个别细胞散为主细胞学标本的细胞块的制备

简介：

这是一个视频描述Shidham的细胞液基细胞学（LBC）使用HistoGelTM（Thermo Scientific的）（HG）的标本块制备方法。相对于其他随机方法，以下是本议定书的两个关键特性，准备从相对松散的细胞（1-5），单分散的hypocellular标本细胞块的。

1. 该协议涉及的步骤集中沿平面平行的细胞块切割面的细胞。
2. 它还包括一个AV标记（图1b），灯塔般的黑暗，以符合以下目的两个：
   1. 要可视化的水平，这些细胞都集中在。现在感兴趣的细胞与细胞块面积可以可视化的histotechnologist时，深色的灯塔是在切割过程中暴露。这种监控能力，防止切割的水平，大部分细胞或不切割样品细胞浓度最高水平太肤浅。
   2. 作为一个不同的幻灯片上串行细胞块段的定位参考点。这个参考点作为一盏明灯，帮助查找特定的细胞或细胞与上海化学工业区的方法（6,7）的坐标免疫模式的评价。

协议（图2）

样品制备。

1. 转移剩余LBC宫颈细胞学标本平底玻璃试管（直径15mm x 45毫米）（图2.1至2.4）。玻璃管放入一个更大的塑料载体管（28 × 85毫米）和离心机。删除从承运人管玻璃底管，倒出上清液。
2. 玻璃管，然后封顶（在下一步的加热水，以防止溢出），并放在一个较大的平底托盘塑料管
3. 托盘塑料管中的玻璃管，然后加盖，放置在离心机旋转杯，并没有固定的角杯，使细胞平底玻璃管垂直下降，并在1805纺G（3000 RPM的，转子半径17厘米）为5分钟（图2.5）。
4. 然后取出管垂直离心机和删除，而不会干扰细胞的沉淀颗粒小玻璃管载体较大的塑料管钳。
5. 与样品的玻璃管是不封顶的，浇灭了上清小心不要打扰底部泥沙细胞平层（图2.6）。

列入参考坐标AV标记此外凝胶

1. 一个黑暗的灯塔AV标记 （约2毫米× 2毫米大小，平面浮出水面，深色，sectionable材料的片段）（图3），增加一条，作为一个玻璃管的路标（图2.7 ）。
2. 熔点为10秒，在中等功率微波液化等分的汞。
3. 加入0.5毫升的熔融汞管，迅速与泥沙混合，并重述（图2.8）（不允许HG的开始巩固的情况下迅速着手进行下一步）。
4. 新增约2.5毫升的温水（45 ° C）水向承运人的塑料管（图2.9）。
5. 较小的上限玻璃管放在里面的塑料管，用温水。 （这一步是必要的，以防止在接下来的步骤巩固了HG）（图2.9）。
6. 承运人塑料管被放置在离心机（ 与旋转杯，并没有固定的角杯，使细胞平底玻璃管垂直下降），和G（3000 RPM的，转子半径17厘米纺在1805年五）分钟。这种离心步骤的目的是推动影音标记，并集中到一个层细胞接近最终石蜡包埋细胞块（图1d）的切割面。
7. 然后取出，轻轻地，垂直管的照顾，不要去打扰样品池底部的沉淀一层薄薄的离心机。
8. 较大的塑料管是不封顶的小玻璃管中删除一个镊子垂直，而不会干扰标本细胞的沉积层。
9. 小玻璃管中冷藏15分钟，冷却和巩固HG（图2.11）的垂直位置。

作为细胞的凝胶标本按钮块进行最终处理的去除

1. 凝固HG磁盘，在底层的集中/沉积物样本，被赶出平底玻璃管喷出通过23号针头与注射器（图2.12）10％的福尔马林。
2. 针插入凝固标本（图2.12）中汞光盘的边缘沿管端。
3. 该needle是旋转沿管的一侧，而甲醛是缓慢通过注射器推。伴随着深色灯塔AV标记集中在从平底的玻璃管（图2.12）标本中汞按钮分离结果。
4. 细胞块（标本细胞的凝胶按钮），然后放置在一个标签的磁带和组织处理提交给准备石蜡包埋细胞块（图2.13 ）。

嵌入和切割试样

1. 磁盘是嵌入在切割面黑暗中的灯塔标记侧（图1）石蜡。
2. 直到深色AV标记块切片，一盏明灯，是揭露和清晰可见。
3. 从这个层面上，它应该包含最分散的单细胞标本3到4微米的部分削减。
4. 部分收集玻片上作进一步的染色，免疫组织化学染色，或作为其它测试。对这些类型的幻灯片，用于安装部分的测试协议可能会有所不同。一般免疫，涂幻灯片，以防止在免疫步骤浮动和幻灯片的部分路段损失。

（按字母顺序）的缩写 ：CISH，显色原位杂交试验，鱼，荧光原位杂交试验FFPE，福尔马林固定石蜡包埋;活检，细针穿刺; HG，HistoGel™（Thermo Scientific的） ; LBC，液基细胞学检查; PCR聚合酶链反应;

图1。细胞块的结构编制Shidham的协议。

图2。Shidham的协议，准备从LBC标本的细胞块的不同步骤的摘要。

图3。香蕉皮AV标记的制备。

图4。AV标记和无细胞块和部分比较。

图5比较有和没有AV标记的细胞块切片的细胞结构。

Cell blocks are a valuable tool for evaluation of various cytology specimens (1). Most importantly, in addition to the architectural details of the specimen, cell blocks allow for evaluation of ancillary studies such as immunocytochemistry, fluorescent/chromogenic in-situ hybridization tests (FISH/CISH) and in-situ PCR. A variety of methods for preparation of cell block are described. However, most of these are suitable for non-gynecologic cytology specimens which contain relatively many cells and with tissue microfragments such as in FNA aspirates and some serous fluids such as effusions (1,3,4,5).

Because cell blocks primarily provide the opportunity for immunocytochemical evaluation, their processing should preferably be similar to that of the formalin-fixed paraffin-embedded (FFPE) tissues. Ultimately the results obtained after immunocytochemical evaluation of cell block sections are compared to those with the published literature performed predominantly on FFPE tissue sections. Any alterations in the protocol potentially compromise and nullify the validity of the results obtained on cell blocks processed through different fixatives and reagent sequences other than that used for routine FFPE. Some commercial techniques may have the drawback of processing through a protocol of exposure to other fixative-reagent exposure.

Various methods of cell block preparation are available for specimens with a significant quantity of sediment and tissue fragments. Principally, the concentrated sediments are supported by some gel or coagulation principle. The maneuverable button is then embedded in paraffin after processing like the surgical pathology specimens/biopsies.

The gels used include gelatin, agar, fibrinogen/plasma-thrombin, and other commercial gels such as HG. The methods of concentration vary from simple pelleting of the sediment by centrifugation to concentration of cells along various types of membranes. Examples include: Milipore, collodin (Celloidin) bags or scraping the cells from the cytology smears on glass slides (1). We also evaluated a variety of gels by trying different combinations of agar and gelatin. None of the combinations achieved the a firm enough consistency to obtain an easily maneuverable disc of solidified gel with embedded cells from the specimen in one piece. HG showed appropriate consistency and in our experience the immunostaining results on HG cell block sections have been excellent.

Liquid based cytology (LBC) specimens for cervicovaginal cytology are generally less cellular than non-gynecologic specimens as mentioned above. In addition, the gynecologic LBC specimens predominantly contain individual scattered exfoliated superficial cells from cervicovaginal mucosa. Due to this, appropriate cellularity within the cell block sections may not be achieved without a special approach. As these singly scattered cellular components in the the block cannot be seen by the histotechnologist during section cutting, the level at which the cells start appearing in the sections cannot be appreciated and may be missed, either by cutting past the level with most cells or not cutting deep enough into the level with highest concentration of sample cells (Figure 4). Shidham’s protocol addresses both of these issues using HG as embedding medium and conventional lab equipments (2) (Figure 2).

This protocol involves following two major features (Figure 1):

1. Concentration and alignment of singly scattered cells in a narrow plane adjacent and parallel to the cutting surface of the cell block (Figure 5).
2. Inclusion of a beacon-like dark AV-marker as a signpost. This is critical for achieving following features:
   1. Identifying and monitoring the level at which the cells are concentrated in the cell block. The exposure of the dark colored signpost highlights the level at which most of the singly scattered cells in the cell block are expected to be located (Figure 4). This prevents the overcutting (cutting past the level with most cells) or undercutting (not cutting deep enough into the level with highest concentration of sample cells) in to the cell block and allow selection of the sections from the level in the cell block corresponding with highest concentration of cells.
   2. The dark colored signpost in the sections also serves as a reference point to survey and identify exactly the same cells in different serial sections of the cell block on different slides (Figure 4). This is critical while interpreting and evaluating the coordinate properties such immunoprofile of particular cells by the SCIP approach to follow the same cells in different sections (6,7).

Although our protocol is standardized and reported for liquid based cervical cytology specimens, it can also be used to enhance diagnostic yield of many other non-gynecologic specimens such as effusion fluids, FNA, brushings, cyst contents etc. In addition the AV marker would facilitate improved application of SCIP approach during immunohistochemical evaluation of cell block sections of these specimens. The embedding medium may be replaced by other reagents with appropriate modifications at relevant steps. HG may be replaced by plasma (Fibrinogen) to be gelled by Thrombin at room temperature (1).