PEI Budapest May 24, 2012

Novartis Diagnostics Symposium IPFA/PEI Budapest May 24, 2012 Addressing Emerging Infectious Threats to the Supply of Blood and Blood Derived Produc...
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Novartis Diagnostics Symposium IPFA/PEI Budapest May 24, 2012

Addressing Emerging Infectious Threats to the Supply of Blood and Blood Derived Products Agenda: 12.45 Welcome and Introduction Susan L. Stramer, Ph.D. Executive Scientific Officer, American Red Cross

12.50 Advancing the NAT Pipeline: Status of Parvo B19/HAV, Dengue and HEV Jeffrey M Linnen, Ph.D. Director Product Development, Gen-Probe Incorporated

13.10 In-Process Testing for Parvo B19 and HAV: the Finnish Experience Anna Kallio, Microbiologist M.Sc Laboratory Operations for Production, Finnish Red Cross Blood Service

13.25 Q&A

Parvovirus B19  Erythrovirus, tropic for erythroid progenitor cells  Non-enveloped; resistant to inactivation, thus plasma for manufacture screened by NAT at a cutoff below which transmission has not been documented

 Some patients at risk for more severe disease  Extremely high viral loads during acute infection, frequently ~1012 DNA copies/mL

 Most adults immune  Viremia precedes symptoms but rarely associated with transfusion; some countries screen donors (NAT, HA, Ab-”safe”)

 DNA prevalence in donors varies (~1%; ultra-sens ID NAT)  Transfusion transmission associated with low and high-titer virus (some with Ab)

HAV  Non-enveloped Hepatovirus (picornavirus)  Resistant to inactivation (heat, pH; persists weeks to months in the environment), thus plasma for manufacture screened by NAT

 Fecal-oral transmission, food and water-borne, or close personal contact; may result in large outbreaks

 Infection and disease are almost always acute; immunity life-long  Vaccine available for children > 1 yr, travelers to high-risk areas  Viremia precedes symptoms but rarely associated with transfusion; some reports of secondary local transmission

 Transmission by clotting factors

HEV  Non-enveloped Hepevirus  4 genotypes (1 serotype) with varying geographic distribution and pathogenicity for humans  Globally, most common cause of acute viral hepatitis

 Fecal-oral transmission, water-borne  Some food-borne cases/outbreaks attributable to consumption of raw or undercooked pork

 20-40% antibody prevalence; high rates attributable to serotypes of low human pathogenicity

 Increased disease severity in pregnant women (3rd trimester)  Transfusion transmission reported in Hokkaido, Japan; NAT implemented

Dengue viruses  Mosquito-borne flavivirus; 4 closely related “types”  Most important “arthropod-borne” virus (arbovirus)  Rapidly expanding global footprint; >2.5 billion people (~1/3 world’s population) live in areas of risk; endemic in >100 countries

 Asia/Latin America – leading cause of hospitalization in children  No vaccine or specific treatment; vector control is the only effective intervention

 Immunity to a given type is lifelong but cross reactivity between types is short lived and increases risk for severe dengue

 3 clusters of transfusion transmission reported (Hong Kong, Singapore, Puerto Rico)

 Kidney, BM transplant, needle-stick and lab infections

Considerations for each agent  Should more be done?  If so, what? • Plasma donor screening – inactivation-resistant viruses • Routine blood donor screening

Advancing the NAT Pipeline: Status of Parvo B19/HAV, Dengue and HEV Assays Jeffrey M. Linnen, Ph.D. Gen-Probe Incorporated

The Procleix Panther System and Procleix Ultrio Elite Assay are currently under development and not available for commercial sale. The performance characteristics of this product (under the IVD Directive, 98/79/EC) have not yet been established. The Procleix Dengue Assay and the Hepatitis E Assay are currently under development and are not available for commercial sale. The performance characteristics of these products in development have not been established. 8

Gen-Probe/Novartis Product Development Activity •

Procleix Ultrio Elite Assay on the Panther System – Assay for screening for HIV-1/2 RNA, HCV RNA, and HBV DNA



Procleix WNV Assay on the Panther System – Assay for screening for West Nile virus RNA (lineages 1 and 2)



Procleix Parvo/HAV Assay on the TIGRIS System – CE mark in January of 2012; available for use in the US since March 2012



Procleix Dengue Virus Assay on the TIGRIS System – Prospective screening under an investigative protocol (IND) will start later this year at the American Red Cross (ARC, Charlotte, NC) and Creative Testing Solutions (Tampa, FL)



Hepatitis E virus (HEV) assay on the Panther System –

Prototype assay under development PROCLEIX and ULTRIO are trademarks of Novartis Vaccines and Diagnostics, Inc. TIGRIS and PANTHER are trademarks of Gen-Probe Incorporated; The Procleix Panther System and Procleix Ultrio Elite Assay are currently under development and not available for commercial sale. The performance characteristics of this product (under the IVD Directive, 98/79/EC) have not yet been established

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Procleix Parvo/HAV Assay on the TIGRIS System Duplex Assay: Quantitative detection of parvovirus B19 and qualitative detection of hepatitis A virus (HAV) – Discrimination between B19 and HAV in initial reaction

• TIGRIS System: – High throughput automation – Fully enclosed and integrated – Process controls: over 250 onboard sensors • All steps tracked and logged

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Assay Design: Quantitative Parvovirus B19/Qualitative HAV Assay •

Quantitative Parvovirus B19 Detection –





Qualitative HAV Detection – –



Quantitative range: 500-100,000 IU/mL (calibrated based on WHO standard); allows variable pool sizes: ~1:16 to 1:512 for screening (valid results outside of reportable range) Customer configurable cutoff (by work list)

95% detection < 1 IU/mL Customer configurable suppression of HAV results (by work list)

Internal Control –

Validates each individual reaction (target capture  amplification  detection)

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In-house Studies Completed at Gen-Probe Performance characteristics evaluated: •

Limit of detection (LOD) for HAV



Sensitivity for detection of HAV subtypes



HAV reproducibility



Accuracy of B19 quantitation



Limit of B19 detection (LOD) and limit of quantitation (LOQ)



Linearity of B19 quantitation



Quantitation of parvovirus genotypes 1, 2, and 3



B19 reproducibility (precision)



B19 clinical sensitivity (neat and in plasma pools)



Effect of high parvovirus levels on HAV sensitivity



Effect of HAV on B19 quantitation at the low end of the dynamic range



Evaluation of donor and donation factors on assay performance

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HAV Limit of Detection HAV WHO (00/560) IU/mL

Lot 1

Lot 2

Lot 3

Combined Lots

Reactive/ Tested

% Reactive (95% CI)

Reactive/ Tested

% Reactive (95% CI)

Reactive/ Tested

% Reactive (95% CI)

Reactive/ Tested

% Reactive (95% CI)

10

60/60

100 (94-100)

60/60

100 (94-100)

60/60

100 (94-100)

180/180

100 (98-100)

3.16

60/60

100 (94-100)

60/60

100 (94-100)

60/60

100 (94-100)

180/180

100 998-100)

1

58/60

97 (89-99)

58/60

97 (89-99)

59/60

98 (91-100)

175/180

97 (94-99)

0.32

39/60

65 (52-76)

37/60

62 (49-73)

34/60

57 (44-68)

110/180

61 (54-68)

0.1

15/60

25 (16-37)

26/60

43 (32-56)

21/60

35 (24-48)

62/180

34 (28-42)

0.03

10/60

17 (9-28)

6/60

10 (5-20)

4/60

7 (3-16)

20/180

11 (7-17)

0

0/60

0 (0-6)

0/60

0 (0-6)

0/60

0 (0-6)

0/180

0 (0-2)

CI: confidence interval by SCORE method; CV: coefficient of variability; S/CO: signal to cutoff

• • •

Panels comprised of serial dilutions of the WHO First International Standard for Hepatitis A Virus RNA (NIBSC code: 00/560) Two lots of the sensitivity panels (each from 2 vials of lyophilized standard) were tested using 3 reagent kit lots on 2 TIGRIS Systems A total of 180 replicates at each concentration 13

Probit Analysis of HAV Analytical Sensitivity Results HAV Detection Probabilities Reagent Kit Lot

50% Limit of Detection in IU/mL (95% Fiducial Limits)

95% Limit of Detection in IU/mL (95% Fiducial Limits)

Lot 1

0.20 (0.15-0.25)

0.96 (0.71-1.45)

Lot 2

0.18 (0.14-0.23)

0.95 (0.70-1.46)

Lot 3

0.22 (0.17-0.27)

0.88 (0.67-1.29)

3 Lots Combined

0.20 (0.17-0.22)

0.93 (0.78-1.16)

• •

Similar performance with the 3 lots evaluated Met design goal of 95% detection at approximately 1 IU/mL

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Limit of Quantitation of Parvovirus B19 B19 WHO (99/802) Targeted Concentrations

Lot 1 (N =60)

Lot 2 (N =60)

Lot 3 (N =60)

3 Lots Combined (N =180)

IU/mL

Log IU/mL

Mean

SD

%CV

Mean

SD

%CV

Mean

SD

%CV

Mean

SD

%CV

10,000

4.00

3.96

0.04

1.0

4.03

0.04

1.0

3.98

0.06

1.5

3.99

0.06

1.5

5,000

3.70

3.66

0.04

1.1

3.73

0.04

1.1

3.68

0.06

1.6

3.69

0.06

1.6

2,000

3.30

3.25

0.05

1.5

3.34

0.07

2.2

3.29

0.07

2.2

3.29

0.07

2.2

1,000

3.00

2.94

0.07

2.4

3.00

0.06

2.0

2.96

0.08

2.7

2.97

0.07

2.4

500

2.70

2.64

0.10

3.8

2.70

0.12

4.5

2.69

0.12

4.5

2.68

0.12

4.5

300

2.48

2.00

0.73

36.5

2.43

0.31

15.5

2.33

0.28

14.0

2.26

0.5

25.0

150

2.18

0.91

0.78

85.7

1.30

0.85

93.4

1.19

0.88

96.7

1.16

0.84

92.3

SD: standard deviation in log IU/mL; CV: coefficient of variability in log IU/mL

• • •

Serial dilutions of the 2nd WHO International Standard for Parvovirus B19 DNA (NIBSC code: 99/802) Two lots of the analytical sensitivity panels were tested using 3 reagent lots on 2 TIGRIS Systems LOB: 44 IU/mL; LOD: 271 IU/mL; LOQ: 271 IU/mL (calculated according to CLSI EP17-A [2004])

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Quantitation of B19 Genotypes 1-3 B19 Genotype (09/110)

Lot 1 (N=10) Target Log IU/mL

Lot 2 (N=10)

Mean Log Mean Log IU/mL IU/mL Difference 5.00 4.95 -0.05 5.01 1 4.00 3.99 -0.01 4.05 2.70 2.74 0.04 2.71 5.00 5.02 0.02 5.03 4.00 3.98 -0.02 3.99 2 2.70 2.68 -0.02 2.62 5.00 4.85 -0.15 4.92 4.00 3.86 -0.14 3.94 3a 2.70 2.60 -0.10 2.58 N: number of replicates tested; SD: standard deviation in log IU/mL



• • •

Mean Log IU/mL

Mean Log IU/mL Difference 0.01 0.05 0.01 0.03 -0.01 -0.07 -0.08 -0.06 -0.12

Lots 1 and 2 Combined (N=20) Mean Log Mean Log IU/mL IU/mL Difference 4.98 -0.02 4.02 0.02 2.73 0.03 5.02 0.02 3.99 -0.01 2.65 -0.05 4.88 -0.12 3.90 -0.10 2.59 -0.11

Panel members of the 1st WHO International Reference Panel for Parvovirus B19 Genotypes for Nucleic Acid Test (NAT) based assays (NIBSC 09/110) were diluted to levels spanning the proposed dynamic range (500 to 100,000 IU/mL or 2.7 to 5 log IU/mL) 2 reagent lots were tested on 2 TIGRIS Systems FDA Guidance (July 2009): Primers and probes selected for parvovirus B19 NAT should detect all known genotypes of the virus  European Pharmacopoeia : “…primers and probes should be designed to detect and quantify consistently the different human parvovirus genotypes.”  16

Reproducibility of B19 Quantitation •

3 reagent kit lots and 3 TIGRIS Systems were used to test a B19 viral panel obtained from DDL Diagnostic Laboratory (Sensitivity Panel Parvo-B19 Genotype 1, Voorburg, The Netherlands) along with an in-house prepared panel member (500 IU/mL) Inter-instrument Mean Observed Log IU/mL SD %CV

B19 IU/mL

N

5.00

54

4.95

0.00

4.48

54

4.45

4.00

54

3.48

Inter-Lot

Inter-Run

Intra-Run

Total

SD

%CV

SD

%CV

SD

%CV

SD

%CV

0.0

0.01

0.3

0.00

0.0

0.05

1.1

0.05

1.1

0.01

0.2

0.05

1.2

0.00

0.0

0.06

1.3

0.08

1.7

3.95

0.01

0.2

0.04

1.1

0.00

0.0

0.06

1.5

0.07

1.8

60

3.45

0.01

0.4

0.02

0.6

0.04

1.3

0.06

1.8

0.08

2.4

3.00

54

2.95

0.01

0.4

0.02

0.6

0.00

0.0

0.08

2.6

0.08

2.7

2.70

180

2.72

0.02

0.8

0.06

2.1

0.03

1.0

0.09

3.2

0.11

4.0

SD: standard deviation in log IU/mL; CV: coefficient of variability in log IU/mL



Largest contributor to overall assay variability was the intra-run factor. Reagent lot, instrument, and inter-run factors showed relatively little contribution to overall assay variability

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Clinical Sensitivity •

165 clinical specimens were obtained from Sanquin Diagnostic Services (Amsterdam, The Netherlands); specimens were previously quantified with Roche LightCycler B19V Test •



B19 values ranged from 1.2x106 IU/mL to 4.2x1012 IU/mL

Specimens were tested neat and in plasma pools (1:16 and 1:512) using 2 reagent kit lots on 3 TIGRIS Systems Lot 1 (n=165) Specimen Dilution

Neat 1:16 1:512

Lot 3 (n=165)

% B19 Reactive

% HAV Reactive

#Invalid (%)

%B19 Reactive*

% HAV Reactive

#Invalid (%)

100 100 100

0 0 0

1 (0.61)** 0 (0) 0 (0)

100 100 100

0 0 0

1 (0.61)** 0 (0) 0 (0)

*B19 cutoff was set at 1,000 IU/mL **Initially invalid reaction yielded valid, reactive result upon retesting 18

Lot-to-Lot Comparison of Quantitation B19 Positive Donations Donations identified during routine screening at Sanquin (Amsterdam, The Netherlands)

• •

Development Lot = DL Bland-Altman Bland difference plot –



Passing-Bablok linear regression method –



Mean bias: 0.02 (95% CI 0.01 to 0.04) Slope: 1.00 (95% CI 0.99 to 1.00); intercept: 0.05 log IU/mL (95% CI 0.00 to 0.09)

Very good correlation between Lot 1 (1st development lot) and Lot 3 (1st commercial lot) 19

Assay Specificity N (%)



Specimens Tested

2,800 (100%)

HAV Reactive (%)

1 (0.04%)

HAV Repeatedly Reactive (%)

0 (0.00%)

B19 Initially Reactive (%)

2 (0.07%)

B19 Repeatedly Reactive (%)

2 (0.07%)*

Initially Invalid (%)

5* (0.18%)

Repeatedly Invalid (%)

0 (0.00%)

HAV Specificity (95%CI)

99.96% (99.80-99.99)

B19 Specificity (95% CI)

100% (99.86-100.00)

2,800 plasma specimens – 1,700 whole blood (WB) donor plasma specimens and 800 source plasma donor specimens and 300 fresh WB donor plasma specimens – Tested using 2 reagent kit lots (1,400 specimens per lot).



*3 initially invalid results due to internal control failure, 2 invalid due to instrument/SW error **confirmed > 1,000 IU/mL by testing at National Genetics Institute (Los Angeles, CA)

HAV reactivity was examined along with B19 reactivity (using a quantitative cutoff of 1,000 IU/mL for B19)

Reactive Specimen ID  (B19> 1,000 IU/mL)

Initial B19 quantitation  (IU/mL)

Repeat B19 quantitation  (IU/mL)

DLS6177

2,971

2,515

DLS7525

1,141

1,469 20

Procleix Dengue Virus Assay •

Based on same technology as FDA-licensed and CE-marked Procleix blood screening assays – Qualitative nucleic acid test (NAT) for the detection of dengue virus RNA – Target capture  transcription-mediated amplification (TMA)  chemiluminescent detection (internal control validates each of these steps)



Design most closely resembles the Procleix WNV Assay – Same base reagent formulations with dengue-specific oligonucleotides – Uses very similar TIGRIS software, with all cutoff calculations and validity criteria identical to WNV Assay



Prospective screening under an investigative protocol (IND) will start later this year

The Procleix Dengue Assay is currently under development and is not available for commercial sale. The performance characteristics of this product in development have not been established

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Dengue Blood Safety Investigations •

Linnen JM et al. Transfusion. 2008 Jul;48(7):1355-62 – Established dengue viremia in asymptomatic donors blood donors in Honduras and Brazil • •



Honduras (2994 tested, 0.3%) Brazil (4858 tested, 0.04%)

Mohammed H et al. Transfusion. 2008 Jul;48(7):1348-54 – Prevalence in blood donors in Puerto Rico (16,521 tested, 0.07%)



Muñoz-Jordán JL et al. J Clin Microbiol. 2009 Apr;47(4):927-31 – Detection of dengue virus nucleic acid in samples from clinically ill patients; ~90% of acute phase samples submitted for dengue diagnosis detected by TMA assay



Stramer SL et al. Transfusion. 2012 Feb 17 – Dengue viremia in blood donors identified by TMA during the 2007 dengue outbreak in Puerto Rico – Detection of dengue transfusion transmission 22

Procleix Dengue Virus Assay Preliminary Analytical Sensitivity Similar Detection of 4 Dengue Virus Types •

Two panels (for each type) made from in vitro synthesized transcripts – –



One panel made by Gen-Probe manufacturing group and the other made by R&D; combined results are shown Panel members: 100, 30, 10, 3, 1, and 0 copies/mL tested (90 replicates at each level) tested with 1 reagent kit lot

Probit analysis to determine 50% and 95% detection levels Dengue Type

50% Detection Probability* (95% Fiducial Limits)

95% Detection Probability* (95% Fiducial Limits)

DENV 1

5.1 (4.1 - 6.0)

20.5 (16.3 - 27.9)

DENV 2

5.8 (4.7-6.8)

22.5 (18.0 - 29.9)

DENV 3

4.8 (3.9 - 5.8)

23.9 (18.7 - 32.9)

DENV 4

6.5 (5.3 - 7.8)

29.4 (23.3 - 39.9)

*Probit analysis was performed with SAS version 9.2; limits of detection determined using combined data from testing 2 RNA transcript lots for each DENV serotype 23

Hepatitis E Virus (HEV) Assay on the Panther System •

Gen-Probe and Novartis are developing a prototype assay to detect HEV RNA on the Procleix Panther System



Design allows detection of all clinically relevant genetic variants of HEV: – Genotypes 1 and 2 (restricted to humans) – Genotypes 3 and 4 (known to infect humans, pigs, and other species)



Performance characterization of current prototype assay is underway – Analytical Sensitivity (LOD) – Specificity – Determine prevalence in blood donors (based on detection of RNA) The Hepatitis E Assay is currently under development and is not available for commercial sale. The performance characteristics of this product in development have not been established 24

Panther System Overview Intended design features •

Fully automated; fully enclosed system with built-in process controls for sample and results integrity



Compact, all-in-one system; fits into standard laboratory configurations



On-board*, off-board pooling & IDT capabilities



Software designed with a guided, taskdriven interface



178 cm

81 81cm cm 122 cm

Planned assay menu: – Ultrio Elite initially, followed by WNV Assay, Parvo/HAV Assay, and other assays

*under development

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Panther System Operational Efficiency Intended design features

• Release of blood with immediate access to reportable results  3.5 hours to first reportable result with 5 results every 5 minutes thereafter  275 samples processed in individual donor testing (IDT) in 8 hours  500 samples processed in IDT in 12 hours

• Two (2) return visits in 8 hours to load additional samples and/or consumables • Automated daily maintenance (can be scheduled) 26

Panther System Workflow Intended design features



Batch and continuous processing – allows operators to load and prioritize samples as needed (STAT testing ability)



Random access capability – Perform multiple tests from a single sample – Obtain on-demand results for high priority samples with



Simultaneous use of multiple tube barcode formats and all industrystandard tube sizes

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HEV Assay on Panther System: Preliminary Analytical Sensitivity Comparison of in vitro synthesized transcript (IVT) and WHO Standard HEV WHO  Standard  (6329/10) IU/mL

#Reactive/#Tested

% Reactive

HEV IVT  (copies/mL)

#Reactive/#Tested

% Reactive

90

108/108

100%

90

108/108

100%

30

106/108

98%

30

108/108

100%

10

87/108

81%

10

87/108

81%

3

36/108

33%

3

47/108

44%

1

12/108

11%

1

14/108

13%

0

0/108

0%

0

0/108

0%

Detection  Probabilities by Probit  Analysis 95% 50%



WHO Std. IU/mL  (95% Fiducial Limits)

IVT Copies/mL  (95% Fiducial Limits)

22.3(17.3‐30.9) 

19.2 (14.9‐26.9) 

4.11 (3.51‐4.80) 

3.50 (2.98‐4.09) 

Ongoing work to optimize analytical sensitivity 28

HEV Assay on Panther: Preliminary Specificity N (%) Specimens Tested

2,000 (100%)

Reactive (%)

1 (0.05%)

Repeatedly Reactive (%)

1 (0.05%)

Specificity (95% CI)

99.95% (99.72-99.99)

CI: confidence interval using SCORE method

• •

Reactive Specimen ID

Initial S/CO

Repeat S/CO

R149449

5.2

4.5

2,000 unlinked frozen normal plasma specimens collected in the US from whole blood donors 3 Panther Instruments used in this study 29

Summary • Parvo/HAV Assay on the TIGRIS System – Very sensitive detection of HAV RNA (LOD patients

● Blood components to hospitals ● Clinical laboratory

services ● Antenatal blood group determination ● Tissue compatibility tests ● Stem cell transplants ● Tissue services ● Pharmaceutical distribution ● Research and development www.veripalvelu.fi www.veripalvelu.fi www.veripalvelu.fi www.bloodservice.fi

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Agenda • Parvo/HAV Assay Validation • Parvo/HAV Assay in Routine use

34

www.veripalvelu.fi www.veripalvelu.fi www.veripalvelu.fi www.bloodservice.fi

34 34 34 34

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Parvo/HAV Assay Validation • Parvo/HAV Assay – Quantitative detection of Parvo B19 – Qualitative detection of HAV

• Validation was performed during October-November 2011 – Reagents: two Development Reagent Lots – Equipment: Parvo/HAV Assay was installed into one Tigris instrument – Two operators

35

www.veripalvelu.fi www.veripalvelu.fi www.veripalvelu.fi www.bloodservice.fi

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Precision of Parvovirus B19 Quantitation Sample

n

runs

Expected Concentration IU/mL

Expected Concentration Log IU/mL

Observed Concentration Log IU/mL

SD

B19 WHO Standard

48

22

500

2,7

2,61

0,12

The 2nd WHO International Standard for Parvovirus B19 DNA for Nucleic Acid Amplification (NAT) Assay, NIBSC 99/802

48

22

600

2,8

2,73

0,11

48

19

800

2,9

2,82

0,08

48

19

1 000

3,0

3,0

0,12

48

19

1 200

3,1

3,05

0,08

78

30

10 000

4,0

3,91

0,05

B19 high viremic plasma sample

48

8

10 000

4,0

4,01

0,08

DDL B19 panel

48

8

1 000

3,0

3,02

0,08

BioQcontrol-DDL P0143 Parvo B19DNA genotype 1 reference panel

48

8

3 000

3,5

3,51

0,07

48

8

10 000

4,0

4,01

0,04

48

8

30 000

4,5

4,5

0,04

48

8

100 000

5,0

4,99

0,03

BioQControl The BioQControl P0105 Check Control Parvo B19-DNA/HAV-RNA

www.veripalvelu.fi www.veripalvelu.fi www.veripalvelu.fi www.bloodservice.fi

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Linear measuring range of B19

Concentration IU/mL

Concentration Log IU/mL

n

runs



300

2,5

9

3



1 000

3,0

9

3

3 000

3,5

9

3

10 000

4,0

9

3

30 000

4,5

9

3

100 000

5,0

9

3

www.veripalvelu.fi www.veripalvelu.fi www.veripalvelu.fi www.bloodservice.fi

Linear range reported by the manufacturer is 500 – 100 000 IU/mL Linearity was determined from DDL Panel B19 samples analysed as three replicates in three separate runs

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37 37 37 37

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HAV Analytical Sensitivity Sensitivity was tested using The 1st WHO International Standard for Hepatitis A Virus RNA, NIBSC 00/560 HAV RNA IU/mL

Proportion of Reactive / Valid results (%)

HAV Sensitivity 95% Limit of Detection in IU/mL (95% CI)

20

27/27 (100%)

FRC

0,76 (0,52-2,04)

12,3

25/25* (100%)

7,6

27/27 (100%)

Gen-Probe**

0,93 (0,78-1,16)

4,7

26/26* (100%)

3,8

27/27 (100%)

1,9

27/27 (100%)

0,95

27/27 (100%)

0,48

20/26* (76.9%)

0,24

21/27 (77.8%)

0

0/26*

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*Invalid results **PROCLEIX Parvo/HAV Assay Package Insert www.veripalvelu.fi www.veripalvelu.fi www.veripalvelu.fi www.bloodservice.fi

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More data from Validation • Parvo Genotypes 1,2 and 3 were all detected –

The 1st WHO International Reference Panel for Parvovirus B19 Genotypes for NAT based assays, NIBSC 09/110

• Parvo/HAV Specificity 100% – – – –

Reference method: Roche LightCycler Parvo/HAV negative plasma pools of 96 samples n = 171 Parvo DNA reactive plasma pools of 96 samples n = 2 Considered reactive if Parvo ≥ 1 000 IU/mL (Procleix® Parvo/HAV Assay)

• Failure rates – 0,89% invalid samples (13 out of 1 457) – 1,79% invalid runs (1 out of 56)

• No cross-contamination • Hemolytic and lipemic sample materials did not cause false interpretation of the results or affect the detection of the 39 viral load www.veripalvelu.fi www.veripalvelu.fi www.veripalvelu.fi www.bloodservice.fi

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Parvo/HAV Assay in Routine use • Requirements for the Parvo/HAV process: – Parvo B19 ≥ 105 IU/mL (individual donation) – Parvo/HAV results ready the following day from donation – The assay meets requirements set by European Pharmacopoeia (2011)

• Parvo/HAV Assay in use since 2nd of January 2012 – Analysed side by side with Ultrio Plus on TIGRIS

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Results from routine use January-April 2012 • 82 664 donor samples • Pools 3 252 – Invalid: 32 (0,98%)

• Worklists n = 160 – Invalid: 3 (1,9%) Pools

Pool size B19 Cut off B19 ”pos” donations

479

96

Jan – Feb

2 773

16

March

HAV pos donations

500

9

0

3 000

7

0

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Parvo/HAV Process Samples Pools of 16 samples Procleix® SP System

Nucleic acid capture, amplification, detection Procleix® Parvo/HAV Assay, Tigris

Parvo < Cut off, Non-Reactive HAV result

Parvo ≥ Cut off, Reactive HAV result Samples from the pool tested individually 42

Sanguin Aurora 2.01 software Stratec Biomedical

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Workflow • Two worklists per day: first worklist ready ~10 am, second worklist ready ~12 pm • Samples from a positive pool and invalid pools are tested the same afternoon or the following night

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Thank You

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Thank You Very Much !