N. Sioutos et al., POLYMERASE CHAIN-REACTION VERSUS SOUTHERN BLOT HYBRIDIZATION - DETECTION OF IMMUNOGLOBULIN HEAVY-CHAIN GENE REARRANGEMENTS, Diagnostic molecular pathology, 4(1), 1995, pp. 8-13
To determine efficiently the clonality of B-cell lymphoproliferative d
isorders, we modified an immunoglobulin heavy-chain (IGH) gene rearran
gement polymerase chain reaction (PCR) assay that requires only a sing
le primer germline variable (V-H) and joining (J(H)) pair and does not
involve nested priming, blot hybridization, radioactivity, or sequenc
ing of the amplified PCR product. This simple PCR technique enabled de
tection of IGH gene rearrangements in as little as 10 pg (one cell equ
ivalent) of DNA or when the clonal-to-polyclonal B-cell ratio was expe
rimentally set at 1:1000. We detected IGH gene rearrangements in 83.5%
(71 of 85) of clonal B-cell processes, a sensitivity approaching that
of more cumbersome multiple primer and nested primer assays. Moreover
, this technique is equally effective with fixed tissues, either B5 or
formalin, and can be performed on minute samples, histologic sections
, fine-needle aspirates, or cerebrospinal fluids. When compared with c
onventional Southern blot analysis using a genomic J, probe, the PCR a
ssay demonstrated IGH gene rearrangements in 82% (37 of 45) of B-cell
processes positive by Southern blot. No false-positive results were ob
served in 29 negative control tissues. We now use IGH gene TCR routine
ly in our laboratory for the detection of clonal B-cells in virtually
any tissue sample as an aid in early diagnosis, staging, and monitorin
g, and the Southern blot procedure is reserved for only a minority of
diagnostic cases.