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![Page 1: EQASs for Blood Borne Virus Genomes and BSE Prions from Cattle Brain and INSTAND Reference Materials H.-P. Grunert 1,2, K.-O. Habermehl 1,2, V. Lindig.](https://reader036.fdocuments.us/reader036/viewer/2022072014/56649e8a5503460f94b8f2c2/html5/thumbnails/1.jpg)
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
![Page 2: EQASs for Blood Borne Virus Genomes and BSE Prions from Cattle Brain and INSTAND Reference Materials H.-P. Grunert 1,2, K.-O. Habermehl 1,2, V. Lindig.](https://reader036.fdocuments.us/reader036/viewer/2022072014/56649e8a5503460f94b8f2c2/html5/thumbnails/2.jpg)
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
![Page 3: EQASs for Blood Borne Virus Genomes and BSE Prions from Cattle Brain and INSTAND Reference Materials H.-P. Grunert 1,2, K.-O. Habermehl 1,2, V. Lindig.](https://reader036.fdocuments.us/reader036/viewer/2022072014/56649e8a5503460f94b8f2c2/html5/thumbnails/3.jpg)
Grunert, SoGAT, Bethesda, 2005 3
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)
![Page 4: EQASs for Blood Borne Virus Genomes and BSE Prions from Cattle Brain and INSTAND Reference Materials H.-P. Grunert 1,2, K.-O. Habermehl 1,2, V. Lindig.](https://reader036.fdocuments.us/reader036/viewer/2022072014/56649e8a5503460f94b8f2c2/html5/thumbnails/4.jpg)
Grunert, SoGAT, Bethesda, 2005 4
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
![Page 5: EQASs for Blood Borne Virus Genomes and BSE Prions from Cattle Brain and INSTAND Reference Materials H.-P. Grunert 1,2, K.-O. Habermehl 1,2, V. Lindig.](https://reader036.fdocuments.us/reader036/viewer/2022072014/56649e8a5503460f94b8f2c2/html5/thumbnails/5.jpg)
Grunert, SoGAT, Bethesda, 2005 5
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]
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Grunert, SoGAT, Bethesda, 2005 6
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%)
![Page 7: EQASs for Blood Borne Virus Genomes and BSE Prions from Cattle Brain and INSTAND Reference Materials H.-P. Grunert 1,2, K.-O. Habermehl 1,2, V. Lindig.](https://reader036.fdocuments.us/reader036/viewer/2022072014/56649e8a5503460f94b8f2c2/html5/thumbnails/7.jpg)
Grunert, SoGAT, Bethesda, 2005 7
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
log10 Copies/ml or IU/ml
+0,5 log-0,5 log
Median
+0,5 log-0,5 log
Median
Copies/ml
IU/ml95,6%
81,8%
Results in copies/ml:median = 28838 copies/ml = 4,46 log10geo. mean = 4,53+/- 0,5 log interval = 3,96 - 4,96+/- 1,0 log interval = 3,46 - 5,46
Results in IU/ml:median = 10200 IU/ml = 4,01 log10geo. mean = 3,97+/- 0,5 log interval = 3,51 - 4,51+/- 1,0 log interval = 3,01 - 5,01
Ntotal = 137
Ncopies/ml = 22
NIU/ml = 115
PCR all [IU/ml]bDNA [IU/ml]PCR all [copies/ml]bDNA all [copies/ml]
![Page 8: EQASs for Blood Borne Virus Genomes and BSE Prions from Cattle Brain and INSTAND Reference Materials H.-P. Grunert 1,2, K.-O. Habermehl 1,2, V. Lindig.](https://reader036.fdocuments.us/reader036/viewer/2022072014/56649e8a5503460f94b8f2c2/html5/thumbnails/8.jpg)
Grunert, SoGAT, Bethesda, 2005 8
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
log10 Copies/ml or IU/ml
+0,5 log-0,5 log
Median
+0,5 log-0,5 log
Median
Copies/ml
IU/ml91,2%
77,3%
Results in copies/ml:median = 3472 copies/ml = 3,54 log10geo. mean = 3,51+/- 0,5 log interval = 3,04 - 4,04+/- 1,0 log interval = 2,54 - 4,54
Results in IU/ml:median = 1217 IU/ml = 3,09 log10geo. mean = 3,09+/- 0,5 log interval = 2,59 - 3,59+/- 1,0 log interval = 2,09 - 4,09
Ntotal = 136
Ncopies/ml = 22
NIU/ml = 114
PCR all [IU/ml]bDNA [IU/ml]PCR all [copies/ml]bDNA all [copies/ml]
![Page 9: EQASs for Blood Borne Virus Genomes and BSE Prions from Cattle Brain and INSTAND Reference Materials H.-P. Grunert 1,2, K.-O. Habermehl 1,2, V. Lindig.](https://reader036.fdocuments.us/reader036/viewer/2022072014/56649e8a5503460f94b8f2c2/html5/thumbnails/9.jpg)
Grunert, SoGAT, Bethesda, 2005 9
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%)
![Page 10: EQASs for Blood Borne Virus Genomes and BSE Prions from Cattle Brain and INSTAND Reference Materials H.-P. Grunert 1,2, K.-O. Habermehl 1,2, V. Lindig.](https://reader036.fdocuments.us/reader036/viewer/2022072014/56649e8a5503460f94b8f2c2/html5/thumbnails/10.jpg)
Grunert, SoGAT, Bethesda, 2005 10
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
log10 Copies/ml or IU/ml
+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
![Page 11: EQASs for Blood Borne Virus Genomes and BSE Prions from Cattle Brain and INSTAND Reference Materials H.-P. Grunert 1,2, K.-O. Habermehl 1,2, V. Lindig.](https://reader036.fdocuments.us/reader036/viewer/2022072014/56649e8a5503460f94b8f2c2/html5/thumbnails/11.jpg)
Grunert, SoGAT, Bethesda, 2005 11
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
![Page 12: EQASs for Blood Borne Virus Genomes and BSE Prions from Cattle Brain and INSTAND Reference Materials H.-P. Grunert 1,2, K.-O. Habermehl 1,2, V. Lindig.](https://reader036.fdocuments.us/reader036/viewer/2022072014/56649e8a5503460f94b8f2c2/html5/thumbnails/12.jpg)
Grunert, SoGAT, Bethesda, 2005 12
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
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Grunert, SoGAT, Bethesda, 2005 13
22ndnd National BSE EQAS June 2003 National BSE EQAS June 2003Variance Analysis of the BioRad Test ResultsVariance Analysis of the BioRad Test Results
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Grunert, SoGAT, Bethesda, 2005 14
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
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Grunert, SoGAT, Bethesda, 2005 15
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.
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Grunert, SoGAT, Bethesda, 2005 16
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