Usefulness of Flow Cytometry for the Detection of Cutaneous Localization in Malignant Hematologic Disorders

Elsa Maitre; Anne‐Laure Le‐Page; Francois Comoz; Florence Truquet; Gandhi Damaj; Edouard Cornet; Laurence Verneuil; Véronique Salaün; Xavier Troussard

doi:10.1002/cyto.b.21784

Engraftment Of Human Polycythemia Vera CD34+ Cells In hSIRPα-Transgenic-Human-TPO-Expressing RAG2-/-, IL2Rγ-/- Immunodeficient Mice

Blood ◽

10.1182/blood.v122.21.2844.2844 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2844-2844

Author(s):

Rouven Müller ◽

Alexandre Theocharides ◽

Renate Looser ◽

Radek C. Skoda ◽

Richard A. Flavell ◽

...

Keyword(s):

Flow Cytometry ◽

Polycythemia Vera ◽

Point Mutation ◽

Peripheral Blood ◽

Mouse Strains ◽

Hematologic Disorders ◽

Immunodeficient Mice ◽

Cd34 Cells ◽

Clonal Composition

Abstract Introduction Xenotransplantation of human hematopoietic malignancies into immunodeficient mice represents the most appropriate in vivo model system for human malignant hematopoiesis. While a diversity of immunodeficient mouse strains on the NOD/SCID and RAG2-/-/IL2Rγ-/--BALB/C background are described for the most aggressive human malignancies like acute leukemias, xenotransplantation models of less aggressive human hematologic disorders like myeloproliferative neoplasms and myelodysplastic syndromes show only limited engraftment levels. We recently developed next generation mouse strains expressing human cytokines and key factors of xenogeneic cell acceptance (e.g. hSIRPα to inactivate mouse macrophage activation by human cells) and hypothesized that these would represent suitable models for the assessment of human less aggressive hematologic disorders in vivo by providing an optimized “humanized” microenvironment. Methods Peripheral blood (PB) and bone marrow (BM) samples of polycythemia vera (PV) patients were collected after informed consent at the Division of Hematology, Zurich University Hospital. Human CD34+ cells were isolated by density gradient centrifugation followed by immunomagnetical selection using anti-CD34 coupled beads. Purity of magnetical selection process was confirmed by FACS analysis. Newborn (24h-48h old) hSIRPα-tg-hTPO-knockin mice on the RAG2-/-/IL2Rγ-/--BALB/C background received sublethal irradiation (split dose of 2x1.5 Gy) and were transplanted intra-hepatically 24 hours later. Transplanted cell dose was dependent on availability of CD34+ stem and progenitor cells isolated from one phlebotomy sample (∼400ml of PB) of the respective patient. Mice were bled 4 weeks after transplantation and chimerism in peripheral blood was analyzed by flow cytometry using a panel of antigens (mCD45.2, hCD45, hCD33, hCD34, hCD3, hCD19). Mice showing positive chimerism in PB (i.e.>0.1% hCD45+ of total MNCs) at week 4 were sacrificed between week 8-16 and engraftment in BM, spleen and PB was analyzed by flow cytometry. To verify engraftment of human malignant hematopoiesis we quantified allele-burden of JAK2V617F point mutation in mouse BM using allele specific (AS)-PCR for the pathognomonic point mutation of the JAK2 gene. Results By transplantation of 4-10x105 CD34+ cells into newborn hSIRPα-tg-hTPO-RAG2-/-/IL2Rγ-/- mice we could detect engraftment of hCD45+ cells in PB at week 4 (median 0.68%, range 0.12-23.8%). At week 8, BM engraftment of hCD45+ cells ranged 0.88-54.1% (median 4.43%) with a high proportion of human myeloid cells detected by hCD45/hCD33 co-staining (median 3.07 %, range 0.6-17.6% of total MNCs). We could detect engraftment until week 16, the latest timepoint assessed. Since all transplanted PV patient samples were positive for the common point mutation JAK2V617F, AS-PCR was used to quantify human malignant hematopoiesis. In tested BM samples of engrafted mice we found JAK2V617F positive alleles with a frequency of 2-12% (median 8%). To further assess the clonal composition of the engrafted population we established single cell sorting of primary and engrafted human PV-CD34+ cells in a 96 well format followed by liquid culture expansion and AS-PCR. In pilot studies we could show the clonal composition of a BM engrafted CD34+ population that split into 80% JAK2 WT expressing, 10% JAK2V617F heterozygous and 10 % JAK2V617F homozygous clones. We are currently extending these findings by side by side comparison of the clonal composition of primary vs. xenografted human PV-CD34+ cells of the same patient to test for the influence of a xenogeneic humanized microenvironment on maintenance of malignant cells in vivo. Conclusions By using hSIRPα-tg-hTPO-RAG2-/-/IL2Rγ-/- mice we could show engraftment of PV-CD34+ cells that extends previous reported engraftment levels in other model organisms. To our knowledge this is the first study assessing the clonal composition of human PV engrafted cells in the xenogeneic environment aiming at identifying components that are critical for the maintenance of human malignant hematopoiesis in vivo. This model will thus be a useful tool to test targeted therapies in vivo. Disclosures: No relevant conflicts of interest to declare.

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Flow cytometry of reticulocytes applied to clinical hematology

Blood ◽

10.1182/blood.v61.6.1091.1091 ◽

1983 ◽

Vol 61 (6) ◽

pp. 1091-1097 ◽

Cited By ~ 53

Author(s):

HJ Tanke ◽

PH Rothbarth ◽

JM Vossen ◽

GJ Koper ◽

JS Ploem

Keyword(s):

Flow Cytometry ◽

Peripheral Blood ◽

Blood Bank ◽

Control Group ◽

Allogeneic Bone ◽

Blood Samples ◽

Frequency Distributions ◽

Hematologic Disorders ◽

Rna Content ◽

Polychromatic Erythrocytes

Abstract Reticulocytes in fixed human blood samples were stained for RNA with the fluorescent dye pyronin Y and measured by flow cytometry. The resulting relative frequency distributions of the RNA fluorescence intensities concurred with the different stages in maturation from early reticulocytes to mature red cells. A computer program was written to calculate from these frequency distributions the relative number of reticulocytes, their relative RNA content, and the median of the reticulocyte population (RNA index). This method was applied to 30 healthy blood bank donors (control group), as well as to patients with various hematologic disorders showing abnormal erythropoietic activity. The measured percentage of reticulocytes, RNA content, and RNA index were found to correlate well with the various hematologic disorders. Changes in erythropoiesis could be clearly followed, as was demonstrated by analyzing blood samples from children with aplastic anemia or acute myeloid leukemia, who were treated with allogeneic bone marrow transplantation. Measurements on blood samples from healthy blood bank donors showed that with this method, small changes in the reticulocyte population, such as the appearance of polychromatic erythrocytes in the peripheral blood 5–8 hr after donation, can be detected. The statistical reliability and the information provided on the maturation stage of the entire reticulocyte population make flow cytometry of peripheral blood reticulocytes a more informative method for the study of hematologic abnormalities than conventional methods for reticulocyte counting and classification.

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Decreased CD10-Positive Mature Granulocytes in Bone Marrow From Patients With Myelodysplastic Syndrome

Archives of Pathology & Laboratory Medicine ◽

10.5858/2000-124-1152-dcpmgi ◽

2000 ◽

Vol 124 (8) ◽

pp. 1152-1156

Author(s):

Chung-Che Chang ◽

Ronald P. Cleveland

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Myelodysplastic Syndrome ◽

Cell Lineage ◽

Surface Marker ◽

Refractory Anemia ◽

Control Group ◽

Light Scatter ◽

Hematologic Disorders ◽

Lymphoma Involvement

Abstract Objective.—We retrospectively examined the maturation of the granulocytic cell lineage in bone marrow specimens from patients with myelodysplastic syndrome (MDS) by flow cytometry using both light scatter and surface marker characteristics, including CD10 and myeloid lineage–associated antigens. Patients.—The 7 MDS cases we studied included 2 patients with refractory anemia (RA), 3 with RA with ringed sideroblasts, 1 with RA with excess of blasts, and 1 unclassified case. Another 7 patients matched for age and sex who received bone marrow aspirates for lymphoma staging (all negative for lymphoma involvement or any other hematologic abnormalities) were selected as the control group. Results.—The percentage of CD10+ mature granulocytes was significantly lower in patients with MDS than in control patients. Additionally, all patients with MDS had less than 50% CD10+ cells in the granulocytic lineage. In contrast, only 1 of the 7 control patients had less than 50% CD10+ cells (P < .01). Conclusions.—These results suggest that flow cytometry might be a useful adjunct in the assessment of patients with suspected MDS. Further studies to correlate CD10+ mature granulocytes from MDS cases with other benign hematologic disorders are indicated to confirm our evaluation.

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Flow cytometry of reticulocytes applied to clinical hematology

Blood ◽

10.1182/blood.v61.6.1091.bloodjournal6161091 ◽

1983 ◽

Vol 61 (6) ◽

pp. 1091-1097

Author(s):

HJ Tanke ◽

PH Rothbarth ◽

JM Vossen ◽

GJ Koper ◽

JS Ploem

Keyword(s):

Flow Cytometry ◽

Peripheral Blood ◽

Blood Bank ◽

Control Group ◽

Allogeneic Bone ◽

Blood Samples ◽

Frequency Distributions ◽

Hematologic Disorders ◽

Rna Content ◽

Polychromatic Erythrocytes

Reticulocytes in fixed human blood samples were stained for RNA with the fluorescent dye pyronin Y and measured by flow cytometry. The resulting relative frequency distributions of the RNA fluorescence intensities concurred with the different stages in maturation from early reticulocytes to mature red cells. A computer program was written to calculate from these frequency distributions the relative number of reticulocytes, their relative RNA content, and the median of the reticulocyte population (RNA index). This method was applied to 30 healthy blood bank donors (control group), as well as to patients with various hematologic disorders showing abnormal erythropoietic activity. The measured percentage of reticulocytes, RNA content, and RNA index were found to correlate well with the various hematologic disorders. Changes in erythropoiesis could be clearly followed, as was demonstrated by analyzing blood samples from children with aplastic anemia or acute myeloid leukemia, who were treated with allogeneic bone marrow transplantation. Measurements on blood samples from healthy blood bank donors showed that with this method, small changes in the reticulocyte population, such as the appearance of polychromatic erythrocytes in the peripheral blood 5–8 hr after donation, can be detected. The statistical reliability and the information provided on the maturation stage of the entire reticulocyte population make flow cytometry of peripheral blood reticulocytes a more informative method for the study of hematologic abnormalities than conventional methods for reticulocyte counting and classification.

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