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When lymphocytes are isolated from blood using the Böyum method a sometimes severe contamination with non-lymphoid cells (NLC) is observed in cancer patients, but also in patients with non-malignant diseases. These non-lymphoid cells consist mainly of monocytes and granulocytes. A large monocytic contamination is found in cancer patients, while the percentages of monocytes in a patient group with benign diseases and in a normal donor group were equally lower than in the group of cancer patients. The granulocytic contamination covered a large range in both patient groups and the mean percentages did not differ. When lymphocyte studies are to be performed in vitro, allowances have to be made for these varying percentages of non-lymphoid cells. In addition a high percentage of non-lymphoid cells makes the presence of a malignant tumor more likely.

In an established double layer clonogenic assay, the PHA-leukocyte feeder colony assay, hairy cell leukemia (HCL) cells formed strong aggregates simulating colonies. After irradiation with 50 Gy, colony formation persisted. Even in a modified colony assay consisting of agar 0.5% overlayered by methylcellulose 0.9%, cell aggregation was still possible due to increasing fluidity of the methylcellulose during the culture period. Time-lapse video recordings confirmed prominent cell motility leading to pseudo-colony formation. Studies with bromodeoxyuridine incorporation showed a low proliferation index (up to 13%) of hairy cells. In conclusion, any assay that facilitates cell motility is unsuitable to study HCL colony growth.

Although follicle center cell (FCC) lymphomas represent mature B cells, a considerable percentage do not have detectable Ig production. We have used Southern blotting and the polymerase chain reaction (PCR) to study the involvement of translocations t(14;18) and t(8;14) in causing defective Ig production in 16 Ig- FCC-derived lymphomas and three Ig- B cell acute lymphoblastic leukemias. In 6 of 19 cases, a t(14;18) was present with the other allele either deleted or in germline. In two cases a t(14;18) and a t(8;14) affected both Ig alleles, as confirmed by karyotyping. In two other cases, rearrangement of both bcl-2 on chromosome 18 and c-myc on chromosome 8 were found as well. Although cytogenetic proof was not available, the latter was probably involved in t(8;14). Restriction map analysis of one more case showed rearrangement on the pseudo-JH3 gene on one allele and t(14;18) on the other. Thus, in 11 of 19 cases, defective Ig H chain production could be explained by the inactivation of both Ig H chain genes due to translocation of one allele, in combination with deletions or defective rearrangements of the other allele. In contrast, in 28 of 30 Ig+ lymphomas, one functional Ig H chain allele was found, either in, or not in, combination with t(14;18). In two cases a single rearranged Ig H chain allele was found in combination with rearrangement of bcl-2. No comigration of the single Ig rearrangement with bcl-2, however, was found both by Southern blotting and PCR, suggesting a variant bcl-2 translocation, which leaves the Ig H chain allele functionally intact.

In the PHA-leukocyte feeder colony assay--a fluid assay on top of an agar underlayer--colonies might not be the product of clonogenic cells but rather from aggregates, as was already shown for hairy cell leukemia (Leukemia Res. 11, 911 (1987)). To study the role of aggregation in this colony assay in other B-cell malignancies, we irradiated cells from B-chronic lymphocytic leukemia, B-non-Hodgkin's lymphoma and multiple myeloma. In nearly all cases, viable "colonies" were seen after irradiation, albeit in lower numbers. These data indicate that in the PHA-leukocyte feeder colony assay, a considerable percentage of colonies from a large variety of B-cell malignancies originate from aggregating rather than from proliferating cells.