Myeloid Leukemia

246 Christopherson et al.
4. Each batch of slides is tested with cell lines and/or frozen peripheral blood leukocytes
or leukemia cells of known immunophenotype to check antibody-binding
activities.
3.2. Binding of Leukocytes to the DotScan Microarray
1. The collection of peripheral blood from patients and normal subjects, purification
of mononuclear leukocytes on Histopaque-1077, and their immunophenotyping on
a CD antibody microarray are described by Belov et al. (3,10).
2. Most lymphoid cell suspensions are tested in PBS containing 1 mM ethylenediamine
tetraacetic acid (EDTA), but cells from patients with AML are tested in
PBS, without EDTA, containing heat-inactivated human AB serum (2% (v/v),
Sigma-Aldrich, St. Louis, MO) to minimize nonspecific Fc receptor binding (see
Note 1).
3. Leukocytes (4–6 ⋅ 10 6 cells, see Note 2) suspended in 300 ∝L of PBS plus AB
serum are incubated at room temperature (20°C) for 60 min.
4. Unbound cells are gently washed off with PBS; then the microarrays are fixed for
at least 20 min in PBS containing 3.7% (v/v) formaldehyde, and washed in PBS.
5. Complete instructions on the use of the DotScan microarray are provided with
the kits available from Medsaic Pty. Ltd. (Australian Technology Park, Eveleigh,
Australia).
3.3. Data Recording and Analysis
Dot patterns of leukocytes captured on the DotScan microarray are recorded
using a DotScan array reader and software (see Note 3). The software quantifies
the intensities of each dot from the digital image file and compiles expression
profiles as bar charts showing the average intensities above background
on an 8-bit grayness scale from 1 to 256 (see Note 4). Fig. 1 shows dot patterns
obtained for M4 AML from peripheral blood and bone marrow from two different
patients. The digital images of the dot patterns obtained with the DotScan
array reader (Fig. 1) were analyzed using the DotScan software to produce the
bar charts shown in Fig. 2. The immunophenotype (see Note 5) of the AML-
Fig. 1. (opposite page) Mononuclear leukocytes from patients with acute myeloid
leukemia captured on the DotScan microarray: (A) antibody location; (B) patient
63047, acute myeloid leukemia (AML)-M4 peripheral blood; (C) patient 63269, AML-
M4 bone marrow. The DotScan microarray contains duplicate antibody microarrays,
and the dot intensities are averaged by the software. The numbers in the key provide
addresses for the cluster of differentiation (CD) antibodies; mIgG1, mIgG2a, mIgG2b
and mIgM are murine isotype control antibodies; TCR α/⇓, TCR . /δ, HLA-DR, FMC7,
. , . , and sIg are antibodies against T-cell receptors α/⇓ and . /δ, HLA-DR, FMC7,
. -, and . -immunoglobulin light chains and surface immunoglobulin, respectively.
Mabthera (Roche, Hertfordshire, UK) is a humanized monoclonal antibody against
CD20. 33 HIM and 34 581 are antibodies 33 HIM 3–4 and 34 581 derived from different
hybridoma clones from those for CD33 and CD34 in the main array.