scholarly journals Importance of Flow Cytometry in the Differential Diagnosis of Hairy Cell Leukemia in the State of Rio Grande Do Norte, Brazil

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 14-15
Author(s):  
Aldair Sousa Paiva ◽  
Alessandra Suelen Jardim Silva ◽  
Victor lima Soares ◽  
Gustavo Henrique de Medeiros Oliveira ◽  
Lenilton Silva DA Silva Júnior ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Differential Diagnosis ◽  
Peripheral Blood ◽  
Complete Blood Count ◽  
Conflicts Of Interest ◽  
Specific Treatment ◽  
Leukemic Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cell Leukemia

Introduction:Hairy Cell Leukemia (HCL) is a B-cell non-Hodgkin's Lymphoma (B-NHL) representing about 2% of chronic leukemias, is manifested in adults with an average age of 55 years old or more and the ratio of male: female is 5:1, being more common among white people. It is characterized by the presence of neoplastic lymphocytes with cytoplasmic projections (villous cells), a characteristic commonly observed in other DLPCs such as variant HCL (HCL-v) and splenic villous cell lymphoma (SVCL), being the immunophenotyping by flow cytometry determinant in the differential diagnosis of these neoplasms. HCL is characterized by splenomegaly, hepatomegaly, pancytopenia in peripheral blood (PB) with leukopenia, anemia, neutropenia, monocytopenia, and thrombocytopenia. It has a low number of circulating tumor cells, spleen, liver, and bone marrow (BM) infiltration.Objective:To investigate, by flow cytometry, patients with lymphocytosis and presence of villous lymphocytes in the characterization of HCL and HCV-v and SMZL.Methodology:Were investigated samples of peripheral blood (SP) and bone marrow (MO) from 27 patients previously diagnosed with DLPC and presence of villous lymphocytes which were by flow cytometry with a panel of monoclonal antibodies (MoAb) conjugated to fluorochromes and targeted to T-lymphocytes: CD1a, CD2, CD3, CD5, CD7, subpopulation T-helper (CD3/CD4) and T-cytotoxic (CD3/CD8), in addition to TCR a/b and TCR g/d; Natural Killer cells: CD16-56; B-lymphocytes: CD19, CD20, CD21, CD22, CD23, CD79b, CD200, IgM, IgG, IgD, anti-kappa and anti-lambda, in addition to CD10, TdT (Terminal deoxynucleotidyl Transferase), CD103, CD123, CD11c, CD25, CD38, CD138, CD45 and CD14. At the same time, a complete blood count with differential white blood cell count and investigation of clinical and demographic data such as age, sex and ethnicity / race were also performed.Results:The distribution of patients according to ethnicity and gender, there was a predominance of white individuals and males. The age group most affected was in patients older than 60 years. All patients expressed pan-B antigens on leukemic cells with expression of CD19, CD22 / CD20 (Forte), sIgH, associated with clonal restriction for immunoglobulin light chain (kappa= 20 and lambda= 7), associated with FMC7 expression, HLADR, CD38 and CD45 strong and negativity to CD10, CD138, CD200, CD23, CD5, TdT and related T antigens. Sixteen cases were categorized as HCL, six HCLv and five SVCL. The immunophenotyping of HCL cases was positive for CD103, CD25, CD123 and CD11c. HCLv was negative for CD103 in three cases and CD25 and SVCL negative for CD103, CD123 and CD11c and CD25 in all cases.Conclusions:The precise diagnosis of HCL has fundamental importance because each NHL-B has a specific treatment, besides emphasizing the sensitivity and speed of the IFC regarding diagnosis and follow-up. Disclosures No relevant conflicts of interest to declare.

Circulating Megakaryocyte-Derived Cells Detected by Flow Cytometry As Marker of Aggressive Neoplasms of Megakaryocytic Lineage in Acute Megakaryoblastic Leukemia and Allied Malignancies Presenting As Primary Myelofibrosis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1461-1461
Author(s):  
Serena Marotta ◽  
Giovanna Giagnuolo ◽  
Giulia Scalia ◽  
Maddalena Raia ◽  
Santina Basile ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Differential Diagnosis ◽  
Cell Population ◽  
Peripheral Blood ◽  
Primary Myelofibrosis ◽  
Giant Platelets ◽  
Megakaryoblastic Leukemia ◽  
History Of ◽  
Blast Cells

Abstract Abstract 1461 The differential diagnosis of myelofibrotic disorders encompasses chronic primary myelofibrosis (PMF), myelodysplastic syndromes with fibrosis (MDS-F), acute panmyelosis with myelofibrosis (APMF) and acute megakaryoblastic leukemia (AMKL). Most of these conditions are recognized as distinct entities by the WHO 2008 revised classification of myeloid neoplasms; however, the WHO admits that often a definitive diagnosis is problematic, mostly because of specimens with insufficient cellularity (e.g., “dry tap”). Nevertheless, the correct identification of the most aggressive fibrotic disorders (APMF and AMKL) remains crucial, given their poor prognosis and subsequent need of intensive treatment (including transplantation). Even the most recent molecular studies did not result in any contribution in the differential diagnosis. Here we report our experience on a cohort of about 300 patients who were admitted in our bone marrow failure unit because of cytopenia in the last 7 years. All these patients were evaluated by standard peripheral blood and bone marrow cytology, karyotype analysis and bone marrow thephine biopsy, aiming to a definitive hematological diagnosis. Flow cytometry analysis was performed at initial presentation and then serially during the follow up on both peripheral blood and bone marrow aspirate. All patients were classified according to the WHO 2008 revised classification of myeloid neoplasms, and received the best standard treatment based on the specific disease, age and comorbidities. This report focuses on 8 patients who shared a unique flow cytometry finding of an aberrant megakaryocyte-derived cell population, which seems associated with a distinct disease evolution. Two of these patients received the diagnosis of AMKL according to bone marrow aspirate and trephine biopsy; the karyotype was complex in one case (monosomal karyotype, including a 5q-), whereas no Jak-2 mutation or any other genetic lesions could be demonstrated. Their blast cells were CD34+, CD38+, CD45+, CD117+, CD33+, CD13+; in addition, in the peripheral blood, we detected the presence of an aberrant cell population which was CD45-, CD42b+ (CD34+ in one case and CD34- in the other one). In the blood smear, we observed megakaryocyte fragments which likely correspond to this aberrant cell population, as identified by flow cytometry. Other three patients presented with a severe pancytopenia: all of them had a dry tap, and their trephine biopsies documented a massive fibrosis. They had no previous hematological disorder (one suffered from Behcet syndrome), normal karyotype and absence of any typical genetic lesion (i.e., wild-type Jak-2). All of them did not show splenomegaly, increased LDH or leukoerythroblastosis; their peripheral blood smear showed abnormal giant platelets, often resembling megakaryocyte fragments. Flow cytometry documented in the peripheral blood the presence of a distinct population of CD45-, CD42b+, CD61+ cells, which was also CD34+ in one case. These 3 patients were initially classified as PMF, even if APMF could not be ruled out; however, within 6 months they all progressed to AMKL. At this stage, typical CD34+, CD45+ blast cells were accompanied by a progressive increase of CD45+, CD42b+, CD61+ cells. This aberrant megakaryocyte-derived cell population (which could not be demonstrated in patients with thrombocytopenia) was also identified in 3 additional patients, who have a previous history of hematologic disorders: two had a history of pure red cell aplasia (successfully treated by immunosuppressive therapy), and one a 5q- melodysplastic syndrome (responding to lenalidomide, even with transient cytogenetic remission). In all of them we observed the appearance of CD45-, CD42b+ cells in the peripheral blood, which appeared as giant platelets/megakaryocyte fragments in the blood film; this finding within a few weeks was followed by progression to AMKL (5q- was detected in 2 of 3 cases). In conclusion, we demonstrate that aberrant circulating megakaryocyte-derived cells detected by flow cytometry may be useful in the differential diagnosis of myelofibrotic disorders. These giant platelets or megakaryocyte fragments, regardless the initial diagnosis, were associated with early evolution into AMKL, likely representing a surrogate marker for aggressive neoplasms of the megakaryocytic lineage. Disclosures: Risitano: Alexion: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Differential Diagnosis of Hereditary and Acquired Thrombocytopenia By the Immature Platelet Fraction and Thrombopoietin Levels

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1049-1049 ◽  
Author(s):  
Fabio Luiz Bandeira Ferreira ◽  
Marina Pereira Colella ◽  
Samuel de Souza Medina ◽  
Maiara Marx Luz Fiusa ◽  
Loredana Nilkenes Gomes da Costa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Differential Diagnosis ◽  
Platelet Count ◽  
Healthy Volunteers ◽  
Complete Blood Count ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Healthy Individuals ◽  
Technical Limitation ◽  
Immature Platelet Fraction

Abstract Introduction: The differential diagnosis of hereditary and acquired thrombocytopenias can be challenging, especially when between immune thrombocytopenia (ITP) and less well characterized hereditary thrombocytopenias (HT) such as MYH9-related disorders. Fundamental differences in the management of these two conditions add clinical relevance to the search for novel parameters that differentiate these conditions. The immature platelet count (IPF) represents the fraction of platelets with higher RNA content, and in analogy to the reticulocyte count for erythropoiesis is a biomarker of thrombopoietic activity. In a recent report (Miyazaki et al, 2015), IPF values that were more than 5-fold higher than those observed in ITP patients were reported in a population of 15 patients with HT. However, whether this increased values represented a real increase in thrombopoietic activity, or reflected a technical limitation of IPF determination in large platelets could not be clarified. Here, we aimed to evaluate the role of IPF determination in the differential diagnosis between HT and several forms of acquired thrombocytopenia in a larger and more diverse population of patients. We also evaluated thrombopoietin (TPO) levels in HT compared to ITP, to further investigate the mechanisms by which extremely large IPF values are observed in HT. Methods: IPF and mean platelet volume (MPV) were prospectively determined using a Sysmex XE5000 hematologic analyzer (as part of the complete blood count) in a cohort of patients with post-chemotherapy thrombocytopenia (n=56), bone marrow failure (myelodysplastic syndromes and aplastic anemia; n=22), ITP (ITP; n=105) and inherited thrombocytopenias (n=27). The latter population consists of a well-defined cohort of individuals with HT thrombocytopenia characterized by clinical, familial, laboratory and molecular data. TPO levels were determined by ELISA (R&D Systems) in 21 HT patients and 22 ITP patients matched for platelet count and age. A group of 178 healthy volunteers were used to determine normal IPF and MPV values. Results: Median platelet counts were similar in post-chemotherapy patients (CTx) (32.0*109/L), bone marrow failure (BMF) (33.5*109/L), ITP (52.0*109/L) and HT (52.0*109/L) (P=0.15). Similar IPF levels were observed in CTx and BMF patients (5.6%; IQR 3.4-8.8% and 6.5%; IQR 3.5-13.7%. Compared to these two groups, higher IPF values were observed in both ITP (12.3%; 7.0-21.0%) and HT patients (29.8%; 17.5-56.4%) (both P values < 0.05). In addition, IPF were significantly higher in HT compared to ITP (Kruskall-Wallis test and Dunn's post test,P=0.001). MPV values were different between HT and CTx/BMF groups, but could not differentiate ITP from HT. TPO levels. The accuracy of IPF to discriminate HT from all other causes of thrombocytopenia estimated by ROC analysis was 0.88 (CI95%0.8-0.96, p<0.0001). Similar TPO levels were observed in platelet count-matched ITP, HT patients and healthy volunteers without thrombocytopenia. Interestingly, TPO presented marked correlations with both platelet count (Rs = - 0.61, P=0.002) and IPF (Rs= 0.59, P=0.003), even with TPO levels in the same range of healthy individuals. In contrast, no significant correlation could be observed between TPO and IPF or platelet count in HT patients. Conclusions: IPF represents an informative biomarker for the differential diagnosis of hereditary and acquired thrombocytopenias, and accurately differentiates ITP from the most common HT. As expected, TPO levels in patients with ITP were not higher than in individuals with normal platelets counts. The inverse correlation between TPO and platelet count in these patients confirm a blunted TPO response to thrombocytopenia in these patients. Similarly, patients with HT did not present increased TPO levels compared to healthy individuals. Accordingly, increased IPF levels in these patients cannot be attributed to higher TPO levels. Disclosures No relevant conflicts of interest to declare.

CML Shows Characteristic Pattern by Flow Cytometry Analysis of Peripheral Blood

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4430-4430
Author(s):  
Kalley Scott ◽  
Wojciech Gorczyca
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Characteristic Pattern ◽  
Differential Count ◽  
Aberrant Expression ◽  
Chromosomal Changes ◽  
Wbc Count

Abstract Abstract 4430 A total of 75 chronic myeloid leukemia (CML) cases from peripheral blood (40 cases) and bone marrow (BM, 35 cases) with adequate flow cytometry (FC) data, smear, blood cell count, and the presence of t(9;22)/BCR-ABL by fluorescence in-situ hybridization (FISH) studies were analyzed for immunophenotypic pattern. The FC pattern in CML was compared with benign (healthy) controls (blood, 20 cases; BM, 20 cases), blood with reactive neutrophilia (15 cases), myelodysplastic syndrome (BM, 15 cases), and blood with eosinophilia (15 cases). CML showed a characteristic pattern by FC in blood, which can be easily differentiated from reactive neutrophilia or eosinophilia, regardless of WBC count. The identification of distinct population of blasts, basophilia, lack of CD10, CD11b, CD13 and/or CD16 on subset of granulocytes, decreased granularity, and/or aberrant expression of CD56 on granulocytes and monocytes, can be easily identified by routine FC analysis. We suggest using FC analysis of blood as a screening tool for patients with leukocytosis (neutrophilia) with follow-up FISH studies in cases with the phenotypic features suggestive of CML. Patients with confirmed CML diagnosis by FISH will undergo marrow biopsy for differential count including blast and basophil enumeration (to exclude accelerated phase or blast crisis), degree of reticulin fibrosis and cytogenetic studies (for additional chromosomal changes present at diagnosis). This approach, in our opinion, allows to diagnose early (unsuspected) CML and eliminates the need for unnecessary cytogenetic/FISH testing, and especially bone marrow biopsy in patients with reactive leukocytosis or eosinophilia. Disclosures: No relevant conflicts of interest to declare.

Do Leukemic Cells and Mesenchymal Stem Cells (MSCs) From AML Patients Share The Same Chromosomal Defect? A Cytogenetics, FISH and aCGH/Snpa Study

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2602-2602
Author(s):  
Paolo Bernasconi ◽  
Irene Dambruoso ◽  
Manuel Gotti ◽  
Marina Boni ◽  
Rita Zappatore ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Gradient Centrifugation ◽  
Leukemic Cells ◽  
Hematopoietic Tissue ◽  
Trisomy 8 ◽  
Commercial Fish ◽  
Monosomy 7 ◽  
Microarray Scanner

Abstract Recent evidence suggests that leukemia is not solely a cancer autonomous process, but rather a disease in which the bone marrow microenvironment, the niche, plays a crucial role too (Raaijmakers, 2011). MSCs are key component of the niche. Thus, several studies have tested whether these cells from haematological patients contain chromosomal defects identical or different from those present in leukemic cells. Based on these findings the principal aim of the present study was to evaluate whether leukemic and MSC from six AML patients shared the same cytogenetic defects after examination with three different technologies, conventional cytogenetics (CC), FISH and aCGH/SNPa. At the onset of the disease and after informed consent all the six patients were submitted to bone marrow (BM) aspiration. BM cells were submitted to CC and FISH analyses. In addition, MSC were isolated from BM cell suspension (10-15 ml) as previously described. Briefly, mononucleated cells were isolated from BM by density gradient centrifugation using Lympholyte®-H and seeded in 75 cm2 cell culture flasks at a cell density of 106 cells/cm2. Cells were cultured at 37°C, 5% CO2 in MEM-alpha medium containing 1% Penicillin/Streptomycin, 1% L-Glutamine and 10% fetal bovine serum. After 48-h adhesion, non-adherent cells were removed and culture medium replaced (Achille et al, 2011). Growth medium was changed every three days. MSCs were examined after the first passage and their phenotype was evaluated by flow cytometry. Cells were detached from culture using Tripsin-EDTA, washed twice with PBS and stained for ten minutes with the following fluorochrome-conjugated antibodies: anti-CD90-FITC, anti-CD105-PE, anti-CD14-FITC, anti-CD73-PE, anti-CD34-FITC, anti-CD80-PE, anti-CD133-APC, anti-CD31-PE and anti-CD45-APC-Alexa750. Stained cells were acquired with a Beckman Coulter Navios instrument and data analyzed with Kalooza software. The commercial FISH probes used were LSI D7S486/CEP7, LSI AMLETO from Abbot Molecular Inc. (Chicago, Il, USA) and ON c-Myc/SE8, SE10(D10Z1) from Kreatech (Amsterdam, NL). These probes were applied according to manufactures guidelines and cut-off values determined by applying a one-sided 95% confidence interval using a binomial distribution. aCGH/SNPa was carried out with the SureScan Microarray Scanner G4900DA (Agilent Technologies Inc. Santa Clara, CA). CC revealed a monosomy 7 in two patients, a del(7)(q31) in one, a trisomy 8 and a trisomy 10 in one patient each, a t(8;21)(q24,q22) translocation in the last patient. All these defects were confirmed by FISH. In order to establish whether leukemic cells and MSCs shared these same abnormalities, MSCs cultures were tested with FISH. MSC purity assessed by flow-cytometry was 50-87%. FISH revealed a normal pattern in all the cultures examined. In contrast, aCGH/SNPa revealed neither gains/losses nor LOH in four patients, a trisomy 5 in one and the LOH of a 3.8 Mb sized region located on 13q31.1 in one patient. This study, the first one that applied aCGH/SNPa to investigate the MSC chromosomal pattern, suggests that i) MSCs from chromosomally abnormal AML patients may show a normal FISH pattern, but may be either normal or contain chromosomal aberrations different from those present in leukemic cells on aCGH/SNPa analysis; ii) these defects are uncommonly seen in AML; iii) MSCs defects may flag that the leukemogenic event targets not only the hematopoietic tissue but also the stromal cell compartment, i.e. the niche; iii) aCGH/SNPa provides an in-depth view of MSC chromosomal pattern allowing the identification of potential clonal markers. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Contribution of Flow Cytometry Immunophenotyping in Diagnostic of Acute and Chronic Leukemias

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5198-5198
Author(s):  
Aldair Sousa Paiva ◽  
Hugo Diogenes De Oliveira Paiva ◽  
Geraldo Barroso Cavalcanti ◽  
Lenilton Silva Silveira ◽  
Lorena KF Silva ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Acute Leukemias ◽  
Cell Chronic Lymphocytic Leukemia ◽  
Bone Morrow

Abstract BACKGROUND: Today immunophenotyping by flow cytometry is an useful adjunct to cytomorphologyc analysis to correct diagnostic of leukemias. It provides objective and quantitative data allowing for a high level of sensitivity detection and better characterization of acute and chronic leukemias. The purpose of this study was to demonstrate the contribution of the immunophenotyping by monoclonal antibodies (Mo.Ab.) in leukemic cells from patients with acute and chronic leukemias. METHODS: Analyzed 76 patients with leukemias before the treatment. The diagnostic of leukemias was based on the morphological and immunophenotyping analysis of leukemic cells from peripheral blood and bone morrow. The cytomorphologycal analysis was based on French - American - Britsh criteria (FAB classification) in blood and bone marrow films stain by leishmann and the immunophenotyping by flow cytometry with a panel of Mo.Ab. specific to acute leukemias as: CD1a, CD2, CD3, CD4, CD5, CD8, CD7, CD10, CD13, CD19, CD20, CD103, CD22, CD33, CD34, CD117, CD38, HLADR, TdT, anti-mieloperoxidase, anti-IgM and anti-kappa and lambda light chain. Further clinical and laboratory information as age, sex, presence of tumoral mass, lymphadenopathy, hepatosplenomegaly, hemoglobin determination, total and specific white cell count and cytomorphological analysis of blood film and bone morrow smear. RESULTS: Thirty four patients presented acute myeloid leukemia (AML), twenty acute lymphoblastic leukemia (ALL), nineteen B-cell chronic lymphocytic leukemia (B-CLL), two T-cell chronic lymphocytic leukemia and one case was hairy cell leukemia (HCL). Males were more frequently found in all types of leukemias. ALL were more observed in children (age < 15 years old) and in AML however were more frequently in adults patients. The chronic leukemias were presented in patients with 50 years old or more in all cases. The correlation between the immunophenotyping and clinical pathological profile of these leukemias demonstrated that ALL were more associated to lymphadenopathy, AML to hepatosplenomegaly, and CLL to lymphadenopathy and high count of white cells in peripheral blood. The thrombocytopenia and anemia were found in more cases of acute leukemia. CONCLUSION: This date suggest that today the immunophenotyping by flow cytometry is an important methodology to diagnostic and classification of leukemias. Disclosures No relevant conflicts of interest to declare.

scholarly journals Down-Regulation of CD25 Antigen in Hairy Cell Leukemia Patients after Treatment

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4143-4143
Author(s):  
Mirela Anghelina ◽  
Narendranath Epperla ◽  
Kerry A. Rogers ◽  
Ling Guo ◽  
Qiuhong Zhao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Hairy Cell Leukemia ◽  
Research Funding ◽  
Leukemic Cells ◽  
World Health ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Cd25 Expression

Abstract Background: Hairy Cell Leukemia (HCL) is a rare hematological malignancy, comprising only of 2% of all leukemias, with an estimated 900 new cases diagnosed each year in the United States. HCL displays a characteristic immunophenotypic profile that include pan-B cell markers including CD103, CD11c, and CD25. World Health Organization guidelines defines two forms of HCL, classic HCL (cHCL) and variant HCL (HCLv) as two distinct clinical entities. Patients with cHCL have a distinct immunophenotypic profile on their malignant leukemic cells including CD20+, CD19+, CD11c+, CD25+, CD103+, and CD123+, while the leukemic cells from patients with HCLv show CD11c+, CD20+ and CD19+, while lacking CD25 and CD123 expression. Some patients with cHCL will retain CD25 positivity while demonstrating negativity for other typical markers, herein termed atypical HCL (aHCL). Presence or absence of CD25 is an important determinant in classifying patients into cHCL and HCLv. Although it has previously been reported that CD25 expression may be lost during treatment with the targeted agent vemurafenib, we sought to identify whether this immunophenotypic change occurs following other treatment types, including standard purine nucleoside analog therapy and with targeted BTK inhibition. Methods: Adult patients (≥18 years) with a diagnosis of HCL whom had immunophenotype data collected before and after treatment between 2010 and 2018 were included in the study. Immunophenotype and morphological characteristics of initial and follow-up peripheral blood, bone marrow aspirate, and core biopsy specimens were reviewed and correlated with the treatment received. Results: We evaluated 30 HCL patients who underwent different therapies. All available specimens were reviewed and showed morphologic features characteristic for cHCL (n=26, 86.7%), and aHCL (n=4, 13.3%). The median age at HCL diagnosis was 50 years (44-76 years) with male predominance (76%). Patients with aHCL were treated with ibrutinib (n=2) and pentostatin (n=2). Patients with cHCL were treated with pentostatin (n=12), ibrutinib (n=8), vemurafenib (n=4), dabrafenib (n=1), and cladribine (n=1). Bone marrow analyses showed that all the patients had leukemic B-lymphocyte co-expression of CD19, CD20, CD103, CD11c, CD25, and CD123 prior to treatment. Some patients also had a smaller percentage of lymphocytes lacking CD25 expression along with the CD25 positive lymphocytes. Follow-up bone marrow and peripheral blood analysis showed that almost half (n=14, 46%) of treated patients had a partial or complete loss of CD25 expression regardless of the treatment type. Leukemic cells continued to express other HCL signature markers. Conclusion: Our study indicates that during the course of disease some patients display a loss of CD25 expression after therapy. This phenomenon was observed across different therapies and is not specific to the type of treatment. This is the first study to show treatment-dependent CD25 variability with pentostatin, ibrutinib and dabrafenib. Our results advocate for caution when using CD25 for the differential diagnosis of cHCL versus HCLv in treated patients. Future studies are needed in larger patient cohorts to determine the overall role and utility of CD25 in the diagnosis of cHCL and HCLv. Disclosures Lozanski: Genentech: Research Funding; Stem Line: Research Funding; BI: Research Funding; Novartis: Research Funding; Beckman: Research Funding; Coulter: Research Funding. Andritsos:Astra Zeneca: Consultancy; HCLF: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Splenectomy in a Child of X-Linked Thrombocytopenia with Thalassemia and Bone Marrow Fibrosis : Hemoglobin and Platelet Count Were Improved

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1367-1367
Author(s):  
Yang Wan ◽  
Xiaofan Zhu ◽  
Xiaojuan Chen ◽  
Wenbin An ◽  
Peihong Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Bone Marrow Biopsy ◽  
Peripheral Blood ◽  
Clinical Characteristics ◽  
Blood Count ◽  
Complete Blood Count ◽  
Conflicts Of Interest ◽  
Bone Marrow Fibrosis ◽  
Marrow Fibrosis

Abstract X-linked thrombocytopenia with thalass emia (XLTT)(OMIM 314050)was first described by Thompson in 1977(Thompson et al. J Blood 1977 50(2):303-16). This rare inherent disorder was caused by a nucleotide change G>A at position 647, which leads to an amino acid substitution of arginine to glutamine (R216Q) in the gene of GATA-1 on the band p11-12 ohuman X chromosome(Raskind et al. Blood 2000, 95(7):2262-8 ;Yu et al.J Blood 2002,100(6): 2040-2045). GATA-1, belonging to the GATA family of transcription factors plays a crucial role in the development of several hematopoietic cell lines ( Ferreira et al. J Mol Cell Biol 2005,25(4): 1215-1227) . The missense mutation(R216Q) in XLTT affects GATA-1 binding to palindromic DNA sites (Yu et al.J Blood 2002,100(6): 2040-2045). The clinical characteristics of XLTT are mild thrombocytopenia, splenomegaly, reticulocytosis, hemolytic anemia and unbalanced hemoglobin (Hb) chain synthesis resembling ¦Â-thalassemia (Raskind et al. Blood 2000, 95(7):2262-8 ; Balduini et al. J Thromb Haemost 2004, Jan;91(1):129-40). About 7 families of XLTT were reported before (Millikan et al.J Semin Thromb Hemost 2011,37(6): 682-689; Danielsson et al. J Lakartidningen 2012 ,109(34-35): 1474-1477).Bone marrow fibrosis is described only in tow Swedish families ( Danielsson et al. J Lakartidningen 2012 ,109(34-35): 1474-1477).But there is limited data about the treament and prognosis of the diesase. Here we describe the full clinical characteristics of a boy of XLTT who was treated by splenectomy. The patient was first admitted at the age of 1year and 8 months in 2011.The chief complain was skin petechia and pale for more than one month. The boy had lower weight but no visible malformation. Feeding difficult and lag of language development were also complained.His Liver was 2.3cm below the right ribs and spleen was 3.2cm below the left. Peripheral blood count showed hemoglobin 8 g/dL, MCV76.7fl, MCH21.8 pg,MCHC284 g/L and reticulocyte count 0.1764¡Á1012/L. Peripheral blood smear demonstrated marked anisopoikilocytosis, polychromasia and nucleated RBCs.Platelet count was 64¡Á109/L with normal morphology.Wight blood cell was normal in number and morphology.elevated to 0.226(normal range 0-0.025) while HBA2 and hemoglobin electrophoresis was normal. Bone marrow biopsy and aspirate smear revealed a hypercellular marrow with dysplasia of erythrocyte series and megakaryoblasts (Figure 1 A). Polynuclear erythroblast ,micromegakaryocytes and hypolobated megakaryocytes could be easily seen (Figure 1 B). Fibrosis proliferation was obvious (Figure 1 A). Genetic analysis discovered a mutantion of GATA-1(R216Q)and excluded mutations of hemoglobin gnens and JAK-2. Patient was treated with dexamethasone and thalidomide which got little effect. The baseline hemoglobin was 6-8 g/dL.Platelet count ranged from 30 to 70¡Á109/L. Splenectomy was done at the age of 5years and 4 months because of constantly RBC transfusion and splenomegaly. Fibrosis proliferation and extramedullary hematopoiesis in spleen were proved by biopsy (Figure 1 C,D).The boy's complete blood count was recovered 4 months after splenectomy. Hemoglobin rose to11.6 g/dL and platelet count was 337¡Á109/L. HBF was still high at 0.226. Multifocal fibrosis proliferation still existed in bone marrow biopsy but with no myelodysplasia (Figure 1 E,F). Hepatomegaly didn't progress. He has good quality of life,and normal growth and intelligence development. Splenectomy can be a therapeutic strategy of X-linked thrombocytopenia with thalassemia. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Importance of Flow Cytometry in the Diagnosis of Sezary Syndrome in the State of Rio Grande Do Norte, Brazil

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 39-40
Author(s):  
Maria das Graças Pereira Araujo ◽  
Victor lima Soares ◽  
Alessandra Suelen Jardim Silva ◽  
Gustavo Henrique de Medeiros Oliveira ◽  
Lenilton Silva DA Silva Júnior ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
B Lymphocytes ◽  
Peripheral Blood ◽  
Blood Count ◽  
Complete Blood Count ◽  
Conflicts Of Interest ◽  
Demographic Data ◽  
Differential White Blood Cell ◽  
Sézary Cells

Introduction:Sézary Syndrome (SS) is a leukemic form of Fungal Mycosis (FM), a rare form of T-cell lymphoma, characterized by erythroderma, generalized lymphadenopathy and infiltration of neoplastic T cells (Sézary cells) with cerebriform nucleus on the skin, lymph nodes and peripheral blood, being observed predominantly in men and individuals over the age of 60 and black. In the diagnosis of SS / FM, at least one of the criteria must be observed: minimum absolute Sézary cells count of 1000/mm3, expansion of TCD4+ cells with a ratio CD4/CD8 &gt;10, loss of at least one mature T cell antigens as CD2, CD3, CD5, CD7 and CD26 in associated with increased lymphocyte count with evidence of a clone of circulating TCD4 cells determined by flow cytometry (CF).Objective:To investigate MF/SS in patients diagnosed with cutaneous lymphoma by CF immunophenotyping. Methodology: Were investigated in samples of peripheral blood (SP) from 11 patients of both sexes with initial history of MF and confection of SS due to the presence of Sézary cells by cytomorphological analysis by CF constituted by a panel of conjugated monoclonal antibodies (AcMo) to fluorochromes and targeted to T lymphocytes: CD1a, CD2, CD3, CD5, CD7, subpopulation T-Helper (CD3+/CD4+) and T-cytotoxic (CD3+/CD8+), in addition to TCR a/b and TCR g/d; Natural Killer cells: CD16-56; B lymphocytes: CD19, CD20, CD21, CD22, CD23, IgM, IgG, IgD anti-kappa and anti-lambda, in addition to CD10, TdT, CD103, CD25, CD38 and CD138, CD45 and CD14. At the same time, a complete blood count with differential white blood cell count and investigation of clinical and demographic data such as age, sex and ethnicity/race were also performed. Results: Of the patients analyzed, 6/11 were male, the age group above 60 years and white individuals were also found in 6/11 patients. The blood count showed lymphocytosis in 9/11 patients with the presence of convoluted cells in all cases. The diagnosis of SS was confirmed by the presence of Sezary cells in PB counting above 1000/mm3, with an immunophenotype confirmed by the predominance of TCD4+ lymphocytes (CD4/CD8 ratio &gt; 10.0), associated with the expression of CD5, CD2, TCR a/b, CD3 weakly expressed. CD7 was absent in 10/11 samples analyzed. Antigens related to B lymphocytes and NK cells were absent in neoplastic cells as well as CD10, TdT and CD1a.Conclusions:SS is a leukemic variant of FM, characterized by exfoliative erythroderma, associated with lymphadenopathy and leukemization of FM with the appearance of Sézary cells in PB. Because it is a rare and essential disease, an accurate diagnosis of these diseases is necessary, and FC is an important diagnostic confirmation tool for SS. Disclosures No relevant conflicts of interest to declare.

scholarly journals Molecular Characterization of Bone Marrow and Peripheral Blood Compartments from Patients with Plasma Cell Leukemia in the Mmrf Commpass Study

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1782-1782
Author(s):  
Sheri Skerget ◽  
Austin Christofferson ◽  
Sara Nasser ◽  
Christophe Legendre ◽  
The MMRF CoMMpass Network ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Plasma Cell ◽  
Peripheral Blood ◽  
Research Funding ◽  
Plasma Cells ◽  
Cell Cycle Control ◽  
Cell Leukemia

Plasma cell leukemia (PCL) is rare but represents an aggressive, advanced form of multiple myeloma (MM) where neoplastic plasma cells (PCs) escape the bone marrow (BM) and circulate in the peripheral blood (PB). Traditionally, PCL is defined by the presence of >20% circulating plasma cells (CPCs), however, recent studies have suggested that PCL be redefined as the presence of >5% CPCs. The Multiple Myeloma Research Foundation CoMMpass study (NCT01454297) is a longitudinal, observational clinical study with 1143 newly diagnosed MM patients. BM-derived MM samples were characterized using whole genome (WGS), exome (WES), and RNA (RNAseq) sequencing at diagnosis and each progression event. When >5% CPCs were detected by flow cytometry, PCs were enriched independently from both compartments, and T-cells were selected from the PB as a control for WGS and WES. This substudy within CoMMpass provides the largest, most comprehensively characterized dataset of matched MM and PCL samples to date, which can be leveraged to better understand the molecular drivers of PCL. At diagnosis, 813/1143 CoMMpass patients had flow cytometry data reporting the percent PCs in PB, of which 790 had <5%, 17 had 5-20%, and 6 had >20% CPCs. Survival analyses revealed that patients with 5-20% CPCs (median = 20 months) had poor overall survival (OS) outcomes compared to patients with <5% CPCs (median = 74 months, p < 0.001), and no significant difference in outcome was observed between patients with 5-20% and >20% (median = 38 months) CPCs. Patients with 1-5% CPCs (median = 50 months, HR = 2.45, 95% CI = 1.64 - 3.69, p < 0.001) also exhibited poor OS outcomes compared to patients with <1% CPCs (median = 74 months), suggesting that patients with >1% CPCs are a higher risk population, even if they do not meet the PCL threshold. Using a cutoff of >5% CPCs, 23/813 (2.8%) patients presented with primary PCL (pPCL) at diagnosis. Of these patients, 7 (30%) were hyperdiploid (HRD), of whom 1 had a CCND1 and 1 had a MYC translocation; while 16 (70%) were nonhyperdiploid (NHRD), all of whom had a canonical immunoglobulin translocation (6 CCND1, 5 WHSC1, 3 MAF, 1 MAFA, and 1 MAFB). Of 124 patients with serial sample collections, 5 (4%) patients without pPCL had >5% CPCs at progression, and thus relapsed with secondary PCL (sPCL). Of the 5 sPCL patients, 2 (40%) were NHRD with a CCND1 or MAF translocation; while 3 (60%) were HRD, 1 with a WHSC1 translocation. Median time to diagnosis of sPCL was 22 months (range = 2 - 31 months), and patients with sPCL (median = 22 months) and pPCL (median = 30 months) exhibited poor OS outcomes as compared to MM patients (74 months, p < 0.001). Sequencing data was available for 15 pPCL and 5 sPCL samples. For 12 patients with WES, WGS, and RNAseq performed on their PCL tumor sample, an integrated analysis identified recurrent, complete loss-of-function (LOF) events in only CDKN2C/FAF1, SETD2, and TRAF3. Five pPCL patients had complete LOF of a gene involved in G1/S cell cycle control, including CDKN2C, CDKN2A, CDKN1C, and ATM. These LOF events were not observed in NHRD t(11;14) PCL patients, suggesting that CCND1 overexpression and LOF of genes involved in G1/S cell cycle control may represent independent drivers of PCL. Comparing WES and WGS data between matched MM and PCL tumor samples revealed a high degree of similarity in mutation and copy number profile. However, differential expression analysis performed for 13 patients with RNAseq data comparing their MM and PCL tumors revealed 27 up- and 39 downregulated genes (padj < 0.01, FDR = 0.1) in PCL versus MM. Pathway analysis revealed an enrichment (p < 0.001) for genes involved in adhesion and diapedesis, including upregulation of ITGB2, PF4, and PPBP, and downregulation of CCL8, CXCL12, MMP19, and VCAM1. The most significantly downregulated gene in PCL (log2FC = -6.98) was VCAM1, which plays a role in cell adhesion, and where loss of expression (TPM < 0.01) was observed across all PCL samples. Upregulation of four S100 genes including S100A8, S100A9, S100A12, and S100P, which have been implicated in tumor growth, metastasis, and immune evasion, was also observed in PCL. Interestingly, a S100A9 inhibitor has been developed and may represent a novel treatment option for PCL patients. In summary, PCL was found to be associated with molecular events dysregulating G1/S cell cycle control coupled with subtle changes in transcription that likely occur in a subclonal population of the MM tumor. Disclosures Lonial: Genentech: Consultancy; GSK: Consultancy; BMS: Consultancy; Janssen: Consultancy, Research Funding; Karyopharm: Consultancy; Takeda: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding; Amgen: Consultancy.

scholarly journals Diagnostic Utility of Flow Cytometric Immunophenotyping in Chronic Myelomonocytic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5526-5526
Author(s):  
Leonor Arenillas ◽  
Ivonne Parraga ◽  
Lourdes Florensa ◽  
Sara Montesdeoca Romero ◽  
Anna Puiggros ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Chronic Myelomonocytic Leukemia ◽  
Nonspecific Esterase ◽  
Age Related ◽  
Hla Dr ◽  
Myelomonocytic Leukemia ◽  
Classical Monocytes

Abstract INTRODUCTION The diagnosis of chronic myelomonocytic leukemia (CMML) according to WHO 2017 requires the presence of ≥1x109/L and ≥10% of monocytes in peripheral blood (PB). Establish an accurate diagnostic is difficult since many clinical situations present persistent monocytosis. The presence of dysplasia, mainly dysgranulopoiesis, is frequent but not always present in CMML. Cytogenetic aberrations are infrequent in this disease (20-25% of cases). Although 85-90% of CMML patients present at least one mutation in TET2, SRSF2 or ASXL1 genes, the use of NGS panels is not widespread. Furthermore, mutations in these genes are among the most frequently observed in age-related clonal hematopoiesis. Therefore, complementary techniques are required to support the diagnosis of this entity. The study of the peripheral monocyte subsets by flow cytometry (FC) has gained special interest due to a high sensitivity and specificity for the diagnosis of CMML (S = 90.6%, E = 95.1%, Selimoglu-Buet et al., Blood, 2015). An increase in the fraction of classical monocytes (Mo1) to >94% of total monocytes is an event frequently observed in CMML. There are no specific bone marrow (BM) FC panels for the diagnosis of CMML and very few have been validated for the diagnosis of MDS. "Ogata score", the only multicenter validated score in MDS, has not been applied in CMML. The aim of our study was to evaluate the usefulness of FC in PB and BM for the diagnosis of CMML. METHODS Twenty-two CMML were prospectively studied from 02/2016 to 04/2018. Patients' characteristics are summarized in Table 1. Diagnostic procedure consisted of morphological, cytochemical (Perls, myeloperoxidase, nonspecific esterase), cytogenetic and FC studies in BM, and morphological and FC studies in PB. "Ogata Score" was applied in BM samples (Table 2). Aberrant coexpression of CD2, CD7 and CD56 in BM monocytes was assessed. Immunophenotypic maturation profile of the monocytic elements in BM distinguishes: promonocytes (CD34-/CD117-/CD64++/CD14- or dim/CD45+/HLA-DR+++), mature monocytes (CD34-/CD117-/CD64++/CD14++/CD45++/HLA-DR++) and mature monocytes in terminal stage (CD300e+). In PB, the monocytes FC subsets (Mo1, Mo2 and Mo3) were studied, as well as the aberrant coexpression of CD2, CD7 and CD56 (Table 3). RESULTSThe presence of ≥2 aberrations in Ogata Score predicted properly the diagnosis of CMML in all patients analyzed (100% sensitivity). Due to the study design, we could not obtain results about specificity.An increase in Mo1 (classical monocytes) > 94% was detected in 18/20 patients (Table 3). This method predicted the diagnosis of CMML with a sensitivity of 91%, a result almost identical to the original study (Selimoglu-Buet et al., Blood, 2015).A good positive correlation was established between the percentage of BM promonocytes detected by morphology and by FC (Rho Spearman 0.61, P = 0.003).A negative correlation was found between the percentage of promonocytes by FC in MO and the expression of CD56 (Rho Spearman -0.612, P = 0.002). Similarly, CD56+ CMML presented a percentage of promonocytes by FC significantly lower than the CD56- CMML group (median: 24.5% (14-40) vs. 41% (23-71), P = 0.005). The expression of CD56 seems to be related to a more mature immunophenotypic profile of the monocytic population. On the other hand, the correlation between the percentage of CD56+ monocytes in BM and PB was almost perfect (Rho Spearman 0.928, P <0.001). CONCLUSION Our findings support the usefulness of flow cytometry in bone marrow and peripheral blood for the diagnosis of CMML. Disclosures No relevant conflicts of interest to declare.

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