Diagnosis of paroxysmal nocturnal hemoglobinuria in peripheral blood and bone marrow with six-color flow cytometry

2013 ◽  
Vol 7 (1) ◽  
pp. 99-111 ◽  
Author(s):  
Hai-Su Yang ◽  
Min Yang ◽  
Xiaoyu Li ◽  
Sorina Tugulea ◽  
Henry Dong
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Color Flow

Immunophenotypic Study of Basophils by Multiparameter Flow Cytometry

2008 ◽  
Vol 132 (5) ◽  
pp. 813-819
Author(s):  
Xiaohong Han ◽  
Jeffrey L. Jorgensen ◽  
Archana Brahmandam ◽  
Ellen Schlette ◽  
Yang O. Huh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Chronic Myelogenous Leukemia ◽  
Peripheral Blood ◽  
Myelogenous Leukemia ◽  
Multiparameter Flow Cytometry ◽  
Color Flow ◽  
Aberrant Expression ◽  
Flow Cytometric ◽  
Hla Dr

Abstract Context.—The immunophenotypic profile of basophils is not yet fully established, and the immunophenotypic changes in chronic myelogenous leukemia are not fully characterized. Objective.—To establish a comprehensive immunophenotypic spectrum of normal basophils and to assess the range of immunophenotypic aberrations of basophils in chronic myelogenous leukemia. Design.—Using 4-color flow cytometry, we compared the immunophenotypic profile of basophils in peripheral blood or bone marrow samples from 20 patients with no evidence of neoplasia to basophils from 15 patients with chronic myelogenous leukemia. Results.—Basophils in control cases were all positive for CD9, CD13, CD22, CD25 (dim), CD33, CD36, CD38 (bright), CD45 (dimmer than lymphocytes and brighter than myeloblasts), and CD123 (bright), and were negative for CD19, CD34, CD64, CD117, and HLA-DR. Basophils in all chronic myelogenous leukemia patients possessed 1 to 5 immunophenotypic aberrancies. The most common aberrancies were underexpression of CD38, followed by aberrant expression of CD64 and underexpression of CD123. CD34 and CD117 were present in cases with basophilic precursors. Myeloblasts showed a distinct immunophenotypic profile, as they typically expressed CD34 and CD117, showed dimmer expression (compared with basophils) of CD38, CD45, and CD123, and lacked expression of CD22. Conclusions.—Flow cytometric immunophenotyping can identify immunophenotypic aberrations of basophils in chronic myelogenous leukemia, and discriminate basophils from myeloblasts.

Flow Cytometric Detection of the Expression of CXCR4 on CD34+ Cells and the Distribution of Lymphocyte Subsets in Allogeneic Hematological Grafts.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5195-5195
Author(s):  
Lulu Lu ◽  
Yongping Song ◽  
Baogen Ma ◽  
Xiongpeng Zhu ◽  
Xudong Wei ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Lymphocyte Subsets ◽  
Color Flow ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Mobilized Peripheral Blood

Abstract Background and objectives: Normal human bone marrow (BM), cord blood (CB) and mobilized peripheral blood (MPB) are the most commonly used sources for allogeneic hematopoietic stem cell transplantation (HSCT). The aim of this study was to detect the expression of CXCR4 on CD34+ cells and to assess the distribution of lymphocyte subsets in each type allograft. Methods: CD34+ cells were separated from BM (n=30), CB (n=30) and MPB (n=30) by the CD34 MultiSort Kit immunomagnetic bead system. The expression of CXCR4 on CD34+cells was assayed by double color flow cytometry. The lymphocyte subsets in each type of allograft were detected by three-color flow cytometry. The groups of monoclonal antibodies were used as the following: CXCR4-PE/CD34−Pecy5, CD8−FITC/CD4−R-PE/CD3−TC, CD45RA-FITC/CD45RO-PE/CD4−Pecy5, CD45RA-FITC/CD45RO-PE/CD8−Pecy5, and CD3−FITC/CD16+56-PE. Isotype-specific antibodies were used as controls. Results: The expression of CXCR4 of cord blood and mobilized peripheral blood CD34+ cells was lower than that of bone marrow cells (BM 40.21%±6.72%, CB 20.93%±3.96%, MPB 20.93%±3.96%, P <0.05). The difference between cord blood and mobilized peripheral blood was not significant (P>0.05). The CD3+CD8low and CD3+CD4−CD8low subsets were higher in BM than that of CB and MPB (BM 8.61%±1.40%, CB 3.31%±0.88%, MPB 5.11%±0.76%,P<0.01). The relative frequencies of the naïve CD45RA+ CD45RO− phenotype among CD4+ and CD8high T cells were highest in CB, and it was higher in MPB than in BM grafts (BM 28.09%±4.52%, 41.86 %±3.31%; CB83.83%±12.24%, 86.69%±6.12%; MPB 43.58%±4.54%, 57.64%±4.77%, P<0.01). Naïve T cells (CD45RA+ CD45RO−) were mobilized preferentially compared to memory T cells (CD45RA− CD45RO+)(P <0.01); The relative frequencies of NKT (CD3+CD16+56+) among lymphocytes were lower in CB than that in BM and MPB (CB 0.77±0.19, BM4.15±1.10, MPB 4.13±0.84, P<0.01). Conclusion: BM, CB and MPB allografts differ widely in cellular makeup of CD34+ cells and lymphocyte subsets, which are associated with the distinct characteristics after allogeneic HSCT from different allogeneic hematological sources.

scholarly journals Identification and comparison of CD34-positive cells and their subpopulations from normal peripheral blood and bone marrow using multicolor flow cytometry

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2591-2596 ◽  
Author(s):  
JG Bender ◽  
KL Unverzagt ◽  
DE Walker ◽  
W Lee ◽  
DE Van Epps ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Hematopoietic Progenitors ◽  
Color Flow ◽  
Normal Peripheral Blood ◽  
Hla Dr ◽  
Cd34 Cells ◽  
Wide Range ◽  
Immature Cells

Four-color flow cytometry was used with a cocktail of antibodies to identify and isolate CD34+ hematopoietic progenitors from normal human peripheral blood (PB) and bone marrow (BM). Mature cells that did not contain colony forming cells were resolved from immature cells using antibodies for T lymphocytes (CD3), B lymphocytes (CD20), monocytes (CD14), and granulocytes (CD11b). Immature cells were subdivided based on the expression of antigens found on hematopoietic progenitors (CD34, HLA-DR, CD33, CD19, CD45, CD71, CD10, and CD7). CD34+ cells were present in the circulation in about one-tenth the concentration of BM (0.2% v 1.8%) and had a different spectrum of antigen expression. A higher proportion of PB-CD34+ cells expressed the CD33 myeloid antigen (84% v 43%) and expressed higher levels of the pan leukocyte antigen CD45 than BM-CD34+ cells. Only a small fraction of PB-CD34+ cells expressed CD71 (transferrin receptors) (17%) while 94% of BM-CD34+ expressed CD71+. The proportion of PB-CD34+ cells expressing the B-cell antigens CD19 (10%) and CD10 (3%) was not significantly different from BM-CD34+ cells (14% and 17%, respectively). Few CD34+ cells in BM (2.7%) or PB (7%) expressed the T-cell antigen CD7. CD34+ cells were found to be predominantly HLA-DR+, with a wide range of intensity. These studies show that CD34+ cells and their subsets can be identified in normal PB and that the relative frequency of these cells and their subpopulations differs in PB versus BM.

scholarly journals Identification and comparison of CD34-positive cells and their subpopulations from normal peripheral blood and bone marrow using multicolor flow cytometry

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2591-2596 ◽  
Author(s):  
JG Bender ◽  
KL Unverzagt ◽  
DE Walker ◽  
W Lee ◽  
DE Van Epps ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Hematopoietic Progenitors ◽  
Color Flow ◽  
Normal Peripheral Blood ◽  
Hla Dr ◽  
Cd34 Cells ◽  
Wide Range ◽  
Immature Cells

Abstract Four-color flow cytometry was used with a cocktail of antibodies to identify and isolate CD34+ hematopoietic progenitors from normal human peripheral blood (PB) and bone marrow (BM). Mature cells that did not contain colony forming cells were resolved from immature cells using antibodies for T lymphocytes (CD3), B lymphocytes (CD20), monocytes (CD14), and granulocytes (CD11b). Immature cells were subdivided based on the expression of antigens found on hematopoietic progenitors (CD34, HLA-DR, CD33, CD19, CD45, CD71, CD10, and CD7). CD34+ cells were present in the circulation in about one-tenth the concentration of BM (0.2% v 1.8%) and had a different spectrum of antigen expression. A higher proportion of PB-CD34+ cells expressed the CD33 myeloid antigen (84% v 43%) and expressed higher levels of the pan leukocyte antigen CD45 than BM-CD34+ cells. Only a small fraction of PB-CD34+ cells expressed CD71 (transferrin receptors) (17%) while 94% of BM-CD34+ expressed CD71+. The proportion of PB-CD34+ cells expressing the B-cell antigens CD19 (10%) and CD10 (3%) was not significantly different from BM-CD34+ cells (14% and 17%, respectively). Few CD34+ cells in BM (2.7%) or PB (7%) expressed the T-cell antigen CD7. CD34+ cells were found to be predominantly HLA-DR+, with a wide range of intensity. These studies show that CD34+ cells and their subsets can be identified in normal PB and that the relative frequency of these cells and their subpopulations differs in PB versus BM.

scholarly journals Bone Marrow as a Source of Cells for Paroxysmal Nocturnal Hemoglobinuria Detection

2018 ◽  
Vol 150 (3) ◽  
pp. 273-282 ◽  
Author(s):  
Alina E Dulau-Florea ◽  
Neal S Young ◽  
Irina Maric ◽  
Katherine R Calvo ◽  
Cynthia E Dunbar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
High Specificity ◽  
Cell Maturation ◽  
Flow Cytometry Analysis ◽  
Clone Size ◽  
Specificity And Sensitivity ◽  
Pnh Clone

Abstract Objectives To determine fluorescently labeled aerolysin (FLAER) binding and glycophosphatidylinositol–anchored protein expression in bone marrow (BM) cells of healthy volunteers and patients with paroxysmal nocturnal hemoglobinuria (PNH) detected in peripheral blood (PB); compare PNH clone size in BM and PB; and detect PNH in BM by commonly used antibodies. Methods Flow cytometry analysis of FLAER binding to leukocytes and expression of CD55/CD59 in erythrocytes. Analysis of CD16 in neutrophils and CD14 in monocytes in BM. Results FLAER binds to all normal BM leukocytes, and binding increases with cell maturation. In PNH, lymphocytic clones are consistently smaller than clones of other BM cells. PNH clones are detectable in mature BM leukocytes with high specificity and sensitivity using common antibodies. Conclusions PNH clone sizes measured in mature BM leukocytes and in PB are comparable, making BM suitable for PNH assessment. We further demonstrate that commonly used reagents (not FLAER or CD55/CD59) can reliably identify abnormalities of BM neutrophils and monocytes consistent with PNH cells.

Evaluation of Minimal Residual Disease in Blood and Bone Marrow for CLL as Assessed by 6 Color Flow Cytometry.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2793-2793 ◽  
Author(s):  
Karim Maloum ◽  
Laurent Sutton ◽  
Magali Le Garff-Tavernier ◽  
Caroline Fiocconi ◽  
Patrick Bonnemye ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Residual Disease ◽  
Biological Response ◽  
Leukemic Cells ◽  
Therapeutic Approaches ◽  
Becton Dickinson ◽  
Color Flow ◽  
New Therapeutic Approaches

Abstract MRD evaluation is becoming critical in CLL patients since the new therapeutic approaches lead to higher and higher biological response. Multicolor flow cytometry is demonstrated to be one of the best technique for assessing MRD. In a recent work, Moreno et al (Blood, 2006) suggested that peripheral blood could be sufficient for MRD evaluation. We here report our experience which compares peripheral blood and bone marrow aspirate for MRD assessment using a 6 color flow cytometry (FC) method. Two months after the end of the treatment, blood and bone marrow were harvested at the same time for 64 CLL patients in complete remission according to the NCI criteria. Patients were treated by chemotherapy followed or not by autologous stem cell transplantation (ASCT). None of them received immunotherapy. Until now 30 out of the 64 couples of blood and marrow samples have been studied. Immunophenotyping was performed using the FacsCanto Device (Becton Dickinson) and the following monoclonal antibody (moAb) combinaisons: k-FITC / l-PE / CD19-PerCP-Cy5.5 / CD5-PE-Cy7 / CD79b-APC / CD20-APC-Cy7; FMC7-FITC / CD38-PE / CD19PerCP-Cy5.5 / CD5-PE-Cy7 / CD20-APC-Cy7 for peripheral blood assessment and k-FITC / l-PE / CD45-PerCP-Cy5.5 / CD5-PE-Cy7 / CD19-APC / CD20-APC-Cy7; FMC7-FITC / CD19-PE / CD45PerCP-Cy5.5 / CD5-PE-Cy7 / CD79b-APC / CD20APC-Cy7 ; CD38-FITC / CD23-PE / CD45-PerCP-Cy5.5 / CD5-PE-Cy7 / CD19-APC. In the bone marrow, we have discriminated hematogons from CLL cells by using moAb combinations containing CD45 and CD38. Between 4500 and 5500 lymphocytes were analyzed in each test. By using a method of serial dilutions, we demonstrated a sensitivity of 10−4 for our 6-color FC technique. CLL cells were defined as CD19+CD5+CD79b low CD20low lymphocytes with low expression of monotypic Ig light chain. Results were expressed as a percentage of leukemic cells among total B lymphocytes. We showed a complete correlation between blood and bone marrow for MRD evaluation using this technique. A complete phenotypic remission defined by the absence of leukemic cells in the blood and in the bone marrow, was observed in 17 out of 30 cases. In the other 13 patients, persistent leukemic cells were detected in the marrow as well as in the peripheral blood with a median rate of 1.56 % [0.15 – 41] and 0.56% [range 0.06 – 36] respectively. Our results showed that, by using 6 color FC, blood assessment for detecting MRD in CLL after chemotherapy or ASCT could provide a simple surrogate to bone marrow assessment. Analysis of the remaining couples of samples for the last 34 patients is ongoing.

scholarly journals Recombinant human interleukin-11 stimulates megakaryocytopoiesis and increases peripheral platelets in normal and splenectomized mice

Blood ◽  
1993 ◽  
Vol 81 (4) ◽  
pp. 901-908 ◽  
Author(s):  
TY Neben ◽  
J Loebelenz ◽  
L Hayes ◽  
K McCarthy ◽  
J Stoudemire ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Color Flow ◽  
Platelet Counts ◽  
In Vivo Treatment ◽  
Interleukin 11 ◽  
Megakaryocyte Progenitors

Abstract The effects of recombinant human interleukin-11 (rhIL-11) on in vivo mouse megakaryocytopoeisis were examined. Normal C57Bl/6 mice and splenectomized C57Bl/6 mice were treated for 7 days with 150 micrograms/kg rhIL-11 administered subcutaneously. In normal mice, peripheral platelet counts were elevated compared with vehicle-treated controls after 3 days of rhIL-11 treatment and remained elevated until day 10. Splenectomized mice treated with rhIL-11 showed elevated peripheral platelet counts that were similar in magnitude to normal rhIL-11-treated mice. However, on day 10 the platelet counts in rhIL-11- treated, splenectomized mice were no longer elevated. Analysis of bone marrow megakaryocyte ploidy by two-color flow cytometry showed an increase, relative to controls, in the percentage of 32N megakaryocytes in both normal and splenectomized animals treated with rhIL-11. In normal mice, the number of spleen megakaryocyte colony-forming cells (MEG-CFC) were increased twofold to threefold relative to controls after 3 and 7 days of rhIL-11 treatment, whereas the number of bone marrow MEG-CFC were increased only on day 7. The number of MEG-CFC in the bone marrow of rhIL-11-treated, splenectomized mice was increased twofold compared with controls on both days 3 and 7 of the study. These data show that in vivo treatment of normal or splenectomized mice with rhIL-11 increased megakaryocyte progenitors, stimulated endoreplication of bone marrow megakaryocytes, and increased peripheral platelet counts. In addition, results in splenectomized mice showed that splenic hematopoiesis was not essential for the observed increases in peripheral platelets in response to rhIL-11 administration.

scholarly journals A two-color flow cytometry assay for detection of hairy cells using monoclonal antibodies

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1063-1068
Author(s):  
JS Kristensen ◽  
J Ellegaard ◽  
P Hokland
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Cell Suspensions ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Morphological Evidence ◽  
Acute Leukemias ◽  
Color Flow ◽  
Hairy Cells

We have developed a simple two-color immunofluorescence assay equally suited for microscopy and flow cytometry detecting hairy cells (HCs) in single cell suspensions, based on the concomitant reactivities with the B cell-specific monoclonal antibody B1 (CD20) and the monocyte/HC- associated antibody SHCL-3 (CD11c). Thus, HCs can be demonstrated in peripheral blood, bone marrow, and spleen specimens from hairy cell leukemia (HCL) patients even when they constitute less than 1% of the cell suspension. Likewise, admixture experiments with normal mononuclear cells and the MOLT-4 T-acute lymphocytic leukemia (ALL) cell line demonstrated that HCs could be detected in amounts as low as 1%. The validity of this assay has been ascertained by the lack of double marker positivity in cell suspensions from B-chronic lymphocytic leukemia (CLL) and acute myelogenous leukemia (AML) patients that only expressed B1 or SHCL-3, respectively. Furthermore, other malignant blood diseases, including malignant lymphomas, acute leukemias, and chronic leukemias disclosed no double marker positive cells. In a clinical setting, this assay was used for purifying HCs (by flow cytometry) from the peripheral blood from patients with no apparent morphological evidence of circulating HC infiltration and for monitoring the effect of interferon therapy. In conclusion, this assay should be of value for both diagnosis and monitoring patients with HCL.

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