IBTS - Nucleic Acid Testing Flashcards
How many people receive BTs every week?
Over 1000 every week
How many donations do we need every week to fulfil requirements?
3,000
How many donations so we get every year?
140,000
Write about using serology and NAT testing to test our donors
NAT and serology complement each other to detect viral infections
We use Alinity analysers in serology but Panther analysers in NAT
What do we test for in NAT?
HIV-1/2
HCV
HBV
HEV
Selective WNV testing
Is HIV RNA or DNA
RNA
Is HBV RNA or DNA
DNA
Is HCV RNA or DNA
RNA
Is HEV RNA or DNA
RNA
Is WNV RNA or DNA
RNA
Why use NAT
NAT detects very low levels of viral RNA or DNA
They are qualitative tests -> either positive or negative (not great for donor as it doesn’t say how much of an infection they have)
What is meant by selective West Nile Virus testing?
Only test on donors who have travelled to a risk area
What is the newest assay introduced
Hepatitis E assay
When can NAT not detect a virus?
Cannot detect viral infections during the eclipse phase (when stored in liver/lymph)
What sample type is used for NAT
Plasma or serum
What is the window period
The time between acquiring an infection and when it can be detected using serological markers (antibodies)
What is the main benefit or NAT?
Detects viral RNA or DNA early in the development of infection (during the window period) before antibodies are detectable
Comment on RNA replication versus DNA replication
RNA replicates rapidly -> substantial increase in viral load <5 days after infection -> doubling time only a few hours e.g. HIV @ 20 hours
DNA replicates slower -> can take several weeks to reach detectable levels e.g. HBV which has a doubling time of 2-3 days
Write about the combination of HBV NAT testing and HBV serology testing
HBV DNA can detect early infection (3 to 12 weeks)
HBV DNA + HBsAg detects acute infection (3 to 12 weeks)
HBV DNA + anti-Hbc total - detects chronic infection (DNA might not be detectable)
What proves to be the biggest risk to donors and why
HBV due to its very slow doubling time making it very difficult to detect
Low DNA levels are undetectable during the window period or are often below the limit of detection by the assay
Low DNA also seen during chronic HBV infections which can also be HBsAg negative -> may only have anti-Hbc positive serology
What is a chronic HBV infection also called?
Occult HBV infection
What Assay method is used
Ultrio Elite Assay Methodology
What are the main steps to the Ultrio Elite Assay method?
- Sample prep -> DNA/RNA extraction
- RNA/DNA target amplification
- Detection of amplified target
How is DNA/RNA amplified using the Panther?
Transcription mediated amplification (TMA)
How do we detect the amplified products using the panther assay?
Using the hybridisation protection assay (HPA) and dual kinetic analysis
Describe how the assay works
The target capture reagent (TCR) contains a detergent which solubilises the viral envelope - DNA/RNA now available
Oligonucleotides (primers) are added which bind to the DNA/RNA
The oligo/NA complex binds to magnetic particles in the TCR which allows for the isolation of the NA
A TER (target enhancer reagent) can be added which enhances the disruption of HBV viral particles
How does transcription mediated amplification work
An enzyme reagent is added containing two enzymes:
- MMLV reverse transcriptase
- T7 RNA polymerase
How does MMLV Reverse transcriptase work?
Generates DNA copy of the target sequence
Containing a promoter sequence for T7 RNA poly
How does T7 RNA polymerase work?
Produces multiple copies of RNA amplicon from the DNA copy template
How does the detection step of the assay work
A probe reagent is added containing a chemiluminescent label which binds to hybridised probe + virus to give a positive result
A selection reagents is added which inactivated any unhybridised probes (probes not bound to viral NA)
The chemiluminescent signal from the probe is measured in a luminometer and reported as relative light units (RLUs)
How does dual kinetic analysis work?
There is 2 results -> an internal control or a ‘flasher’ signal and the sample ‘glower’ signal -> both are required for the test to be valid
Results are graded as reactive, non reactive (negative/no virus), invalid (control failed - no ‘flasher’)
How do we test for multiple viruses at once
Multiples UE assay
Tests for all three viruses at once
What do we do if we get an initial reactive (IR)/positive result?
Our first test is a multiplex UE assay -> tests for 3 viruses at once
If positive we need to run a disciminatory assay
What is a discriminatory assay?
An assay whereby we use discriminatory probes to determine which virus is present i.e. which are repeat reactive in the sample
Tells us if its HIV, HCV or HBV
Why do we not have disciminatory probes for HEV and WNV?
These two assays only test for HEV or WNV, => no need for discrimination
What do we do if all three disciminatory probe assays come up positive?
Need to retest the donation -> very low chance that donor is positive for all three at once -> if still positive probably reacting against some component of the assay
What is West Nile Virus
A mosquito borne flavivirus transmitted in wild and captive birds
A neuropathogen which affects humans, equine and avians
Single stranded RNA virus lineage 1 and 2
When do we decide to test for WNV
We check the ECDC map every week to determine which countries are considered high risk
Anyone coming from these countries needs to be tested
In general when do we test for WNV
Seasonal testing done between may and december on those from high risk areas
We have not had a positive test yet
Why do we test for WNV even if we havent seen a single positive yet?
We can accept additional blood products collected from donors who otherwise would have been deferred
- think about rare phenotypes in SCD that could need this blood
Talk about HEV testing
The HEV assay is the only assay directly funded by the department of health - brought in from 2016
Incidence of about 1 in 5,000 Irish donations
1-4 genotypes
Transmitted through domestic pigs, consumption of undercooked pork and shellfish
Why is HEV testing so important in Ireland?
in 2020 there was 58 repeat positives -> meaning there was a 1 in 2,314 chance of HEV
There is no consistent seasonal pattern of HEV infection so all donors are tested
What sample types other than plasma/serum are seen in NAT lab?
Tissue samples
Maternal/Cord samples
Bone marrow samples
Virology positive follow ups
