HRS-like and B-CLL cells were analyzed together with T cells, and the controls were aspirated samples of the buffer covering the tissue sections during the micromanipulation. The data on patient 1 represent a summary of three independent micromanipulation experiments, and one repeat experiment was performed for patient 2. The clonalV_H and V_γ genes from HRS-like cells were coamplified in one cell from patient 1 (V_H4 and V_λ3gene 3a), in three cells from patient 2, and in one cell from patient 3. Coamplification of the V_H andV_L gene rearrangements from single cells was also seen for most B-CLL V region genes. In addition, amplification of the V region genes obtained from single B-CLL cells (and no additional other ones) and also from whole tissue DNA confirmed assignment of these genes to the B-CLL clones. Cells were analyzed with several different primer combinations, as outlined in Table 1.

The efficiency of the amplification of a V region gene from a single cell is usually below 50%,8 and therefore, sometimes two B cells of the CLL were micromanipulated and transferred into a PCR tube to minimize PCR work. We did not sequence 9V_H4 and 9 V_λ3gene rearrangements from the B-CLL cells of patient 1, 1V_κ2 gene rearrangement from the B-CLL cells of patient 2, and 1 V_H3 gene rearrangement from B-CLL cells of patient 3. The V gene segments used in the rearrangements are given in brackets. Identical V gene designations indicate clonally related V gene rearrangements. In patient 3,1c-1 and 1c-2 represent clonally unrelated rearrangements of the V_λ1 gene1c, amplified from HRS-like and B-CLL cells, respectively (but in both cases repeatedly obtained from the HRS-like and B-CLL cells).

Indicates that one sequence is identical to theV_H4 gene rearrangement repeatedly amplified from B-CLL cells.

Indicates that this rearrangement is identical to theV_H4 gene rearrangement of the B-CLL cells.

Indicates that one product (V_H5-51, in-frame, 19% mutation) was obtained only once. IgD-positive B-cells were analyzed to control the efficiency of the single-cell PCR (data not shown).