Immunohistochemical analysis
All FL cases were stained for CD20 (DAKO, Hamburg, Germany; dilution 1:500), CD10, CD23 (both Novocastra, Berlin, Germany; both dilution 1:30) CD3 (DCS, Hamburg, Germany; dilution 1:100), BCL6 (Zytomed, Berlin, Germany; dilution 1:25), MIB1 (Ki67; DAKO, Hamburg, Germany; dilution 1:200) and BCL2 (clone 100/D5 against residues aa 41–54; DAKO, Hamburg, Germany; dilution 1:50). Cases that were negative in the staining with BCL2 (100/D5) were additionally stained with the two alternative BCL2 antibodies clone E17 (Zytomed, Berlin, Germany; dilution 1:50, against residues aa 61–76) and SP66 (Cell Marque, Rocklin, California, USA; dilution 1:100, against the N-terminal portion of the protein). Cases of “in situ follicular neoplasia” (ISFN) were stained with BCL2 (100/D5) and MIB1. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue sections on an automated immunostainer (Ventana Medical Systems©, Tucson AZ, USA) following the manufacturer’s protocols.
Fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION)
In this study, FICTION, used as a combination of immunohistochemistry for the cell typing and FISH (fluorescence in situ hybridization) for the detection of the t(14;18), was carried out on 4-μm-thick paraffin sections. For the identification of mantle zone B-cells, endothelial cells and follicular dendritic cells, we used IgD (DAKO, FLEX polyclonal Rabbit Anti-Human IgD, Cat.-No.: A0093), CD34 (Clone DBEnd-10, DAKO Denmark, Code M7165), and clusterin antibodies (Apolipoprotein J) (Novocastra™/LEICA MICROSYSTEMS, Newcastle, UK).
Paraffin sections were de-waxed (3× times for 10 min in Xylene) and rehydrated (2× times for 5 min in ethanol). Endogenous peroxidase activity was blocked using 3% H2O2 for 10 min. After antigen retrieval by pressure cooking in sodium citrate buffer (pH 6.0) for 3.5 min and rinsing with TBS (pH 7.4), the slides were incubated for 3 min at 37 °C with 250 μl/ml Pepsin (Sigma-Aldrich/Merck, Sant-Louis/Missouri, Cat. No.: P6887, Pepsin stock solution 25 mg/ml, pepsin final working solution 250 μg/ml diluted in 37 °C pre-heated sodium chloride). After treatment with blocking solution for 15 min primary antibody incubation for 60 min at room temperature was performed. All antibody mixes, including the secondary antibodies, were diluted with the ready to use Antibody Diluent from DAKO/Agilent Technologies (Cat. No. S0809).
For IgD staining, a dilution of 1: 400 was used for the preparation of the antibody mix and a total volume of 800 μl was applicated per slide. Thus, 2 μl of IgD antibody plus 798 μl of antibody diluent were used for the IgD mix.
For the clusterin staining a dilution of 1: 30, for the CD34 staining a dilution of 1: 50 was used. The secondary antibodies were labelled with Cy™2 and Cy™3 antibodies. The dilution of the Rabbit Anti-Mouse Cy™3 antibody was 1: 25 and the Goat Anti-Mouse Cy™2 and Cy™3-labelled antibodies were used in a 1: 50 dilution (Table
1).
Table 1
Primary and secondary antibodies for the FICTION method
Polyclonal Ab Rabbit anti-Human IgD Code: A0093 | DAKO/Agilent Technolo. | 1:400 | 60 min/RT | Goat anti Rabbit IgG (H + L) | Jackson ImmunoResearch (Dianova distrib.) | 1:25 | 60 min/RT | Cy™3, Code:111–165-144 |
mAb Mouse anti-Human CD34 Class II Clone QBEnd-10 Code: M7165 | DAKO/Agilent Technolo. | 1:50 | 60 min/RT | Goat anti Mouse IgG (H + L) | Jackson ImmunoResearch (Dianova distrib.) | 1:50 1:25 1:50 | 60 min/RT | Cy™2, Code:115–228-003 Cy™3, Code:115–165-003 Alexa Flourʘ 488, Code: 115–545-003 |
mAb Mouse anti-Human IgG1 kappa Clusterin (Apolipoprotein J) Code: NCL-CLUSTERIN | Novocastra (LEICA distribut.) | 1:30 | 60 min/RT | Goat anti Mouse IgG (H + L) | Jackson ImmunoResearch (Dianova distrib.) | 1:50 1:25 1:50 | 60 min/RT | Cy™2, Code:115–228-003 Cy™3, Code:115–165-003 Alexa Flourʘ 488, Code: 115–545-003 |
After incubation of the secondary antibody, the slides were washed in graded alcohol series and dried on air. Before the application of the Vysis LSI BCl2 dual color break apart rearrangement probe (Abbott Molecular/Vysis, Illinois/USA, Cat. No.: 05 N51–020) was denatured for 5 min at 73 °C. After applying the probe and sealing the coverglass with Fixogum, a rubber cement special adhesive (Fixogum, Tamm/Germany, Cat. No. 290117000), the mixture was incubated in a humid chamber (ThermoBrite from Abbott Molecular/Vysis, Cat. No. 07 J91–020) for 17 h at 37 °C. On the second day, adhesive and coverglass were carefully removed and the slides were washed with 2xSSC at room temperature for 5 min. After an additional wash step with the detergent 0.3% NP-40 at 73 °C in a 2xSSC solution, a final washing with pure 2xSSX was carried out for 5 min to remove the non-(specific)-bound probe. After quick dehydration series in ethanol and air-dried the fluorescence dye DAPI (4′, 6-diamidine-2-phenylindole, component of the BCl2 probe kit from Abbott/Vysis) was used for counter-staining.
Appropriate positive controls from FL and negative controls from hyperplastic tonsils were evaluated simultaneously. In a translocation negative cell the expected signal pattern is two orange/green (yellow) fusion signals. Translocation positive cells showed one separated orange and green signal pair in addition to an intact fusion signal of the second allele.
The cut-off value for the presence of the t(14;18) was calculated with 10%. The evaluation of the signals was carried out using a Zeiss Axio Scope A1 microscope (HXP 120C) (Zeiss, Oberkochen, Germany). The fluorescence filters used for the two signals from the FISH for spectrum green and orange were Cyanine Cy™2 (excitation = 493 nm / emission = 519 nm), Indocarbocyanine Cy™3 dye (excitation = 550 nm / emission = 570 nm) and DAPI (excitation = 350 nm / emission = 470 nm).
For the evaluation as a first step the intensity of the immunostaining of the respective case was compared to the controls. Several high power fields (600× magnification) were screened and the IgD-, CD34- and clusterin-positive cells, respectively were evaluated. A total of at least 100 positively stained cells containing both signal types and a DAPI stained cell nucleus were evaluated per case. Analyses of all cases were performed in triplicates.
For photo documentation the supplied Zeiss software and the Zeiss Monochromator camera (Zeiss, Oberkochen, Germany) were used.