Pharmacodynamic monitoring of BAY 43-9006 (Sorafenib) in phase I clinical trials involving solid tumor and AML/MDS patients, using flow cytometry to monitor activation of the ERK pathway in peripheral blood cells

Abstract In primates and rodents, platelets originate from the bone marrow megakaryocytes through a unique differentiation process with nuclear polyploidization, cytoplasmic maturation and proplatelet formation. In contrast, circulating thrombocytes of most non-mammalian vertebrates are particularly distinctive; the cells are large and nucleated. Adult Xenopus laevis may be an useful non-mammalian model for analyzing dynamic hematopoiesis because they are individually tolerable for time lapse analysis in vivo with sequential blood sampling, whereas classification of cell types has not been established yet. Microstructures of Xenopus thrombocytes observed with electron microscope exhibited structural characteristics largely resembling zebrafish thrombocytes with nucleated spindle cellular features (Thattaliyath et al., Blood 2005), and they had lobulated nuclear chromatin, granules, microparticles and open canalicular system-like-structures as in mammalian megakaryocytes. Since thrombocyte identification based on the morphological aspect was not sufficient, chemical staining with acetylecholinesterase and thiazole orange were performed. Additionally, mice were immunized by Xenopus peripheral blood cells to generate monoclonal antibodies, and two hybridomas producing IgG, respectively T12 and T5, were screened. T12+ (T12 positive) cells were morphologically typical thrombocytes. Flow cytometric analysis revealed that T12+ cells were also positive to anti-human GpIIb/IIIa polyclonal antibodies, and approximately 2-3% of whole peripheral blood cells were T12+/GpIIb/IIIa+ that distributed in FSClow/SSClow fraction. When T12 was injected into Xenopus to deplete T12+ cells in vivo, the detectable level of T12 in the circulation lasted for more than several weeks. Peripheral thrombocyte counts predominantly began to decrease immediately and reached their nadir at day 3, but white blood cell counts were not changed. RNA-rich blood cells considered as younger cells were then increasingly appeared, and finally the cell counts recovered to normal levels at day 10–15, indicating that in vivo depletion of T12+ cells induced thrombopoiesis and/or release of mature thrombocytes from the pool. T5 recognizing cells were classified into two populations by immunostaining and flow cytometry; T5+/GpIIb/IIIa+ cells were morphologically thrombocytic as the cells recognized by T12, while T5+/GpIIb/IIIa− cells were spherical and similar appearance to lymphocytic cells. These observations raised some possibilities e.g.; antigen of T5 was a membrane protein common to both lymphocytes and thrombocytes, or T5+/GpIIb/IIIa− cells were thrombocyte progenitors at earlier development stage than T12+/GpIIb/IIIa+ cells. Nevertheless only a few percent of T12+ and T5+ cells resided in peripheral blood, immunostaining revealed that the proportions of T12+/T5+ and T5+ cells in spleen were 10% and 70%, and T12+/T5+ and T5+ cells in liver were 5% and 20%, respectively. These suggest that spleen is predominantly involved in thrombopoiesis and/or thrombocyte storage in adult Xenopus. As T12 and T5 can be used successfully in flow cytometry and magnetic cell sorting, they should contribute us directly to elucidate the origin of circulating Xenopus thrombocytes and their cellular development process.

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Subpopulations of human peripheral blood cells: Analysis of granulocytic progenitor cells by flow cytometry and immunologic surface markers

American Journal of Hematology ◽

10.1002/ajh.2830070209 ◽

1979 ◽

Vol 7 (2) ◽

pp. 163-172 ◽

Cited By ~ 7

Author(s):

R. L. Drapkin ◽

M. Adreeff ◽

B. Koziner ◽

A. Strife ◽

D. Wisniewski ◽

...

Keyword(s):

Flow Cytometry ◽

Progenitor Cells ◽

Peripheral Blood ◽

Blood Cells ◽

Human Peripheral Blood ◽

Surface Markers ◽

Peripheral Blood Cells

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Peripheral blood cells are predominantly chimeric of affected and normal cells in patients with paroxysmal nocturnal hemoglobinuria: simultaneous investigation on clonality and expression of glycophosphatidylinositol-anchored proteins

Blood ◽

10.1182/blood.v83.3.853.bloodjournal833853 ◽

1994 ◽

Vol 83 (3) ◽

pp. 853-859 ◽

Cited By ~ 1

Author(s):

H Ohashi ◽

T Hotta ◽

A Ichikawa ◽

T Kinoshita ◽

R Taguchi ◽

...

Keyword(s):

Flow Cytometry ◽

Peripheral Blood ◽

Blood Cells ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Southern Hybridization ◽

Peripheral Blood Cells ◽

Chain Reaction ◽

Normal Cells ◽

Clonality Analysis ◽

Polymerase Chain

To investigate clonal compositions of hematologic cells in paroxysmal nocturnal hemoglobinuria (PNH), we analyzed peripheral blood (PB) cells of 12 female patients with PNH, by clonality analysis using X-chromosome inactivation and assessment of expression of glycophosphatidylinositol-anchored proteins (GPI-APs) by flow cytometry. Southern hybridization showed that granulocytes were monoclonal in three and polyclonal in eight patients, respectively, whereas lymphocytes were polyclonal in all nine patients examined. Expressions of CD16 and CD59 on granulocytes varied greatly in seven patients examined. Clonality analysis of granulocytes by the polymerase chain reaction showed that CD59-and CD59low+ cells were monoclonal, whereas CD59+ cells were polyclonal. It was shown that PB cells are predominantly chimeric of clonal (GPI-AP-or GPI-APlow+) and nonclonal (GPI-AP+) cells in PNH, and that degrees of chimerism differ greatly from patient to patient.

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Flow Cytometry to Determine Serotonin Transporter Function in Human Peripheral Blood Cells

Neuromethods - Serotonin Receptor Technologies ◽

10.1007/978-1-4939-2187-4_8 ◽

2014 ◽

pp. 151-167

Author(s):

Brendan S. Beikmann ◽

Anne M. Andrews

Keyword(s):

Flow Cytometry ◽

Serotonin Transporter ◽

Peripheral Blood ◽

Blood Cells ◽

Human Peripheral Blood ◽

Peripheral Blood Cells

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Faculty Opinions recommendation of Detection of Chlamydia in the peripheral blood cells of normal donors using in vitro culture, immunofluorescence microscopy and flow cytometry techniques.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13674.471167 ◽

2006 ◽

Author(s):

Steven Witkin

Keyword(s):

Flow Cytometry ◽

In Vitro Culture ◽

Peripheral Blood ◽

Blood Cells ◽

Immunofluorescence Microscopy ◽

Peripheral Blood Cells

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Peripheral blood cells are predominantly chimeric of affected and normal cells in patients with paroxysmal nocturnal hemoglobinuria: simultaneous investigation on clonality and expression of glycophosphatidylinositol-anchored proteins

Blood ◽

10.1182/blood.v83.3.853.853 ◽

1994 ◽

Vol 83 (3) ◽

pp. 853-859 ◽

Cited By ~ 15

Author(s):

H Ohashi ◽

T Hotta ◽

A Ichikawa ◽

T Kinoshita ◽

R Taguchi ◽

...

Keyword(s):

Flow Cytometry ◽

Peripheral Blood ◽

Blood Cells ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Southern Hybridization ◽

Peripheral Blood Cells ◽

Chain Reaction ◽

Normal Cells ◽

Clonality Analysis ◽

Polymerase Chain

Abstract To investigate clonal compositions of hematologic cells in paroxysmal nocturnal hemoglobinuria (PNH), we analyzed peripheral blood (PB) cells of 12 female patients with PNH, by clonality analysis using X-chromosome inactivation and assessment of expression of glycophosphatidylinositol-anchored proteins (GPI-APs) by flow cytometry. Southern hybridization showed that granulocytes were monoclonal in three and polyclonal in eight patients, respectively, whereas lymphocytes were polyclonal in all nine patients examined. Expressions of CD16 and CD59 on granulocytes varied greatly in seven patients examined. Clonality analysis of granulocytes by the polymerase chain reaction showed that CD59-and CD59low+ cells were monoclonal, whereas CD59+ cells were polyclonal. It was shown that PB cells are predominantly chimeric of clonal (GPI-AP-or GPI-APlow+) and nonclonal (GPI-AP+) cells in PNH, and that degrees of chimerism differ greatly from patient to patient.

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Phase I trial of recombinant fusion protein PIXY321 for mobilization of peripheral-blood cells.

Journal of Clinical Oncology ◽

10.1200/jco.1996.14.9.2521 ◽

1996 ◽

Vol 14 (9) ◽

pp. 2521-2526 ◽

Cited By ~ 19

Author(s):

M R Bishop ◽

J D Jackson ◽

B O'Kane-Murphy ◽

K Schmit-Pokorny ◽

J M Vose ◽

...

Keyword(s):

Bone Marrow ◽

Fusion Protein ◽

Phase I ◽

Malignant Lymphoma ◽

Peripheral Blood ◽

Blood Cells ◽

Phase I Trial ◽

Peripheral Blood Cells ◽

Hematopoietic Recovery ◽

Dose Levels

PURPOSE Mobilization of peripheral-blood cells (PBC) with cytokines alone results in rapid hematopoietic recovery and avoids the potential morbidity associated with mobilization by chemotherapy. PIXY321, a fusion protein that consists of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), has enhanced hematopoietic colony-forming activity as compared with individual or equimolar combinations of the two cytokines. A phase I trial of PIXY321 for mobilization of PBC in patients with malignant lymphoma was performed. PATIENTS AND METHODS Thirteen patients with malignant lymphoma who were eligible for high-dose therapy (HDT) were enrolled onto the trial. All patients were ineligible for autologous bone marrow transplantation due to overt metastatic disease in the marrow or to severe marrow hypocellularity. PIXY321 was administered at three dose levels of 250, 500, and 750 micrograms/m2/d by continuous infusion until completion of PBC collections. Collections were initiated when the WBC count was greater than 10 x 10(9)/L or 4 days after the initiation of PIXY321, whichever came first. Collections were continued until a minimum of 6.5 x 10(8) mononuclear cells (MNC)/kg patient weight were obtained. RESULTS PIXY321 was generally well tolerated. Side effects associated with PIXY321 administration did not exceed grade 2 and included fever (85%), chills/sweats (54%), myalgias (38%), fatigue (31%), nausea/vomiting (31%), headache (31%), edema (23%), and rhinorrhea (23%). The median numbers of colony-forming units-granulocyte/macrophage (CFU-GM) in the graft products for the three dose levels were 0.31, 2.94, and 2.88 x 10(4)/kg, respectively; the median numbers of burst-forming units-erythroid (BFU-e) were 0.20, 6.94, and 12.78 x 10(4)/kg, and the median numbers of CD34+ cells were 2.30, 0.74, and 0.39 x 10(6)/kg. Following transplantation, the median times to an absolute neutrophil count (ANC) > 0.5 x 10(9)/L were 12, 15, and 12 days, respectively, and the median times to platelet transfusion independence were 30, 19, and 15 days. CONCLUSION PIXY321 can be safely administered and effectively mobilizes PBC in patients with bone marrow defects. PIXY321-mobilized PBC autotransplants result in rapid and sustained hematopoietic recovery.

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