| Abstract|| |
Acute erythroid leukemia in children is very rare. Here is a case of erythroleukemia in a child of age 1.5 years, which was diagnosed on peripheral smear, bone marrow examination, cytochemistry but was confirmed on immunophenotyping. CD45 versus side scatter demonstrated blast population (29%) expressing CD45 of variable intensity (dim to negative). The myeloid nature of blast population showed bright expression of cytoplasmic myeloperoxidase (MPO), heterogenous positivity of CD117 and dim expression of CD13, CD33. These blasts also showed bright positivity for CD71 which showed erythroid nature of blasts. Flow cytometry can be comprehensive enough to completely subtype cases of leukemias/myelodysplastic syndromes, polycythemia rubra vera, non-neoplastic conditions like reactive erythroid hyperplasia following immunosuppressive therapy or viral infections or nutritional deficiencies, unlyzed RBCs or thrombocytosis which may mimic acute erythroid leukemia on flow cytometry.
Keywords: Acute erythroid leukemia, CD45, CD71, childhood, flow cytometry
|How to cite this article:|
Sharma A, Buxi G, Walia R, Yadav RB, Sharma S. Childhood acute erythroleukemia diagnosis by flow cytometry. Indian J Pathol Microbiol 2011;54:173-5
|How to cite this URL:|
Sharma A, Buxi G, Walia R, Yadav RB, Sharma S. Childhood acute erythroleukemia diagnosis by flow cytometry. Indian J Pathol Microbiol [serial online] 2011 [cited 2013 May 26];54:173-5. Available from: http://www.ijpmonline.org/text.asp?2011/54/1/173/77395
| Introduction|| |
Acute erythroid leukemia is characterized by a predominant immature erythroid population and accounts for approximately 2-5% of all cases of acute leukemia.  Two subtypes are recognized based on the presence or absence of a significant myeloid component: erythroleukemia and pure erythroid leukemia. Erythroleukemia is predominantly a disease of adults, while pure erythroid leukemia can be seen in any age including children. 
Exact incidence of erythroleukemia in children is not known. Only very few case reports are available.  Here is a very rare case of erythroleukemia in a child of age 1.5 years, which was diagnosed on peripheral smear, bone marrow examination, cytochemistry but was confirmed on immunophenotyping.
| Case Report|| |
A 1.5-year-old male child presented with the complaints of increasing pallor and red spots all over the body for 1 month. He had black colored stool and moderate fever for 8 days. He had past history of pneumonitis 1 month before, for which he was treated. On examination, he had marked pallor without icterus, clubbing and cyanosis. Multiple cervical lymph nodes were present, measuring 1-2 cm, which were free and mobile. On per abdominal examination, there was hepatomegaly of 2 cm and splenomegaly of 8 cm. There were multiple purpuric spots present all over the body.
Peripheral smear revealed RBCs showing moderate anisocytosis with few macrocytic normochromic cells along with few microcytes. Approximately 10 nucleated RBCs/100 WBCs were seen. Leukocytosis (total leukocyte count 25,000/mm 3 ) showing blasts with differential count (polymorphs 17%, lymphocytes 48%, blasts 33%, myelocytes 02%) was seen. These blasts were two to four times the size of small mature lymphocytes, with scant to moderate amount of pale blue cytoplasm. Most of these cells showed cytoplasmic vacuolation and had fine granules. Nuclei were round with opened up chromatin and 0-3 nucleoli. Platelets were reduced.
Serum uric acid was increased to 10 mg/dl. His liver function tests and kidney function tests were normal. Mantoux, Widal and HIV were negative. Serum vitamin B12, folic acid and iron profile were normal.
Bone marrow (BM) aspirates were diluted with peripheral blood, showing predominantly erythroid population with marked megaloblastic change and dyserythropoiesis. Blasts, as mentioned in peripheral smear, were also seen [Figure 1]. Few maturing cells of myeloid series were seen. Exact differential count was not possible. No megakaryocytes were seen.
|Figure 1: Bone marrow aspirate showing erythroid population with marked megaloblastic change and dyserythropoiesis. Blast cells with round and opened up chromatin and scant to moderate amount of pale blue cytoplasm. Most of these cells showed cytoplasmic vacuolation and had fine granules. (Inset) PAS staining on peripheral blood showing globular as well as diffuse PAS positivity in blasts as well as the nucleated RBCs|
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Cytochemistry on peripheral smear showed globular as well as diffuse Periodic acid Schiff (PAS) positivity in the blasts as well as the nucleated RBCs. Myeloperoxidase (MPO) and Sudan Black B were negative. Thus, diagnosis of acute leukemia was made with the probability of acute erythroleukemia and acute megakaryocytic leukemia.
Multiparametric flow cytometery was performed on peripheral blood prepared by stain-lysis-wash technique. CD45 gating [CD45 versus side scatter (SS)] demonstrated blast population (29%) expressing CD45 of variable intensity showing R1 (red) CD45 dim positive and R2 (Green) CD45 negative blast population [Figure 2]a. R1 blasts showed cytoplasmic MPO positivity in 49% of R1 gated cells [Figure 2]e, dim positivity for myeloid markers (CD33 and CD13) in 26% [Figure 2]b and heterogenous positivity for CD117 [Figure 2]d, expressed by early myeloid precursors. R2 blasts were negative for myeloid markers [Figure 2]c. These blasts also showed bright positivity for CD71 which showed erythroid nature of the blasts [Figure 2]f. However, 39% of the blasts showed expression of CD41 which is a megakaryocytic marker [Figure 2]g, but it is mentioned that expression of CD41/CD61 can be seen in acute erythroid leukemia due to possible adherence of platelets to blast cells.  Other markers for B/T-ALL (CD10, CD19, CD22, HLADR, CD5, CD7) and monocytic markers (CD14, CD11c) were negative in both, i.e., R3 (R1 + R2; orange).
|Figure 2: Immunophenotyping: (a) CD45 versus SS gating showing two blast populations: CD45 dim positive R1 (red) and CD45 negative R2 (green). (b) R1 blasts dim positive for myeloid markers. (c) R2 blasts myeloid markers negative. (d) R1 blasts CD117 positive (heterogenous). (e) c-MPO positive in 49% R1 gated cells. (f) CD71 positivity seen in all the blasts, R3 (orange, R1 + R2). (g) CD41 (megakaryocytic marker) 39% R3 blasts. (h) R3: CD10, CD34 negative. (i) R3: T-cell markers negative. (j) R3: HLADR, CD5 negative. (k) R3: monocytic markers negative. (l) R3: B-cell marker negative|
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Thus, based on peripheral smear, BM examination and immunophenotyping, a confirmed diagnosis of erythroleukemia was made.
| Discussion|| |
Erythroleukemia is a rare heterogenous disease with an increase in red cell and myeloid precursors.
It is important to distinguish erythroleukemia from childhood myelodysplastic syndrome (MDS), reactive erythroid hyperplasia following immunosuppressive therapy or following viral infections or nutritional deficiencies. Thus, BM differential count of all nucleated cells should be performed, if the overall percentage of blasts is >20% and multilineage dysplasia is present in >50% of the cells of two or more lineages; diagnosis of acute myeloid leukemia (AML) with myelodysplasia related changes should be made. If blasts are <20%, diagnosis is usually MDS. 
Barnard et al. have shown that childhood erythroleukemia and acute megakaryocytic leukemia resemble MDS in presentation. In contrast, Honda et al. observed in their study that erythroblast-rich refractory anemia with excess blast and erythroleukemia in children have similar characteristics and may belong to a single disease entity. According to a recent study, AML is considered a continuum of MDS and AML with erythroid hyperplasia, where karyotype rather than an arbitrary blast cut-off percentage represents prognostic factor.  In our case, aspirates were diluted with peripheral blood; hence, exact blast percentage could not be given on morphology.
At times, because of the variability of the effects of vitamin B12 and folic acid deficiency on hematopoiesis, a preliminary look at the bone marrow may masquerade as an acute leukemia to the unsuspecting eye.  In our case, serum levels of both were normal, excluding the diagnosis of megaloblastic anemia.
It may be difficult to distinguish it from other types of AML and also from ALL or lymphoma on morphology.  Distinction from acute megakaryocytic leukemia is most difficult as both resemble on morphology and cytochemistry.
Flow cytometry (FC) is the primary tool for phenotyping acute erythroid leukemia and other related conditions. The data generated by FC can be comprehensive enough to completely subtype cases of leukemias/MDS into their myriad categories. This wealth of information thus provides phenotype, reproducible enumeration of blasts and can reveal the presence of cell population with aberrant antigenic profiles.
In acute erythroid leukemia, the blast population is accompanied by a conspicuous erythroid cluster. The relative proportion of erythroid cells by FC is often not as dramatic as the degree of erythroid hyperplasia seen on the aspirate as many of the late precursors are likely to be eliminated at red cell lysis stage during specimen preparation.  Few antibodies currently available for evaluating erythroid cells include CD71 (transferrin receptor) and anti glycophorin (GlyA). The maturation process from early erythroid precursors to the erythrocyte stage is accompanied by a loss of CD45 while the GlyA level remains unchanged after reaching its peak at the basophilic erythroblast stage. 
GlyA negativity and CD71 positivity are consistent with the proerythroblast stage of erythroid differentiation. 
Inspection of CD71/CD45 dot plots may disclose evidence of abnormal antigenic maturation in erythroid precursors.  In our case, glyA was not used. Instead, CD71/CD45 dot plots showed CD45-negative, CD71-positive erythroid population.
In benign conditions with erythroid hyperplasia such as vitamin B12/folate deficiency or RBC hemolysis, the erythroid clusters seen on dot plot become more conspicuous. The presence of either unlyzed RBC recognizable CD71 negative population or pronounced thrombocytosis with platelet clumping can also produce a substantial cluster in CD45 negative region of SSC/CD45 dot plot. 
Other preneoplastic/neoplastic conditions with erythroid hyperplasia such as low grade MDS/polycythemia rubra vera, in which most abnormalities are in the erythroid series, can yield a similar picture on FC. These disorders may display no other abnormalities on FC because the blast level is usually within the normal range and myeloid antigenic maturation is often devoid of overt abnormalities. 
Distinction with acute megakaryocytic leukemia is difficult as FC is unable to demonstrate a clear megakaryoblast population by CD41 because of aberrant expression of megakaryocytic markers in myeloblast and some erythroblasts.  In our case also, CD41 was expressed on 39% of blasts but it does not suffice to diagnose acute megakaryocytic leukemia as at least half of the blasts must be of megakaryocytic lineage for the diagnosis of acute megakaryocytic leukemia.  Most commonly, CD41 and CD61 are examined for surface expression but cytoplasmic staining eliminates the quandary concerning whether the staining is real or represents platelet membrane adherent to the blasts. 
| Conclusion|| |
Thus, immunophenotyping is a valuable tool in diagnosing childhood erythroleukemia when morphological examination and cytochemistry of the blasts on peripheral smear and bone marrow are not sufficient to confirm the diagnosis as was seen in our case.
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Department of Pathology, Dr. Ram Manohar Lohia Hospital and PGIMER, New Delhi - 110 001
[Figure 1], [Figure 2]