bluuebunny/crossref_metadata_2025 huggingface dataset:
Paroxysmal coughing, subdural and retinal bleeding: a computer modelling approach
Authors: Geddes J. F. , Talbert D. G.
Published: September 2006

Unexplained subdural and retinal haemorrhages in an infant are commonly attributed to ‘shaking’, the mechanism of which is believed to be traumatic venous rupture. However, the haemorrhagic retinopathy reported as a result of Valsalva manoeuvres and the subdural bleeding that is a rare complication of pertussis together demonstrate that if a sustained rise in intrathoracic pressure is transmitted to cerebral and retinal vessels, it may result in bleeding, similar to that reported in inflicted injury. Such haemorrhages would be expected to occur whenever severe paroxysmal coughing were induced, whatever the cause. This study used a computer modelling approach to investigate feeding accidents as the trigger for bleeding. A dynamic circulatory model of a 3‐month‐old infant was induced to ‘cough’, and the response to changes in physiological variables monitored. It showed that coughing causes intracranial pressures to build up exponentially to approach a maximum, proportional to the amount of pressure the musculature of the thorax can produce, as venous return is impeded. They do not have time to become dangerous during individual coughs, as blood quickly returns after the cough is over, reestablishing normal pressures. Paroxysmal coughing, however, does not allow blood to return between coughs, with the result that very high luminal pressures may be generated, sufficient to damage veins. A history of coughing, vomiting or choking is not uncommon in otherwise normal infants with retinal and subdural bleeding. Our findings suggest that paroxysmal coughing could account for such bleeding in some cases.
SI06 Protein Microarrays for Blood Typing and Testing
Authors: Petrik J.
Published: September 2006

Microarrays, thanks to high multiplexing power, have a potential to accommodate various assays within one testing format. There are numerous variations of microarrays in relation to surfaces, probe deposition, inclusion of microfluidics, detection method etc. However, two key choices relate to microarray matrix (flat surface versus microparticle based microarrays) and, perhaps most importantly to the character of probe (DNA versus protein), determining genotypic or phenotypic character of the assay. There are two basic configurations for blood grouping and pathogen testing–antigen and antibody detection. Antigen detection–blood typing and pathogen antigen detection, are primarily phenotypic assays but can be determined by blood group antigen genotyping and pathogen nucleic acid amplification techniques (NAT). Detection of antibodies against relevant transfusion‐transmitted pathogens and rare antibodies against blood group antigens are exclusively phenotypic assays. Protein microarrays are capable of accommodating all types of assays. However, in some cases genotypic assays have higher precision and sensitivity. It remains to be seen if either approach (genotyping versus phenotyping) could solve the potential discrepancies completely, but a combination of the two types of arrays on one platform seems feasible in the future. Our initial efforts focused on ABO and Rh typing and resulted in the development of nonagglutination blood grouping array and label‐free detection of specifically bound erythrocytes on gold slides. Direct antiglobulin test has been shown to work on this array, too. In a parallel development we have investigated antibody and antigen microarrays for pathogen detection, focusing on mandatory targets – HIV, HCV, HBV and TP. While a gold surface is well suited for antibody–cell interactions, it is less so for antibody–antigen interactions taking place closer to the surface. A variety of slide surfaces and assay conditions were studied and applied successfully to antibody detection in a clean (spiked) assay. It was evident that pathogen antigen detection requires an increase in sensitivity. We have chosen signal amplification for this purpose and applied it successfully in a preliminary set of experiments. Although certain assays need further improvement the proof of concept studies confirmed suitability of protein arrays as the basis for potential next generation comprehensive blood testing platform.
SI22 The BSHI Diploma in Histocompatibility and Immunogenetics Training Scheme
Authors: Robson A. J. ,   on behalf of the British Society for Histocompatibility and Immunogenetics BSHI Training Executive
Published: September 2006

The BSHI Diploma in Histocompatibility and Immunogenetics (H&I) training scheme is a carefully structured, in‐service training plan for Healthcare Scientists working in H&I laboratories. The aim of the Scheme is to provide a solid practical training in the skills necessary to undertake the work in a clinical H&I laboratory and to underpin this with a sound theoretical knowledge base. The 10 requirements of the Scheme include the successful completion of two essays, a casebook and a project/dissertation. These and other requirements in addition to the many theoretical ‘Key Tasks’ in the syllabus, ensure that the Training Scheme has an appropriate academic and practical balance. The role of a trainee's Training Manager is pivotal to the successful running of the Scheme. Among the Training Manager's responsibilities is the continuous management of the Trainee throughout the training period. The H&I Training Manager is an experienced member of staff working full time in a H&I laboratory. Every Training Manager has completed the BSHI ‘Training for Training Managers’ course or an approved equivalent course. The Trainee's work and progress is also regularly externally assessed by the BSHI Training Executive and their appointed assessors. In this way the Executive aims to maintain a standardised approach to basic training in the discipline. Where the facilities and resources are available Trainees attend an appropriate part‐time MSc course to augment their post‐graduate training. It is stressed however, the Scheme is self‐contained. Its successful completion equips the Trainee with a thorough grounding in Histocompatibility and related Immunogenetics which, for some individuals, will act as the core requirement for Clinical Scientist State Registration and a useful stepping‐stone to the Royal College of Pathologists’ Part I Examination in H&I. The Training Scheme is also recognised by the IBMS as providing the training required for Biomedical Scientists to attain the IBMS Specialist Diploma in H&I.
YS03 Gene Expression Profiling of Platelet RNA Identifies Differentially Expressed Genes Associated with Collagen Signalling Responses
Authors: Carr P. , Gusnanto A. , Macaulay I. C. , Dudbridge F. , Langford C. , Ouwehand W. H. , Watkins N. A.
Published: September 2006

Platelet Glycoprotein VI (GPVI) is a lineage specific collagen signalling receptor that transmits outside‐in signalling via the FcR γ‐chain. Two GPVI haplotypes have been identified, ‘a’ and ‘b’, that differ by five amino acids. These substitutions have a profound effect on signalling downstream of GPVI and are also associated with variation in the expression level of GPVI on the platelet surface. The aim of this study was to screen the transcriptomes of platelets from ‘aa’ and ‘bb’ homozygous individuals and determine differences at the gene expression level between individuals with different haplotypes. Platelet RNA was isolated from leukocyte‐depleted apheresis concentrates obtained from eight ‘aa’ and eight ‘bb’ donors. Using a template‐switching PCR based amplification protocol the mRNA in 100 ng of total RNA was amplified and then labelled with either Cy3 or Cy5‐dCTP. The eight ‘aa’ and eight ‘bb’ samples were randomly paired and compared on a 15 552 feature DNA microarray. In total 32 competitive hybridizations were performed, with four replicates for each pairing to control for technical variability. The microarray data were analyzed using a combination of statistical approaches and a list of significantly differentially expressed (DE) genes was identified. The DE genes had small fold‐changes, typically two to three‐fold, however we were able to verify differential expression by qRT‐PCR. Of the 10 genes selected for validation, two were confirmed as significantly differentially expressed between the two haplotypes (TUBB1, P‐val 0.0004; VWF, P‐val 0.0126) while a further four showed a trend in the same direction as the microarray result. Three DE transcripts were not confirmed by qRT‐PCR and the final transcript could not be accurately quantified. The results of this study suggest that differences in GPVI signalling are associated with differential gene transcription. The strategy applied here will be used to compare the transcriptomes of platelets from individuals identified using functional tests as high and low responders to a number of agonists. The identification of differentially expressed genes and correlation with functional responses may identify critical proteins involved in determining the extent of platelet response in vivo.
PL04 Tissue Retrieval and Processing Initiatives to Reduce the Risk of vCJD Transmission
Authors: Poniatowski S.
Published: September 2006

Risk reduction concerning potential tissue allograft vCJD transmission starts with effective donor selection and screening measures. Even as tests are developed and introduced, there are still risks regarding window period, which may result in potentially infective units being transplanted. Methodology can be implemented to reduce this risk by attempting to remove the infectious agents. NBS introduced blood leucodepletion as a risk reduction measure. For many highly processed tissue products, this is already achieved by washing blood and marrow from the graft. However the bone graft of preference for UK orthopaedic surgeons is fresh frozen femoral heads (containing blood/marrow) which have not been irradiated to 25 kGy. NBS have developed a bone washing protocol to achieve >99% marrow and blood depletion and are assessing whether the irradiation dose can be reduced as a result of bioreduction events included in the process. This is achieved without pooling tissues from multiple donors in turn reducing recipient exposure. Clinicians may find this a suitable alternative risk‐reduced product. Other initiatives have been introduced to reduce the risk of abnormal PrP contamination of equipment, in turn cascading to subsequent grafts. Disposable equipment is now used wherever possible. Where not possible (e.g. bone saws, skin dermatomes etc.) the equipment is washed and sterilized to best practice (HTM2010 and HTM2030), is fully traced through subsequent graft exposures, and when a suitable screen test is performed, kit is quarantined until a negative result is received.
YS06 The Use of the MAIEA Assay for Solving Serological Problems and Identifying Novel Blood Group Antigens
Authors: Warke N.
Published: September 2006

Background The monoclonal antibody‐specific immobilisation of erythrocyte antigens (MAIEA) assay was devised mainly for locating blood group antigens on specific red cell membrane proteins. The assay can also be used to map epitopes on protein molecules when used as a competitive binding assay, using monoclonal antibodies to different areas of a protein. The MAIEA assay has proved to be a very useful additional technique in the red cell reference laboratory when serological results are inconclusive and for assigning novel antigens to particular blood group systems.

Methods The MAIEA assay detects trimolecular complexes formed by the reaction of an unknown human antibody and a known monoclonal antibody with a particular red cell protein. The trimolecular complex is captured on a microtitre plate coated with goat anti‐mouse immunoglobulin (Ig) and detected by the addition of peroxidase conjugated goat anti‐human Ig. A positive reaction indicates that the epitopes recognised by the human and monoclonal antibodies are present on the same membrane protein and are binding at different regions. A negative reaction indicates that the epitopes are either not on the same protein or the two antibodies are competing for the same or similar epitopes resulting in inhibition.

Results During a 39 month period we carried out the MAIEA assay on 65 samples using the following monoclonal antibodies: CR1 (Knops) [54]; CD44 (Indian) [3]; AChE (Yt) [2]; DAF (Cromer) [3]; Kell [2]; Lutheran [1]. A suspected specificity was confirmed on 28 occasions: CR1 [21]; CD44 [3]; AChE [1]; DAF [1]; Kell [1]; Lutheran [1]. The Kell, Lutheran and two of the CD44 antibodies were to novel specificities which have since been defined by molecular analysis.

Conclusions The MAIEA assay is a useful technique in the identification of unknown and novel specificities. Assigning a specificity to a particular blood group protein, followed by competitive binding assays, can indicate the region of the gene for molecular analysis. In addition to solving many difficult and complex serological problems, four novel antigens have been identified using the MAIEA assay over a 39 month period.
SI32 Management of Anaemia in MDS
Authors: Killick S.
Published: September 2006

The myelodysplastic syndromes (MDS) are clonal disorders of haemopoeisis. They share characteristic morphological abnormalities of the blood and bone marrow and a risk of evolution to acute leukaemia which varies according to the subtype of MDS and the prognostic score (IPSS). Overall annual incidence rates based on European studies are between 3 and 4 per 100 000 but the rate dramatically increases with age, exceeding 30 per 100 000 for patients over 80 years. It is therefore mainly a disease seen in older patients, where curative treatments are not an option, and supportive care is the reality. The majority of patients are anaemic at diagnosis and red cell transfusions are the mainstay of supportive care in those symptomatic. The aim is to achieve a haemoglobin level to maximise the patient's quality of life (QOL), which should be tailored to the individual. Red cell transfusions do however have risks associated, including the development of red cell antibodies and iron overload in multi‐transfused patients. Iron chelation therapy, due to its mode of delivery, further impacts on QOL. Alternatives to transfusions include growth factors and disease modifying agents. The aim of using Erythropoietin (EPO), is to improve the overall QOL by avoiding fluctuations in the haemoglobin levels. It also has the benefit therefore of avoiding the risks of transfusion. Unfortunately, it has been shown that those who would benefit most, are least likely to respond. Overall response rates (100% reduction in transfusion requirements) are in the order of 16–24%. Other options to avoid multiple transfusions are by modifying the disease. There are a number of approaches available including stem cell transplantation, immunosuppression, demethylating agents (azacitidine, decitabine) and thalidomide analogues (Lenalidomide). These should be tailored to the individual patient and where possible patients should be treated within clinical research protocols.
SI40 Conflicting Direct Antiglobulin Test Results when Investigating a Delayed Haemolytic Transfusion Reaction
Authors: Stamps R. , Tomlinson T. , Scott M. , Lumley A. , Ward D.
Published: September 2006

Background  A post‐transfusion sample from a patient suffering a delayed haemolytic transfusion reaction due to anti‐Jka was found to have a negative direct antiglobulin test (DAT) when the test was performed manually in our laboratory. The DAT on the same sample performed by the hospital transfusion laboratory using a Diamed gel station was strongly positive.

Methods  The 6 mL EDTA sample was centrifuged (RCF 2665) for 4 minutes prior to sampling of the red cells. Two manual DAT tests were carried out using 10 μL aliquots of concentrated red cells removed from just below the buffy layer and from the bottom portion of red cells and resuspended in 1 mL of Diamed diluent II. The sample was also tested on the Diamed gel station with the probe aspirating cells from the bottom portion of the red cells. Both manual and automated tests were performed using the Diamed monospecific DAT card containing anti‐IgG, IgA, IgM, C3c, C3d and an inert control reagent. Jka phenotyping was performed using monoclonal anti‐Jka (M15‐Imumed). An eluate was prepared from the mixed sample using an acid elution method (Gamma Elu‐kit II).

Results  The manual DAT on the cells from the upper layer was negative. The automated and manual DAT's performed on the cells taken from the bottom of the sample were strongly positive for C3d and weakly positive for IgM. No IgG was detected on the red cells. Jka phenotyping did not reveal any evidence of surviving Jka+ cells in either layer. The eluate did not contain any detectable antibody using IAT. There was no evidence of cold autoagglutination.

Discussion  The separation of immunoglobulin coated and uncoated red cells at the centrifugal force used has not, to our knowledge, been reported previously. The population of cells that were strongly C3d positive were not Jka+ and it is postulated that activated complement was binding to autologous red cells. These findings indicate that when investigating a possible haemolytic transfusion reaction it may be necessary to use cells taken from the bottom of a centrifuged sample or only aspirate cells after thorough mixing of the concentrated cells.
SI03 The Standing Advisory Committee on Care and Selection of Donors
Authors: Hutton D.
Published: September 2006

The Standing Advisory Committee on Care and Selection of Donors (SAC‐CSD) advises the UK Blood Services on all matters related to the care and selection of whole blood and component donors. It works very closely with the SAC on transfusion transmissible infections and also with the SACs dealing with tissue and stem cell donors. It has responsibility for relevant chapters in the Guidelines for the Blood Transfusion Services in the United Kingdom (the Red Book) and for the Whole Blood and Components Donor Selection Guidelines, which include the Geographical Disease Risk Index used by all the Donor Selection Guidelines. Membership is drawn from a wide geographical base and includes donor care doctors together with nonmedical staff involved in donor care. Topics for discussion include subjects referred to the SAC by the UK Forum of the Blood Services and the Joint Professional Advisory Committee (JPAC). Members are required to keep a watching brief of developments around the world that may affect donor care and selection and to respond to concerns from both donors and staff related to donor selection issues. In recent years a great deal of its time has been spent dealing with the increasing amount of legislation relating to the running of donor sessions.
P15 Transfusion Related Acute Lung Injury (TRALI) or Other Acute Lung Injury (ALI)?
Authors: Michala S. , De Lord C. , MacRate E. , Botfield C. , Brown R. , Win N.
Published: September 2006

Transfusion related acute lung injury (TRALI) is a form of acute lung injury (ALI) occurring within 6 h of transfusion. Patients presenting with TRALI are often indistinguishable from other causes of ALI. It is an increasingly recognized severe complication of transfusion. We present two cases where TRALI was strongly clinically suspected but only one case was serologically proven to be TRALI. Case 1: A 51 year old lady sustained a massive haemorrhage following an elective gynaecological procedure. 48 h later she returned to theatre and received 4 units of FFP and developed ALI. Case 2: A 28 year old had a postpartum haemorrhage following an emergency caesarian section. After receiving 3 units of RBC she developed respiratory failure. Both patients required ventilation but made a full recovery. Fluid overload, cardiogenic pulmonary oedema, sepsis and pulmonary thromboembolism were excluded and the cases were referred to the NBS for investigation of TRALI. In both cases, the expert panel consulted by the NBS was doubtful about the likelihood of TRALI since all 4 FFP units were collected from male donors and red cells are thought less likely to be implicated. However, all donors were contacted and investigated for WBC antibodies in their serum. Serology did not confirm the diagnosis in Case 1 but TRALI was confirmed in the second case where anti‐HNA1 antibodies were identified in one of the red cell donors and the patient typed as HNA‐1a positive. The diagnosis of TRALI is one of exclusion of other causes of ALI. Here we illustrate that despite the clinical similarities between the two patients, both strongly suspected of TRALI, only one was serologically confirmed to have TRALI. This highlights the difficulties in deciding which cases should be referred for further investigation. Although the referral process is complicated and requires a great deal of clinical input, TRALI needs to be considered as a cause of ALI following transfusion with all blood products, including RBCs. Finally, it is important to reach a diagnosis for both the patient concerned, as this may impact on their future management, and to exclude the implicated donors from further donations.
P18 Which Patients Receive Platelets Transfusions in the South West Region and Why?
Authors: Birchall J. , Webb M. , Copplestone A. ,   on behalf of the South West Regional Blood Transfusion Committee
Published: September 2006

Introduction  Although the use of red cells in England has declined since 2000 platelet transfusions have continued to increase and this rise has been more pronounced in the South West (issue figures between April 02 and March 05 identify a 22.5% increase compared with 7.5% nationally). To investigate this the South West Regional Blood Transfusion Committee organised a study to determine which patients are being transfused platelets and why.

Methods  Information was collected for one month, 14th November to 11th December 2005, by transfusion laboratory staff and a simple data set was defined to identify ‐ age of the recipient, directorate/specialty, use for prophylaxis/bleeding, operation (if relevant), platelet count prior to issue and number of doses requested and number used.

Results  A total of 21 out of 28 hospitals returned satisfactory information. Overall 1323 units were requested and 1226 (93%) units used for the initial request. Using NBS issue data this represents 86% (1323/1535) of all platelets issued to participating hospitals. Variation in usage between hospitals was large with 4 hospitals requesting more than 100 and 2 hospitals requesting more than 200 units. Platelet use increased with age and over 500 units were requested for patients 65 years or over. 779 units (59% total), were requested for haematology patients. Oncology and cardiac surgery were the next highest users requesting 149 (11% total) and 139 (11% total) respectively. When prophylactic or therapeutic use was known, 904 units (74%) were requested for prophylaxis. 75% of these were for haematology patients and 73% when the preceding platelet count was greater than 10 × 109 L−1. 310 (24%) units were requested for bleeding. 30% of these were for cardiac surgery and 53% when the previous count was more than 50 × 109 L−1.

Conclusion  This study indicates that around 40% of all platelet requests are to prevent bleeding in haematology patients when the preceding platelet count is greater than 10 × 109 L−1. The increasing age profile of the population is likely to put greater demands on the National Blood Service to collect platelets with increasing cost to the hospitals unless platelet use alters. The current British Society of Haematology guidelines should be followed whenever possible and further work to assess the need for prophylactic platelet transfusions should be performed.

Acknowledgements  The Executive working group of the RTC would like to thank all the regional Blood Transfusion Laboratory managers for their assistance with this audit.
P19 Appropriateness of Red Cell Transfusion in Medical patients
Authors: Mold D. , Allard S.
Published: September 2006

There is now increasing attention focused on promoting appropriate use of blood transfusion on the background of significant concerns regarding safety. Wide variations have been described in surgical usage of blood but there is relatively little data available on red cell transfusion in medical patients with anemia.

Methods We retrospectively audited transfusion practice in general medical patients over a 1 month period. Appropriateness of transfusion was assessed against national guidance (British Journal of Haematology 2001; 113, 24). Documentation of transfusion episodes was measured against the criteria given in Better Blood Transfusion (HSC 2002/009). Over‐transfusion was classified as a post transfusion haemoglobin (Hb) increment of >2 g dL−1 above transfusion threshold.

Results Out of 72 consecutive cases audited, 18 were excluded (three patients transferred to surgical care, five on long term transfusion regimes, 10 case notes unavailable). Data was available on 54 patients (31F;23M) aged 23 to 91 years (mean 67 years) with a wide variety of medical diagnoses. 66 cross‐matches were undertaken resulting in 56 transfusions. 201 units of red cells were cross‐matched with 15 units transfused giving a transfusion ratio of 1.3 and a transfusion index of 2.8. 55 cross‐matches (83.5%) were assessed as appropriate and of these four were not transfused. The indication for transfusion was not recorded in 21% (n = 14) of cases and the outcome was not recorded in 20% (n = 11) of cases. Pre‐transfusion Hb levels ranged between 5.4 g dL−1 and 12.7 g dL−1 (mean 8.2 g dL−1). Post transfusion Hbs ranged between 7.0 g dL−1 and 15.2 g dL−1 (mean 10.7 g dL−1); there were no post transfusion Hb samples taken in five cases (7.6%). The post transfusion increment was found to be >2 g dL−1 in 51.5% (n = 34) of transfusions.

Conclusions The documentation of transfusions remains poor with indications and outcome not being recorded in a significant proportion of medical cases audited. Over 15% of cross‐matches were found to be inappropriate against objective audit criteria and there was evidence of overtransfusion. Transfusion practice for medical indications warrants further scrutiny with a view to promoting appropriate use of red cells and better documentation to improve safety.
P28 Storage Study Of Red Blood Cells Treated With The Macopharma P‐CAPT™ Prion Removal Filter.
Authors: Bégué S. , Sumian C. , Walicka I.
Published: September 2006

Background  Three occurrences of probable blood transfusion transmitted cases of vCJD have recently been reported in the UK. The P‐CAPTTM Prion Removal Filter (MacoPharma) has been developed to remove infectious prions potentially present in leucodepleted red blood cell concentrates (RBCC's). In order to be accepted for use in routine blood processing practice, the filter must retain the quality of RBCC's following treatment, in addition to conforming to blood service operational evaluation and manufacturing specifications.

Aims  The aim of this study was to investigate if filtration through the P‐CAPT™ filter has any deleterious effect on the quality of RBCC's stored in SAG‐M or plasma‐CPD.

Methods  RBCC's issued from leucodepleted Whole Blood (WB), either prepared in SAG‐M or plasma, were treated with the P‐CAPT™ filter at 20°C on day 0 or day 1 following a 4° storage after blood collection. To ensure comparative trials, a series of 3 WB units were pooled, mixed and then split into 3 equal units before being treated at either day 0 or at day 1. For each pool, one unit was not filtered through the P‐CAPT filter to serve as a reference. RBC Plasma‐CPD units (12) were stored upto 28 days and RBC SAG‐M units (18) were stored upto 42 days at +2°C/+6°C. Stability of the red cell concentrates was evaluated every 7 days by measuring hemolysis, pH, K+, pO2, pCO2, ATP, 2,3 DPG, glucose, lactate, and other parameters according to standard protocols.

Results  All the final product specifications (Hb g/unit, hematocrit, residual leucocytes, total volume) as well as end of shelf life product parameters (Hemolysis, ATP, 2,3 DPG, K+, pH) were within the routine specifications acceptable to the UK blood services (Guidelines for the Blood Transfusion Services in the UK “Red Book”) and met the Council of Europe Guidelines (Guide to the preparation, use and quality assurance of blood components). These results indicate that red blood cell stability is not adversely affected by exposure to the P‐CAPT™ filter, whether they are treated in SAG‐M solution at day 0 or day 1 or treated in plasma‐CPD at day 1.
P35 Two Cases of Anti‐D in DAR Individuals
Authors: Lee E. , Redman M. , Malde R.
Published: September 2006

Introduction  Current monoclonal anti‐D reagents will react with most D‐variant RBCs. A small number of individuals with D variant phenotypes (e.g. IIIa, IV, V, VI, VII, DAU) appear to be stimulated to produce allo anti‐D more readily than others. DAR is characterised by the complete loss of at least nine of 37 RhD epitopes. We are reporting two cases of allo anti‐D in patients who have the DAR phenotype.

Case history  Case 1: An African woman aged 20 was admitted for termination of pregnancy with no clinical history available. She was grouped as B, Ro. Her D phenotype using BioVue was weakly positive and also with anti‐D reagents RUM1 and MS201 by tube. The antibody screen was positive, and suggestive of anti‐D. Our laboratory found her RBCs strongly positive with RUM1 and MS201 anti‐Ds by tube and with DiaMed grouping cards. The Diagast D‐screen suggested a weak D antigen. No plasma was supplied for antibody identification. A weak anti‐D by tube LISS IAT was detected in a subsequent sample. The D status was identified by IBGRL as DAR. Case 2: A pregnant African female, aged 35 was grouped by the referring hospital as O, D+; the antibody screen was suggestive of anti‐D by Capture R. Our laboratory had tested her D status in Feb. 2003 after a miscarriage. We reported her as weak D with a negative antibody screen. No anti‐D Ig prophylaxis was given. In our laboratory the most recent sample grouped as O, Ro. Her RBCs gave strongly positive reactions with RUM1 and MS201 by tube and with DiaMed grouping cards. The Diagast D‐screen suggested weak D. Anti‐D was detected with a level of <0.1 IU mL−1. The D status was identified by IBGRL as DAR.

Conclusion  Immunisation risk for DAR is estimated to be >1% per D+ transfusion, which is less than for anti‐K and anti‐c but similar to anti‐Fya and anti‐Jka. The incidence of DAR has been reported as one in 4233 from a population with ‘mixed ethnicity’ in Canada. There is no routine serological method to identify all partial D types. In these two cases the D‐screen indicated both were weak D, and the current monoclonal anti‐D typing reagents used gave positive reactions. Although both of the patients were immunised, the anti‐D in both cases was weak which concurs with our previous experience of anti‐D in partial D individuals. Molecular studies may help to provide further information about the likelihood of the risk of immunisation.
P04 In Vitro Evaluation of Buffy Coat Derived Platelet Concentrates in SSP+ Platelet Storage Medium
Authors: Beard M. , Garwood M. , Cookson P. , Bashir S. , Hancock V. , Pergande C. , Smith K. , Turner C. , Wiltshire M. , Thomas S. , Cardigan R.
Published: September 2006

Introduction  As part of the process to improve the safety and quality of the platelet supply, the NBS is evaluating the use of platelet storage media (PSM). The objective of this study was to produce a platelet concentrate (PC) containing 70% SSP+ and 30% plasma, whilst maintaining platelet yield, and to evaluate their in vitro function. Study Design. Buffy coats were prepared using the standard NBS method and four (ABO matched) were pooled together with 250 mL of SSP+. Centrifugation was performed using optimised conditions for PSM and an Optipress used to express the PC through a Pall Autostop LD filter into a Pall ELX storage bag. Ten PCs were prepared, and platelet quality was assessed using in vitro assays for functionality and activation on days 1, 5, 7 and 9 of storage.

Results 

Parameter
Preparation day 1
Specification

Volume (mL)
307 (299–317)
150–350 (Local)

Residual WBC (×106 U‐1)
0.1 (0–0.2)
<1 (EU)

Platelet Content (×109 U‐1)
326 (259–387)
>240 (Red Book)

Median (range) n = 10.

The assay results were compared to platelets stored in 100% plasma from a previous study.

Day
SSP+
Plasma

1
5
7
9
1
5
7
9

pH
7.17‡
7.35
7.36†
7.34‡
7.13
7.36
7.32
7.24

pO2 (kPa)
4.9
5.8
6.4
6.0
6.3
5.4
5.8
6.6

HSR (%)
65
60‡
62
55†
76
71
65
71

ATP (μmol/1011 platelets
4.20
4.35
4.26
4.37
3.9
3.8
Not tested
3.6

%CD62P positive
15.6†
17.8
24.4‡
32.0‡
22.1
16.6
31.3
43.1

s‐CD62P (ng mL‐1)
35‡
56‡
67‡
80‡
79
97
106
121

%PMP
1.6†
1.8‡
2.1‡
2.9
5.6
6.0
5.9
5.1

C3ades arg (ng mL‐1)
67‡
250‡
360‡
481‡
34
1399
3206
5072

Median, n = 10, †P < 0.05, ‡P < 0.01 versus plasma (Mann‐Whitney U‐test).

HSR in SSP+ was 85% of day 1 value on day 9, predicting satisfactory in vivo viability.

Conclusion  Buffy coat derived PC in 70% SSP+ met the required specifications for platelet yield, residual leucocyte count, pH and volume. Platelet activation was lower in PCs stored in SSP+ and units stored in SSP+ maintain acceptable in vitro function during storage.
P40 A Novel Variant in the Ok Blood Group System
Authors: Karamatic Crew V. , Thomas R. , Gillen B. , Poole J. , Daniels G.
Published: September 2006

Introduction  Oka (OK1), the only antigen of the Ok blood group system, occurs with a very high frequency in all populations studied. Oka is located on the immunoglobulin superfamily glycoprotein CD147, also known as M6 leukocyte activation antigen, basigin (BSG) or extracellular matrix metalloproteinase inducer (EMMPRIN), which is encoded by the CD147 gene located on chromosome 19p13.3. The only known polymorphic forms of Oka are the extremely rare Ok(a‐) phenotypes described in eight families of Japanese origin (Spring et al. (1997) Eur. J. Immunol. 27, 891–897) and one Caucasian individual of Iranian background (Karamatic Crew et al. (2003) Transfusion Med 13(1), P32). In both cases, the molecular basis of the Ok(a‐) phenotype were unique missense point mutations leading to amino acid changes within the carrier protein.

Case Study and Methods  We investigated a case of a prenatal patient of Hispanic origin whose serum contained an antibody to an unknown high frequency antigen, with a suspected Oka specificity. There was no known history of transfusion or previous pregnancies. Serological tests were performed by standard agglutination techniques. Genomic DNA was isolated from whole blood of the patient and all the exons of the CD147 gene were amplified by PCR and directly sequenced.

Results  The patient's serum reacted with all tested cells of rare and null phenotypes, with the exception of three examples of Ok(a‐) cells. Her red cells were positive with all examples of anti‐Oka tested, indicating a novel Oka phenotype. Sequencing of CD147 revealed a novel homozygous mutation in exon 2 178G>A, causing Val60Met change in CD147 protein.

Conclusion  We report the second non‐Japanese individual with a variant Oka phenotype. We have confirmed that patient's serum contained an antibody associated with Oka, although it was different from the anti‐Oka examples described previously. This rare novel Oka variant results from a single point mutation in CD147 leading to a Val60Met change. This new Oka variant and the distribution of the mutations within CD147 indicate a more complex blood group system.
P44 Review of Hospital Transfusion Committee Membership and Attendance in the West Midlands
Authors: Watkins E. , Baker C. E.
Published: September 2006

Introduction  Hospital transfusion committees (HTC) are now well established. Over the last 5 years blood usage has declined by 15%. It is now well established that surgical patients utilise less than 50% of red cells despite initiatives to reduce the number of patients waiting for surgery. It was perceived that physicians were under represented on HTCs. As physicians are responsible for an increasing proportion of blood used we decided to quantify attendance at HTCs.

Methods  Minutes from 18 Trusts’ HTC meetings held in 2005 and 2006 within the West Midlands were reviewed and job title was recorded for members of and those attending their HTC. Sufficient data was available from 14 HTCs to review their last three meetings.

 The most common posts/departments represented are as follows:

Member
Members of X % of HTCs

Consultant Haematologist
100

Transfusion Practitioner/Co‐ordinator
89

Anaesthetics
89

Blood Bank Manager/Chief BMS
83

Orthopaedics
61

Obs and Gynae
67

Haematology Lab/Pathology Manager
56

Clinical Risk
61

Consultant Surgeon
56

Matron
44

 Remaining categories/groups

Member
Members of X % of HTCs

Paediatrics
50

Clinical Governance
39

A and E Consultant
33

Medical Director
33

Physician
28

Director of Nursing (and Midwifery)
28

Theatres/Recovery
22

PCT Representative
22

Quality Manager
17

Clinical Audit
11

‘Others’
89

 Invitation and Attendance at HTCs by each group

Group
% of 42 HTCs to which each group was invited
% of 42 HTCs to which each group attended

C. Haematologist
100
90

TP/Co‐ordinator
79
76

BB Manager
100
88

Anaesthetics
93
69

Clinical Risk
64
31

Clinical Governance
36
19

C. Surgeon
50
21

Obs and Gynae
64
43

Matron
43
21

Haematology Lab
43
29

C. Physician
7
2

Orthopaedics
64
17

 Percentage of 14 Trusts with ≥1 attendance in the last 12 months, by group/member

Group
% of 14 Trusts with ≥1 attendance in the last 12 months

C. Haematologist
100

TP/Co‐ordinator
79

BB Manager
100

Anaesthetics
86

Clinical Risk
36

Clinical Governance
29

C. Surgeon
36

Obs and Gynae
64

Matron
36

Haematology Lab
36

C. Physician
7

Orthopaedics
29

Results  Conclusions  Of medical staff, physicians are members of only 28% of the HTCs. More worrying is that in the period under review for which there was comprehensive data only once did a physician attend an HTC meeting. There is an urgent need to engage more physicians on to the membership of HTCs and to ensure that they attend, review their current practice and disseminate best practice to their colleagues.
P47 Inherited Familial Thrombocytopenia due to an Autosomal Dominant Mutation in the Cytoplasmic Domain of Platelet β3‐Integrin that is Associated with the Expression of Activation‐Dependent Epitopes
Authors: Watkins N. A. , Ghevaert C. , Schaffner‐Reckinger E. , Rankin A. , Garner S. F. , Stephens J. , Smith G. A. , Kieffer N. , Laffan M. , Ouwehand W. H.
Published: September 2006

Background  Glanzmann's Thrombasthenia (GT) and Bernard Soulier Syndrome (BSS) are the two most common inherited forms of thrombasthenia. They are caused by alterations in expression or function of integrin alphaIIb/β3 or glycoprotein (GP) Ib/IX/V respectively. We identified an individual with chronic mild macrothrombocytopenia (range 59–100 × 109 L−1) but no overt bleeding diathesis. The propositus was found to have atypical binding of monoclonal antibodies (mAb) to β3 integrin and increased expression of GPIbVIX.

Methods and Results  The exons and flanking intronic sequences of ITGB3, ITGA2B GPIBA, GPIBB, GP9 and GP5 genes were sequenced. Heterozygous, non‐synonymous single nucleotide polymorphisms (SNP) were identified in the ITGB3 and the GPIBA genes. These result in the substitution of Histidine for Aspartate at residue 723 (D723H) in ITGB3 and Leucine for Proline at residue 69 (P69L) in GPIBA. Asp723 in ITGB3 is known to form a critical salt‐bridge with ITGA2B and charge reversal at residue 723 causes constitutive activation of the integrin in vitro. In agreement with this, mutant alphaIIb/β3‐His723 expressing CHO cell lines showed an increased expression of activation dependent epitopes relative to wild‐type as measured by mAb PAC‐1 binding. Increased PAC‐1 binding was also observed on resting platelets of affected individuals compared with non‐affected family members. Further investigation of three generations of the propositus’ family identified several individuals with both SNPs each of whom had an identical platelet phenotype. However, in the one family member identified who carried the GPIBA mutation alone, normal platelet size and count were observed suggesting that the ITGB3 D723H substitution is the causative mutation. Genotyping a panel of 1127 unrelated controls for the two SNPs identified five individuals with the GPIBA mutation and none with the ITGB3 mutation.

Conclusions  We report a novel autosomal dominant cause of a mild form of macrothrombocytopenia caused by a D723H substitution in the beta3 integrin. We postulate that the low platelet count is due to increased consumption of platelets due to enhanced levels of surface expressed, activated GPIIbIIIa.
P50 Novel Activatory and Inhibitory Platelet Receptors Identified Through Comparative Gene Expression Profiling of Megakaryocytes and Erythroblasts
Authors: Macaulay I. C. , Tijssen M. R. , Thijssen‐Timmer D. , Gusnanto A. , Carr P. , Steward M. , Langford C. , Ellis P. , Dudbridge F. , Zwaginga J. , Watkins N. A. , Van Der Schoot E. , Ouwehand W. H.
Published: September 2006

Summary  Platelets are derived from a polyploid precursor cell, the megakaryocyte (MK). The MK and the precursor of red cells, the erythroblast (EBs) share a common bipotent progenitor and have closely linked differentiation pathways. We hypothesized that the subset of genes that are differentially expressed between MKs and EBs would contain novel, previously uncharacterized, ‘lineage specific’ genes. We have used cDNA microarrays to identify genes differentially expressed between CD34+ Haematopoietic Stem Cell (HSC) derived MKs and EBs.

Methods  CD34+ HSCs were purified from Umbilical Cord Blood by magnetic cell separation. For differentiation into MKs, HSCs were cultured for 7 days in serum‐free medium containing Thrombopoietin and IL‐1β. For differentiation into EBs, HSCs were cultured for 10 days with Erythropoietin, IL‐3 and Stem Cell Factor. Differentiated cells were purified by Fluorescence Activated Cell Sorting to >95% purity, RNA isolated and then compared on Sanger Hver2.1.1 15K cDNA arrays. The data was statistically and bioinformatically analysed to identify novel transcripts encoding proteins with transmembrane domains. Taqman Real‐time PCR was used to validate the differential expression of both known and novel transcripts and to investigate blood cell distribution. Murine polyclonal antisera were raised against recombinant protein and used to confirm expression of these genes at protein level in platelets and MKs by western blot.

Results  Known lineage specific markers, including CD41, CD42a and CD42b were upregulated in MKs relative to EBs. A number of novel transmembrane proteins were also identified as being upregulated in MKs. These include the immunoglobulin fold domain containing proteins c6orf25 (G6b) and c6orf21 (G6f). Transcript profiling of blood cells demonstrated that G6b and G6f expression is restricted to the platelet and megakaryocyte lineages. Western blotting confirmed the presence of G6b and G6f as glycosylated proteins of the platelet plasma membrane. Functional studies have shown that cross‐linking of G6b with polyclonal antisera can inhibit platelet aggregation in response to both collagen and ADP, suggesting that this protein has an inhibitory role in vivo. In addition, we have identified a novel G‐protein coupled receptor in platelets that may have a co‐stimulatory role in platelet aggregation.

Conclusions  The comparative analysis of the MK and EB transcriptomes provides an overview of gene expression in these two cells and has identified novel platelet‐specific transmembrane proteins. Experiments are underway to characterise the role of these novel proteins in the regulation of platelet function.
P52 Characteristics of HLA Class I and Class II Antigens of the Somali Population
Authors: Mohamoud A. M.
Published: September 2006

HLA antigens of the Somali population are not categorised as well as those of other international ethnic groups. We analysed the HLA antigens of 76 unrelated Somalis who lived in the west of England. HLA ‐A, ‐B, ‐C and DRB1 typing was performed by polymerase chain reaction using sequence‐specific oligonucleotide probes (PCR‐SSOP) at a low‐intermediate resolution level. Phenotype frequency, gene frequency and haplotype frequency were used to study the relationship between Somalis and other relevant populations. The antigens with highest frequencies were HLA ‐A1, A2, and A30; B7, B51 and B39; Cw7, Cw16, Cw17, Cw15 and Cw18; DR 13, DR17, DR8 and DR1. HLA haplotypes with high significance and characteristics of the Somali population are B7‐Cw7, B39‐Cw12, B51‐Cw16, B57‐Cw18. The result of HLA class I and class II antigen frequencies show that the Somali population appear more similar to Arab or Caucasoid than to African populations. The results are consistent with hypothesis, supported by cultural and historical evidence, of common origin of the Somali population. This study will serve as a reference for further anthropological studies, as well as studies of associations between HLA and disease.
P54 Production and Characterisation of Monoclonal Antibodies Against Human Platelet Prion Protein
Authors: Jones M. , McLoughlin V. , Norrby K. , Connolly J. , Farquhar C. , Head M. , MacGregor I.
Published: September 2006

To date all of the existing monoclonal antibodies which react with human prion protein have been produced following immunisation with recombinant PrP, PrP from other species or synthetic peptide fragments. The aim of this project was to produce monoclonal antibodies raised against the native form of PrPc purified from human platelets. Following immunization of PrP‐/‐ Edinburgh mice and the use of conventional hybridoma techniques a panel of 12 monoclonal antibodies has been established. These antibodies have been characterized by epitope mapping; binding to both native/denatured platelet PrP and native/denatured α‐helical/β‐sheet recombinant mouse PrP; immunoprecipitation of PrPc, PrPsc and PrPres from neurological control/vCJD brain homogenates and as probes for Western blotting, immunohistochemistry and flow‐cytometry studies. A range of epitopes was recognised by the panel, with discrimination between normal α‐helical and abnormal β‐sheet forms. Several of the antibodies specifically captured abnormal forms of prion protein (PrPsc and PrPres ) found in vCJD‐infected CNS and lymphoreticular tissues. It is hoped that these antibodies will aid in the early diagnosis and classification of human prion disease and the detection of vCJD infectivity in tissues for transplant and blood donations.
P57 How Many Donors with Transfusion Microbiology False Positive Results Return to Give Samples for Re‐entry.
Authors: Howell D. R. , Moore C.
Published: September 2006

Donors who falsely react in any of the transfusion microbiology screen tests are sent a standard letter and asked to give a sample after a suspension of twelve weeks [84 days]. The donors can then be considered for re‐entry to the active panel. We are not aware of any work showing how many donors re‐attend to give samples and how soon, so have analysed our national figures. 1301 donors who donated in the 3 months from 1st Oct 05 [434 per month] gave a false positive reaction and were sent the standard letter. 2269 donors returned in the period 1st Jan 06 to 31st May 06 [453 per month] following the standard letter and gave samples to be considered for re‐entry. Of these: 769 had donated in the 3 months from 1/10/05, 393 females and 376 males. The average number of days between initial and repeat samples was 130 days for females and 129 days for males with standard deviations of 27.4 and 27.9. Two donors attended and were sampled at less than 84 days! We recorded a maximum time interval between sample and donation of 231 days ‐ with each testing centre having donors attending at over 150 days. 755 donors were returned to the active panel [400 using the alternative assay re‐entry algorithm]. We have shown that 59% of false positive donors have given further samples for re‐entry. The figure is constrained by the length of time for which data is currently available. It may be higher if data were available for a longer period. When more data are available it may be possible to investigate how many false positive donors do not return and compare the results with those for first time donors, currently approximately 30%.