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Biomedical subjects

M Keeney

Publications and source records attributed to M Keeney.

At least 19 recordsLinked to original sources
PubMed2005

Effect of activated recombinant human factor 7 (Niastase) on laboratory testing of inhibitors of factors VIII and IX.

Activated recombinant human factor VIIa (rFVIIa) has been used as a hemostatic agent in patients with hemophilia and acquired inhibitors. Other indications for rFVIIa may include liver disease, warfarin sodium (Coumadin) overdose, or trauma. Monitoring patients on this treatment with standard laboratory testing is problematic. Bleeding risk does not correlate well with the prothrombin time (PT) or the activated partial thromboplastin time (aPTT) during therapy with rFVIIa. In addition, there is no identifiable literature on the effect of rFVIIa on assays of inhibitors in this patient group. Monitoring inhibitors may be important during interventions aimed at acutely reducing inhibitor levels, such as during plasma exchange or protein adsorption. We performed factor assays and evaluated inhibitor levels in plasma from 3 patients with deficiencies in FVIII (2 patients) or FIX (1 patient) and inhibitors (titer range, 5.8-17.4 Bethesda units) before and after adding rFVIIa (range, 0.25-8 microg/mL) in vitro. Additionally, we performed assays of factors of both intrinsic and extrinsic systems to determine the impact of rFVIIa on these tests. We found that both factor levels and inhibitor titers from patients with hemophilia A or B could be measured accurately, even in the presence of suprapharmacologic doses of rFVIIa (8 microg/mL). We also obtained accurate measurements for other assays of the intrinsic coagulation system (FXI and FXII) based on the aPTT. Conversely, we found that assays of the extrinsic system based on the PT (FII, FV, and FX) produced results that were unreliable. FVII results were very high but reproducible. These results suggest that assays based on the PT are inaccurate and should be avoided during FVIIa treatment. Conversely, FVIII and FIX levels and inhibitor titers can be accurately monitored in hemophilia patients receiving rFVIIa according to results of aPTT-based coagulation tests.

Antibodies↗
PubMed2003

Effect of 3.2 vs. 3.8% sodium citrate concentration on anti-Xa levels for patients on therapeutic low molecular weight heparin.

In this study, we compared the effect of sodium citrate, a sample collection variable, on the anti-Xa levels of patients (n = 28) on dalteparin, a low molecular weight heparin. The median anti-Xa level for 3.2% sodium citrate was 0.235 U/ml while the median level for 3.8% sodium citrate was 0.230 U/ml. We conclude that different sodium citrate concentrations give statistically equivalent anti-Xa levels for the same samples. This conclusion is in contrast to the findings of the effect of sodium citrate concentration on International Normalized Ratio (INR) and activated partial-thromboplastin time (aPTT). In accordance with previous recommendations, we advocate the exclusive use of 3.2% sodium citrate in an effort to standardize coagulation testing.

Anticoagulants↗
PubMed2003

Validation of the single-platform ISHAGE method for CD34(+) hematopoietic stem and progenitor cell enumeration in an international multicenter study.

BACKGROUND: Flow cytometric enumeration of CD34+ hematopoietic sterm and progenitor cells (HPC) is the reference point for undertaking apheresis and evaluation of adequacy for PBSC engraftment. An external quality assurance (EQA) scheme for CD34+ HPC enumeration has been operational in Belgium, Netherlands and Luxemburg (Benelux) since 1995. Within this group, a multicenter survey was held to validate the state-of-the-art methodology, i.e., multiparametric definition of HPC based on light scatter, expression of CD34 and CD45, and counting beads (i.e., 'single platform ISHAGE' method). METHODS: 'Real-time' EQA was used to monitor the application of the single-platform ISHAGE method by 36 participants. Three send-outs of stabilized blood with CD34+ cell counts 35-60 cells/microl were distributed to 36 participants, who were required to assay the samples on three occasions using the standard assay and their local techniques. These results were compared with thosed obtained by 111-116 UK NEQAS participants testing the same specimens. RESULTS: Using the single platform ISHAGE methods, between-laboratory coefficients of variations (CVs) as low as 10% were achieved. Intra-laboratory CVs were < 5% for approximately 50% of the participants. Local single-platform techniques yielded between-laboratory CVs as low as 9% in both Benelux and UK NEQAS cohorts. In contrast, the lowest between-laboratory CVs using dual-platform techniques were 17% (Benelux) and 21% (UK NEQAS), respectively. CONCLUSION: The single-platform ISHAGE method for CD34+ cell enumeration has been validated by an international group of 36 laboratories. The observed varation between laboratories allows a meaningful comparison of CD34+ cell enumeration.

Antigens, CD34↗
PubMed2002

Number of viable CD34(+) cells reinfused predicts engraftment in autologous hematopoietic stem cell transplantation.

Reduced CD34(+) cell viability due to cryopreservation has unknown effects on subsequent hematopoietic engraftment in autologous transplantation. Thirty-six consecutive autologous peripheral stem cell collections were analyzed for absolute viable CD34(+) cell numbers at the time of stem cell collection and prior to re-infusion. Viable CD34(+) cells were enumerated using single platform flow cytometry and the molecular exclusion dye 7-amino actinomycin D. The median number of viable CD34(+) cells was 3.6 x 10(6)/kg at the time of harvest and 2.0 x 10(6)/kg after thawing. When viable CD34(+)cells enumerated after thawing were <2.0, 2.0-5.0, or >5.0 x 10(6)/kg, the median time to platelet engraftment was 17, 12 and 10 days, respectively (P < 0.05 for comparison of the group with <2.0 x 10(6)/kg and the other two groups), and the median time to neutrophil engraftment was 13, 14 and 12 days, respectively (P = NS). A minimum of 2.0 x 10(6) CD34(+) cells/kg was harvested in 33 of 36 patients (92%) but only 19 of 36 (52%) patients met this threshold at the time of reinfusion. The reduced numbers of viable CD34(+) cells measured prior to re-infusion is associated with time to platelet engraftment and may be useful in monitoring stem cell loss during processing and identifying patients at risk of graft failure.

Adolescent↗
PubMed2002

Optimising parameters for peripheral blood leukapheresis after r-metHuG-CSF (filgrastim) and r-metHuSCF (ancestim) in patients with multiple myeloma: a temporal analysis of CD34(+) absolute counts and subsets.

Patients (n = 69) with multiple myeloma undergoing peripheral blood stem cell collection (PBSC) were treated with cyclophosphamide and a combination of recombinant methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF, filgrastim) and recombinant methionyl human stem cell factor (r-metHuSCF, ancestim). The objectives of this study were to determine: (1) The proportion of patients reaching a target yield of >or=5 x 10(6) CD34(+) cells/kg in one or two successive large-volume (20 liter) leukapheresis procedures; (2) the optimal collection time for leukapheresis; (3) mobilization kinetics of CD34(+) subsets in response to G-CSF/SCF. All patients were mobilized with cyclophosphamide (2.5 g/m(2)) on day 0 followed by filgrastim (10 microg/kg ) plus ancestim (20 microg/kg) commencing day 1 and continuing to day 11 or 12. Of the 65 evaluable patients, 57 were considered not heavily pretreated and 96.5% obtained a target of >or=5 x 10(6)/kg in one collection. The median CD34(+) cells/kg was 39.5 x 10(6) (range: 5.2-221.2 x 10(6)). Subset analysis demonstrated the number of CD38(-), CD33(-), and CD133(+) peaked at day 11; and CD34(+), CD90(+) cells peaked at day 10. The optimum day for leukapheresis was determined to be day 11. The median absolute peripheral blood CD34(+) cell numbers on day 11 was 665 x 10(6)/l (range: 76-1481 x 10(6)/l). Eight of the 10 heavily pretreated patients were evaluable: three achieved the target dose in one leukapheresis (37.5%) and three (37.5%) achieved the target dose with two leukaphereses. Use of this mobilization strategy allowed the collection of high numbers of CD34(+) cells and early progenitors and the ability to predictably schedule leukapheresis.

Adult↗
PubMed2001

Inactivation of p53 sensitizes U87MG glioma cells to 1,3-bis(2-chloroethyl)-1-nitrosourea.

We examined the effect of p53 inactivation on the response of U87MG glioma cells to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). These studies were motivated by three observations: (a) some human astrocytomas are sensitive to BCNU and some are resistant; (b) chemosensitive astrocytomas are more likely to be found in young adults whose tumors are more likely to harbor a p53 mutation; and (c) mouse astrocytes lacking the p53 gene are more sensitive to BCNU than wild-type cells. Here, we observed that p53 inactivation by transfection with pCMV-E6 sensitized U87MG cells to BCNU. Compared with control U87MG-neo cells with intact p53 function, the clonogenic survival of U87MG-E6 cells exposed to BCNU was reduced significantly. In U87MG-E6 cells, sensitization to BCNU was associated with failure of p21(WAF1) induction, transient cell cycle arrest in S phase, accumulation of polyploid cells, and significant cell death. In contrast, resistance to BCNU in U87MG-neo cells was associated with up-regulation of p53, prolonged induction of p21(WAF1), sustained cell cycle arrest in S phase, and enhancement of DNA repair. U87MG cells with disrupted p53 function were less able to repair BCNU-induced DNA damage and survive this chemotherapeutic insult. The question arises of whether p53 dysfunction might be a chemosensitizing genetic alteration in human astrocytic gliomas.

Antineoplastic Agents, Alkylating↗
PubMed2001

Flow cytometric enumeration and immunophenotyping of hematopoietic stem and progenitor cells.

Flow cytometric enumeration of CD34(+) hematopoietic stem and progenitor cells (HPC) is widely used to evaluate the adequacy of peripheral blood stem cell grafts and is also useful for planning the apheresis sessions needed to obtain these grafts. A state-of-the-art method to enumerate CD34(+) cells has been developed that makes use of a multiparameter definition of HPC, based on their light scatter characteristics and dim expression of CD45, utilizing fluorescent counting beads. This approach allows the absolute CD34(+) cell count to be determined directly from a flow cytometer. The method can be extended with a viability stain and additional markers for further immunologic characterization of CD34(+) cells, and has been successfully implemented in multicenter trials. Using such a standardized assay, it should be possible to define more accurately the lower threshold for a safe HPC graft in terms of short- and long-term hematopoietic reconstitution. Semin Hematol 38:139-147.

Animals↗
PubMed2001

A comparison of three rapid D-dimer methods for the diagnosis of venous thromboembolism.

We compared three rapid D-dimer methods for the diagnosis of venous thromboembolism. Patients presenting to four teaching hospitals with the possible diagnosis of deep vein thrombosis or pulmonary embolism were investigated with a combination of clinical likelihood, D-dimer (SimpliRED) and initial non-invasive testing. Patients were assigned as being positive or negative for deep vein thrombosis or pulmonary embolism based on their three-month outcome and initial test results. The three D-dimer methods compared were: (a) Accuclot D-dimer (b) IL-Test D-dimer (c) SimpliRED D-dimer. Of 993 patients, 141 had objectively confirmed deep vein thrombosis or pulmonary embolism. The sensitivity of SimpliRED, Accuclot and IL-Test were 79, 90 and 87% respectively. All three D-dimer tests gave similar negative predictive values. The SimpliRED D-dimer was found to be less sensitive than the Accuclot or IL-Test. When combined with pre-test probability all three methods are probably acceptable for use in the diagnosis of venous thromboembolism.

Fibrin Fibrinogen Degradation Products↗
PubMed2001

Comparison of five platforms for enumeration of residual leucocytes in leucoreduced blood components.

The need for quality control of leucoreduction of blood products has led to the development of various methods to count low levels of residual leucocytes. We compared five platforms side-by-side: the Nageotte haemocytometer and four based on fluorescent staining of nuclei: two flowcytometers (Beckman Coulter, BD Biosciences) with methods based on counting beads, a volumetric flow cytometer (Partec) and the microvolumic fluorimeter ImagN2000 (BD Biosciences), all according to their manufacturers' recommended methods. Analysis of double-filtered red cell concentrates (RCCs) and platelet concentrates (PCs), spiked with various numbers of leucocytes, revealed good linearity for all methods over the range of 1.6-32.7 leucocytes/microl, all with r(2) > 0.99. At the rejection level of leucocyte-reduced blood components, i.e. 1 x 10(6) per unit corresponding with approximately 3.3 leucocytes/microl, the Nageotte haemocytometer had low accuracy (0% for RCCs, 56% for PCs), and was relatively imprecise [coefficient of variance (CV) of 34% and 30% respectively]. The Partec flow cytometer gave good results for RCCs (accuracy 67%, CV 22%), but not for PCs (accuracy 0%, CV 25%). The ImagN2000 had an accuracy of 44% for RCCs and 89% for PCs, but the precision was variable (CV 32% for RCCs, 15% for PCs). The best results were obtained with the Beckman Coulter (RCCs: accuracy 86%, CV 13%, PCs: accuracy 67%, CV 16%), and BD Biosciences platforms (RCCs: accuracy 100%, CV 10%; PCs: accuracy 89%, CV 11%). We conclude that, at the rejection level of 1 x 10(6) leucocytes per unit, the widely used Nageotte haemocytometer performs poorly in terms of inaccuracy and imprecision, and that both counting-bead-based, flow cytometric methods performed best.

Flow Cytometry↗
PubMed2000

The human hematopoietic stem cell compartment is heterogeneous for CXCR4 expression.

The chemokine stromal derived factor-1alpha (SDF-1alpha) has been implicated recently in the chemotaxis of primitive human hematopoietic cells, suggesting that pluripotent human stem cells express the SDF-1alpha receptor, CXCR4. By using flow cytometry and confocal microscopy, we have identified and isolated primitive subsets of human CXCR4(+) and CXCR4(-) cells. Distinctions in the progenitor content and response to SDF-1alpha in vitro indicate that CXCR4(+) and CXCR4(-) cells represent discrete populations of primitive blood cells. The i.v. transplantation of these subfractions into immune-deficient mice established that both possess comparable engraftment capacity in vivo. Human myeloid, lymphoid, and primitive CD34(+) CXCR4(+) cells were present in chimeric animals transplanted with either subset, indicating that CXCR4(+) and CXCR4(-) stem cells have equivalent proliferative and differentiative abilities. Our study indicates that the human stem cell compartment is heterogeneous for CXCR4 expression, suggesting that the relationship between CXCR4 expression and stem cell repopulating function is not obligatory.

Animals↗
PubMed2000

Isolation and characterization of human CD34(-)Lin(-) and CD34(+)Lin(-) hematopoietic stem cells using cell surface markers AC133 and CD7.

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34(-)Lin(-)). A major barrier in the further characterization of human CD34(-) stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34(-)CD38(-)Lin(-) and CD34(+)CD38(-)Lin(-) cells derived from human cord blood. Although the majority of CD34(-)CD38(-)Lin(-) cells lack AC133 and express CD7, an extremely rare population of AC133(+)CD7(-) cells was identified at a frequency of 0.2%. Surprisingly, these AC133(+)CD7(-) cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34(+) stem cells, and they were the only subset among the CD34(-)CD38(-)Lin(-) population capable of giving rise to CD34(+) cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo-cultured AC133(+)CD7(-) cells isolated from the CD34(-)CD38(-)Lin(-) population, whereas 400-fold greater numbers of the AC133(-)CD7(-) subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34(-) cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34(+) cells. (Blood. 2000;95:2813-2820)

AC133 Antigen↗
PubMed2000

Characterization of chemokine receptors expressed in primitive blood cells during human hematopoietic ontogeny.

Chemokines are capable of regulating a variety of fundamental processes of hematopoietic cells that include proliferation, differentiation, and migration. To evaluate potential chemokine signaling pathways important to the regulation of primitive human hematopoietic cells, we examined chemokine receptor expression of highly purified subpopulations of uncommitted human blood cells. CXCR1-, CXCR2-, CXCR4-, and CCR5-expressing cells were detected by flow cytometry among human blood subsets depleted of lineage-restricted cells (Lin(-)) derived from adult bone marrow, mobilized peripheral blood, cord blood (CB), and circulating fetal blood. Although these chemokine receptors could be detected on Lin(-) cells throughout human development, only CXCR4 could be detected in CD34(-)CD38(-)Lin(-) and CD34(+)CD38(-)Lin(-) subfractions enriched for stem cell function, suggesting that independent of ontogeny, CXCR4-mediated signals are critical to primitive hematopoiesis. Distinct to other stages of human hematopoietic development, primitive CB cells expressed higher levels of CXCR1, CXCR2, CCR5, and CXCR4 on both CD34(-)CD38(-)Lin(-) and CD34(+)CD38(-)Lin(-) subsets. Isolation of these fractions revealed expression of additional chemokine receptors CCR7, CCR8, and Bonzo (STRL133), whereas BOB (GPR15) could not be detected. Our study illustrates that rare uncommitted hematopoietic cells express chemokine receptors not previously associated with primitive human blood cells. Based on these results, we suggest that signaling pathways mediated by chemokine receptors identified here may play a fundamental role in hematopoietic stem cell regulation and provide alternative receptor targets for retroviral pseudotyping for genetic modification of repopulating cells.

Adult↗
PubMed1999

Cerebral hemispheric mechanisms linking ambiguous word meaning retrieval and creativity.

The deferral of ambiguity resolution has been thought to be an important component of creativity. The time course of priming of dominant and subordinate meanings of ambiguous words was investigated using a divided visual field priming paradigm with subjects that varied on a measure of creativity. The Wallach-Kogan similarities subtest was used to group 72 subjects into three levels of verbal creativity to compare their performance on the ambiguity resolution task (Burgess & Simpson, 1988a). Results suggest that both the left and right hemispheres contribute to the maintenance of multiple word meanings in highly creative subjects, while less creative subjects show sustained subordinate priming only in the right hemisphere or no sustained subordinate priming. These results support an interactive, collaborative theory of verbal creativity (Bogen & Bogen, 1969) and suggest that there are important individual differences that expand on the basic time course model of hemispheric processing (Burgess & Simpson, 1988a).

Brain↗