Nat testing

NAT

1. Dr. RAFIQ AHMAD
Regional Laboratory & Blood Bank
Dammam, K.S.A.
NAT Testing in Blood Banks

2. Blood Transfusion Safety
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)
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

3. 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

4. Viral Safety in Blood Transfusion

5. 1/100
1/1,000
1/10,000
1/100,000
1/1,000,000
Viral Safety in Blood Transfusion

6. Transfusion transmitted risks
 In 1994, several cases of HCV infection were attributed
to IVIG 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)
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

7. 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)
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

8. Window Period
It is the period that 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
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 sensitivity exceeds the minimum infective dose for that
virus (window period closure)

9. Window Period

10. 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
Risk is calculated by multiplying the incidence rate in blood
donor by the length of the window period
Determine the incidence of seroconversion among donors
who donate more than once (multiple time donors)
Note the prevalence rate in donor population
Under-reporting

11. 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

12. NAT in Regional Blood Bank
NAT System used in RBB:
1.PCR-based assays (Roche Cobas Ampliscreen)
2.Transcription mediated amplification assay (Prism
Grifols)

13. 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
4.Hybridization of products to oligonucleotide peroxidase conjugated
probe
5.Detection of probe-bound products
by colorimetric determination

14. Prism Grifols
Approved by FDA and EU for donor screening
Three main steps
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
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. 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
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

16. 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

17. 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

18. 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
Very amenable to detection by pooled NAT
NAT theoretically reduce the window period by 41-60
days

19. HCV

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

21. HIV

22. 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

23. HBV

24. 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
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
In Saudi Arabia ID-NAT testing started in 2008

25. Will NAT Close the Window?
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
Current US minipool HIV NAT screening protocols fail to
detect very low level viraemia
Ling AE, et al. JAMA 2000;284:210-214

26. Cost-effectiveness
NAT is a intensive process to perform, requiring specially
ventilated & clean laboratory, expensive equipment and
reagents
In K.S.A. Under SFDA rules and CBAHI guidelines the
IND-NAT testing is recommended

27. Conclusion
Despite cost-effective issues, based on public perception and
political pressure, NAT screening of the blood supply has
become a standard in transfusion medicine
Draft Guidance on Use of NAT to identify HIV and HCV in
Whole Blood and Blood components is issued by FDA in
March 2002, Replacing p24 antigen
More and more countries will require NAT non-reactive
results before release of blood products
Automated and high-throughput system
Individual NAT testing in K.S.A. Since 2008

28. 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

29. The End!!!
Thank you