080429 APHM

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