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  Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 53  |  Issue : 3  |  Page : 349-352
 

Applicability of a single 5 color cytoplasmic markers tube as primary panel for immunophenotyping of acute leukemia: A Gujarat Cancer and Research Institute experience


1 Department of Pathology, Gujarat Cancer and Research Institute, Ahmedabad, Gujarat, India
2 Department of Cancer Biology, Gujarat Cancer and Research Institute, Ahmedabad, Gujarat, India

Date of Web Publication24-Feb-2017

Correspondence Address:
BP Parikh
Department of Pathology, Gujarat Cancer and Research Institute, Ahmedabad, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-509X.200659

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 » Abstract 

INTRODUCTION: Flow cytometry is highly sensitive for detection and quantitative analysis of surface and intracellular antigens in malignant hemopoietic cells. Immunophenotyping is a routine practice for classification and lineage assignment of acute leukemia. In the present study, our aim is to identify the role of a single 5 color, CD45, myeloperoxidase (MPO), cCD79a, cCD3, and Tdt, cytoplasmic markers combination as a primary tube. We compared with final diagnosis on the basis of morphology, cytochemistry, and primary and secondary panels of immunophenotyping and also with other study. MATERIALS AND METHODS: We have included 455 new cases of acute leukemias with applied primary and secondary panels of markers for immunophenotyping. We analyzed sensitivity and specificity of different subsets with combination of positive and negative markers. RESULTS: MPO was positive in 61.4% of acute myeloid leukemia (AML) cases. All 184 (100%) cases of the AML were negative for cCD3 and cCD79a co-expression. cCD79a expression was highly sensitive as 98.5% B-acute lymphoblastic leukemia (B-ALL) expressed it. cCD3 expression was detected in 100% cases of T-ALL, and its co-expression was not seen in B-ALL and AML. CONCLUSION: Our study indicates that there was very good correlation of 5-color cytoplasmic tube-based diagnosis versus final diagnosis based on morphology, cytochemistry, and flow cytometry. We can use this 5-color cytoplasmic tube method to make immunophenotyping cost-effective.


Keywords: Acute leukemia, cytoplasmic antigens, flow cytometry, immunophenotyping


How to cite this article:
Parikh B, Patel S, Raiya B, Vora H, Jetly D. Applicability of a single 5 color cytoplasmic markers tube as primary panel for immunophenotyping of acute leukemia: A Gujarat Cancer and Research Institute experience. Indian J Cancer 2016;53:349-52

How to cite this URL:
Parikh B, Patel S, Raiya B, Vora H, Jetly D. Applicability of a single 5 color cytoplasmic markers tube as primary panel for immunophenotyping of acute leukemia: A Gujarat Cancer and Research Institute experience. Indian J Cancer [serial online] 2016 [cited 2017 Apr 26];53:349-52. Available from: http://www.indianjcancer.com/text.asp?2016/53/3/349/200659



 » Introduction Top


Acute leukemias are a heterogeneous group of malignancies with varying clinical, morphological, immunological, and molecular characteristics.[1] Leukemias account for 0.15–0.6% of the total medical admissions in many general hospitals in India.[2] Acute myeloid leukemia (AML) accounts for approximately 20% of acute leukemia in children and 80% acute leukemia in adults.[3] Immunophenotypic panel of acute leukemia based on as panel of lineage-related markers including cell surface and cytoplasmic markers. Many studies have shown cytoplasmic markers are the earliest identifiable marker of a particular cell development and later come on surface of the cell.[4],[5],[6],[7],[8] Several advances in flow cytometry, including availability of new monoclonal antibodies, improved gating strategies, and multiparameter analytic techniques, have all improved the utility of flow cytometry in the diagnosis and classification of leukemia.[9] Morphological diagnosis of acute leukemia may be incorrect up to 9% in comparison to flow cytometry.[10] Myeloperoxidase (MPO) detection is probably the most specific technique for differentiating myeloid from lymphoid antigens.[11] The current study tried to identify the role of the flow cytometry study of acute leukemias by a single 5 color cytoplasmic markers tube, containing the markers MPO, cCD79a, cCD3, and Tdt (along with CD45-gating marker) as primary panel of acute leukemia immunophenotyping. We also compared sensitivity and specificity of the markers with the final diagnosis based on morphology, cytochemistry, and flow cytometry.


 » Materials And Methods Top


The study was conducted in the Department of Pathology, Gujarat Cancer and Research Institute, Ahmedabad, India, from January 2015 to December 2015. All the 455 cases of acute leukemia, i.e., acute lymphoblastic leukemia (ALL: B-ALL and T-ALL), AML, and mixed phenotypic acute leukemia (MPAL) were enrolled in this 1-year period.

Sample collection and preparation

The bone marrow or peripheral blood was collected in ethylenediaminetetraacetic acid vacutainer for peripheral smear examination and immunophenotyping. A morphological evaluation was done from the Wright-stained peripheral smears and bone marrow aspirates using French–American–British classification of acute leukemias. Special relevant cytochemical stains were performed on the bone marrow aspirates in all cases. Final diagnosis of acute leukemia was based on morphological examination, cytochemistry along with full panel of flow cytometric immunophenotyping. All the samples were processed within 24 h.

Multicolor monoclonal antibody combination

The monoclonal antibodies used in the primary panel were CD45 (PerCP), CD22 (FITC), CD34 (PE), CD5 (PE Cy7), CD10 (APC), CD19 (APC-H7), CD7 (FITC), CD13 (PE), CD33 (PE Cy7), CD117 (APC), HLA-DR (APC-H7), MPO (FITC), cCD79a (PE), cCD3 (PE Cy7), and Tdt (APC) and in the secondary panel were CD11b (PE Cy7), CD11c (PE), CD14 (APC-H7), CD15 (FITC), CD2 (FITC), CD4 (PE Cy7), CD8 (APC-H7), CD1a (PE), CD41a (PE), CD41b (FITC), and CD61 (FITC). The CD45 was used for blast gating for both surface and cytoplasmic markers. The antibodies were procured from BD Biosciences, USA.

Flow cytometric immunophenotyping

For surface markers, respective antibody (20 µl) mentioned above was added in six-color combination to the bone marrow or peripheral blood (100 µl, 1 × 106) and incubated for 15 min. After incubation, 2 ml of erythrocyte lysing solution (1:10 dilution with double distilled water; BD Biosciences, USA) was added and incubated for 15 min at room temperature. Then, cells were centrifuged at 400 g for 5 min and supernatant was discarded. Remaining pellet was washed twice with phosphate-buffered solutions (PBS) and then resuspended in 500 µl of PBS. For cytoplasmic markers, 2 ml lysing solution was added to 100 µl of bone marrow or peripheral blood to lyse red blood cells and incubated for 15 min. After centrifugation, to the pellet, 1 ml perm/wash buffer was added to permeabilize the cells for intracellular staining and incubated for 20 min. After centrifugation, to the pellet, respective antibody (20 µl) was added to the pellet and incubated for 15 min. Then, 2 ml PBS was added and the samples were centrifuged at 1500 rpm for 5 min. The supernatant was discarded and the pellet was resuspended in 500 µl of PBS. For surface and cytoplasmic markers, negative control tubes were run simultaneously with the addition of sample and CD45 antibody.

Acquisition and data analysis

The cytometer setup and tracking beads were (BD Biosciences, USA) used for daily calibration of the instrument. The samples were then acquired in FACSCanto II flow cytometer (6-color, 2-Laser, BD Biosciences, San Jose, CA 95131, USA) and analyzed using FACSDiva software (BD Biosciences, San Jose, CA 95131, USA). At least 30,000 total cells were acquired, and the side scatter versus CD45 PerCP dot plot was used for blasts gating. The percentage of positive cells more than 20% was considered positive for that surface or intracellular markers.


 » Results Top


In this study, 455 acute leukemia patients were enrolled, of which 184 (40.4%) diagnosed as AML, 214 (47%) as B-ALL, 55 (12.1%) as T-ALL, and 2 (0.4%) as MPAL by morphology, cytochemistry, and immunophenotyping. These patients were subgrouped according to 11 different subsets listed in [Table 1] based on marker expression.
Table 1: Different subset of tubes with variable positive and negative expression of cytoplasmic markers

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Acute myeloid leukemia subgroup

Out of 184 AML patients diagnosed, 113 (61.4%) were positive for MPO and remaining 71 (38.6%) were negative for MPO. Further, MPO + cCD79a cCD3 Tdt phenotype was detected in 112 (61%) and MPO cCD79a cCD3 Tdt in 61 (33.1%) of AML patients [Table 1]. Tdt positivity was noted in 11 (5.4%) patients with a phenotype of MPO cCD79a cCD3 Tdt + in ten patients and MPO + cCD79a cCD3 Tdt + in one patient. Hence, MPO + cCD79a cCD3+/− Tdt +/− phenotype was detected in 113 (61.4%) cases. These AML patients did not show co-expression of cCD3 and cCD79a. It was found that MPO has 61.4% sensitivity and 99.63% specificity for the diagnosis of AML [Table 3].
Table 2: Different subset of cytoplasmic tubes with variable positive negative expression after ignoring Tdt status

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Table 3: Frequency of different cytoplasmic markers in their particular lineage

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B-acute lymphoblastic leukemia subgroup

Out of 214 B-ALL patients diagnosed, 211 (98.6%) were positive for cCD79a and remaining 3 (1.4%) were negative for cCD79a. Further, MPO cCD79a + cCD3 Tdt + phenotype was detected in 179 (83.6%) and MPO cCD79a + cCD3 Tdt phenotype in 32 (15%) of B-ALL patients. MPO cCD79a cCD3 Tdt + was detected in 2 (0.9%) and MPO cCD79a cCD3 Tdt in 1 (0.5%) of B-ALL patients. Hence, MPO cCD79a + cCD3 Tdt +/− was noted in 211 (98.6%) and MPO cCD79a cCD3 Tdt +/− in 3 (1.4%) of B-ALL patients. These B-ALL patients did not show co-expression of MPO and cCD3 [Table 3]. It was found that cCD79a has 98.62% sensitivity and 99.59% specificity for the diagnosis of B-ALL.

T-acute lymphoblastic leukemia subgroup

Out of 55 T-ALL patients, MPO cCD79a cCD3+ Tdt + phenotype was detected in 38 (69.1%) and MPO cCD79a cCD3+ Tdt phenotype in 17 (30.9%) of T-ALL patients. cCD3 expression was detected in 100% cases of T-ALL. Tdt co-expression was found in 38 (69.1%) cases of T-ALL, with a phenotype of MPO cCD79a cCD3+ Tdt +. It was found that cCD3 has 100% sensitivity and 99.75% specificity for T-ALL diagnosis [Table 3].

Mixed phenotypic acute leukemia subgroup

Out of 455 cases of acute leukemia, two cases were diagnosed as MPAL. One case was T-myeloid and another B/T-ALL leukemia. MPO + cCD79a cCD3+ Tdt phenotype was detected in T-myeloid acute leukemia. MPO cCD79a cCD3+ Tdt + phenotype was detected in single case of B/T-ALL. cCD3 was expressed in both the cases of MPAL while Tdt and MPO expression were found in single case. None of the cases of MPAL expressed cCD79a in our study.


 » Discussion Top


Flow cytometry-based immunophenotyping has important role in accurate classification and diagnosis of acute leukemia. Various cell surface and intracellular CD markers are required for primary and secondary panels. Intracellular markers expressed earliest phase of disease and had good specificity for myeloid and lymphoid lineage determination in acute leukemia blasts.[4],[5],[6] In this current study, we tried to identify the applicability of a single 5 color cytoplasmic markers tube of the combination of MPO, cCD79a, cCD3, and Tdt as primary panel. We correlated the diagnosed based on the cytoplasmic tube with final diagnosis. Our final diagnosis was based on morphology, cytochemistry, and surface and cytoplasmic markers on immunophenotyping. We found that cCD3 and MPO positivity with cCD79a negativity provides 100% specific diagnosis of T-ALL and AML, respectively. Single intracellular combination MPO cCD3+ cCD79a +/− Tdt +/− independent had 100% sensitivity and 99.75% specificity for T-ALL. A single tube of MPO + cCD79a cCD3+/− Tdt +/− has 61.4% sensitivity and 99.63% specificity. MPO-negative AML was 71 (38.6%) cases. All the cytoplasmic markers negativity was 38.6% specific for AML diagnosis. MPO + cCD79a cCD3+ Tdt was 100% sensitive and 100% specific for MPAL (T-myeloid).

Acute myeloid leukemia subgroup

In our study of AML subset, MPO + cCD79a cCD3 Tdt was 61% comparable with Sharma et al. study (56.7%).[12] MPO + cCD79a cCD3 Tdt + was very low 0.5% in our study against their study (23.4%). In AML, all four markers were negative, MPO cCD79a cCD3 Tdt was 33.15% in our study against 5.4% in their study [Table 1]. When we ignore Tdt positivity/negativity in AML, MPO + cCD79a cCD3+/− Tdt +/− was 61.4% in our study while 80.1% in their study [Table 2]. However, there was significant difference in AML subset; MPO cCD79a cCD3 Tdt +/− was 38.6% in our study against 8.1% in their study.[12] [Table 4] depicts that in AML, we detected 61.4% positivity of MPO which is comparable with Salem et al. (67.2%) and Renate et al. (70.5%).[1],[2],[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13] The current study shows good correlations of the expression of cCD79a, cCD3, and Tdt in AML cases with various authors [Table 4].
Table 4: Comparison of various cytoplasmic markers in particular lineage

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B-acute lymphoblastic leukemia

In our study of B-ALL, MPO cCD79a + cCD3 Tdt + was 83.6% comparable with Sharma et al. study (86.9%).[12] MPO cCD79a + cCD3 Tdt was found in 15% in the current study against 11.9% of their study. Hence, both subsets were correlated very well. When we ignore Tdt positivity/negativity sensitivity, MPO cCD79a + cCD3 Tdt +/− was 98.62% in our study while 98.8% in their study.[12] MPO cCD79a cCD3 Tdt +/− was 1.4% in our study against 1.1% in their study. It shows good correlation.[12] In total B-ALL cases, individual expression of MPO, cCD79a, cCD3, and Tdt in the present study which shows very good correlation with vzarious authors [Table 4].

T-acute lymphoblastic leukemia

In T-ALL subset, MPO cCD79a cCD3+ Tdt + was detected in 69.1% in the present study against 25% as per Sharma et al.[12] Our study shows MPO cCD79a cCD3+ Tdt in 30.9% cases against 18.1% of their study. In T-ALL, if we ignore cCD79a and Tdt positivity/negativity, sensitivity of subset MPO cCD3+ cCD79a +/− Tdt +/− was 100% as same as their study 100%. We found 69% Tdt expression in T-ALL while it was 63.64% in their study.[12] It gives good correlation. In our study, cCD3 positivity was 100% sensitive and specific for diagnosis of T-ALL similar to Sharma et al. In total T-ALL cases, individual expression of MPO, cCD79a, cCD3, and Tdt in the present study which shows very good correlation with various authors [Table 4].[1],[12],[14] Various studies show that intracellular expression of MPO, cCD3, cCD79a, and CD22 is the earliest expression myeloid, B- and T-cell markers.[14],[15],[16],[17],[18],[19]


 » Conclusion Top


In the present study, we detected role and applicability of a single 5 color cytoplasmic markers tube-based method to use as primary panel in routine acute leukemia immunophenotyping. There was very good correlation of 5 color cytoplasmic markers tube-based diagnosis versus final diagnosis based on morphology, cytochemistry, and flow cytometry. We can use this single 5 color cytoplasmic markers tube method to make immunophenotyping cost-effective; however, further study is required from India.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 » References Top

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Sharma RK, Puri V, Mutereja D, Kumar S, Chauhan P, Purohit A, et al. Applicability of a single 5 color cytoplasmic tube as primary panel in routine immunophenotyping of acute leukemia. J Blood Disord Transfus 2015;6:1-5.  Back to cited text no. 12
    
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Renate TS, Mitterbauer G, Simonitsch I, Jaeger U, Lechner K, Schneider B, et al. The immunophenotype of 325 adult acute leukemias: Relationship to morphologic and molecular classification and proposal for a minimal screening program highly predictive for lineage discrimination. Am J Clin Pathol 2002;117:380-9.  Back to cited text no. 13
    
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Sartor M, Bradstock K. Detection of intracellular lymphoid differentiation antigens by flow cytometry in acute lymphoblastic leukemia. Cytometry 1994;18:119-22.  Back to cited text no. 14
    
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van der Schoot CE, Daams GM, Pinkster J, Vet R, von dem Borne AE. Monoclonal antibodies against myeloperoxidase are valuable immunological reagents for the diagnosis of acute myeloid leukaemia. Br J Haematol 1990;74:173-8.  Back to cited text no. 15
    
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van der Schoot CE, von dem Borne AE, Tetteroo PA. Characterization of myeloid leukemia by monoclonal antibodies, with an emphasis on antibodies against myeloperoxidase. Acta Haematol 1987;78 Suppl 1:32-40.  Back to cited text no. 16
    
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Dworzak MN, Fritsch G, Fröschl G, Printz D, Gadner H. Four-color flow cytometric investigation of terminal deoxynucleotidyl transferase-positive lymphoid precursors in pediatric bone marrow: CD79a expression precedes CD19 in early B-cell ontogeny. Blood 1998;92:3203-9.  Back to cited text no. 17
    
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Mason DY, Cordell JL, Tse AG, van Dongen JJ, van Noesel CJ, Micklem K, et al. The IgM-associated protein mb-1 as a marker of normal and neoplastic B cells. J Immunol 1991;147:2474-82.  Back to cited text no. 18
    
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Rani S, De Oliveira MS, Catovsky D. Different expression of CD3 and CD22 in leukemic cells according to whether tested in suspension or fixed on slides. Hematol Pathol 1988;2:73-8.  Back to cited text no. 19
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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