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  1. 1.  Nucleic Acid Technology IN Blood Transfusion MODERATOR- DR. POONAM NANWANI
  2. 2.  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
  3. 3.  A nucleic acid test, often called a "NAT", (or nucleic acid amplification test - "NAAT") is a molecular technique used to detect a virus or a bacterium These tests were developed to shorten the window period a time between when a patient has been infected and when they show up as positive by antibody tests.
  4. 4.  It reduces the window period by detecting low levels of viral genomic materials that are present soon after infection but before the body starts producing antibodies in response to a virus
  5. 5.  Tests used for TTI  Front-line screening :  • Rapid tests  • ELISA tests  • Chemiluminescence  Tests for residual risks:  • NAT test methods  • Supplemental marker
  6. 6.  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
  7. 7.  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. 8.  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. 9.  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. 10.  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
  11. 11.  Determine the incidence of seroconversion among donors who donate more than once (multiple time donors)  Not the prevalence rate in donor population
  12. 12. Recently approved by FDA (Feb 2002) for donor screening Three main steps 1)Sample preparation & target capture
  13. 13. 1)Sample preparation & target capture RNA hybridized to target-specific oligonucleotides and then captured onto magnetic microparticles which are separated from plasma in a magnetic field
  14. 14. 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
  15. 15. 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.
  16. 16.  Five main steps: 1) Sample preparation by ultra-centrifugation 2) Reverse transcription of target RNA to cDNA 3) Polymerase chain reaction amplification of cDNA
  17. 17. 4) Hybridization of products to oligonucleotide peroxidase conjugated probe 5) Detection of probe-bound products by colorimetric determination
  18. 18.  Choice of anticoagulant  Nucleic acid stability in sample during transportation  PCR inhibitors in the sample  False positive result and cross-contamination  Internal control
  19. 19.  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
  20. 20.  Very amenable to detection by pooled NAT  NAT theoretically reduce the window period by 41-60 days
  21. 21.  Short doubling time of 21 hours  Window period of 16 days (p24 antigen) may be reduced to 11 days by NAT
  22. 22. HIV
  23. 23.  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
  24. 24.  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)
  25. 25.  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
  26. 26.  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
  27. 27.  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
  28. 28.  Current US minipool HIV NAT screening protocols fail to detect very low level viraemia
  29. 29.  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?
  30. 30.  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
  31. 31.  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
  32. 32.  • In Singapore, among the 466,779 samples tested by NAT since October 2007 they were able to pick 9 HCV and 10 HBV NAT yield samples (1 in 24,567). • Similarly in Thailand, Hong Kong and in Korea the NAT yield rate is 1 in 11, 676, 1 in 202,500 and 1 in 1, 46,628 respectively. Despite these countries having a very stringent donor counseling and screening process, a high rate of regular repeat voluntary donation, and use of the most sensitive serological tests, they were able toidentify a significant number of samples which were NAT reactivebut sero-negative.
  33. 33.  • In India, Indraprastha Apollo Hospitals, Delhi has taken the initiative for NAT implementation for the first time in the country. In the first nine months of implementing NAT, they were able to pick five (3HBV and 2 HCV) NAT yield samples among 13,331 samples test  (1 in 2,666).
  34. 34.  Chemiluminescent Immunoassay  Enzyme Immunoassay (EIA)  Immunofluorescent assay (IFA)  Nucleic Acid Testing (NAT)  Polymerase chair reaction (PCR)  Transcription Mediated Amplification (TMA)  Western Blot  Rapid Immunoassay (kit tests such as OraSure)
  35. 35.  Others  – Nucleic acid sequence-based amplification (NASBA),  ligase chain reaction (LCR),  branched DNA signal amplification (bDNA)
  36. 36.  – NO,  – Small percentage of Antibody positive donors have been tested negative by NAT tests.  – It is possible that an antibody positive and NAT Negative donation might transmit infection to the recipient.  – Therefore NAT Testing will not replace current serology tests in blood screening  – So far no country has discontinued the serology screening for HBsAg, Anti HIV and Anti HCV after implementation of NAT screening
  37. 37.  DHANYWAD