In all patients, 45 to 56 buffer aliquots covering the Ki67-stained sections were taken as negative controls. In patient 2 the same V_H 3-23 rearrangement was amplified from one cell and a buffer control. This cell was excluded from further analysis. In each of patients 4 and 5, a single unmutated V_H rearrangement unrelated to the rearrangements amplified from micromanipulated cells was amplified. In addition, for all patients, 20 to 24 CD3⁺cells were micromanipulated from adjacent CD3-stained sections. Eight buffer aliquots taken from each of these sections were always negative. From one CD3⁺ cell of patient 3, an unmutated V_κ and a unmutated V_λ rearrangement were amplified.

All sequences have been deposited in the EMBL database under accession numbers AJ406595-925, AJ298845-863, and AJ299840-944.

Pt indicates patient, PCR, polymerase chain reaction; PTGC, progressively transformed germinal center.

PCR was performed for V_H, V_κ, and V_λ gene rearrangements. Cells from which combinations of rearrangements indicative of cellular contamination were obtained (more than 2 rearrangements per locus or combinations of informative unmutated rearrangements with rearrangements mutated more than 5%) were excluded from further analysis (one Ki67⁺ cell of patient 2; 3 cells of PTGC1 of patient 5; one cell of PTGC2 of patient 5).

Cells from which only unmutated V_κ rearrangements were obtained are uninformative regarding somatic hypermutation.12 13 

Patient 1: 6 of 18 informative rearrangements mutated, obtained from 6 of 14 informative samples. Patient 2: 7 of 40 informative rearrangements mutated, obtained from 6 of 23 informative samples. Patient 3: 3 of 21 informative rearrangements mutated, obtained from 3 of 16 informative samples.

From one Ki67⁺ cell and a Ki67⁻ sample, the same unmutated and potentially functional V_λ 3h rearrangement with 2 N-nucleotides was amplified. These 2 cells likely represent members of a clone, though it cannot be excluded that the 2 cells are clonally unrelated.