Spectrum of acute leukemias diagnosed on flow cytometry : Analysis from tertiary care centre from North India

RESULT: Over a period of year, 422 individuals were diagnosed as AL. There were 287 males and 135 females with M:F = 2.1:1. There were 237 adults & 185 children. 36.3% were AML and 60.4% were ALL, while 3.3% of cases were mixed phenotypic acute leukemia (MPAL). The commonest WHO subtype in AML group was AML with maturation being 31%. In case of ALL there were 83.9% B-ALLs and 16.1% TALLs. In MPAL B-Myeloid was 71.4%, whereas TMyeloid was 28.6% of cases. Both AML and MPAL were more frequently seen in adults accounting to 83% and 92.9% respectively of all ALs cases. In contrast, 62% of ALLs were children and only 38% were adults. Out of all ALs, 37.6% of showed cross lineage antigen expression. In AML, B-ALL and T-ALL cross lineage antigen expression were 26.14%, 39.71% and 82.9% respectively.


Introduction
Acute leukemias are a heterogeneous group of malignancies with varying clinical, morphologic, immunologic, and molecular characteristics.Flow cytometric immunophenotyping is a valuable tool for the diagnosis, classification, staging, and monitoring of acute leukemia.Differentiation between myeloid and lymphoid leukemias, most often made by flow cytometry, is important [1,2].Several advances in flow cytometry, including availability of new monoclonal antibodies, improved gating strategies, and multiparameter analytic techniques, have all dramatically improved the utility of flow cytometry in the diagnosis and classification of leukemia.Detailed understanding of phenotypic patterns of differentiation, particularly in myeloid leukemia, allows for more precise classification of leukemia than does morphology alone [3].
2008 World Health Organization (WHO) classification of hematolymphoid malignancy requires a multiparametric approach to diagnosis and outlines the morphologic, immunophenotypic, and genotypic features characteristic of each disease entity.Many of genetically distinct subgroups of leukemia have been found to be closely associated with distinct immunophenotypes.Thus, in addition to classification into differentiation-based subtypes, detailed flow cytometric studies can define complex antigenic profiles that are associated with specific molecular defects and well-defined biology.In summary, multiparameter flow cytometry is an invaluable tool in the diagnosis, classification, and monitoring of patients with acute leukemia.
The purpose of the study was to evaluate the role of flow cytometry in diagnosis and proper classification of acute leukemias.This study aims to find out the frequency of cross lineage antigen expression in AML, B-ALL and T-ALL.

Methods
Four hundred and twenty two patients of adult and paediatric acute leukemias, diagnosed in the Department of Hematology, PGIMER, Chandigarh, during June 2012 to May 2013 were retrospectively analysed.In addition to routine evaluation by complete blood counts, the patients were evaluated with peripheral blood films, bone marrow aspirate & trephine biopsy, using May Grunwald Giemsa, Hematoxylin & Eosin and cytochemical stains.Bone marrow aspirate and/or peripheral blood samples collected from all the patients were processed with standardized "lyse-stain-wash" technique, stained with 4 colors combination of antibody cocktails and acquired on dual laser BD FACS Canto II flow cytometer.All samples were processed within 24 hour of collection.
Immunophenotyping was done in mononuclear cell obtained by lysing whole blood by BD FACS lysing solution.For immunophenotyping various combination of flurochrome conjugated monoclonal antibodies (MoAbs) were added per tube in sample.They were conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), Allophycocyanine (APC) or peridinin chlorophyll protein (PerCP), and were directed to antigen of myeloid cell, B cell, T cell, monocyte or immature precursor cells.Data were acquired and blast gating strategy included using dot plots of CD45 expression versus intracellular complexity (side scatter angle, SSC) and also a second gate was based on cell forward scatter angle, (FSC) versus SSC.Demographic profile of the patients, including age, sex was recorded for statistical analysis.Immunophenotypic profiles of all patients were analysed for cross lineage antigen expression.
Out of 422 cases of acute leukemia 37.6% (159/422) showed cross lineage antigen expression.Out of 153 cases of AML, 40 cases showed cross lineage antigen expression.B lineage antigens were expressed in 22.5% (9/40), T lineage markers were seen in 75% (30/40) and both B & T lineage markers were present in 2.5% (1/40) cases.Similarly, in B-ALL, 85 out of 214 cases showed cross antigen expression comprising of myeloid lineage, T-lineage and combined myeloid-T-lineage cross expression in  5, Figure 1).

Discussion
Diagnosis of acute leukemia was tradionally based on morphological and cytochemical features (FAB classification) [4], but now, flow cytometry is also one of an indispensable tool for the proper classification and diagnosis of acute leukemia.Flow cytometry differentiate types of acute leukemia based on precursor cell expression of surface molecule that is called as cluster of differentiation (CD) antigen.Flow cytometry has also great importance in identification of biphenotypic leukemia and identification of unusual co-expression of antigen or abberent expression of CD antigen [5].
The core of monoclonal antibodies (MoAbs) investigated comprised: anti-MPO, CD13, CD33 and CD117 for the myeloid lineage cytCD3, CD2 and CD7 for the T-cell lineage, and CD19, CD10, CD20, and cytCD79a for the B-lymphoid lineage.All cases were also investigated for the expression of nuclear TdT, CD34, and HLA-DR and a substantial proportion for CD14, CD 64, CD 11c, CD41 and CD61.
Acute Myeloid leukemia is most common in adult whereas Acute lymphoid leukemia is common in children [6].In our study also we observed the same frequency of acute leukemia based on age group.
In this present study, incidence of ALL was 60.4%, AML was 36.3% and remaining 3.3% was MPAL.In total AML cases, 15 % cases were APML while remaining 85% were non APML.Most of the other studies also found APML ranges as 5-14 %, and few study stated as 23% [7].The commonest AML subtype was AML M2 that account for 31% which is similar to the study done by Ghosh et al [8].
Similarly, in case of ALL B-ALL (83.90%) is predominant to T-ALL (16.10%).Similar study was observed in the West, the predominant immunophenotype observed in ALL was B-ALL, accounting for 60-80% of total cases, whereas T-ALL comprised only of 15-20% [9].In one study from eastern India, both T-ALL (50.4%) and B-ALL (49.6%) incidence was almost equal in distribution [10].
Biphenotypic acute leukemia or MPAL is a rare type of leukemia which probably arises from hemopoietic pluripotent stem cell with the capability of differentiating along both myeloid and lymphoid (T or B) lineages of antigen expression [11].In our study, MPAL accounted for 3.3% (14/422).
In present study, the aberrant cross lineage expression of myeloid antigens on lymphoid leukemias was more common than expression of myeloid antigen on lymphoid leukemias.While in other study, cross lineage antigen expression of lymphoid lineage was common in AML (12).

Conclusion
Flow cytometry is useful in correct diagnosis and subclassification of acute leukemia and is essential in cytochemical myeloperoxidase (MPO) negative cases, as well as for the diagnosis of MPALs.In present study, majority of cases were cytochemically MPO negative (including B & T ALLs, AML-M0, M7 and MPALs).The incidence of AML and MPALs was higher in adults but ALL was predominantly seen in paediatric patients.Cross antigen lineage expression is a common phenomenon, most frequent in T-ALLs, and hence, lineage specific intra-cytoplasmic antibodies including anti-MPO and cytoplasmic-CD3 are essential for correct categorization of acute leukemias.

Fig. 1 .
Fig. 1.Cross Lineage antigen expression in different acute leukemias.B: B lineage cross Ag expression in AML and T-ALL.T: Tlineage cross Ag expression in AML and B-ALL.Myeloid: Myeloid lineage cross Ag expression in B-ALL and T-ALL.Both: Combination of B & T, myeloid & T and Myeloid & B cross lineage Ag expression in AML, B-ALL and T-ALL, respectively.

Table 2 .
Distribution of Acute Leukemias according to age group

Table 3 .
Distribution of AML cases according to immunophenotyping