1. Anti-B19 screening for safe cellular blood products for at–risk patients SoGAT XVIII, USA, May 24-25th, 2005 Harry Bos on behalf of Project Group Parvo B19 Safe Blood Components (Theo Cuijpers, Marco Koppelman, John Jongerius, Maarten Koot, et al.)

2. SoGAT, May 25th, 2005 Parvovirus B19 Small non enveloped virus; 23 nm. Single-stranded DNA genome; approx. 5500 nucl. Encoding 3 proteins: VP1: ± 5% of capsid VP2: ± 95% of capsid NS1: non-structural for viral replication Erythrovirus Highly thropic for erythroid progenitor cells Infected cells undergo lysis caused by expression of NS1 Decline in red blood cell count and haemoglobin

3. SoGAT, May 25th, 2005 Clinical manifestation of infection Epidemiology - Seroprevalence in adults world-wide approx. 50-80% - Seroconversion rate for adults is about 0.8% per year Healthy individuals - Erythema infectiosum (fifth disease) - Chills - Headache - Rash - No clinical symptoms

4. SoGAT, May 25th, 2005 Risk groups for B19 infections Women during second trimester of pregnancy - Intra uterine fetal death (10%) - Hydrops fetalis (3%) Patients with congenital or acquired haemolytic anaemia Cellular immunodeficient patients - Aplastic crisis - Prolonged bone marrow damage and aplasia - Bone marrow transplantation

5. SoGAT, May 25th, 2005 Health Council of the Netherlands* B19 may cause serious complications in risk groups B19 can be transmitted by blood tranfusion B19 infection in healthy individuals: - Formation of protective Antibodies - Sometimes persistance of B19 virus for some time B19 safe blood components from: - Donors with IgG Antibodies - Interval at least six months *) Council of the Netherlands Bloodproducts and Parvovirus B19. 2002; publication no. 2002/07

6. SoGAT, May 25th, 2005 Health Council of the Netherlands Plasma products The risk-group approach can not be used for plasma products because of their large-scale production and use. Approach for blood derivatives: - Lowering the load in manufacturing pools by NAT screening and removal of high load units (>5 x 10 6 IU/ml)

7. SoGAT, May 25th, 2005 Estimation of demand of Parvo safe blood products in the Netherlands Erythrocyte concentrates54,800 (8%) Plasma 9,950 (8%) Thrombocyte concentrates11,000 (22%) equivalent to 56,000 donor units  Delivery of thrombocyte concentrates is the determing factor in the number of donors to be screened for B19  Total numbers of donors to be screened: 250,000 = 50% of the donorpopulation

8. SoGAT, May 25th, 2005 B19 IgG antibody screening Screening Assay: - Biotrin Parvovirus B19 Sandwich ELISA - α-VP2 IgG - FDA cleared B19 ELISA Automation of the screening assay: - Automatic pipetting device: Tecan Genesis - ELISA back-end processor: Ortho Summit Processor

9. SoGAT, May 25th, 2005 OD/CO for reactive α-Parvo B19 result 25,500 donations first time tested Reactive OD/CO > 1.080.1 % Reactive OD/CO > 1.578.7%

10. SoGAT, May 25th, 2005 Status Anti-parvo B19 Testing April 1st, 2005 Reactive OD/CO > 1.575.4 % First time tested Realised number244,572 Reactive (OD/CO > 1.5)184,468 Firist and second time tested Reactive (OD/CO > 1.5)124,932 Desired number first and second time reactives 125,000

11. SoGAT, May 25th, 2005 Status demand of Parvo safe cellular blood products in the Netherlands, April 1th, 2005 Explicit demand of hospitals  : Division Northeast< 10 Division Southeast15 Division Southwest16 Division Northwest10-20 thrombocyte concentrates weekly  ) The Parvo status of the product is printed standard on the label of the bloodproduct

12. SoGAT, May 25th, 2005 Limitations of the B19 screening method A) False positive reactions - 1 donor: low pos OD/CO ratio B)Persistance of B19 virus in infected donors for a period of more than six months - 2 donors: high pos OD/CO ratio, low B19 DNA load Remark: Blood products will probably not be infectious, because the high titer of B19 antibodies will neutralise the virus present in a low concentration.

13. SoGAT, May 25th, 2005 Persistent B19 infection (B) Donor 1

14. SoGAT, May 25th, 2005 Alternative Screening Based on Natural History of Infection. Further R&D on the testing system in place. Follow up of acute infected donors found in plasma- screening Sensitive B19 NAT of 5000 donations 6 month bleeding (2 nd IgG anti-Parvo screening). % False positive rate IgG test? Possible Consequences Extension of the 6 month-interval period to the length of the observed viremic period Parvo-NAT screening of donation for safe product IgM and IgG screening of donation for the safe product Or combination of these approaches

15. SoGAT, May 25th, 2005 Projects under consideration to improve the screening method -Determine the false positive rates of the different brands of IgG anti-Parvo assays -Determine the in vitro neutralising effect of B19 antibodies (e.g. α-VP1, α-VP2, α-NS1), using cohort of follow up samples of acute infected individuals.

16. SoGAT, May 25th, 2005 Conclusions Parvo B19 safe blood components can be derived from donors with IgG Antibodies tested at an interval of at least six months. Parvo B19 safe cellular blood products can be in case of a transfusion used for risk groups for B19 infection. This approach will reduce serious complications due to transfusion related B19 infection. Further research is in consideration to improve the screening method.