Double-staining chromogenic in situ hybridization as a useful alternative to split-signal fluorescence in situ hybridization in lymphoma diagnostics.
BACKGROUND
Malignant lymphomas are classified based on morphology, immunophenotype, genetics and clinical features. The pathological diagnosis is generally considered difficult and prone to mistakes. Since non-random chromosomal translocations are specifically involved in specific entities, their detection is an important adjunct for increasing the reliability of the diagnosis. Recently, split-signal fluorescence in situ hybridization has become available as a robust method to detect chromosomal breaks in paraffin-embedded formalin-fixed tissues. A bright field approach would bring this technology within the reach of every pathology laboratory.


METHODS
Our study was initiated to determine the consistency between chromogenic in situ hybridization and fluorescence in situ hybridization, both using split-signal probes developed for the detection of chromosomal breaks. Five hundred and forty cases of 11 lymphoma entities and reactive, benign lymphoid tissues, collected from eight different pathology laboratories, placed on 15 fluorescence in situ hybridization pre-stained tissue microarray slides, were double stained for the chromogenic hybridization. For each core morphology and actual signal were compared to the original fluorescence hybridization results. In addition, hematoxylin background staining intensity and signal intensity of the double-staining chromogenic in situ hybridization procedure were analyzed.


RESULTS
With respect to the presence or absence of chromosomal breaks, 97% concordance was found between the results of the two techniques. Hematoxylin background staining intensity and signal intensity were found to correspond. The overall morphology after double-staining chromogenic in situ hybridization had decreased compared to the initial morphology scored after split-signal fluorescence in situ hybridization staining.


CONCLUSIONS
We conclude that double-staining chromogenic in situ hybridization is equally reliable as fluorescence in situ hybridization in detecting chromosomal breaks in lymphoid tissue. Although differences in morphology, hematoxylin staining and chromogenic signal intensity vary between the tumor entities none of the entities appeared more easy or difficult to score.
van Rijk
Anke
A
Department of Pathology-824, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. a.vanrijk@pathol.umcn.nl
Svenstroup-Poulsen
Tim
T
Jones
Margaret
M
Cabeçadas
José
J
Cigudosa
Juan Cruz
JC
Leoncini
Lorenzo
L
Mottok
Anja
A
Bergman
Christiane Copie
CC
Pouliou
Evi
E
Dutoit
Stephen Hamilton
SH
van Krieken
Han J
HJ
D002863:Chromogenic Compounds; D019457:Chromosome Breakage; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence; D008223:Lymphoma / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D046888:Tissue Array Analysis
D002863:Chromogenic Compounds