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Volume 2
     

Blood grouping and genotyping: Improving patient safety and blood transfusion compatibility (BloodGen)

   
Project

QLK3-2002-01772

Cell factory area

3.1.1

EU Contribution

2,350,000 Euro

Duration

36 months

Type

DM

Starting date

Foreseen 01 January 2003 (contract not yet signed at date of printing)

Keywords
allele screening
alloimmunisation
phenotyping
polymorphism
ABSTRACT

Currently, all blood utilised in transfusion is phenotyped by serological means. As a consequence, blood is not phenotyped for all clinically significant antigens and there is a real risk of patients becoming alloimmunised to incompatible blood units.

The BloodGen consortium proposes to demonstrate the use of molecular genetic techniques to genotype a large cohort of individuals drawn from across the EU in order to demonstrate the accuracy and improvement of this technology over standard serological testing. The project will utilise two novel technologies involving single-nucleotide polymorphism detection, on biochips and by using real-time fluoro-sequence-specific primer PCR techniques. The project will use these two different technologies as one provides the ability to screen for large numbers of blood group alleles, whilst the other is able to test for a smaller number of alleles but with much higher throughput.

OBJECTIVES

The aim of this demonstration proposal is to validate and standardise molecular genotyping approaches to large-scale blood group diagnosis, to prove its superiority over currently applied serological testing. This aim will be achieved by attaining three scientific objectives:

  1. enhancing the safety of blood products, including enhanced detection of weak antigens;
  2. improving the efficiency and efficacy of blood group pre-transfusion compatibility testing;
  3. providing European standards for the diagnosis of blood group status.

There are also three technical objectives:

  1. demonstrating novel nucleic acid based diagnostic tests to reduce the instance of alloimmunisation;
  2. providing an innovative approach to blood group genotyping on a large scale, which is easily extendable to other alleles;
  3. providing a platform technology for future clinical genotyping.
DESCRIPTION OF THE WORK

The first phase of the project is divided into three work packages. Work packages 1 and 2 will fabricate the prototype technologies for blood genotyping, respectively biochip arrays and fluorescence SSP. Complementary to this area of fabrication work package 3 will standardise the DNA extraction and PCR techniques to be used throughout the project. Close collaboration between these three work packages will be required to achieve their stated objectives. To specify the breadth of the project the end-user participants will provide a review, based on their expert knowledge, of the current knowledge of the molecular basis of the blood group systems in Caucasian, Negroid and Asian populations. The DNA sequences of the selected blood group system will be provided. This information will allow the selection of a small number of single nucleotide polymorphisms (SNPs) (approximately 100-150) for the development of the first prototypes, which will be evaluated within work packages 1 and 2. Feedback from this exercise will then drive the fabrication of the second prototype, with the extended range of selected SNPs required for the preliminary clinical trial (work package 4).

The second phase of the project is the clinical trial which has an initial preliminary stage (work package 4) followed by the more comprehensive full trial (work package 5). The preliminary trial will allow the experts on a specific blood system to first evaluate the technology in their own area of expertise. The inter-laboratory trial will provide the mechanism by which all end-user participants analyse known samples for the full range of selected SNPs using the prototype technologies. Open and full discussion with the consortium of any issues arising from this study will define the corrective measures to be undertaken prior to the full clinical trial (work package 5).

DELIVERABLES
  1. description of the combination of various SNPs necessary for genotyping of blood groups;
  2. optimised nucleic acid extraction protocol from a variety of blood samples of differing qualities;
  3. evaluation report of the performance of the first prototype;
  4. optimisation of the amplifluor uniprimer system;
  5. a robust series of multiplex PCRs capable of amplifying polymorphic regions of genes;
  6. protocols that utilise the above multiplex PCRs in the chip analysis procedure;
  7. working prototype test kit for blood group genotyping based on an automated SSP method;
  8. report of the samples with rare phenotype or specific ethnic origin amenable for analysis. Ethical approval for all trial sites;
  9. written report with overview of the samples analysed;
  10. robust SOP for the genotyping of all clinically significant blood group antigens;
  11. written report with recommendations for the full clinical trial;
  12. compilation of nucleic acid blood grouping and serological blood grouping data sets;
  13. DNA micro-array for high-throughput genotyping to be validated in WP 5;
  14. written report documenting the functioning of the developed automated SSP technique;
  15. identification and reasons for discrepancies between nucleic acid and serological blood group testing;
  16. statistical analysis of the results;
  17. a written final report detailing the outcomes of the demonstration project.
CONSORTIUM
COORDINATOR
 
Dr Neil Avent
Centre for Research in Biomedicine,
Faculty of Applied Sciences
University of the West of England, Bristol
BS16 1QY, Bristol, United Kingdom
Tel: +44-117-344 3819
Fax: +44-117-344 2904
Neil.Avent@uwe.ac.uk
 
PARTNERS
 
Dr Masja De Haas
Sanquin Blood Supply Foundation
Central Laboratory of the Netherlands
Red Cross Blood Transfusion Service
1006 AD Amsterdam, The Netherlands
Tel: +31-20-512 3377
Fax: +31-20-512 3474
M_de_haas@clb.nl

Dr Marion Scott
Bristol Institute for Transfusion Sciences
National Blood Service
Southmead Rd.
BS10 5ND, Bristol, United Kingdom
Tel: +44-117-991 2112
Fax: +44-117-959 1660
marion.scott@nbs.nhs.uk

Dr Willy A. Flegel
Blood Group Serology & Immunohaematology
DRK-Blutzentrale Ulm
89081 Ulm, Germany
Tel: +49-731-150 600
Fax: +49-731-150 600
willy.flegel@medizin.uni-ulm.de

Dr Martin L. Olsson
Dept. of Blood Group Genetics,
University Hospital, Blood Centre
221 85 Lund, Sweden
Tel: +46-46-173 207
Fax: +46-46-173 226
Martin_L.Olsson@transfumed.lu.se

Dr Anna Ribera
Serveis sanitaris de referčncia
Centre de Transfusió i Banc de Teixits
08035 Barcelona, Spain
Tel: +34-93-274 9025
Fax: +34-93-274 9161
8976arc@comb.es

Martin Pisaćka MD
Institute of Haematology and Blood Transfusion
128 20 Prague 2, Czech Republic
Tel: +420-2-2197 7205
Fax: +420-2-2197 7366
pisacka@uhkt.cz

Dr Dietmar Pfeifer
Business Unit Diagnostics
Biochip Technologies GmbH
79108 Freiburg, Germany
Tel: +49-761-5038 315
Fax: +49-761-5038 111
d.pfeifer@genescan.com

Dr Bodo Holtkamp
R & D Department Diagnostics
BIOTEST AG
63266 Dreieich, Germany
Tel: +49-6103-801 116
Fax: +49-6103-801 135
bodo_holtkamp@biotest.de