eqass for blood borne virus genomes and bse prions from cattle brain and instand reference materials...

EQASs for Blood Borne Virus GenomesEQASs for Blood Borne Virus Genomes

and BSE Prions from Cattle Brainand BSE Prions from Cattle Brain

and INSTAND Reference Materialsand INSTAND Reference Materials

H.-P. Grunert1,2, K.-O. Habermehl1,2 , V. Lindig1 and H. Zeichhardt1,3

1Charité University Medicine Berlin, Campus Benjamin Franklin, Institute for Infectious Diseases Medicine, Dept. Virology, Berlin2Institute for Biotechnological Diagnostics (GBD), Berlin3Institute for Standardization and Documentation in Medical Laboratory (INSTAND), Düsseldorf (WHO Collaborating Center)

Collaborating Centers ofInternational Consortium for Blood Safety (ICBS), New York

Grunert, SoGAT, Bethesda, 2005 2

Serology PCR/NAT

HIV-1/2 Cytomegalovirus HIV-1 (RNA) Enterov. (PCR/NAT)

HIV-1 p24 Epstein-Barr Virus Hepatitis B Virus Adenov. (PCR/NAT)

Hepatitis A Virus Varicella Zoster V. Hepatitis C Virus

Hepatitis B V. Prg. I Herpes simplex V. Cytomegalovirus

Hepatitis B V. Prg. II Measlesvirus Varicella Zoster V.

Hepatitis C Virus Mumpsvirus Herpes simpl. V. 1

Hepatitis D Virus Resp. Sync. V. AG Herpes simpl. V. 2

Hepatitis E Virus Influenzav. A/B AG Influenzavirus A/B

Rubellavirus TBE Virus Parvovirus B19

Parvovirus B19 BSE (PrPsc) HPV (PCR/NAT)

Samples total: > 549.000

National and WHO International EQASsNational and WHO International EQASsin Immunology/Virologyin Immunology/Virology


EQASs in VirologyEQASs in VirologyVirusserology

since 1988: > 446,000 samples total (20 programs)

Virusgenome detection

since 1997: > 103,000 samples total (12 programs)

Organization INSTAND (Düsseldorf)

Authorized by Bundesärztekammer (BÄK)World Health Organization (WHO)International Consortium for Blood Safety (ICBS)

Scientific societies Deutsche Vereinigung zur Bekämpfung der Viruskrankheiten (DVV)Gesellschaft für Virologie (GfV)Deutsche Gesellschaft für Hygiene und Mikrobiologie (DGHM)


Experiences from EQAS Experiences from EQAS

for HIV, HCV and HBV:for HIV, HCV and HBV:

Evaluation of quantitative PCR/NATEvaluation of quantitative PCR/NAT

by median +/- 0.5 logby median +/- 0.5 log1010

IU/ml versus copies/mlIU/ml versus copies/ml


EQAS Nov 2004 HIV sample EQAS Nov 2004 HIV sample 1005410054 median: 1,110 copies/ml; median: 1,110 copies/ml; 1,033 IU/ml1,033 IU/ml

2,10-2,19

2,2- 2,3- 2,4- 2,5- 2,6- 2,7- 2,8- 2,9- 3,0- 3,1- 3,2- 3,3- 3,4- 3,5- 3,6- 3,7- 3,8- 3,9- 4,0- 4,1- 4,2-

NAT all [Copies/ml]; N=100

NAT all [IU/ml]; N=6

0

2

4

6

8

10

12

14

16

18

20

Nu

mb

er

log10 Copies/ml or IU/ml

+0,5 log-0,5 logMedian

+0,5 log-0,5 logMedian

Copies/ml

IU/ml

Results in copies/ml:median = 1110 copies/ml = 3,05 log10geo. mean = 3,05+/- 0,5 log interval = 2,55 - 3,55+/- 1,0 log interval = 2,05 - 4,05

Results in IU/ml:median = 1033 IU/ml = 3,01 log10geo. mean = 3,07+/- 0,5 log interval = 2,51 - 3,51+/- 1,0 log interval = 2,01 - 4,01

Ntotal = 106

Ncopies/ml = 100

NIU/ml = 6

100%

96%

NAT all [copies/ml]

NAT all [IU/ml]


Quantitative PCR/NAT Quantitative PCR/NAT Confidence Interval: Median +/- 0.5 logConfidence Interval: Median +/- 0.5 log

* without results for bDNA for samples <200,000 copies/ml (Nov. 1999 - Nov. 2000)& without results for bDNA for samples <3,000 copies/ml (Apr. 2004) without results for bDNA for samples <1,220 IU/ml (Apr. 2004)

Virus Date No. of samples

Median(copies/ml)

(IU/ml)

Numberof

analyses

Results in interval:

median+/-0.5 log

HIV-1 Nov 99-Nov 04 35 700-21,700 3498 93%(88%-99%)

Nov 02-Nov 04 16 380-14,000 105 87%(57%-100%)

HCV*,& Nov 99-Nov 04 32 3,000-1,351,000 1117 80%*,&

(67%-91%)

Apr 00-Nov 04 29 1,220-400,000 2683 93%&

(85%-100%)


EQAS Nov 2004 HCV sample EQAS Nov 2004 HCV sample 1205412054 median: 28,838 copies/ml; median: 28,838 copies/ml; 10,200 IU/ml10,200 IU/ml

<3,0 3,00 -3,09

3,1 3,2 3,3 3,4 3,5 3,6 3,7 3,8 3,9 4,0 4,1 4,2 4,3 4,4 4,5 4,6 4,7 4,8 4,9 5,0 5,1 5,2 5,3 5,4 >5,5

PCR all [IU/ml]; N=104

bDNA all [IU/ml]; N=10

PCR all [Copies/ml]; N=13

bDNA all [Copies/ml]; N=9

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

Nu

mb

er


+0,5 log-0,5 log

Median

+0,5 log-0,5 log

Median

Copies/ml

IU/ml95,6%

81,8%



Ntotal = 137

Ncopies/ml = 22

NIU/ml = 115

PCR all [IU/ml]bDNA [IU/ml]PCR all [copies/ml]bDNA all [copies/ml]


EQAS Nov 2004 HCV sample EQAS Nov 2004 HCV sample 1205312053 median: 3,472 copies/ml; median: 3,472 copies/ml; 1,217 IU/ml1,217 IU/ml

<2,1 2,10-2,19

2,2- 2,3- 2,4- 2,5- 2,6- 2,7- 2,8- 2,9- 3,0- 3,1- 3,2- 3,3- 3,4- 3,5- 3,6- 3,7- 3,8- 3,9- 4,0- 4,1- 4,2- 4,3- 4,4- 4,5- >4,6

PCR all [IU/ml]; N=104

bDNA all [IU/ml]; N=10

PCR all [Copies/ml]; N=13

bDNA all [Copies/ml]; N=9

0

2

4

6

8

10

12

14

16

18

20

22

Nu

mb

er


+0,5 log-0,5 log

Median

+0,5 log-0,5 log

Median

Copies/ml

IU/ml91,2%

77,3%



Ntotal = 136

Ncopies/ml = 22

NIU/ml = 114

PCR all [IU/ml]bDNA [IU/ml]PCR all [copies/ml]bDNA all [copies/ml]


Quantitative PCR/NAT Quantitative PCR/NAT Confidence Interval: Median +/- 0.5 logConfidence Interval: Median +/- 0.5 log

* without results for bDNA for samples <200,000 copies/ml (Nov. 1999 - Nov. 2000)& without results for bDNA for samples <3,000 copies/ml (Apr. 2004) without results for bDNA for samples <1,220 IU/ml (Apr. 2004)# without results for bDNA and hybridization for samples <1,000,000 copies/ml (Nov. 1999 - Apr. 2003)$ without results for hybridization for samples <1,000,000 copies/ml (Nov. 2003 - Apr. 2004)§ without results for bDNA for samples <500,000 IU/ml (Apr. 2002 - Apr. 2003)

Virus Date No. of samples

Median(copies/ml)

(IU/ml)

Numberof

analyses

Results in interval:

median+/-0.5 log

HIV-1 Nov 99-Nov 04 35 700-21,700 3498 93%(88%-99%)

Nov 02-Nov 04 16 380-14,000 105 87%(57%-100%)

HCV*,& Nov 99-Nov 04 32 3,000-1,351,000 1117 80%*,&

(67%-91%)

Apr 00-Nov 04 29 1,220-400,000 2683 93%&

(85%-100%)

HBV#,$,§ Nov 99-Nov 04 33 8,300-1,668,000 2399 79%#,$

(67%-91%)

Nov 01-Nov 04 21 1,350-933,000 419 74%§

(55%-92%)


EQAS Nov 2004 HBV sample EQAS Nov 2004 HBV sample 1105511055 median: 1,500,000 copies/ml; median: 1,500,000 copies/ml; 439,000 IU/ml439,000 IU/ml

<4

,6

4,6

0-4

,69

4,7

4,8

4,9

5,0

5,1

5,2

5,3

5,4

5,5

5,6

5,7

5,8

5,9

6,0

6,1

6,2

6,3

6,4

6,5

6,6

6,7

6,8

6,9

7,0

7,1

>7,

2

PCR (Roche Copies); N=29

PCR (in house Copies); N=3

Taqman (Roche Copies); N=3

Lightcycler (Artus Copies); N=5

bDNA (Bayer Copies); N=5

PCR (Artus IU); N=1

Taqman (Artus IU); N=1

Taqman (in house IU); N=4

Lightcycler (in house IU); N=2

bDNA (Bayer IU); N=5

0

2

4

6

8

10

12N

um

ber


+0,5 log-0,5 log

Median

+0,5 log-0,5 log

Median65,7%

77,1%

Ntotal = 118

Ncopies/ml = 70

NIU/ml = 48

Copies/ml

IU/ml

PCR (Roche Copies); N=29PCR (Abbott Copies); N=1PCR (in house Copies); N=3nested PCR (in house Copies); N=1

Taqman (Roche Copies); N=3Taqman (in house Copies); N=13Lightcycler (Artus Copies); N=5Lightcycler (in house Copies); N=10

bDNA (Bayer Copies); N=5PCR (Roche IU); N=3PCR (Artus IU); N=1PCR (in house IU); N=1

Taqman (Artus IU); N=1Taqman (Roche IU); N=17Taqman (in house IU); N=4Lightcycler (Artus IU); N=12Lightcycler (in house IU); N=2other PCR (Artus IU); N=2bDNA (Bayer IU); N=5


National External Quality Assessment National External Quality Assessment Schemes (EQASs) for BSE TestingSchemes (EQASs) for BSE Testing

Cooperations:

K.-O. Habermehl Inst. f. Biotechnologische Diagnostik, Berlin

H. Zeichhardt Charité Universitätsmedizin BerlinH.-P. Grunert Campus Benjamin Franklin

Institut für Infektionsmedizin, Abt. Virologie

M. H. Groschup Bundesforschungsanstalt f. ViruskrankheitenU. Ziegler der Tiere, Insel Riems

Financial support by: BMBF, BMGes, DVV

Performed: since May 2002

Participants: total 40 - 56

Samples total: > 1000


National BSE EQASs: Summary of the ResultsNational BSE EQASs: Summary of the ResultsDate

Sample Correct Results

No. Properties % No. of analyses

May 2002

1 neg. 100 57/57

2 neg. 98,2 56/57

3 pos. (1:12) 89,5 51/57

4 pos. (1:6) 96,4 54/56

5 pos. (1:4) 96,5 55/57

Jun. 2003

1 neg. 92,7 38/41

2 neg. 92,7 38/41

3 pos. (1:12) 100 41/41

4 pos. (1:8) 100 41/41

5 pos. (1:6) 100 41/41

Mar. 2004

1 neg. 100 44/44

2 neg. 100 44/44

3 pos. (1:16) 95,5 42/44

4 pos. (1:12) 100 44/44

5 pos. (1:12) 100 44/44

6 pos. (1:6) 100 44/44

Feb. 2005

1 neg. 97,5 39/40

2 neg. 97,5 39/40

3 neg. 100 40/40

4 neg. 100 40/40

5 pos. (1:16) 97,5 39/40

6 pos. (1:12) 100 40/40

7 pos. (1:12) 97,5 39/40

8 pos. (1:6) 97,5 39/40


22ndnd National BSE EQAS June 2003 National BSE EQAS June 2003Variance Analysis of the BioRad Test ResultsVariance Analysis of the BioRad Test Results


Global Network Global Network for External and Internal Quality Controlfor External and Internal Quality Control

External Quality Control (EQASs)

• EQASs (Germany, Europe, WHO) with >990 laboratories with all routine tests• 3x per year (2006: 4x per year)

Internal Quality Control

Surplus samples from EQASs• "Run" controls• daily

National reference materials• "Run" controls in coop. with PEI• daily

Identical controls for: Diagnostic labs

Blood banks Industries Licensing organizations (e.g. PEI) Scientific labs


National Reference MaterialsNational Reference Materials

Virus Properties Copies/ml

HIV-1 Subtype B pos. (++) 40.000 adjusted tointernationalreference materials and standards (WHO)

pos. (+) 4.000

HCV Genotype 1b pos. (++) 30.000

pos. (+) 3.000

HBV pos. (++) 160.000 developed in cooperation with PEI and reference laboratories pos. (+) 8.000

Negative control neg. 0

Influenza A pos. Bejing/262/95 (H1N1) available fromINSTAND and Institut für Biotechnologische Diagnostik (GBD)

pos. Sydney/5/97 (H3N2)

Influenza B pos. Yamanashi/166/98

Negative control neg.


INSTAND Reference MaterialsINSTAND Reference Materials(www.instand-ev.de)(www.instand-ev.de)

INSTANDINSTITUTE FOR STANDARDIZATION AND DOCUMENTATION

IN MEDICAL LABORATORIES e.V.P.O. Box 250211, D-40093 Düsseldorf, Germany

Tel.: +49-211-159213 0, Fax: +49-211-159213 30, E-mail: [email protected]

Samples for Test Assessment of Virus Diagnostics

National Reference Samples

(National Working References)

Surplus Samples of External Quality Assessment Schemes

in Virus Immunology and Virus PCR/NAT

As of February 2005

eqass for blood borne virus genomes and bse prions from cattle brain and instand reference materials...

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