Mixed phenotypic acute leukemia with two immunophenotypically distinct blast populations: Report of an unusual case


  • How to cite this article: Rahman K, George S, Tewari A, Mehta A. Mixed phenotypic acute leukemia with two immunophenotypically distinct blast populations: Report of an unusual case. Cytometry Part B 2013; 84B: 198–201.


Mixed phenotypic acute leukemia (MPAL) is a rare disorder with an incidence of less than 2% of all acute leukemia using the recent 2008 WHO criteria. Common subtypes encountered are the B/myeloid and T/myeloid; B/T or trilineage MPAL being an exception. We discuss here a case of 20-year-male patient who presented with pallor and generalised lymphadenopathy. Peripheral blood smear examination showed presence of 61% blasts of lymphoid morphology. Immunophenotyping by multicolor flow cytometry showed two distinct populations of blasts with T and B phenotype respectively. He was diagnosed as MPAL with two distinct blast lineages. Conventional karyotyping done on bone marrow sample showed t(9;22)(q34;q11)(Ph +). Induction was started using ALL based protocol. The patient is on follow up with post induction marrow being in morphological remission. © 2013 International Clinical Cytometry Society


Mixed phenotypic acute leukaemia (MPAL) is a rare disease known previously by a variety of names like mixed lineage leukaemia, biphenotypic leukaemia and bilineal leukemia. Recent studies using the scoring system employed by European group for immunological classification of leukaemia (EGIL) have found the incidence rate of MPAL to be 2–5% of all acute leukemia (1–6). Frequency of MPAL, assessed using the 2008 WHO (7) criteria, was found to be less than 2% (8).

At the cytogenetic level, MPAL with Philadelphia chromosome and t (v, 11q23) have been found frequently enough to be considered as separate entities (7). In absence of these recurrent genetic rearrangements, MPAL is categorized based on the lineage of blasts. Common subtypes seen are the B& myeloid and T& myeloid, while B&T and trilineal, MPAL are exceptions (1, 7–9).

We are presenting a rare case of MPAL with two separate lineages of blasts comprising of B and T immunophenotypes, and also exhibiting the Philadelphia chromosome.


A 20-year-male patient presented to our institute with complaints of intermittent fever of 2 months along with pain and swelling of right knee radiating to right ankle of 20 days duration. His physical and systemic examinations were non contributory but for mild pallor and generalised lymphadenopathy.

Complete blood count and peripheral blood smear examination revealed a haemoglobin of 70g/L, total leukocyte count of 14.5×10*9/L, platelet count of 18×10*9/L along with 61% blasts with high N:C ratio, scanty agranular cytoplasm, and moderately condensed chromatin with occasional nucleoli. These blasts were negative for myeloperoxidase (Fig. 1).

Figure 1.

Peripheral blood smear showed presence of blasts with high N:C ratio, scant agranular cytoplasm, moderately condensed chromatin and prominent nucleoli[A] (Leishman stain, 100×). These blasts were negative for Myeloperoxidase stain. [B] (Myeloperoxidase, 20×). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Immunophenotyping was done on the peripheral blood by multicolor flowcytometry using standard stain-lyse-wash technique. Briefly, 100 μl of homogenized peripheral blood was stained with cocktails of monoclonal antibodies as described below. Following incubation for 15 min with the antibodies in dark, the samples were washed with phosphated buffered saline-bovine serum albumin.(PBS-BSA.) This was followed by RBC lysis using commercially available FACS lyse. After lysis the cells were washed and cell pellet was resuspended in a final volume 0.5 mL PBS. For intracellular staining commercially available Perm 2 reagent from BD biosciences were used for permeabilization.

Acute leukemia differentiation panel with 14 antibodies in a four colour combination using FITC/PE/PerCP/APC fluorescent conjugates were used. Six combinations were as follows : (1) IgG1/IgG1/CD45/IgG1, (2) CD10/CD34/CD45/CD19, (3) CD7/CD5/CD45/HLADR, (4) CD33/CD13/CD45/CD117, (5) antiMPO/cCD79/CD45/cCD3, and (6) TdT/-/CD45/-. CD45 vs. side scatter strategy was used to gate the blast population. Two distinct populations of blasts were noted on CD45 vs. SSC plot. One population with “moderate CD45”(‘T’ Blasts) expressed CD34, Tdt, CD5, CD7, cCD3, and CD13(weak), and were negative for other B and Myeloid markers. The other population with “dim CD45”(‘B’ Blasts) expressed CD34, Tdt, CD10, CD19, cCD79, HLADR, and CD13, and were negative for other T and Myeloid markers (Fig. 2). Based on the above immunophenotypic profile, this case was diagnosed as mixed phenotypic acute leukaemia (MPAL) with two separate population of blasts belonging to ‘B’ and ‘T’ immunophenotype.

Figure 2.

Multicolour flow cytometry shows three distinct populations in CD45 vs. Side scatter plot. One population of mature lymphocyte (high CD45 expression, green color) and two separate populations of blasts (moderate CD45, blue color, and dim to negative CD45, red color) were noted. The blue population expressed CD34, Tdt, CD5, CD7, CD13, and cyto CD3, indicating T lymphoid blast with aberrant CD13 expression. The red population expressed CD34, Tdt, CD10, CD19, cytoCD 79, and CD13, indicating B lymphoid blast with aberrant CD 13 expression. Immunophenotypic features suggested a diagnosis of MPAL with two separate blast populations. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Bone marrow aspirate smears demonstrated a hypercellular marrow with 85% blasts of lymphoid morphology. Conventional karyotyping of this marrow aspirate revealed t(9;22)(q34;q11) (Ph +ve) rearrangement. Cerebrospinal fluid was clear and bereft of blast.

The patient was administered chemotherapy with augmented Berlin-Frankfurt-Münster (BFM) protocol and demonstrated good response, with morphological remission seen on Day 22 marrow.2


Most acute leukaemias can readily be assigned to a myeloid or lymphoid (B or T) ancestry. However, a small fraction defy being categorised into a specific lineage and are designated as “Acute leukaemia of ambiguous lineage”. This in turn can either be a MPAL combining phenotype of two or more lineages or “Undifferentiated Leukaemia” without clear evidence of any lineage commitment. In addition, these MPAL may either comprise of a single population of blast expressing both myeloid and lymphoid phenotypes, or be made up of two distinct populations, each expressing different lineage specific antigens.

Earlier, a diagnosis of biphenotypic acute leukemia was made using the few available lineage associated markers, which had spuriously shown an incidence of acute mixed leukemia to be as high as 20% (10). As more antibodies became available, it became clear that a significant proportion of AML and ALL cases expressed aberrant antigens (11–14). It became necessary to distinguish aberrant expression of an antigen(s) from true “mixed phenotype acute leukaemia”. Specific criteria were first enunciated by Catovsky et al. (15) in the form of a scoring system based on weighted expression of antigens by blasts. This scoring system was further modified by EGIL where a score of over 2 was required to assign a lineage. The current WHO (2008) classification however, has dismissed these scoring systems and relies upon lineage assignment based on specific features (7). Using this recent WHO criterion, prevalence of MPAL has been found to be 1.6% of acute leukaemia (8).

MPAL usually are either T/Myeloid or B/Myeloid. B/T MPAL is rare and represents less than 4% of all MPAL (3). Most of the cases of B/T MPAL found in literature describe biphenotypic blasts rather than bilineal blasts, as seen with our case. Weir et al. (1) described 19 cases of acute bilineal leukaemia over a 10-year-period, but they did not report any case of B/T bilineal leukaemia. To assign ‘T’ lineage to a blast population, cytoCD3 or rarely surface CD3 is required. To assign “B” lineage,multiple antigenic expressions are required, which includes a strong CD19 along with strong expression of any one of CD79, CD10 and cytoCD22.However, if CD19 is weakly expressed, then, strong expression of at least 2 of the above is necessary. Expression of CD79 or CD10 in a T cell ALL is not assigned as a feature of “B” lineage, because these can be aberrantly expressed in T leukemia (7).

In the present case, two separate populations of blasts were observed. While the first population expressed cytoCD3, CD5, CD7 without any evidence of CD10 or CD79, fulfilling the WHO criteria for “T” lineage assignment, the second population strongly expressed CD19 and CD10 in addition to moderate expression of CD79, without any evidence of cytoCD3 or other “T” antigens, thus fulfilling the criteria for B lineage FSC vs. SSC plot was not able to differentiate the two different populations of blasts. But, CD45 vs. SSC clearly discriminated the two populations with T blasts expressing CD45 stronger than B Blasts

The study conducted by Weir et al. (1) included only T/myeloid or B/myeloid blasts, where definite morphological differences existed between the two populations. The light scatter properties of the these populations were sufficiently different to allow a clear cut recognition amongst them. The larger myeloid blasts had higher scatter as compared to smaller lymphoid blasts with a lower scatter. In contrast, our case had lymphoid blasts only, which made it difficult to interpret the difference in scatter properties.

Although the putative cell of origin for MPAL is unknown, it is suggested that these arise from very early hematopoietic progenitors with a potential to grow either as myeloid or lymphoid. According to the myeloid-based model suggested by Katsura et al. (16), stem cells initially generate a common myelo-erythroid progenitor and common myelo-lymphoid progenitors. T and B lymphoid progenitors subsequently arise from myeloid-T and myeloid-B progenitor (16). The recognition of MPAL with B and T cell lineages would support the proposed model of adult bone marrow hematopoiesis, where a common lymphoid progenitor is present (17).

Cytogenetic alteration reported in MPAL are t(9;22)(q34;q11) and 11q23 abnormalities. These two abnormalities have been observed so frequently that newer WHO classification has acknowledged these as separate entities (7). The present case was positive for BCR-ABL translocation with no preceding CML. If the patient has an antecedent history of CML, then a diagnosis of CML blast crisis with special note describing the mixed phenotype population of blasts should rendered, rather than MPAL.

Controversies regarding the treatment of MPAL ranges from, which induction protocol to be used (myeloid or lymphoid) to, whether induction is to be followed by stem cell transplantation or not. ALL based treatments have been found to be more effective with higher response rate as compared to AML or AML+ ALL schedule (9, 18). Rubnitz et al. (4) in their study of 35 cases of MPAL in paediatric patients, found a better response rate with ALL based induction protocol compared with AML-based protocol(CR of 83% vs. CR of 52%). In addition, 8 out of 10 patients who failed to respond or had PR with AML protocol, attained CR after receiving standard ALL therapy. In spite,of this it is proposed that treatment for biphneotypic acute leukemia should begin with one course of AML type induction therapy with a provision for a switch to lymphoid type induction therapy, if patient responds poorly. Hematopoietic stem cell transplantation has been recommended only for the patients with greater than 1% blasts by flow cytometry at the end of induction. The role of HSC in patients with low level of disease (0.01–1%) is uncertain (4). The present case was treated with ALL-based protocol as the two populations of blasts were lymphoid only.

Cases of MPAL have shown poor outcomes probably due to adverse cytogentic features associated with it, especially the presence of BCR-ABL traslocation (2). In addition, high incidence of CD34 positivity, high incidence of extramedullary infiltrate, lack of optimised guidelines for induction and high incidence of relapse after remission has led to a lower overall survival and disease free survival in patients of MPAL (3). Our case responded well with ALL based treatment and post induction marrow was in remission. But a longer follow up is required to draw the inference on the treatment effect.