1. Detection of Genetic Alterations by ImmunoFISH Analysis of Whole Cells Extracted from Routine Biopsy Material 
Göran Mattsson, Soo Yong Tan, David J.P. Ferguson, Wendy Erber, Susan H. Turner, Teresa Marafioti, David Y. Mason 
The Journal of Molecular Diagnostics 
Volume 9, Issue 4, Pages (September 2007)
DOI: /jmoldx
Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2. Figure 1
Ultrastructural features of cells isolated from paraffin-embedded tonsil. a: A cluster of five cells, all of which appear to be partially or completely intact. Scale bar = 1 μm. b: Example of a nucleus with a small amount of attached cytoplasm. Scale bar =1 μm. c: Enlargement of the enclosed area in b showing the periphery of the nucleus (N) and rough endoplasmic reticulum (arrowed)within the attached cytoplasm. Scale bar = 0.5 μm. d: Two adjacent cells are illustrated, one of which (on the left) is partially intact, containing both endoplasmic reticulum and mitochondria (arrows), whereas the other (on the right) consists of a nucleus surrounded by a very thin rim of residual cytoplasm. Scale bar = 1 μm. Inset: Detail showing a mitochondrion. Scale bar = 0.5 μm. e: Details of the cytoplasm of a plasma cell showing well-preserved endoplasmic reticulum (ER) and mitochondria (Mi). Scale bar = 0.5 μm.
The Journal of Molecular Diagnostics 2007 9, DOI: ( /jmoldx )
Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3. Figure 2
Double immunofluorescence labeling of cells isolated from paraffin-embedded tonsil. The different antigens detected are indicated in colors corresponding to the fluorochrome used. All images show blue DAPI nuclear counterstaining. Top row: Many cells are positive for markers of B cells (CD20 and the Oct2 transcription factor) or T cells (CD3 and the adaptor protein SLP76), and there is no overlap between these markers. A plasma cell is recognizable because of its cytoplasmic labeling for CD79a. Bottom row: Immunofluorescence staining for Ki-67 in combination with CD3, CD20, and CD79a allows assessment of the proliferation status of T and B cells. Magnification, ×1000.
The Journal of Molecular Diagnostics 2007 9, DOI: ( /jmoldx )
Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

4. Figure 3
Flow cytometric analysis of cells isolated from paraffin-embedded tonsils with markers for B cells (CD20) and T cells (CD3 and the adapter protein SLP76) showing log fluorescence versus cell counts. In the negative control sample, the primary antibody was omitted.
The Journal of Molecular Diagnostics 2007 9, DOI: ( /jmoldx )
Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

5. Figure 4
Cells isolated from paraffin-embedded lymphoma biopsies analyzed by the immunoFISH technique in which a specific marker is visualized using a blue fluorochrome and gene configuration is assessed using conventional red and green FISH probes. Cells lacking a marker are outlined in red (non-neoplastic cells) or white (neoplastic cells). Note that the neoplastic cells (detected by Ki-67 labeling) in a diffuse large B-cell lymphoma sample (but not the non-neoplastic cells) in the fourth row carry multiple copies of the BCL2 gene, three copies of the BCL6 gene, and a split IGH gene. Magnification, ×1000.
The Journal of Molecular Diagnostics 2007 9, DOI: ( /jmoldx )
Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

6. Figure 5
A: ImmunoFISH analysis of cells isolated from a chronic myeloid leukemia trephine biopsy, using dual fusion probes for the t(9;22) translocation involving ABL and BCR genes. This anomaly is seen not only in myeloid cells (CD15- and MPO-positive) but also in erythroid (glycoprotein-positive) and megakaryocytic (LAT-positive) cells. The white outlines indicate marker-negative cells. B: A tissue section and an isolated cell preparation from a case of mantle cell lymphoma analyzed for the t(11;14) translocation using CCND1-IGH dual-fusion probes (DAPI counterstain). The background cytoplasmic fluorescence and overlapping/adjacent nuclei in thick parts of the tissue section would render interpretation difficult. These problems are less evident in a thinner part of the section but are essentially absent from the isolated cell preparation. ImmunoFISH labeling of a diffuse large B-cell lymphoma (DLBCL) shows triple copies of C-MYC in neoplastic lymphoid cells (circled white) and a normal pattern in an endothelial cell (immunostained by antibodies against and von Willebrand factor CD34). Magnification, ×1000.
The Journal of Molecular Diagnostics 2007 9, DOI: ( /jmoldx )
Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

7. Figure 6
A: A six-spot cellular “miniarray” of cells isolated from six different paraffin blocks immunostained for the proliferation marker Ki-67 (blue) and probed with a BCL2 split apart probe (green and red). Ki-67-negative neoplastic cells are outlined in white. There is sufficient separation between each sample to allow immunoFISH analysis of each sample with different antibody/probe combinations. B: A higher density, 50-spot cellular array for screening multiple biopsies with a single antibody/probe combination. Magnification, ×1000.
The Journal of Molecular Diagnostics 2007 9, DOI: ( /jmoldx )
Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions