bluuebunny/crossref_metadata_2025 huggingface dataset:
Injection of dsRNA and Electroporation in Avian Embryos
Authors: Stoeckli Esther T.
Published: September 2006

INTRODUCTION

This protocol describes a method for in ovo transfection of avian embryos with double-stranded RNA (dsRNA). The dsRNA is injected into the spinal cord of the embryo. Subsequent electroporation facilitates the cellular uptake of the dsRNA molecules. It may be necessary to optimize the stage of the embryo and the electroporation procedure to improve the effectiveness of in ovo RNAi—cell competence changes with differentiation. The half-life of the target protein product may also affect the time point of injection and electroporation—proteins with slow turnover may require RNAi at earlier stages, preferably before the onset of expression. Check the quality of the dsRNA used for injections and make sure that the buffer used for injection does alter development or survival of the embryo. Tris buffers and buffers containing glycerol are not compatible with in vivo injections. Salt concentration and pH should be in the physiological range. Always use the same buffers for injection and electroporation of test and control embryos.
Staining Yeast
Authors: Harlow Ed , Lane David
Published: September 2006

INTRODUCTION

Staining yeast cells for the presence and localization of antigens has been particularly challenging because of several factors. The yeast cells are small, making the resolution of any antigen difficult; they have a thick cell wall that antibodies cannot penetrate and that is difficult to remove; and they grow in suspension, making handling difficult. Background problems can be especially severe, particularly with polyclonal antibodies, because many antisera contain antibodies to yeast cell-wall components. The difficulties encountered in localizing antigens in yeast can be overcome by using fluorochrome-labeled secondary antibodies, controlled enzymatic removal of the cell wall, and by binding the cells to a solid phase (e.g., poly-L-lysine-coated slides).
Fixing Caenorhabditis elegans in Bouin’s fixative
Authors: Harlow Ed , Lane David
Published: September 2006

INTRODUCTION

Bouin’s fixative is a particularly good choice for worms because it penetrates dense tissues well and is extremely good for fixing antigens. Like all strong fixatives, however, it is unsuitable for some antibody-antigen pairs. In such cases, the length of time in the Bouin’s fixative can be shortened, or paraformaldehyde fixation can be used instead.
Antibody Addition and Detection for Staining Caenorhabditis elegans
Authors: Harlow Ed , Lane David
Published: September 2006

INTRODUCTION

The most common method of detection for staining worms is to use fluorochrome-labeled reagents, because of the desire for high resolution of the antigen location. Extreme care should be used to identify and verify positive reactions, however, because cross-reactions are common. Counterstaining is essential for examining worms by immunofluorescence and is used to identify the exact cell in which an antigen appears. Methods for counterstaining include labeling all cells with a fluorescent dye that is specific for nucleic acids (e.g., DAPI or propidium iodide) and using GFP driven by tissue-specific promoters.
Preparing Late Whole-Mount Drosophila Embryos for Immunostaining
Authors: Harlow Ed , Lane David
Published: September 2006

INTRODUCTION

The choice of fixation and permeabilization methods for examining flies depends on the stage of development. Early and late embryos are treated with a mixture of organic solvents, formaldehyde, and alcohols. The cuticles of late-stage embryos (17-22 hours or until hatching) are usually opened by sonication, as described here. Tissues from later stages of development are normally dissected by hand and then fixed and stained in a standard paraformaldehyde/detergent combination.
Unmasking Hidden Epitopes Using the Microwave Oven
Authors: Harlow Ed , Lane David
Published: September 2006

INTRODUCTION

Several methods have been developed to “retrieve” antigens that have been masked by fixation. The principle behind using the microwave oven method described here is to use extended periods of heat to break some of the subcellular structures that block antibody access. Be aware that any of the antigen retrieval methods should be avoided wherever possible, because they may introduce artifactual false-positive staining. Methods for antigen retrieval are specific for each antibody/antigen combination. No attempts to establish quantitative data following these methods should be considered.
Immunoprecipitation: Purifying the Immune Complexes
Authors: Harlow Ed , Lane David
Published: September 2006

INTRODUCTION

In this protocol, immune complexes are formed by the addition of antibodies to cell lysates. The antibodies bind to their cognate antigen from a lysate. Protein A or protein G beads are then added to the solution containing antibody-antigen complexes, and the proteins that do not bind to the beads are removed by washing. The correct controls for immunoprecipitation should include nonimmune antibodies that are as close as possible to the specific antibody; for example, polyclonal serum should be compared to polyclonal serum from the same species (ideally a prebleed from the same animal used for immunization). For monoclonal antibodies, the control must be from the same source as the specific antibody. Do not use tissue culture supernatants from parental myeloma cells as they do not contain antibodies. Suitable control hybridoma cell lines can be obtained from the American Type Culture Collection and the European Collection of animal cell cultures.
Drying SDS-Polyacrylamide Gels
Authors: Sambrook Joseph , Russell David W.
Published: September 2006

INTRODUCTION

This protocol describes a method for drying SDS-polyacrylamide gels. Gels containing proteins radiolabeled with 35S-labeled amino acids must be dried before autoradiographic images can be obtained. Nonradioactive gels can also be preserved by drying.
FNA of breast fibroadenoma: observer variability and review of cytomorphology with cytohistological correlation
Authors: Kollur S. M. , El Hag I. A.
Published: September 2006

Objective:  To determine the observer variability in reporting fibroadenoma of the breast by fine needle aspiration (FNA) and to review the cytomorphological features of the lesion with cytohistological correlation.

Methods:  Retrospective analysis of FNA smears from 110 cases diagnosed as fibroadenoma of which surgical pathology follow‐up was available in 33. Two pathologists were asked to categorize smears from 67 cases of breast lesions while blinded to the clinical finding as fibroadenoma, epithelial hyperplasia (usual and atypical) and malignant. All fibroadenoma (33) and cancer (15) cases were biopsy‐proven. The same set of slides was re‐circulated to one of the pathologists, and his first and second round results were compared.

Results:  Pre‐review cytohistological correlation was attained in 32 of 33 cases of fibroadenoma (97%). The overall agreement between the two observers was 87% [Kappa = 0.74, 95% confidence interval (CI) 0.72–0.76]. Cytohistological correlation was achieved in 26 of 33 (79%) cases. Intra‐observer agreement was 91% (Kappa = 0.82, 95% CI 0.89–0.93) with cytohistological correlation in 29 of 33 (87%) cases. Causes of diagnostic errors included marked dissociation, pleomorphism, poorly cellular smears from hyalinized fibrodenoma, lacational changes and apocrine metaplasia with cystic changes. Multinucleated giant cells were frequently encountered in FNA smears from fibroadenoma (31.8%), but in none of the lumpectomy specimens. Their histiocytic nature was suggested by immunohistochemistry.

Conclusion:  FNA was a highly sensitive method for the diagnosis of fibroadenoma. Current cytological criteria were reliable and gave high inter‐ and intra‐observer reproducibility.
The diagnostic value of on‐site cytopathological evaluation and cell block preparation in fine‐needle aspiration cytology of liver masses
Authors: Ceyhan K. , Kupana S. A. , Bektaş M. , Coban S. , Tuzun A. , Cınar K. , Soykan I. , Ormeci N. , Erdogan N. , Erekul S. , Kose K.
Published: September 2006

Objective:  The aims of this study were to evaluate the typing accuracy of conventional smear (CS), cell block (CB) preparations and combined use of both procedures (CS + CB) for the diagnosis of hepatic malignancies and to determine whether immediate on‐site cytopathological evaluation improves the diagnostic yield of liver fine‐needle aspiration cytology (FNAC).

Methods:  Ultrasound‐guided FNABs were performed on 323 consecutive cases with liver masses between December 2002 and December 2004. Histologically and/or clinically correlated 167 cases were included in the study. Preliminary FNAB results, results of CS, CB, and combined use of CS and CB were compared regarding diagnostic sensitivity, specificity, and accuracy for the diagnosis of malignancy. Subtyping accuracies of different methods were also compared.

Results:  The sensitivity of on‐site cytopathological examination and CS were both 92.8%. The sensitivity of CS + CB was slightly better than that of CB (93.5% versus 84.8%). Specificity of all procedures was achieved 100%. Diagnostic accuracy of on‐site cytopathological evaluation, CS, CB, and CS + CB were 93.9%, 93.9%, 87.2%, and 94.5%, respectively. A specific subtype diagnosis of malignant tumours could be rendered accurately on the basis of preliminary diagnosis in 71%, CS in 75.4%, CB in 78.3% and combined approach in 92% of cases. In terms of typing accuracy, 87.5% of HCCs, 93.2% of adenocarcinomas, 92.3% of neuroendocrine carcinomas, 100% of lymphomas and 100% of other malignant tumours were correctly subclassified in the final cytopathological diagnosis. The agreement between preliminary diagnosis and final cytopathological diagnosis was 77.2%.

Conclusion:  With use of on‐site cytopathological evaluation and combined use of CS and CB, the diagnostic accuracy of liver tumours approaches 100% and also significantly improve diagnostic and subtyping accuracy of liver malignancies.
Diagnosing amyloid goitre with thyroid aspiration biopsy
Authors: Özdemir B. H. , Uyar P. , Özdemir F. N.
Published: September 2006

Objective:  The aim of the study was to evaluate the value of fine needle aspiration biopsy of the thyroid as a tool for diagnosing amyloid goitre and assess how amyloidosis affects thyroid tissue and thyroid function.

Methods:  Clinical and laboratory evaluation of 50 patients with secondary systemic amyloidosis was done, and goitre was found in 38 of them. All 38 patients underwent thyroid aspiration biopsy. Tissue samples were stained with haematoxylin and eosin, May‐Grünwald–Giemsa, crystal violet and Congo red.

Results:  Of the 38 cases of amyloid goitre, 10 showed euthyroid sick syndrome, two showed primary hyperthyroidism, two showed hypothyroidism and one showed subacute thyroiditis. The serum levels of thyroid hormones and thyroid‐stimulating hormone were normal in the remaining patients. Thirty‐five of the 38 patients (92%) showed amyloidosis after thyroid aspiration. One of these patients had papillary carcinoma in addition to amyloid goitre. Ten patients underwent subtotal thyroidectomy, and one patient underwent total thyroidectomy after aspiration. Microscopic evaluation revealed that the thyroid parenchyma in all patients was largely replaced with amyloid and adipose tissue.

Conclusion:  Fine needle aspiration of the thyroid is a valuable and sensitive method for diagnosing amyloid goitre, especially because it is a safe and easily performed procedure. Further, amyloid goitre has no significant influence on thyroid function even when it causes extensive parenchyma replacement.
Acute and chronic hypoxia as well as 7‐day recovery from chronic hypoxia affects the distribution of pulmonary mast cells and their MMP‐13 expression in rats
Authors: Vajner Luděk , Vytášek Richard , Lachmanová Věra , Uhlík Jiří , Konrádová Václava , Novotná Jana , Hampl Václav , Herget Jan
Published: September 2006

Chronic hypoxia results in pulmonary hypertension due to vasoconstriction and structural remodelling of peripheral lung blood vessels. We hypothesize that vascular remodelling is initiated in the walls of prealveolar pulmonary arteries by collagenolytic metalloproteinases (MMP) released from activated mast cells. Distribution of mast cells and their expression of interstitial collagenase, MMP‐13, in lung conduit, small muscular, and prealveolar arteries was determined quantitatively in rats exposed for 4 and 20 days to hypoxia as well as after 7‐day recovery from 20‐day hypoxia (10% O2). Mast cells were identified using Toluidine Blue staining, and MMP‐13 expression was detected using monoclonal antibody. After 4, but not after 20 days of hypoxia, a significant increase in the number of mast cells and their MMP‐13 expression was found within walls of prealveolar arteries. In rats exposed for 20 days, MMP‐13 positive mast cells accumulated within the walls of conduit arteries and subpleurally. In recovered rats, MMP‐13 positive mast cells gathered at the prealveolar arterial level as well as in the walls of small muscular arteries; these mast cells stayed also in the conduit part of the pulmonary vasculature. These data support the hypothesis that perivascular pulmonary mast cells contribute to the vascular remodelling in hypoxic pulmonary hypertension in rats by releasing interstitial collagenase.
Differences in the quantitative and qualitative performance of a calcium‐specific food frequency questionnaire across age and sex
Authors: Magkos F. , Manios Y. , Babaroutsi E. , Sidossis L. S.
Published: September 2006

Objective  To examine putative differences in the quantitative and qualitative performance of a food frequency questionnaire (FFQ) for assessing dietary calcium intake across age and sex in the Greek population.

Materials and methods  A total of 351 children (189 girls and 162 boys, aged 11.9 ± 1.2 years), 260 adults (192 women and 68 men, aged 29.6 ± 2.7 years) and 390 elderly individuals (317 women and 73 men, aged 68.6 ± 4.6 years) were recruited. Estimates of calcium intake from the 30‐item FFQ were compared with those from a multi‐pass 24‐h recall.

Results  The FFQ significantly underestimated mean calcium intake in all age groups and both sexes (P < 0.05). The magnitude of underestimation, however, was greater in adults (−207 ± 344 mg day−1), less in the elderly (−137 ± 310 mg day−1) and even less in children (−74 ± 340 mg day−1; P < 0.025), with no differences between sexes. Calcium intakes by the two methods were positively and significantly correlated in all study groups (r = 0.536–0.739, P < 0.001). Cohen's weighted kappa statistic ranged from 0.39 to 0.57, indicating moderate agreement between the two methods. The 95% limits of agreement were comparably wide across age and sex (boys: −762, 585 mg day−1; girls: −747, 624 mg day−1; adult men: −972, 505 mg day−1; adult women: −867, 412 mg day−1; elderly men: −858, 486 mg day−1; elderly women: −732, 480 mg day−1). A significant association between age, sex and the classification of individuals as true/false positive/negative was detected (P < 0.001), implying that sensitivity, specificity, positive and negative predictive values of the FFQ were not independent of the age and sex of the participants. Gross misclassification by the FFQ ranged from 0% to 4.2%, whereas 75.3–87.3% of the subjects were correctly classified. In this respect, the FFQ performed similarly across the study groups (P = 0.065). Without controlling for age, however, gross misclassification appeared to be higher in females than in males (3.2% versus 0.7%, respectively, P = 0.048).

Conclusions  There may be several significant differences in the quantitative and qualitative performance of a calcium‐specific FFQ across age and sex. This should be taken into account when attempting to evaluate dietary calcium intake in men and women or in different age groups, as some of the differences between study groups may actually be due to the different response of these groups to the FFQ.
Unidentified under‐nutrition: dietary intake and anthropometric indices in a residential care home population
Authors: Leslie W. S. , Lean M. E. J. , Woodward M. , Wallace F. A. , Hankey C. R.
Published: September 2006

Background  Research investigating the nutritional status of older people in residential care homes is scant.

Objective  To determine the anthropometric measures and dietary intakes of older people in this setting as a basis for future intervention studies.

Methods  Dietary intake was assessed using 3‐day‐weighed food records, nutritional status was evaluated using anthropometric measurements (knee height to predict standing height, and body weight). Catering provision was assessed using a computer‐based menu assessment tool (CORA).

Results  Mean body mass index (BMI) for the 34 participants was 22.2 kg m2 (range 14.5–34.4). Six participants (17.6%) had a BMI ≤18.5 kg m2 with a further seven identified as having a BMI >18.5 but <20 kg m2. Only two subjects with BMI <18.5 kg m2 were prescribed oral supplements. In both men and women, recorded mean energy intakes were below current estimated average requirements by 24% and 22% respectively.

Conclusion  Despite adequate food provision, under‐nutrition was prevalent and, in the majority of cases, unidentified and untreated. A larger study is warranted to investigate whether improved nutritional intake is achievable through dietary modification. These data indicate that a sample size of around 60, with 90% power and at the 5% significance level, is required to detect a difference of 1674 kJ between groups of residents in an intervention study following a cluster randomized design.
Effects on mortality of abciximab in ST‐elevation myocardial infarction treated with percutaneous coronary intervention including stent implantation
Authors: TORNVALL P. , NILSSON T. , LAGERQVIST B.
Published: September 2006

Objectives.  To investigate the effects of abciximab on mortality in ST‐elevation myocardial infarction (STEMI) treated with percutaneous coronary intervention (PCI) including stent implantation.

Design.  Meta‐analysis of three selected randomized studies and analysis of data from the Swedish Coronary Angiography and Angioplasty Registry (SCAAR).

Subjects.  Pooled data from randomized studies containing in total 1736 patients undergoing PCI with stent implantation because of STEMI with duration between symptom and treatment <12 h, and 7436 patients from SCAAR treated with PCI because of STEMI (52% treated with abciximab) in Sweden 2000–2004.

Results.  Analyses of pooled data showed that abciximab was associated with a decreased risk of reinfarction [odds ratio (OR) 0.38] and urgent target vessel revascularization (OR 0.38) at 30 days. No effect was seen on mortality at 30 days or 6 months. Multivariate analysis of data from SCAAR showed that abciximab reduced the risk of death during 14 months of follow‐up (hazard ratio 0.82).

Conclusions.  The results are encouraging and support the ACC/AHA and ESC recommendation to use abciximab in treatment of STEMI with PCI including stent implantation. Considering that the pooled results from previous trials showed no effect of abciximab on mortality and the registry part of the present study was observational, the results encourage carrying out new randomized studies of abciximab in STEMI treated with PCI, including stent implantation, with sufficient size and length of follow‐up.
The solution structure of the invasive tip complex from Afa/Dr fibrils
Authors: Cota Ernesto , Jones Celine , Simpson Peter , Altroff Harri , Anderson Kirstine L. , du Merle Laurence , Guignot Julie , Servin Alain , Le Bouguénec Chantal , Mardon Helen , Matthews Stephen
Published: September 2006

Afa/Dr family of adhesins are produced by pathogenic Escherichia coli strains that are especially prevalent in chronic diarrhoeal and recurrent urinary tract infections. Most notably, they are found in up to 50% of cystitis cases in children and 30% of pyelonephritis in pregnant women. Afa/Dr adhesins are capped surface fibrils that mediate recognition of the host and subsequent bacterial internalization. Using the newly solved three‐dimensional structure of the minimal invasive complex (AfaDE) combined with biochemical and cellular assays, we reveal the architecture of the fibrillar cap and identify a novel mode of synergistic integrin recognition.
SI08 Protein Misfolding Cyclic Amplification of Disease‐Associated Prion Protein Using Human Autopsy Tissues
Authors: Jones M. , Peden A. , Ironside J. , Prowse C. , MacGregor I. , Head M.
Published: September 2006

The protein misfolding cyclic amplification (PMCA) technique allows for the in vitro production of protease‐resistant prion protein (PrPres) using a seed of disease‐associated prion protein (PrPsc) to convert normal cellular prion protein (PrPc). This system has been developed using brain tissue from scrapie infected hamsters to provide the seed and normal uninfected hamster brain to provide PrPc substrate and the required conversion factors. These are mixed and subjected to repeated rounds of sonication and incubation. Extremely low concentrations of PrPsc, such as those present in blood, have been detectable. In order to adapt this method for use in CJD research and in the development of diagnostic tests for human prion diseases it will be necessary to identify a source of human PrPc and co‐factors that support amplification of human PrPsc. We have tested whether human autopsy tissues held at the National CJD Surveillance Unit support PMCA. Using a seed extract from variant CJD brain mixed with homogenates of nonCJD neurological control brain we have been able to effect a >600‐fold increase in the detection limit of PrPres, as determined by Western Blotting. Further development of this technique in concert with high sensitivity PrPsc detection methods, such as conformation dependent immunoassay, may result in a method capable of acting as a confirmatory assay for disease‐associated prion protein in human tissues and biological fluids such as blood.
SI14 Stripped Red Cells–Efforts to Eliminate A and B Antigens from the Blood Supply
Authors: Olsson M. L.
Published: September 2006

Eliminating the risk for ABO‐incompatible transfusion errors and simplifying blood logistics by creating an ABO‐universal blood inventory is a challenging idea but not a new one. In the early 1980s Goldstein and co‐workers at the New York Blood Centre pioneered the field of enzymatic conversion of blood group B red blood cells (RBC) to group O. They used native or recombinant α‐galactosidase from green coffee beans to remove the immunodominant galactose residues terminally located in the carbohydrate chains found on group B RBC. These enzyme‐converted O (ECO) RBC survived normally in the circulation of individuals independent of the blood group of the recipient. Small infusions were escalated to single, multiple and repeated RBC units. A successful phase II clinical trial in patients was reported in 2000 by Kruskall et al. However, economically unfavourable doses of enzyme were required to convert a unit of blood due to the low pH optimum of the enzyme. Conversion of blood group A cells was never achieved due to lack of A‐converting enzymes working at RBC‐friendly conditions. Also, the chemical nature of group A antigens is more complex due to the A1/A2 subgroups. Following screening of >2500 microorganisms we recently identified a novel family of bacterial exoglycosidases with specificities for the blood group A or B antigens and remarkably improved kinetic properties. Enzymatically converted group A and B RBC type as group O and can now be achieved under cost‐efficient and optimised conditions including neutral pH and room temperature incubation. Clinical trials to evaluate the safety and efficacy of ECO RBC are currently ongoing. Of the different strategies envisioned to create a universal blood component supply, the ECO concept is the only one for which clinical trials in humans have been performed. The presentation will review the current status of this exciting technology and its potential for introduction in the clinical component preparation laboratory.

The speaker is a Visiting Associate Professor at Harvard Medical School, Boston, MA, USA and also a scientific consultant to the R&D biotech company ZymeQuest, Inc., Beverly, MA, USA. He gratefully acknowledges the significant and dedicated work performed by numerous members of the ECO team at Copenhagen University, Harvard Medical School, Lund University Hospital Blood Centre and the scientific staff at ZymeQuest.
SI15 Operational Evaluation of a Prion Reduction Device
Authors: Nicholson G. S. , Thomas S. , Wiltshire M. , Haines M. , Scott J.
Published: September 2006

The National Blood Service has performed an operational evaluation of the Pall Prion Reduction Filter (PRF1BU) on behalf of the UK and Ireland Prion Reduction Working Group. The filter is a device for use on red cells and requires sterile connection to the red cell component. The evaluation took the form of three individual pieces of work which included: (i) an evaluation of in vitro immunohaematological effects on red cells following prion filtration; (ii) an operational assessment of the routine use of the Pall prion reduction filter within a blood processing department; and (iii) an evaluation of the storage characteristics of red cells after prion reduction, including irradiated and washed red cells. The results of these evaluations indicate that, with the exception of a significant loss of haemoglobin caused by retention of red cells in the prion filter, the process of filtration through the PRF1BU does not appear to be detrimental to either the functional or immunological characteristics of red cells as discernible from in vitro testing. It is clear from these results however, that prion reduction of red cells derived from ‘Top and Bottom’ (TAB) collection packs yields a final red cell component that will not comply with the EU Directive requirements for 40 g haemoglobin per pack. This is because the normal processing of TAB packs requires the removal of a buffy coat that directly reduces the haemoglobin content of the final red cell component. Prion reduced red cell components derived from ‘Top and Top’ (TAT) systems do not undergo buffy coat removal and prion filtered red cells produced from such starting material are able to comply with this EU Directive specification. In addition, 60% of prion reduced, thawed and washed TAT red cells did not comply with the EU Directive specification for haemoglobin content (36 g per pack).
SI16 Standardising the Red Cell Dose: Pros and Cons
Authors: Prowse C.
Published: September 2006

Unlike other prescription medicines, blood components are issued in variable doses. For example, last year's SNBTS data indicates a mean 55 g haemoglobin in additive red cell product but with a range from 36 to 88 g. With the advent of metered anticoagulant addition it is possible to collect donations to a given dose of Hb rather than a fixed volume using various apheresis equipment or the Macopharma ABC machine. Hogman and others have advocated that there would be advantages in providing red cells as a standard dose product, while Arslan has shown it may be possible to match red cell dose to patient size. Since patients as well as product vary in size at present pursuing a standard dose may have less impact than hoped. The usual rule of thumb that a single unit of red cells transfused to an adult should result in an increment in circulating haemoglobin of 1 g dL‐1 will be assessed against a range of patient blood volumes and the likely impact of a reduction in dose variation discussed.

References  Hogman C.F. & Meryman H.T. (2006) Red blood cells intended for transfusion: quality criteria revisited. Transfusion, 46, 137–42.

Arslan O, Toprak S, Arat M & Kayalak Y. (2004) Hb content‐based transfusion policy successfully reduces the number of RBC units transfused. Transfusion, 44, 485–8.
YS01 Protective Effect of HLA‐B57 on HCV Genotype 2 Infection in a West African Population
Authors: Chuang W. C.
Published: September 2006

Background  Recovery from Hepatitis C virus (HCV) infection is considered infrequent (<20%) in western populations but reaches 50% in West Africa where genotype 2 infection is predominant (Candotti J Virol 2003; 77: 7914).

Aim  To investigate cellular immune responses as alternative diagnostic of HCV infection and the possible involvement of viral or host genetic factors in recovery.

Methods  Samples from 104 Ghanaian blood donors screened with anti‐HCV rapid tests and enzyme immunoassay were collected between 2000 and 2005. HCV antibody screening was confirmed using genotype 2 recombinant core, E2 and NS3 proteins by Western blot. HCV viral load and genotype were determined. Cellular response to recombinant HCV genotype 2 proteins was tested by IFN gamma ELISpot on frozen cells. HLA genotype was determined by sequence specific oligonucleotide probes.

Results  A total of 104 donors were stratified in 37 chronic, 35 recovered infections and 32 false positive. 81% of subjects with chronic infections with RNA detected carried genotype 2 HCV RNA. Cellular immune response was investigated in 35 frozen peripheral blood mononuclear cell (PBMC) samples suitable for interferon‐gamma ELISpot assay. 12 confirmed recovered, one chronically infected and no false positive controls reacted to at least one recombinant protein. The magnitude of response was considerably higher in recovered cases. HLA‐B*57 was significantly more frequent in the group which had recovered from HCV infection compared with chronically infected subjects (P < 0.01, OR = 8.02). This allele is significantly more frequent in West Africa than in Europe or North America.

Discussion and conclusions  Cases classified as recovered by serological means were confirmed by the presence of T‐cells stimulated by viral proteins. Genotype 2 HCV tends to have lower viral load and better response to anti‐viral treatment suggesting that the local genotype might be more susceptible to immune control. However, genetic factors of the host might also influence the clinical outcome. It could be hypothesised that HLA‐B*5703 is the HLA molecule most efficient at presenting peptides from genotype 2 HCV in this population.
YS02 Screening and Characterisation of Novel Platelet Candidate Genes Derived from Genome‐Wide Association Studies for Function in Relation to Haemostasis and Thrombosis in the Model Organism Danio Rerio
Authors: O'Connor M. N. , Salles I. I. , Stemple D. , Deckmyn H. , Ouwehand W. H.
Published: September 2006

Complementary approaches of microarray, proteomics, genome wide association and platelet functional studies have identified novel platelet proteins which are candidates for playing a role in thrombosis or conferring risk of myocardial infarction. The function of many of the proteins encoded by these candidate genes is unknown, and will need to be determined. To address the need for high‐throughput screening of candidate genes in a model organism, we have investigated the suitability of the zebrafish (ZF; Danio rerio), a genetically tractable vertebrate with nucleated thrombocytes. It is known that mouse knockouts of critical pro‐thrombotic genes [e.g. coagulation factor (F) VIII, integrin αIIb (ITGA2B), or glycoprotein VI] have subtle phenotypes and do not bleed spontaneously. We hypothesised that the sensitivity of the ZF model to screen for loss of platelet function could be enhanced by concurrent knock down of the FVIII gene and a pro‐thrombotic platelet gene. To test this, the genes for FVIII, ITGA2B or both were silenced by antisense oligonucleotides (morpholinos). Embryos were then assessed for body plan and bleeding at 3 days post‐fertilization (dpf). In parallel, a thrombosis model using laser injury to initiate thrombus formation was developed. This permits time to adhesion (TTA), time to occlusion (TTO) and time to thrombolysis (TTL) to be determined in morpholino‐injected and wild type ZF. As expected, individual knockdown of the FVIII or ITGA2B genes did not lead to spontaneous bleeding. Knockdown of the ITGA2B gene did, however, result in morphological malformations. Simultaneous knockdown of both genes resulted in a clear and reproducible bleeding phenotype visible in ∼20% of embryo's. Using the laser injury model, venous thrombosis could be reproducibly induced in 3 and 6 dpf wild‐type ZF larvae with partial but not complete thrombolysis occurring within 5–10 min. In conclusion, ZF are a fast and informative way to screen for platelet function modifying genes.
YS04 Recombinant Mini‐Β3 Integrin Fragments for the Detection of HPA Antibodies
Authors: Stafford P. , Ghevaert C. , Campbell K. , Smith G. , Williamson L. , Huntington J. , Watkins N. , Ouwehand W.
Published: September 2006

Background  The β3 integrin [Glycoprotein (GP) IIIa] gene is highly polymorphic and encodes eight of the Human Platelet Antigen (HPA) systems. HPA immunisation is of clinical relevance in neonatal alloimmune thrombocytopenia (NAIT), post‐transfusion purpura (PTP) and platelet refractoriness. However, the current methods of antibody detection are cumbersome and rely upon the use of platelets from genotyped donors. In the case of the rare HPA antigens, obtaining donor platelets is particularly problematic. We therefore set out to develop an antibody screening assay that uses recombinant, soluble β3‐integrin fragments expressing the HPA‐1, –4, –6, –7, –10, –11 and –16 epitopes.

Methods  A total of four integrin fragments of different lengths (ΔSDL, ΔβA, PSI‐Hybrid and PSI) were designed using structural data and expressed in Drosophila S2 cells. HPA‐1a (33) and HPA‐1b (P33) forms of all four proteins were expressed as were the fragments ΔSDL‐4b and ΔSDL‐SuperRare, the latter two for the detection of HPA‐4b and the rare HPA antigens (HPA‐6, 7, 10, 11 and 16) respectively. The recombinant proteins were then screened for reactivity with monoclonal antibodies (mAb) and polyclonal antisera containing anti‐HPA in an ELISA based assay.

Results  The reactivity pattern seen with murine mAbs Y2/51, SZ21 and CRC54 was as expected. Two of three human monoclonal HPA‐1a antibodies (19–7, 23–15, both from a PTP patient) reacted with the four L33 fragments but the third (CamTran007, derived from a NAIT patient) only reacted with the ΔSDL‐L33 and ΔβA‐L33 fragments, defining an epitope split. The reactivity of both potency (NIBSC‐03/152) and sensitivity (NIBSC‐93/710) standards for anti‐HPA‐1a detection were comparable or better than those seen by MAIPA. In addition, samples from 132 NAIT patients with anti‐HPA‐1a [15 with cerebral haemorrhage (CH), 49 with platelet counts <20 × 109 L−1 but no CH, 35 with counts ≥ 20 × 109 L−1 and 33 with antenatal treatment] were tested. Anti‐HPA‐1a was detected in 126 samples including all CH cases. The reactivity patterns against the four different β3‐integrin fragments did not correlate with clinical outcome. Finally, mini‐β3 integrins Δ SDL‐4b and ΔSDL‐SuperRare allowed the detection of 14 of the 15 anti‐HPA samples tested (5 HPA‐4a, 5 HPA‐4b, 1 HPA‐6b, 1 HPA‐7b and 2 of 3 HPA‐11b).

Conclusions  Recombinant fragments of the β3‐integrin expressing HPA antigens can be used to successfully detect platelet alloantibodies. These fragments with be of use in the screening and diagnosis of suspected cases of NAIT.
YS05 In Vitro Evaluation of Platelet Concentrates Prepared Using the Automated Gambro OrbiSac System
Authors: Wiltshire M. , Bashir S. , Beard M. , Cookson P. , Garwood M. , Hancock V. , Pergande C. , Smith K. , Turner C. , Thomas S. , Cardigan R.
Published: September 2006

Introduction  The Gambro OrbiSac device is designed to automate the preparation of platelet concentrates (PC) from buffy coats (BC). The aim of this study was to evaluate the in vitro function of PC produced using the OrbiSac system and stored in plasma.

Study design  Ten PC were produced by automated pooling of four ABO matched BC and a single unit of plasma. The ring‐shaped pooling bag was centrifuged and the platelet rich supernatant automatically expressed, through a leucodepletion filter, into a Gambro ELP storage bag. All PC were stored at 22°C with agitation and sampled on days 1, 5, 7 and 9. Samples were evaluated for platelet count and standard markers of platelet quality.

Parameter
OrbiSac PC
Specification (Manual PC)

Volume (mL)
366 (327–396)
150–350 mL (Local)

Platelet Content (×109 U−1)
297 (251–419)
>240 (UK)

Median (range) n = 10.

The assay results were compared to platelets produced by manual methods and stored in a Pall ELX bag in a previous study.

Parameter
OrbiSac PC
Manual PC

Day 1
Day 5
Day 7
Day 9
Day 1
Day 5
Day 7
Day 9

pH (22 °C)
7.23
7.45
7.38
7.29
7.13
7.36
7.32
7.24

pO2 (kPa)
9.1
7.1
7.2
8.6
6.3
5.4
5.8
6.2

pCO2 (kPa)
4.5
1.8
1.7
1.7
5.9
2.6
2.5
2.4

HSR (%)
61.8†
54.3†
55.6
58.1
76.3
71.4
64.5
70.6

% +ve CD62P
25.5
21.9
28.9
36.2
22.1
16.6
31.3
43.1

% +ve CD63
12.9
18.5
15.9
16.9
9.4
11.3
14.2
17.2

Median n = 10.

†
P < 0.001 versus manual (T‐test).

Results  Hypotonic shock response (HSR) values were found to be significantly lower in this study when compared to manually produced PC. This was due to increased red cell contamination in these units. HSR was further investigated during a larger scale trial and all units (n = 25) showed improved day 1 values (78%, range: 61–93%). Values from these additional PC were comparable with those from manually produced PC.

Conclusion  PC produced with the OrbiSac system achieved the local specification for platelet count. However, these units were higher in volume than those produced by manual methods, with 70% of units exceeding the locally specified maximum volume of 350 mL. This problem could be addressed during later trials. OrbiSac PC have acceptable pH, blood gases, HSR levels and platelet activation markers and results are broadly comparable to data generated in a previous study of manually produced PC. These results suggest that platelets produced with the Gambro OrbiSac system are comparable to those produced using the current manual methods.
SI29 What is an Acceptable Donor?
Authors: Gillon J.
Published: September 2006

Donor deferral rates continue to rise inexorably. Currently in Scotland more than a third of new donors are deferred, and nearly 20% of all donors attending. This reflects not only some major precautionary measures, e.g. the exclusion of donors with a history of transfusion since 1980, but also the increasing stringency and effectiveness of our selection procedures. The CSD SAC has been working to make the donor selection guidelines more evidence based, but often there is little or no evidence to guide decision making. Donors are frequently upset and angry at what they see as arbitrary decisions. While they are not always right, and cannot be considered to have a right to donate, their reaction may be triggered by the paternalistic nature of our approach to this issue. While accepting that we must do nothing to jeopardise recipient safety, an argument can be constructed for putting our relationship with potential donors on a more ‘modern’ footing, perhaps by defining a ‘contract’ which allows for a definition of, and acceptance of, the risks of donation on a ‘partnership’ basis. It is also fair to say that improvements in recipient safety due to the extreme sensitivity of donation testing have as yet had no impact on donor selection. This is not unreasonable but as and when pathogen inactivation becomes a reality it will perhaps provide an opportunity to review donor selection criteria for which we should be prepared. The threat that testing for vCJD might drastically reduce the available donor base should provide the stimulus for us to begin to contemplate a ‘paradigm shift’ in donor selection.
SI33 Implications and Management of Iron Overload in Myelodysplastic Syndromes
Authors: Malcovati L.
Published: September 2006

According to evidence‐ and consensus‐based practice guidelines, the majority of patients with myelodysplastic syndrome (MDS) should receive supportive therapy at present. Once anemia is symptomatic, red cell transfusions are the mainstay of therapy. The life expectancy of patients with isolated erythroid dysplasia aged 70 years or older is not significantly shorter than that of the general population; avoiding disease complication is therefore very important in these individuals. Recently we demonstrated that the development of red cell transfusion‐dependency significantly affects survival of MDS patients (Malcovati et al. J Clin Oncol 2005; 23: 7594–603). This partly reflects the severity of bone marrow failure; however, development of secondary iron overload significantly worsens survival of transfusion‐dependent patients. Considering patients with transfusion needs, iron overload (defined as serum ferritin above 1000 ng mL−1) is developed after receiving a median number of 20–25 RBC units. The effect of iron overload is mainly noticeable among patients with refractory anemia, who have a longer survival and therefore are more prone to develop the adverse effects of iron overload. In contrast, secondary iron overload does not affect the survival of patients with refractory cytopenia, who have a median survival of about 50 months. The negative effect of iron overload is significantly associated with serum ferritin levels, with a 40% increase in hazard for every 500 ng mL‐1 of increase in serum ferritin. According to the currently available therapeutic guidelines iron chelation should be considered once a patient has received 20–50 units of red cells, but only in patients for whom long‐term transfusion therapy is likely, or when an allogeneic stem cell transplantation is planned. The target serum ferritin concentration should be <1000 ng mL‐1. Desferrioxamine 20–40 mg kg‐1 per day should be administered by 12 h subcutaneous infusion or by two daily subcutaneous bolus administrations for 5 days per week. Recently, deferasirox (ICL670) has proved to be an effective oral iron chelator in patients with thalassemia major and in those with MDS. In 2005, the USA Food and Drug Administration has approved deferasirox for treatment of chronic iron overload due to multiple blood transfusions. A European Medicines Agency (EMEA) decision is waited in 2006.
S134 Anaemia and Heart Failure
Authors: Kalra P. R.
Published: September 2006

Anaemia is common in patients with chronic heart failure, with prevalence ranging from 15‐55% depending upon the population studied and definition for anaemia used. The very fact that anaemia is so common and that the clinical symptoms of anaemia closely mirror those of heart failure may account for the fact that the potential for an important pathophysiological link has largely been ignored until recently. The evidence used to guide the management of chronic heart failure is amongst the strongest for any chronic medical condition. Yet it remains a leading cause of hospitalization in the Western World and a substantial financial burden. The benefits of ‘add on neurohormonal antagonism’ appear to have reached a plateau. Therefore, identification of novel therapeutic strategies to improve symptoms and/or survival is of clinical and economic importance. This presentation will discuss the data that lend support to an important pathophysiological role for low haemoglobin in patients with chronic heart failure, and how it may be amenable to therapeutic correction. This is of particular relevance in hospitalised patients with marked fluid retention and concomitant anaemia, in whom it may be difficult to promote a satisfactory diuresis without correction of haemoglobin. The use of blood transfusion and its avoidance in this setting will be specifically considered.
SI35 Blood Group Antigen Typing Using DNA Microarray BeadChipsTM–First UK Report
Authors: Armstrong‐Fisher S. , Varzi A. M. , Urbaniak S. J. , Cavanagh G.
Published: September 2006

Background  The determination of human blood group phenotypes has routinely involved red cell agglutination techniques. The genes for all but one of the 29 human blood group systems have now been cloned and the molecular bases of all clinically significant blood group polymorphisms are known. Consequently these DNA sequences can be used to predict blood group phenotypes. Molecular typing as an adjunct to serology offers improved accuracy and can save both rare reagents and laboratory time while screening for compatible donor units. This study determined the feasibility of analysis of single nucleotide polymorphisms (SNPs) associated with a variety of blood group antigens using a DNA microarray system with a beadchip technology platform.

Method  SNPs were tested by elongation‐mediated analysis of polymorphisms (eMAPTM) using allele‐specific oligonucleotides with variable 3’‐terminal sequences attached to colour‐encoded beads. The beads are assembled into arrays on semiconductor chips (BeadChipTM), available from Bioarray Solutions. Elongation products for SNPs or small deletions/insertions were simultaneously detected by instant imaging of fluorescence signals from the entire array. Seventeen polymorphisms associated with Human Erythrocyte Antigens and one mutation associated with hemoglobinopathies (CO, DI, DO, FY, JK, KEL, LU, LW, MNS, SC and HgbS) were included in the HEA BeadChipTM Kit.

Results  The system was validated using a reference DNA panel of known phenotype (n = 30) and genotype, established by conventional DNA‐PCR SSP assays. Replicate testing (n = 6 reps for eight samples) gave 100% consistent data demonstrating the Bioarray system to be both reliable and reproducible. The initial concordance rate with serology was 99.2%.

Conclusion  The BeadChip System represents the first integrated flexible array solution for high complexity transfusion testing, accommodating ‘multiplex’ analysis of polymorphisms. This high throughput, microarray system offers the potential to determine extended genotypes of patients and maintain an extended‐genotype, national panel of donors to provide compatible blood for sensitised patients. The initial high concordance rate and the rarity of CE marked phenotyping reagents justifies its further development.
SI37 Two Novel D Genes of the Rh Blood Group System Producing D Variant Phenotypes
Authors: Etheridge W. , Tilley L. , Poole J. , Daniels G.
Published: September 2006

Introduction  The D antigen is the most clinically significant of the Rh blood group system. Many variants of D exist, which are often classified as weak or partial D. D variants usually result either from nucleotide changes encoding amino acid substitutions or from the replacement of sections of RHD by the equivalent region of RHCE. Amino acid substitutions within the cytoplasmic or transmembrane domains of the RhD protein often produce weak D phenotypes with quantitative changes to D antigen expression, whereas substitutions in the extracellular loops of the RhD protein often result in qualitative changes associated with partial D phenotypes. This dichotomy, however, is not definitive.

Samples and Methods  Molecular analysis was performed on 14 samples referred for investigation of RhD status. Genomic DNA was extracted from whole blood. Exons 1–10 of RHD were sequenced for all samples and compared to reference sequences. Serological data were obtained where possible.

Results  Nucleotide mutations were found in all samples. Most could be assigned to known D variant types, but two novel mutations were detected. In four out of five samples identified as a possible DOL D variant by serological testing, an extra mutation was found. DOL variants have T>C509 in exon 4 (M170T) and T>G667 in exon 5 (F223V). In addition a C>G1132 mutation in exon 8, encoding L378V in the twelfth transmembrane domain, was present. This variant of DOL has subsequently been reported as DOL‐2 (http://www.uni‐ulm.de/~wflegel/RH). The second novel mutation, A>G485 in exon 3 encoding N162S, was found in one person with alloanti‐D. The substitution is predicted to be in extracellular loop three of the RhD protein.

Conclusions  Two novel D variants were identified. One resembles DOL and is called DOL‐2. The other, which we propose to name DNS, was found in a person with anti‐D.