Hong Kong Association of Blood Transfusion & Haematology NAT and Viral Safety in Blood Transfusion Dr. P.H. Yu Medical Officer Department of Pathology.

Hong Kong Association of Blood Transfusion & Haematology

NAT and Viral Safety inBlood 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)


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


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

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

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


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


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?

Commonly used systems are

I. 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 captureRNA 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)


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


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


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



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



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)

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

HBV 1:543,500 1:60,759

Yield of NAT Screening on Blood Donors

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

Hong Kong Association of Blood Transfusion & Haematology NAT and Viral Safety in Blood Transfusion Dr. P.H. Yu Medical Officer Department of Pathology.

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