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

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)

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

• ....

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

`

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

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

Question:

Do we need a specific International Standard

for each pathogen?

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

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

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

Inter-laboratory variation inpathogen quantitatition

Quantitative Results Variation QCMD CMV

2010 Distribution

Note: QCMD CMV 2002: SD 0.5 - 0.8 log

Quantitative Results Variation QCMD CMV

2010 Distribution (by technology)

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.

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)

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.

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

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)

-

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

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