Levels of circulating CD19+ cells in patients with multiple myeloma
T Rasmussen, L Jensen… - Blood, The Journal of the …, 2000 - ashpublications.org
T Rasmussen, L Jensen, HE Johnsen
Blood, The Journal of the American Society of Hematology, 2000•ashpublications.org4020 CORRESPONDENCE BLOOD, 15 JUNE 2000• VOLUME 95, NUMBER 12 least one
staining procedure, showing that the MoAbs were not competing for the same binding site
(epitope). In addition, the B4, Leu-12, and FMC63 epitopes were tested for sensitivity to
neuraminidase treatment. No effects were seen on the single staining or when MoAbs were
used in combinations. As a positive control for neuraminidase treatment, the CD34 molecule
expressed on KG1 cells was used. The median channel value of the neuraminidase …
staining procedure, showing that the MoAbs were not competing for the same binding site
(epitope). In addition, the B4, Leu-12, and FMC63 epitopes were tested for sensitivity to
neuraminidase treatment. No effects were seen on the single staining or when MoAbs were
used in combinations. As a positive control for neuraminidase treatment, the CD34 molecule
expressed on KG1 cells was used. The median channel value of the neuraminidase …
4020 CORRESPONDENCE BLOOD, 15 JUNE 2000• VOLUME 95, NUMBER 12 least one staining procedure, showing that the MoAbs were not competing for the same binding site (epitope). In addition, the B4, Leu-12, and FMC63 epitopes were tested for sensitivity to neuraminidase treatment. No effects were seen on the single staining or when MoAbs were used in combinations. As a positive control for neuraminidase treatment, the CD34 molecule expressed on KG1 cells was used. The median channel value of the neuraminidase-sensitive class I epitope was significantly lowered in all experiments, whereas the neuraminidase-insensitive class III epitope was unaltered.
In contrast to the Leu-12 FITC/PE, B4 PE, and FMC63 PE MoAbs, the B4 FITC and FMC63 FITC MoAbs showed an additional stained population originating from the monocyte gate when serum levels in the PBS staining buffer were 0%. But this population was reduced with increasing serum concentration. The effect of using PBS with different serum concentrations was identical on B cells from healthy donors and MM patients. To test the possibility that the identification of CD19 stained cells originating from the monocyte gate was due to unspecific binding of the CD19 MoAbs, these CD19 stained cells were flow-sorted as single cells and CD19 RT-PCR was performed. For both healthy donors and MM patients, the frequency of CD19 (mRNA) cells within this population was between 1: 100 and 1: 1000, documenting the unspecific binding of the B4 and FMC63 FITC MoAbs. When 10% serum was included in the PBS incubation and washing buffer to avoid unspecific binding of the B4 FITC MoAb, a good correlation (R 0.977) between the numbers of CD19 cells detected by B4 and Leu-12 was found in 59 MM patients, thus illustrating that under optimal conditions a reliable quantitation is achieved with both MoAbs. When CD19 cell levels in untreated MM patients (n 9) with healthy donors (n 20) were compared, there was no significant difference in the mean levels (P. 803), but a pro-
ashpublications.org