Immunophenotypic study of acute leukemia by fl ow cytometry at BPKMCH

Background: Immunophenotyping of acute leukemia is one of the most important clinical applications of fl ow cytometry. The aim of this study was to determine the immunophenotyping profi le of acute leukemia, by means of a fl ow cytometric method, using monoclonal antibodies all marked with a fl uorochrome, in four colour systems to assess their distribution according to type of leukemia (lymphoid B or T / myeloid). Materials and Methods: We retrospectively collected data of immunophenotyping from 52 acute leukemia patients at the department of pathology in B.P. Koirala Memorial Cancer Hospital from January 2010 to December 2011. Diagnosis was based on peripheral blood and bone marrow examination for morphology, cytochemistry and immunophenotypic studies. Results: Out of total 52 cases of acute leukemia diagnosed by fl ow cytometry over a two year period, there were 31 cases (59.6 %) of acute lymphoblastic leukemia, 20 cases (38.4 %) of acute myelogenous leukemia and one case (1.9 %) of bi-phenotypic acute leukemia. Leukemia was diagnosed among adults in 44.2 % whereas among children with age less than or equal to 15 years in 55.7 %. Thirty eight (73%) were male and 14 (27 %) were female with a male: female ratio of 2.7:1. For acute myelogenous leukemia, it was found that M0 (5.0 %), M1 (20%), M2 (60%), M3 (15%), M4 (5.0 %) were detected. CD13 and CD33 were the most useful markers in the diagnosis of acute myelogenous leukemia. The most common subtype was AML-M2. Of the 31 cases with acute lymphoblastic leukemia, 20 cases (64.5 %) were identifi ed as B-ALL and 11 cases (35.5%) as T-ALL. Aside from cytoplasmic CD3 (cCD3) and CD7 were the most sensitive antigens present in all cases of T-ALL. All cases of B-ALL showed expression of pan B-cell markers CD19 and CD22, but 15 (75 %) of 20 cases expressed CD10. Conclusion: Flow cytometric immunophenotyping was found to be especially useful in the correct identifi cation and diagnosis of acute myeloid or lymphoblastic leukemia and its subtypes. In combination with French-American-British (FAB) morphology and immunophenotyping, we were able to diagnose and classify all patients with acute leukemia in this study. Journal of Pathology of Nepal (2013) Vol. 3, No.1, Issue 5, 345-350 DOI: http://dx.doi.org/10.3126/jpn.v3i5.7856

expansion of progenitor cells of the hematopoietic system. 1 Hallmark for the diagnosis of acute leukemia, until recent past, has been the morphology and cytochemistry.They provide correct diagnosis in about 80% of the cases.For instance, Jawaid et al. in their series reported that 11% cases of acute leukemia's were unidentifi able in terms of their phenotype while 9% were identifi ed incorrectly on morphological basis, all of which were correctly allocated to their lineages after fl ow cytometric analysis. 2Thus, major developments in the fi eld of immunology have now brought an era of diagnosing acute leukemias by means of flow cytometry.The ability of immunophenotyping to identify myeloid versus lymphoid differentiation approaches 98%. 3 The immunophenotypic studies of acute leukemia by flow cytometry have become a powerful tool for proper identification of myeloid or lymphoid lineage.Therefore, it has great prognostic and therapeutic implications. 4Not only acute myeloid leukemia can be differentiated from the acute lymphoblastic leukemia (ALL) but B-cell or T-cell lineages can also be determined which cannot be achieved by morphology and cytochemistry alone. 2,5mmunophenotyping is also essential for recognizing several subtypes of acute myeloid leukemia and biphenotypic acute leukemias.
The flow cytometry study of acute leukemia samples started first time in Nepal at the department of pathology, BPKMCH Bharatpur, Chitwan from January 2010.Since then there has been continuous evaluation of the panel of the antibodies used to study the leukemic cells.

MATERIALS AND METHODS
A total of 214 cases of acute leukemia were diagnosed at BP Koirala Memorial Cancer Hospital between over a period of two years between Jan. 2010 and Dec.2011.However, immunophenotyping was performed on only 52 cases.Acute leukemia was classified on the basis of standard morphological and cytochemical criteria of the French-American-British cooperative study groups. 6Immunological classification of acute leukemia was assessed using a four line panel of monoclonal antibody for phenotying leukemic blasts cells. 7We found 20 cases of B-cell lineage acute lymphoblastic leukemia , 11 cases of T-cell ALL, 20 cases of acute myeloid leukemia and one case of biphenotypic leukemia.

Morphology and Cytochemistry
All specimens were obtained and prepared for morphologic examination using standard techniques.Bone marrow aspirate smear and peripheral blood specimen were air dried and stained with May-Grünwald Giemsa (MGG) stain technique and examined under light microscopy.Cytochemical staining of myeloperoxidase (MPO) and periodic acid-shiff (PAS) stain were done in all cases of acute leukemia.
Membrane and intracytoplasmic labeling was performed using 1 x 10 6 cells per tube.For membrane labeling, the samples were incubated with each antibody for 10 to 15 minutes.The erythrocytes were lysed with 2 ml of FACS lysing solution (Becton Dickinson, California, U.S), diluted to 1:10 and then washed with 2 ml of phosphate-buffered saline (PBS; pH = 7.2).For intracytoplasmic labeling, FACS permeabilizing solution (Becton Dickinson) was used in accordance with the manufacturer's instructions.
Data acquisition and sample analysis was performed in a BD FACS Caliber (4-color, Becton Dickinson, USA), using the Cell Quest software (Becton Dickinson), after calibration with the Calibrate bead kit (Becton Dickinson).
The blast gating strategy included using dot plots of CD45 expression versus intracellular complexity (side scatter angle, SSC) and also a second gate considering cell size (forward scatter angle, FSC) versus SSC.A total of 10,000 events were acquired in the target gate.Antigen was considered as positive if 20% or more of the blast cells reacted with a particular antibody.
There was also lack of expression of B and T cell makers like cyCD3 and cyCD79a in AML M0.HLA-DR is present in most acute leukemias, except T-ALL and AML M3.The myelomonocytic leukemia (AML-M4) shows expression of CD14 and CD36 (not shown in Table 2).
Detection of intracelullar myeloperoxidase (MPO), cyCD13, cyCD79a, cyCD22, and cyCD3 has become the most specific tool for the assignment of myeloid (MPO and cyCD13), B (cyCD79a and cyCD22) and T lymphoid

DISCUSSION
][10] Acute lymphoblastic leukemia is a major subtype of leukemia in children, where as AML is as common in adults. 11 this study cyCD79a, cyCD22, cyCD3, and cyMPO were highly sensitive, specific B, T, and myeloid markers that were expressed in virtually all cases of B and T cell ALL and in all subtypes of AML.By definition, the diagnosis of AML-M0 require less than 3% MPO+ and / or SBB+ blasts, expression of myeloid-associated markers, and lack of B/Tlineage-associated antigens (i.e.negativity for cyCD22/ or cyCD79a and cyCD3). 6The most significant distinctive immunologic markers between AML-M0 and ALL are CD13, CD33 and CD117, which are typical of M0. 12 In our study, one AML was classified as M0.The CD34 antigen is expressed by immature blast cells and is thus associated with less differentiated forms of leukemia, whereas the CD13, CD15 and CD33 antigens are expressed by more mature cells and is associated with the more differentiated leukemias. 13Identification of AML-M3 is important because it has specific therapy.Having a classical morphology, the promyelocytes show a strong MPO positivity.FC shows a myeloid leukemia (CD13, CD33 and CD117 positivity) along with HLADR negativity.However cytogenetics/ FISH test is must for a confirmation and management of AML-M3. 14The combined use of CD34 and HLADR is much helpful in distinguishing AML-M3 from Non-M3 AML's than either of these antigens alone. 5Expression of CD14 and CD36 was most often seen in myelomonocytic leukemia.In cases of monocytic differentiation CD13 and CD33 are most commonly expressed while CD34 and CD117 are generally absent. 3 present study, incidence of B-ALL (64.5%) was high in comparison to the Western population 3.0 % (Germany), Ludwig et al. 0.5 % (USA) Rivera et al. 15,16 An ALL of the B-lymphocyte lineage is assumed if CD22 or CD79a expression is found either cytoplasmic or on the cell surface with the expression of CD19 and HLA-DR. 17n childhood ALL, patients with Common-ALL which is CD10+ve ALL do best; while those with Null type ALL which is CD10-ve ALL {without Common ALL antigen (CALLA)} and T cell ALL do less well; In adult ALL, patients with Null or Common immunologic subtypes have a poor prognosis.[18][19][20] An ALL of the T-lymphocyte lineage is assumed if CD3 expression is found either cytoplasmically or on the cell surface with the simultaneous expression of CD7.T-lineage ALL subtypes can be defined based on the surface expression of CD1a, CD2, CD3, CD4 and CD8. 17 The aberrant expression of lymphoid antigens on myeloid leukemias was more promiscuous than expression of myeloid antigen on lymphoid leukemias.5 The most commonly expressed lymphoid associated antigen on AML was CD7 and similarly myeloid associated antigen on ALL was CD13.This was similar to the results of Putti et al, Den Boer et al, Bachir et al and Zheng J et al. [21][22][23] Biphenotypic acute leukemia is an uncommon type of leukemia which probably arises in a multipotent progenitor cell with the capability of differentiating along both myeloid and lymphoid (T and B) lineages where both types are associated with poor outcomes.Diagnosis of biphenotypic acute leukemia is based on immunophenotyping. 3,24

CONCLUSION
A combined classification of the immunophenotype and FAB morphology/cytochemistry was devised for AML and ALL subtyping.Flow cytometric immunophenotyping can be uniquely useful in the diagnosis of AML M0, its differentiation of acute promyelocytic leukemia (APL/ AML M3) from AML M1/M2, correct identification of T and B cell lineage of ALL and diagnosis of biphenotypic leukemia.

Figure 1 :
Figure 1: B Cell ALL.The blasts express the B cell markers CD10, CD19 CD22 and HLA-DR.The Cy MPO is negative.

Figure 2 :
Figure 2: T cell All.The blasts express the T cell markers CD3, cy CD3 and CD7.The B cell marker CD79a is negative.