Variability of polymerase chain reaction detection of the bcl-2-IgH translocation in an international multicentre study

Abstract

The polymerase chain reaction to detect bcl-2-IgH rearrangements is presently carried out with widely disparate results. Further effort is required to bring forward a standard PCR protocol which can be re-tested in different laboratories to improve accuracy and reproducibility. The application of quantitative techniques such as real-time PCR may resolve many of the problems presently encountered.

Quantification of bcl-2/JH Fusion Sequences and a Control Gene by Multiplex Real-Time PCR Coupled with Automated Amplicon Sizing by Capillary Electrophoresis

“…5,6 Since our original report, 6 several investigators have designed real-time PCR assays to quantify the number of cells carrying the t (14;18), and these assays have proved to be equally sensitive and quantitatively more reliable than conventional single-round or nested PCR assays. 7,8 Real-time TaqMan PCR assays are based on the 5Ј33Ј exonuclease activity of Taq polymerase, and employ a non-extendable probe labeled with a fluorescent reporter dye at its 5Ј end and a quencher dye at its 3Ј end. 9 -13 The use of labeled probes complementary to target permits identification of specific PCR products.…”

Sensitivity and reproducibility of conventional qualitative and quantitative PCR assays for detection of the t(14;18)(q32;q21) chromosomal translocation in biopsy material from patients with follicular lymphoma

“…These restricted breakpoint localizations have allowed the design of primers specific for 5' regions of the MBR and mcr clusters on chromosome 18, and for conserved portions of the J H gene segments on chromosome 14, which have been widely used for standard polymerase chain reaction (PCR) assays (7,10). The combination of standard MBR and mcr primers with a J H consensus primer enables the detection of approximately 70% of BCL-2/J H rearrangements (7,10).…”

“…The combination of standard MBR and mcr primers with a J H consensus primer enables the detection of approximately 70% of BCL-2/J H rearrangements (7,10). Various methodologies in performing PCR with different primer sequences, methods of amplification and detection of amplified products have been described (7,8,(10)(11)(12).…”

“…The combination of standard MBR and mcr primers with a J H consensus primer enables the detection of approximately 70% of BCL-2/J H rearrangements (7,10). Various methodologies in performing PCR with different primer sequences, methods of amplification and detection of amplified products have been described (7,8,(10)(11)(12). Recently, quantitative real-time PCR has been extensively employed in most laboratories, particularly for detection of minimal residual disease (MRD) in follow-up of patients (13)(14)(15)(16)(17)(18)(19)(20)(21)(22).…”

“…Recently, quantitative real-time PCR has been extensively employed in most laboratories, particularly for detection of minimal residual disease (MRD) in follow-up of patients (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). The sensitivities of described methods vary greatly, mostly depending on the methodology (qualitative vs quantitative, single-round vs nested PCR) and choice of primers (10). Most previous studies were performed using DNA isolated from fresh peripheral blood or bone marrow aspirates (12)(13)(14)(15)(16)(19)(20)(21)(22)(23) and only a few studies used DNA derived from formalin-fixed, paraffin-embedded tissue specimens (11,21,23,24).…”