Technical Notes

B cell Clonality Analysis

Clinical Significance:

When the immunoglobulin heavy chain gene rearranges, a V (variable), D (diversity), and J (joining) region are brought together to form a functional unit. As part of the V-D-J joining process, random numbers of DNA basepairs are inserted between the V-D and D-J junctions, forming the third complementarity-determining region (CDRIII) that is involved in antigen specificity. Consequently, all non-neoplastic B cell populations exhibit a random distribution of distances between the V and J segments ⁽¹⁾.
The polymerase chain reaction allows one to produce large amounts of DNA, the ends of which are determined by the primers used. For B cell clonality analysis, the primers chosen are such that one corresponds to a consensus sequence common to most V segments, and the other a consensus sequence common to most J segments ⁽²⁾.
When the DNA is amplified, the length of the PCR product is determined by the number of random nucleotides added at the time of VDJ joining. In a non-neoplastic population of B cells, each PCR product is slightly different in size and appears as a smear on the gel while neoplastic B cell proliferations exhibit specific bands on a background of smear ^{(3, 4)}.
The assay would be useful in assignment of presumptive lineage in mature monoclonal lymphoproliferative disorders and in determination of clonality in atypical lymphoproliferations ^{(5, 6)}.

---

Methodology:

Patient DNA would be isolated, purified, and subjected to PCR amplification using oligonucleotide primers specific for the immunoglobulin heavy chain gene-joining region (chromosome 14) and a consensus primer specific for the immunoglobulin heavy chain variable regions (chromosome 14). An additional PCR amplification directed at a GAPDH gene segment would be performed as a control for sample DNA quality. PCR products would be analyzed by electrophoresis and UV transillumination of ethidium bromide-stained gels. The assays would be validated by a positive control reaction using DNA from a B-cell lymphoma with a clonal immunoglobulin heavy chain gene rearrangement and a negative control reaction using DNA from a reactive tissue sample with no evidence of a clonal immunoglobulin heavy chain gene rearrangement.

---

References:

1) Sklar J. and Longtine J. (1992) Cancer Suppl. 70:6, 1710.
2) Diss T.C., Pan L., Peng H. et al (1994) J. Clin. Pathol. 47, 493.
3) Taylor J.M.E., Spagnolo D.V., Kay P.H. (1997) Pathology 29, 309.
4) Philippe B., Delfau-Larue M.H., Epardeau B. et al (1999) Chest 115, 1242.
5) Segal G.H., Jorgensen T., Scott M. et al (1994) Hum Pathol. 25, 1276.
6) Wickert R.S., Weisenburger D.D., Tierens A. et al (1995) Blood 86, 2312.