PubMed Health⌕ Search

Biomedical subjects

D Barnett

Publications and source records attributed to D Barnett.

At least 19 recordsLinked to original sources
PubMed2004

Evaluation of leukocyte stabilisation in TransFix-treated blood samples by flow cytometry and transmission electron microscopy.

In this report, we have evaluated the effects of a TransFix-based stabilisation technique on leukocyte scatter characteristics, immunophenotyping, membrane permeability, absolute cell counting and morphology to extend previously reported flow cytometric data focused on the lymphocyte population. We show that scatter characteristics, immunophenotyping and absolute cell counting are well preserved, particularly in the lymphocyte population. Nevertheless, a general increase in membrane permeability, evaluated by propidium iodide (PI) uptake, was observed in TransFix-treated leukocyte subsets. Ultrastructural observations show selective morphological preservation (up to 10 days of storage) of lymphocytes and, to a lesser extent, of monocytes. In contrast, granulocytes have necrosis-like features, although the plasma membrane seems well preserved. Therefore, electron microscopy observations reflect modifications induced in different cell populations as evidenced by flow cytometry (FC). The data indicate that this short-term stabilisation method is particularly suitable for the analysis of human lymphocytes and it is a good procedure for quality control programmes for inter- and intra-laboratory performance evaluation; good results are obtained with respect to antigen definition and absolute cell counting procedures. Any apoptotic pathways in leukocyte subsets are blocked for at least 10 days.

Adult↗
PubMed2004

Diagnosis of plasma cell leukaemia: findings of the UK NEQAS for Leucocyte Immunophenotyping scheme.

The diagnosis of plasma cell leukaemia, a rare disorder with an aggressive clinical course and poor prognosis, is not always straightforward and may be dependent on the results of immunophenotyping. Samples from two cases of plasma cell leukaemia have been issued by the UK NEQAS for Leucocyte Immunophenotyping Scheme during the last 5 years and on each occasion a significant number of laboratories failed to make the correct diagnosis. The details of the two samples issued and the results of both surveys are presented. The data highlights the need to adhere to guidelines for immunophenotyping, with respect to using the correct antibody panels, the importance of data interpretation in conjunction with morphological appearance as well as the need to participate in external quality assurance schemes.

Antigens, Surface↗
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

The development of a national standardized approach for the enumeration of residual leucocytes in blood components.

BACKGROUND AND OBJECTIVES: The UK Blood Transfusion Services implemented universal leucocyte depletion of the blood supply in November 1999. To provide statistical process monitoring of these processes, automated methods were introduced to count residual leucocytes (white blood cells) in blood components. MATERIALS AND METHODS: Initially in the National Blood Service (NBS) England, protocols were standardized on the use of LeucoCount reagents with either Becton-Dickinson or Beckman Coulter flow cytometers. RESULTS: Standardization of protocols resulted in a decreased intersite variability of red cell samples (from 36% to 9% at a level of 11 and 10 cells/ micro l, respectively), and 100% of sites (n = 11) fulfilled the validation criteria. However, we also evaluated the use of alternative reagents with the result that reagents from either Becton-Dickinson or Beckman Coulter, used on either a Becton-Dickinson or Beckman Coulter flow cytometer, passed our validation criteria. CONCLUSIONS: It is critical to include samples from filtered products containing white blood cells in validations of leucocyte enumeration methodology, as results may differ between methods using these samples but not using spiked or fixed material. Standardized gating strategies and optimization methods for flow cytometers are critical for obtaining equivalent results with different reagents and instruments.

Blood Banks↗
PubMed2001

Clinical effectiveness and cost effectiveness of zanamivir (Relenza): translating the evidence into clinical practice, a National Institute for Clinical Excellence view.

The UK National Institute for Clinical Excellence (NICE) is charged with the duty of providing informed guidance on clinical practice (clinical effectiveness and cost effectiveness) to patients and health professionals. The Appraisal Committee through its process of review of evidence advises NICE on the clinical effectiveness and cost effectiveness of new and existing technologies and their appropriate use within the National Health Service in England and Wales. The appraisal process takes into account both published and unpublished evidence as well as input from professional and patient and carer groups when coming to its decisions. The appraisal of a new technology often has to bridge the gap between the evidence required for licensing purposes and that needed to provide pragmatic advice to practising clinicians. The appraisal of zanamivir (Relenza) is an excellent working example of this difficult and important process.

Antiviral Agents↗
PubMed2001

Affordable CD4+ T cell counts by flow cytometry. II. The use of fixed whole blood in resource-poor settings.

We tested the feasibility and precision of affordable CD4+ T cell counting in resource-poor settings using a recently standardised fixative, TransFix in whole blood (WB) by flow cytometry (FCM). The precision of the assays was established under optimal conditions for single-platform FCM such as the volumetric CytoronAbsolute and the bead-based FACSCan. Fresh WB samples from HIV-seropositive and seronegative patients were tested in Tanzania and South Africa, fixed and sent to the UK for reanalysis 7 days later. Correlation, bias and limits of agreements were analysed by linear regression and the Bland-Altman test. Absolute CD4+ T cell counts remained stable for at least 10 days when TransFix was added to WB in 1:10 dilution at 20-25 degrees C, and for 7 days when added in 1:10 or 1:5 dilution to samples stored to mimic 'tropical' conditions at 37 degrees C. Higher temperatures such as 42 degrees C were tolerated for only short periods since the recovery had decreased to 63% by day 3. The reproducibility of lymphocyte subset analysis remained unchanged by TransFix with coefficient of variations <6% for all T cell subsets. Absolute CD4+ T cell counts and CD4+ T cell % values on fixed samples in the UK showed a high correlation with the results using fresh samples in Tanzania (r=0.993 and 0.969, respectively) and with the samples handled in Johannesburg (r=0.991 and 0.981) with minimal bias. Primary CD4 gating using only a single CD4 antibody also remained accurate in TransFixed samples (r=0.999). Thus, TransFix permits optimal fixation and transport of WB samples in the developing world for FCM to local regional laboratories and for quality assurance in international centres. When used together with inexpensive primary CD4 gating, TransFix will allow reliable and affordable CD4+ T cell counting by FCM in resource-poor settings.

Adult↗
PubMed2001

Sex with children is abuse: Comment on Rind, Tromovitch, and Bauserman (1998).

B. Rind, P. Tromovitch, and R. Bauserman (1998) reported a meta-analysis of the relation between sexual abuse in childhood and adolescence and psychological functioning among college students. Several aspects of their work have proven to be highly controversial, including their assertion that the relation between child sexual abuse and adjustment is quite small and their questioning of whether child sexual abuse should be labeled abuse in scientific inquiry. In this commentary, the authors summarize the controversy that has ensued, place it in a historical context, discuss the limitations of B. Rind et al.'s findings, and critique the manner in which those findings are presented. The authors also argue for the appropriateness of the term abuse and for scientific terminology that reflects rather than contradicts consensual public morality.

Child↗
PubMed2001

Low level leucocyte counting: a critical variable in the validation of leucodepleted blood transfusion components as highlighted by an external quality assessment study.

Leucocyte counts of < 5 x 106 per blood transfusion product are currently recommended in the UK in order to reduce transfusion-related infections and febrile reactions. Routine leucocyte depletion, however, requires the development of reliable internal and external quality assurance (EQA) programmes. We report preliminary findings from the UK NEQAS for Low-Level Leucocyte Counting from 18 UK Transfusion Centres over a four month period. Data analysis showed that the IMAGN 2000 had the lowest CVs (range 7.5-36%, mean 16.7) for samples with counts of 5-30 cells/microl when compared to the flow cytometric (range 13.8-88%, mean 29.5) and Nageotte methods (range 20.6-117%, mean 61.8). In addition, laboratories using commercial nuclear stains (LeucoCOUNTTM) had consistently lower CVs than those using 'in-house' propidium iodide staining methods. Important differences in flow cytometric gating strategies were also identified. This study highlights the current variability in low level leucocyte counting, especially within the critical range of 5-30 cells/microl (equating to < 5 x 106/l). The acceptance of consensus protocols, including gating strategies and nuclear staining techniques, is required to reduce the observed interlaboratory variation. Finally, we demonstrate that stabilized blood preparations can be successfully used to provide a national/international low-level leucocyte EQA scheme.

Blood Component Transfusion↗
PubMed2001

UK NEQAS for leucocyte immunophenotyping: the first 10 years.

In the past decade, cellular immunophenotyping has become a new discipline in diagnostic haematology and immunology, and is invaluable in the rapid diagnosis of leukaemia and monitoring disease progression in human immunodeficiency virus infected individuals. The introduction of bench top flow cytometers has meant that immunophenotyping is now also used for the quantitation of CD34(+) peripheral blood stem cells (PBSCs) to ensure the correct timing and adequacy of haematopoietic progenitor cell harvests. Furthermore, flow cytometry has become an important tool for the counting of leucocytes in blood components after leucocyte depletion. Because this new discipline is now such a major diagnostic and prognostic tool in the clinical arena, its use must be subject to both internal and external quality control. Such a requirement was first recognised as early as 1986 when an Inter-Regional Quality Assessment Scheme (IRQAS) was initiated for laboratories that undertook the immunocytochemical diagnosis of leukaemia using the alkaline phosphates anti-alkaline phosphatase technique. This programme began with around 25 UK laboratories. In 1990, after the introduction of two more programmes (one for leukaemia diagnosis using UV microscopy and latterly flow cytometry, and one for the enumeration of CD4(+) T cells) the IRQAS achieved UK National External Quality Assessment Scheme (UK NEQAS) status and changed its title to UK NEQAS for Leucocyte Immunophenotyping. In the past decade the once small IRQAS programme has evolved into the largest international scheme of its kind, providing EQA to over 650 laboratories world wide for leukaemia immunophenotyping, lymphocyte subset analysis, PBSCs, and more recently low level leucocyte counting. Over the years, this EQA programme has highlighted important problems, such as the inappropriate use of fluorochromes and antibody titre, and the identification of effective gating strategies, all of which have contributed directly to the high interlaboratory variations seen in cellular immunophenotyping. Furthermore, particularly in absolute counting of lymphocyte subsets, PBSCs, and the enumeration of low numbers of leucocytes, UK NEQAS for Leucocyte Immunophenotyping programmes have been instrumental in highlighting the differences that occur between single and dual platform flow cytometric technologies. As a result of these findings, UK NEQAS for Leucocyte Immunophenotyping has helped to reduce the variation seen on an interlaboratory basis and enabled greater standardisation both in the UK and internationally. These advances have been attributable to the development, by UK NEQAS for Leucocyte Immunophenotyping, of a unique whole blood stabilising process that ensures the retention of the physical characteristics (both light scatter and antigenic profile) required of cells to ensure successful cellular immunophenotyping. This major technological advancement has enabled the distribution of specimens for EQA purposes on a global scale that have minimal matrix effect and behave in a manner identical to fresh blood for several months after stabilisation.

Antigens, CD34↗
PubMed2000

Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis.

The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)+ individuals (CD4+ T- lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34+ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single- or multiple-color cell staining and logical gating strategies. These can be accomplished using single- or dual-platform counting technologies employing cytometric methods. Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies. Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized. Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources.

Antigens, CD34↗
PubMed2000

Negative reactivity and attachment: Down syndrome's contribution to the attachment-temperament debate.

This longitudinal study investigated the relation between negative reactivity and attachment in children with Down syndrome (DS). Specifically, we examined whether extremely low negative reactivity is associated with the enactment and organization of attachment behaviors. To address this issue 30 infants with DS (13 female, 17 male) were observed in Ainsworth's Strange Situation at 19 and 27 months. The intensity and duration of distress reactions were coded in 15-s intervals throughout each Strange Situation episode. Children's distress intensity during separations from caregivers was used to place children into Low versus Moderate/High Negative Reactivity groups. In addition, ratings of infants' social interactive behaviors (contact maintenance, proximity seeking, distance interaction, resistance, and avoidance) were made for each episode and used to assess the quality of infants' attachment relationships. The data indicated that there are high rates of Type A and D insecure attachments within the DS population at each age studied. However, low negative reactivity was not consistently related to the activation of the attachment behavioral system, or associated with insecure attachment relationships. Alternative explanations for the high rate of insecure attachments are discussed.

Affect↗
PubMed2000

Reduction of intra- and interlaboratory variation in CD34+ stem cell enumeration using stable test material, standard protocols and targeted training. DK34 Task Force of the European Working Group of Clinical Cell Analysis (EWGCCA).

The European Working Group on Clinical Cell Analysis (EWGCCA) has, in preparation for a multicentre peripheral blood stem cell clinical trial, developed a single-platform flow cytometric protocol for the enumeration of CD34+ stem cells. Using this protocol, stabilized blood and targeted training, the EWGCCA have attempted to standardize CD34+ stem cell enumeration across 24 clinical sites. Results were directly compared with participants in the UK National External Quality Assessment Scheme (NEQAS) for CD34+ Stem Cell Quantification that analysed the same specimens using non-standardized methods. Two bead-counting systems, Flow-Count and TruCount, were also evaluated by the EWGCCA participants during trials 2 and 3. Using Flow-Count, the intralaboratory coefficient of variation (CV) was </= 5% in 39% of the laboratories (trial 1), increasing to 65% by trial 3. Interlaboratory variation was reduced from 23.3% (trial 1) to 10.8% in trial 3. In trial 2, 70% of laboratories achieved an intralaboratory CV </= 5% using TruCount, increasing to 74% for trial 3; the interlaboratory CV was reduced from 23.4% to 9.5%. Comparative analysis of the EWGCCA and the UK NEQAS cohorts revealed that EWGCCA laboratories, using the standardized approach, had lower interlaboratory variation. Thus, the use of a common standardized protocol and targeted training significantly reduced intra- and interlaboratory CD34+ cell count variation.

Antigens, CD34↗
PubMed2000

Standardization of lymphocyte antibody binding capacity - a multi-centre study.

As quantitative flow cytometry is being increasingly used to characterize non-malignant and malignant disorders, interlaboratory standardization becomes an important issue. However, the lack of standardized methods and process controls with predefined antibody binding capacity values, limits direct interlaboratory comparison. The present study has addressed these issues using a stable whole blood product and a standardized antigen quantification protocol. It was demonstrated that: (i) a standard technical protocol can result in a high degree of interlaboratory concordance; (ii) interlaboratory variation of less than 12% can be achieved for CD4 antibody binding capacity values; and (iii) stable whole blood can be used as a process control with predefined antibody binding capacity values. Furthermore, using such an approach, a normal range was established for CD3, CD4 CD8 and CD19. These antigens appear to be expressed in a hierarchical manner, a factor that could be used as a procedural quality control measure.

Antigens, CD↗
PubMed2000

Phase I study of CBT-1 and Taxol in patients with Taxol resistant cancers.

CBT-1, a natural product, was studied as an MDR modulator with Taxol (135 mg/m2) in an escalating dose Phase I clinical trial. CBT-1 was administered orally at doses from 300 mg/m2 to 500 mg/m2 daily x 7. The MTD was determined to be 500 mg/m2 with moderate nausea and occasional emesis. Side effects were mainly attributable to Taxol rather than the study drug. A total of 18 patients were registered on study with only one patient determined to be intolerant of CBT-1 due to nausea and emesis. In this Phase I study four patients (3 breast, 1 NSCLC) remained stable for greater than two cycles of treatment. No complete or partial responses were seen in this Taxol resistant population of patients with advanced cancer.

Adult↗
PubMed2000

The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection.

We report the cloning of the gene encoding the 32-kDa lipoprotein, designated LipL32, the most prominent protein in the leptospiral protein profile. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment to design an oligonucleotide probe. A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 5.0-kb DNA fragment which contained the entire structural lipL32 gene was identified. Several lines of evidence indicate that LipL32 is lipid modified in a manner similar to that of other procaryotic lipoproteins. The deduced amino acid sequence of LipL32 would encode a 272-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by a lipoprotein signal peptidase cleavage site. LipL32 is intrinsically labeled during incubation of L. kirschneri in media containing [(3)H]palmitate. The linkage of palmitate and the amino-terminal cysteine of LipL32 is acid labile. LipL32 is completely solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL32 exclusively into the hydrophobic, detergent phase, indicating that it is a component of the leptospiral outer membrane. CaCl(2) (20 mM) must be present during phase separation for recovery of LipL32. LipL32 is expressed not only during cultivation but also during mammalian infection. Immunohistochemistry demonstrated intense LipL32 reactivity with L. kirschneri infecting proximal tubules of hamster kidneys. LipL32 is also a prominent immunogen during human leptospirosis. The sequence and expression of LipL32 is highly conserved among pathogenic Leptospira species. These findings indicate that LipL32 may be important in the pathogenesis, diagnosis, and prevention of leptospirosis.

Acylation↗