Table 2.
Patient | Issue | Exp. | Primer set | Cells positive | PCR products | Rearrangements
|
|
---|---|---|---|---|---|---|---|
Repeated | Unique | ||||||
6104 | Lymph node | 1 | VH, Vκ FRI | 3/18 | 3 | 3 Vκ 1 | |
Lymph node | 2 | VH, Vκ FRI | 5/12 | 5 | 4 Vκ 1 | 1 Vκ 3 | |
Cytospin | 1 | VH, Vκ FRI | 5/16 | 6 | 5 Vκ 1 | 1 VH3 | |
6444 | Lymph node | 1 | VH, Vκ FRI | 0/6 | |||
VH, Vκ FRI | 1/6 | 1 | 1 VH3 6 | ||||
VHL | 4/17 | 4 | 4 VH3 | ||||
Cytospin | 1 | VHL | 10/18 | 10 | 10 VH3 | ||
Controls | |||||||
6104 | Lymph node | 1 | VH, Vκ FRI | 1/3 B cells | 1 | 1 VH4 | |
0/8 neg. controls | |||||||
Lymph node | 2 | VH, Vκ FRI | 3/3 B cells* | 6 | All 6 | ||
0/5 neg. controls | |||||||
Cytospin | 1 | VH, Vκ FRI | 2/10 CD30 neg. | 2 | 1 Vκ1, 1 VH3 | ||
0/4 buffer | |||||||
6444 | Lymph node | 1 | VH, Vκ FRI | 0/1 B cells | |||
0/2 neg. controls | |||||||
VH, Vλ FRI | 1/2 B cells | 1 | 1 VH3 | ||||
0/3 neg. controls | |||||||
VHL | 1/4 B cells | 2 | 1 VH3, 1 VH4 | ||||
0/8 neg. controls | |||||||
Cytospin | 1 | VHL | 0/12 neg. controls |
Three different primer sets were used, as indicated: (i) six VH and four Vκ family-specific primers hybridizing to sequences within FRI (VH, VκFRI), (ii) The same six VH primers together with eight Vλ family-specific FRI primers (VH, Vλ FRI), and (iii) five VH leader family-specific primers (VH). Controls consisted of B and T cells micromanipulated on the same day from neighboring sections or independent cytospins. In one experiment, CD30-negative cells were micromanipulated as control from the same cytospin that was used for the isolation of HRS cells. Neg. controls consist of micromanipulated T cells and aliquots of the buffer covering the sections or cytospins. Repeated rearrangements indicates clonally related rearrangements; unique rearrangements are rearrangements obtained only once. The three B cell samples of the second experiment of case 6104 each contained two B cells.