个别细胞散为主细胞学标本的细胞块的制备

Published 7/21/2009
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Summary

Shidham准备从单独分散的细胞和小细胞群体的细胞学标本AV标记的细胞块的方法。

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Varsegi, G. M., Shidham, V. Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells. J. Vis. Exp. (29), e1316, doi:10.3791/1316 (2009).

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Abstract

这个视频演示Shidham的宫颈液基细胞学标本含有单独分散的细胞和小细胞群体的细胞块的制备方法。这种技术使用HistoGel(Thermo Scientific的)与传统的实验室设备。

使用细胞块切片是一种宝贵的的评价非妇科细胞学检查的辅助工具。他们使cytopathologist研究额外的形态标本的细节,包括病变的架构。最重要的是,他们允许为辅助研究的评价,如免疫细胞化学,原位杂交试验(FISH / CISH)和原位聚合酶链反应(PCR)。传统的细胞块制备技术大多被应用于非妇科细胞学标本,通常用于津液积液和细针穿刺活检。

液基宫颈标本相对较少,比他们的非妇科同行与许多单个细胞散细胞。正因为如此,足够的内细胞块切片的细胞​​结构是难以实现的。此外,histotechnologist切片块不能想像细胞在浓度最高的是在哪一级。因此,它是难以监测的适当水平,在这部分可以选择要进行测试,以玻片。因此,感兴趣的细胞与细胞块面积可能会被错过,无论是过去或切割不切割深度不够。目前“议定书”Shidham的方法解决这些问题。虽然这个协议是标准化和妇科液基细胞学标本报道,它也可以被应用于非妇科标本,如液体积液,活检,刷检,囊肿内容质量的提高细胞块切片的诊断材料等。

Protocol

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
图1。细胞块的结构编制Shidham的协议。

图2
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
图3。香蕉皮AV标记的制备。

图4
图4。AV标记和无细胞块和部分比较。

图5
图5比较有和没有AV标记的细胞块切片的细胞结构。

Discussion

细胞块是一个有价值的工具,评估各种细胞学标本(1)。最重要的是,除了建筑细节的标本,细胞块允许配套的研究,如免疫细胞化学,荧光/色原位杂交试验(FISH / CISH)和原位PCR的评价。多种方法制备细胞块。然而,这些最适合非妇科细胞学标本,其中含有较多的细胞和组织microfragments如活检抽吸和一些浆液性液体,如积液(1,3,4,5)。

,由于细胞块,主要提供免疫评价的机会,他们的处理最好的福尔马林固定石蜡包埋(FFPE)组织的类似。最终,免疫细胞块切片评价后得到的结果进行了比较FFPE组织切片主要出版的文献。在协议的任何改变可能的妥协和否定了通过不同的固定剂和试剂序列比常规FFPE使用的其他处理的细胞块上所取得的成果的有效性。一些商业技巧,可能已通过其他固定液试剂曝光的曝光的协议处理的缺点。

细胞块准备的各种方法都提供相当数量的泥沙和组织碎片标本。主要支持一些凝胶或凝血原则,集中沉积物。机动按钮,然后像手术病理标本/活检的处理后,石蜡包埋。

所用的凝胶包括明胶,琼脂,纤维蛋白原/血浆凝血酶,和其他商业凝胶,如HG。浓度的方法不尽相同,从简单的泥沙离心造粒沿膜的不同类型细胞的浓度。例子包括:Milipore,collodin(Celloidin)袋或刮从细胞学涂片的玻片上(1)细胞。我们还评估了各种凝胶,通过尝试不同的琼脂和明胶组合。组合没有实现的坚定足够的一致性,以获得在一块标本嵌入细胞凝固凝胶容易机动光盘。 HG表现出适当的一致性和HG细胞块切片的免疫组化结果在我们的经验已经很好的。

液基细胞学(LBC)宫颈细胞学标本一般比非妇科标本较少的细胞,如上所述。此外,妇科LBC标本主要包含个别分散脱落宫颈粘膜表层细胞。由于这个原因,适当的细胞结构内的细胞块切片可能不能没有一种特殊的办法来实现。由于这些单分散的的块细胞成分可以不被的剖切期间histotechnologist看到,在该细胞开始出现在路段的水平不能赞赏并可能会丢失,或者由过去的水平切割与大多数细胞或不切割到足够深的层次最高浓度的样品细胞(图4)。 Shidham的协议地址使用嵌入中型和常规实验室设备(2)(图2)HG的这些问题都。

该协议涉及以下两个主要特点(图1):

  1. 相邻和平行的切割表面的细胞块(图5)在一个狭窄的飞机的单分散细胞的浓度和对齐。
  2. 包括像一盏明灯,黑暗 AV作为一个路标标记。这是实现以下功能的关键:
    1. 确定和监测细胞集中在细胞块的水平。深色路标曝光突出分散的单细胞在细胞块的大部分预计将位于(图4)的水平,这可以防止过度切削 (切割过去的水平大部分细胞)或伤人 (不削减到足够深的层次最高浓度的样品细胞)的细胞块,并选择部分允许在相应的最高浓度的细胞块水平细胞。
    2. 黑暗的部分彩色路标,也可作为一个参考点,进行调查和确定不同的序列在不同的幻灯片上的细胞块(图4)部分完全相同的细胞。同时解释和评估的坐标属性,特定的细胞由上海化学工业区的做法等immunoprofile遵循相同的细胞在不同的部分(6,7),这是非常重要的。

虽然OUř协议标准化和液基宫颈细胞学标本报告,它也可以用于其他许多非妇科标本,如积液液,活检,刷检,囊肿内容等,以提高诊断率,此外AV标记将有助于改善上海化学工业区的方法应用在这些标本的细胞块切片的免疫组织化学评价。包埋剂,可能会被替换其他试剂与适当的修改,在有关步骤。 HG可能会被替换血浆(纤维蛋白原)在室温下(1)凝血酶凝胶。

Acknowledgements

作者感谢克里斯Chartrand,HT(ASCP)展示了部分与AV标记的细胞块切割。

References

  1. Shidham, V. B., Epple, J. Chapter 14, Appendix I: Collection and processing of effusion fluids for cytopathologic evaluation. Cytopathologic Diagnosis of Serous Fluids. Shidham, V. B., Atkinson, B. F. Elsevier. Forthcoming.
  2. Varsegi, G., D'Amore, K., Shidham, V. p16INK4a Immunocytochemistry as an Adjunct to Cervical Cytology - Potential Reflex Testing on Specially Prepared Cellblocks from Residual Liquid Based Cytology (LBC) Specimens. Modern Pathology 22: 98th Annual Meeting of United States and Canadian Academy of Pathology. 2009 Mar 7-13, Boston, Ma, Abstract 424 97a-97a (2009).
  3. Nigro, K., Tynski, Z., Wasman, J., Abdul-Karim, F., Wang, N. Comparison of cell block preparation methods for nongynecologic ThinPrep specimens. Diagn Cytopathol. 35, 640-643 (2007).
  4. Saleh, H. A., Hammoud, J., Zakaria, R., Khan, A. Z. Comparison of Thin-Prep and cell block preparation for the evaluation of Thyroid epithelial lesions on fine needle aspiration biopsy. CytoJournal. 5, 3-3 (2008).
  5. Kyroudi, A., Paefthimiou, M., Symiakaki, H., Mentzelopoulou, P., Voulgaris, Z., Karakitsos, P. Increasing diagnostic accuracy with a cell block preparation from thin-layer endometrial cytology: a feasibility study. Acta Cytol. 50, 63-69 (2006).
  6. Atkinson, B. F. Chapter 5: Immunocytochemistry of effusion fluids: introduction to SCIP approach (Chapter 5. Cytopathologic Diagnosis of Serous Fluids. Shidham, V. B., Atkinson, B. F. Elsevier. (2007).
  7. Shidham, V. B. Chapter 15, Appendix II: Immunocytochemistry of effusions processing and commonly used immunomarkers. Cytopathologic Diagnosis of Serous Fluids. Shidham, V. B., Atkinson, B. F. Elsevier. (2007).

Comments

15 Comments

  1. This is a very interesting protocol and we would like to try it with tissue culture cells. Can the authors please share the vendor and product number for the flat bottom glass tubes and a protocol to make an AV marker? Thanks!

    Reply
    Posted by: Anonymous
    August 2, 2010 - 6:16 PM
  2. Details on supplies as requested (April 16, ²011)-




    Flat bottom glass tubes with cap-

    Ordered from Fisher (about $ 60 for 144)

    Catalog # 03-339-²6B

    Vials, shell with seal closure (lead), unattached

    Size: 15 x 45 mm, 1 Dr.

    Qty: 144



    Dark colored AV marker

    Ordered from

    BioInnovation

    456²0 Elmwood Circle

    Canton, MI 48188

    (²6²) 797 03²3
    http://bioinnovationllc.com/uploads/Oreder_form_11-6-09N__PDF_.pdf

    Catalog # 09-991-AVM100

    Dark colored AV marker for cell block depth perception and SCIP mapping

    Qty: 100



    The flat bottom plastic tube could be any- we use plastic Slide container with slots similar to Coplin jar (can be reused) from Fisher-

    Fisher Scientific* PROTOCOL* Cytology Transport System
    http://www.fishersci.com/wps/portal/PRODUCTDETAIL?prodcutdetail='prod'&productId=649503&catalogId=²910²&matchedCatNo=²3034803&pos=13&catCode=SA_SC&endecaSearchQuery=%²3store%3DSafety%²3N%3D0%²3rpp%3D15&fromCat=yes&keepSessionSearchOutPut=true&fromSearch=Y&searchKey=container||slide||plastic&highlightProductsItemsFlag=Y



    Cat No. ²3-034803

    Case of 100 for $109.18

    Reply
    Posted by: Vinod S.
    April 16, 2011 - 1:13 PM
  3. Although we would like to try it with cell lines, There are problems. Can the authors please share the vendor and product number for the flat bottom glass tubes and an AV marker? Thanks!

    Reply
    Posted by: Anonymous
    March 30, 2011 - 3:31 AM
  4. Details on supplies (April 16, ²011)-

    Flat bottom glass tubes with cap-

    Ordered from Fisher (about $ 60 for 144)

    Catalog # 03-339-²6B

    Vials, shell with seal closure (lead), unattached

    Size: 15 x 45 mm, 1 Dr.

    Qty: 144



    Dark colored AV marker

    Ordered from

    BioInnovation

    456²0 Elmwood Circle

    Canton, MI 48188

    (²6²) 797 03²3
    http://bioinnovationllc.com/uploads/Oreder_form_11-6-09N__PDF_.pdf

    Catalog # 09-991-AVM100

    Dark colored AV marker for cell block depth perception and SCIP mapping

    Qty: 100



    The flat bottom plastic tube could be any- we use plastic Slide container with slots similar to Coplin jar (can be reused) from Fisher-

    Fisher Scientific* PROTOCOL* Cytology Transport System
    http://www.fishersci.com/wps/portal/PRODUCTDETAIL?prodcutdetail='prod'&productId=649503&catalogId=²910²&matchedCatNo=²3034803&pos=13&catCode=SA_SC&endecaSearchQuery=%²3store%3DSafety%²3N%3D0%²3rpp%3D15&fromCat=yes&keepSessionSearchOutPut=true&fromSearch=Y&searchKey=container||slide||plastic&highlightProductsItemsFlag=Y



    Cat No. ²3-034803

    Case of 100 for $109.18

    Reply
    Posted by: Vinod S.
    April 16, 2011 - 1:15 PM
  5. What can be applications, limitations, sensitivity and specificity of a cell block?

    Reply
    Posted by: Anonymous
    April 14, 2011 - 1:33 AM
  6. This will be a long discussion. Some aspects are stated in the discussion of the article.
    About sensitivity and specificity- arbitrarily the method has high yield of diagnostic representative material if present in the specimen.

    Reply
    Posted by: Vinod S.
    April 16, 2011 - 1:15 PM
  7. Is it necessary for your specimen to be totally fixed prior to actually making the gel-block. For example we have a 6 hour minimum fix time --is this done prior to making, or dŒs the NBF actually permeate the gel block> How will this type of material effect prognostic markers like Her ²? Thanks Deb

    Reply
    Posted by: Anonymous
    April 15, 2011 - 4:45 PM
  8. The specimen may be prefixed prior to embedding in Histogel or the cell block may be prepared from unfixed specimen to be postfixed after embedding in Histogel.
    The total fixation time will be same as any other protocol applicable for a particular immunomarker including Her².

    Reply
    Posted by: Vinod S.
    April 16, 2011 - 1:11 PM
  9. what are the samples suitable for cell block?

    Reply
    Posted by: Anonymous
    April 19, 2011 - 4:48 AM
  10. Although this protocol is published with reference to cervical cytology specimens, we have used it for similar other comparable specimens with singly scattered cells and small groups/tissue fragments.

    In brief, this protocol may be applied to all cytology specimens or other specimens with dispersed isolated cells or small groups of cells. Some of the examples of the specimens in clinical practice include- cervical cytology, endocervical curretings, serous fluids and washings, various brushes & washings (e.g.- bronchial, biliary e.t.c.), urine, FNA needle rinses. This will also be an excellent method for preparing cell blocks from various tissue cultures.

    The current protocol is optimized for specimens with relatively low cellularity, due to this it should be adjusted appropriately for hypercellular specimens by adjusting the sample volume. For example, for some highly cellular serous fluids, the volume of sediments should be appropriately reduced.

    Reply
    Posted by: Vinod S.
    April 19, 2011 - 11:22 AM
  11. Update on Dark colored AV marker

    BioInnovation LLC
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    Phone: (²6²) 797 03²3

    Order from at:
    http://bioinnovationllc.com/uploads/01_Order_form__GI-MI_-_5-13-²01².pdf Catalog # 09-991-AVM100

    Dark colored AV marker for cell block depth perception and SCIP mapping

    Qty: 100

    Reply
    Posted by: Vinod S.
    November 9, 2012 - 1:31 PM
  12. Hello,
    We are trying to use HistoGel to create cell blocks from cultured cells and we're having some trouble. We are considering using the suggested protocol, and so I have a few questions:
    1. Do you fix the cells prior to their inclusion in the HistoGel button? If so, how do you do it?
    2. Which protocol do you recommend for the paraffinization and embedding of the cell blocks?
    Thanks so much in advanced for your help,
    Bat S.

    Reply
    Posted by: Shachaf B.
    July 25, 2013 - 8:10 AM
  13. Responding to your questions (sorry for the delayed response):

    1. Do you fix the cells prior to their inclusion in the HistoGel button? If so, how do you do it?
    Response: No, there is no need to fix (rather if they are not fixed it is better). The final cell block as solidified Gel is then put in the fixative for suitable time (E.g. about 2-3 hours in 10% formalin).

    2. Which protocol do you recommend for the paraffinization and embedding of the cell blocks?
    Response: Any protocol suitable to your lab or the tissue processor in use is OK!

    ____________________________________________

    Additional Details on supplies provided previously are also updated below;
    (10-22-2014)

    Flat bottom glass tubes with cap-

    Ordered from Fisher

    Catalog # 03-339-26B

    Vials, shell with seal closure (lead), unattached

    Size: 15 x 45 mm, 1 Dr.
    Pack of 144 for $37.88
    http://www.fishersci.com/ecomm/servlet/fsproductdetail?storeId=10652&productId=674157&catalogId=29103&matchedCatNo=0333926B&fromSearch=1&searchKey=26B||339||03&highlightProductsItemsFlag=Y&endecaSearchQuery=%23store%3DHealthcare%23nav%3D0%23rpp%3D25%23offSet%3D0%23keyWord%3D03-339-26B%23searchType%3DPROD%23SWKeyList%3D%5B%5D&xrefPartType=From&savings=0.0&xrefEvent=1413999575562_0&searchType=PROD&hasPromo=0
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    Ordered from

    BioInnovation LLC
    http://www.bioinnovationllc.com
    26277 E River Rd
    Grosse Ile, MI 48138

    (262) 797 0323

    Order form:
    http://www.bioinnovationllc.com/uploads/01_Order_form__GI-MI_-_5-13-2012.pptx

    Info:
    http://bioinnovationllc.com/uploads/00_Protocols__GI-MI_-_5-13-2012.pdf

    Catalog # 09-991-AVM100

    Dark colored AV marker for cell block depth perception and SCIP mapping

    Qty: 100

    __________________________________________

    The flat bottom plastic tube could be any- we use plastic Slide container with slots similar to Coplin jar (can be reused) from Fisher-

    Fisher Scientific* PROTOCOL* Cytology Transport System

    Cat No. 23-034803
    http://www.fishersci.com/ecomm/servlet/fsproductdetail?storeId=10652&productId=649503&catalogId=29103&matchedCatNo=23034803&fromSearch=1&searchKey=034803||23&highlightProductsItemsFlag=Y&endecaSearchQuery=%23store%3DHealthcare%23nav%3D0%23rpp%3D25%23offSet%3D0%23keyWord%3D23-034803%23searchType%3DPROD%23SWKeyList%3D%5B%5D&xrefPartType=From&savings=0.0&xrefEvent=1413999345377_0&searchType=PROD&hasPromo=0#
    _________________________________________

    Reply
    Posted by: Vinod S.
    October 22, 2014 - 1:45 PM
  14. Thanks for sharing the video and detailed information. Cell blocks set up from residual tissue fluids and fine-needle aspirations might be very helpful add-on to establish perfect cytopathologic diagnosis. Especially for the categorization of tumors, this will be highly useful. Its identification goes through various medical instruments that you can see on http://www.ilexmedical.com/products.php?act=cat for successful testing. This improved research provides fine cytomorphologic features which correspond closely to cells in Papanicolaou-stained mark and ensures finest preservation of histochemical and immunocytochemical functionalities. It is an interesting protocol for finding tissue culture cells; however can you share the vendor and product number.

    Reply
    Posted by: Shawn P.
    January 16, 2015 - 7:32 AM
  15. On consistent demand with many enquirers including phone calls, I am providing following details:
    One of the company (AV BioInnovation) is coming up withe kit based on this principle.

    Will send additional information including website details when available.
    In the meanwhile, please check workshop presented at 19th International Congress of Cytology, Pacifico Yokohama (Tokyo), Japan, May 28 - June 1, 2016:
    Shidham VB. Cell block & beyond: High yield goal- How to? (Workshop# 16)
    http://www.slideboom.com/presentations/1482461/00-CellBlock-%28ICC-2016--Japan%29-5-23-2016

    Reply
    Posted by: Vinod S.
    June 11, 2016 - 7:40 PM

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