Identification of acute myeloid leukemia in dogs using flow cytometry with myeloperoxidase, MAC387, and a canine neutrophil-specific antibody
Vet Clin Pathol. March 2006;35(1):55-71.
Elizabeth Villiers, Stephen Baines, Anne-Marie Law, Victoria Mallows
I. Introduction
a. Immunophenotyping by flow cytometry is now used routinely in the diagnosis of leukemia in human medicine, and is becoming more widely available in veterinary medicine as increasing numbers of antibodies become available for this purpose
b. Canine lymphocytes can be identified readily using antibodies against:
i. CD3 (expressed by all T cells)
ii. CD4 (expressed by helper T cells, but also by canine neutrophils)
iii. CD5 (expressed by T cells and a subset of B cells)
iv. CD8 (expressed by cytotoxic T cells)
v. CD21 (expressed by B cells)
vi. CD79a (expressed by B cells)
c. Fewer antibodies are available that are specific for cells of monocytic and granulocytic lineage
i. CD11b is expressed principally on granulocytes, monocytes, and dendritic cells, but also may be expressed by subpopulations of lymphocytes
ii. CD11c integrin is expressed by monocytes, neutrophils, dendritic cells, and macrophages
iii. Antibodies recognizing canine CD14 are available and are specific for cells of monocytic lineage
d. The CD34 antigen is expressed by stem cells of any lineage
e. The aim of the study reported here was to evaluate three antibodies:
i. Canine neutrophil-specific antibody (NSA)
ii. Mouse anti-human macrophage antibody (MAC387)
iii. Mouse anti-human myeloperoxidase (MPO) antibody
f. NSA has been shown to label only neutrophils and not other granulocytes, monocytes, or lymphoid cells
i. It is not known whether this molecule is expressed by myeloblasts and other neutrophil precursors or whether expression is confined to more mature neutrophils
g. MAC387 recognizes the intracytoplasmic antigen calprotectin expressed in granulocytes, monocytes, and macrophages, and demonstrates cross-reactivity in the dog
h. MPO recognizes the peroxidase present in azurophilic granules in neutrophils and their precursors
i. It is expressed by cells of monocytic and granulocytic lineages
II. Materials and Methods
a. Animals and samples
i. 6 control dogs
1. Presented for orthopedic conditions
ii. Blood and/or bone marrow samples from dogs with suspected leukemia were sent to the laboratory from private veterinary practices in the UK, or were obtained from patients in the University of Cambridge Veterinary Hospital between January 2002 and June 2003
iii. A diagnosis of acute leukemia was based on the finding of >30% blasts in bone marrow or >30% blasts in peripheral blood, with cytopenias
iv. A diagnosis of AML was based on the morphology of blast cells, together with lack of staining reaction to antibodies recognizing the lymphoid antigens CD3, CD5, CD8, CD21, and CD79a, and by positive staining reaction to CD14 or CD4 with no reaction to other lymphoid markers
v. Staining reaction to a given antibody was regarded as positive if ≥20% of the blast cells stained
vi. The cutoff for positive staining to MPO was set at 10%
b. Flow cytometric analysis
i. Antibody staining was performed in plastic tubes containing 106 nucleated cells in whole blood or anticoagulated bone marrow
ii. The antigens recognized by MPO and MAC387 are intracellular
1. Aliquots of whole blood containing 106 nucleated cells were first permeabilized using a cell permeabilization kit that also lyses the red cells
iii. Cytometric analysis was performed using a FACScan flow cytometer with CellQuest software
iv. Cell populations were identified on the basis of their side light-scatter vs. forward light-scatter characteristics
v. The permeabilization step altered the scatter plot from that for nonpermeabilized cells; in some sample, this made accurate gating of monocytes and neutrophils difficult, as the two regions overlapped by variable amounts
III. Results
a. Control blood
i. Scatter plots for control blood samples indicated three distinct populations:
1. Monocytes
2. Neutrophils
3. Lymphocytes
ii. Neutrophils stain strongly with NSA and MAC387
iii. Monocytes did not stain with NSA
iv. Lymphocytes did not stain with any of the antibodies
b. Leukemic blood
i. In all cases, acute leukemia was suspected based on circulating blast cells in the peripheral blood, bi- or pancytopenia, and clinical signs of disease
ii. Propidium iodide staining revealed <5% of dead cells in all cases except case 7
iii. Case 1
1. Hematologic abnormalities
a. Neutropenia
b. Mild anemia
c. >30% circulating blast cells
2. The blast cells were:
a. Slightly larger than small lymphocytes
b. Had a round nucleus
c. Moderate amount of pale cytoplasm
d. Sometimes contained nucleoli
3. Flow cytometric analysis revealed positive staining with CD4 in 85% of the blast cells
4. The staining reaction to all other lymphoid markers was negative
5. The blast cells also expressed CD11b and CD11c
a. Suggested the cells were of neutrophil lineage
6. The blast cells strongly expressed NSA, MPO, and MAC387, again consistent with AML-M1
iv. Case 2
1. Hematologic abnormalities included:
a. Marked monocytosis
b. Numerous circulating blast cells
2. Bone marrow Cytologic examination revealed 80% blast cells with large indented or convoluted nuclei containing 1-3 nucleoli
3. The cells were considered most consistent with monoblasts
4. The predominance of monoblasts over maturing monocytes was consistent with AML-M5a
5. The cells did not express any lymphoid markers with the exception of CD4
6. There was strong staining reaction to CD14 as well as to CD11a, 11b, and 11c, consistent with AML-M5a
7. The CD34 integrin was expressed by 35% of the smaller cells, consistent with acute leukemia
8. The permeabilization procedure for staining intracellular antigens resulted in a change in the scatter plot such that, aside from the neutrophil population, only one distinct population was visible
a. Within this population, MAC387 stained 56% of the cells positive and MPO staining was negative
v. Case 3
1. Hematologic findings were similar to those in case 2
a. Marked monocytosis
b. Numerous circulating blast cells that had morphologic features suggestive of monoblasts
2. Blasts had highly convoluted nuclei containing one ore two nucleoli
3. Flow cytometric analysis resulted in a scatter plot with a discrete population of blasts/monocytes
4. 71% of cells stained positive for CD14, consistent with monocytic lineage
5. Substantial staining reaction to CD11b and CD11c, consistent with granulocytic or monocytic lineage
6. 31% of the cells stained positive for CD4
a. Some of this positivity may have been due to staining of neutrophils
vi. Case 4
1. Hematologic abnormalities included:
a. Neutropenia
b. Thrombocytopenia
c. Anemia
2. No monocytosis, but a high proportion of blast cells had large, round, or reniform nuclei
3. The blasts were identified in two distinct regions on the scatter plots
a. Did not express lymphoid markers, but did express CD14 and beta-2 integrins, consistent with monocytic leukemia
b. 53% of the cells in R1 expressed CD4
i. This was not thought to be consistent with neutrophil contamination because the dog was neutropenic and because the location of cells in the scatter plot was not consistent with that of neutrophils
c. 50% of the blast cells expressed MAC387, but none expressed MPO
vii. Case 5
1. Severe anemia
2. Thrombocytopenia
3. Neutropenia
4. Numerous circulating blasts
5. Blast cells formed a discrete region on the scatter plot
a. Expressed CD14 (68%) and CD34 (40%)
b. Did not express NSA, MPO, MAC387, or CD4
viii. Case 6
1. Anemia
2. Thrombocytopenia
3. Marked monocytosis
4. Numerous circulating blast cells
5. Blasts appeared in two regions on the scatter plot
a. Cells in R3 expressed only CD34
b. Cells in R1 expressed CD14, CD34, MPO, MAC387, and CD4
6. NSA results were negative
ix. Case 7
1. Hematologic findings included:
a. Anemia
b. Neutropenia
c. Thrombocytopenia
d. Numerous circulating atypical blasts
2. Bone marrow Cytologic examination revealed 99% blast cells
3. On the basis of morphology alone, it was not clear whether these were granulocytic/monocytic or lymphoid cells
4. Flow cytometric analysis of bone marrow revealed two populations of cells
5. Staining with propidium iodide in the isotype control tubes revealed a large number of dead cells in the small region (R3)
a. Only staining beyond this peak was considered positive
6. The population of larger cells in R1 contained few dead cells
a. The cells stained with CD14, MPO, MAC387, and CD34
b. Staining reaction for CD4 was negative
x. Case 8
1. Hematologic abnormalities included:
a. Anemia
b. Thrombocytopenia
c. Mild neutrophilia
d. High percentage of circulating blasts
2. Bone marrow Cytologic evaluation revealed 80% blast cells
3. There was moderate generalized lymphadenopathy due to an infiltrate of blast cells in the lymph nodes
4. Cells expressed CD14, CD4, NSA, MPO, and MAC387, as well as CD11a, CD11b, and CD11c
5. All stained negative for lymphoid-specific markers and CD34
IV. Discussion
a. In human medicine, AML is classified on the basis of blood and bone marrow morphology, and the results of cytochemical, immunophenotypic, and cytogenetic analyses
i. AML with recurrent genetic abnormalities
ii. AML with multilineage dysplasia
iii. Therapy-related AML
iv. AML not otherwise categorized
b. The most significant change is that the requisite blast percentage for a diagnosis of AML should be >20% myeloblasts in the blood or bone marrow (reduced from 30%)
c. In AML, monoblasts and promonocytes are counted as blast equivalents, and in acute promyelocytic leukemia, abnormal promyelocytes are counted as blast equivalents
d. In veterinary medicine, AML is analogous to the human category, AML not otherwise categorized
e. The presence of >20% or 30% myeloblasts or monoblasts in the peripheral circulation can only be due to AML
f. In healthy individuals, CD34 is expressed by hematopoietic stem cells
i. In humans and animals, expression of CD34 in Hematologic malignancies is exclusive to blast cells in acute leukemia and is absent form mature cells in chronic disorders
ii. Expression of CD34 does not indicate cell lineage; it may be expressed in ALL and AML
g. MPO is an important antibody for identifying myeloid leukemia
i. Leukemia is defined as being of myeloid (i.e., granulocytic or monocytic) lineage when ≥3% of blast are positive fro MPO by use of enzyme cytochemistry
ii. Flow cytometry is more sensitive than cytochemistry in detecting the presence of MPO when the lower threshold is used
iii. Cytochemical staining is limited to the contents of MPO granules rather than the perigranular membrane
iv. In flow cytometry, a monoclonal antibody is used to detect the presence of MPO within permeabilized granulocytes and is not dependent on enzyme activity or microscopic resolution
v. MPO is the only myeloid-specific antibody from the first-line human panel that is available for use in dogs and is makes a useful addition to a panel of antibodies for Immunophenotyping canine leukemia
h. Study of normal blood indicated that NSA was specific for neutrophils
i. Study of normal blood indicated that MAC287 stained both monocytes and neutrophils
j. CD4 may be expressed by monocyte precursors
i. This contrasts with findings in humans, in which CD4 is not expressed by mature neutrophils and is absent or weakly expressed on monocyte precursors, but is expressed by neutrophil precursors and mature monocytes
k. CD14 expression develops at an early stage of monopoiesis
l. Positive staining with CD11b is suggestive of myeloid lineage, although it also is expressed in disseminated Histiocytic sarcoma and in a minority of lymphoid leukemias
m. Expression of CD11c is relatively specific for myeloid lineage, although again, it may stain disseminated Histiocytic sarcoma cells in dogs and a small proportion of cells in B-cell lymphoid leukemias
n. Cytoplasmic CD79a is known to stain canine B lymphocytes from and early stage of development through that of mature plasma cells
o. The CD3 integrin is present on the cell surface only in more mature T cells
p. MPO is known to stain canine myeloblasts
q. Results of this case series suggest that CD14, CD4, MPO, MAC387, and NSA are expressed at an early stage of myeloid cell development
r. Immunophenotypic classification of leukemia should never be based on expression/lack of expression of one particular marker, but should be determined by the pattern of expression of a wide panel of antibodies
