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Biomedical subjects

D Barnett

Publications and source records attributed to D Barnett.

At least 55 records · Page 3Linked to original sources
PubMed1998

Quality assessment of CD34+ stem cell enumeration: experience of the United Kingdom National External Quality Assessment Scheme (UK NEQAS) using a unique stable whole blood preparation.

CD34+ peripheral blood stem cell (PBSC) mobilization and harvesting has rapidly replaced autologous bone marrow as a source of stem cells for transplantation. Timing and adequacy of harvests rely upon the accurate enumeration of circulating CD34+ cells. However, previous EQA programmes have reported interlaboratory CVs as high as 284%, suggesting the need for greater standardization. In addition the routine use of fresh and/or frozen cells as analytes also introduces antigen instability as a variable factor. To circumvent this problem and achieve a true reflection of interlaboratory variation, we have used a novel whole blood preparation in which the antigenic profiles of PBSCs, as determined by flow cytometry, are retained for > 200 d. This international scheme, currently the largest in the world, distributes aliquots of stabilized whole blood bi-monthly to 91 laboratories in 20 countries (44 U.K., 47 overseas). Participants are required to determine the percentage and absolute values for CD34+ PBSCs using in-house techniques. Adopting such a preparation, a more accurate determination of interlaboratory variation has been possible when compared to previous EQA studies, with CVs as low as 22% and 24% for percentage and absolute counts. In addition the programme has established that a wide range of methods are in routine use, emphasizing the urgent requirement for national/international consensus guidelines.

Antigens, CD34↗
PubMed1998

Determination of leucocyte antibody binding capacity (ABC): the need for standardization.

The flow cytometric determination of antigen density, or cellular antibody binding capacity, is now an accepted technique for the characterization of cells in health and disease. In HIV infection, for example, antigen density changes in CD38 expression may be an important indicator of disease progression. Our experience of using one such method, Quantum Simply Cellular, which measures antibody binding capacity (ABC), has highlighted several technical factors which can affect the results. We report the influence of pH, incubation temperature and time, antibody fluorochrome and titre, as well as lysing reagent (FACS Lysing Solution v. Ortho-mune Lysing Reagent) on the ABC of anti-CD3, CD4 and CD8 of normal lymphocytes. In addition, the effect of single, double or triple-staining was assessed. The results indicate that the ABC values are influenced by all the variables studied. The pH range tested (6.0-9.0) demonstrated that pH 7.4 gave maximal binding. Furthermore, temperature also influenced the pH of the two lysing solutions, and thus potentially the ABC. Antibody concentration, fluorochrome and staining technique are also important factors with an observed difference of up to 458,855 ABC between the various fluorochromes. In addition a maximal difference of 130,119 ABC was observed between single and triple staining techniques. In conclusion, if antigen quantification is to be used in the clinical setting, an internationally standardized method is required to ensure the reproducibility of results from centre to centre. Our data suggests that single staining, using fluorescein isothiocynate (FITC) conjugated antibodies with all reagents at pH 7.4 + 0.1, with incubation and lysing carried out at 20 + 1 degrees C, could be used as a 'benchmark' method for ABC determination using the QSC system.

Antigen-Antibody Reactions↗
PubMed1998

A phase I and pharmacokinetic study of CBT-1 as a multidrug resistance modulator in the treatment of patients with advanced cancer.

CBT-1, a natural product, was studied in an escalating dose Phase I clinical trial with doxorubicin at 60 mg/m2. CBT-1 was administered by mouth at doses from 200 mg/m2 to 600 mg/m2. The drug was given for 7 days and doxorubicin administered intravenously on day 6. The MTD was determined to be 500 mg/m2 although some patients did tolerate 600 mg/m2 with moderate nausea and occasional vomiting. Side effects were otherwise mild in the 23 patients treated. Pharmacokinetic determinations in an additional 11 patients demonstrated that CBT-1 did not significantly alter the pharmacokinetics of doxorubicin. In this Phase I study, 25 of 34 patients were evaluable for response and 5 patients demonstrated tumor shrinkage.

Administration, Oral↗
PubMed1998

Gene amplification as a prognostic factor in primary brain tumors.

The most reliable prognostic factors for patients with primary malignant brain tumors remain histology, age, and functional status. Management of these individuals might be improved by quantifying pertinent molecular markers. We have measured the gene dosage of the epidermal growth factor receptor (EGFR), mouse double minute 2 (MDM2), and cyclin-dependent kinase 4 (CDK4) genes in a series of brain tumor specimens and correlated their amplification status with standard prognostic factors and survival. Individual tumor DNA was successively hybridized with probes for EGFR, MDM2, and CDK4. The signal was quantified by densitometry, and amplification was defined as gene signal > or = 2 times normal. Survival, age, Karnofsky performance status, and histology were correlated with gene amplification. Nineteen astrocytomas, 20 anaplastic astrocytomas, and 70 glioblastomas had complete data available. Median survival with and without any form of gene amplification was 70.7 and 88.6 weeks, respectively (P = 0.0369). For the EGFR gene alone, those with and without amplification had a median survival of 58.9 and 88.6 weeks, respectively (P = 0.0104). By Cox analysis, only tumor histology (P = 0.04) and Karnofsky performance status (P = 0.0157) were significant independent predictors of survival. Gene amplification by itself was not predictive of survival, even for glioblastomas (P = 0.8249). The lack of correlation between gene amplification and survival for patients with primary malignant brain tumors may be because EGFR, MDM2, and CDK4 are only portions of larger signaling systems. Therefore, the lack of a direct correlation between a single gene and outcome is not entirely unexpected.

Adult↗
PubMed1998

Parenting and preschooler attachment among low-income urban African American families.

This study examined the parental correlates of child attachment in a preschool-aged, economically disadvantaged, urban, African American sample. Sixty-nine 4- to 5-year-olds and their primary caregivers participated in the Strange Situation assessment procedure. Based on Cassidy and Marvin's classification system for preschoolers, 61% of the children were classified as securely attached, with girls being significantly more likely to be securely attached than boys (74% versus 45%). The majority of the insecure attachments were of the avoidant variety. Consistent with attachment theory, parents of securely attached children were rated as significantly more warm and accepting and less controlling with their children than were parents of insecurely attached preschoolers. Relative to parents of securely attached preschoolers, parents of children judged to be insecurely attached reported being more likely to use corporal punishment and less likely to use verbal reminders when their children misbehaved. Parenting was associated with attachment over and above the effects of child sex.

Black or African American↗
PubMed1997

Isolation and amino acid sequence of a new long-chain neurotoxin with two chromatographic isoforms (Aa el and Ae e2) from the venom of the Australian death adder (Acanthophis antarcticus).

The amino acid sequence of a previously undescribed toxin from Australian death adder venom (Acanthophis antarcticus) has been elucidated. It appears to exist in two forms which are separated by reverse-phase high-performance liquid chromatography, but which have the same sequence and mol. wt. It has 79 amino acid residues and is therefore longer than other long postsynaptic neurotoxins. It shows homology with the conserved regions of the other long postsynaptic neurotoxins except for three unique substitutions of conserved residues, which are Arg-29 instead of Trp or Phe, Leu-33 instead of Arg and Thr-43 instead of Ala.

Amino Acid Sequence↗
PubMed1997

Relationship of Beck Depression Inventory factors to depression among adolescents.

Beck Depression Inventory (BDI) scores of 328 adolescents referred to a depression clinic were factor analyzed to test the discriminant validity of each factor. Three of the four factors (Negative Self Attitude. Performance Difficulty, and Somatic Symptoms) discriminated depressed adolescents from those with a behavior disorder or no diagnosis; the Negative Self Attitude and Performance Difficulty factors also discriminated depressed from anxious adolescents. The fourth factor, Physical Worry, failed to discriminate diagnostic groups. Diagnostic efficiency statistics are reported for both the BDI and for items comprising the 13-item BDI Short Form. Results indicate the BDI is a valid screening tool for adolescent depression in a clinical setting, regardless of the presence of comorbid conditions.

Adolescent↗
PubMed1996

Evaluation of a novel stable whole blood quality control material for lymphocyte subset analysis: results from the UK NEQAS immune monitoring scheme.

The UK NEQAS immune Monitoring Scheme (UK NEQAS) evaluates the performance of laboratories routinely performing T-lymphocyte subset analysis on HIV-infected individuals. The scheme originally issued fresh whole blood, but a significant problem was that of analyte stability, especially 36 h postphlebotomy. To circumvent this problem, we have developed a novel stabilisation procedure that ensures retention of leucocyte light scatter and immunological staining characteristics for up to 300 days. In addition, the stabilised whole blood preparation is fully compatible with flow cytometer technology, incorporating either whole blood lysis or "no wash, no lyse" techniques. The ranges of interlaboratory coefficient of variation for the stabilised material are now tighter than those previously obtained with fresh whole blood. Development of this novel material has enabled overseas laboratories to participate in the UK NEQAS immune Monitoring Scheme and could in the future lead to the production of reference and/or calibration reagents for leucocyte immunophenotyping.

B-Lymphocytes↗
PubMed1996

Performance evaluation of the FACSCount System: a dedicated system for clinical cellular analysis.

Flow cytometers are instruments that can determine multiparameter data simultaneously and have a great potential in providing unique information about cells. The FACSCount System is designed as the first dedicated flow cytometer for the clinical laboratory. Its current configuration provides CD4, CD8, and CD3 absolute counts from 100 microliters of whole blood. Adapting the FACSCount System to the clinical setting are minimal sample handling, lysis free cell preparation, automated fluorescence gating, built-in calibrated reference beads, and appropriate error code reporting. Quality control checks ensure that reported CD4 counts, important for clinical follow-up and patient management, are accurate, precise, and reproducible across instruments over time.

Adolescent↗
PubMed1996

Self-perceptions, motivation, and school functioning of low-income maltreated and comparison children.

Maltreated children are at risk for disturbances and delays in their socioemotional and scholastic functioning. This study examined the impact of child maltreatment and age on perceptions of competence, and the relations among perceived competence, motivation, and school functioning. The sample included 76 school children living in poverty, approximately two-thirds of whom had been victims of child maltreatment. Results indicated that both maltreated and nonmaltreated children exhibited maladaptive motivational orientations toward scholastic tasks and poor academic performance, supporting the idea that threats to scholastic functioning reside as much within the ecology of poverty as in that of maltreatment. Over and above the general effects of poverty, maltreatment was found to disrupt the psychological processes accounting for children's scholastic performance. Results revealed that younger maltreated children (6- and 7-year-olds) reported more inflated self-perceptions of competence and social acceptance than nonmaltreated children. In contrast, older maltreated children (8- through 11-year-olds) reported lower perceived social acceptance than nonmaltreated children. Among older nonmaltreated children, perceived competence was positively related to teacher's ratings of their effort, intrinsic motivation, and grades. For older maltreated children, these relations among self-perceptions and school functioning were in the opposite direction from those of nonmaltreated children, suggesting that the determinants of academic engagement are different for maltreated and nonmaltreated children.

Achievement↗
PubMed1995

Analysis of epidermal growth factor receptor gene amplification and alteration in stereotactic biopsies of brain tumors.

In recent years, results from molecular-based investigations of central nervous system neoplasms have indicated that the occurrence of specific genetic alterations correlates with tumor malignancy and clinical behavior. One such example is epidermal growth factor receptor gene amplification and alteration in association with tumors histologically classified as glioblastoma multiforme. As our understanding of the implications of these changes develops, detection of epidermal growth factor receptor alteration and amplification may have diagnostic, prognostic, and ultimately, therapeutic value. The goal of this communication is to demonstrate the usefulness of stereotactic specimens for molecular genetic analysis, in this case, the analysis of the epidermal growth factor receptor gene for amplification and alterations. The techniques used here will maximize the number of cases from which useful information can be obtained. Properly adapted, the value of these techniques for expanding our understanding of these lesions is enormous.

Biopsy↗
PubMed1995

Circulating CD20dim T-lymphocytes increase with age: evidence for a memory cytotoxic phenotype.

The CD20 antigen has been regarded as a B lineage specific, 35 kDa, non-glycosylated membrane phosphoprotein, which functions as either a Ca2+ ion channel or as a regulatory protein of such a channel. Weak expression of CD20 (CD20dim), however, has recently been reported on a sub-population of T lymphocytes. We present results which confirm the existence of a CD20dim T lymphocyte population and show that such cells have a reduced antibody-binding capacity, when compared to CD20bright B-cells (10337 +/- 642 and 346311 +/- 24264 respectively). In addition, CD20dim cell counts vary with age, with the highest levels occurring in octogenarians: cord blood 0.3 +/- 0.1% (n = 13), 20-60 year-old group 2.1 +/- 1.1% (n = 18) and individuals > or = 61 years of age 6.9 +/- 3.2% (n = 10) (P < 0.001). Further characterization of CD20dim T cells, using three colour flow cytometry, demonstrated a predominantly memory cytotoxic phenotype, in that the cells were CD8+CD28+CD45RO+T-CR alpha beta +CD38-HLA-DR-.

Adult↗
PubMed1994

Karyotypic and ras gene mutational analysis in idiopathic myelofibrosis.

Karyotypic analysis was performed in a total of 69 patients with well-characterized idiopathic myelofibrosis. Karyotypic abnormalities were detected in 46% of cases examined during the chronic phase (29/63); with three abnormalities, del(13q), del(20q) and partial trisomy 1q, accounting for 75% of all abnormalities at diagnosis. The absence of del(5q), trisomy 8 and 21, as well as the rarity of monosomy 7, contrasts with pooled published data and may reflect our exclusion of closely related disorders, in particular MDS with fibrosis. Chromosomal aberrations increased to approximately 90% (8/9) in patients analysed during acute transformation. Mutational activation of codons 12, 13 and 61 of N-, Ha- and Ki-ras genes were assessed by polymerase chain reaction and hybridization with synthetic non-radioactive digoxigenin-labelled probes. Three mutations were detected in samples of peripheral blood DNA taken from 50 patients during the chronic phase of their disease: one N12 Asp (GGT-->GAT) and two N12 Ser (GGT-->AGT) mutations. The results from this study indicate that karyotypic abnormalities are present in at least 29% of cases at diagnosis and that del(13q), del(20q) and partial trisomy 1q are the most frequent findings. Ras mutations were relatively infrequent (6%) and appeared restricted to the N-ras gene. Karyotypic analysis at diagnosis was found to be of prognostic significance.

Adult↗
PubMed1994

Expression of the costimulatory molecule B7/BB1 in autoimmune thyroid disease.

Efficient antigen presentation requires the provision of a costimulatory signal, the best characterized of which is B7/BB1. It is unclear whether thyroid cells expressing class II molecules can present autoantigens to T cells, although this has been suggested as an important mechanism in the initiation of Graves' disease and Hashimoto's thyroiditis. We have found that thyroid cells from patients with thyroid autoimmunity do not express B7/BB1 in vivo or in vitro, even after activation with the cytokines interleukin-1 or gamma-interferon, or with a phorbol ester. Increased numbers of CD20+ B cells and CD14+ dendritic cells expressing B7/BB1 were found in intrathyroidal lymphocyte preparations from such patients compared to peripheral blood. These results suggest that conventional antigen-presenting cells rather than thyroid cells provide B7/BB1 costimulatory activity in autoimmune thyroid disease, and argue against a role for the thyroid cells themselves in autoantigen presentation to T cells via the B7/BB1 pathway.

Antigens, CD↗
PubMed1993

IL-2 receptor-positive intrathyroidal lymphocytes in Graves' disease. Analysis of V alpha transcript microheterogeneity.

There has been considerable interest recently in the possible restriction of the TCR repertoire in autoimmune disorders, because such restriction would have important therapeutic implications. Reports of restriction of the TCR V alpha but not V beta repertoire in the thyroid in Graves' disease could not be repeated in an earlier study. Using RNA derived from matched peripheral blood, thyroid tissue, intrathyroidal lymphocytes (ITL), and IL-2R+ and IL-2R- subpopulations of ITL from Graves' patients, we conducted reverse transcription polymerase chain reaction/Southern blot analysis of TCR V alpha family usage. No evidence was found for V alpha restriction in the IL-2R+ subpopulation of ITL from eight patients, one of whom was operated on within 1 mo of diagnosis. We have further analyzed samples from seven of these patients by resolution on denaturing polyacrylamide gels. Typically, a single dominant band was amplified with surrounding minor bands in a normal distribution. Dominant and minor species were both the result of amplification from in frame V alpha transcripts, and the minor bands were separated in size by increments of 3 bp. We found no evidence for reduced heterogeneity of the V alpha transcripts in ITL or IL-2R+ ITL populations relative to peripheral blood in the vast majority of samples. This therefore suggests that there is little difference between the blood lymphocyte population and the activated T cell population in the thyroid in patients with Graves' disease, and indicates that in autoimmune thyroid disease, this method of analysis is not sufficient to distinguish between autoreactive and bystander T cell populations.

Amino Acid Sequence↗
PubMed1993

Characterization of an acute micromegakaryocytic leukaemia: evidence for the pathogenesis of myelofibrosis.

The current hypothesis for the pathogenesis of myelofibrosis involves the intramedullary release of growth factors from defective or abnormal megakaryocytes. We describe a case of an acute micromegakaryocytic leukaemia, in a patient with chronic myelofibrosis, that provides additional evidence for this concept. The micromegakaryocytes, which reached 223 x 10(9)/l, were characterized morphologically by both light and electron microscopy, immunocytochemically and by platelet peroxidase activity. The cells were shown to have a mature cytoplasm, containing alpha granules and the associated proteins; vWF:Ag, fibrinogen, fibronectin and protein S. DNA analysis, by both a Seescan Solitaire Plus image analysis system and flow cytometry, revealed nuclear immaturity, with 92% of cells being diploid. Serum markers of connective tissue synthesis, namely carboxy terminal peptide of procollagen I (PICP), procollagen terminal peptide III (PIIIP) and laminin all increased significantly following transformation and were associated with an increase in platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta). These observations support the current hypothesis for bone marrow fibrosis formation and provide, for the first time, a link between in vivo growth factor release, bone marrow stromal turnover and megakaryocyte mass. In addition, the release of biologically active TGF-beta may explain both the increased fibronectin and angiogenesis characteristic of myelofibrotic bone marrow.

Female↗