1.  Nucleic Acid Technology IN
Blood Transfusion
MODERATOR- DR. POONAM NANWANI

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.  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.  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.  Tests used for TTI
 Front-line screening :
 • Rapid tests
 • ELISA tests
 • Chemiluminescence
 Tests for residual risks:
 • NAT test methods
 • Supplemental marker

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.  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.  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.  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.  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.  Determine the incidence of seroconversion
among donors who donate more than once
(multiple time donors)
 Not the prevalence rate in donor population

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

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. 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. 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.

17.  Five main steps:
1) Sample preparation by ultra-centrifugation
2) Reverse transcription of target RNA to
cDNA
3) Polymerase chain reaction amplification of
cDNA

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

19.  Choice of anticoagulant
 Nucleic acid stability in sample during
transportation
 PCR inhibitors in the sample
 False positive result and cross-contamination
 Internal control

20.  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

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

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

24. HIV

25.  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

27.  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)

28.  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

29.  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

30.  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

31.  Current US minipool HIV NAT screening
protocols fail to detect very low level
viraemia

32.  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?

33.  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

34.  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

35.  • 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.

36.  • 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).

37.  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)

38.  Others
 – Nucleic acid sequence-based amplification
(NASBA),
 ligase chain reaction (LCR),
 branched DNA signal amplification (bDNA)

39.  – 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

40. 
DHANYWAD