scholarly journals Accurate quantification of fourteen normal bone marrow cell subsets in infants to the elderly by flow cytometry

2018 ◽  
Vol 94 (5) ◽  
pp. 783-792 ◽  
Author(s):  
Julie Pont ◽  
Alice Souvignet ◽  
Lydia Campos ◽  
Adriana Plesa ◽  
Bénédicte Bulabois ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Bone Marrow Cell ◽  
Marrow Cell ◽  
The Elderly ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Normal Bone Marrow Cell ◽  
Accurate Quantification

171 Complex effects of azacitidine in primary myelodysplastic and normal bone marrow cell cultures

2011 ◽  
Vol 35 ◽  
pp. S67
Author(s):  
M. Grövdal ◽  
M. Karimi ◽  
M. Nikpour ◽  
L. McGovern ◽  
M. Jansson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cell ◽  
Cell Cultures ◽  
Marrow Cell ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Normal Bone Marrow Cell

Diagnosis of Neonatal Bacterial Infection: Hematologic and Pathologic Findings in Fatal and Nonfatal Cases

PEDIATRICS ◽  
1979 ◽  
Vol 64 (1) ◽  
pp. 60-64 ◽  
Author(s):  
Edward Squire ◽  
Blaise Favara ◽  
James Todd
Keyword(s):  
Bone Marrow ◽  
Bacterial Infection ◽  
Normal Values ◽  
Marrow Cell ◽  
Blood Cultures ◽  
Normal Bone Marrow ◽  
Bacterial Sepsis ◽  
Normal Bone ◽  
Normal Bone Marrow Cell ◽  
Pathologic Findings

Consecutive newborn autopsy cases were divided into infected and noninfected groups on the basis of pathologic findings and cultures, and were compared to a concomitant consecutive group of neonatal survivors with proven bacterial sepsis. Newborns dying with bacterial infection often demonstrated leukopenia, neutropenia, and thrombocytopenia, usually associated with normal bone marrow cell production. Those with nonfatal sepsis frequently had neutrophiia with an increase in absolute band counts. Of infected newborns 80% showed one or more hematologic abnormalities as did 43% of newborns dying without bacterial infection. Of newborns dying with bacterial infection 13% had no hematologic abnormality. Blood cultures were negative in 18% (seven) of the infants dying with bacterial infection. Abnormalities of the white blood cell, differential and platelet counts are not invariably specific for bacterial infection nor do normal values adequately exclude it. Blood cultures may be negative in newborns dying with significant foci of bacterial infection.

Imaging Mass Cytometry Reveals the Spatial Architecture of Myelodysplastic Syndromes and Secondary Acute Myeloid Leukemias

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 44-45
Author(s):  
Karolyn A. Oetjen ◽  
Diane E. Bender ◽  
Marianna B. Ruzinova ◽  
Daniel A.C. Fisher ◽  
Stephen T. Oh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Marrow Cell ◽  
Cell Populations ◽  
Normal Bone Marrow ◽  
Mass Cytometry ◽  
Normal Bone ◽  
Lineage Markers ◽  
Spatial Architecture ◽  
Acute Myeloid

Histologic review of bone marrow trephine biopsies is a central component of the diagnostic and treatment response evaluation of hematologic malignancies. Well-validated antibody reagents are routinely used for immunohistochemistry of these samples to provide additional insight into abnormal antigen expression. However, current immunohistochemistry staining protocols are typically limited to only one or two markers simultaneously. Dysplastic changes in cellular morphology and dyssynchronous expression of lineage markers are common features of myelodysplastic syndromes, myeloproliferative neoplasms and secondary acute leukemias. We have integrated the use of multiple diagnostic validated antibody clones with additional antibodies for hematologic lineages and structural proteins to create a 30-marker panel for imaging mass cytometry (IMC). Antibodies included in this panel identify myeloid, lymphoid, erythroid, macrophage, vascular, megakaryocyte and stromal markers as well as markers of cellular proliferation and apoptosis. Through conjugation to elemental metal tags, the entire panel is stained simultaneously on the tissue sample, then acquired by time-of-flight mass spectrometry on a Hyperion instrument (Fluidigm). Antibody staining concentrations and antigen retrieval conditions were optimized for formalin-fixed paraffin-embedded (FFPE) bone marrow to obtain consistent staining for all markers on the panel. Redundant markers for cell populations were selected to provide further internal validation of the observed staining patterns. After data acquisition, cell segmentation algorithms using CellProfiler and ilastik were applied to quantify marker expression in single cells and Phenograph in HistoCAT was used for cell population clustering. Cluster identities for all cells are associated with the original image location in order to plot the spatial arrangement of populations. Using this highly multiplexed panel, we have imaged sets of bone marrow specimens from patients with normal bone marrow morphology and those with myeloid malignancies. We initially confirmed the staining patterns expected for each antibody patterns of co-expression of lineage markers in normal bone marrow samples. We then extended this panel to examine biopsies from patients with myelodysplastic syndrome, myelofibrosis, and secondary acute myeloid leukemia. We found a clear population of CD71+ CD235a+ erythroid cells with strong expression of the proliferative marker Ki67 located within erythroid islands in normal bone marrow samples and MDS. Cell markers of apoptosis and DNA damage are scattered at low frequency throughout the bone marrow in samples with normal bone marrow morphology, but increased clusters of the DNA damage marker phospho-H2AX are observed in selected cases of myelodysplastic syndromes. Overall, this IMC imaging approach is able to extend the current clinical immunostaining for myeloid malignancies by identifying all major bone marrow cell populations. Through highly multiplexed analysis of bone marrow cell populations, the spatial architecture of cell populations and stromal structures can be elucidated, including erythroid islands, lymphoid aggregates and changes in vascular structures with increasing severity of myelofibrosis. In ongoing studies, the development of these imaging techniques for analysis of archived FFPE bone marrow samples is being applied to translational research on hematologic diseases. Disclosures Oh: Kartos Therapeutics: Consultancy; Disc Medicine: Consultancy; PharmaEssentia: Consultancy; Constellation: Consultancy; CTI Biopharma: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; Blueprint Medicines: Consultancy; Novartis: Consultancy; Gilead Sciences: Consultancy; Incyte Corporation: Consultancy.

Novel Multiparametric Quantitative Immunocytomorphological Characterisation of Human Bone Marrow Precursor Differentiation and of Haematopoietic Neoplasms Using ImageStream® Cytometry

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4871-4871
Author(s):  
Catherine Claude Martin ◽  
Chantal Jayat-Vignoles ◽  
Jean-Luc Faucher ◽  
Thaddeus George ◽  
Vidya Venkatachalam ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Blood Cells ◽  
High Speed ◽  
Tumour Cells ◽  
Leukemic Cells ◽  
Normal Bone Marrow ◽  
Normal Cells ◽  
Normal Bone ◽  
Abnormal Cells

Abstract The ImageStream technology performs high speed acquisition of brightfield, laser scatter and up to four fluorescent images per cell for several thousands of cells in suspension, thereby enabling simultaneous immunophenotyping and morphology-based measurements. This is the only technology combining cytology and flow cytometry in one single platform. Our aim was to study normal and tumour cells of the haematopoietic lineage with this new technology in order to improve diagnosis of haematological disorders. We have defined cytomorphological criteria of normal bone marrow (n=4) and circulating blood cells (n=40). Cells were multi-colour labelled with both DRAQ5 nuclear stain and CD45 ECD-mAb, and additionally labeled with a combination of mAbs against either CD3/CD19, CD11b/CD16, CD14/CRTH2, or CD71/CD235. Results for normal cells were compared to those obtained by classical cytometry and cytology. We then applied these criteria to samples with patients with circulating leukemic cells, including 1 myelodysplatic syndrome (MDS), 1 myeloproliferative syndrome (MPS), 3 acute lymphoblastic leukaemia (ALL), 2 follicular lymphomas (FL) and 20 chronic lymphocytic lymphomas (CLL). We have created completely new quantitative cytomorphological criteria for classifying blood cells using parameters that measure cellular size and shape, nuclear to cytoplasmic area ratio, nuclear lobe count, SSC texture, the ratio between the size and the major axis of CD45, the ratio between the intensity and the compactness of SSC signal, and the intensity of DRAQ5 labelling, to name a few. Using these criteria, we have characterised normal bone marrow differentiation and normal circulating blood cells. We have obtained a perfect correlation with classical cytology and flow cytometry. Analysis of pathological samples showed that abnormal cells were recognized in all cases. We found an abnormal blast cell compartment and an abnormal monocytic differentiation branch in the case of MDS. We have also defined specific cytomorphological properties that distinguish ALL, FL and CLL tumour cells from normal cells. We also provide data that enumerates the proportion of large cells, of atypical CLL cells and of cells in the G2/M phase. Altogether, these results show that a technology combining cytology and flow cytometry in a single platform leads to the discovery of completely new and quantitative cytomorphological parameters defining each stage of normal cell and each category of abnormal cells of the haematopoietic lineage, opening completely new perspectives for the diagnosis of haematopoietic neoplasms.

A CD123-Targeting Antibody-Drug Conjugate (ADC), IMGN632, Designed to Eradicate Acute Myeloid Leukemia (AML) Cells While Sparing Normal Bone Marrow Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 768-768 ◽  
Author(s):  
Yelena Kovtun ◽  
Gregory Jones ◽  
Charlene Audette ◽  
Lauren Harvey ◽  
Baudouin Gerard ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Cell Populations ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Healthy Donors ◽  
Marrow Cells ◽  
Myeloid Progenitors

Abstract Current AML therapies are effective in a subset of patients, but often lead to prolonged myelosuppression. CD123 is an attractive AML target due to its elevated expression on AML compared to normal bone marrow cells. Still, severe myelosuppression and myeloablation have been reported in preclinical studies for some CD123-targeted therapies. Here, we present a novel ADC which selectively kills CD123-positive AML cells over normal bone marrow cells. A novel humanized anti-CD123 antibody with two engineered cysteines for payload conjugation was generated. Indolinobenzodiazepine dimers, termed IGNs, were chosen as payload molecules for the antibody due to their high potency against AML cells. The IGN dimers containing mono-imines alkylate DNA, whereas the di-imine containing IGNs can both alkylate and crosslink DNA. To select an optimal IGN payload, we compared the cytotoxicity of an ADC with a mono-imine IGN (A-ADC) to one with a di-imine IGN (C-ADC) on AML cells, as well as normal bone marrow cells in vitro. Potency of the ADCs was evaluated using AML cell lines that have CD123 levels similar to patient cells and carry markers of poor prognosis (FLT3-ITD , MDR1, EVI1, DNMT3A and TP53), as well as on samples from 11 AML patients. AML cells exposed to either ADC displayed markers of DNA damage, cell cycle arrest and apoptotic cell death by flow cytometry. Both ADCs were highly cytotoxic, generating IC50 values between 0.4 to 60 pM on the cell lines in WST-8 assays and killing 90 percent of progenitors from AML patients between 2 to 46 pM in CFU assays. The C-ADC was, on average, two-fold more active than the A-ADC. The cytotoxicity of both ADCs was CD123 dependent, since masking CD123 with a competing anti-CD123 antibody reduced the potency by more than 100-fold. Toxicity of the ADCs was assessed using bone marrow cells from a healthy human donor. The cells were exposed to the ADCs at 100 pM (a concentration highly potent against all AML samples) for 72 hours, and then markers of apoptosis were detected in different cell populations by flow cytometry. Neither ADC affected the viability of monocytes, lymphocytes and multipotential progenitors, consistent with low CD123 levels in these cell populations. In contrast, an apoptotic signal was detected in myeloid progenitors, the population with the highest CD123 level, following exposure to the C-ADC, but not to the A-ADC. The toxicity of the ADCs was also tested in CFU assays on bone marrow cells from 7 healthy donors, as the assays have been reported to predict clinical myelosuppression. Surprisingly, the C-ADC was, on average, 50-fold more cytotoxic to normal myeloid progenitors than the A-ADC (40 pM vs 2,000 pM IC90 values, respectively) (Figure 1). Finally, we compared CD123 independent toxicity of the ADCs in CD-1 mice. The C-ADC showed significantly reduced tolerability, and unlike the A-ADC, was associated with delayed toxicity manifested by weight loss 30 days after administration. Based on its potent yet highly selective toxicity to AML cells and more favorable tolerability profile, the A-ADC was selected for further study, and renamed as IMGN632. To compare IMGN632 to an ADC previously approved for the treatment of AML, the potency of IMGN632 and gemtuzumab ozogamicin (GO) was tested on bone marrow cells from 11 healthy donors and 17 AML patients, including 4 relapsed/refractory and 8 with strong multidrug resistance (Figure 1). Only 6 of 17 AML samples were sensitive to GO at concentrations that did not impact normal progenitors. In contrast, AML progenitors from all 17 patients were highly sensitive to IMGN632. Importantly, normal progenitors were only affected by IMGN632 at 150-fold higher concentrations. The pronounced difference between AML and normal progenitors in their sensitivity to IMGN632 likely reflects both higher CD123 levels on AML progenitors and the lower sensitivity of normal progenitors to the mono-imine IGN payload we observed in CFU assays. In conclusion, through use of a mono-imine IGN payload, IMGN632 demonstrated potent activity in all tested AML samples at concentrations far below levels that impact normal bone marrow cells, suggesting the potential for efficacy in AML patients in the absence of or with limited myelosuppression. These findings together with strong efficacy in multiple AML xenograft models (Kovtun et al., 21st EHA congress, 2016; Adams et al., 58th ASH annual meeting, 2016) support advancing IMGN632 into clinical trials. Disclosures Kovtun: ImmunoGen, Inc.: Employment. Jones:ImmunoGen, Inc.: Employment. Audette:ImmunoGen, Inc.: Employment. Harvey:ImmunoGen, Inc.: Employment. Gerard:ImmunoGen, Inc.: Employment. Wilhelm:ImmunoGen, Inc.: Employment. Bai:ImmunoGen, Inc.: Employment. Adams:ImmunoGen, Inc.: Employment. Goldmacher:ImmunoGen, Inc.: Employment. Chari:ImmunoGen: Employment. Chittenden:ImmunoGen, Inc.: Employment.

scholarly journals CELLS INVOLVED IN THE IMMUNE RESPONSE

1969 ◽  
Vol 130 (1) ◽  
pp. 141-163 ◽  
Author(s):  
Nabih I. Abdou ◽  
Maxwell Richter
Keyword(s):  
Bone Marrow ◽  
Immune Response ◽  
Glass Bead ◽  
Mononuclear Cells ◽  
Marrow Cell ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Immunological Specificity ◽  
Rabbit Bone ◽  
Free Antigen

The antigen-reactive cells in normal rabbit bone marrow could be isolated from a suspension of marrow cells by passage of the cells through an antigen-sensitized glass bead column. The cells which passed through the column were deficient in antigen-reactive cells directed to the antigen used to sensitize the glass beads, whereas the cells eluted from the column could transfer antibody-forming capacity to irradiated recipients only with respect to the specific sensitizing antigen. Separation of the bone marrow antigen-reactive cells could not be achieved by passage of the cells through nonsensitized glass bead columns or in the presence of excess free antigen in the column. Cells which were retained by, and later eluted from, the antigen-sensitized glass bead columns were mostly small mononuclear cells, whereas cells which passed through the columns were morphologically similar to the original unfractionated bone marrow cell suspension. The data indicate the presence of an antibody or antibody-like structure, with defined immunological specificity, on the surface of the normal bone marrow antigen-reactive cell.

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