The morphologic analysis of acute leukemia begins with the examination of Wright-Giemsa stained smears of
peripheral blood and bone marrow aspirates. The universally used French-American-British (FAB) system for
classification of acute leukemia is in fact based on the cytological and cytochemical features of the
leukemic cells as seen on smear preparations. Immunophenotypic features of the leukemic cells, which can be
detected by flow cytometry or immunohistochemistry, are not integrated into the FAB system but are now in
the process of being included alongside genetic and clinical features in the new WHO classification of
hematologic malignancies (Harris et al, J Clin Oncol, 1999). Immunophenotypic information by
immunohistochemistry is of course of particular value when interpreting bone marrow biopsy material in
patient with "dry tap" marrow aspirates as well as extramedullary leukemic infiltrates.
A limited correlation between the immunophenotype and the various morphologic (FAB, WHO) or genetic subtypes
(WHO) can be obtained by using the following paraffin reactive antibodies:
A polyclonal antibody which identifies with a high degree of sensitivity and specificity myeloid cells in
paraffin sections. It is the stain of choice in this regard. In normal tissues the stain is positive in
myeloid cells of both neutrophilic and eosinophilic types at all stages of maturation. The staining is
cytoplasmic. Cells of monocytic derivation are weakly positive or non reactive. Mast cells, plasma cells,
lymphoid cells, megakaryocytes, and erythroblasts are negative.
The reactivity parallels in general the cytochemical staining. However, the antibody seems to be more
sensitive than cytochemistry in recognizing the "minimal" myeloid differentiation of cases of AML-M0. No
published information is available on the presence or absence of myeloperoxidase staining in cases of ALL
with expression of myeloid associated markers (e.g. CD13/33) by flow cytometry. However, in our limited
experience, it is negative in these cases.
A polyclonal antibody reactive with hemoglobin A and F in tissue sections is a useful tool to demonstrate
the erythroid nature of blasts in cases of poorly differentiated acute erythremia In AML-M6 (acute
erythroleukemia) the hemoglobin stain facilitates recognizing the presence within the marrow nucleated cells
of >50% erythroblasts (see FAB definition of AML-M6). We also use the stain for hemoglobin to confirm the
erythroid nature of the sometimes "worrisome looking" proerythroblasts observed in patients with
megaloblastic anemia, myelodysplastic syndromes, and chemotherapy-induced megaloblastoid changes.
Glycophorin A can also be used as a pan-erythroid marker in tissue sections, but we prefer hemoglobin.
Although AML-M7 comprises only 5% of AML, it is probably the most frequent type of AML in children with
Down's syndrome. It is also seen in patients with myeloproliferative disorders where M7 occurs as a rare
subtype of blastic transformation It has also been reported in association with mediastinal non-seminomatous
germ cell tumors. One of the characteristic features of AML-M7 is marrow fibrosis, which precludes the
possibility of using flow cytometry or other bone marrow aspirate-based techniques to confirm the
megakaryocytic derivation of the abnormal cells . Immunoperoxidase staining of bone marrow biopsies is,
therefore, an essential diagnostic step in these patients. CD61, an antibody which recognizes platelet
glycoprotein gpIIb/IIIa can also be applied to biopsy material for the demonstration of megakaryocytic cell
origin . However, CD61 produces satisfactory staining only in formalin fixed material and, even in
well-fixed cases, is often weaker than Factor VIII. CD31, a vascular endothelial antigen, has also been
shown to stain normal megakaryocytes in biopsy sections although no information is available on a its
potential value in diagnosing acute megakaryoblastic leukemia.
CD68 is expressed throughout the monocytic differentiation pathway, usually more intensely in macrophages
than in monocytes. Mast cells may also exhibit CD68 (KP-1) positivity, while Langherans' cells and other
dendritic cells are negative. In biopsy specimens, CD68 is used to identify cases of AML with a monocytic
component (i.e. AML-M4, M5). There are 3 epitopes of CD68 available for immunohistology. They are KP-1,
PG-M1, and HAM-56. In our experience PG-M1 is the most specific, although not very sensitive. KP-1 and
HAM-56 are positive in many cases of AML M1, M2, and M3, in addition to being positive in AML-M4 and M5. The
CD68 stain is particularly recommended for diagnosing cases of AML-M5a, a subtype which is usually negative
by myeloperoxidase. CD68 is strongly expressed in marrow macrophages in cases of infection associated
hemophagocytic syndrome, in T-cell lymphoma with associated "histiocytosis", as well as in cases of true
malignant histiocytosis (MH) including MH cases associated with mediastinal germ cell tumors. KP-1 is
expressed in both cases of systemic mastocytosis and Langherans cell histiocytosis.
Lysozyme CD43, Elastase
These have been used as second line myeloid/myelomonocytic markers. Lysozyme stains both granulocytes and
macrophages, and has a higher degree of nonspecific background staining than the CD68 antibodies. CD43 has
been touted by others as a valuable myeloid marker, but it is not lineage specific, being expressed in both
T & B lymphocytes as well as myeloid cells. Elastase is less sensitive than other markers such as
myeloperoxidase, and does not work well in acid decalcified paraffin-embedded material.
CD56 is employed predominantly to identify natural killer cells in biopsy material. It stains nasal type
NK/T-cell lymphomas and rare case of acute leukemia including a subset of AML-M2.
CD34, TdT, CD99
These markers, although not lineage specific, have a restricted expression which is limited to immature
cells. Although most useful in the diagnosis of acute lymphoblastic leukemia they may be also useful in the
diagnosis of AML. These antibodies can also be of value when assessing the proportion of blasts present in
a bone marrow biopsy specimen (see discussion on ALL and detection of minimal residual leukemia). CD34 is
discussed at length in the sections that cover myelodysplastic syndromes and myeloproliferative disorders.
Acute panmyelosis with myelofibrosis (acute myelofibrosis)
Acute myelofibrosis is distinct from chronic idiopathic myelofibrosis by abrut onset with fever and bone
pain, and the absence of both splenomegaly and tear drops in the peripheral blood. Histology of the marrow
shows marked fibrosis (+3) associated with numerous micromegakaryocytes , an increased number of blasts
(>20%) and severe dysplasia. A large proportion of the blasts shows reactivity with vWF and CD61
antibodies. The differential diagnosis is with AML-M7 which overlaps and with aggressive subtypes of
myelodysplastic syndromes with fibrosis (less than 20% of blasts).
TdT, CD99, CD79a,CD20, CD10, CD3,CD1a
These paraffin reactive antigens are used to diagnose precursor B-ALL, mature B-cell ALL (Burkitt type), and
T-cell ALL. TdT positivity confirms the precursor status of the leukemic cells. TdT is poorly preserved in
B5 fixed, decalcified material and, as a result, may be more advantageously used in particle sections. CD99
which is relatively fixative-independent can be used as an effective "substitute" for TdT. It is, however,
less sensitive than TdT, being positive in about 2/3 to 3/4 of TdT positive cases.
CD79a is more frequently positive than is CD20 in precursor B- ALL. They are both positive in mature B-cell
ALL (Burkitt-type). CD79a stain a proportion of poorly differentiated AML. CD10 is positive in precursor
and mature B-cell ALL and, rarely, in T-ALL. TdT and CD99 are usually negative in mature B-cell ALL. The
latter disorder is strongly positive with CD20.
CD3 and CD1a are employed, in conjunction with TdT, to identify cases of T-cell ALL. The newer paraffin
reactive pan-T cell antibodies CD2, CD5, CD7, and CD1a can also be added in selected cases. CD43 should not
be used on its own to confirm a T-cell derivation, since it is totally nonspecific.
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