NUCLEIC ACID AMPLIFICATION TESTING

VIRAL SAFETY IN BLOOD TRANSFUSION

Risk of transmitting infection to recipients has been drastically reduced in the past decades, due toa)Improved donor selection

b)Sensitive serologic screening assays

c)Application of viral inactivation procedures during manufacturing of plasma products

RESIDUAL RISK

Major sources of remaining risk are:

1. Window period donation

2. Viral variants not detected 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

NUCLEIC ACID AMPLIFICATION TESTING

Amplifying a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

APPLICATIONS IN TRANSFUSION MEDICINE

INFECTIONS

GENOTYPE/PHENOTYPE ANTIGENS

HLA TESTING

PARENTAGE

NUCLEIC ACID TESTING

Hybridisation based methods Probes with markers

Solid phase Liquid phase Enzymatic

Disadvantage Limited sensitivity Abundant quantity Inadequate for donor screening Less amenable for automation Good for research,not diagnosis

Amplification Based methods Small quantity More sensitive

PCR Real Time PCR TMA NASBA MICROARRAY SNP

PCR-POLYMERASE CHAIN REACTION

1983-Kary Mullis(Nobel Prize in 1993) Thermal cycling Amplify a specific region of a DNA strand (the DNA

target) typically ~10 kilo base pairs (kb) Cycles of repeated heating and cooling of the reaction

DNA melting  Enzymatic  replication of the DNA

DNA generated is itself used as a template for replication Chain reaction  -DNA template is exponentially amplified

COMPONENTS

1. DNA template that contains the DNA region (target) to be amplified

2. Two primers   complementary to the 3' (three prime) ends of each of the sense

and anti-sense  strand of the DNA target.

3.DNA polymerase (Taq polymerase from thermaus aquaticus or another) with a temperature optimum at around 70 °C.

Discovering a thermostable enzyme was crucial

4. Deoxynucleotide triphosphates (dNTPs), the building blocks from which the DNA polymerase synthesizes a new DNA strand

5. Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase

6.Divalent cations, magnesium or manganese ions; Generally Mg2+ is used Mn2+ used for PCR-mediated DNA mutagenesis, Higher Mn2+ concentration increases the error rate during DNA synthesis

7. Monovalent cation potassium ions

DNA ISOLATION

Cell disruption or cell lysis Removing membrane lipids by adding a detergent. Removing proteins by adding a protease (optional

but almost always done). Precipitating the DNA with an alcohol — usually

ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will

aggregate together, giving a pellet upon centrifugation.

PCR-STEPS Reaction volume of 10–200 μl Eppendorf tubes-Small reaction tubes (0.2–0.5 ml volumes) Thermal cycler- heats and cools the reaction tubes to achieve

the temperatures required at each step of the reaction   Peltier effect - Heating and cooling of the block holding the

PCR tubes by reversing the electric current Heated lids to prevent condensation at the top of the reaction

tube Older instruments needed layering.

PROCEDURE

. PCR consists of a series of 20-40 repeated

temperature changes, called cycles each cycle commonly consists of 3 discrete

temperature steps The cycling is often preceded by a single temperature

step (called hold) at a high temperature (>90°C), One hold at the end for final product extension or

brief storage.

Initialization step: heating the reaction to a temperature of 94–96 °C, which is held for 1–9 minutes. (hot-start PCR)

Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causesDNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.

Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing

annealing of the primers to the single-stranded DNA template. 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very

closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

Extension/elongation step: Depends on the DNA polymerase used;  Taq polymerase-optimum activity temperature at 72–

80 °C, DNA polymerase synthesizes a new DNA strand

complementary to the DNA template strand (by adding dNTPs that are complementary to the template in 5' to 3' direction)

At each extension step, the amount of DNA target is doubled,

Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.

Agarose gel electrophoresis is employed for size separation of the PCR products.

The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker)

PCR STAGES

Exponential amplification: At every cycle, the amount of product is doubled (assuming 100%

reaction efficiency). The reaction is very sensitive: only minute quantities of DNA need to

be present.

Levelling off stage: The reaction slows as.. DNA polymerase loses activity Consumption of reagents such as dNTPs and primers-limiting

Plateau: No more product accumulates Exhaustion of reagents and enzyme.

PCR-OPTIMISATION-CONTAMINATION

The PCR method is extremely sensitive, Requires only a few DNA molecules in a single

reaction for amplification across several orders of magnitude

Adequate measures to avoid contamination from any DNA present in the lab environment (bacteria, viruses, or human sources)

CONTROL OF CONTAMINATION

2 work areas Preparation and handling of pre-PCR reagents and the setup of the PCR reaction post- PCR processing, such as gel electrophoresis or PCR product purification

Pipettes with filter tips Fresh  laboratory gloves, A  laminar flow cabinet with UV lamp as a work station A negative control PCR reaction. dUTP and Uracil DNA Glycosylase Control reaction is set up in the same way as the experimental

PCRs, but without template DNA added, and is performed alongside the experimental PCRs.

Inhibitors Heparin Hemoglobin Lactoferrin Protocols should be followed

PRIMER DEFECTS

Cross annealing to unintended targets Primer dimer Hairpin

HAIR PINS

Secondary structures in the DNA Result in folding or knotting of DNA template or

primers- decreased product yield or failure of the reaction.

Correction Primer design that includes a check for potential secondary

structures in the primers Addition of DMSO or glycerol to the PCR to minimize

secondary structures in the DNA template

POLYMERASE ERRORS

Taq has no error-proof-reading activity Excision of any newly misincorporated nucleotide base from the

nascent DNA strand that does not match with its opposite base in the complementary DNA strand.

High error rate (mutations per nucleotide per cycle) of approximately 1 in 10,000 bases, which affects the fidelity of the PCR,

More if errors occur early in the PCR with low amounts of starting material,

Accumulation of a large proportion of amplified DNA with incorrect sequence in the final product.[2]

Several "high-fidelity" DNA polymerases, having engineered 3' to 5' exonuclease activity, have become available.

KOD DNA polymerase, a recombinant form of Thermococcus kodakaraensis 

KOD1 extracted from Thermococcus litoralis;  Pfu DNA polymerase, which is extracted from Pyrococcus

furiosus; Pwo, which is extracted from Pyrococcus woesii.

MAGNESIUM CONCENTRATION

Primers which bind to incorrect template sites are stabilized in the presence of excessive magnesium concentrations

Stabilize double stranded DNA and prevent complete denaturation of the DNA during PCR reducing the product yield.[3][4] 

Inadequate thawing of MgCl2 may result in the formation of concentration gradients within the magnesium chloride solution

Reduce the amount of free magnesium present hence reducing the activity of the enzyme Inc .template concentration, dNTPs and the presence of chelating agents (EDTA) or proteins .[

VARIANTS

Multiplex-PCR uses several pairs of primers annealing to different target sequences

Variable Number of Tandem Repeats (VNTR) PCR targets areas of the genome that exhibit length variation

Asymmetric PCR Nested PCR Hot-start PCR

Helicase-dependent amplification Nicking Enzyme Amplification Reaction Inverse PCR Thermal Asymmetric InterLaced

PCR (or TAIL-PCR)

RT PCR

For amplification of RNA Reverse transcriptase enzyme used to generate c

DNA from RNA Then PCR done

TMA-TRANSCRIPTION MEDIATED AMPLIFICATION

HIV,HCV,WNV TARGET OF AMPLIFICATION RNA RT AND DNA POLYMERASE IN SAME

REACTION

NUCLEIC ACID SEQUENCE BASED AMPLIFICATION

Single mixture ISOTHERMAL 410 –NO DENATURING RNA template is given to the reaction mixture, the first primer

attaches to its complementary site at the 3' end of the template Reverse transcriptase synthesizes the opposite, complementary

DNAstrand RNAse H destroys the RNA template (RNAse H only destroys

RNA in RNA-DNA hybrids, but not single-stranded RNA) The second primer attaches to the 5' end of the DNA strand T7 RNA polymerase produces a complementary RNA strand

which can be used again in step 1, so this reaction is cyclic.

STRAND DISPLACEMENT AMPLIFICATION LIGASE CHAIN REACTION PROBE HYBRID CAPTURE CLEAVASE INVADER ASSAY

REAL TIME PCR

FLOURESCENT-QUENCHER PROBE DEGRADATION

FLOURESCENT QUENCHER PROBE HAIRPIN UNFOLD

FLOURESCENT QUENCHER WORKING IN PROXIMITY

CYBER GREEN DYE AND MELTING CURVE

FLOURESCENCE PLOTTED-REAL TIME ANALYSIS POSSIBLE

MICRO ARRAYS

MULTIPLE PROBES IN GENE CHIPS COMPOSITION OF SPECIMEN

SNP-SINGLE NUCLEOTIDE POLYMORPHISM

Not needed for infectious disease screening Red cell antigen detection RFLP

IMPLICATIONS IN BLOOD BANKING

Work flow design Space Sample integrity Technical expertise Storage Control of contamination Cost

FIRST STEP

1998 Transmission of HCV by an intravenous

immunoglobulin preparation NAT for hepatitis C virus (HCV) RNA in plasma

pools recommended by the Committee for Proprietary Medicinal Products (CPMP)

Accepted by the European Agency for the Evaluation of Medicine (EMEA)

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

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

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

INTERNATIONAL FORUM ON IMPLEMENTATION OF DONOR SCREENING FOR INFECTIOUS AGENTS TRANSMITTED BY BLOOD BY NAT

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

MINI POOL NAT

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