Myeloid Leukemia

Classification of AML by Monoclonal Antibody Microarray 243
entities, and these are incorporated in the WHO classification with morphological
differences.
The DotScan microarray also has considerable potential for identifying prognostic
factors for leukemia subtypes (e.g., stable or progressive CLL), and
whether a leukemia will be susceptible or refractory to a drug, but many more
samples are required to define the consensus patterns of CD antigen expression
that correlate with these attributes. When myeloid leukemias are analyzed using
the DotScan microarray, human AB serum is included to eliminate nonspecific
binding of leukocytes to immobilized antibodies via Fc receptors on cells
(CD16, CD23, CD32, CD64, and CD89). Data of excellent quality have been
obtained for AML samples from panels of patients, but more samples are
required to define some of the rarer subtypes of this heterogeneous leukemia.
1.2. Immunophenotyping Techniques
1.2.1. Flow Cytometry
Immunophenotyping of hematological malignancies is typically performed
by flow cytometry with gating to identify the leukemic population and then
study by fluorescence the antigen profile of these cells (7,8). The subjective
placement of electronic gates to establish positive and negative antigen expression
results in some variation between laboratories. Less commonly now,
immunophenotyping may be performed using a slide-based immunofluorescence
or cytochemical technique (9) as in cytochemistry. Immunophenotyping
of hematological malignancies by flow cytometry typically involves testing
for 15–20 antigens to determine cell lineage and leukemic classification.
1.2.2. DotScan Microarray
We have devised a novel microarray of CD antibody dots (10 nL) bound to
nitrocellulose on a glass slide, that enables testing for 80–100 antigens in a
single, simple assay. The DotScan microarray has been successfully used to
immunophenotype lymphoid (B- and T-) and myeloid leukemias. We have
analyzed many B-lymphoid leukemias, using mononuclear leukocytes purified
on Histopaque, and the results have been definitive, especially for B-cell
chronic lymphocytic leukemia (B-CLL), where we have data for 100 patients
and a characteristic immunophenotype (3,10). For these leukemias, an extensive
immunophenotype (82 CD antigens) should be sufficient for diagnosis.
We believe the microarray has considerable potential for identifying prognostic
factors in B-CLL, but to date insufficient cases have been analyzed for
meaningful correlation, and we are continuing to build the database. In general,
there are insufficient data at present to enable discrimination between the
rare forms of lymphoid leukemia, but this work is ongoing.