Cell block

Farhood16 specimens paired in Pap smear and cell block for comparison Not de-stained solution, pH 8, steamed for 40 min 8G7G3/1, Dako) Pap-stained smears gave identical positive rate as that obtained by cell block, but air-dried slides were unreliable. 

Liu J, Farhood A. Immunostaining for thyroid transcription factor-1 on fine-needle aspiration specimens of lung tumors a comparison of direct smears and cell block preparations. Cancer 2004 102 109-114. 

We recently conducted experiments to extract mRNA from a cell model (MBA-MB-486 cell line of human breast cancer) processed in both frozen and FFPE blocks in a comparable fashion (unpublished data). The cell model system was prepared in three ways for comparison (1) Positive Control Fresh Cell Pellet Two flasks of cells were collected in a pellet, and stored at -70°C until use (2) Frozen Cell Block Two flasks of cells was embedded in OCT... 

Compound (Miles Laboratories, Elkhart, IN), snap-frozen, and cut into sections for comparison with paraffin-embedded cell sections (3) FFPE Cell Blocks Six cell pellets were fixed in 10% neutral buffered formalin immediately after harvest, at room temperature for 6,12,24h, 3,7, and 30 days, respectively. For further comparison with the cell model system, recently collected sample of human breast cancer tissues were processed by OCT-embedding and snap-freezing the corresponding routine FFPE block that was obtained from the Norris Cancer Hospital and Research Institute at the University of Southern California Keck School of Medicine (USC). This tissue block was processed routinely (formalin-fixed 24h and processed by automatic equipment). 

However, ICC is not used as frequently in cytology as IHC in surgical pathology due to some technical issues, such as scanty or lack of diagnostic material in cell blocks or cytospins, and questionable reliability of immunos-taining on smears. 

In essence, the basic steps of making cell blocks consist of fixation, centrifugation to make cell pellet, transfer the pellet to a labeled tissue cassette which then is processed and embedded in paraffin. The most challenging component of this technique is the methods to harden the cell pellet so it can be easily picked up from the tube without losing precious material. With only a simple sedimentation technique, the cell pellet is usually small and friable. In order to harden the cell pellet, several technical modifications have been reported. The most popular methodology includes plasma-thrombin clot technique, agar technique, and fixation with Bouin s solution. 

Bouin s solution is one of the traditional ways to harden cell pellet. Some cytologists believe it provides the best cellular details, especially nuclear features in cell blocks.28 The major steps are (1) After centrifugation, fix the cell pellet with Bouin s solution. (2) After 2h, discard the solution. (3) Remove the hardened cell pellet from the tube, wrap it with lens paper, and transfer it into a cassette for further processing. We have been using this method for many years. In our experience, most of the time, ICC results are consistent with IHC from the surgical specimen. The biggest drawback of this method is the toxicity of Bouin s fixative which creates biohazard and safety issues for the laboratory. We also found cell blocks gave poor fluorescence in situ hybridization (FISH) results after Bouin s fixation. 

Recently, Hologic, Inc. developed a fully automated cell block system, Cellient system (Bedford, MA), expecting to improve capture, presentation, consistency, and efficiency of cell block preparation. This system is built on ThinPrep technology with vacuum-assisted filtration to maximize cell collection. The cell block can be produced in less than an hour.39 However, the biggest concern of this methodology is the fixation issue. Cellient system adopts alcohol instead of formalin for fixation, which unfortunately creates problems for ICC analysis. 

In summary, it is recognized that cell block technique provides a valuable ancillary cytopreparation for diagnostic cytopathology and ICC. There are many ways to prepare cell blocks. So far, there is no universally accepted method. As emphasized by Fowler and Lachar, it is advisable to validate any new cell block methodology by comparing its immunostaining results with IHC results from surgical samples in order to avoid misinterpretation.19... 

Even though cell block is the first choice for ICC, there is not always sufficient cell sample for cell block preparation. Therefore, it is necessary to develop techniques that allow multiple immunostaining on limited smears. 

Shin SJ, Chen B, Hyjek E, et al. Immunocytochemistry and fluorescence in situ hybridization in Her-2/neu status in cell block preparations. Acta Cytol. 2007 51 552-557. 

Harris M. Cell block preparation three percent bacterial agar and plasmathrombin clot methods. Cytotech. Bull. 1979 15 25-27. 

Liu H, Shi J, Wilkerson M, et al. Immunohistochemical detection of pl6INK4a in liquid-based cytology specimens on cell block sections. Cancer (Cancer Cytopathol.) 2007 111 74-82. 

Kulkarni M, Desai S, Ajit D, et al. Utility of the thromboplastin-plasma cell-block technique for fine-needle aspiration and serous effusions. Diagn. Cytopathol. 2009 37 86-90. 

Nigro K,Tynski Z, Wasman J, et al. Comparison of cell block preparation methods for nongynecologic ThinPrep specimens. Diagn. Cytopathol. 2007 35 640-643. 

Bhatia P, Dey P, Uppal R, et al. Cell blocks from scraping of cytology smear comparison with conventional cell block. Acta Cytol. 2008 52 329-333. 

Mayall F, Chang B, Darlington A. A review of 50 consecutive cytology cell block preparations in large general hospital. J. Clin. Pathol. 1997 50 985-990. 

Musso C, Silva-Santos M, Pereira F. Cotton block method one-step method of cell block preparation after fine needle aspiration. Acta Cytol. 2005 49 22-26. 

Krogerus L, Anderson L. A simple method for the preparation of paraffin-embedded cell blocks from fine needle aspirates, effusions and brushings. Acta Cytol. 1988 32 585-587. 

Cellient automated cell block system. Hologic, Inc., Bedford, MA, USA. http // www.cellientsystem.com/. 

STUDIES WITH FFPE CELL BLOCKS AND GEL-EMBEDDED PROTEINS... 

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