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1.
Reg Anesth Pain Med ; 43(6): 616-620, 2018 Aug.
Article in English | MEDLINE | ID: mdl-29659439

ABSTRACT

BACKGROUND: The American Society of Regional Anesthesia and Pain Medicine guidelines recommend discontinuation of warfarin and an international normalized ratio (INR) of 1.2 or less before a neuraxial injection. The European and Scandinavian guidelines accept an INR of 1.4 or less. We evaluated INR and levels of clotting factors (CFs) II, VII, IX, and X 5 days after discontinuation of warfarin. METHODS: Patients who discontinued warfarin for 5 days and had an INR of 1.4 or less had activities of factors II, VII, IX, and X measured. The primary outcome was the frequency of subjects with CF activities of less than 40%. RESULTS: Twenty-three patients were studied; 21 (91%) had an INR of 1.2 or less. In these 21 patients, the median (interquartile range) activities of factors II, VII, IX, and X were 66% (52%-80%), 114% (95%-132%), 101% (84%-121%), and 55% (46%-63%), respectively. Ninety-five percent (99% confidence interval, 69%-99%) had CF activities of greater than 40%. The patient who did not CF activities greater than 40% had end-stage renal disease. Two subjects had an INR of greater than 1.2; the activities of factor II, VII, IX, and X were 37% and 46%, 89% and 105%, 66% and 78%, and 20% and 36%, respectively. Neither patient had CF activities of greater than 40%. CONCLUSIONS: Based on 40% activity of CFs, patients with INRs of 1.2 or less can be considered to have adequate CFs to undergo neuraxial injections. The number of patients with an INR of 1.3 and 1.4 is too small to make conclusions.


Subject(s)
Anticoagulants/administration & dosage , Blood Coagulation Factors/metabolism , International Normalized Ratio/trends , Warfarin/administration & dosage , Aged , Aged, 80 and over , Cross-Sectional Studies , Female , Humans , Male , Middle Aged , Time Factors
2.
J Biomol Tech ; 22(3): 90-4, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21966256

ABSTRACT

MALDI-TOF mass spectrometry is used here to differentiate different glycoisoforms of normal and variant hemoglobins (Hbs) in nonenzymatic in vitro glycation. Single, double, and/or multiple glycation of the α-globin, ß-globin, and/or γ-globin is observed. Different glycation rates are observed for various Hbs, and the normal Hb A has the slowest rate. Although the Hb A is relatively stable upon condensation with glucose at 37°C, the variants Hb C, Hb E, Hb F, Hb Leiden, and Hb San Diego are less stable. In addition, data reveal that the number of glucose attached/Hb molecule (state of glycation) increases with longer incubation time, higher glucose concentration, and higher temperature. The pH dependence of the state of glycation is more complex and varies for different Hbs. Although pH has little effect on the state of glycation for Hb C, Hb E, and Hb Leiden, it increases for Hb A and Hb F upon changing the pH of the solution from phosphate buffer saline (pH 7.4) to carbonate buffer (pH 10). Results obtained in this study could lead to the inference that the linkage of Hbs with glucose occurs in diabetic conditions in vivo (37°C, ∼neutral pH, ∼0.007 M glucose), and the state of glycation is more severe in the individuals who carry abnormal Hbs.


Subject(s)
Hemoglobins/chemistry , Diabetes Mellitus/blood , Glucose/chemistry , Glycosylation , Hemoglobins/isolation & purification , Humans , Hydrogen-Ion Concentration , Molecular Weight , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
3.
Hemoglobin ; 34(2): 145-50, 2010 Jan.
Article in English | MEDLINE | ID: mdl-20353349

ABSTRACT

Under culture conditions that promote hematopoietic differentiation, human embryonic stem cells (huESC) give rise to primitive erythroid cells that closely resemble the nucleated erythrocytes of early-stage human embryos. The globin chain distribution of these cells is similar to that seen during the embryonic and fetal stages of development. Here we show that huESC-derived erythroid cells produce substantial quantities of homotetrameric hemoglobin (Hb) composed exclusively of gamma-globin-containing subunits. The globin synthesis of these erythroid cells was also significantly unbalanced, with a substantial decrease of alpha-like globin chain synthesis in relation to that of their beta-like globins, a pattern characteristically associated with alpha-thalassemia (alpha-thal). This pattern of unbalanced globin synthesis appears to be an inherent feature of human erythroid cells that synthesize predominantly embryonic-stage globins.


Subject(s)
Embryonic Stem Cells/cytology , Erythroblasts/metabolism , Erythropoiesis/genetics , Gene Expression Regulation, Developmental , alpha-Globins/biosynthesis , alpha-Thalassemia/genetics , beta-Globins/biosynthesis , gamma-Globins/biosynthesis , Cells, Cultured/cytology , Cells, Cultured/metabolism , Hemoglobins, Abnormal/biosynthesis , Hemoglobins, Abnormal/genetics , Humans , alpha-Globins/genetics , beta-Globins/genetics , gamma-Globins/genetics , zeta-Globins/biosynthesis , zeta-Globins/genetics
4.
Blood ; 112(12): 4475-84, 2008 Dec 01.
Article in English | MEDLINE | ID: mdl-18713948

ABSTRACT

Human erythropoiesis is a complex multistep process that involves the differentiation of early erythroid progenitors to mature erythrocytes. Here we show that it is feasible to differentiate and mature human embryonic stem cells (hESCs) into functional oxygen-carrying erythrocytes on a large scale (10(10)-10(11) cells/6-well plate hESCs). We also show for the first time that the oxygen equilibrium curves of the hESC-derived cells are comparable with normal red blood cells and respond to changes in pH and 2,3-diphosphoglyerate. Although these cells mainly expressed fetal and embryonic globins, they also possessed the capacity to express the adult beta-globin chain on further maturation in vitro. Polymerase chain reaction and globin chain specific immunofluorescent analysis showed that the cells increased expression of beta-globin (from 0% to > 16%) after in vitro culture. Importantly, the cells underwent multiple maturation events, including a progressive decrease in size, increase in glycophorin A expression, and chromatin and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to more than 60% of the cells generating red blood cells with a diameter of approximately 6 to 8 mum. The results show that it is feasible to differentiate and mature hESCs into functional oxygen-carrying erythrocytes on a large scale.


Subject(s)
Cell Nucleus/physiology , Embryonic Stem Cells/physiology , Erythrocytes/physiology , Animals , Cell Differentiation/physiology , Cell Fractionation , Cells, Cultured , Embryonic Stem Cells/cytology , Erythrocytes/cytology , Erythrocytes/metabolism , Erythroid Cells/cytology , Erythroid Cells/metabolism , Flow Cytometry , Humans , Mice , Rh-Hr Blood-Group System/metabolism , Tissue Engineering/methods
5.
Blood ; 103(11): 4134-41, 2004 Jun 01.
Article in English | MEDLINE | ID: mdl-14962900

ABSTRACT

Gene expression patterns of CD34(+)CD38(-) cells derived from human embryonic stem cells (ESCs) were compared with those of cells isolated from adult human bone marrow (BM) using microarrays; 1692 and 1494 genes were expressed at levels at least 3-fold above background in cells from BM and ESCs, respectively. Of these, 494 showed similar levels of expression in cells from both sources, 791 genes were overexpressed in cells from BM (BM versus ESCs, at least 2-fold), and 803 genes were preferentially expressed in cells from ESCs (ESCs versus BM, at least 2-fold). The message of the flt-3 gene was markedly decreased in cells from ESCs, whereas there was substantial flt-3 expression in cells from BM. High levels of embryonic epsilon-globin expression were observed-but no adult beta-globin message-in CD34(+)CD38(-) cells from ESCs, whereas high levels of beta-globin expression-but no embryonic epsilon-globin message-could be detected in cells from BM. Furthermore, high levels of major histocompatibility complex (MHC) gene expression were demonstrated in cells from BM but very low levels of MHC message in corresponding cells from ESCs. These observations demonstrate that CD34(+)CD38(-) cells derived from ESCs correspond consistently to an early developmental stage at which the yolk sac and fetal liver are the primary sites of hematopoiesis.


Subject(s)
ADP-ribosyl Cyclase/metabolism , Antigens, CD34/metabolism , Antigens, CD/metabolism , Gene Expression Regulation, Developmental , Hematopoietic Stem Cells/physiology , Oligonucleotide Array Sequence Analysis , ADP-ribosyl Cyclase 1 , Cells, Cultured , Cytokines/genetics , Hematopoietic Stem Cells/cytology , Hemoglobins/genetics , Histocompatibility Antigens/genetics , Humans , Membrane Glycoproteins , Mitogen-Activated Protein Kinases/genetics
7.
Stem Cells ; 20(5): 428-37, 2002.
Article in English | MEDLINE | ID: mdl-12351813

ABSTRACT

Rhesus monkey embryonic stem (ES) cells undergo differentiation in vitro to generate hematopoietic progenitor cells. Our previous studies demonstrated a high degree of similarity in the expression of genes associated with hematopoietic differentiation, homing, and engraftment in CD34(+) and CD34(+)CD38(-) cells from rhesus monkey ES cells and from fresh or cultured bone marrow (BM). In the present study, we compared the expression patterns of cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors (CDIs) in these cells. The expression of genes for cyclins, CDKs, and CDIs was similar among the hematopoietic progenitor cells of different origins, with only minor differences. Differentially expressed genes were also analyzed in CD34(+) lineage-negative cells derived from mouse ES cells and from BM. No difference or totally divergent results were obtained with the latter system, suggesting that this variation may be species specific. We observed, however, that CD34(+) and CD34(+)CD38(-) cells derived from ES cells expressed embryonic epsilon and zeta as well as alpha, beta, and gamma globin genes, whereas no expression of embryonic globins could be detected in the cell preparations from BM. Moreover, erythroblast-enriched CD34(-) cells derived from 4- or 5-week ES cell differentiation cultures also expressed embryonic, fetal, and adult globin genes, with greater beta gene expression, but otherwise were identical to those of the more primitive CD34(+) cells derived from 2-week ES cultures. These latter observations may reflect the presence of heterogeneous cell populations within the cell fractions that were compared, or they may represent variability among ES-cell-derived hematopoietic stem cells.


Subject(s)
Cell Cycle Proteins/genetics , Cell Differentiation/genetics , Embryo, Mammalian/immunology , Gene Expression Regulation, Developmental/genetics , Hematopoietic Stem Cells/immunology , Neoplasm Proteins , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Animals , Antigens, CD34/genetics , Antigens, CD34/immunology , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p18 , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinase Inhibitor p27 , Cyclin-Dependent Kinases/genetics , Cyclins/genetics , Embryo, Mammalian/cytology , Enzyme Inhibitors , Globins/genetics , HSP40 Heat-Shock Proteins , Hematopoietic Stem Cells/cytology , Homeodomain Proteins/genetics , Humans , Macaca mulatta , Mice , Repressor Proteins/genetics , Transcription Factors/genetics , Tumor Suppressor Proteins/genetics
8.
Exp Hematol ; 30(1): 58-66, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11823038

ABSTRACT

OBJECTIVE: The aim of this study was to characterize at the molecular level the hematopoietic progenitor cells derived from rhesus monkey embryonic stem (ES) cell differentiation. MATERIALS AND METHODS: We purified CD34(+) and CD34(+)CD38(-) cells from rhesus monkey ES cell cultures and examined the expression of a variety of genes associated with hematopoietic development, by semiquantitative polymerase chain reaction analysis. For comparison, we examined cell preparations from fresh or cultured rhesus monkey bone marrow (BM) and from mouse ES cells and BM. RESULTS: We observed a high degree of similarity in the expression patterns of these genes, with only a few exceptions. Most notably, the message of the flt3 gene was undetectable in rhesus monkey ES cell-derived CD34(+) and CD34(+)CD38(-) cells, whereas substantial flt3 expression was observed in the corresponding cells from fresh BM and in CD34(+) cells from cultured BM. The integrin alphaL and interleukin-6 (IL-6) receptor genes also were expressed in CD34(+)CD38(-) cells from BM, but there was little or no expression of these genes in CD34(+)CD38(-) cells derived from ES cells. Parallel analyses, using CD34(+)Lin(-) cells derived from murine ES cell cultures, showed no apparent expression of flt3, integrin alphaL, or IL-6 receptor, whereas corresponding cell preparations isolated from mouse BM expressed high levels of all of these genes. CONCLUSIONS: ES cell-derived hematopoietic progenitors, both from the rhesus monkey and from the mouse, exhibited the same alterations in gene expression compared with BM-derived cells from these animals. These observations could reflect the presence of different subpopulations in the cell fractions that were compared, or they may represent altered biologic properties of ES cell-derived hematopoietic stem cells.


Subject(s)
Antigens, CD , Cell Differentiation/genetics , Hematopoietic Stem Cells/physiology , Stem Cells/physiology , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , Animals , Antigens, CD34 , Antigens, Differentiation , Cell Lineage/genetics , Gene Expression Profiling , Gene Expression Regulation , Macaca mulatta , NAD+ Nucleosidase , Polymerase Chain Reaction
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