Immunoguided laser assisted microdissection techniques for DNA methylation analysis of archival tissue specimens.
Author(s): Eberle FC, Hanson JC, Killian JK, Wei L, Ylaya K, Hewitt SM, Jaffe ES, Emmert-Buck MR, Rodriguez-Canales J
Publication: J Mol Diagn, 2010, Vol. 12, Page 394-401
PubMed ID: 20413681 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of slide pretreatment, slide type, antigen retrieval method, and IHC protocol on specimen loss and IHC staining of lymphomas.
Summary of Findings:
When slides were not pretreated, the tissue sections were lost during antigen retrieval, but the problem was eliminated by ultraviolet (UV) irradiation and poly-L-lysine coating of the slides prior to tissue mounting and incubation of the slides at 60 degrees C after tissue mounting. When antigen retrieval was performed with pepsin or HIER at 60 degrees C, none of the tissue sections were lost, but the authors report that pepsin applied on a horizontal surface at 37 degrees C for 10 min led to the best results. The authors report that longer pepsin treatments led to some tissue destruction, and longer HIER resulted in bleb formation underneath the membrane, however, the blebs did not affect subsequent IHC. HIER solution had no effects on CD20 staining, but CD30 staining was better using the Dako Target Retrieval solution as opposed to the Dako Cytomation retrieval solution. For CD20, the best results were obtained with 16 h of antigen retrieval, but for CD30, 42 h were necessary. The quality of CD20 and CD30 IHC staining was comparable in tissues mounted on PEN and glass slides.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Lymphoma
Platform:
Analyte Technology Platform Protein Immunohistochemistry Morphology Macroscopic observation Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Antigen retrieval Pepsin in vertical bath
Pepsin pipetted on horizontal slide
60 degrees C in Dako Retrieval solution
60 degrees C in Dako Cytomation retrieval solution
10 min
15 min
20 min
16 h
24 h
42 h
48 h
Immunohistochemistry Specific Targeted peptide/protein CD20
CD30
Immunohistochemistry Specific Antibody dilution/concentration 1:400
1:200
1:50
1:20
Biospecimen Aliquots and Components Type of slide Not pretreated
Pretreated
PEN
Glass
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Study Purpose
The purpose of this study was to determine the effects of using ILAM rather than H&E guided LAM for cell capture on DNA yield, quality and methylation status of lymphomas. DNA was extracted using the QIAamp DNA micro kit.
Summary of Findings:
Manual microdissection of tumor cells was successful using all three systems, but the LMD 6000 and PALM MicroBeam allowed for easier cell recovery and the software with the Arcturus XT was better suited for automatic cell recognition when IHC was visualized using DAB. 9-64% as much DNA was obtained from 75,000 ILAM tumor cells than from the same number of H&E guided LAM cells. However, amplification of 152 bp and 268 bp fragments of beta-globin was similarly successful from LAM and ILAM cells, and amplification of a 676 bp fragment of beta-globin was not possible with DNA obtained from ILAM or LAM cells. CpG methylation was strongly correlated between the ILAM and LAM stained cells (r>0.94). Of 1505 identified methylation sites, 1501 had the same status in ILAM and LAM cells, but 4 targets did show significant differences in methylation (p<0.0001-0.044).
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Lymphoma
Platform:
Analyte Technology Platform DNA PCR DNA Spectrophotometry Morphology H-and-E microscopy Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) PCR Specific Targeted nucleic acid Beta-globin
PCR Specific Length of gene fragment 152 bp
268 bp
676 bp
PCR Specific Type of tissue stain H&E stained
Immunostained
Biospecimen Aliquots and Components Cell capture method Leica LMD 6000
Arcturus XT
PALM MicroBeam