Cytometric detection of DNA amplified with fluorescent primers:
applications to analysis of clonal bcl-2 and IgH gene rearrangements in
malignant lymphomas
RL Barker, CA Worth and SC Peiper
Department of Pathology, University of Louisville, KY 40202.
Follicular lymphomas comprise almost two thirds of the US adult non-
Hodgkin's lymphomas (NHL) and are the most common malignancy of B- lineage
lymphocytes. Polymerase chain reaction (PCR) protocols have been developed
to detect the t(14;18) translocation, which juxtaposes the bcl-2
proto-oncogene to the Ig heavy-chain (IgH) gene in 85% of follicular
lymphomas and monoclonal rearrangements of the IgH gene in B- cell NHL that
lack bcl-2 rearrangements. We used PCR to amplify bcl-2 and IgH
rearrangements in DNA from patients with lymphoproliferative disorders and
analyzed the products in parallel by gel electrophoresis and flow
cytometry, which detected PCR products incorporating fluoresceinated
oligonucleotide primers by sequence-specific capture to
oligonucleotide-coated magnetic beads. Overall, flow cytometry was superior
to electrophoresis of ethidium-bromide-stained agarose gels for detection
of products of nested PCR to detect intergenic rearrangements involving
bcl-2 and single primer-pair amplification of clonal rearrangement of IgH.
Flow cytometric analysis detected bcl-2 translocations in 12 of 13 CD10+
B-cell lymphomas and clonal IgH rearrangements in 14 of 17 monoclonal
B-cell populations. In contrast, analysis by gel electrophoresis detected
bcl-2 translocations in only 10 of 13 CD10+ and clonal IgH gene
rearrangements in only 9 of 17 monoclonal B-cell populations. Flow
cytometric analysis was more sensitive than gel electrophoresis and could
detect a 16-fold greater dilution of a bcl-2-amplified product than gel
electrophoresis. Similarly, flow cytometry could detect an amplification
product when template DNA was diluted 10,000-fold, whereas gel
electrophoresis only detected amplification products when template was
subjected to dilution between 100- and 1,000-fold. This shows the utility
of flow cytometry for the analysis of DNA amplification products
incorporating fluorochrome-labeled primers as a rapid, objective
alternative to conventional strategies. Because current-generation clinical
laboratories emphasize automation, flow cytometric analysis of PCR-
amplified products shows increased analytic sensitivity and offers a
vehicle for automation of DNA amplification tests.
Volume 83,
Issue 4,
pp. 1079-1085,
02/15/1994
Copyright © 1994 by The American Society of Hematology
