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TRANSCRIPT
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
Blood Safety
Transfusion Safety
BTS
Potential Hazards of Transfusion
1. Inherent infectious risks from donor2. Risks introduced at blood collection3. Risks introduced in blood processing4. Risks introduced during blood
administration5. Risks that are dependent on blood
component-recipient interactions6. 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
Most transfusion recipients are battling serious disease and have weakened immune systems
Screening limitations Gaps in current defenses exist, due to the window period and
limited screening sensitivity
Known pathogensRoutine testing covers only
a limited number
Bacteria The most frequent
transfusion-transmitted infection
LeukocytesResidual cells and cytokines
can cause harmful post-transfusion reactions
New and emerging pathogensA risk that current safety
measures cannot eliminate
Current blood safety risks to patients
1/100
1/1,000
1/10,000
1/100,000
1/1,000,000
Adapted from Transfusion 2000; 40:143-159
Risk of TTI
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
NAT
HIV
HCV
2007
2002
NAT
HBV
Infectious Diseases Screening at HKRCBTS
Mandatory Serological Tests
Primary screening
Supplementary Test
Confirmatory Test
NAT
HBsAg ChLIA - Specific Antibody
Neutralization Technique
IDT
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 testsTheoretical 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
Vi
rus:
RN
A / D
NA
Antib
odie
s
Front Antibody negative window
NAT negative window
INFECTION Detection minipool
SeroconversionDetectionIn Singledonation
Sero- reversion
Occasionally
DNA/RNA
Positive
(HBV/HCV)Late Antibody negative window
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 Amplificati
on - Ultrio/TIGRIS Roche Molecular Systems - Polymerase Chain Reacti
on - 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
NAT Testing Algorithms for Single Donation Testing
TMA MultiplexSingle Donation Testing
TMA MultiplexSingle Donation Testing
Non-ReactiveNon-ReactiveReactiveReactive
ReactiveReactive
Serological Test NegativeSerological
Test Negative
Non-ReactiveNon-Reactive
Serological Test PositiveSerological
Test Positive
ReleaseReleaseDispose DonationDispose Donation
Permanent Deferral
Permanent Deferral
Follow-Up Donor
Follow-Up Donor
TMA DiscriminatoryTMA Discriminatory
NAT TestingAlgorithmsfor Pooled Samples
TMA Multiplex in poolsTMA Multiplex in pools
Non-ReactiveNon-Reactive
ReactiveReactive
ReactiveReactive
Serological Test NegativeSerological
Test Negative
Non-ReactiveNon-Reactive
Serological Test PositiveSerological
Test Positive
Release if seronegative
Release if seronegative
Dispose DonationDispose Donation
Permanent Deferral
Permanent Deferral
Follow-Up Donor
Follow-Up Donor
TMA DiscriminatoryTMA Discriminatory
TMA Multiplex in singlesTMA Multiplex in singles
Non-ReactiveNon-Reactive
Repeat pool X2Repeat pool X2
ReactiveReactive
HIV
HCV
3.4
9.0 11.3
ID-NAT MP-NAT WB
ID-NAT MP-NAT
50.9
EIA 3.0
5.6
1 copy/20 mls
1 copy/20 mls
4.9 2.5
7.4
Window Periods Days for HIV, HCV & HBV
Source: Busch, AABB, 2006, & Kleinman and Busch, J Clin Virol. 2006;36:S23-S29, Assal ISBT &AABB
-------------------------------------------------------------------------------------------------------------------
1 copy / 20 mls Auszyme HBsAg (6,800 copy/ mls)Prism HBsAg
(1,664 copy / mls)
5.3
15
38.3
HBV
-------------------------------------------------------------------------------------------------------------------
19
ID NAT 1:8 - NAT
4
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
Sample Dilution
Results (S/C)
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
HIV-1 NAT on Window Period Sample
NAT Yield of HCV and HIV
CountryHIV-1 NAT Yield
(per 100,000)HCV-NAT Yield (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
? Anti-HBc Screening
L Comanor & P Holland. Hepatitis B virus blood screening: unfinished agendas. Vox Sang 2006; 91: 1-12.
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.
2. To estimate the prevalence of HBV DNA Positive/HBsAg negative Hong Kong Blood Donors (HBV NAT yields).
3. 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 detecte
d 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, Performa
nce qualification Compliance with tender specification requirement Performance check: Sensitivity, Specificity, Precision, LOD,
Carry over, Sample matrix effect 95% detection limit
WHO HIV-1 RNA 99/636 (IU/mL)
WHO HCV RNA 96/798 (IU/mL)
WHO HBV DNA 97/746 (IU/mL)
Package Insert 26 (*WHO 97/656)
4.6 (*WHO 96/790)
11.0
ARCBS Study 42.2 2.0 12.2
HKRCBTS Validation
24.1 (14.5-59.9) 2.72 (2.10-3.89) 7.29 (5.48-10.7)
Commissioning
Communicate with hospital clients Prospective policy A list of Q&A Switching over on 16 April 2007
April – December 2007Apr May June July Aug Sept Oct Nov Dec Total %
No of Reactive Ultrio 61 110 90 86 68 82 136 145 126 904 0.6247
No of Non-Reactive Ultrio 8,307 17,814 16,43
2 16,414 17,839 16,378 17,89
0
16,348 16,39
4
143,816
Total no of samples tested 8,368 17,924 16,522 16,500 17,907 16,460 18,026 16,493 16,520 144,720
No of samples with reactive Ultrio and non-reactive discriminatory results
35 36 33 16 24 30 53 56 65 348 0.2405
No of samples with reactive Ultrio and reactive discriminatory results
26 74 57 70 44 52 83 89 61 556 0.3842
No of samples with reactive discriminatory and positive serology results
24 70 53 63 40 48 75 82 58 513 0.3545
No of samples with reactive HBVNAT and negative HBV serology results
2 4 5 7 4 3 6 6 2 39 0.0269
No of samples with reactive HCVNAT and negative HCV serology results
0 0 0 0 0 1 2 1 1 5 0.0035
No of samples with reactive HIVNAT and negative HIV serology results
0 0 0 0 0 0 0 0 0 0 0.0000
Average down time 3.42%
Summary of resultsRoutine (July 2002 - April 2007)
Head to Head Comparative Study
16 April 2007 - 31 Dec 2007
Total tested ~930,000 10,397 144,720
NAT Assay Chiron PROCLEIX® HIV-1 & HCV,
pooled (16 ) and IDT
Chiron PROCLEIX® ULTRIO® Assay, IDT
Roche Cobas Taqscreen MPX Test, pooled (6)
Chiron PROCLEIX® ULTRIO® Assay, IDT
HIV Yield 0.32 / 100,000 donations
0 0 0
HCV Yield 0 0 0 0
HBV Yield NA 19.2/100,000 donations
19.2/100,000 donations
24.2/100,000 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 deter
mine 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 re
active 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