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Preparation of compact agarose cell blocks from the residues of liquid-based cytology samples.

Choi SJ, Choi YI, Kim L, Park IS, Han JY, Kim JM, Chu YC - Korean J Pathol (2014)

Bottom Line: The resulting agarose cell blocks were trimmed and represented on a CMA for high-throughput analysis using immunocytochemical staining.The SurePath residues were effectively and entirely incorporated into compact agarose cell buttons and embedded in paraffin.This agarose-based compact cell block technique enables preparation of high-quality cell blocks by using up the residual SurePath samples without loss of diagnostic material during cell block preparation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Inha University Hospital, Inha University School of Medicine, Incheon, Korea.

ABSTRACT

Background: Inevitable loss of diagnostic material should be minimized during cell block preparation. We introduce a modified agarose cell block technique that enables the synthesis of compact cell blocks by using the entirety of a cell pellet without the loss of diagnostic material during cell block preparations. The feasibility of this technique is illustrated by high-throughput immunocytochemistry using high-density cell block microarray (CMA).

Methods: The cell pellets of Sure- Path residues were pre-embedded in ultra-low gelling temperature agarose gel and re-embedded in standard agarose gel. They were fixed, processed, and embedded in paraffin using the same method as tissue sample processing. The resulting agarose cell blocks were trimmed and represented on a CMA for high-throughput analysis using immunocytochemical staining.

Results: The SurePath residues were effectively and entirely incorporated into compact agarose cell buttons and embedded in paraffin. Sections of the agarose cell blocks revealed cellularities that correlated well with corresponding SurePath smears and had immunocytochemical features that were sufficient for diagnosis of difficult cases.

Conclusions: This agarose-based compact cell block technique enables preparation of high-quality cell blocks by using up the residual SurePath samples without loss of diagnostic material during cell block preparation.

No MeSH data available.


Related in: MedlinePlus

Agarose cell block preparation. The residue of a SurePath sample is placed in Eppendorf reaction tubes and fixed in formalin (A). Then, the material is pelleted and resuspended with a minimal volume of 3% (w/v) ultra-low gelling temperature agarose solution at room temperature and the resulting agarose cell suspension is allowed to gelate in the refrigerator at 4°C (B). Each of the solidified agarose cell buttons is transferred into the cap of the tube (C). Then, the cap with an agarose cell button at the bottom is filled with 3% standard agarose solution (D). The resulting agarose gel disk is removed from the cap with the aid of a 23-gauge needle (E) and then subjected to tissue processing for paraffin embedding. When the agarose gel disk is fractured during this step, it is carefully reconstructed and put in a tissue embedding mold and re-embedded in additional 3% standard agarose solution (F). The agarose cell block is trimmed to expose the agarose cell button (G). If a small piece of redundant formalin-fixed tissue (arrows) is embedded in advance in parallel with the cell button, it can be effectively used as a visible marker indicating the optimal cutting level of the cell block (H). Reconstructed gel disks are processed in the same way and embedded in paraffin blocks (I).
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f2-kjpathol-48-5-351: Agarose cell block preparation. The residue of a SurePath sample is placed in Eppendorf reaction tubes and fixed in formalin (A). Then, the material is pelleted and resuspended with a minimal volume of 3% (w/v) ultra-low gelling temperature agarose solution at room temperature and the resulting agarose cell suspension is allowed to gelate in the refrigerator at 4°C (B). Each of the solidified agarose cell buttons is transferred into the cap of the tube (C). Then, the cap with an agarose cell button at the bottom is filled with 3% standard agarose solution (D). The resulting agarose gel disk is removed from the cap with the aid of a 23-gauge needle (E) and then subjected to tissue processing for paraffin embedding. When the agarose gel disk is fractured during this step, it is carefully reconstructed and put in a tissue embedding mold and re-embedded in additional 3% standard agarose solution (F). The agarose cell block is trimmed to expose the agarose cell button (G). If a small piece of redundant formalin-fixed tissue (arrows) is embedded in advance in parallel with the cell button, it can be effectively used as a visible marker indicating the optimal cutting level of the cell block (H). Reconstructed gel disks are processed in the same way and embedded in paraffin blocks (I).

Mentions: We recruited consecutive 37 cytology case samples to be included in this study. There were cases that were from residual LBC samples after rendering the cytodiagnosis based on SurePath smears (SurePath, TriPath Care Technologies, Burlington, NC, USA), including 30 ultrasound-guided thyroid fine needle aspirates (FNAs), two lymph node FNAs and five serous effusions. Each SurePath residue was subjected to centrifugation and the cell pellet was resuspended in 200–300 µL buffered formalin. After incubation for 1 hour at room temperature, the suspension was entirely transferred to a 1.5 mL Eppendorf reaction tube and was centrifuged on a table-top centrifuge for 30 seconds at 15,000 rpm (Fig. 2A). Then, the supernatant was carefully discarded by pipetting, leaving a formalin-fixed cell pellet.


Preparation of compact agarose cell blocks from the residues of liquid-based cytology samples.

Choi SJ, Choi YI, Kim L, Park IS, Han JY, Kim JM, Chu YC - Korean J Pathol (2014)

Agarose cell block preparation. The residue of a SurePath sample is placed in Eppendorf reaction tubes and fixed in formalin (A). Then, the material is pelleted and resuspended with a minimal volume of 3% (w/v) ultra-low gelling temperature agarose solution at room temperature and the resulting agarose cell suspension is allowed to gelate in the refrigerator at 4°C (B). Each of the solidified agarose cell buttons is transferred into the cap of the tube (C). Then, the cap with an agarose cell button at the bottom is filled with 3% standard agarose solution (D). The resulting agarose gel disk is removed from the cap with the aid of a 23-gauge needle (E) and then subjected to tissue processing for paraffin embedding. When the agarose gel disk is fractured during this step, it is carefully reconstructed and put in a tissue embedding mold and re-embedded in additional 3% standard agarose solution (F). The agarose cell block is trimmed to expose the agarose cell button (G). If a small piece of redundant formalin-fixed tissue (arrows) is embedded in advance in parallel with the cell button, it can be effectively used as a visible marker indicating the optimal cutting level of the cell block (H). Reconstructed gel disks are processed in the same way and embedded in paraffin blocks (I).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215960&req=5

f2-kjpathol-48-5-351: Agarose cell block preparation. The residue of a SurePath sample is placed in Eppendorf reaction tubes and fixed in formalin (A). Then, the material is pelleted and resuspended with a minimal volume of 3% (w/v) ultra-low gelling temperature agarose solution at room temperature and the resulting agarose cell suspension is allowed to gelate in the refrigerator at 4°C (B). Each of the solidified agarose cell buttons is transferred into the cap of the tube (C). Then, the cap with an agarose cell button at the bottom is filled with 3% standard agarose solution (D). The resulting agarose gel disk is removed from the cap with the aid of a 23-gauge needle (E) and then subjected to tissue processing for paraffin embedding. When the agarose gel disk is fractured during this step, it is carefully reconstructed and put in a tissue embedding mold and re-embedded in additional 3% standard agarose solution (F). The agarose cell block is trimmed to expose the agarose cell button (G). If a small piece of redundant formalin-fixed tissue (arrows) is embedded in advance in parallel with the cell button, it can be effectively used as a visible marker indicating the optimal cutting level of the cell block (H). Reconstructed gel disks are processed in the same way and embedded in paraffin blocks (I).
Mentions: We recruited consecutive 37 cytology case samples to be included in this study. There were cases that were from residual LBC samples after rendering the cytodiagnosis based on SurePath smears (SurePath, TriPath Care Technologies, Burlington, NC, USA), including 30 ultrasound-guided thyroid fine needle aspirates (FNAs), two lymph node FNAs and five serous effusions. Each SurePath residue was subjected to centrifugation and the cell pellet was resuspended in 200–300 µL buffered formalin. After incubation for 1 hour at room temperature, the suspension was entirely transferred to a 1.5 mL Eppendorf reaction tube and was centrifuged on a table-top centrifuge for 30 seconds at 15,000 rpm (Fig. 2A). Then, the supernatant was carefully discarded by pipetting, leaving a formalin-fixed cell pellet.

Bottom Line: The resulting agarose cell blocks were trimmed and represented on a CMA for high-throughput analysis using immunocytochemical staining.The SurePath residues were effectively and entirely incorporated into compact agarose cell buttons and embedded in paraffin.This agarose-based compact cell block technique enables preparation of high-quality cell blocks by using up the residual SurePath samples without loss of diagnostic material during cell block preparation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Inha University Hospital, Inha University School of Medicine, Incheon, Korea.

ABSTRACT

Background: Inevitable loss of diagnostic material should be minimized during cell block preparation. We introduce a modified agarose cell block technique that enables the synthesis of compact cell blocks by using the entirety of a cell pellet without the loss of diagnostic material during cell block preparations. The feasibility of this technique is illustrated by high-throughput immunocytochemistry using high-density cell block microarray (CMA).

Methods: The cell pellets of Sure- Path residues were pre-embedded in ultra-low gelling temperature agarose gel and re-embedded in standard agarose gel. They were fixed, processed, and embedded in paraffin using the same method as tissue sample processing. The resulting agarose cell blocks were trimmed and represented on a CMA for high-throughput analysis using immunocytochemical staining.

Results: The SurePath residues were effectively and entirely incorporated into compact agarose cell buttons and embedded in paraffin. Sections of the agarose cell blocks revealed cellularities that correlated well with corresponding SurePath smears and had immunocytochemical features that were sufficient for diagnosis of difficult cases.

Conclusions: This agarose-based compact cell block technique enables preparation of high-quality cell blocks by using up the residual SurePath samples without loss of diagnostic material during cell block preparation.

No MeSH data available.


Related in: MedlinePlus