text:
A method for enriching rare cell populations from a whole blood sample by separating rare cell fractions from whole blood sample according to the relative charge density and/or the relative binding affinity for a leukocyte depletion solid phase matrix is described. The enrichment method may be operated stand alone, or as a pre or post-processing step in conjunction with a charge-flow separation method.
1. A method for enriching a whole blood sample for a desired cell population selected from the group consisting of nucleated cells, erythroid progenitor cells and fetal nucleated erythrocytes within the whole blood sample, comprising the steps of:
a) density gradient centrifugation of the whole blood sample in the presence of a Ficoll gradient having a density of at least about 1.119 g/ml at less than or equal to about 500 g;
b) recovering a desired fraction from the density gradient centrifigation and passing the desired fraction containing the desired cell population through a counterflow stabilized charge-flow separator apparatus, in which the mobility of each cellular component in the blood sample in the presence of an applied electrical field is opposed by a buffer counterflow, thereby concentrating the desired fraction;
c) retrieving at least one fraction eluting from the charge-flow separator apparatus having a concentrated desired fraction from step (b);
d) depleting unwanted cells from the concentrated desired fraction; and
e) recovering an enriched desired cell population for analysis.
2. The method of claim 1, wherein said step of depleting unwanted cells further comprises the step of ammonium lysis under conditions which lyse a substantial quantity of enucleate erythrocytes and without lysing substantial quantities of the desired cell population.
3. The method of claim 2, wherein said step of depleting unwanted cells further comprises the step of complement lysis after the step of ammonium lysis under conditions which lyse a substantial quantity of lymphoid and myeloid cells and without lysing substantial quantities of the desired cell population.
4. The method of claim 1 further comprising the step of obtaining a separated cell population of nucleated blood cells from the charge-flow separator apparatus and passing the separated cell population sample containing nucleated blood cells through a leukocyte depleting affinity matrix.
5. The method of claim 1 further comprising the step of obtaining a separated cell population of nucleated blood cells from the charge-flow separator apparatus and passing the separated cell population sample containing nucleated blood cells through a leukocyte depleting adsorption-filtration matrix.
6. A method for recovering a desired cell population selected from the group consisting of nucleated cells, erythroid progenitor cells and fetal nucleated erythrocytes from a whole blood sample, comprising the steps of:
a) depleting a major fraction of unwanted blood cells from the whole blood sample, thereby concentrating the desired cell population;
b) passing the concentrated desired cell population through a counterflow stabilized charge-flow separator apparatus, in which the mobility of each cellular component in the blood sample in the presence of an applied electrical field is opposed by a buffer counterflow, thereby causing the desired cell population to concentrate from the other cellular blood components;
c) retrieving at least one fraction from the charge-flow separator apparatus having a concentrated desired cell population;
d) recovering an enriched desired cell population for analysis.
7. The method of claim 6, wherein said step of depleting unwanted blood cells further comprises the step of ammonium lysis under conditions which lyse a substantial quantity of enucleate erythrocytes and without lysing substantial quantities of the desired cell population.
8. The method of claim 7, wherein said step of depleting unwanted blood cells further comprises the step of complement lysis after the step of ammonium lysis under conditions which lyse a substantial quantity of lymphoid and myeloid cells and without lysing substantial quantities of the desired cell population.
9. The method of claim 7, wherein said step of depleting unwanted blood cells further comprises the step of adsorbing the desired cell population onto an solid adsorption matrix having a greater binding affinity for the desired cell population than for the unwanted cell populations in the whole blood sample.
10. The method of claim 7, wherein said step of depleting unwanted blood cells further comprises the step of adsorbing the unwanted cell populations onto a solid-phase adsorption matrix having a greater binding affinity for the unwanted cell populations in the whole blood sample than for the desired cell population.
This application is a continuation of application Ser. No. 08/401,131 filed Mar. 8, 1995 which application is now issued as U.S. Pat. No. 5,676,849, which is a continuation-in-part of application Ser. No. 08/327,483 filed Oct. 21, 1994, issued as U.S. Pat. No. 5,662,813, which is commonly assigned.
The present invention relates generally to separation of rare cell populations from whole blood samples. More particularly, the present invention provides a system and non-invasive method for enriching the populations of rare cells, such as nucleated fetal erythrocytes or nucleated fetal red blood cells ("NRBCs") obtained from maternal blood samples by separating the NRBCs from the mother's erythrocytes, leukocytes and other blood components. Those skilled in the art will appreciate that the present system and method are not confined, in their utility, to enrichment of fetal NRBCs. Rather, a wide variety of rare cell populations present in the peripheral blood circulation may be separated according to the present method, as will be explained more fully herein.
In accordance with a preferred embodiment thereof, the present invention offers a system and method for enriching a population of fetal-origin NRBCs from a maternal blood sample by a negative selection process which concentrates the NRBCs by lysis and yields viable rare cell populations which may be cultured or otherwise analyzed. While enrichment of viable fetal-origin NRBCs is described herein as an operative model of the present system and method, the present invention is not so limited in its utility.
Physicians have long sought to develop non-invasive methods for prenatal diagnosis because the available methods, amniocentesis and chorionic villus sampling (CVS) are potentially harmful to the mother and to the fetus. The rate of miscarriage for pregnant women undergoing amniocentesis is increased by 0.5-1%, and that figure is be slightly higher for CVS. Because of the inherent risks posed by amniocentesis and CVS, these procedures are offered primarily to older women, i.e., those over 35 years of age, who have a statistically greater probability of bearing children with congenital defects.
Some non-invasive methods have already been developed to diagnose specific congenital defects. For example, maternal serum alpha-fetoprotein, and levels of unconjugated estriol and human chorionic gonadotropin can be used to identify a proportion of fetuses with Downs syndrome, however, it is not one hundred percent accurate. Similarly, ultrasonography is used to determine congenital defects involving neural tube defects and limb abnormalities, but is useful only after fifteen weeks gestation.
The presence of fetal cells in the maternal peripheral circulation has been known for some time. However, fetal cells represent a rare cell population in maternal circulation present in estimated numbers ranging from 1 in 100,000 to 1 in 1,000,000. Fetal lymphocytes (Walknowska, et al., Lancet 1:1119 (1969), Schroder, et al., Blood 39:153 (1972; Schroder, Scand J. Immunol. 4:279 (1975)), syncytiotrophoblasts (Douglas et al., Am. J. Obstet. Gynecol. 78:960 (1959); Goodfells, et al., Brit. J. Obstet. Gynecol. 89:65 (1982); Covone, et al., Lancet 2:841 (1984); Kozma, et al., Hum. Reprod. 1:335 (1986)), cytotrophoblasts (Mueller, et al., Lancet 336:197 (1990)), and erythrocytes (Freese, et al., Obstet. Gynecol., 22:527 (1963); Clayton, et al., Obstet. Gynecol. 23:915 (1964); Schroder, J. Med. Genet. 12:230 (1975; Medaris, et al., Am. J. Obstet. Gynecol. 148:290 (1984)) have been identified in the maternal peripheral blood circulation.
Separation of nucleated fetal erythrocytes from maternal blood has been identified as a desirable method for facilitating prenatal diagnosis of genetic disorders. Fetal NRBCs have been separated from maternal blood by flow cytometry using a lysing reagent (European Published Patent Application No. 582736, published Feb. 16, 1994); by triple gradient discontinuous gradient gel electrophoresis (Bhat, et al, U.S. Pat. No. 5,275,933, issued Jan. 4, 1994); by separation from nucleated cells using leukocyte depletion and ammonium chloride lysis of enucleated erythrocytes (Goldbard, PCT Publication WO 9417209, published Aug. 4, 1994); by use of anti-CD71 monoclonal antibody and magnetic beads and in-situ fluorescence hybridization (FISH) (Ahlert, et al, German Published Patent Application No. 4222573, published Aug. 12, 1993) or by other antibodies specific to a fetal erythrocyte antigen (Bianchi, PCT Publication WO 9107660, published May 30, 1991). Unfortunately, to date, there are no clinically acceptable methods for prenatal genetic diagnosis using maternal peripheral blood samples. Amniocentesis and CVS continue to be the only options available to a small percentage of pregnant women. These women represent the group statistically at risk of conceiving a fetus having genetic abnormalities.
Substantial attention has been given to methods of prenatal sex diagnosis using fetal cells in the maternal peripheral blood. Because obtaining a maternal blood sample is far less invasive than amniocentesis, CVS or fetal blood sampling, methods which are capable of enriching fetal cells from maternal peripheral blood samples have become a paramount focus of neonatology. Many studies have been made on prenatal sex diagnosis by means of the polymerase chain reaction (PCR) amplifying the Y-chromosome-specific DNA sequence in maternal blood. See, e.g., Bianchi, D. W., et al., Proc. Natl. Acad. Sci. USA, 87:3279-3283 (1990), Bianchi, D. W., et al. Prenat. Diagn., 13:293-300 (1993), Wachtel, S., et al., Hum. Reprod., 6:1466-1469 (1991), Suzemori, K., et al., Obstet. Gynecol., 80:150-154 (1992). In some of the studies using PCR amplification, fetal cells in the maternal blood were enriched and analyzed by PCR. While the sensitivities and specificities of the methods reported in the PCR studies is high, e.g., 64-100 and 80-100 percent, respectively, and the positive and negative predictive values were 75-100 and 67-100 percent, respectively, the diagnostic accuracy reported in each study was insufficient to be acceptable for routine clinical use. Hamada, H., et al., Prenat. Diag., 15:78-81 (1995).
Flow cytometry has also been used to separate fetal NRBCs on the basis of positive CD71 antibody (transferrin receptor) and glycophorin-A antibody binding. PCR conducted on the flow-sorted cells identified male fetuses at a rate of 100% accuracy and female fetuses at a rate of 83% accuracy. Simpson, J. L., et al., J. Am. Med. Ass'n. 270:2357-2361 (1993). Immunogenetic procedures have been combined with fluorescence-activated cell sorting (FACS) procedures to enrich fetal cells in whole blood from pregnant women. Herzenberg, L. A., et al., Proc. Natl. Acad. Sci. USA, 76:1453-1455 (1979) employed rabbit anti-HLA-A2 antibodies in conjunction with fluorescein-conjugated goat anti-rabbit immunoglobulins and analyzed the fluorescein-bound cells using FACS.
Fluorescence in situ hybridization (FISH) offers a method for identifying desired DNA in a cell at the interphase stage. FISH with conventional cytogenetic methods has been used on fetal cells recovered from maternal blood samples for sex determination, Wessman, M., et al., Prenat. Diag., 12:993-1000 (1992), and to detect chromosomal abnormalities, Simpson, J. L., et al., Prenat. Diag., 12:S12 (Supp. 1992) fetal trisomy 21!, Bianchi, D, et al, Prenatal Diagnosis through the Analysis of Fetal Cells in the Maternal Circulation, Genetic Disorders and the Fetus 3d Ed., Milunsky, A., ed., pp. 759-770 (1992) Fetal trisomy 18!.
Fetal progenitor cells have also been enriched from maternal blood by ligand binding onto an immobilization medium. CellPro, Inc., International Publication No. WO 94/25873, published Nov. 10, 1994, discloses an immunoselection method for enriching fetal erythroid progenitor cells from maternal blood by separating a large fraction of maternal erythrocytes from the blood sample, such as by density gradient centrifugation on a Ficoll gradient or by preferential lysis of maternal erythrocytes in the presence of ammonium chloride, potassium chloride and a carbonic anhydrase inhibitor, then incubating a sample of maternal blood with a ligand to bind fetal progenitor cells and then removing unbound blood products, leaving the enriched, ligand-bound fetal erythroid progenitor cells. The ligands disclosed include antibodies, erythropoietin or transferrin. The ligand is immobilized on any of a variety of solid supports, such as hollow fibers, magnetic beads, plates, dishes, flasks, meshes, screens, solid fibers, membranes or dipsticks.
The CellPro International Publication, discloses, for example, the use of first member-second member binding pairs, including biotin-avidin, biotin-streptavidin, biocytin-avidin, biocytin-streptavidin, methotrexate-dihydrofolate reductase, 5-fluorouracil-thymidylate synthetase and riboflavin-riboflavin binding protein, wherein the first binding member is linked to a ligand capable of binding fetal nucleated erythroid cells, the second binding member is linked to an immobilization medium, and the second member has a binding affinity constant for the second member of greater than about 108 M-. The preferred embodiments disclosed include methods for positive and negative selection of fetal cells, useful independently or in conjunction with one another. The positive selection process using a binding pair consisting of biotinylated anti-CD34 antibody and immobilized avidin. The CD34 antigen is expressed on fetal progenitor cells and hematopoietic progenitor cells, however, in normal adults, the hematopoietic progenitor cells reside in the bone marrow and CD34 positive cells are found in the peripheral circulation at a rate of less than 0.1%. The negative selection process using a binding pair consisting of biotinylated anti-CD45 and immobilized avidin. The CD45 antigen is expressed on maternal erythrocytes, but not fetal progenitor cells. The immunoselected fetal cells then may be subjected to analysis by karyotyping, PCR, RFLP, SSCP or FISH to provide genetic or biochemical information. The CellPro method, however, suffers from a clinically unacceptable yield of enriched cells. While this published application states enrichment of fetal progenitor cells to a concentration greater than 1%, the highest level of enrichment supported by the examples was attained by a dual positive selection process using sequential CD34 antibody binding steps yielding a sample containing about 1 in 2,000 or 0.5% fetal cells, representing about a 500-fold enrichment from the starting sample.
To date, however, no clinically acceptable method for enrichment of rare cell populations, particularly fetal nucleated erythrocytes, from peripheral blood samples has been devised which yields cell populations sufficient to permit clinical diagnosis. The clinical need for a method capable of producing higher yields of rare fetal cell populations separated from maternal whole blood was recently underscored by Hamada, H., et al., Prenat. Diag. 15:78-81 (1995) who stated that "The data obtained in this study suggest that fetal sex determination using maternal peripheral blood with FISH is possible and that this diagnostic method will be clinically useful when more cells are analyzed."
The present invention provides a system and method for separation of viable, culturable fetal nucleated red blood cells from maternal whole blood constituents at clinically useful levels. The enriched fetal NRBCs are present in numbers sufficiently large for effective cell culture and subsequent genetic diagnosis with conventional and cytogenetic methods, as well as emerging DNA methods such as PCR, RFLP, SSCP, FISH chromosome banding and karyotyping.
The present invention achieves its objectives by a)density gradient centrifugation of whole blood to provide a gross separation of a major fraction of maternal erythrocytes and blood plasma from whole blood, b) charged-flow separation of the gross separated whole blood to separate fetal erythroid progenitor cells from maternal myeloid and lymphoid cells, c) ammonium lysis of remaining maternal erythrocytes and d) complement mediated lysis to remove myeloid and lymphoid cells present in the charged flow separated fractions. The inventive system and method achieves enrichment of rare cell populations, fetal erythroid progenitor cells, specifically, CFU-GEMM (colony forming unit, granulocyte, erythroid, megakaryocyte, monocyte), BFU-E (burst-forming unit, erythroid), CFU-E (colony-forming unit, erythroid), pronormoblasts, normoblasts (erythoblasts), and reticulocytes by negative selection for the desired cell population.
The charged-flow separation (CFS) apparatus described in the co-pending application Ser. No.: 08/327,483 filed Oct. 21, 1994 (hereinafter "Twitty, et al."), issued as U.S. Pat. No. 5,662,813 which is hereby incorporated by reference, or as described in our U.S. Pat. Nos. 5,336,387 and 5,173,164, which are also hereby incorporated by reference, is ideally suited for separation rare cell populations from whole blood. The CFS apparatus is preferably run under sterile conditions to yield culturable separated cells. Under charge-flow separation conditions, as described in the Twitty, et al application, or as described in U.S. Pat. Nos. 5,336,387 or 5,173,164, incorporated by reference, fetal erythroid progenitor cells exhibit consistent migration patterns in an electric field according to surface charge density which are different and distinct from the migration patterns of mature enucleated erythrocytes. By using the CFS apparatus coupled with the pre- and post-processing methods described more fully hereinafter, the present invention has achieved enrichment of up to 12 percent of rare cell populations in the final sample.
The CFS system and method has been successfully used to recover nucleated red blood cells from the peripheral blood circulation of pregnant women. The recovered NRBCs were identified histologically. The NRBCs exhibited consistent migration patterns whether they came from maternal blood or from umbilical cord blood collected at birth. No NRBCs were found in blood from nulliparous women.
Because the inventive system and method of charge-flow separation of NRBCs from maternal blood is based on the intrinsic physical properties of the NRBCs, there is little need for extensive preparation of the maternal blood sample with antibodies or ligands. The cells may be processed at greater than or equal to 60,000 cells per second and specialized training is not required. When the inventive charge-flow separation system and method is used, the recovered cells are viable, thus raising the possibility of further enrichment by cell culture.
In conjunction with or in addition to the charge-flow separation system and method, the present invention also includes an affinity separation method for separating NRBCs from other cell populations in a maternal blood sample. The adsorption-filtration affinity method of the present invention entails layering a maternal blood sample onto a fibrous adsorption-filtration filter medium having a nominal pore size of about 8 microns and being capable of 40-80% leukocyte immobilization, with a 70-80% post-wash leukocyte retention rate, and which is extremely hydrophilic, being capable of wetting with solutions having surface tensions of up to 85-90 dynes, which has a hold up volume of 40-70 μl/cm2 for a single layer of adsorption-filtration filter medium and which is characterized by low to medium protein binding. The preferred adsorption-filtration separation filter medium is that sold by Pall Corporation under the trademarks "LEUKOSORB" TYPES A and B or that described in U.S. Pat. Nos. 4,923,620, 4,925,572, or European Patent No. 313348, each of which is hereby incorporated by reference.
The charge-flow separation system and method and the adsorption-filtration separation system and method of the present invention may be used separately or may be used in conjunction with one another to achieve enrichment of the nucleated fetal red blood cell population in a maternal blood sample.
FIG. 1 is a flow diagram of representing hemopoiesis and differentiation between erythroid, myeloid and lymphoid cell lines.
FIG. 2 is a flow diagram of the inventive enrichment process for separating rare cell populations from whole blood.
FIG. 3 is a graph representing fetal hematopoietic development and sites during gestation.
FIG. 4. is a karyotype image of a 51 year old male obtained from cultured erythroid progenitor cells isolated from a whole blood sample using the method of the present invention.
FIG. 5 is a flow diagram illustrating a first embodiment of the adjunct adsorption-filtration supports in accordance with of the present invention.
FIG. 6 is a flow diagram illustrating a second embodiment of the adjunct adsorption-filtration supports in accordance with of the present invention.
FIG. 7 is a flow diagram illustrating a third embodiment of the adjunct adsorption-filtration supports in accordance with of the present invention.
FIG. 8 is a flow diagram illustrating a fourth embodiment of the adsorption-filtration supports used as an adjunct to the enrichment method of the present invention.
FIG. 1 is a diagram depicting the hematopoietic system and progenitor hierarchy starting from a common pool of pluripotent hematopoietic stem cells, through commitment to erythroid, myeloid and lymphoid cell lines, to unique morphology cell lines. As those skilled in the art will understand, the process of mature blood cell production from stem cells is a complex series of irreversible cellular differentiation and amplifying proliferation events. The hematopoietic system provides the backdrop against which the system and method for separating rare cell populations from whole blood may be viewed.
It is well known in the art that as stem cells differentiate during hematopoiesis, the maturing cells undergo morphological changes. Concomitant with these morphological changes, the cells develop different sets of surface molecules, i.e., proteins, polysaccharides, etc., such as those exemplified by the cluster differentiation (CD) series to classify the surface molecules. Antibodies specific for different surface molecules, such as the CD series, may then be used to identify the presence of specific cell populations in a blood sample. It has been found, however, that various erythroid, myeloid and lymphoid cells share some common CD surface molecules, as do individual cell types within each development family, e.g., erythropoietic cells. These shared CD surface molecules make selection processes cumbersome. A good summary of principal features of known CD molecules present in hematopoietic cell lines is found in Hoffbrand, A. V., et al., Essential Haematology, 3d ed., Blackwell Scientific Publications, pp. 417-418, (1993), hereby specifically incorporated by reference thereto.
In accordance with a first preferred embodiment of the present invention, and with particular reference to FIG. 2, there is provided a method for enriching rare cell populations from whole blood. It is axiomatic that all healthy human blood samples, whether male, female, child or adult, young or aged, irrespective of blood type and Rh factor, is constituted of a full range of blood cell types along the hematopoietic hierarchy depicted in FIG. 1. When a person is afflicted with a particular cellular disorder, such as breast cancer, prostate cancer, lung cancer, leukemia, or the like, abnormal cells representative of the disorder are usually found, in varying degrees, as rare populations in the peripheral blood circulation. similarly, it is well known that fetal-origin cells may be found at very low levels, i.e., 1�10-6 to 1�10-7, in the maternal peripheral blood circulation. By using alternating negative selection steps (defined herein as selecting the undesired cell populations by any means, including antibody or ligand binding, filtration, or manual selection) and positive selection steps (defined herein as selecting the desired cell populations, including antibody or ligand binding, filtration, or manual selection), the system and method of the present invention isolates rare cells from peripheral blood, by enriching the desired rare cell population from whole blood.
As depicted in FIG. 2, a peripheral blood sample 12 is obtained from a subject 10, by any conventional technique known in the art, such as by venipuncture, usually of the antecubital vein. The volume of blood collected typically ranges between 20-40 ml, but may be more or less. The blood is collected in vacuum containers, typically in the presence of one or more anticoagulants, as desired, including acid-citrate-dextrose (ACD), ethylenediaminetetraacetic acid (EDTA), heparin, and citrate-phosphate-dextrose-adenine (CPDA). The collected blood sample may be stored for up to 4 days at 4� C. It is preferable, however, to process and analyze the blood sample within 24 hours of collection. It has been found that after 24 hours, the blood sample will undergo changes, such as maturation of some hematopoietic cells present in the blood sample.
The whole blood sample is layered onto Ficoll/Hypaque-1119 (polysucrose type 400/sodium diatrizoate, 1.119 g/ml) (HISTOPAQUE-1119, Sigma Chemical Company) density gradient and centrifuged at about 400�G for about 40 minutes. After centrifugation, plasma forms a top layer, a white band of nucleated cells, including nucleated erythroid cells and lymphocytes, is immediately below the plasma layer, the Ficoll/Hypaque-1119 containing a broad diffuse band containing other nucleated erythroid cells and immature red cells with densities heavier than the white layer but lighter than the mature erythrocyte pellet is immediately below the nucleated cells and the erythrocyte pellet is present at the very bottom of the tube.
The plasma fraction is discarded and the nuclear fraction is removed by decanting or aspiration without disturbing the nuclear fraction. The nucleated cell portion is removed to a separate sterile centrifuge tube, washed in phosphate buffered saline and prepared for loading onto the charged flow separator apparatus 20 and subjected to charged flow separation as more fully disclosed in the Twitty, et al. patent application and U.S. Pat. Nos. 5,336,387 and 5,173,164, incorporated by reference, under conditions to maintain a sterile environment.
It has been found that the mobility of fetal NRBCs in an electric field is intermediate between the fastest enucleated red blood cells and the slowest white blood cells. Thus, for example, where enucleated red blood cells 22 elute in fractions 4-7, and leukocytes elute from fractions 6-8, it has been found that the nucleated fetal red blood cells elute in fractions 5-8 with the peak in fraction 7. Based upon these findings, it has been determined that fetal NRBCs 26 have electrophoretic mobilities more similar to many mononuclear cells of lymphoid origin 28, such as T lymphocytes and B lymphocytes, than to erythrocytes 22 or a majority of granulocytes 29 or other cells of myeloid origin. It is desirable, therefore, to collect not only the peak fraction, but also the two fractions adjacent the peak fraction, e.g., fractions 6 and 8. In this manner, the nuclear fraction eluting from the CFS apparatus is separated from a major fraction of mature erythrocytes, granulocytes and other cells of myeloid origin in the original blood sample.
To test the suitability of CFS-separation as a clinically acceptable separation tool for providing cells for clinical diagnosis, sex determination, fluorescence in situ hybridization (FISH) was performed on the CFS-separated nucleated cell portion. FISH analysis confirmed that the CFS enrichment method, as described above, obtains enrichment levels sufficient to make clinically reliable diagnoses. Ficoll/Hypaque-1119 density gradient centrifugation, followed only by CFS-separation, yielded fetal NRBC frequencies in the range of 1/20 to 1/50, or about 2-5% fetal NRBCs. Table 1, below, summarizes data from blood samples obtained from five pregnant women at 20-30 weeks gestation, together with blood samples from a nulliparous female control and a male control. All samples were processed as described above and analyzed by FISH using a DYZ1/DYZ3 probe specific for the Y chromosome.
TABLE 1______________________________________ Gestation Fetal Nuclei Labeled/ PercentCase (Weeks) Sex Nuclei Counted Labeled______________________________________1 NR Male 15/1000 1.52 20.5 Male 15/1000 1.53 30 Male 46/2500 1.84 20 Male 13/1000 1.35 23 Male 29/1000 2.9Male 988/1000 98.8ControlFemale 3/1000 0.3Control______________________________________
The suitability of the CFS-enriched fetal NRBCs for clinical prenatal genetic diagnosis was determined by processing whole blood samples from one woman taken two days post-partum with a stillborn delivery and two pregnant women who had previously undergone amniocentesis at 16 and 19 weeks, respectively, with known results. Karyotyping of the still-born fetal blood sample confirmed a positive fetal trisomy 18 with karyotype 47,XX,+18, while amniocentesis on second subject indicated a fetal karyotype 47,XX,+21 and third subject indicated a fetal karyotype 47,XY,+21+m, both confirming positive fetal trisomy 21. Blood samples from normal adult males were used as controls. An example of a karyotype from a normal adult male is presented as FIG. 4.
The subjects' blood samples and the controls were processed by Ficoll-1119 gradient centrifugation followed by CFS-separation as described above. Following charged flow separation, the trisomy 18 positive sample and the first male control sample were analyzed by FISH using a chromosome-18-specific probe, and the trisomy 21 positive samples and the second male control sample were analyzed using a chromosome-21-specific probe. The data for the known fetal trisomy 18 sample is presented below in Table 2 and the data for the known fetal trisomy 21 sample is presented below in Table 3.
TABLE 2______________________________________ Percent nuclei containing n signalsSample n = 0 1 2 3 ≧4______________________________________Trisomy 18 3 26 70 2 0Control A 0 5 95 0 0Control B 0 21 79 0 0______________________________________
TABLE 3______________________________________ No. Cells Percent nuclei containing n signalsSample Scored n = 0 1 2 3 ≧4______________________________________Case 1 1000 6 16 69 7 2Case 2Sample 2(a) 1000 11 23 61 5 0.4Sample 2(b) 200 0 1 93 4 2Sample 2(c) 500 26 36 36 2 0Control 500 3 24 73 0.4 0______________________________________
Table 4, below, summarizes the results of genetic detection of CFS-processed fetal cells analyzed by FISH in accordance with the FISH protocol published and distributed by Oncor, Inc. as of the date of this application for use with the Oncor, Inc. FISH kit, which is hereby incorporated by reference.
TABLE 4______________________________________ No. Mean % CellsProbe Samples Sample Type Labeled (SD)______________________________________DYZ1/DYZ3 5 Experimental 1.8 (0.6)(Y Chromosome) 2 Male Control 98.8 (0.8) 2 Female Control 0.3D18Z1 1 Experimental 2.0(Chromosome 18) 2 Male Control 0D21S1219/ 2 Experimental 5.3 (2.4)D21S1220 1 Female Control 0.4(Chromosome 21)______________________________________
Because the relative mobilities of lymphocytes and fetal NRBCs are similar, it was deemed desirable to further enrich the fetal NRBC population obtained by charge flow separation. The charge flow separated sample was then further enriched by negative selection of the remaining erythrocytes and myeloid cell populations.
The enriched nucleated cell fraction eluting from the CFS apparatus typically contains a small fraction of mature erythrocytes, a major fraction of mononuclear lymphoid cells, such as B and T lymphocytes, a minor fraction of fetal NRBCs and a minor fraction of myeloid-lineage cells.
Ammonia hemolysis 30 preferentially eliminates mature erythrocytes from the enriched nucleated CFS fetal cell eluate. The rate controlling factor in ammonium hemolysis is the level of erythrocytic carbonic anhydrase present in the cell. Fetal erythroid cells have carbonic anhydrase levels one-fifth those of adult erythroid cells. As a result of this difference in carbonic anhydrase levels, selective ammonium hemolysis of mature erythroid cells is controlled as a time-rate function, i.e., for any given time period, adult red cells will lyse five times faster than fetal cells.
The enriched nuclear CFS eluate is incubated in the presence of ammonia or ammonia salts, for example ammonia chloride, chloride ions, for example sodium chloride, potassium chloride, magnesium chloride or calcium chloride, and a carbonic anhydrase inhibitor, such as acetazolamide, cyanide, cyanate, monovalent sulfides or sulfonamides, under conditions and for a time sufficient to allow ammonium ion transport within the cells, as described in Jacobs and Stewart, J. Gen. Physiol. 25:539-552 (1942), or Maren and Wiley, Molec. Pharmacol. 6:430-440 (1970), followed by incubation in the presence of ammonia and carbon dioxide, to preferentially lyse adult erythrocytes in the sample.
The lysed erythrocytes are separated by centrifugation and the pellet is resuspended in PBS. The resulting product is now substantially free of maternal erythrocytes, but contains fetal nucleated erythroid cells and maternal myeloid and lymphoid cells. To remove a substantial number of maternal myeloid and lymphoid cells remaining in the sample, compliment lysis 32 of the myeloid and lymphoid cells is undertaken. For example, anti-human CD37 IgM is added to the PBS suspension and allowed to bind to human lymphocytes present in the sample. After binding, rabbit complement is added and the resulting samples are allowed to incubate. Following incubation, incomplete Iscove's medium with only fetal calf serum (FCS) is added to the sample and the sample is centrifuged. The resulting pellets are may be prepared for culture 34, PCR 36, FISH 38 or other analytical method as desired in accordance with analytical protocols well known in the art. It is desirable to resuspend the pellet in Iscove's medium with only FCS and remove and treat aliquots using 3% acetic acid and crystal violet to lyse erythrocytes and stain nuclei. Nucleated cell counts of the aliquots may then be made under light microscopy.
In accordance with a further preferred embodiment of the invention, the resuspended cells are prepared for cell culture by diluting the cells with incomplete Iscove's medium in the presence of FCS so that each 0.1 mL contains 4�105 nucleated cells. 0.1 mL of incomplete Iscove's medium containing 4�105 nucleated cells is then added to 1.0 mL of complete Iscove's medium (containing growth factors and methylcellulose), which is then added to a 35 mm Petri dish and cultured according to standard culture protocols as described in Colony Assays of Hematopoietic Cells Using Methylcellulose Media. An Introductory Technical Manual, Terry Fox Laboratory Media Preparation Service, Vancouver, B.C., Canada, 1992 ed., hereby incorporated by reference.
The plates are scored for colonies at day 10-12 and again at day 18-21. At day 10-12, CFU-E and mature BFU-E colonies are noticeable, and at day 18-21, primitive BFU-E, CFU-GM and CFU-GEMM colonies are present and may be scored. At any desired point in colongy growth, so long as mitotic cells are present, growth is synchronized via 5-fluorouracil block and thymidine release of the 5-fluorouracil block. See, e.g., Fraser, et al., Cancer Genetics, Cytogenetics, 24:1-6 (1987), hereby incorporated by reference for the 5-fluorouracil block procedures. Cellular growth is subsequently arrested in the metaphase poriton of mitosis, using colecemid colcemid, colchicine, or similar substance, so that high mitotic indices may be obtained and karyotypes prepared. Growth synchronization is an optional, but preferred stem and usually yields higher mitotic indices.
40 mL of whole blood was collected from a pregnant female by venipuncture. The blood sample was anticoagulated with CPD (2.55 g d-glucose, 2.63 g sodium citrate, 0.327 g citric acid and 0.222 monobasic sodium phosphate in 100 mL distilled water) and placed in a 50 mL sterile centrifuge tube. A 50-100 ul aliquot was removed to take erythrocyte and lymphocyte counts. HISTOPAQUE-1119 (Ficoll/Hypaque-1119, polysucrose type 400/sodium diatriazote, 1.119 g/ml, Sigma Chemical Company, Catalog Number 1119-1) was warmed to 37� C. in a water bath. The blood may be held at room temperature or in the water bath at 37� C.
20 mL of HISTOPAQUE-1119 was aseptically placed into each of two sterile 50 mL centrifuge tubes and 20 mL of blood is carefully layered on top of the HISTOPAQUE-1119 in each tube. If there is an insufficient blood volume available to yield two 20 mL fractions, sterile phosphate buffered saline may be used to bring the blood volume in each tube to 20 mL. The blood-HISTOPAQUE layered tubes are centrifuged at 430�G in a Sorvall HS-4 swing bucket rotor for 40 minutes at room temperature. After centrifugation, the upper plasma layer in each tube was decanted then discarded. The the white nucleated cell band immediately above the HISTOPAQUE-1119 along with the diffuse erythroid band in the HISTOPAQUE-1119 were removed with a pipette and combined in a separate sterile 50 mL centrifuge tube. The mature erythrocyte pellet was discarded.
Sterile PBS at 37� C. was added to the nuclear fraction pipetted to a separate sterile 50 mL centrifuge tube to a volume of 50 mL, and centrifuged at 1750 RPM for 20 minutes. The supernatant was decanted and the resulting pellet resuspended in 50 mL sterile PBS and centrifuged at 1500 RPM for 20 minutes. The supernatant was decanted and the pellet resuspended in 5 mL of buffer (triethanolamine, 5 mM; glycine, 280 mM; glucose, 22 mM, acetic acid to pH 7.3). The re-suspended cells were processed the a CFS apparatus configured with 12 separation channels, 10 counterflow input channels and 4 counterflow output channels. 5 mL of the cells resuspended in buffer were introduced in a sample input flow, with the sample buffer input having a flow rate of 0.220 ml/min/channel, with a buffer output flow rate of 0.370 ml/min/channel and a counterflow pump rate of 4.0 ml/min, in the presence of an applied electric field of 325 V at 50-55 mA.
After the CFS run, 12 fractions were collected in 50 mL centrifuge tubes. Fractions 4-6 were determined to contain cells and the fractions collected in centrifuge tubes 4-6 were centrifuged at 1750 RPM for 20 minutes, the supernatant was decanted and the resulting pellet was resuspended in 5-10 mL of sterile PBS and recentrifuged at 1500 RPM for 10 minutes.
The supernatant was decanted and 10 mL of ammonium lysate (1 mL 10-3 acetazolamide, 9 mL NaCl and 90 mL of 0.1844 NH4 Cl) was added to each tube and incubated at room temperature for 3 minutes. After incubation, 2 mL of a 3 mM solution of NH4 HCO3 was added to each tube and incubated for an additional 4 minutes at room temperature. The tubes were then centrifuged at 1500 RPM for 10 minutes and the supernatant decanted. The pellets were resuspended in 0.9 mL of sterile PBS and 1 ug of anti-human CD37 IgM (Research Diagnostics) was added to each tube and each tube was agitated until antibody was completely disbursed. After the antibody is dispersed, 100 ul of Low Tox Rabbit Complement for human lymphocytes (Accurate Antibody, Cedar Lane Laboratories, Hornby, Ontario, Canada) was added to each tube and incubated at 37� C. for 30 minutes.
Following incubation with antibody and complement, 12 mL of incomplete Iscove's medium with 2% FCS (Stem Cell Technologies, Vancouver, B.C., Canada) was added to each tube and the tubes centrifuged at 1500 RPM for 10 minutes. The supernatant was removed and the pellets were resuspended in 0.5-5 mL of incomplete Iscove's medium with 2% FCS. Aliquots were removed and fixed with 3% acetic acid and crystal violet to lyse erythrocytes and stain nuclei. Nucleated cell counts were made on each aliquot.
The resuspended cells were then re-diluted with incomplete Iscove's medium with 2% FCS to a concentration of 4�105 nucleated cells per 0.1 mL. 0.1 mL of re-diluted cells was added to 1.0 mL of complete Iscove's medium, containing growth factors and methyl cellulose), and this amount, 1.1 mL, was added to a 35 mm Petri dish and placed into culture in accordance with Colony Assays of Hematopoietic Cells Using Methylcellulose Media. An Introductory Technical Manual, Terry Fox Laboratory Media Preparation Service, Vancouver, B.C., Canada, 1992 ed.
The plates were scored for colonies at day 10 and again at day 18. At day 10, CFU-E and mature BFU-E colonies were present and at day 18, primitive BFU-E, CFU-GM and CFU-GEMM colonies were present.
At day 18 50 ul of colcemid (Gibco, diluted 1/10) were evenly added to the culture dishes using an automatic pipette. The colcemid-added culture dishes were incubated for 45-60 minutes at 37� C. in 5% CO2. 100 ul of hypotonic solution were added to several microfuge tubes. Colonies were pipetted from the culture dishes and individual colonies were mixed in each microfuge tube containing hypotonic solution and incubated for 20 minutes. The contents of each microfuge tube were then placed on a poly-1-lysine coated slide and incubated for 5 minutes.
20 ul of 20% fixative (3:1 methanol/acetic acid) was then added to each slide. The slides where then immersed in two changes of 5 minutes each of full strentgh fixative, followed by drying at 37� C. overnight.
The cultured cells were then analyzed by FISH using the Y-chromosome probes DYZ1/DYZ3 (Oncor, Inc.) in accordance with the FISH protocol published and distributed as of the date of this application by Oncor, Inc. for use with the Oncor, Inc. FISH kit. The results of FISH analysis are detailed in Table 5, below:
TABLE 5______________________________________ Y Metaphase MitoticSlide Colony Slide Sig- (No. Index TotalNo. Type Date nal Cells) (%) Cells______________________________________CS1/P1/T1 Red 1/30/95 0 0 0.00 158CS1/P1/T1 Myeloid 1/30/95 5 1 0.21 476CS2/P1/T1 Mixed 1/30/95 0 3 0.78 383CS2/P2/T1 Red 1/30/95 0 3 2.8 107CS3/P1/T2 Myeloid 1/30/95 2 1 0.26 380CS3/P1/T3 Mixed 1/30/95 0 17 5.25 324CS3/P2/T2 Red 1/30/95 0 4 0.52 644ControlCFS100/F6 Cytospin 11/29/94 495 98.61 502______________________________________
Sister cells obtained from one subject (CS3) to those cultured and analyzed by FISH, as summarized in Table 5, above, were enriched and cultured in accordacne with the above-described protocol. Twelve colonies were harvested on day 18 of culture from the sister cell line. PCR testing for Y-sequences in the CFS-eluates was conducted with SRY2 and SRY3 primers, each consisting of a 258 base pair sequence in SRY conserved motif. PCR was conducted in accordance with Perkin-Elmer's published PCR protocol, hereby incorporated by reference. Lanes 3 and 19 contained a 123 base pair marker. The presence of SRY-positive fetal cells in two colonies, 6 and 8, of the twelve colonies was confirmed by PCR. The presence of two positive colonies in twelve total colonies represents an enrichment level of 1 in 6, or 16.67%. The results of the PCR are summarized in Table 6, below:
TABLE 6______________________________________Lane Sample ID SRY Band Sex Indicated______________________________________2 CS3-2 Tube 1 - F4 CS3-2 Tube 2 - F5 CS3-2 Tube 3 - F6 CS3-2 Tube 4 + M7 CS3-2 Tube 5 - F8 CS3-2 Tube 6 + M9 CS3-2 Tube 7 - F10 CS3-2 Tube 8 - F11 CS3-2 Tube 9 - F12 CS3-2 Tube 10 - F13 CS3-2 Tube 11 - F14 CS3-2 Tube 12 - F15 Male Control M16 Blank - na17 Female Control - F18 NO DNA - na______________________________________
Finally, both the ammonia lysis and complement lysis procedures may be carried out in solution, as described above, or may be carried out using negative selection non-rare cell binding immobilization media, such as LEUKOSORB A and LEUKOSORB B (Pall Corporation). As described more fully in the Twitty, et al. patent application, incorporated herein by reference, LEUKOSORB A and LEUKOSORB B have binding affinities primarily for nucleated cells, but not mature enucleated erythroid cells. Thus, the complement lysing step, describe above, will first be applied to rid the matrix of bound unwanted cells, then the ammonum lysis step will be carried out on an affinity medium having a binding affinity for nucleated cell populations. The wanted cells are then removed from the matrix by increasing the salt concentration or with proteases.
The adjunct use of adsorption-filtration affinity matricies as soild phase supports for ammonium lysis and complement lysis, entails layering a CFS-separated fraction onto a fibrous adsorption-filtration filter medium having a nominal pore size of about 8 microns and being capable of 40-80% leukocyte immobilization, with a 70-80% post-wash leukocyte retention rate, and which is extremely hydrophilic, being capable of wetting with solutions having surface tensions of up to 85-90 dynes, which has a hold up volume of 40-70 μl/cm2 for a single layer of adsorption-filtration filter medium and which is characterized by low to medium protein binding. The preferred adsorption-filtration separation filter medium is that sold by Pall Corporation under the trademarks "LEUKOSORB" TYPES A and B or that described in U.S. Pat. Nos. 4,923,620, 4,925,572, or European Patent No. 313348, each of which is hereby incorporated by reference.
Turning now to FIGS. 5-8, there is disclosed the use of at least one leukocyte-depleting matrix which preferentially captures leukocytes by at least one of adsorption, filtration and affinity as described in U.S. Pat. No. 4,925,572 or PCT Publication No. WO 94/17209, incorporated herein by reference. The preferable leukocyte-depleting matrices are "LEUKOSORB A" and "LEUKOSORB B" both manufactured and sold by Pall Corporation. For purposes of illustration only, the leukocyte-depleting matrices depicted in FIGS. 5-8 are identified by LEUKOSORB A and LEUKOSORB B, but any type of adsorption, filtration or affinity matrix which functions to preferentially pass nucleated red blood cells or white blood cells and red blood cells in a manner as described in FIGS. 6-9 is contemplated as being useful.
FIG. 5 illustrates a first embodiment 100 of the adsorption-filtration process in accordance with the present invention. A whole blood sample containing erythrocytes (RBC), leukocytes (WBC) and nucleated erythrocytes (NRBC) is layered onto a Ficoll gradient 102 as described earlier. Density gradient centrifugation on the Ficoll gradient separates a major fraction containing NRBC and WBC with few RBC components 104. The NRBC/WBC fraction is passed 106, 108 either over a LEUKOSORB A 110 or a LEUKOSORB B 112 adsorption-filtration medium. Passing the NRBC/WBC fraction 104 over the LEUKOSORB A 110 in step 106, passes 116 a fraction which includes a major fraction of NRBC 124 and an accompanying minor fraction of WBC and RBC, while a major fraction of WBC 122 and an accompanying minor fraction of NRBC and RBC 122 is adsorbed 114 on the LEUKOSORB A 110. Alternatively, in step 108, the NRBC/WBC fraction 104 may be passed over a LEUKOSORB B 112 medium, and a fraction of NRBC, WBC and RBC 128 is passed, while a major fraction of NRBC and WBC 126, with an accompanying minor fraction of RBC is adsorbed.
The NRBC fraction 124 from step 106 is then passed through the CFS 60, described above, to further concentrate the desired rare cell population product 140. Further, the adsorbed fraction 130 may be extracted 130 by desorbing with an extraction medium appropriate for the adsorbed cell population to yield the desired cell population product 140.
Alternatively, if step 108 is followed, the fraction 128 passing through the LEUKOSORB 112 is either discarded as waste 132 or is concentrated by CFS 60, as described above. The adsorbed fraction 126 may be desorbed 130 to yield the desired cell population product 140, or concentrated by CFS 60, as described above, to yield the desired cell population product 140.
It is to be noted that in the FIGS. 5-8, major fractions are denoted by larger typeface legends and minor fractions are denoted by smaller typeface legends. Thus, for example, in FIG. 5, the whole blood sample 100 contains a major fraction or erythrocytes (RBC) and minor fractions of leukocytes (WBC) and nucleated erythrocytes (NRBC). After the density gradient centrifugation at step 102, the fractions containing a major fraction of nucleated erythrocytes (NRBC) and leukocytes (WBC) and a minor fraction of erythrocytes (RBC) is recovered. Thus, density gradient centrifugation removes a major fraction of erythrocytes and passes the minor fraction of WBC and NRBC.
FIGS. 6-8, set forth a second embodiment of the adsorption-filtration process 200, a third embodiment of the adsorption-filtration process 300, and a fourth embodiment of the adsorption-filtration process 400, respectively. In each of embodiment 200, 300 and 400, the basic process steps are identical, except that the order of performing the process steps has been altered between the embodiments as illustrated in the Figures. For example, in FIG. 6, the sample is placed onto a LEUKOSORB A medium 110, then passed either to a Ficoll gradient for density gradient centrifugation and separation of the desired cell population, or the fraction passing is placed onto a LEUKOSORB B medium 112, and the fraction passing is subject to density gradient centrifugation 210 to yield the product 240. The adsorbed fraction on the LEUKOSORB B medium 112, is extracted 222 to yield the desired product 240. The fraction adsorbed on the LEUKOSORB A medium 110 is extracted 222, and may be concentrated by CFS 60, to yield the desired cell population 240.
In FIG. 7, the sample is placed onto a LEUKOSORB B medium 112, the fraction passed is subjected to density gradient centrifugation 310 or is waste 312, while the adsorbed fraction 304 is extracted 314 and subjected to CFS 60 to yield the product 340, or the extracted fraction is passed onto LEUKOSORB A medium 100 for further adsorption 318 or filtration 316 to either positively or negatively select the desired product 340.
Finally, in FIG. 8, as illustrated in the Example set forth above, the sample either first subjected to CFS 60, or is subjected to density gradient centrifugation 404 followed by CFS 60. The concentrated sample from CFS 60, is then passed onto either LEUKOSORB A medium 110 and LEUKOSORB B medium 112, each either positively (passing) or negatively (adsorbing) the desired cell population 440.
It has been found that enrichment of rare fetal nucleated red cells from a maternal whole blood sample using Ficoll/Hypaque-1119 followed by charge flow separation yielded rare cell populations at a level between 2-7%. By using the subsequent steps of ammonium lysis followed by complement lysis, the system and method of the present invention further increased rare cell population yield to a level up to about 17%. These levels of enrichment represent substantial and significant advances in recovery of rare cell populations from the peripheral blood circulation and achieve, for the first time, sufficent numbers of separated rare cells to make clinical diagnosis possible and meaningful.
Those skilled in the art will appreciate, from the foregoing, that while the present invention has been described with reference to its preferred embodiments, that the spirit and scope of the present invention is not intended to be limited thereto, but by the claims appended hereto.
Patent Citations
Non-Patent Citations
Referenced by
Legal Events
Original Image


text:
Youth Summit on tap for May 3
Apr. 17, 2014 @ 09:30 AM
The Nu Chi Omega and Rho Tau Omega chapters of Alpha Kappa Alpha Sorority will host this year's Emerging Youth Leaders Community Youth Summit on May 3 from 9 a.m. to noon at Red Bud Church in Henderson.
The theme will be "Empowering Our Youth for Global Leadership." Workshops will be held on leadership development and bullying. There will also be a youth-led panel discussion.
The summit is free and open to students in grades six through eight.
Door prizes and snacks will be provided.
For more information, contact Audrey Davis at (252) 492-3433 or firstname.lastname@example.com or Dorothy Perry at (252) 492-9996 or firstname.lastname@example.com.

subset: commoncrawl


text:
Rookies of the Year: Jose Fernandez and Wil MyersBy MIKE FITZPATRICK
The Associated Press | November 12,2013
Wil Myers Tampa Bay RaysNEW YORK Jose Fernandez of the Miami Marlins and Wil Myers of the Tampa Bay Rays were selected baseballs Rookies of the Year on Monday.
Fernandez stood out in a very deep National League class, and the pitcher received 26 of 30 first-place votes from a Baseball Writers Association of America panel. His debut season was so superb that hes one of three finalists for the NL Cy Young Award, with the winner to be announced Wednesday.
Myers took home the American League prize after putting up impressive offensive numbers in barely half a season. The right fielder was chosen first on 23 of 30 ballots, beating out Detroit shortstop Jose Iglesias and Rays teammate Chris Archer.
Myers became the third Tampa Bay player in six years to be selected Rookie of the Year, joining Jeremy Hellickson (2011) and Evan Longoria (2008).
Its awesome, Myers said in an interview on MLB Network. Its a huge honor to win this. Im very excited about it.
Fernandez easily topped runner-up Yasiel Puig of the Los Angeles Dodgers. The right-hander was the fourth Marlins player in 11 years to win, following Chris Coghlan (2009), Hanley Ramirez (2006) and Dontrelle Willis (2003).
After the Marlins dumped nearly all of their high-priced stars last winter, Fernandez was a rare bright spot in Miamis 62-100 season. He made the All-Star team at age 20 and went 12-6 with a 2.19 ERA and 187 strikeouts in 172 2-3 innings.
The 22-year-old Myers batted .293 with 13 home runs and 53 RBIs in only 88 games after he was called up from the minors June 18. He immediately added much-needed power to the middle of the Rays lineup, helping them reach the playoffs as an AL wild card.
Myers was rated one of baseballs best hitting prospects when he was traded from Kansas City to Tampa Bay last December in a seven-player deal that sent pitchers James Shields and Wade Davis to the Royals.
The Rays were 36-33 before Myers arrived this season and went 56-38 the rest of the way. They won a tiebreaker at Texas for the final AL playoff berth and beat Cleveland in the wild-card game before getting eliminated by eventual World Series champion Boston in the division series.
Myers finished with 131 points in the balloting to 80 for Iglesias, traded from the Boston Red Sox to Detroit Tigers just before the deadline in late July. Archer and fellow pitcher Dan Straily of the Oakland Athletics each had one first-place vote.
The announcements marked the beginning of awards week in baseball. NL and AL Manager of the Year will be revealed today.MORE IN Wire SportsGREEN BAY, Wis. Clay Matthews understood why everyone was so concerned. Full StoryTORONTO David Price was blown away by the reception he got in his Toronto debut. Full Story
- Most Popular
- Most Emailed
- RICHARD'S POOR ALMANACK: On this day 1739, 'Richard Palmer' identified in prison at York Castle as the notorious outlaw DICK TURPIN; IN 1836, Battle of the Alamo begins near San Antonio de Bexar, Texas; 1896, the Tootsie Roll invented by LEO HIRSCHFELD.
- RICHARD'S POOR ALMANACK: On this day in 1472, Orkney, Shetland islands put up as collateral by Norway to Scotland in lieu of dowry for MARGARET OF DENMARK on her marriage with JAMES III, king of Scotland; 1962, JOHN GLENN first American to orbit Earth.
- TOMORROW'S HEADLINES TODAY: City mayoral candidates debate campaign issues; Hartford, Conn., woman still missing; Neal Goswami reports attempts to legislate suicide; local woman loses 100 pounds through TOPS program.
- RICHARD'S POOR ALMANACK: On this day in 1878, JOHN TUNSTALL murdered near Lincoln, New Mexico, by the outlaw JESSE EVANS; in 1930, ELM FARM OLLIE first cow to fly in aircraft, first to be milked airborne; 1955, nuke test WASP; '79, snow in Sahara.
- TOMORROW'S HEADLINES TODAY: Rutland Herald News Editor Alan J. Keays and staff writer Gordon Dritschilo discuss stories planned for the February 18, 2015, edition of the newspaper: Winter budgets maxed, legal marijuana, Springfield bank job, USPS slowdown
- RICHARD'S POOR ALMANACK: On this day in 1249 AD, ANDRE of LONGJUMEAU is dispatched by LOUIS IX of France to meet the KHAGAN, ruler of the Mongol Empire; in 1804, during 1st Barbary War, STEPHEN DECATUR scuttles the pirate-held USS Philadelphia in Tripoli.


text:
Service accommodating and professional
A few Unappreciative guests
proximity to downtown core and other better-appearance hotels
sloping floor in room. washroom needs lots of work.
nice large clean corner room with balcony with beautiful view. Only problem was that the hotel is under construction and we had scaffolding outside our windows.
furniture is outdated
It had a great view of the mountains
It was an ugly building, poorly kept up and rooms were poorly cleaned.
Was very expensive for what you got. The top dining area sounded great, but was dated and smelled like old cheese.
Interlaken was beautiful & was the one redeeming factor about this place.
Room was spacious, clean and location was an easy walk to main part of town. Fantastic authentic Swiss breakfast with lovely coffee.
The room was gigantic!
No coffee/tea in room
Clean, great rooms, nice bar and restaurant, friendly staff, great location near the train station.
No Gym
Location and price. Staff was wonderful, especially the front desk staff.
The free parking was only 6 spots. Plan on paying 20 Francs to park.
Good breakfast, helpful staff, nice room
There wasn't any wow factor that made me feel like it was worthy of 4 stars. The rate seemed high for the complete off season.
The only thing they need is Air Conditioning but that's rare in Europe
It was over 90 but we had brought fans so wasn't terrible
The place made me feel like I was at my home, with the small pantry and balcony. The views are priceless and breathtaking. The staff were superb they smiled all the time and offered their help. The Internet and parking is big thing to me, they were available at all time.
Taking the bags from car to the room was a nightmare. There is no bell captain to help you. Neither do they have at least a trolley to be able to make your trip easier.
Here's why:
We're not the seller
At KAYAK we don't set prices, so it's not possible for us to guarantee what other companies are selling.
We aggregate tons of data for you
Our service lets you quickly and easily compare results from hundreds of travel sites at once. In this sense, KAYAK is a search engine and we don't guarantee prices we find.
Why aren't prices accurate 100% of the time?
Prices on airline seats, hotel rooms and car rentals can change frequently. Several people may also be trying to buy the same travel option simultaneously. As a result, you may find on occasion that certain prices are no longer available.
More questions or if you have a bad price to report to us
Please send us an
and someone from our team will get back to you promptly.
KAYAK is part of The Priceline Group, the world leader in online travel & related services.


text:
Labour can beat the fear factor
by Staff blogger Published
In an ideal world, democratic elections would be characterised by a kind of Socratic dialogue. Rival politicians would put forward cogent arguments, quoting unadorned and carefully sourced facts. Questioners would seek further details and probe weaknesses. Occasionally, somebody would say "you've got a point there" or "I must admit our ideas for improving [say] hospitals didn't quite work". A candidate might even say "I don't know" or "I'll have to look that up". Wise citizens would debate the parties' rival merits in pubs and workplaces, having made notes on the previous night's political broadcasts.
This world has never existed. Most people used to vote according to class interest; as social boundaries have eroded, they increasingly do so according to individual interest - what's in it for me? Now, they no longer expect much of a government. They do not look to either party to provide jobs or houses. They have their own transport and their own pensions. Telephones, gas, electricity, water, TV and radio are all available from a multitude of private suppliers. For all the clamour about standards in schools and hospitals, when faced with a bad service many simply go and find a better one (inside or outside the state sector), as politicians have encouraged them to do. Once the idea is accepted - as it now is by all three main parties - that the consumer is sovereign, even in state-financed services, why should most people bother about the outcome of an election? They do not get to vote for the chief executive of Tesco. As public services are increasingly customised and fragmented, their accountability to us collectively diminishes. Why vote for an MP who may well be told that the affairs of quasi-independent hospitals and schools are no business of his? Smart individuals will in future get the services they want, not by voting but by exercising their consumer rights.
Even self-interest, therefore, is no longer much of a basis for voting. Nothing has the straightforward appeal of a proposal for a free National Health Service, an old-age pension for everyone, or secondary education for all. Arguments about foundation hospitals, means-tested credits or city academies become complex and technical. The voters are, in any case, convinced that if politicians promise something there must be a catch in it somewhere. But as any marketing executive knows, if you can't appeal to greed, you had best appeal to fear: fear of crime; fear of terrorist attack; fear that immigrants will swamp the country; fear of hefty tax rises; fear of the NHS or pensions being abolished.
The appeal to fear is nothing new. The Conservatives tried to stop Labour in 1945 by warning it would introduce a British Gestapo. Fear of nuclear annihilation (or Soviet invasion) won many western elections in the cold war era. And fear is the dominant theme of this election, as one side (usually, but not always, the Tories) tries to create fear and the other either to allay it, or to argue that its own policies will do a better job of holding the monster at bay. Fear will surely grow in importance in future, as politicians become more desperate to hold the voters' attention. And fear, more than self-interest, is the enemy of rational debate and, ultimately, of democracy.
Take immigration. Here there is certainly room for discussion. One side might highlight the possible effects of increased population on, for example, traffic congestion and the demand for greenbelt housing development. The other might stress how young migrants boost economic growth. Yet, as the Optimum Population Trust reveals in a new report, none of the parties even mentions crowding or overpopulation in its manifesto, while, as Richard Reeves points out (page 23), Labour fails to make the link with economic prosperity. The argument is not linked, as it could be, to the environment or the economy, but to law and order: the Tories appeal to fears of potential terrorists entering the country, while Labour, conceding this as a major worry, insists it has the problem under control. It has long been the same story over Europe, so that issues about how to democratise the EU have given way to a sterile debate about a potential Brussels tyranny. Likewise, health issues are dominated by fear of the MRSA bug, with Michael Howard sounding as though he would personally tour the country with a bottle of Dettol. Again, a better debate would concern which kinds of treatment the NHS should continue to provide free of charge. This is bad ground for Labour to fight on, because the Tories have always been better at fear, and their cheerleaders at the Daily Mail are masters of it. To borrow from Franklin D Roosevelt, the party has nothing to fear but fear itself.
Christian dialogue
Following our cover story two weeks ago highlighting the late pope's role in the spread of Aids in Africa, the editor of the Catholic Herald, Luke Coppen, provided an address and phone number so his readers could convey their "views" about "the nastiest invective . . . in decades" directly to the NS editor, Peter Wilby. To maintain the flow of debate, NS readers may wish to comment on John Paul II's newly elected successor, Joseph Ratzinger. Homosexuals - described by the former cardinal Ratzinger as an "intrinsic moral evil" - may have something to say. So may Anglicans ("not a proper church"), devotees of rock music (a "vehicle of anti-religion") and women who aspire to become priests (banned "to protect true doctrine"). Views should in the first instance be addressed directly to Benedict XVI at the Vatican. But if you can't get through, Mr Coppen on 020 7588 3101 would no doubt be happy to pass on your opinions.


text:
This one has to be simple, but I can't find the words to Google this.
I have a script attached to an object in Unity. From that script, how do I access the object's name or other properties? I've read this page, but it doesn't seem to address my question.
I've tried Gameobject.name and this.name, but I can't make them work.
asked Jun 21, 2013 at 05:54 AM
Dubious Drewski
Use 'gameObject'...'gameObject.name'...'gameObject.tag'. See the reference page for GameObject for all the properties you can access.
Jun 21, 2013 at 05:58 AM robertbu
Hmm. This is my code:
if(gameObject.name="RightFeeler"){ Debug.Log ("Right feeler collided"); }
And this is the error: Cannot implicitly convert type 'string' to 'bool'
I don't know what that means. Any ideas?
Jun 21, 2013 at 06:14 AM Dubious Drewski
Comparison is '==', so it should be:
Jun 21, 2013 at 06:18 AM robertbu
Incorrect operator in your conditional
== , not = (is equal to, not equals)
what do people have against using spaces, there is no optimization in not using spaces, and makes code so much easier to read
Jun 21, 2013 at 06:20 AM alucardj
You attatch the script to the object then use the inspector tab in unity. You can also edit the name of the object in inspector.
answered Jun 21, 2013 at 06:19 AM
Oh my God, I needed a '==' instead of a single '='. What a rookie mistake. Thank you, everyone. You're always so helpful.
answered Jun 21, 2013 at 06:27 AM
Dubious Drewski
according to your code here, you are accessing the name of the gameObject that you attach this script to, not to another gameObject
this wouldn't really work in a collision situation as you want to find out what your current gameObject collided with, it cannot collide with itself.
you may want to look up triggers too http://docs.unity3d.com/Documentation/ScriptReference/Collider.OnTriggerEnter.html
ive found this a good method for finding WHAT you have collided with.
answered Jun 21, 2013 at 06:31 AM
bubzy: I have a reason for this strange setup.
I am making an insect. I have a body and two child objects acting as a left feeler and a right feeler. The feelers are not physical objects - they simply guide the body away from obstacles when they 'feel' (collide with) something.
So each feeler has a variable for if Collided (true/false), and the body (somehow) pays attention to this and responds to the input.
I don't yet know how to code this, but this is why I'm practicing.
Jun 21, 2013 at 07:07 AM Dubious Drewski


text:
ISPs iiNet, Optus and AAPT are pressuring the competition watchdog to reject fresh revisions to the way Telstra proposes to handle confidential NBN information.
The three access seekers have written to the Australian Competition and Consumer Commission (ACCC) to complain that Telstra's latest NBN information security plan does not provide enough assurance about how NBN data received under the definitive agreements can be shared by Telstra's business units.
The plan has been under close scrutiny since November last year.
The ACCC knocked back Telstra's first plan in February. Telstra resubmitted the plan with some concessions, but critics say these do not go far enough.
The plan is meant to prevent confidential NBN migration information being passed to Telstra Retail, where it could be used for anti-competitive behaviour, such as marketing.
iiNet welcomed Telstra's "general commitment" to information usage in the revised plan, but said without detail "this general commitment is of little value" and should not be approved. (pdf)
AAPT called the revised commitments "positive" but did not think the current plan would "sufficiently prevent Telstra from using or disclosing NBN Co migration information to gain or exploit an unfair commercial advantage over Telstra's wholesale customers". (pdf)
Optus also called the current revisions "welcome" but said they "fall short of providing the industry with sufficient comfort regarding appropriate use and disclosure of NBN Co Migration information by Telstra". (pdf)
Copyright © iTnews.com.au . All rights reserved.
Processing registration... Please wait.
This process can take up to a minute to complete.
A confirmation email has been sent to your email address - SUPPLIED GOES EMAIL HERE. Please click on the link in the email to verify your email address. You need to verify your email before you can start posting.
If you do not receive your confirmation email within the next few minutes, it may be because the email has been captured by a junk mail filter. Please ensure you add the domain @itnews.com.au to your white-listed senders.


text:
Home ≫ Virtual Library ≫ Crop Management
Crop Management
- Fertilizer Checklist
A one page checklist on the Do's and Don'ts of Soil Fertility. This portion of the Back to Basics program offers seven steps to fertility management.
- Nutrient Management in Irrigated Peas
Specific to irrigated peas, this discussion paper focuses on the effects of potassium and phosphorus placement on yields and quality.
- Crop Nutrient Removal Calculator
All crop species remove unique amounts of the nutrients necessary for growth. A comprehensive nutrient management system requires knowledge of a crop's needs to determine if additional nutrients are necessary for crop growth. Use this handy removal calculator to find how crops stack up.
- Guidelines for Nitrogen Fertilizer
Making fertilizer available to the plant can do more harm than good if it is too close to the plant. Learn what the risks are for nitrogen placement in the seed row, and what the recommendations are for effective use of the nutrient.
In This Section:
- Virtual Library


text:
- Bladder
- Ureters—tubes that carry urine from the kidneys to the bladder
- Urethra—the tube that carries urine from the bladder to outside of the body
|The Urinary Tract
|Copyright © Nucleus Medical Media, Inc.
Reasons for Procedure
- Vesicoureteral reflux —urine flows from the bladder back towards the kidneys
- Bladder distention—enlargement of the bladder
- Bladder irregularities, such as bladder cancer and incomplete voiding
Possible Complications
- Urinary tract infection due to the catheter being inserted
- Bleeding due to the catheter being inserted
- Discomfort during urination, which may last several hours
- Allergic reaction to the contrast dye
What to Expect
Prior to Procedure
Description of the Procedure
How Long Will It Take?
Will It Hurt?
- The catheter is placed into the urethra
- The contrast dye goes into the bladder
Post-procedure Care
- Resume your normal activities.
- Drink plenty of fluids. This will help flush the contrast dye from your bladder.
Call Your Doctor
- Blood in the urine that lasts longer than expected
- Discomfort during urination that lasts longer than expected
- Pain
- Fever
- Inability to urinate
National Kidney and Urologic Diseases Information Clearinghouse http://kidney.niddk.nih.gov
Urology Care Foundation http://www.urologyhealth.org
Canadian Urological Association http://www.cua.org
Health Canada http://www.hc-sc.gc.ca
Cystogram. Children's Hospital of Chicago website. Available at: https://www.luriechildrens.org/en-us/care-services/specialties-services/medical-imaging-radiology/diagnosis-services/nuclear-medicine/Pages/cystogram.aspx. Accessed January 12, 2015.
Cystogram. PeaceHealth website. Available at: http://www.peacehealth.org/peace-harbor/services/imaging-services/radiology/Pages/cystogram.aspx. Accessed January 12, 2015.
Cystogram or voiding cystogram (VCUG). Conemaugh Health System website. Available at: http://www.conemaugh.org/template%5Farticle.aspx?id=8210. Accessed January 12, 2015.
Radionuclide cystogram. American Urological Association website. Available at: http://www.urologyhealth.org/urology/index.cfm?article=81. Updated January 2011. Accessed January 12, 2015.
Schedule test: cystogram, voiding cystouretrhrogram or incontinence cystogram. PennMedicine website. Available at: http://www.uphs.upenn.edu/radiology/patient/docs/Cystogram.pdf. Accessed January 12, 2015.
Your urinary system and how it works. National Kidney and Urologic Diseases Information Clearinghouse (NKUDIC) website. Available at: http://kidney.niddk.nih.gov/kudiseases/pubs/Yoururinary. Updated January 22, 2014. Accessed January 12, 2015.
Revision Information
- Reviewer: Adrienne Carmack, MD
- Review Date: 01/2015 -
- Update Date: 04/29/2014 -
This content is reviewed regularly and is updated when new and relevant evidence is made available. This information is neither intended nor implied to be a substitute for professional medical advice. Always seek the advice of your physician or other qualified health provider prior to starting any new treatment or with questions regarding a medical condition.
Copyright © EBSCO Publishing
All rights reserved.
Quick Links
Find a Physician
Use our online search physicians by specialty, name, city, or zip code. find now
Call 972-566-7111 if you need help with a Physicians referral
Schedule an Appointment
Request a new patient appointment online
Join Our Team
Find job opportunities at Medical City
Medical City Children's Hospital
Built by Specialists, Loved by Kids


text:
CPGC Application Deadline: Winter Break Migration Field Study in Mexico CityCPGC Application Deadline: Winter Break Migration Field Study in Mexico Cityhttp://www.haverford.edu/calendar/details/1876712011-11-132011-11-13
With Casa de los Amigos, a Quaker Center for Peace and International Understanding
Mexico is one of the pivotal global crossroads and the quintessential border between the global north and south. It is also one of the busiest migration corridors in the world, and tens of thousands of migrants cross it each year trying to get to the United States and Canada. This movement has created transnational communities and upheaval, as well as atrocities like the massacre of San Fernando in the state of Tamaulipas in August, 2010. Even though undocumented immigration of Mexicans to the United States is at a 10 year low, Mexico is still the world's most prolific producer of migrants. Mexico is also an important destination country for many people: political refugees from many countries in Africa, the Middle East, South Asia, and elsewhere; and economic refugees from Central America and the Caribbean. In the teeming cultural capital of Mexico City, all of these currents converge. However, migration is a not only a national concern for Mexicans, but a regional concern for North and Central American countries, especially the United States.
The Casa de los Amigos Migration Field Study, a partnership between the Center for Peace and Global Citizenship and Casa de los Amigos, is a one-week program from January 8-15, 2012, that exposes participants to the wide ranging dynamics of migration in Mexico. The field study intends to introduce participants to the complexities of the migratory phenomenon in Mexico. Students will interact directly with a diversity of relevant social actors and agencies. The week also hopes to provide a human face and a living context to help shape an understanding of peoples and communities in movement.
Students and faculty who attend the field study will hear speakers from various NGOs and international agencies who work both with policy and legal issues and who interact directly with migrants, refugees, asylum seekers and human trafficking victims. They will visit non-profit and government offices, a refugee cultural center and a "Casa de Migrante" (migrant house) in the State of Mexico.
The field study is coordinated by the Casa de los Amigos, a Quaker center for peace and international understanding in Mexico City. Participants will stay at the Casa's social justice-oriented guest house in the heart of the city, and also have the chance to visit several important cultural sites and a number of Mexico City's diverse neighborhoods.
Participants will be provided with advanced readings on relevant topics. There will be debriefing seminar-style discussions each evening integrating reading materials and site visits.
Participating students will also be required to enroll in one of the following courses for Spring 2012:
History 209 Modern Latin America
GNST B145 Introduction to Latin American, Latino, and Iberian Peoples and Cultures
Spanish 222 Rethinking Latin America in Contemporary Narrative
Spanish 322 Mexican Narratives after 1968
To apply, fill out the application on CPGC's home page under Announcements, www.haverford.edu/cpgp, by November 13, 2011 at midnight.
For More Info
Stephanie Zukerman


text:
Out of my own desires, and the lack of true swingers clubs in the Montgomery, Alabama area, I have taken on the task of making a club dedicated to everyone with open minds and a desire to explore!
This club is a members only club, and there are rules and regulations to keep anonymity and safety for those that join. Currently, this is a free club to join, but as I see a need and desire for the group, I will be charging a small fee to assist with the cost of mingling parties and other preplanned activities such as demonstrations, seminars, etc., that will be free to attend for members.
You must be at least 19 years of age to join due to the adult nature of this club. Only single females and couples (male and female / female and female) will be admitted at this time. Harassment of any type to any member or the group as a whole will call for an immediate ban, remember that no means no. Use discretion when chatting with any member to avoid uncomfortable situations, and remember that some people do not like their private lives becoming public.
I hope you enjoy!


text:
Bark: Confessions of a Dog Trainer
What we love about dogs, and how we can help them become even better.
Note: The Times Union is not responsible for posts and comments written by non-staff members.
Posts filtered on Category
Dog-Related Events
- Aggression
- Animal Advocacy
- Attention
- Barking
- Body language
- Breeds
- Car Anxiety
- Cats and Dogs
- Chasing
- Chewing
- Children
- Destructive Behavior
- Digging Dogs
- Dog Food
- Dog Parks
- Dog Sports
- Dog Tricks
- Dog-Related Events
- Doggie day care
- Fears and Phobias
- Food Aggression
- Foster Dogs
- General
- Groomers
- Home dog training
- Housebreaking
- Humane Society
- India
- Jumping
- Leadership
- Mouthing
- Multiple dogs
- Nutrition
- Obedience
- Outdoor dogs
- Pet supply stores
- Pulling on the Leash
- Puppies
- Quiz
- Reactive dogs
- Rescue dogs
- Resource guarding
- Senior dogs
- Separation Anxiety
- Service dogs
- Shelter dogs
- Sibling rivalry
- Socialization
- Spay/Neuter
- Stray dogs
- Street dogs
- Training methods
- Traveling with dogs
- Walking
Rain or shine, it's Paws in the Park!
Posted by Rachel Baum on Friday, June 7 at 8:31am
What's a little rain? You're going to walk your dog anyway, right? So come on over to Siena College in Loudonville tomorrow, Saturday, June 8th at 9:00AM to register for Paws in the Park and strut your mutt to benefit the Mohawk Hudson Humane Society. Bring the family, and the family dog, of course. There […] [Read More]
Categories: Dog-Related Events
90 percent
Posted by Rachel Baum on Friday, October 26 at 7:53am
I learned something shockingly obvious at a dog trainers' conference I went to recently. This was the annual Association of Pet Dog Trainers (APDT). Sue Sternberg, a well-known dog trainer and advocate based in Roundout Valley, New York, works with shelter dogs to help them become more adoptable. During her presentation, she remarked that dogs, […] [Read More]
Categories: Animal Advocacy, Attention, Dog-Related Events, General, Obedience
See you there!
Posted by Rachel Baum on Friday, August 31 at 1:10am
Don't even think about leaving town in September. Get out the leash and bring your best friend to these upcoming events! APF's Fireplug 500 Saturday, September 8, 2012 beginning at 9:30 AM in Central Park, Schenectady, NY Join me for the 20th annual walk to raise funds for the Animal Protective Foundation. _________________________________________________________________________________________________ Pet Spas […] [Read More]
Categories: Dog-Related Events, General, Rescue dogs, Shelter dogs
Tags: Adoption clinics | Animal Protective Foundation | APF | Benson's Pet Center | family fair | More
Pet Appreciation Week
Posted by Rachel Baum on Friday, June 8 at 10:04pm
June 10 – 17, 2012 is Pet Appreciation Week. I KNOW that you appreciate your dog every single day. So do I. In honor of this celebration, I'd like to talk about separation anxiety. Nearly 15 percent of dogs suffer from this condition when their owners leave the house. I've talked about this behavioral issue […] [Read More]
Categories: Dog-Related Events, General, Separation Anxiety
Tags: Greg Kleva | Martha Stewart | Siriux Radio | VueZone
Wanted: dog lovers
Posted by Rachel Baum on Tuesday, May 29 at 4:03pm
If it feels like June in May (my car registered 99° today!) that's because June is right around the corner. Most Saturdays in June, you will find me at a dog-related event. I hope you plan to attend one or all of these dog walks, advocacy adoption clinics, puppy workshops, or just plain good times. […] [Read More]
Categories: Animal Advocacy, Dog-Related Events, Humane Society
Tags: Bark in the Park | Curtis Lumber | Hudson Mohawk Humane Society | NYS Animal Advocacy Day | Paws in the Park | More
Any dog can bite
Posted by Rachel Baum on Thursday, May 17 at 9:00pm
Readers know that I'm not one to preach, pontificate, or otherwise bore you to pieces. So I will try to make this post brief and to the point. May 20-26, 2012 is National Dog Bite Prevention Week. Nearly five million Americans are bitten by dogs on average each year. Almost 50 percent are children bitten […] [Read More]
Categories: Aggression, Dog-Related Events, General, Reactive dogs
Not Mother's Day yet (again)
Posted by Rachel Baum on Thursday, May 10 at 7:17am
I confess I've been as busy as a dog in a yard with a family of gophers. No time to do anything but dig in. So I am running my Mother's Day blog post from last year. The way I figure it, I am ALWAYS trying to come up with new ideas for a great […] [Read More]
Categories: Dog-Related Events, General, Rescue dogs
Tags: American Cancer Society | Anna Quindlen | Cadbury bars | Dogs and Divas | EcoMaids | More
- Tags:
- Less
Fall Means FIREPLUG!
Posted by Rachel Baum on Wednesday, September 7 at 7:53am
If its September, it must be the Fireplug 500! Please join me in Schenectady's Central Park from 9:30 AM – 1:00 PM this Saturday, September 10 for the Animal Protection Foundation's annual dog walk and all-around good time for dogs and their owners. This is one of the most fun dog-related events in the Capital […] [Read More]
Categories: Animal Advocacy, Dog-Related Events, General, Humane Society, Shelter dogs
Tags: Animal Protective Foundation | B95.5 FM | Blessing of the Animals | CBS 6 News | Central Park | More
- Tags:
- Less
Show More


text:
Apple has just released its first update for iOS 7 beta, bringing the firmware to beta 2. This is just two weeks after Apple announced iOS 7 at the World Wide Developers Conference. If you are a developer you can download iOS 7 beta 2 over-the-air (OTA) or through the iOS Dev Center.
The update rings in at just over 200MB and should include a wide range of performance improvements and bug fixes. There could very well be some cosmetic changes and new features as well, but we will have to wait and see until we start digging around the update. And for everyone wondering if the iPad beta of iOS 7 has been released,
so far the answer is no (it has, see Update #1).
iOS 7 includes a wide-range of new features such as a completely re-designed user interface, AirDrop, Control Center, new Siri functionality and so much more. It is expected to be released in the Fall alongside new hardware. In case you missed our hands-on demo with iOS 7 be sure to check it out, it is the best way to familiarize yourself with all the new features.
If you are going to be testing out iOS 7 beta 2 be sure to let us know what you find in the comments section below.
Update #1: iOS 7 beta 2 has also been released for the Apple iPad. This means developers can start testing iOS 7 on the iPad. Notably iOS 7 beta 2 also includes the new male and female Siri voices and the stock Voice Memos app has returned.


text:
Trial for teen accused of shooting his mother continued to January
The case of a teenager charged with shooting his mother to death is now scheduled for January rather than next month.
Judge George Collins Jr. placed the case on the docket for Monday, Jan. 6, in Vance County Superior Court. Jaquarius Thomez Holden, just 15 years old when 33-year-old Letitia Renee Hammond was shot to death on June 15, 2011, is facing a first-degree murder charge.
The new date replaces a Dec. 9 date set by Judge Robert Hobgood during case status hearings in October.
The deadline for all evidence from the state labs was due this past Friday. Speaking for the prosecution, assistant district attorney Allison Capps said she could not comment on whether evidence came from labs in time, or if more came since Friday.
"We had to continue the case," she said.
According to documents added to the case file last week, defense attorney Paul Stainback sought to be released from representing any other cases in the month before a December trial date. Because of the delay to January, he withdrew that request.
Stainback did receive an order that he requested to obtain the service of a forensics cellphone expert to examine a seized cellphone in the case.
Stainback said Holden maintains his innocence, and he seeks "evidence that would help substantiate his claim that he did not kill the decedent," Stainback wrote.
According to Stainback, the focus for a forensic study of the cellphone would be on certain records that may have been erased or not copied while it was being kept by Henderson police.
Stainback filed a motion in October notifying prosecutors of his intent to possibly introduce an alibi defense.
Prosecutors last month asked for a rush order on the SBI lab testing for firearm ballistics, latent findings, DNA samples and further trace evidence.
Hammond died from six gunshots at the 710 South Carolina Avenue home where they lived. At least one was close range, according to autopsy reports.
She was last known to be alive at 3 p.m., on the day she died. A Henderson police incident report listed 6:30 p.m. as the time of the call to police.
No other murder cases were heard during the special trial court session. According to Assistant District Attorney Bill Griffin, prosecutors obtained guilty pleas on two smaller cases this week.
Contact the writer at email@example.com.


text:
607 First Amendment (3)
An examination of a series of First Amendment issues under the speech, press, and religion clauses, not dealt with in detail in Constitutional Law II. The following are representative of the topics to be treated: the vagueness and overbreadth doctrines; prior restraint; expression restrictions in connection with national security, the administration of justice, the electoral process, and common law torts; First Amendment issues in the regulation of and access to the news media; government secrecy and inquiry into beliefs; access to government facilities and benefits by religious organizations; and First Amendment rights in specialized government facilities such as schools, prisons, and military bases.
Prerequisite: Constitutional Law II (600)
This elective course is normally offered every year.


text:
Below is the poem entitled sweet torture which was written by poet
Pawlicka. Please feel free to comment on this poem. However, please remember, PoetrySoup is a place of encouragement and growth.
Read Poems by
Zuzanna Pawlicka
to cherish
your body
like a cake with a cherry
on top
to lick it
to try it
to love not deny it
but stop
to lick it once more
to kiss and adore
to give you a f.uck
but just once
to bite
just a piece
to touch you and tease
and then dance
to squeeze
your breasts tight
to make you feel right
and f.uck like a whore
to tell how I love you
and beg you a pardon
for torture of waiting before.
A comment has not been posted for this poem. Encourage a poet by being the first to comment.


text:
Best Casino St. Louis 2005 - i-Bar
777 Casino Center Drive
Maryland Heights, MO 63043
Readers' Choice: Harrah's St. Louis
Remember when you first walked into a casino? Weaned on images of Robert De Niro playing high stakes games in bespoke suits, you were crushed to discover that in reality today's casinos are populated by blue-hairs who alternate breaths between an oxygen tank and a Misty cigarette as they deposit their pensions, one credit at a time, into video slot machines. But take heart, gamblers and Scorsese devotees. The suits at Harrah's are trying to make the pastime sexy again. Exhibit A: the i-Bar. With its illuminated amber counter and massive stock of booze, this oval island bar nestled within the casino bids to lure young gamers away from their on-line Texas Hold 'Em tourneys and back to the mother ship. The strategy seems to be working: On any given night, the i-Bar boasts a respectable throng of fresh young things sipping sidecars as they sidle up to the card tables. Friendly as it is, the i-Bar won't up your chances of winning. But scoring? The odds are decidedly in your favor.


text:
Dr. Richard Haass, Director of Foreign Policy Studies at the Brookings Institution in Washington, D.C., was a speaker at the first annual Harry and Bernice Gockel International Symposium in April, 1997. So popular was he, that by repeated requests from those present that evening, he returned for this year's Symposium.
Since last year, his book The Reluctant Sheriff: The United States After the Cold War has been published. The New York Times called it "thought-provoking" and analyzed it in depth. Dr. Haass, in the book, is deeply concerned that "there is less interest in and consensus over foreign policy than at any time in the past half-century." He says clearly: "What the United States does and does not do can help determine history." His previous book was Intervention: The Use of American Military Force in the Post-Cold War World and was selected by Choice Magazine as one of the outstanding academic books of 1995. A previous book is The Power to Persuade, which was described by Tom Peters, co-author of In Search of Excellence, as "an extraordinarily thoughtful examination of personal success and failure in the public sector and a 'how to' guide of the first order." The book has been described by reviewers as "a business bible" on how to perform more effectively whenever persuasion, rather than direct command, is the rule.
Dr. Haass has extensive government experience. From 1989 to 1993 he was special assistant to President George Bush and senior director for Near East and South Asian Affairs on the staff of the National Security Council. In 1991, Haass was awarded the Presidential Citizens Medal for his contributions to the development and articulation of U.S. foreign policy after the Iraqi invasion of Kuwait. Previously he served in various posts in the Departments of State and Defense and was a legislative aide in the U.S. Senate.
Haass has also been the Director of National Security Programs and a senior fellow of the Council of Foreign Relations, the Sol M. Linowitz Visiting Professor of International Studies at Hamilton College, a senior associate at the Carnegie Endowment for International Peace, a Lecturer in Public Policy at Harvard University's Kennedy School of Government, and a research associate at the International Institute for Strategic Studies. He also has consulted for numerous governmental agencies and corporations.
A Rhodes scholar, Richard Haass holds a B.A. from Oberlin College and both the master's and doctor of philosophy degrees from Oxford University. He is also the author of Conflicts Unending: The United States and Regional Disputes (1990), Beyond the I.N.F. Treaty: Arms, Arms Control and the Atlantic Alliance (1988), and Congressional Power: Implications for American Security Policy (1979) as well as co-editor of Superpower Arms Control: Setting the Record Straight (1987). He is the author of numerous shorter pieces on international affairs.
Dr. Haass resides in the Washington, D.C., area with his wife and two children.


text: The Vashti Center, a mission of the United Methodist Women, has provided services to children and adolescents at its Thomasville address since 1903. Driven by its mission to improve the physical, social, emotional, educational, and spiritual development of children and adolescents, Vashti has successfully served thousands of children over its century of operation. While the type of children served and the nature of services have changed since inception, the mission and values remain. Today, reflecting the national trend to deinstitutionalize residential care, Vashti has undertaken extensive growth in community mental health services, while reducing the number of children treated in our residential programs. Fundamental to all services delivered by Vashti employees is the faith-based nature of our services. Ultimately, the mind and spirit can be addressed simultaneously and we at Vashti believe our treatment must support The Lord's service.


text:
Hang tight!
Recipe by dsgoldens
"By far the best chopped chicken liver ever! This is from the recipes used at the famous Grossinger's Resort in the Catskills, NY. Please use real rendered chicken fat; it makes a big difference in the flavor. See the recipe for rendered chicken fat in the footnote of this recipe. Serve with crackers or on lettuce or on a bagel with a tomato slice."
Hmm. None of these ingredients are on sale today.
Show ingredients on sale
Sort stores by
Save money at local stores when ingredients are on sale!
Watch video tips and tricks
2 tablespoons
rendered chicken fat
onions, diced
1 pound
chicken livers, rinsed
hard-cooked egg yolks
1 teaspoon
1/4 teaspoon
freshly ground black pepper
I've been making chopped liver for decades and my recipe is similar to this and everyone loves it. My grandmother was a good friend of Jennie Grossinger and I spent many happy weekends there in my young, single days.
Since I do not keep kosher, I tend to substitute no trans fat margarine for the chicken fat. I also use about a dozen hard-boiled eggs to one pound of chicken livers to make it taste less like liver, plus it makes a much larger quantity. For those, like me, who dislike liver of any kind, you may find you enjoy this version.
I made this recipe today and I can honestly say it's the best ever. I followed it exactly as posted.
3 Ratings
* Percent Daily Values are based on a 2,000 calorie diet.
Chopped Chicken Liver a la Jennie Grossinger
Serving Size: 1/8 of a recipe
Servings Per Recipe: 8
Amount Per Serving
Calories: 170
Calories from Fat: 97
We have over 100 cucumber salads to help you keep your cool.
Meatloaf is the hearty, family-pleasing meal that makes the greatest leftovers ever.
Delicious recipes, party ideas, and cooking tips! Get a year of Allrecipes magazine for $7.99!
Learn how to make an easy chicken and curry appetizer.
See Chef John's secrets for making the best game-day dip ever!
See how to make succulent baked chicken with a crispy, corn flake crust.


text:
Tomorrow the Elbert Theatre will feature the reading of a new play from a local play writer.
Playwright Michael Blackston's new play is titled Mr. Long Said Nothing. Elbert Theatre Director Toni King said that Blackston has spent a lot of time coming up with the new play.
"It's actually based on true life events. He created it from a story that his wife told him from her childhood. He has been working on this play for nearly twenty years, and it is finally done", said Elbert Theatre Director Toni King. "He would like the opportunity to share it with us for the very first time in public, and we are very excited to do that."
There will be no cost to attend the reading. It will be held at the Elbert Theatre on Saturday beginning at 2:00pm, and lasting until 4:00pm. Following the reading, there will be chance for the audience to ask questions.
For more information on the play reading you can contact the Elbert Theatre Office at (706) 283-1049 or (706) 213-3109.