流式荧光强度分级

2014-5-9 16:48| 发布者: lipc| 查看: 2827| 评论: 10|原作者: niwanmao

摘要: 流式中文网倪万茂简译＋个人总结（本章节图片较多，有8张，请耐心等待图片显示）：评估荧光强度是区分恶性细胞的一个重要步骤，在区分抗原表达相似的类似疾病（如CLL/SLL和MCL等）时，荧光强度就显得尤为重要。 ...

流式中文网倪万茂简译＋个人总结（本章节图片较多，有8张，请耐心等待图片显示）：
评估荧光强度是区分恶性细胞的一个重要步骤，在区分抗原表达相似的类似疾病（如CLL/SLL和MCL等）时，荧光强度就显得尤为重要。
荧光强度的评估还需要考虑以下因素：（1）荧光素（如FITC的荧光一般比PE的要弱一点）；（2）抗体的结合能力；（3）细胞表面的抗原结合位点。
用绝对数值来表示荧光强度，是定量流式细胞术的一个优势，然而，在实际应用中，异常细胞与正常细胞的区别通常是表现在一个或多个抗原表达方式的异常（例如表达下调或过度表达等），所以，在免疫分型中，用模糊表达方式，更能体现细胞的抗原表达情况。
荧光强度通常用对数坐标表示，10E0－10E1作为第一档，10E1－10E2为第二档，10E2－10E3为第三档，10E3－10E4为第四档。通常用第一档代表阴性，而第二、三、四档分别代表弱（＋）、中等（＋＋）、强（＋＋＋）阳性。
衡量阳性和阴性，我们通常用同型对照来作为阴性MARKER设置的标准，然而，在临床标本中，由于细胞本身特性和抗体生物学特性等影响，并不能完全依赖同型对照来判断。
在实际应用中，有时候由于某些细胞偏大或颗粒偏多，造成阴性细胞的荧光向右偏移，进入第二档，这时，我们判断＋、＋＋、＋＋＋的标准也需要依次右推一个数量级；同样，如果在CLL等小细胞、少颗粒细胞中，阴性细胞的荧光可能会向左偏移，我们判断阳性水平的标准也需要稍微调低。
另外，还有一种极弱（dim）阳性，这种阳性判断的标准就是，将该细胞的荧光与阴性细胞的荧光重叠，如果较阴性细胞右移，但未进入下一数量级（即＋），可判断为dim，看图（(Figure 3.16).)。

Assessing fluorescence intensity is an important step in characterizing and classifying hematopoietic malignancies. This is even more critical when dealing with closely related disorders that share the expression of several antigens (e.g., CLL/SLL and MCL). The level of fluorescence detected is dependent on (1) the fluorochrome employed, (2) the binding capacity of the particular fluorochrome–antibody conjugate, and (3) the number of antigenic epitopes on the cell surface. To this end, efforts have been spent on quantitative FCM, with an aim to standardize and measure absolute fluorescence intensity. It is still not clear that this approach is widely applicable, however, or whether absolute fluorescence intensity measurements are criteria sufficient to characterize abnormal populations. In practice, a cell population is recognized as abnormal because of the pattern of one or more aberrancies (e.g., downregulated expression or overexpression) in comparison with normal cells, rather than absolute fluorescence measurements. Therefore, for diagnostic purposes, fluorescence intensity determinations utilize relative units rather than absolute ones. The relative intensities are usually estimated based on the difference between the mean, median, or modal channel (channel number with the most events) of normal and neoplastic cells, and the results are reported in a semiquantitative/ qualitative manner (e.g., negative, positive, dim, and bright).
Because of the wide range of fluorescence intensities spanning over four decades (i.e., 10,000 : 1), flow cytometers employ logarithmic amplifier systems for compressing the data over 1024 channels into a log scale of four decades of 256 channels each. On the dot plots, fluorescence intensities are often displayed on a scale of 10[sup]0[/sup], 10[sup]1[/sup], 10[sup]2[/sup], 10[sup]3[/sup], and 10[sup]4[/sup]. In general, the first decalog (between 10[sup]0[/sup] and 10[sup]1[/sup]) represents negative fluorescence, and signals falling in the second, third, and fourth decalogs represent weak (+), moderate (++), and strong (+++) fluorescence intensity, respectively.
The evaluation of fluorescence intensities takes into account the isotype negative controls and the presence of normal cells in the sample. The intensity of a given antigen on the cells of interest is determined by visually comparing the pattern produced by the critical cells reacting with the corresponding antibody to that obtained with the isotype-matched control. Placing of the cursor is not useful because, in clinical samples, there is often an overlap between the negative and positive cells. It is important not to rely solely on isotype controls, however, as they may not necessarily match the biochemical properties of every antibody in the panel, despite an identical concentration and protein:fluorochrome ratio. The background normal cells in the sample are important internal controls. In a large panel, there will always be cells or antibodies serving as negative controls. Monocytic cells tend to have nonspecific staining of immunoglobulins, however (Figures 3.9 and 3.10). It is also important not to be too rigid about the above-mentioned scale division, especially when the cell population exhibits a significant degree of autofluorescence. This feature is typical of leukemic blasts in AML-M3 (Figure 3.11).
Fig 3.9

In straightforward situations, the fluorescence intensity of a given marker on the negative population falls within the first decalog, and the cells of interest form a clear-cut cluster in the positive region. This is most easily seen when the sample is composed almost entirely of abnormal cells. The lack of staining of this population with a given antibody serves as its own negative control when compared with the positive staining with another antibody (Figure 3.12). In many instances, however, an increase in the cell size of the malignant cells is accompanied by a higher degree of background staining, as the large cells tend to be more “sticky” (presumably from increased Fc receptors) or have more autofluorescence. The fluorescence signals of the negative isotype control may then extend well into the region usually considered to be positive staining (i.e., into the second decalog and occasionally beyond). A mental correction of this right shift is necessary to assign the appropriate fluorescence intensity to the positive population (Figure 3.11). For example, if the expression of a given antigen on the abnormal cells falls within the third decalog (moderate) but the negative population is rightshifted well into the second decalog (Figure 3.13), the fluorescence intensity on the positive population should be appropriately reported as weak instead of moderate. It is important not to confuse this phenomenon with poor instrument calibration. When the background shift is caused by increased “stickiness” or autofluorescence, it should not be present in other specimens (e.g., neoplasms with small cell size, or normal specimens) run the same day. The reverse phenomenon can also be seen; that is, the negative population does not fill the entire first decalog but is significantly shifted close to 100. This situation is relatively infrequent, however. It may be seen in samples composed of a nearly pure population of small malignant lymphoid cells (Figure 3.14).
Fig 3.12

A common phenomenon associated with large B-cell lymphomas with intense expression of CD20 is the shedding of CD20, which then coats the residual T-cells. As a result, the T-cell cluster appears to be CD20-positive when viewed on any of the dot plots that feature CD20 (e.g., FSC/CD20, CD5/CD20). This phenomenon is also observed in FCC lymphomas (see Section 3.6.3.1).
A difficult but sometimes important task in assessing fluorescence intensity is to distinguish very weak (dim) positive staining from negative cells. This occurs frequently when evaluating surface light chain expression, especially in CLL. With regard to light chains, this difficulty can be resolved in most instances by including two different sets of kappa and lambda reagents in the FCM panel (Figure 3.15). Irrespective of the methods applied to describe fluorescence intensity, either quantitative or qualitative, the distinction between dim positive and negative staining ultimately involves arbitrary criteria. In general, the population is considered positive for an antigen if its histogram is shifted by a certain number of fluorescence channels in comparison with an internal negative control. Regardless of the exact number of channels chosen to define the minimum shift arbitrarily, it is important that this shift can be clearly visualized when the histograms or the cluster displays are superimposed on one another (Figure 3.16).
Fig 3.15