Parvovirus B19 and HAV Screening of Whole Blood Donations

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Parvovirus B19 and HAV Screening of Whole Blood
Donations

• SL Stramer, KL Kane, ML Beyers, RY Dodd,American
  Red Cross andRIF Smith, National Genetics
  Institute (NGI)
• AABB, 2001
• Presented at FDA NAT Workshop, December 2001
• and modified for
• Blood Products Advisory Committee, December 12,
  2002

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Background 1

• Manufacturers of plasma derivatives have
  implemented NAT for nonenveloped viruses and
  such testing will likely be implemented for
  recovered plasma
• Most parvovirus B19 (B19V) NAT programs target
  the elimination of units ³1 x 106 copies/mL
• Studies of HAV and B19V frequencies in recovered
  plasma are limited. Dodd et al., 1997 (AABB) from
  screening pools of 512 (NGI) reported
• 020,000 for HAV RNA
• 710,000 for B19V DNA

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Background 2

• Three-year experience at Vitex for NAT screening
  final product (2,500 donations, NGI) for HAV or
  minipools of 100 for B19V (in-house) report
• 1476,000 for HAV RNA
• 1800 for B19V DNA
• ARC has obtained units from positive subpools of
  20 from positive minipools of 100 for
  identification/characterization of B19V-positive
  units
• Of gt1000 units tested from 72 positive subpools,
  only 23 tested B19V positive at NGI (from 16
  subpools)
• 77 (56/72) were false positive minipool results

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Background 3
Quant. and Ab Levels in B19V() Units
Copies/mL IgM IgG lt100 100
200 14,000 54,000 430,000 920,
000,000
Biotrin
5
Background 4
Quant. Levels in 23 B19V() Units
Copies/mL lt100-2 14,000 2,000,000
100-4 54,000 3,200,000 200-4 96,000 290,000,000
300 160,000 300,000,000 450 430,000 920,000,000
1,150
5 of 16 pools contained multiple low level
positives suggestive of contamination
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Background 5

• Therefore, we know that
• HAV is infrequent
• B19V NAT false positivity may be common
• Low level B19V DNA positive, IgG positive
  samples occur
• Individuals with early acute B19V infection have
  high viral titers

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Methods

• Unlinked study to determine HAV/B19V frequencies
  in recovered plasma
• HIV/HCV NAT-neg, seroneg surplus plasma in PPTs
  NGI
• 2 primers for HAV and B19V, each tested in
  duplicate at NGI

Estimated 95 sensitivity 1.2 x 107 copies/mL
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Results 1

• 0100 pools HAV 051,200 frequency
• 3100 pools B19V 112,800 frequency_at_ 11000
  dilution4 donations

Copies/mL IgM IgG IgG IU/mL 2.6 x 105
lt310 1.0 x 105 lt310 4.4 x 109
lt310 1.7 x 1011 lt310
Reactive in same diluted pool
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Results 2

• 3497 pools B19V 1528 frequencywithout
  dilution95 donations

No. of Samples Copies/mL IgM IgG IgG IU/mL
3 2.9 x 106 1 / (510) 9.2 x 104 1 1
(1,600) 1.2 x 104 1 1 (4,800) 60 102-103
34 34 26 26 14 lt102 9 9
2 2 3 3 18 QNS N/A N/A
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Parvovirus B19 PrevalenceUsing Gen-Probe Assay

• 2,547 pools of 16 (April-May 2002 collections,
  N40,752) tested at Gen-Probe using a
  combination B19V/HAV NAT assay
• 23 (0.9) pools of 16 repeat reactive and disc.
  B19V NAT reactive no reactives for HAV
• Assume one B19V-pos donation/reactive pool of 16
  23/40,752 11,772 versus 112,800 for NGI
  study (7-fold higher)
• Product loss, due to discard of all members of a
  reactive pool of 16 1111 (unacceptable)
• Based on distribution of quant. results of 23
  positive pools, the addition of a 11,000
  predilution would result in a prevalence
  comparable to NGI (3/40,752 113,584)

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Parvovirus B19 Titers for23 Gen-Probe Positive
Samples
(Copies/mL at NGI for Reactive Pools of 16)
100 200-2 300-2 350
3,700 4,100 5,200-2 5,600 6,300 7,100 8,100 9,500-
2 9,900
10,000 13,000 17,000
1,300,000 51,000,000 55,000,000
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HAV PCR Test Results onManufacturing Pools of
Plasma

• 3,250-L mfg. pool (11,500 donations)
• Time period covered 05/08/01 - 11/12/02
• 512 pools tested
• 6 (3 pending) positive pools identified
• If initial test is reactive, retest two
  independent samples
• If either of two retests is reactive, consider
  positive and discard pool
• Frequency
• 6 (9) pos. HAV RNA mfg. pools per 512 mfg. pools
  tested 1 pos. HAV RNA sample per 981,333
  (654,222) donations
• Estimated titers of positive samples 1 x 105 to
  ³1.1 x 107 copies/mL

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Conclusions

• Blood collectors considering implementation of
  B19V screening will have to evaluate NAT methods
  that are relatively insensitive to prevent issues
  from contamination and detection of low level
  NAT positives
• 112,800 frequency using insensitive method
• 125,600 high-titer, acute viremic donors,
  IgG-negative
• 1528 frequency using sensitive method
• 117,000 moderate-titer, IgG/, IgM
• 1539 low-titer, IgG, IgM/

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Conclusions

• High-titer screening methods may not capture all
  infectious B19V-positive units however,
  infectivity of Ab-reactive, low-titer positives
  is unknown
• This study defines expected yields of B19V if
  sensitive and insensitive NAT methods are used
• This study also demonstrates the infrequent
  occurrence of HAV in recovered plasma
• 1476,000 to 1981,333 million donations

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Strategies for HAV/B19V NAT for Plasma for
Further Manufacturing
Phase I Outsource testing process time
exceedsthe dating of labile components
NGI pools to 512and tests by HAV
UltraQual2000 RT PCR and Parvovirus
B19UltraQual1000 PCR(Following a11000
dilution)
Identify individualNAT tubes correspondingto
recovered plasma
Pools of 16(recoveredplasma only)
Following completionof HIV-1/HCV NAT
Neg
Plasma for Mfg.
Resolve to RxPool of 16
Pos
Discard all in-date frozen products(no FFP will
exist)
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Strategies for HAV/B19V NAT for Plasma for
Further Manufacturing
Phase II HAV/B19V Testing In-House (commercial
kit)

• Real-time pool testing (pool size TBD)
• Reactives resolved to individual donation
• No product release unless HAV/B19V NAT neg
• B19V sensitivity level initially set for the
  removal of high-titer units (³106 copies/mL) for
  plasma only
• No claims for labile products
• Determine needs for recipients of labile products
• Donor notification, management of products from
  NAT-Rx donors previous donations and recipient
  tracing TBD
• Timeline dependent on regulatory
  policy/availability of test kit