Immunophenotyping Of Acute Leukemia Research Articles

Guidelines for the use of flow cell sorting in diagnosis and monitoring of acute leukemia

Flow cell sorting is an advanced laboratory technique that combines the analytical capabilities of flow cytometry with the ability to isolate pure cell populations from heterogeneous samples. It has tremendous potential both for fundamental research and laboratory diagnosis. For example, the combination of cell sorting and molecular genetic studies can be used to clarify ambiguous results of acute leukemia immunophenotyping obtained both at diagnosis and during minimal residual disease monitoring. These guidelines are based on years of experience in incorporating cell sorting into the diagnostic and monitoring processes at the Leukemia Immunophenotyping Laboratory of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. They include methods used for the confirmation of flow cytometry data depending on the type of leukemia, the stage of a flow cytometry assay and previous therapy. They also describe cell sorting algorithms for disease diagnosis and the specifics of sample preparation for cell sorting in different molecular genetic studies.

Immunophenotyping of Acute Leukemia in Pediatric Patients: Tertiary Care Centre Experience from Lahore

Acute Leukemia is the most prevalent malignancy of childhood, globally. Immunophenotyping by flowcytometry has developed as an important technique for its outstanding contributions in diagnosis and management of leukemia patients. Objective: To determine the frequency and immunophenotyping by flowcytometry of Acute leukemia in pediatric age group. Methods: The study was conducted at The University of Child health sciences and Children’s Hospital, Lahore from July-2021 to Feb-2022. Data were gathered from 101 consecutive patients, of age from 0.5 to 15 years, that had acute leukemia immunophenotyping using flowcytometry. Results: Flowcytometric immunophenotyping of 101 leukemia patients over 8-month period showed that 67.3% and 33.6% male were females respectively. AML, T-ALL, and B-ALL prevalence was highest in patients with 5 to 10 years of age. Out of 101 leukemia patients, 16 (15.8%) and 85 (84.1%) had AML and ALL diagnosis respectively. Of these 85 ALL cases, 72 (84.7%) were B-ALL and 13 (15.2%) were T-ALL. Study founded that most frequent CD markers in B-ALL, were CD79 and CD19 (100%) whereas CD3 and CD5 in T-ALL and CD13 (93.8%) and CD34 (87.5%) in AML. CD34 was a common marker among B-ALL, T-ALL and AML. Among B-ALL and T-ALL, AntiTdt was a common CD marker whereas HLA-DR was common among AML and B-ALL. Conclusions: Results of Acute leukemia immunophenotyping were homologous to worldwide published research. For accurate leukemia lineage, immunophenotyping of AML and ALL is essential since, if therapy is started based solely on morphological diagnosis, approximately 25% of patients may not respond or recure.

Molecular Cytogenetics and Immunophenotypic Characterization of Childhood Acute Leukaemia Patients in Tertiary Care Center of Bangladesh – An observative study

Background: Childhood leukemia is genetically a heterogeneous disease. Various types of cytogenetic abnormalities and immunophenotypic character are present in leukaemia which are important for risk stratification, treatment and play as significant prognostic factor. Pediatric acute leukaemia presents with varying clinical, morphological, immunological and molecular characteristics. It is very highly curable if diagnosed and treated properly. For detail typing and subtyping of acute leukemia immunophenotyping and cytogenetics are crucial. The aim of this study was to find out the genetic abnormalities and immunophenotypic characterization of childhood acute leukaemia patients in Bangladesh. Material & Methods: This was a retrospective observational study and was conducted in the Department of pediatric hemato-oncology of Combined Military Hospital, Dhaka and Ahsania Mission Cancer Hospital, Mirpur, Dhaka, Bangladesh during the period from February,2014 to March, 2022. There was total 98 cases of acute leukaemia. Results: In total 98 patients completed the study. We found that 79.59% patients were ALL and 20.41% patients were AML. Among ALL 80.64% were B cell type, 6.40% were T cell Type ; 12.82 % had TEL/AML1 or ETV6/RUNX1 t(12;21)(p13;q22), 5.13% patient had TCF3/PBX1 or E2A/PBX1 t(1;19)(q23;p13). In AML30.00% patient had PML/RARAt(15;17)(q22;q21), 10.00% patient had AML1/ETO or RUNX1/CBFA2T1 t(8;21) (q22;q22), 5.00% patient had FLT3/ ITD. In case of B-ALL highest expression of antigen was CD19 (91.64%) followed by CD10 (80.58%), HLADR (67.94%), CD22 (72.68%), CD79a (72.68%), TdT (52.14%) and CD34 (48.98%). In 44.24% cases there was co-expression of CD10 and CD19 and there was 11.6% expression of myeloid marker CD13 and 1.58% expression of T cell marker CD5. In case of T-ALL there was 100% expression of CD3. Expression of other antigen CD4, CD5, CD7, CD8, CD4/8 co-expression, TdT was 60%. There was 40% expression of CD1a and CD2. There was 20% expression of CD10, CD34 and TCRab also. In case of AML highest expression was MPO (93.75%) followed by CD33 (87.50%), CD13 (81.25%), CD117 (75%), HLADR (43.75%) and CD64 (50%). There was 6.25% aberrant expression of B-ALL marker CD19 and T-ALL marker CD3, CD4, CD5, CD7 also. Conclusion: Depending on this study we can say that except few variations distribution of immunophenotypical subtypes and genetic abnormalities of childhood acute leukaemia are almost similar to other literature published from neighboring countries.This study will serve as a guideline for future study in our country in this aspect.

CD72 Is a Pan-Tumor Antigen Associated with Childhood Acute Leukemia

Immunotherapy has recently gained increasing attention as a new paradigm shift in cancer and has revolutionized the course of cancer treatment. The identification of ideal tumor-associated antigens (TAA) is a challenging process, since antigens have to be specific for the target cells and assure no off-target effects. We exploited a gene expression dataset of acute leukemias previously generated in our lab to identify potentially interesting TAAs. Among the most interesting hyper- and over-expressed genes with respect to normal bone marrow (BM) and with a surface protein localization, the CD72 was selected because of its specific association with childhood B-cell precursor acute lymphoblastic leukemias (BCP-ALL) and with a large proportion of pediatric acute myeloid leukemias (AML) cases. Based on the findings described below, we here propose CD72 as a novel and robust pan-acute leukemia TAA. Even though CD72 is known to be expressed by B-lineage cells, we profiled its expression in a variety of cell lines and primary samples, providing a map of its expression. CD72 was expressed by BCP-ALL cell lines (NALM-6, SEM, RS-4;11, RHC-ACV) and various AML cell lines (SHI-1, SKNO-1, MV4;11, MOML-13). Conversely, T-ALL cell lines (DND-41, TALL-1, RPMI-8402, ALL-SIL, JURKAT) and non-hematopoietic tumor cell lines (DAOY, NB-1, HT-29, A549, HeLa, MCF7) did not express the antigen. This supports the idea that CD72 is specific for hematopoietic tumors. Furthermore, we tested CD72 expression on major populations within normal BM specimens and on other healthy tissues: skin fibroblasts, mesenchymal stromal cells, skeletal myoblasts, endothelial cells. We confirmed that the antigen is expressed on B-lineage cells, whereas in other cell types we did not detect any signal. Then, to assess the interest of CD72 for the development of chimeric antigen receptor (CAR)-based immunotherapy approaches, we documented that CD72 was not expressed on CD34-positive/CD38-negative hematopoietic stem and progenitor cells (HSPCs) from both mobilized peripheral blood cells and healthy-regenerating BM samples (Figure 1). We profiled CD72 expression by flow-cytometry on 289 pathological pediatric BM samples, including: 93 BCP-ALL at diagnosis and 44 at relapse; 38 T-ALL at diagnosis and 3 at relapse; 68 AML at diagnosis and 12 at relapse; blast cells from 6 myelodysplastic syndromes; 14 non-hematopoietic solid tumors invading BM (4 rhabdomyosarcomas, 6 neuroblastomas, 4 Ewing's sarcomas); blast cells from 3 T-cell Lymphoblastic Lymphomas and 8 B-lineage Non-Hodgkin Lymphomas (NHL) with BM involvement. Data from clinical samples confirmed the in silico and in vitro prediction. CD72 was positive in 134/137 (98%) of the examined BCP-ALL cases and in all B-lineage NHL and 52/80 (65%) of AML cases. Conversely, CD72 was expressed only in 2/44 (4%) of T-ALL cases and was absent in T-cell NHL and in non-hematopoietic solid tumors. Notably, CD72 was expressed by all 12 BCP-ALL patients that were previously treated with CD19-directed immunotherapies, including four patients who lost CD19 expression. Moreover, CD72 was positive on two rare cases of CD19-negative BCP-ALL at diagnosis (Figure 2). Within our AML cohort, we did not detect any difference in CD72 expression across AML genetic subgroups. Taken together, the preliminary data here reported highlight the potential clinical usefulness of CD72 for advanced diagnostics, including the ability to identify blast cells in case of CD19 loss, and provide support for developing novel immunotherapy approach directed at CD72 that may be endowed with the unique potential of targeting both acute lymphoid and myeloid leukemia. Figure 1. CD72 expression by flow-cytometry on healthy CD34+ and CD34+/CD38- cells from healthy-regenerating bone marrow. CD72 expression was evaluated according to AIEOP-BFM Consensus guidelines, comparing the fluorescence shift and distribution pattern of the cells to the appropriate negative control. Figure 2. CD72 expression on CD19-negative BCP-ALL by flow-cytometry at first diagnosis and at time of relapse after CD19-directed immunotherapy. B-lineage was assigned with the contribution of other antigens relevant in immunophenotyping of acute leukemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Diagnosis of Acute Leukemia by Multiparameter Flow Cytometry with the Assistance of Artificial Intelligence.

We developed an artificial intelligence (AI) model that evaluates the feasibility of AI-assisted multiparameter flow cytometry (MFC) diagnosis of acute leukemia. Two hundred acute leukemia patients and 94 patients with cytopenia(s) or hematocytosis were selected to study the AI application in MFC diagnosis of acute leukemia. The kappa test analyzed the consistency of the diagnostic results and the immunophenotype of acute leukemia. Bland–Altman and Pearson analyses evaluated the consistency and correlation of the abnormal cell proportion between the AI and manual methods. The AI analysis time for each case (83.72 ± 23.90 s, mean ± SD) was significantly shorter than the average time for manual analysis (15.64 ± 7.16 min, mean ± SD). The total consistency of diagnostic results was 0.976 (kappa (κ) = 0.963). The Bland–Altman evaluation of the abnormal cell proportion between the AI analysis and manual analysis showed that the bias ± SD was 0.752 ± 6.646, and the 95% limit of agreement was from −12.775 to 13.779 (p = 0.1225). The total consistency of the AI immunophenotypic diagnosis and the manual results was 0.889 (kappa, 0.775). The consistency and speedup of the AI-assisted workflow indicate its promising clinical application.

Spectrum of immunophenotypic aberrancies in acute leukemia along with their correlation with adverse hematological parameters

BACKGROUND: The identification of aberrant phenotypes in acute leukemia is crucial for treatment monitoring and minimal residual disease analysis. Flowcytometric immunophenotyping in acute leukemia is an important tool for the detection of these aberrancies. There is wide variation in the incidence of aberrant antigens in acute leukemia in different studies and its correlation with prognostic markers.AIMS AND OBJECTIVES: The aim and objective of this study is to assess the frequency of aberrant markers in acute leukemia and determine any association with hematological parameters.MATERIALS AND METHODS: A total of 150 newly diagnosed cases of acute leukemia during the period January 2018–December 2020 were included. The flowcytometric immunophenotyping of all the above cases was done using BD FACS Canto II.RESULTS: Out of the total 150 cases of acute leukemia, 56.7% expressed aberrant phenotype. The proportional frequency of aberrant antigen expression in B-acute lymphoid leukemia (ALL), T-ALL and acute myeloid leukemia (AML) was 56.6%, 50%, and 58.5%, respectively. CD13, CD13, and CD7 were the most common aberrancies in B-ALL, T-ALL, and AML, respectively. Aberrant myeloid phenotype in B-ALL was associated with lower mean total leukocyte count (TLC) and blast percentage in peripheral blood as compared to the B-ALL with conventional phenotypes. Aberrant lymphoid phenotype in AML was associated with a higher TLC and greater blast percentage in peripheral blood than the AML with conventional phenotypes.CONCLUSION: Aberrant lymphoid phenotype in AML is associated with unfavorable hematological features. However, aberrant myeloid phenotype in B-ALL is not associated with adverse features.

Health technology assessment-based approach to flow cytometric immunophenotyping of acute leukemias: a literature classification.

Acute leukemia (AL) is a broad, heterogeneous group of malignant diseases. The diagnostic workup of AL is based on several clinical and laboratory findings, including flow cytometric immunophenotyping. However, the role of this assay in the diagnosis of AL has not been systematically investigated. The aim of this study was to determine the accuracy and utility of flow cytometric immunophenotyping in the identification, characterization, and staging of AL. We performed a systematic selection and classification of the literature since 1980, focused on flow cytometric immunophenotyping of AL. We applied a 6-variables model to cover both the technical capabilities and the clinical value of flow cytometric immunophenotyping in the diagnosis of AL. Using 3 key words (acute leukemia, immunophenotyping, flow cytometry), we screened the literature from January 1985 to April 2015 in PubMed and Embase databases and found 1010 articles. A total of 363 were selected and submitted to the expert panel, which selected a final data set of 248 articles to be analyzed. Of these, 160 were focused on clinical and biological issues, 55 were technical articles, and 31 were reviews. These 248 articles were then analyzed according to the 6-variables model and definitively classified. We assessed the literature on flow cytometric immunophenotyping of AL over 3 decades as the first step toward an evidence-based analysis of the impact of this technology on the clinical management of patients with AL.

Characterization of Immunophenotypic Aberrancies in Adult and Childhood Acute Lymphoblastic Leukemia: Lessons from Regional Variation.

Background & Objective:Although the antigen expression patterns of acute lymphoblastic leukemia (ALL) are well known, this study attempted to evaluate commonly used immune markers for immunophenotyping of acute leukemia to set the minimum of necessary diagnostic panels by flow cytometry.Methods:This study evaluated 89 patients referred from all over the country to the Iranian Blood Transfusion Organization (IBTO) in Tehran from 2013 to 2015. We compared the immunophenotype patterns of childhood and adult ALLs including 69(77.5%) B-cell lymphoblastic lymphoma (B-LBL), 2(2.2%) Burkitt’s lymphoma (BL), and 18(20.2%) T-cell lymphoblastic lymphoma (T-LBL) cases using flowcytometry with broad antibody panel.Results:CD19 and CD79a were the most frequent markers for B-LBL while CD7 was the most sensitive marker in T-LBL; the frequency of CD7, CD3, and CD5 antigens were 100%, 38.9%, and 88.9%, respectively. TdT+/CD34+ was significantly higher in adult B-LBLs than children, which indicates blast cells are more immature in adults. In addition, CD10 and cCD79a were significantly higher in children with B-LBL like as CD5 and CD8 in children with T-LBL. Aberrant phenotypes including CD13, CD33, CD7, and CD117 were found in 7(10.1%) cases of B-LBL. These phenotypes were CD117, HLA-DR, and CD33 in 7(38/9%) cases of T-LBL. Expression of CD117 aberrant myeloid antigen was significantly more associated with T-LBL than with B-lineage ALL.Conclusion:Significant differences were observed in antigen-expression patterns between adult and childhood ALLs. Further studies are needed to correlate specific markers with recurrent cytogenetic abnormalities and prognosis with therapeutic response.

Clinically useful flow cytometry approach to identify immunophenotype in acute leukemia.

ObjectivesAcute leukemia (AL) is a highly heterogeneous malignant disease caused by hematopoietic cell abnormalities. Our study investigated the potential for immunophenotyping of leukemic cells via flow cytometry and the clinical usefulness of this approach in treatment of AL.MethodsBone marrow (BM) specimens were collected to detect antigen expression on hematopoietic cells in pre-treatment samples from patients with AL. In addition, fraction survival curves were calculated using the Kaplan-Meier method to explore the effect of markers on prognosis in AL.ResultsExpression levels of immunophenotypic markers in patients with acute lymphoblastic leukemia (ALL) were significantly different from those in patients with acute myeloid leukemia (AML). In addition, there was a potential association between the surface marker, cluster of differentiation 2 (CD2), and fraction survival in AML. However, no similar result was found in ALL. Moreover, genetic tests showed greater positive variation of the break point cluster-Abelson tyrosine kinase (BCR-ABL) fusion gene in samples from patients with ALL than in samples from patients with AML.ConclusionsWe have shown a rapid and effective flow cytometry method that enables the identification of immunophenotype in AL. Moreover, CD2 may constitute a predictive marker for prognosis in patients with AML.

Importance of Detection of Intracellular Myeloperoxidase, CD13, CD79a, CD22, CD3 and Terminal Deoxynucleotidyl Transferase By Flow Cytometry Diagnosis of Acute Leukemias

Background: The detection of Intracellular (IC) antigens by flow cytometry (FC) such as myeloperoxidase (MPO), cCD13, cCD79a, cCD22, cCD3 and Terminal deoxynucleotidyl Transferase (TdT) has become the useful tool in the differential diagnosis between acute myeloid leukemias (AML) and acute lymphoid leukemias (ALL). Through detection of myeloid antigens (MPO and cCD13), B cells precursors (cCD79a and cCD22) and precocity T-cells (cCD3) it has been possible to confirm the diagnosis of these acute leukemias. The detection of intracellular cell markers by FC usually requires previous permeabilization of fresh cell suspensions. TdT, also known as DNA nucleotidylexotransferase (DNTT) or terminal transferase, is a specialized DNA polymerase expressed in immature, pre-B, pre-T lymphoid cells, and acute lymphoblastic leukemia/lymphoma cells. TdT adds N-nucleotides to the V, D, and J exons of the TCR and BCR genes during antibody gene recombination, enabling the phenomenon of junctional diversity. In humans, terminal transferase is encoded by the DNTT gene. This antigen is expressed mostly in the nucleus cells from primary lymphoid organs, like the thymus and bone marrow. The TdT detection has also been shown to be useful in confirming the acute forms of B and T-lineage lymphoproliferative diseases by FC. The aim of this study was to demonstrate the importance of this cell markers' detection by FC in the differential diagnostic of acute leukemias. Methods: Bone marrow and/or peripheral blood leukemic cells from 50 cases of acute leukemia: 16 ALL and 36 AML. The cells were fixed and permeabilized in briefly exposed to Becton & Dickinson Lyse Solution at concentration of 10%, and subsequently labeled with monoclonal antibodies anti-MPO, TdT, CD3, CD13, CD22 and CD79a. Results: The MPO expression was observed in 35/36(97,22%) and cCD13 in all cases of AML and in none ALL patients. Three cases of MPO-positive ALL (FAB-L2) could be reclassified as M0-AML. These cases were CD34+;HLADR+;CD33-;CD13-;CD7+ and cCD13+. The intensity of TdT expression was observed in 15/16 (93.8%) of ALL and 5/36 (13.9%) of AML. The cCD22 and cCD79a were positive in 15/16 (93.8%) and all of pre-B ALL respectively and cCD3 was expressed in one case of Pre-T ALL that initial phenotype was CD34+/HLADR+/TdT+/CD7+ and sCD3-). Conclusions: These results indicate that monoclonal antibodies anti-MPO, cCD13, cCD79a, cCD22, cCD3 and TdT were excellent cell markers for the diagnosis and classification of acute leukemias and can be reliably detected by FC. This rapid and specific technique should be a valuable addition to routine immunophenotyping of acute leukemia. DisclosuresNo relevant conflicts of interest to declare.

Flow Cytometric Immunophenotyping of Acute Leukemia the Essential Considerations

Objective: This article serves to promote the interest of participants involved in management of hematological malignancies in the efficacy of flow cytometry in the immunophenotypic characterization of leukemias and lymphomas. This well-defmed characterization plays a crucial role in diagnosis, classification, prognostic evaluation, and detection of minimal residual disease, in the context of clinical features and morphological diagnosis.Methodology: Relevant literature was retrieved to highlight the principles of operation of flow cytometly, applications, derivable clinical information, sources of errors, necessary antibodies and general considerations towards definitive diagnosis of acute leukemia.Conclusion: Hematological malignancy is an extremely heterogeneous disorder. Use of flow cytometric immunophenotyping in these disorders is already proven over two decades and allowed clinicians for proper classification along the latest WHO classification guidelines and will pave the way for targeted therapy and better patient management. The flow cytometric immunophenotyping services in the country will significantly outweigh the cost by reducing the duration of hospital stay and patient management in right way.Pulse Vol.9 January-December 2016 p.27-36

Immunophenotyping of Acute Leukemias: Insight into Prognosis since the Outset

Immunophenotyping of Acute Leukemias: Insight into Prognosis since the Outset

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

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. 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. 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. 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.

Expert consensus on 4 color flow cytometry for immunophenotyping of acute leukemia(2015)

Expert consensus on 4 color flow cytometry for immunophenotyping of acute leukemia(2015)

Applicability of a Single 5 Color Cytoplasmic Markers Tube as Primary Panel in Routine Immunophenotyping of Acute Leukemia

Background: Immunophenotyping in acute leukemia is a routine practice for lineage assignment. Conventionally a primary panel with surface markers are applied first followed by cytoplasmic markers as secondary panel in the diagnosis of acute leukemia.We in this present study aim to assess the relevance of a single 5 color “CD45, MPO, CD79a, CD3, Tdt” cytoplasmic markers combination to be utilized as primary panel. Methods: Sensitivity and specificity of different subset of positive negative combination of these markers were retrospectively analyzed in the 458 acute leukemia cases. Results: MPO or cCD3 positivity alongwith cCD79a negativity was 100% specific diagnosis for AML and T-ALL respectively. Furthermore, cCD79a positivity along with MPO and cCD3 negativity was 97.2% specific for B-ALL diagnosis. MPO and cCD79a dual positivity was found 100% sensitive and 92.6% specific for MPAL (B-My) diagnosis. MPO and cCD3 dual positivity was 100% sensitive and specific for MPAL (T-My) diagnosis. Conclusion: We found a good correlation of this single tube diagnosis when compared with standard morphology, cytochemistry, and flow cytometry based diagnosis. We hope our this cytoplasmic panel may help to design a precise extended immunophenotypic panel for acute leukemia diagnosis and may also be a cost effective approach in resource constrained developing countries.

Presence of MLL Gene Rearrangements in Infant Acute Leukemia Could be Predicted By Tumor cells’ Immunophenotype

Acute leukemia (AL) in children less than 1 year old is the relatively rare disease with specific biological features and poor outcome. It is also characterized by high incidence of MLL gene rearrangements. Immunophenotype of infants’ leukemia varies due to presence or absence of MLL gene rearrangements.Aim of the study –description of immunophenotype in infant acute lymphoblastic and acute myeloid leukemia (ALL and AML respectively) due to presence of MLL gene rearrangements.Methods. Totally 540 cases of pediatric AL were studied. 113 patients (59 boys and 54 girls) aged from 5 days to 11 months were included in the study group. Their data was compared to 427 cases of acute leukemia in older children. Tumor cells immunophenotyping was performed by 6-8-color flow cytometry. Detection of various types of MLL-gene rearrangements was done by fluorescence in-situ hybridization, reverse-transcriptase polymerase chain reaction (PCR) and long-distance inverse PCR.Results. ALL was found less frequently in infants than in older children (68.1% and 86.9% respectively, p<0.001) while percentage of acute myeloid leukemia cases was higher in infants (27.4% and 11.5% respectively, p<0.001). Significant immunophenotypic differences were observed in patients with and without MLL gene rearrangements in both ALL and AML. Number of ALL cases in those tumor cells expressed CD10, CD20, CD45, CD133, CD15, CD65 NG2 significantly varied between MLL-positive and MLL-negative groups (p<0.001, p<0.001, p=0.002, p<0.001, p=0.004, p=0.019 and p<0.001 respectively). NG2-positivity represented the highest overall correct prediction (OCP) rate for presence of MLL-rearrangements (90.6%). Diagnostic accuracy of CD20-negativity and CD45-positivity was lower (81.2% and 81.9% respectively) while OCP for CD10-negativity (76.4%), CD133-positivity (76.5%) CD15-positivity (67.7%) and CD65-positivity (53.7) was not sufficient enough. Nevertheless CD10-positive BCP-ALL with MLL-rearrangements differed from CD10(+) cases in MLL-germline group. CD10 homogeneous expression was noted frequently in MLL-germline cases than in MLL-rearranged ones (p=0.001). Although there were found no significant differences in CD22-positive patients’ number, CD22(+)-cells percentage was significantly lower in MLL-positive cases (median 89.9%, range 25.2-99.7% and median 99.9%, range 96.0-99.9% respectively, р=0.003). Thus CD20-negativity, CD10-negativity/low expression, high CD45, CD15, CD65, CD133 and NG2 expression, decreased CD22-expression are immunophenotypic signatures of MLL-rearranged infant ALL, although NG2 has the highest diagnostic efficacy. Interestingly CD10-negativity and positivity for CD34, CD15 and CD65 could be able to distinguish MLL-AF4-positive cases from patients carrying other types of MLL-rearrangements. Number of AML cases in those tumor cells expressed CD99, CD133, CD15, CD65, CD4, CD11b, CD61, NG2 varied between MLL-positive and MLL-negative groups ((p=0.019, p=0.012, p=0.002, p=0.004, p=0.005, p<0.001, p=0.015 and p<0.001 respectively). Thus CD61-negativity, high CD99, CD15, CD133, CD65, CD11b, CD4 and NG2 expression were immunophenotypic signatures of MLL-rearranged infant AML, although CD11b and NG2 had the highest diagnostic efficacy (95.0% and 89.7% respectively).Conclusion. Thus immunophenotype of AL in children less than 1 year old differs significantly from patients of older age groups. Infants’ ALL and AML immunophenotype varies greatly due to the presence of MLL gene rearrangements. Complex diagnostic immunophenotyping of infants’ AL allows predicting presence of MLL rearrangements while NG2 and CD11b are the most applicable single markers for ALL and AML respectively. DisclosuresNo relevant conflicts of interest to declare.

Immunophenotypic study of acute leukemia by flow 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

Correlation of morphologic and cytochemical diagnosis with flowcytometric analysis in acute leukemia

The classification of acute leukemias has revolutionized over the years. Immunophenotyping of acute leukemia has gained popularity because of its influence on treatment and prognosis of the disease. The various antigens expressed by the leukemic cells can be assessed by flowcytometry (FCA) and can be used in rendering specific treatment and predicting the outcome of the different types of acute leukemia. The main aim of this study was to compare the morphologic and cytochemical diagnoses with flowcytometric diagnoses in acute leukemia and to analyze the usefulness of FCA over morphology. In this study we analyzed 50 cases of acute leukemia and found concordance rate as high as 86% between morphologic/cytochemical diagnosis and flowcytometric diagnosis. Of these, complete concordance was seen in 58% of the cases and partial concordance was seen in 22% of the cases. Non-concordance was seen in only 4% of our cases. In remaining 16% of our cases FCA helped in sub classifying the acute leukemia where morphology and cytochemistry had failed to do so. CD19 and 20 were found to be consistent B-cell markers and CD3 was a very specific marker for T-cell leukemia. CD13 and 33 were important myeloid markers and were aided by other secondary panel of markers like CD14, CD117 and CD41. FCA not only helps in confirming morphologic diagnosis in acute leukemia but also helps in assigning specific lineage to the blasts, particularly in acute lymphoid leukemia. Immunophenotyping is of utmost importance in classifying acute leukemia as it greatly influences the treatment and the prognosis.

Prevalence of Immunophenotypes in Neoplastic Hematology At Laboratorios Fatima De Michoacan

AbstractAbstract 4829 Introduction:Medical indication of flow cytometric immunophenotyping includes diagnosis, classification, prognosis and disease monitoring. This is an important tool that each time is more used in hematology. Third world countries could not stay away from this technology, effort should be made to get access to it and not only make diagnosis in morphology. Objective:To determinate the prevalence of immunophenotypes in neoplastic hematology in our region (Central-West Mexico). Material and Methods:Bone marrow samples referred to Laboratorios Fatima de Michoacan for flow cytometry immunophenotyping for neoplastic hematological disease. The protocol is based on the Report on the Second Latin American Consensus Conference for Flow Cytometric Immunophenotyping of Hematological Malignancies (Cytometry Part B (Clinical Cytometry) 2005;70B:39–44), and Immunophenotyping of acute leukemia and lymphoproliferative disorders: a consensus proposal of the European LeukemiaNet Work Package 10 (Leukemia 2011;25:567–574). Results:One hundred and seventy two cases were diagnosed.Fourteen myelodisplastic syndromes were detected.Forty five acute myeloid leukemia; M0-M1 twenty two cases, 5 with aberrant expression of CD7, one with CD19 and one CD20; M2 four cases, 1 with aberrant expression of CD 2 and 1 with CD7; M3 four cases; M4 one case; M5 twelve cases, 3 aberrant expression of CD7; M6 and M7 one case each.Sixty five B cell precursor acute lymphoblastic leukemia; BI twenty three cases, 2 with aberrant expression of CD7, and 2 aberrant expression of CD13 and one with aberrant expression of CD 33; BII twenty cases, 1 aberrant expression of CD2, 1 aberrant expression of CD5, and 4 aberrant expression of CD33; BIII twenty one cases, 1 aberrant expression of CD3; BIV one case.Five T cell precursor acute lymphoblastic leukemia.Thirty four chronic lymphoproliferative disorders; eighteen B cell chronic lymphocytic leukemia, two T cell chronic lymphocytic leukemia, seven follicular lymphoma, three mantle cell lymphoma, three splenic lymphoma with villous lymphocytes and one hairy cell leukemiaOne adult T cell leukemia/lymphoma.One natural killer cell leukemia (CD94+, perforin +, granzyme +)Seven monoclonal gammapathies. Conclusions:It is important to create the experience with new diagnostic tools based on the regional protocols. Low prevalence of AML M2 presumably because of classic morphologic features. Low prevalence of monoclonal gammapathies because for recent incorporation to diagnosis, treatment and response criteria; it is expected that in future this prevalence will arise. This data is complemented, whenever it is possible, with chromosomal analysis to determinate the risk and treatment. Disclosures:No relevant conflicts of interest to declare.

PTK7: A new biomarker for immunophenotypic characterization of maturing T cells and T cell acute lymphoblastic leukemia

Protein tyrosine kinase-7 (PTK7) was recently identified as a surface protein expressed on hematopoietic cells. To determine if PTK7 is a useful biomarker in clinical practice for acute leukemia immunophenotyping and detection, we examined the PTK7 expression in human bone marrow and thymic specimens. Our results show that PTK7 expression in normal thymic T cells is tightly regulated during the maturational process, but in T cell acute lymphoblastic leukemia (T-ALL) the expected temporal relationship of expression between PTK7 and other maturational T cell markers is lost or disrupted. In addition, nearly all T-ALL cases expressed higher PTK7 levels than mature T cells in the human bone marrow specimens. Therefore, in conjunction with other T cell markers, PTK7 has utility as a biomarker for detecting minimal residual disease of T-ALL in the bone marrow.