Southern Blot Analysis

The first widely used molecular method for detection of B-cell clonality was SBA, and it remains the molecular gold standard for detection of B-cell clonality. This is because the results of interlaboratory surveys testing for IGH clon-ality by SBA have consistently shown close to 100% agreement among participating laboratories, while marked interlaboratory variability has been seen when the same samples were analyzed by IGH PCR.38 SBA also is useful in testing for genes with extensive breakpoint heterogeneity (MYC) and for genes with multiple translocation partners (IGH, BCL6). SBA may be used to identify comigrating translocation partners, such as the IGH and BCL2 genes involved in t(14;18)(q32;q21) translocations (illustrated in Figure 32-4).

SBA for BCL detects changes that occur in a gene's germline structure during Ig gene rearrangement, which delete or create restriction endonuclease sites and lead to changes in the bands on the Southern blot. SBA of cells with nonrearranged Ig genes produces bands of characteristic size for a given restriction enzyme (germline bands). Cells with rearrangements or mutations in restriction sites will have bands of different sizes from the germline bands due to alterations of the relative positions of restriction sites (rearranged bands)(Figure 32-4).

The criteria existing for interpretation of antigen receptor SBA require that rearrangements are seen with two of three restriction enzymes for a positive monoclonal interpretation. Up to two rearrangement bands can be seen in any one digest lane, which can represent two rearranged IGH alleles (if the first rearrangement was unsuccessful

Figure 32-4. Parallel autoradiography of SBA of IGH(JH) and BCL2gene rearrangements in a patient with follicular lymphoma (FL). The negative control is a T-lymphoblastic cell line and shows a germline configuration of the IGH gene. A reactive lymphoid hyperplasia (RLH) shows a polyclonal smear of variably sized IGH rearrangements in normal B cells, while the biopsy from the patient with FL shows a decrease in the polyclonal background and the presence of two clonal IGH rearrangement bands, one above and one below the germline bands seen in all three specimens.The same membrane was stripped and reprobed with a BCL2 probe, and the arrows show the comigrating IGH and BCL2 rearrangement bands in the FL, indicating a translocation between these two genes.

and the second allele was also rearranged), or can be due to the creation of a restriction site within the region of the probe so different ends of the probe hybridize to different fragments of genomic DNA. Complete digestion of the genomic DNA is essential to proper interpretation, since partial digestion can create bands at nongermline positions.

The probes used for IGH SBA are usually against the IGH joining region (JH), rather than the constant region (C||), because heavy-chain switching deletes C| but not JH. For light-chain genes, Jk is also less frequently deleted than Ck in ^-expressing BCL. There is seldom a clinical use for CX probes. Rare BCL will lack a detectable JH rearrangement on IGH SBA, but these will typically show Ck rearrangement. Polyclonal B cells,each with a unique IGH rearrangement, produce a weak background smear of different-sized rearrangement bands on the SBA (Figure 32-4). The sensitivity of SBA for a monoclonal population is approximately 3% to 5% of the total cell population.

Successful SBA also requires high-molecular-weight DNA and thus can be reliably performed only on fresh or frozen tissue or cells; DNA from paraffin-embedded tissue is usually too degraded for successful SBA. SBA also requires large amounts of DNA, as 5 to 10 |g of DNA is used per restriction endonuclease. Typical IGH SBA using three restriction endonucleases therefore requires 15 to 30 |ig of DNA, which can be obtained from 106 cells or approximately 0.5 cm3 of fresh or frozen tissue. Because tissue requirements are more strenuous and SBA is more laborintensive, usually taking 2 to 3 weeks to obtain results, many clinical laboratories no longer perform SBA or use SBA infrequently, only as a backup for equivocal IGH PCR assays.

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