bert niesters - nibsc - home · 2011-09-13 · rob schuurman umcu, utrecht, nl bert niesters umcg,...
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Universal controls to calibrate quantitative real-time PCR based
assays; Overcoming the need for specific International
Standards?
Rob Schuurman
UMCU, Utrecht, NL
Bert Niesters
UMCG, Groningen, NL
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International Standards
• Role International Standards
• To harmonize laboratory results
• Inter-laboratory variation
• Inter-technology variation
• Commercial kits vs home-brew assays
• Quantitative results (viral load)
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International Standards
Need for quantitative MDX assays versus availability of IS:
• HIV √ subtype B
• HCV √ type 1
• HBV √
• CMV √
• B19 √
• EBV (√)
• ADV
• HHV-6
• BKV
• JCV
• Flu
• ....
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Copies/ml
Genome equivalents/ml
Mega-equivalents/ml
Log10 copies/ml
SuperQuant copies/ml
International units/ml
Number of HCV RNA CopiesPlethora of units used for reporting HCV RNA viral loads
`
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Number of HCV RNA Copies per
International Unit*
NGI SuperQuant 1 IU/mL = 3.4 copies/ml(NGI Product License Application to FDA)
Roche COBAS Amplicor v2.0 1 IU/mL = 2.4 copies/ml
Roche COBAS TaqMan 1 IU/mL = 2.7 copies/ml
LCx HCV RNA Assay 1 IU/mL = 3.8 copies/ml(Abbott Diagnostics)
Bayer bDNA 3.0 1 IU/mL = 5.2 copies/ml(Bayer Development Group)
According to CE guidelines and IVD directive, values should be related to an accepted standard: WHO HCV international standard for NAT 96/790 genotype 1A.
* WHO IU only genotype 1
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Why is this completely different for HIV-1:
All data provided in copies per ml for
most commercial assays?
There is an international standard
WHO HIV-1 RNA, subtype B for NAT 97/656
But hardly any results are reported in IU/ml.
Use of IS: HCV versus HIV
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Question:
Do we need a specific International Standard
for each pathogen?
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Assay SetupIn house Realtime PCR assays
Clinical samples Run control Negative control
Spiked with
PhHV (DNA) or
EMCV or BDV(RNA)
(generic internal control)
Automated extraction
Realtime Amplification
Diagnostic Target PhHV EMCV / BDV
Monoplex or multiplex format
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MDx state of the art in many labs
• All assays run in same format and amplification profile
• Each run tailored to the (panel of) requested tests
• Flexibility needs (low and high volumes)
• Standardized setup
• Standardized reagents
• ...
1 2 3 4 5 6 7 8 9 10 11 12
A Inf AB RSV Rhino Para 1/3 Para 2/4 Corona C.pneum M.pneum L.pneum Adeno Entero IC EMC
B Inf AB RSV Rhino Para 1/3 Para 2/4 Corona C.pneum M.pneum L.pneum Adeno PhHV
C Inf AB RSV Rhino Para 1/3 Para 2/4 Corona C.pneum M.pneum L.pneum Adeno Entero IC EMC
D Inf AB RSV Rhino Para 1/3 Para 2/4 Corona C.pneum M.pneum L.pneum Adeno PhHV
E RC Inf RC RSV RC Rhino Rc Para RC Para Rc Corona RC C.pneum RC M.pneum RC L.pneum RC Adeno X
F RC Inf RC RSV RC Rhino Rc Para RC Para Rc Corona RC C.pneum RC M.pneum RC L.pneum RC Adeno X
G NC Inf NC RSV NC Rhino NC Para NC Para NC Corona NC C.pneum NC M.pneum NC L.pneum NC Adeno X
H NTC Inf AB NTC RSV NTC Rhino NTC Para 1/3 NTC Para 2/4 NTC Corona NTC C.pneum NTC M.pneum NTC L.pneum NTC Adeno X
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Calculation of PCR Efficiency – (E = 10 -1/slope)
(based on the EUROHEP HBV standard, related to the WHO IU)
E = 10 -1/slope E = 10 -1/-3.440 E = 10 0.291 E = 1.953 HBV
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Inter-laboratory variation inpathogen quantitatition
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Quantitative Results Variation QCMD CMV
2010 Distribution
Note: QCMD CMV 2002: SD 0.5 - 0.8 log
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Quantitative Results Variation QCMD CMV
2010 Distribution (by technology)
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Standardization independent of target
analyzed using real-time technology.
Select laboratories with a good performance over the years from
the QCMD database.
Select them based on an efficient real time based amplification
assay.
Essential to receive quantitative data.
Ask them to perform the analysis on the identical proficiency
panels in triplicate, or more.
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25
27
29
31
33
35
37
39
41
43
2 3 4 5 6
Log copies DNA per ml
Ct
valu
e
Lab R HSV1 Lab V HSV1 Lab R HSV2 Lab V HSV2 Lab S HSV2
Standardization independent of target
analyzed using real-time technology
(each lab has its own standards)
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15
20
25
30
35
40
45
2 3 4 5 6 7 8 9
Log copies DNA per ml
Ct va
lue
Lab R HSV1 Lab V HSV1 Lab R HSV2 Lab V HSV2
Lab S HSV2 HBV VQC
Standardization independent of target
analyzed using real-time technology.
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Generic Standard for harmonization
quantitative results in plasma (?)
• For CMV IS has recently been defined:
• WHO / NIBSC study
• Strain:AD169
• IS: 1 geq = 1 IU
• Straightforward correlation between copies and IU
• Widely applied diagnostic pathogen (assays operational in
many labs)
• Supports harmonized (semi-)quantitative) viral load
values in the absence of a dedicated IS
• Pre-requisite:
• Amplification dynamics highly comparable between
generic standard and pathogen of interest
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Standardcurves for each pathogenplasma
40 data-points per concentration
y = -3,5904x + 42,897R2 = 0,9998 (HHV6)
y = -3,5826x + 42,536R2 = 0,9998 (EBV)
y = -3,5975x + 43,752R2 = 0,9994 (CMV)
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5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
- 2.00 4.00 6.00 8.00
Th
res
ho
ld C
yc
le (
Ct)
Log input (copies/ml)
Standard Curves Quantitative Assays uponMP96 extraction
(Large Volume TNAI protocol, 500ul))
HHV6 EBV CMV
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Conclusions
• Specific IS’s avaliable for limited number of pathogens
• Assay set up and performances for various pathogens are
highly comparable (“standardized”)
• Quantitive results are requested for increasing number of
pathogens
• Generic IS could serve as a surrogate standard for
harmonization of quantitative test results