Nucleic Acid Technology IN Blood Transfusion

MODERATOR- DR. POONAM NANWANI

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

"NAAT"

A nucleic acid test, often called a "NAT", (or nucleic acid amplification test - "NAAT") is a molecular technique used to detect a virus or a bacterium These tests were developed to shorten the window period a time between when a patient has been infected and when they show up as positive by antibody tests.

It reduces the window period by detecting low levels of viral genomic materials that are present soon after infection but before the body starts producing antibodies in response to a virus

Tests used for TTI Front-line screening :

• Rapid tests • ELISA tests • Chemiluminescence

Tests for residual risks:

• NAT test methods • Supplemental marker

VIRAL SAFETY IN BLOOD TRANSFUSION Risk of transmitting infection to

recipients has been drastically reduced in the past decades, due toa)Improved donor selectionb)Sensitive serologic screening assaysc)Application of viral inactivation

procedures during manufacturing of plasma products

RESIDUAL RISK Major sources of remaining risk are:1. Window period donation2. Viral variants not detect by current

assays3. Immunosilent donor4. 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

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

GENPROBE Recently approved by FDA (Feb 2002) for donor screening

Three main steps1)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

GENPROBE2) 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-

centrifugation2) Reverse transcription of target RNA to

cDNA3) Polymerase chain reaction

amplification of cDNA

ROCHE COBAS AMPLISCREEN4) Hybridization of products to

oligonucleotide peroxidase conjugated probe

5) Detection of probe-bound products by colorimetric determination

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

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 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

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 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 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

NAT EXPERIENCE OF VARIOUSCOUNTRIES IN THE SE ASIA • In Singapore, among the 466,779 samples

tested by NAT since October 2007 they were able to pick 9 HCV and 10 HBV NAT yield samples (1 in 24,567).

• Similarly in Thailand, Hong Kong and in Korea the NAT yield rate is 1 in 11, 676, 1 in 202,500 and 1 in 1, 46,628 respectively. Despite these countries having a very stringent donor counseling and screening process, a high rate of regular repeat voluntary donation, and use of the most sensitive serological tests, they were able toidentify a significant number of samples which were NAT reactivebut sero-negative.

• In India, Indraprastha Apollo Hospitals, Delhi has taken the initiative for NAT implementation for the first time in the country. In the first nine months of implementing NAT, they were able to pick five (3HBV and 2 HCV) NAT yield samples among 13,331 samples test

(1 in 2,666).

TESTING OF DONOR BLOOD – FORMATS Chemiluminescent Immunoassay Enzyme Immunoassay (EIA) Immunofluorescent assay (IFA) Nucleic Acid Testing (NAT)

Polymerase chair reaction (PCR) Transcription Mediated Amplification

(TMA) Western Blot Rapid Immunoassay (kit tests such

as OraSure)

Others – Nucleic acid sequence-based

amplification (NASBA), ligase chain reaction (LCR), branched DNA signal amplification

(bDNA)

WILL NAT TESTING REPLACE EXISTING IMMUNOASSAY SCREENING TESTS IN BLOOD SCREENING?

– NO, – Small percentage of Antibody positive

donors have been tested negative by NAT tests.

– It is possible that an antibody positive and NAT Negative donation might transmit infection to the recipient.

– Therefore NAT Testing will not replace current serology tests in blood screening

– So far no country has discontinued the serology screening for HBsAg, Anti HIV and Anti HCV after implementation of NAT screening

DHANYWAD