Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 4020-4020
CORRESPONDENCE
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To the Editor: |
Levels of circulating CD19+ cells in patients with
multiple myeloma
Clonotypic B cells at different stages of differentiation are
present in patients with multiple myeloma (MM). But the nature and
frequency of the less mature clonotypic B cells expressing CD19 is a
standing controversy in myeloma research.1,2
Currently, the numbers of CD19+ cells are
determined by flow cytometry using monoclonal antibodies
(MoAbs) specific for the CD19 surface molecule. It has been suggested
that the controversy concerning the levels of CD19+ cells
in the peripheral blood (PB) of MM patients stems from the use of
different CD19 MoAbs that recognize different epitopes with a
heterogeneous pattern for clonal and polyclonal B-cells.2,3 In addition, it has been reported that the CD19 epitope defined by the
Leu-12 MoAb (Becton Dickinson Immunocytometry Systems, San Jose, CA),
not normally recognized on these clonal cells, is exposed when they are
treated with neuraminidase.3
The frequency of CD19+ cells in MM patients have become
clinically important because clonal CD19+ cells may contain
the drug-resistant compartment in myeloma,4 and
the CD19+ cell levels at diagnosis have been shown to be a
prognostic factor.5 We therefore investigated whether the
CD19 MoAbs B4 (Coulter Clone, Miami, FL), Leu-12, and FMC63 (Serotech,
Oxford, England) recognized different epitopes and whether these
epitopes displayed a different pattern on B cells from healthy donors
when compared to MM patients.
In brief, 3 different CD19 double stainings were used. The 2 MoAbs were added together; cells were incubated for 30 minutes with
MoAb-1, followed by the addition of MoAb-2; and cells were incubated for 30 minutes with MoAb-2, followed by the addition of
MoAb-1. With each experiment, single staining of the actual MoAbs and
an isotype-matched control were performed. All combinations of MoAbs
including the FITC and the PE-conjugated MoAbs were tested in this setting. For all combinations maximum fluorescence intensity was obtained for both CD19 MoAbs in at 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 pronounced heterogeneity in MM
patients was observed, a result similar to that reported by Kay et
al.1.
The CD19 epitopes defined by the B4, Leu-12, and FMC63 MoAbs were
similarly expressed on B cells from healthy donors and MM patients, and
neuraminidase treatment had no effect on these CD19 epitopes. In
addition, the level of circulating B cells in MM patients was not
significantly different from healthy controls. Thus unspecific binding
of the B4 and FMC63 FITC-conjugated MoAbs to monocytes creating false
high CD19+ cell levels may explain the controversy
regarding the frequency of PB-localized CD19+ cells.
Thomas Rasmussen
Linda Jensen
Hans Erik Johnsen
Department of Hematology L
Herlev Hospital
University of
Copenhagen,
Herlev, Denmark
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References |
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Kay NE, Oken MM, Bone N, et al.
Circulating CD19+ blood cell levels in myeloma [letter].
Blood.
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