NAT Implementation –

the Hong Kong Experience

Dr Wai-Chiu Tsoi
Hong Kong Red Cross Blood
Transfusion Service
 Outline
 Overview of transfusion & blood safety
 Global & regional NAT implementation
 NAT implementation experience in Hong
Kong
The vein to vein transfusion chain
Transfusion Safety

Blood Safety

BTS
Potential Hazards of Transfusion
1. Inherent infectious risks from donor
2. Risks introduced at blood collection
3. Risks introduced in blood processing
4. Risks introduced during blood
administration
5. Risks that are dependent on blood
component-recipient interactions
6. Risks that are dependent entirely on
recipient characteristics
Factors Affecting Blood Safety
 Infection prevalence and incidence
 Donor selection and screening
 Laboratory testing and its performance
 Quality assurance and control
 Research and development
 Transfusion Medicine
 Policy decision and execution (pathogen
inactivation)
 Public health education
 Current blood safety risks to patients
Bacteria
The most frequent
transfusion-transmitted infection

Known pathogens
Routine testing covers only New and emerging pathogens
a limited number A risk that current safety
measures cannot eliminate

Screening limitations Leukocytes

Gaps in current defenses exist, Residual cells and cytokines
due to the window period and can cause harmful post-
limited screening sensitivity transfusion reactions

Most transfusion recipients are battling serious disease and have

weakened immune systems
 Risk of TTI
1/100

1/1,000

1/10,000

1/100,000

1/1,000,000

Adapted from Transfusion 2000; 40:143-159

 Donor issues
 There are donors at risk that still donate blood
- do not understand questionnaire
- do not understand implication of donating
- ‘Blood Bank will test the blood anyway &
would eliminate TTI’
- Seeking blood testing intentionally
 Higher Prevalence of TTI in some countries

⇒ Donor criteria, selection & declaration do not

always work
NA
T
NA
HIV T
HC HB
V V

2 2
002 007
Infectious Diseases Screening at HKRCBTS
Mandatory Serological Tests
Primary Supplementary Confirmatory NAT
screening Test Test
HBsAg ChLIA - Specific IDT
Antibody
Neutralization
Technique
Anti-HCV ChLIA ELISA RIBA-3 IDT
Anti-HIV 1 + 2 ChLIA EIA RIBA-3 IDT
Anti-HTLV I & II ChLIA - WB -
Anti-TP (Syphilis) ChLIA - TPHA
Additional Test
Anti-CMV MEIA - -
 Limitations of current screening tests
Theoretical infection transmission if
 The donor is in the “window period” of an infection
(e.g. HCV and HIV)
 The donor is a “low level carrier” in whom the level of
markers of chronic infection is below the sensitivity
of currently used assays (e.g. HBV)
 Rare / variant strains not detected by current routine
tests
 Possibilities of technical or clerical errors in
screening or quarantining blood components (very
very low in the present technology and laboratory
automation)
 Blood transfusion residual risks
(HBV/HCV)
Late
Antibody
Front Antibody negative window negative
window

NA
/D
NA

ies
NAT negative

:R
window

d
bo
us
Vir

ti
An
Sero- reversion

Occasionall
y
INFECTION Detection Detection Seroconversion
In Single minipool DNA/RNA
donation Positive
 Factors To Be Considered in the Decision to
Implement a new testing strategy e.g. NAT
 Local prevalence of infection
 Effectiveness of existing measures
 Impact in causing / reducing unnecessary donor
loss
 Availability and allocation of healthcare resources
 Political priority and public demand
 Ability to train and maintain skilled staff
 Availability of technology and access to technical
support
 NAT Systems
 Two multiplex NAT assays and automated testing
platforms available :
Gen-Probe/Chiron - Transcription-Mediated
Amplification - Ultrio/TIGRIS
Roche Molecular Systems - Polymerase Chain
Reaction - cobas MPX/cobas s201
 Reported sensitivities for TMA system of 26 IU/ml (16-58)
for HIV-RNA, 4.6 IU/ml (3.7-10.5) for HCV RNA and 11
IU/ml (7.3-22) for HBV DNA
(Kopelman et al. Multicenter performance evaluation of a transcription-mediated
amplification assay for screening of human immunodeficiency virus-1 RNA, hepatitis C
virus RNA, and hepatitis B virus DNA in blood donations. Transfusion 2005; 45:1258-
1266)
 NAT Assay Sensitivity for HIV, HIV and HBV

AR Margaritis et al. Comparison of two automated nucleic acid testing systems for
simultaneous detection of human immunodeficiency virus and hepatitis C virus RNA and
hepatitis B virus DNA. Transfusion 2007; 47:1783-1793
 Mini-pool versus Single Donation
Testing
• Single donation testing
 Follows usual testing algorithms – less disruptive,
faster component release
 Higher testing cost
 Require more facilities for large number of samples
• Minipool testing
 Able to handle large number of samples
 Lower cost
 Dilution factor may affect detection of infectious
donations with low viral load
 May delay availability of products involved in
positive pools
 TMA Multiplex
NAT Testing Single Donation Testing
Algorithms for
Single Donation
Reactive Non-Reactive
Testing

TMA Discriminatory

Reactive Non-Reactive

Serological Serological
Test Positive Test Negative

Permanent Follow-Up Dispose Release

Deferral Donor Donation
 TMA Multiplex in pools

NAT Testing Reactive Non-Reactive

Algorithms
for Pooled TMA Multiplex in singles

Samples
Reactive Non-Reactive

TMA Discriminatory Repeat pool X2

Reactive Non-Reactive
Release if
seronegative
Serological Serological
Test Positive Test Negative

Permanent Follow-Up Dispose

Deferral Donor Donation
 Window Periods Days for HIV, HCV & HBV
1 copy/20 mls ID-NAT MP-NAT WB

5.6 3.4

HIV 9.0 11.3

-------------------------------------------------------------------------------------------------------------------
1 copy/20 mls ID-NAT MP-NAT EIA 3.0

4.9 2.5

HCV 7.4 50.9

-------------------------------------------------------------------------------------------------------------------
1 copy / 20 mls ID NAT 1:8 - NAT Auszyme HBsAg
Prism HBsAg (6,800 copy/ mls)
(1,664 copy / mls)

19 15 4
HBV
38.3 5.3
Source: Busch, AABB, 2006, & Kleinman and Busch, J Clin Virol. 2006;36:S23-S29, Assal ISBT &AABB
Conclusions : Transmission of HIV from a blood donor to a
platelet recipient and a red blood cell recipient occurred in the
preseroconversion infectious window period. The viral load in the
implicated donation was estimated to be less than 40 copies/ml of
plasma. Current US minipool HIV NAT screening protocols may not
be sufficiently sensitive to detect all window period donations.
JAMA (2000) Vol 284 No 2
 HIV-1 NAT on Window Period Sample

Sample Results (S/C)

Dilution Tube 1 Tube 2 Tube 3

1:2 12.5 17 13.47

1:4 8.41 9.99 12.71

1:8 3.54 8.27 9.07

1:16 9.45 0.44 7.55

1:32 Insuff error 0.36 10.88

 NAT Yield of HCV and HIV
HIV-1 NAT Yield HCV-NAT Yield
Country
(per 100,000) (per 100,000)
USA 0.032 0.425

Canada 0 0.028

England 0 0.07

France 0.033 0.049

Germany 0.028 0.067

Netherlands 0 0.025

Spain 0 0.243
Adapted from: Coste et al. Implementation of donor screening for infectious agents
transmitted by blood by nucleic acid technology: update to 2003. Vox Sang 2005:88,
289-303
 NAT implementation experience in
Hong Kong
 Submit NAT implementation plan with estimated
budget in 1999.
 Funding support for routine NAT screening was
obtained in 2001.
 Need to test 200,000 donations annually
 Seroprevalence/residual risk (100,000 donations)
HIV - 3.05 / 0.11
HCV - 18.33 / 1.16
HBV - 450.76 / 29.78
 As NAT technology still evolving and fully
automated was expected to be available
soon, it was considered a better option to
outsource the testing until NAT is fully
automated. The option had the benefits of
saving in capital investments and
implementation time for a nearly outdated
technology.
 In July 2002, through an open tendering
process, contracted Australian Red Cross
Blood Service to provide routine NAT HIV-1
and HCV screening for donated blood using
a combined testing strategy of IDT and
PDT.
 Summary of results
July 2002 - April 2007

Total tested ~930,000 samples

NAT Assay Chiron PROCLEIX® HIV-1 &

HCV, pooled (1:16 ) and IDT
HIV Yield 0.32 / 100,000 donations

HCV Yield ＜ 0.1/100,000

HBV Yield Not performed

Advance in NAT Technology
 Consideration of HBV NAT
 Limitation of HBsAg as a HBV marker

L Comanor & P Holland. Hepatitis B virus blood screening: unfinished agendas. Vox Sang 2006;
91: 1-12.
 ? Anti-HBc Screening
 A joint study to compare the latest NAT blood
screening technology offered by Chiron Blood
Testing and Roche Molecular Systems
1. To compare “head to head” performance of
multiplex assays (including HBV DNA) on the two
available automated screening platforms.

3. To estimate the prevalence of HBV DNA

Positive/HBsAg negative Hong Kong Blood Donors
(HBV NAT yields).

5. To determine the impact of pool size on HBV DNA

detection.

CHIRON
 AR Margaritis et al. Transfusion 2007; 47:1783-1793

 Degree of automation
 Analytical Sensitivity
 Invalid test: 0.05% Vs 2.39 %
 Failed run rate: 2.92% Vs 5.53%
 Throughput
 Estimated Yield: 38 per 100,000 donations
 AR Margaritis et al. Transfusion 2007; 47:1783-1793

 Effect of pooling
Ultrio: 71 samples RR by both systems: Ultrio
detected 67/71 (94%) when tested in pool of 4; one
yield case become NR
Cobas MPX: all 4 yield cases are reactive
 Local Implementation of Automated
NAT Testing System

 Subsequent to the study, the HKRCBTS has,

through an open tender for an automated NAT
system, implemented the Chiron PROCLEIX®
ULTRIO® Assay (HIV-1, HCV & HBV) on the
PROCLEIX® TIGRIS® System in April 2007.
 Samples are tested individually.
 Shipper validation
Temperature, transit time
 Sample integrity
Enzymes
Cross contamination
Dust, glove powder,
solutions, microscopic
blood splashes
 Validation and Acceptance
 Installation qualification, Operational qualification,
Performance qualification
 Compliance with tender specification requirement
 Performance check: Sensitivity, Specificity, Precision, LOD,
Carry over, Sample matrix effect
 95% detection limit
WHO HIV-1 RNA WHO HCV RNA WHO HBV DNA
99/636 (IU/mL) 96/798 (IU/mL) 97/746 (IU/mL)

Package Insert 26 (*WHO 4.6 (*WHO 11.0

97/656) 96/790)
ARCBS Study 42.2 2.0 12.2

HKRCBTS 24.1 (14.5-59.9) 2.72 (2.10-3.89) 7.29 (5.48-10.7)

Validation
 Commissioning
 Communicate with hospital clients
 Prospective policy
 A list of Q&A
 Switching over on 16 April 2007
 April – December 2007
Ap r Ma y Ju ne Ju ly Aug S ept Oc t Nov De c To ta l %
1 1
No of Reactive Ultrio 61 110 90 86 68 82 136 7 14 5 12 6 6 90 0.6 24 7
, , 4
No of Non-Reactive Ultrio 8,307 17,814 16, 16 , 17,83 9 16 ,37 8 16,3 48 14 3, 816 　
8 3
432 41 4
Total no of samples tested 8,368 17,924 16,522 16,500 17,907 16,460 18,0269 16,493 9
16,520 144,720 　
0 4
　 　 　 　 　 　 　 　 　 　 　 　

No of samples with reactive Ultrio

and non-reactive 35 36 33 16 24 30 53 56 65 34 0.2 40 5
discriminatory results 8

No of samples with reactive Ultrio

and reactive 26 74 57 70 44 52 83 89 61 55 0.3 84 2
discriminatory results 6

　 　 　 　 　 　 　 　 　 　 　 　

No of samples with reactive

discriminatory and positive 24 70 53 63 40 48 75 82 58 51 0.3 54 5
serology results 3

No of samples with reactive

HBVNAT and negative 2 4 5 7 4 3 6 6 2 0.0 26 9
39
HBV serology results

No of samples with reactive

HCVNAT and negative 0 0 0 0 0 1 2 1 1 0.0 03 5
5
HCV serology results

No of samples with reactive

HIVNAT and negative HIV 0 0 0 0 0 0 0 0 0 0 0.0 00 0
serology results

Average down time 3.42%

 Summary of results
Routine Head to Head Comparative 16 April 2007 -
(July 2002 - Study 31 Dec 2007
April 2007)

Total tested ~930,000 10,397 144,720

NAT Assay Chiron Chiron Roche Cobas Chiron

PROCLEIX® PROCLEIX® Taqscreen PROCLEIX®
HIV-1 & ULTRIO® MPX Test, ULTRIO® Assay,
HCV, Assay, IDT pooled (6) IDT
pooled (16 )
HIV Yield and
0.32 IDT
/ 0 0 0
100,000
donations
HCV Yield 0 0 0 0

HBV Yield NA 19.2/100,000 19.2/100,000 24.2/100,000

donations donations donations
 HBV NAT Yield Donor & Donation
(Apr 07 – Jan 08)
 39 of these 43 potential yields have been confirmed true
positives by Prof J P Allain’s laboratory in Cambridge;
four were confirmed as negative.
 30 of all the 39 confirmed yield cases were anti-HBc
positive, and represent donors with occult HBV infection.
35 of them are repeat donors.
Some had donated blood over a span of 22 years.
In total, they had made some 700 donations.
 1 confirmed window period donation
 QPCR: 5-2910 IU/ml
 Most were genotype B. Type A2 and B were also found.
 Anti-HBs >100mIU/ml : 4 (all anti-HBc Positive)
 Comprehensive look-back study will be required to
determine the infectivity of these occult HBV donations.
 Potential Size of the Problem
 Annual blood collection = 200,000 units
 Estimated number of occult HBV donors
per year = 53 (200,000 X 0.0265%)
 Estimated number of occult HBV
donations to look-back per year = 2000
units
 Estimated number of recipients to look-
back for per year = 4000
 Importance of HBV NAT
 Of the 3 viruses, HBV has highest prevalence in our population
and therefore highest risk of transfusion transmission

 Anti-HBc screening by itself would lead to deferral of too many

otherwise acceptable donors in our population

 Current HBV NAT offers window period reduction of 10-23 days

over well known sensitive test such as the PRISM HBsAg test

 Occult HBV infections, common in our population and potentially

infectious, characteristically have very low viral levels and are
often HBsAg negative
 Japanese Red Cross Blood Centre
 2000 – 2004: Lookback for NAT, HBsAg and/or anti-HBc
reactive cases: 15721 cases
 ID-NAT
 158 positivities identified
 95 (60%) OBI, 60(38%) Window period donation
 Infectivity: 11/22 components (50%) cause sero-conversion
in WP cases; 1/33 (3%) in OBI
 Conclusion
 Regions with high HBV prevalence
Benefit most from HBV NAT testing
ID testing most practical
Extensive lookback study required to ascertain
infectivity of the yield cases
The End