Russell Cable

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NUCLEIC ACID TESTING (NAT)
An introduction to the Technology and Changes it
created
Russell Cable September 2005
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WPBTS collects 10,000 donations per month and
supplies tested units to the whole of the Western
Cape This means 500 donations collected and
tested daily Blood is collected daily from
Worcester, Paarl and George regions and from our
HQ clinics. All units and sample tubes are
transported to Pinelands for testing and
component preparation The introduction of NAT
testing has meant some changes to collection
methods, transporting of samples, sample
preparation, laboratory facilities and hours of
testing at WPBTS
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OLD Sample collection method 1 citrate tube Used
on Olympus for Blood grouping, syphilis and
irregular antibody testing 1 dry tube Used on
Prism for HIV HCV antibodies and HBsAg
testing and Used on Tecan for P24 Antigen
testing
NEW sample collection method 1 citrate tube Used
on Olympus for Blood grouping, syphilis and
irregular antibody testing 1 PPT gel tube 8,5
ml Used on Prism for HIV HCV antibodies, HBsAg
testing and archiving 1 PPT gel tube 6mlUsed
on Tigris for NAT testing
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New blood Packs/ sample taking method at Clinics
from end Nov
connector
Sampling pouch
Vaccutainer holder
Snap seal
Needle guard
2 GEL TUBES TO BE USED IN PLACE OF DRY TUBE

• Serial numbers
• At top of tube
• Must be straight

New tubes in place
Tubes must be inverted 8 times (minimum) to
activate gel
Gel in tube
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New automated sample preparation, sorting and
Archiving The Olympus - OLA 2500
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Input trays
De-capping
Output trays (Olympus, Prism Tigris)
Archiving
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HIV Testing Time-line
Relative concentration
2005
Time
1985
1996
HIV infection
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(No Transcript)
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The Tigris
1.83m
Mass 581 kg
0.91m
1.75m
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NAT Reduces Window of Detection
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Flow of information (genetic code)
Genetic code refers to the sequence of base pairs
in genes
mRNA is made complementary to the DNA
(transcription)
Proteins are formed from the mRNA (translation)
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How does it work?
A. Primer / Probe
Short length of ssDNA with a specific base
sequence, complementary to the target DNA or RNA
of the virus under investigation.
B. Enzymes

• Reverse Transcriptase (RT)
• Usually makes complementary DNA (cDNA) from RNA.
• But can also make cDNA from DNA.
• RNAse H activity degrades RNA

• RNA Polymerase
• Makes complementary RNA from DNA. (mRNA)

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How does it work ? (cont.)
Three steps are involved
1. Target Capture
2. Amplification (TMA)
3. Detection (HPA DKA)
Multi-tube Unit (MTU)
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Step 1 - Target Capture
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Target Capture
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Step 2 - Amplification
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Transcription Mediated Amplification (TMA)
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Step 3 - Detection
Hybridization Protection Assay (HPA)
Dual Kinetic Assay (DKA)
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Detection
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Interpretation of Results
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Testing Algorithm
Ultrio Reactive (flash glow)
HBcAb (total)
Discriminatory assays
HIV
HCV
HBV
PCR / Viral Load at a Reference. Laboratory
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Contamination
One of the major concerns when performing nucleic
acid amplification tests, is contamination.
To reduce this problem we have adopted certain
techniques and procedures

• Having a dedicated tube for the Tigris.

• Having the tubes decapped / uncorked using
  automated equipment.

• Having a dedicated area for receiving Tigris
  racks from the OLA.

• Using gloves at all times and changing them
  appropriately.

• Area around instruments is a no-go area for
  unauthorized personnel.

• Only trained staff allowed to operate instruments.

• Strictly following daily instrument QC and
  maintaining a clean laboratory environment.

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Contamination
The Tigris reduces the chance of contamination by

• Sample

Reaction tube (MTU).

• Target capture, TMA, HPA and DKA are all
  performed in
  the same reaction tube.

• Oil is used as an overlay in each tube.

• Tigris automatically decontaminates and destroys
  amplicon after each assay (MTUs are
  deactivated and discarded).

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SAMPLE THROUGHPUT

• 1000 samples in 14 hours
• First result in 5 hours
• 100 results per hour after the first set of
  results
• One operator, two instruments

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THANK YOU!
QUESTIONS?