydrated with ethanol, cleared with xylene, and mounted in a resinous medium.
DNA should be stained red. The background, if counterstained, is green.
The Feulgen reaction is a semi-quantitative technique. If the only aldehydes remaining in the cell are those produced from the hydrolysis of DNA, then the technique is quantitative for DNA. It is possible to use an instrument known as a microdensitometer or microspectrophotometer to actually measure the intensity of the pink Feulgen reaction for a given organelle. Using this procedure, it was early determined that interphase cells were composed of two populations, those with diploid DNA and those with tetraploid DNA (two complete genomes). The nuclei looked identical, but one contained twice as much DNA. This gave rise to the division of the interphase period of the cell cycle to G1, S, and G2 phases based on the synthesis of that extra DNA
- 掌心0164
| 以下是引用法师在2010-4-2 16:45:00的发言： 宫颈癌的DNA筛查据资料介绍是对细胞制片用DNA染色（Feulgen染色），然后在专用的机器进行自动扫描，扫描的原理是利用细胞核的大小，染色的深浅其光学反应不一样从而间接推断DAN的多少，仪器事先设置好了各种参数。这种技术又被称为定量细胞学，这个技术可能工作效率比较高，敏感性高，特异性可能比较低，据说筛查出的异常涂片还是要做巴氏染色确认。掌心好像对这个应该很了解，武汉兰丁好像是最早成规模开展此技术的。 |
法师不愧为是法师,短短不到200字,就把这一方法讲得准确而又精辟.
赞赏!
- 王军臣
| 以下是引用掌心0164在2010-4-2 17:21:00的发言：
Feulgen stain
Feulgen stain is a staining technique discovered by Robert Feulgen and used in histology to identify chromosomal material or DNA in cell specimens. It depends on acid hydrolysis of DNA, therefore fixating agents using strong acids should be avoided. The specimen is subjected to warm (60 °C) hydrochloric acid, then to Schiff reagent. In the past, a sulfite rinse followed, but this is now considered unnecessary. Optionally, the sample can be counterstained with Light Green SF yellowish. Finally, it is dehydrated with ethanol, cleared with xylene, and mounted in a resinous medium. DNA should be stained red. The background, if counterstained, is green. The Feulgen reaction is a semi-quantitative technique. If the only aldehydes remaining in the cell are those produced from the hydrolysis of DNA, then the technique is quantitative for DNA. It is possible to use an instrument known as a microdensitometer or microspectrophotometer to actually measure the intensity of the pink Feulgen reaction for a given organelle. Using this procedure, it was early determined that interphase cells were composed of two populations, those with diploid DNA and those with tetraploid DNA (two complete genomes). The nuclei looked identical, but one contained twice as much DNA. This gave rise to the division of the interphase period of the cell cycle to G1, S, and G2 phases based on the synthesis of that extra DNA |
掌心果然非凡,一切尽在掌握中.把该方法的每一个实验细节都述说得那么简明扼要.
- 王军臣
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本帖最后由 于 2010-04-03 09:47:00 编辑
上面说了DNA的染色原理，剩下的所谓的DNA的检测就是一个全自动显微镜的图像分析系统对DNA（Feulgen）染好色的细胞片子进行分析。全自动图像分析系统由：一个带马达控制三轴显微镜（这个容易做到也容易模仿）+图像分析软件（这个软件的参数和软件的实现水平决定最后结果的好坏）；这个软件的原理正如法师老师说的由于细胞通过DNA（Feulgen）染色之后，由于细胞的DNA全部在核中，所以浆不着色；由于每个核的DNA含量不一样、大小不一样、面积、直径、光学密度等等参数，不同的软件有不同的参数，多的120多个，少的软件也有几十个，再通过显微镜用不同了滤光片特定的波长通过染色的核之后让光学的参数转换到计算机的图像分析系统转换成每个核的一个相对DNA含量的参数（这里面有一个外对照：软件中储存有大量的正常和异常的细胞信息，同时在读每个片子的时候还有内对照：一般以成熟淋巴细胞或正常中层细胞的核作为对照）来判断细胞的异常。整个原理上就是一个DNA的含量（染色体的含量）、一个细胞增殖周期和特定波长下细胞核的光学密度的差异的结合产物。这个检测的优点：容易标准化、当时我们做的数据特异性（83%以上）和敏感性（92%）以上、在我们细胞水平不高的时候可以做好帮手，可以作为宫颈癌筛查结合TBS一起使用，做两项检测同时进行可以作为互补。缺点：对2倍体肿瘤（DNA含量不改变）或缺乏对照的肿瘤（细胞全部来源于肿瘤细胞，因为找不到内对照）不敏感、造价高和费时（染色时间长约5个小时）。
- 掌心0164
