1. Hong Kong Association of Blood Transfusion & Haematology NAT and Viral Safety in Blood Transfusion Dr. P.H. Yu Medical Officer Department of Pathology Tseung Kwan O Hospital

2. Viral Safety in Blood Transfusion  Public concern was heightened by the disastrous consequences of HIV epidemic in 1980s  In France, government officials and minister were charged with manslaughter for allowing HIV-contaminated blood to be used for transfusion at a time when screening test were available (1985)

3. Viral Safety in Blood Transfusion  Red Cross officials in Belgium, Switzerland, Canada were also convicted for distributing contaminated blood during the same period  Public perception – blood transfusion should involve absolute no risk of transmitting viral infection

4. 1/100 1/1,000 1/10,000 1/100,000 1/1,000,000 Adapted from Transfusion 2000; 40:143-159

5. Viral Safety in Blood Transfusion  Risk of transmitting infection to recipients has been drastically reduced in the past decades, due to a)Improved donor selection b)Sensitive serologic screening assays c)Application of viral inactivation procedures during manufacturing of plasma products

6. Residual Risk  In 1994, several cases of HCV infection were attributed to IVIg (Gammagard) in Europe  In 1999, an Australian schoolgirl contracted HIV via blood transfusion during surgery in Melbourne (first reported case of TT HIV in Australia since testing for HIV in 1985)

7. Residual Risk  Major sources of remaining risk are: 1.Window period donation 2.Viral variants not detect by current assays 3.Immunosilent donor 4.Laboratory testing error

8. Residual Risk  The greatest threat to the safety of blood supply is the donation by seronegative donors during the infectious window period  Window period donation account for 90% or more of the residual risk (Report of the Interorganization Task Force on NAT Testing of Blood Donors, 2000)

9. Window Period  Period precedes the development of antibodies during the initial infection  Eclipse phase of the window period - the very initial phase after exposure when virus replication is restricted to tissue sites and there is no detectable viraemia  Infectious phase of window period is after eclipse and before seroconversion

10. Window Period  Animal study in chimpanzees (Murthy KK et al, Transfusion 1999) suggested that the eclipse phase is non- infectious for HIV  Direct detection of virus by very sensitive method theoretically eliminate the infective window phase if the assay sensitive exceeds the minimum infective dose for that virus (window period closure)

12. Residual Risk  Risk of acquiring a transfusion-transmitted viral infection depends not only on the length of specific window period but also on the incidence of the infection among blood donors

13. Determination of Residual Risk  Study the rate of infection prospectively in transfusion recipients  Some pathogens, HIV & HCV, the risk is so low that exceeding large number of recipients & lengthy period are required for the risk to be measured accurately  Under-reporting

14. Determination of Residual Risk  Incidence/Window Period Model  First applied in France and US (Courouce & Pillonel 1996; Schreiber 1996)  Risk is calculated by multiplying the incidence rate in blood donor by the length of the window period

15. Determination of Residual Risk  Determine the incidence of seroconversion among donors who donate more than once (multiple time donors)  Not the prevalence rate in donor population

16. USAUKItalyAustraliaHK HIV1:493,000< 1: 2,000,000 1:408,0001:1,200,0001:877,147 HCV1:103,000< 1 : 200,000 1:230,0001:250,0001:86,137 HBV1:63,0001 : 50,000 – 170,000 1:63,4001:160,0001:3357 Source: (1) Muller-Breitkreutz K for the EPFA Working Group on Quality Assurance. Results of viral marker screening of unpaid donations and probability of window donations in 1997. Vox Sang 2000;78:149-157 (2) Aubuchon JP, Birkmeyer JD, Busch MP. Safety of the blood supply in the United States: opportunites and controversies. Ann Int Med 1997;127:904-909. (3) Regan FAM, Hewitt P, Barabara JAJ, Contreras M.on behalf of the current TTI Study Group Prospective investigation of transfusion in transmitted infection in recipients of over 20000 units of blood, Br Med J 2000;320:403-406. (4) Tosti ME, et al, An estimate of the current risk of transmitting blood-borne infections through blood transfusion in Itly. Br J Haemat, 2002;117:215-219.

17. Residual Risk  Chung HT, Kee JS, Lok AS (Hepatology 1993 Nov;18(5):1045-9)  Among 45 HBV serology negative patients received blood of blood products during open-hear surgery in HK  3 recipients (6.7%) developed asymptomatic hepatitis B seroconversion

18. What is NAT?  Nucleic Acid Technology (Nucleic Acid Amplification Testing)  A generic term that include a number of different technologies  All involve extraction or capture of nucleic acid, amplification, and detection

19. What is NAT?  Commonly used systems are I.PCR-based assays (Roche Cobas Ampliscreen) II.Transcription mediated amplification assay (GenProbe) III.Others, including in-house PCR preparations

20. GenProbe  Recently approved by FDA (Feb 2002) for donor screening  Three main steps 1)Sample preparation & target capture

21. GenProbe 1)Sample preparation & target capture RNA hybridized to target-specific oligonucleotides and then captured ontomagnetic microparticles which are separated from plasma in a magnetic field

22. GenProbe 2)Transcription Mediated Amplification - single-step isothermal amplification - initial synthesis of cDNA from the target RNA followed by in-vitro transcription of cDNA into many copies of RNA amplicon 3)Detection by a chemiluminescent probe which hybridized to the amplicon

23. The system includes a robotic pipettor (Tecan; Durham, NC), the Chiron Procleix target capture system, and the Procleix Leader HC+ with the Procleix system software.

25. Roche Cobas Ampliscreen  Five main steps: 1)Sample preparation by ultra-centrifugation 2)Reverse transcription of target RNA to cDNA 3)Polymerase chain reaction amplification of cDNA

26. Roche Cobas Ampliscreen 4)Hybridization of products to oligonucleotide peroxidase conjugated probe 5)Detection of probe-bound products by colorimetric determination

27. Pooling Strategies  Short time frame for implementation and lack of high throughput automated system  The only option is to implement NAT screening in pools of aliquots form several donations (16-512 individual donations)  Sensitivity decreases as pool size increases

28. Pooling Strategies  Automated pipetting system to prepare the pools  Overlapping three-dimensional pools or straight-line pools  Retesting of subpools is slow and will delays the release of final products

29. Standardisation  Different units, eg. genome equivalent/ml, copies/ml, PCR detectable units/ml  WHO Collaborative Study Group has established the reference sample for HCV(1997), HIV(2001), HBV(2001), and Parvovirus B19(2002); and standardised the unit of measurement as IU/ml

30. Other Technical Issues in NAT  Choice of anticoagulant  Nucleic acid stability in sample during transportation  PCR inhibitors in the sample  False positive result and cross-contamination  Internal control  Turnaround time – impact on product release

31. HCV  Prolonged high-titre viraemic phase before seroconversion and elevation of ALT, 7-12 weeks after infection  Very short doubling time of 2-3 hours, therefore high viral load titres are achieved

32. HCV  Very amenable to detection by pooled NAT  NAT theoretically reduce the window period by 41-60 days

33. HCV

34. HIV  Short doubling time of 21 hours  Window period of 16 days (p24 antigen) may be reduced to 11 days by NAT

35. HIV

36. HBV  HBsAg become positive 50-60 days after infection  Preceded by a prolonged phase (up to 40 days) of low-level viraemia  Long doubling time of 4 days  NAT pooling will only detect a small proportion of this pre-HBsAg window period

37. HBV

38. History of NAT Implementation  European Committee for Proprietary Medicinal Products required that by July 1999 all fractionated plasma products should be negative for HCV RNA by NAT technique  Required sensitivity: able to detect 100 IU/ml of HCV-RNA in the final pool (about 230 viral particles/ml)

39. History of NAT Implementation  Paul Erlich Institute in Germany required all blood products should have a negative HCV NAT result before release by April 1999  Required sensitivity: able to detect 5,000 IU/ml of HCV-RNA of individual donation

40. History of NAT Implementation  US blood centres implement NAT testing of blood donors for HIV and HCV in April 1999, under the Investigational New Drug applications  Studying GenProbe and Roche systems only  Canadian Blood Services implemented NAT since October 1999

41. History of NAT Implementation  Australia started NAT testing of blood donors for HIV and HCV since June 2000  Japanese Red Cross Society started NAT screening for HBV, HCV, and HIV since July, 1999

42. International Forum on Implementation of donor screening for infectious agents transmitted by blood by NAT  Vox Sang 2002;82:87-111  Countries screening HBV DNA: Japan, Germany (some plasma manufacturers)  Countries screening HCV RNA: Australia, New Zealand, Japan, USA, Canada, Germany, France, Austria, Italy, Netherlands, UK, Finland, Norway, Spain(partial), HK

43. International Forum on Implementation of donor screening for infectious agents transmitted by blood by NAT  Countries screening HIV RNA: Australia, New Zealand, Japan, USA, Canada, France, Netherlands, Spain (partial), Germany (plasma products only), HK  Still considering: Sweden, Brazil, Greece, South Africa

44. International Forum on Implementation of donor screening for infectious agents transmitted by blood by NAT  Turn Around Time: the blood products can be released within 1-2 days  Australia Red Cross: TAT is 10-26 hours  In most laboratories, invalid results or false positive are significant (2-8%); take longer time to resolve

45. Yield: North America  pool sizes of 16- 24 donations  GenProbe and Ampliscreen  Results from the US and Canadian programs starting from 1999 are:  113 HCV NAT-only positive donations identified from over 29 million donations screened (1/259,000)

46. Yield: North America  6 HIV NAT-reactive, p24 antigen-negative, and seronegative donations identified from 26 million donations screened (1/4,390,000)

47. Yield: Germany  Institute of Transfusion Medicine and Immunohaematology, Frankfurt  From 1997-2000, tested 1,087,000 donations  pool size of 96 donors  Roche Amplicor for HCV  In-house TaqMan PCR for HIV & HBV

48. Yield: Germany  2 HCV RNA-only positive donations identified (1:543,500)  1 HIV RNA-only positive donation identified  2 HBV DNA-only positive donations identified

49. Yield: Japan  Japanese Red Cross NAT Screening Research Group (Microbiol Immunol 2001;45(9):667-672)  From Feb 2000 – April 2001  Minipool of 50 donations  Multiplex HBV/HCV/HIV reagent  Excluded all serological positive cases

50. Yield: Japan  Total donations screened: 6,805,010  HIV-RNA positive: 4 (1:1,701,253)  HCV-RNA positive: 25 (1:272,200)  HBV-DNA positive: 112 (1:60,759)

51. USAAustraliaGermanyJapan HIV1:4,390,0000:1,440,0001:1,087,0001:1,701,253 HCV1:259,0001:360,0001:543,5001:272,200 HBV1:543,5001:60,759 Yield of NAT Screening on Blood Donors

52. Will NAT Close the Window?  Ling AE, et al. JAMA 2000;284:210-214  Transmission of HIV from a blood donor to a platelet recipient and a red blood cell recipient occurred in the window period  viral load in the implicated donation was estimated to be less than 40 copies/mL

53. Will NAT Close the Window?  Current US minipool HIV NAT screening protocols fail to detect very low level viraemia

54. Cost-effectiveness  NAT is a intensive process to perform, requiring specially ventilated & clean laboratory, expensive equipment and reagents  In US under the IND protocol, the cost per donation is US$8

55. Cost-effectiveness  Cost per case detected is estimated at US$1.7 million  After FDA approval of GenProbe, it is estimate that the cost will reach US$15-20 a donation (America Blood Centers Newletter March 8, 2002)  How much we are willing to pay to produce further marginal improvements in safety?

56. Future  Despite cost-effective issues, based on public perception and political pressure, NAT screening of the blood supply is expected to become a standard in transfusion medicine  Draft Guidance on Use of NAT to identify HIV-1 and HCV in Whole Blood and Blood components is issued by FDA in March 2002

57. Future  Replacing p24 antigen  More and more countries will require NAT non-reactive results before release of blood products  Automated and high-throughput system  Individual testing

58. Future  Screening other virus for specific blood products for specific patient group, eg. screening Parvovirus B19 for Anti-D Ig  Screening for new transfusion-transmitted viruses