The Usefulness of CD71 Expression by Flow Cytometry in the Diagnosis of Acute Leukemia.

Abstract 2533

CD71 (transferring receptor 1) is an integral membrane glycoprotein that plays an important role in cellular uptake of iron. It is well known as a marker for cell proliferation and activation. Although all proliferating cells in hematopoietic system express CD71, however, CD71 has been considered as a useful erythroid-associated antigen. The expression proportion on nucleated red blood cells was significantly higher than other cells, approximately 80% of all CD71 positive cells were of CD71 positive erythroid cells in normal bone marrow. CD71 was usually considered as the representative marker for differentiating erythroblasts and diagnosing acute erythroid leukemia (AEL) by flow cytometry. At the ISAC 2000 Congress, most experts agreed that at least one or more B, T, myeloid, erythroid and megakaryocytic reagents should be included in the essential panel. The reagents recommended for erythroid cells included CD36, CD71 and glycophorin A (GlyA). However, there was no agreement on how to choose and group these antibodies. In the practical analysis of immune phenotypes of leukemic cells we noted that no CD71 expression was detected on blasts of some cases of AEL with typical morphological and cytochemical findings, but other types of acute myeloblastic leukemia (AML) cells may express CD71. Thus, we speculated that CD71 expression may associate with the abnormal antigen expression resulting from hematopoietic disorders. In this study, we evaluated CD71 expression on different acute leukemia cells in association with a variety of other antibodies. In this study we aimed to define CD71 as a flow cytometric marker for the diagnosis of acute leukemia.

Bone marrow samples were collected from 82 newly diagnosed acute leukemia patients as well as 13 normal controls. The diagnosis were made according to the WHO 2008 diagnostic criteria. All 6 cases of AEL were erythroid/myeloid subtype (acute erythroid/myeloid leukemia, M6a). The samples were then analyzed using a four-color flow cytometer with antibody panels against a variety of lymphoid, myelomonocytic, erythroid and megakaryocytic antigens. The antibodies included anti-CD3, CD7, CD10, CD11b, CD13, CD14, CD15, CD16, CD19, CD20, CD33, CD34, CD45, CD56, CD61, CD64, CD71, CD117, GlyA, HLA-DR, IgG, IgM, MPO. Subpopulations of bone marrow cells were gated based on CD45 intensities and side scatter (SSC) value to further analyze the expression of antigens in different cell populations.

Positive CD71 expression were identified on bone marrow blast cells of 41 (50%) acute leukemia patients and 9 (69.23%) normal controls. The mean expression level on normal controls was 35.99±19.06%. The mean CD71 expression level on blasts of AML with blasts differentiation at early stage of myelopoiesis (including FAB-M0/M1/M2/M4) was significantly higher than AML with partial differentiation of leukemic cells (FAB-M3/M5) and acuteB lymphoblastic leukemia (B-ALL) (p<0.05), with the mean expression level of 38.78±26.65%, 13.25±8.75% and 10.12±11.65%, respectively, and the latter two lower than normal controls (p<0.05). The percentage of CD71 expression level on blasts of acute megakaryocytic leukemia (FAB-M7) was 80.16±8.23%, significantly higher than normal controls, partial differentiation of leukemic cells (FAB-M3/M5), and B-ALL (p<0.05). The percentage of CD71 expression level on blasts of mixed lineage leukemia was 49.66±22.69%, significantly higher than B-ALL (p<0.05). Positive CD71 expression was found on bone marrow blast cells of 4 (66.67%) AEL cases, with the mean level percentage of 25.68±11.63% that was significantly lower than acute megakaryocytic leukemia (FAB-M7) (p<0.05) and was indifferent from normal controls and other types of acute leukemia. Using CD71 expression levels, we identified different abnormal cell clones simultaneously existing within bone marrow of 2 patients of AML with maturation (FAB-M2) and AEL, implicating the clonal evolution process from normal blasts to leukemic cells.

CD71 is an important marker for diagnosing acute leukemia, and is useful for distinguishing the differentiation stages of AML. However, CD71 may not be the specific diagnostic marker for AEL. CD71 is also valuable for the observation of clonal evolution process of acute leukemia, which may be informative to the etiology of leukemia.