CLINICAL MEDICINE AND PHARMACY VollO-Apr/2015
Study on the immunophenotype of 43 patients with acute leukemia by flow cytometry at 108 MiUtary Central Hospital
Ly Tuan Khai, Nguyen Thanh Binh 108 Military Central Hospital
Summary
Objective: Study the immunophenotype of acute leukemias using fiow cytometry method. Subject and method: We retrospectively collected data of immunophenotyping from 43 acute leukemia patients at the Hematology Department in 108 Military Central Hospital from October 2013 to February 2015. Pa- tients were diagnosed and classified based on peripheral blood and bone marrow examination for mor- phology, cytochemistry and immunophenotypic studies. Result: All patients were adults with male/female ratio of 3.3/1. Out of total 43 cases of acute leukemia diagnosed by flow cytometry, there were 36 cases (83.7%) of acute myelogenous leukemia, 6 cases (14%) of acute lymphoblastic leukemia and one case (2.3%) of bi-phenotypic acute leukemia, when using morphology and cytochemistry alone, there were still 4 cases which could not be classified clearly that they were acute myelogenous leukemia or acute lymphoblastic leukemia. Out of that 4 cases, 3 cases were acute myelogenous leukemia and 1 case was bi-phenotype when diagnosed by immunophentyping. Conclusion: Flow cytometric immuno- phenotyping is power tool for classification of acute leukemias in to acute myelogenous leukemia or acute lymphoblastic leukemia. Particularly, it can correct identification of T and B cell lineage of acute lymphoblastic leukemia and diagnosis of biphenotypic acute leukemia
Keywords: Acute leukemia, flow cytometry, immunophenotype.
1.Background correctly allocated to their lineages after flow cy- tometric analysis [4]. Thus, major developments in Acute leukemia is a cancer of t h e blood and t ^ e field of i m m u n o l o g y have n o w brought an era of bone m a r r o w and characterized by rapid and un- j j j j g n o s i n g acute leukemias by means of flow cy- controlled clonal expansion of progenitor cells of ,(,n,etry. The ability of i m m u n o p h e n o t y p i n g t o iden- the hematopoietic system. Forms of acute leukemia ^.^ myeloid versus lymphoid differentiation ap- includes acute myeloid leukemia (AML) and acute p e a c h e s 98% [10].
lymphoblastic leukemia (ALL). Hallmark for t h e d i - -^^^^ immunophenotypic studies of acute leu- agnosis of acute leukemia, until recent past, has i^^^ia by flow cytometry have become a powerful been t h e m o r p h o l o g y and cytochemistry. They pro- j ^ ^ | ^^^ proper identification of myeloid or lymphoid vide correct diagnosis in about 80% of the cases. For ijpgag^ Therefore, it has great prognostic and t h e - instance, Jawaid et al. in their series reported that f g p g ^ , , ^ implications [7]. Not only acute myeloid 1 1 % cases of acute leukemia's were unidentifiable in igyi^e^ia can be differentiated from t h e acute lym- terms o f their p h e n o t y p e while 9% were identified p^,(,[,|astic leukemia but B-cell or T-cell lineages can incorrectly o n morphological basis, all of w h i c h were ^|^^ j ^ ^ determined which cannot be achieved by morphology and cytochemistry alone [10]. Im- Z ^u • uomatninnv Pio m u h o p h e n o t y p i n g is also essential for recognizing Correspondence to: Ly Tuan Kha, - Hematology De- ^^^^^^P ^P^^ ^^ ^^^^^ ^ ^ ^ ^ ^ ^ . ^ ^^^ ^,_
r¥ nt 10''I iilitaryCentral Mospiiai /r ^
^ T , , . ' ,aihh108(Sigmail.com phenotypic acute leukemias.
Email: ly'
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JOURNAL OF 108 - CLINICAL MEDICINE AND PHARMACY VonO-Apr/201S
The flow cytometry study of acute leukemia samples started first time in Hematology Depart- ment, 108 Military Central Hospital from October 2013. Since then there has been continuous evalua- tion of the panel of the antibodies used t o study t h e leukemic cells. Objective of this research is t o study the immunophenotype of acute leukemias using flow cytometry m e t h o d .
2. Subject and method Subject
From October 2013 t o February 2015 consecu- tively admitted, untreated acute leukemia adult pa- tients w h o were diagnosed at Hematology Depart- ment - 1 0 8 Military Central Hospital, were enrolled in this study. Acute leukemia was diagnosed and classi- fied on the basis of standard morphological and cy- tochemical criteria of t h e French-American-British (FAB) cooperative study groups [3].
Material and method Morphology and Cytochemistry
Bone marrow aspirate smear and peripheral blood specimen were prepared and stained w i t h May-Griinwald Giemsa (MGG) stain technique and examined under light microscopy. Cytochemical staining of myeloperoxidase (MPO), periodic acid- shiff (PAS) and non-specific esterase stain were done in all cases of acute leukemia.
Methodology for immunophenotyping Samples of bone marrow w i t h volume of 0.5-1 mililiter were collected in ethylene diamine tetraace- tic acid (EDTA) for i m m u n o p h e n o t y p i n g . The sam- ples were processed within twenty four hours of col- lection. For the immunophenotypic diagnosis of acute leukemias, various combinations of fluoro- chrome-conjugated monoclonal antibodies (MoAb) per t u b e were added to t h e samples. All the MoAbs were obtained from Becton Dickinson (California, United States). They were conjugated with fluo- rescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP) or APC and were directed t o antigens for T cells (CD2, cytoplas- mic (c) CD3, CD4, CD5, CD7 and CDS), B cells {CD10, GDI 9, cCD22 and cCD79a), myeloid cells [CD13,
CD33, CD117 and cyto myeloperoxidase (MPO)]-, monocytes (CD14 and CD64), non-specific lineage pan-leukocytes (CD45) and precursor cells [CD34 and human leukocyte antigen-DR (HLA-DR)]. In some case, we added MoAbs for Glycoporin A (anti- gen for erythrocyte precusors), CD 4 1 , CD61 (antigen for megakaryocytes).
Membrane and Intracytoplasmic labeling was performed using 50 [i\ bone marrow sample (with cell concentration less than lOVL) per tube. For membrane labeling, t h e samples were Incubated w i t h each antibody for 10 to 15 minutes. The eryth- rocytes were lysed w i t h 2 ml of FACS lysing solution {Becton Dickinson, California, U.S), diluted to 1;10 and t h e n washed w i t h 2 ml of phosphate-buffered saline (PBS; pH = 7.2). For intracytoplasmic labeling, FACS permeabilizlng solution (Becton Dickinson) was used in accordance w i t h t h e manufacturer's in- structions. Data acquisition and sample analysis were performed in a BD FACS Caliber (4 - color, Bec- t o n Dickinson, USA), using t h e Cell Quest software (Becton Dickinson), after calibration w i t h the Cali- brate bead kit (Becton Dickinson).
The blast gating strategy included using dot plot o f CD45 expression versus intracellular com- plexity (side scatter angle, S5C) and also a second gate considering cell size (forward scatter angle, FSC) versus SSC. A total of 10,000 events were ac- quired in t h e target gate. Antigen was considered as positive if 20% or more of t h e blast cells reacted w i t h a particular antibody. A cytoplasmic marker was considered positive w h e n more than 20% of the blast cells expressed it. These values were selected by reference t o previous published immunopheno- t y p i n g AML studies and t o the recent proposals of t h e EGIL group [2].
3. Result
Table 1 . Patient distribution according to age and sex
Male Female
Total 33 10 43
%
76.7 23.3 100
Age (Year) Mean ± SD 55.0±21.3 49.6 ± 1 8 1 53.8 + 20.6
P
>0.05
- CLINICAL MEDICINE AND PHARMACY VollO-Apr/2015
From c. • f 2013 t o February 2015, 43 pa- tients w i t h newiy diagnosed acute leukemia have been collecting in t o t h e study. Patient ages are from 18 t o 85 year old (53.8 ± 20.6, mean ± SD).
Among t h e m , there were 33 (76.7%) males and 10 (23.3%) females. Ratio b e t w e e n male and female patient was 3.3/1.
Table 2 . Patient distribution according t o FAB classification
Diagnosis by morphology and
cytochemistry AML ALL Undefined
Total
Male n 26 3 4 33
Feinale n 7 3 0 10
All n 33
6 4 43
%
76.8 14 9.2 100 Based o n FAB criteria, there were 33 patients who have diagnosed AML, 6 patients w h o were d i - agnosed ALL and 4 patients (9.3%) w h o could not been diagnosed as AML or ALL.
Table 3. Patient distribution according t o i m - m u n o p h e n o t y p e
Diag nosis by imminophenotyping
AML ALL B-lineage
T-lineage Bi-phenotype
Total
Male n 29 2 1 1 33
Female n 7 3 0 0 10
All n 36 5 1 1 43
%
83.7 11.6 2.4 2.3 100
Table 4. Comparison between classification of acute leukemias by FAB criteria (based on mor- phology and cytochemistry) and by Immuno-
phenotyping method
Using i m m u n o p h e n o t y p i n g methods, all of acute leukemia cases can be classified: 36 cases (83 7%) were AML; 6 cases (14%) were ALL (5 cases were B- ALL and 1 case was T -ALL); 1 case (2.3%) was b i - p h f ' -•
Diagnosed by im- munophenotype ALL
AML
Bi-phenotye
All
6
36
1
43
Diagnosed by mor- phology and cyto-
chemistry 6 0 0 33 0 3 0 0 1 43
ALL AML Undefined
AML ALL Undefined
AML ALL Undefined
Ail There was no mis-matched diagnosis w h e n us- ing i m m u n o p h e n o t y p e and using morphology and cytochemistry. However, w h e n using morphology and cytochemistry alone, there were still 4 cases which could not be classified clearly that they were AML or A L L In that 4 cases, 3 cases were AML and 1 case was bi-phenotype when diagnosed by im- m u n o p h e n t y p i n g .
4 . Discussion
Besides morphological and cytochemical stud- ies, i m m u n o p h e n o t y p i n g has become an essential diagnostic t o o l for classification, prognosis, patient management and for disease monitoring of acute leukemias. In this study, 43 adult patients (33 males and 10 females) were enrolled in t h e period of t i m e from October 2013 to February 2015 (table 1). Based on i m m u n o p h e n o t y p n g , 36 patients (83.7%) were diagnosed AML, 6 patients (account for 14%) were ALL, only one patient was bi-phenotype acute leu- kemia. This result was reasonable because acute lymphoblastic leukemia is a major subtype of leu- kemia in children, whereas AML is as c o m m o n in adults [6]. In present study, incidence of B-ALL was 83.3% (5/6 patients) which was high in comparison t o some other studies [ 1 , 9]. An ALL of t h e B-
JOURNAL OF 108 - CLINICAL MEDICINE AND PHARMACY VoilO-Apr/2015
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lymphocyte lineage is assumed if CD22 or CD79a expression is f o u n d either cytoplasmic or on t h e cell surface w i t h the expression of CDl 9 and HLA-DR. An ALL of t h e T-lymphocyte lineage is assumed if CD3 expression is f o u n d either cytoplasmically or on t h e cell surface w i t h t h e simultaneous expression of CD7. T-lineage ALL subtypes can be defined based on the surface expression of CDl a, CD2, CD3, CD4 and CDS [8].
There were 4 cases w h o could not been classi- fied as AML or ALL if only based on morphology and cytochemistry (FAB criteria). But by i m m u n o p h e n o - typing, 3 patients were AML, 1 patient was bi- phenotype acute leukemia. By definition, t h e diag- nosis of AML require expression of myeloid- associated markers, and lack of B/T~ lineage- associated antigens (i.e. negativity for cyCD22/ or cyCD79a and cyCD3). The most significant distinc- tive immunologic markers between AML-MO and ALL are CDl 3, CD33 and CDl 17, which are typical of MO. In our study, one AML was classified as MO. The CD34 antigen is expressed by immature blast cells and is thus associated w i t h less differentiated forms of leukemia, whereas the CD13, CD15 and CD33 an- tigens are expressed by more mature cells and is associated w i t h t h e more differentiated leukemias.
Biphenotypic acute leukemia Is an u n c o m m o n type of leukemia which probably arises in a multipo- tent progenitor cell w i t h t h e capability of differenti- ating along b o t h myeloid and lymphoid tj and B) lineages where b o t h types are associated with poor outcomes. Diagnosis of biphenotypic acute leukemia is based on immunophenotyping [5].
5. Conclusion
Flow cytometric immunophenotyping is power t o o l for classification of acute leukemias in t o AML or ALL. Particularly, it can correct identification of T and B ceil lineage of ALL and diagnosis of biphenotypic acute leukemia.
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