[Analysis of bone marrow by flow cytometry: morphologic and immunologic aspects].
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Transferrin receptor
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This study was aimed to explore the expression of CD34 in patients with biphenotypic acute leukemia (BAL) and its relation with the prognosis of BAL. The flow cytometry was used to detect leukemia-associated antigen. The used monoclonal antibodys (McAb) included CD10, CD19 and CD34 for B lymphocyte lineage, CD2, CD3 and CD5 for T lymohocyte lineage, MPO, CD13 and CD33 for myeloid lineage. The finally results were respectively analyzed. The results indicated that 9 out of 216 cases of leukemia was diagnosed as BAL (4.2%). Among 9 cases of BAL, 6 cases showed the common expression of myeloid and T lymohocyte lineages (66.7%), 3 cases showed the common expression of myeloid and B lymohocyte lineages (33.3%). 4 cases of BAL displayed CD34 positive expression (44.4%). As compared with acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL), the BAL patients showed higher CD34 positive expression (P < 0.05). It is concluded that the BAL patients show a poor prognosis, as compared with AML or ALL patients. The therapeutic effect of BAL may negatively correlate with the CD34 positive expression.
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The aberrant expression of antigens (Ag) in lymphoproliferative disorders may cause a diagnostic problem when single parameter immunohistochemical assays are performed on frozen or paraffin sections because coexpression by relevant cells is not determined. This aberrant expression also raises the question as to whether mixed lineage (biphenotypic) lymphoid proliferations exist. Marrow (6) and extramedullary (20) tissues from 26 patients with diffuse, intermediate and high grade, B-cell lymphomas (IWF E=l, F=l, G=19, H=l and J=4) were analyzed with 19 markers using 3-olor flow cytometry. The percentages (%) of patients with double Ag coexpression in at least 20% of the CD19+ or CD20+ lymphoma cells were: stem cell(SC) Ag: CDlO = 58 and CD34 = 15; T-cell Ag: CD2 = 38, CD5 = 19 and CD7 = 19; myeloid(My) Ag: CD13 = 19 and CD33 = 8. The corresponding % with unusual triple Ag coexpression in at least 10% of the CD19+ B-cells were SC+T+ Ag: CD10CD2 = 50, CD10CD5 = 27, CD10CD7 = 38, CD34CD2 = 31, CD34CD5 = 19 and CD34CD7 = 27; T+T+ Ag: CD2CD5 = 35, CD2CD7 = 42 and CD5CD7 = 31; T+My+ Ag: CD2CD13 = 35 and CD2CD33 = 12; and My+My+ Ag: CD13CD33 = 12. Ten of 12 lymphomas tested showed clonal immunoglobulin (Ig) heavy chain gene rearrangements in the absence of clonal T-cell receptor (TCR) gene rearrangements. None(0%) of the My Ag positive cases showed immunoreactivity for myeloperoxidase. We conclude that the anomalous T and My Ag expression seen in the above B-cell lymphomas is not indicative of mixed lineage proliferation but represents the aberrant expression of these antigens by the malignant cells.
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Immunophenotypic characterization of the leukemic cells has been widely used as a tool for diagnosis, classification, stratification and prognosis of leukaemia.To investigate the immunophenotypic subtype profiles of Iranian patients with acute lymphoblastic leukemia (ALL) and its association to disease outcome.In this study, a total of 60 Iranian patients with ALL were immunophenotyped by flow cytometry using a panel of monoclonal antibodies specific for CD2, CD3, CD5, CD10, CD13, CD14, CD19, CD20, CD33, CD34, CD45, HLA-DR and TdT molecules.The samples were initially categorized into T-ALL (n=9), B-ALL (n=50) and mixed lineage (n=1) based on the expression patterns of CD3 and CD19 molecules. B-ALL patients could further be classified into four subtypes, including Pro-B (n=7, 11.7%), Pre-B I (n=28, 46.7%), Pre-B II (n=13, 21.7%) and immature/mature B cells (n=2, 3.3%) on the basis of expression of CD10, CD19, CD20, HLA-DR and TdT. Clinical manifestations and laboratory findings of the patients did not reveal association with immunophenotypic subtypes of ALL, with the exception of mediastinal mass and WBC count at the time of diagnosis which were found to be significantly higher in patients with T-ALL compared with B-ALL (p=0.001 and 0.014), respectively.Our results indicate that overall the immunophenotypic profile of Iranian ALL patients is similar to previous reports and it might be used for monitoring of minimal residual disease and prognosis.
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Objective To evaluate the immunophenotyping specificity of acute lymphoblastic leukemia.Methods A total of 58 patients with morphologically diagnosed acute lymphoblastic leukemia were immunophenotyped by flow cytometry using a panel of monoclonal antibodies specific for CD2,CD3,CD4,CD5,CD7,CD8,CD10,CD13,CD19,CD20,CD22,CD33,CD34,CD38,CD45,CD56,CD117,HLA-DR,sIgM,cyIg,cyCD79a,MPO and TdT molecules.Results The samples were initially categorized into T-ALL(n=8,13.79%),B-ALL(n=49,84.48%) and mixed lineage(n=1,1.72%) based on the expression patterns of CD3,CD19,CD33 and MPO molecules.B-ALL patients were further classified into four subtypes,including Pro-B(n=5,8.62%),common B(n=34,58.62%),Pre-B(n=9,15.52%) and mature B cells(n=1,1.72%) on the basis of expressions of CD10,CD20,cyIg and sIgM.Abnormal cells showed cross-lineage antigen expression,asynchronous antigen expression,antigen overexpression and antigen lack expression.The immunophenotypes presented no significant difference between children and adults.Conclusion The abnormal bone marrow cells in acute lymphoblastic leukemia show obvious lineal features,blast markers and cross-lineage expressions.The immunophenotyping based on abnormal expressions is applicable for the detection of minimal residual disease.
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This study reports the immunophenotypic features of a series of 62 selected acute leukemia patients with increased incidence of argyrophilic proteins (AgNORs) at the time of initial diagnosis. Peripheral blood and bone marrow cells of patients with T-ALL, B-precursor ALL and AML were studied. The method of silver staining was used to determine the number of AgNORs per cell. Cell surface markers were detected by a standard immunofluorescence assay. To demonstrate the relationship between AgNOR quantity and cell proliferation, the expression of activation and proliferation antigens CD38 and CD71 was investigated. To characterize the immunophenotype and the discrete stages of differentiation, the wide panel of antibodies against lymphoid, myeloid and non-lineage specific antigens was used. The number of AgNORs at diagnosis ranged from 3.05 to 6.70. Immunophenotypic analysis showed a variation in CD38 and CD71 expression among different leukemia subtypes. CD71 antigen was more expressed in T-ALL than in B-precursor ALL or in AML. Notable was the relationship between increased AgNOR quantity and antigens that characterize the immaturity of leukemic cells. The association with CD7, CD2, CD5 (without CD3 membrane expression) and CD34 in T blasts was evident. High positivity of CD19, CD10, CD34 and HLA-DR in relation to the increased amount of AgNORs in B-lineage ALL was observed. The vast majority of AML patients with high numbers of AgNORs simultaneously expressed CD13, CD33, CD34 and HLA-DR. One third of AML cases coexpressed T cell marker CD7. In conclusion, the presence of increased numbers of AgNORs at diagnosis might reflect the dependence on an early stage of leukemia cell differentiation.
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To assess the presence and phenotype of B-lineage cells in the bone marrow (BM) of rheumatoid arthritis (RA) patients after rituximab therapy.Six patients were studied. BM aspirates were collected 3 months after the treatment and analysed using the four-colour flow cytometry.CD19+ (B-lineage) cells in BM samples varied from 0.1 to 3.25% in the lymphoid gate. CD34+ cells varied from 1.23 to 4.86%. The proportion of CD34+ cells committed to the B-lineage varied between 0 and 42.19%. Pro-B-cells were undetectable in one case. The majority of B-cell precursors were pro-B-cells in Patients 5 and 6 (50 and 62% of CD19+ cells, respectively), pre-B-cells in Patients 3 and 4 (64 and 70%) and immature B-cells in Patient 1 (44%). Detectable CD20 expression on CD19+ cells was either low or absent. Plasma cells varied from 0.01 to 0.36% of the total nucleated cells. There was a trend towards longer duration of clinical response in patients with evidence of more complete depletion in BM.In this small cohort of RA patients treated with rituximab, differences in proportion and phenotype of CD19+ BM cells were detected. These differences suggest variation in the degree of depletion achieved and correlate with time to relapse. Although pro-B-cells are not targeted directly by rituximab as they do not express CD20, the levels were unexpectedly low.
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The diagnosis of acute undifferentiated leukemia (AUL) is made when the leukemic cells do not have cytologic or cytochemical features of myeloid cells, and do not express myeloid antigens (CD13, CD14, CD33, CD41 etc.) or lymphoid antigens (CD2, CD3, CD19, CD20, Sm Ig etc.). Most of these cells are reported to be positive for CD7, CD34, TdT and HLA-DR, either alone or in combination. Cell lineage can be suspected in most AUL cells by genotypic analysis, phenotypic analysis after culturing with TPA, or peroxidase activity by ultrastructural or immunohistochemical analysis, which indicates the heterogeneity of AUL. The patients with AUL appear to have a poor prognosis with conventional chemotherapy.
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Aberrant CD2 expression in childhood precursor-B ALL is rare and has recently been reported with an incidence of 3.6% in a study by Cantu-Rajnoldi et al. [Haematologica 77:384, 1992]. There was no association of the CD2 co-expression with other known prognostic factors. Our study represents the second one in the literature. Out of 60 childhood acute lymphoblastic leukemias, analyzed morphologically and by flow cytometric immunophenotyping, 49 were of precursor B origin. Of these 49 cases, CD2 co-expression was detected in 2, yielding an incidence of 4.1%. The complete immunophenotypic profiles of these two cases were as follows, respectively: (1) CD19+, CD20−, CD24+, CD10+, slg−, CD2+, CD3−, CD5−, CD7−, CD13−, CD33−, CD34+, Tdt+; and (2) CD19+, CD20−, CD24+, CD10+, slg−, CD2+, CD3−, CD5−, CD7−, CD13+, CD33−, CD34+, Tdt+. Cytogenetic analysis revealed a normal male chromosome pattern in case 1 and an abnormal female chromosome pattern [4 cells: 46, XX, del (6q) (q21 q23) and 9 cells: 46, XX, del (11q) (q14 q23)] in case 2. Both patients were in continuous complete remission at last follow-up. © 1996 Wiley-Liss, Inc.
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