Transcript NAT and Viral Safety in Blood Transfusion

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
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)
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
1/100
1/1,000
1/10,000
1/100,000
1/1,000,000
Adapted from Transfusion 2000; 40:143-159
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
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)
Residual Risk

1.
2.
3.
4.
Major sources of remaining risk are:
Window period donation
Viral variants not detect by current assays
Immunosilent donor
Laboratory testing error
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)
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
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)
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
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
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
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
USA
UK
Italy
Australia
HK
HIV
1:493,000
< 1:
2,000,000
1:408,000
1:1,200,000
1:877,147
HCV
1:103,000
<1:
200,000
1:230,000
1:250,000
1:86,137
HBV
1:63,000
1 : 50,000 –
170,000
1:63,400
1:160,000
1:3357
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.
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
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
What is NAT?

I.
Commonly used systems are
PCR-based assays (Roche Cobas
Ampliscreen)
II. Transcription mediated amplification
assay (GenProbe)
III. Others, including in-house PCR
preparations
GenProbe
 Recently approved by FDA (Feb 2002)
for donor screening
 Three main steps
1)Sample preparation & target capture
GenProbe
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
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
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.
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
Roche Cobas Ampliscreen
4) Hybridization of products to
oligonucleotide peroxidase conjugated
probe
5) Detection of probe-bound products by
colorimetric determination
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
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
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
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
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
HCV
 Very amenable to detection by pooled NAT
 NAT theoretically reduce the window period
by 41-60 days
HCV
HIV
 Short doubling time of 21 hours
 Window period of 16 days (p24 antigen) may
be reduced to 11 days by NAT
HIV
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
HBV
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)
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
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
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
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
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
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
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)
Yield: North America
 6 HIV NAT-reactive, p24 antigen-negative,
and seronegative donations identified from
26 million donations screened (1/4,390,000)
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
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
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
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)
Yield of NAT Screening on Blood Donors
USA
Australia
Germany
Japan
HIV
1:4,390,000
0:1,440,000
1:1,087,000
1:1,701,253
HCV
1:259,000
1:360,000
1:543,500
1:272,200
1:543,500
1:60,759
HBV
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
Will NAT Close the Window?
 Current US minipool HIV NAT screening
protocols fail to detect very low level
viraemia
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
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?
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
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
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