# Presentation: 197978

## Parvovirus B19 and HAV Screening of Whole Blood Donations

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

## 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 1 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:
***0:20,000 for HAV RNA***
***7:10,000 for B19V DNA***

## 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:
***1:476,000 for HAV RNA***
*** ************1:800 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 >1000 units tested from 72 positive subpools, only 23 tested B19V positive at NGI (from 16 subpools)
***77% (56/72) were false positive minipool results***

## Background – 3

**	****Copies/mL****	****IgM*****	****IgG***
-        **<100****	****–****	****+**
**	**** ****100****	****–****	****+**
**	**** ****200****	****–****	****+**
**	****14,000****	****+****	****+**
**	****54,000****	****+****	****+**
**	****430,000****	****+****	****+**
**	****920,000,000****	****–****	****–**
**	**

**Quant. and Ab Levels in B19V(+) Units**

- * Biotrin

## Background – 4

**	****	****Copies/mL****	**** **
-        **<100-2****	****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****	****	**

**Quant. Levels in 23 B19V(+) Units**

- 5 of 16 pools contained multiple low level positives suggestive of contamination

## 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

## Methods

***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

## Results – 1

- 0:100 pools HAV	**+**	=	0:51,200 frequency
- 3:100 pools B19V	**+**	=	1:12,800 frequency@ 1:1000 dilution4**+** donations

**	****Copies/mL****	****IgM****	****IgG****	****IgG IU/mL**
**	**** ****2.6 x 10****5 ********* ****	****+****	****–**** ****	****<310**
**	**** ****1.0 x 10****5 ********* ****	****–****	****–**** ****	****<310**
**	****4.4 x 10****9****	****+****	****–****	**** <310**
**	****1.7 x 10****11****	****–****	****–****	**** <310**

- * Reactive in same diluted pool

## Results – 2

- 34:97 pools B19V	**+**	=	1:528 frequencywithout dilution95**+** donations

**No. of Samples****	****Copies/mL****	****IgM****	****IgG****	**** IgG IU/mL**
**	****  ****3****	****2.9 x 10****6****	****1 +****	**** +/–****	****(510)****	****	****9.2 x 10****4****	****1 +****	****1 +****	****(1,600)****	****	****1.2 x 10****4****	****1 +****	****1 +****	****(4,800)**
**	****60****	****10****2****-10****3****	****34 +****	****34 +****	****	****	****26 –****	****26 +**
**	****14****	****<10****2****	****  9 –****	****  9 +****	****	****	****  2 –****	****  2 –****	****	****	****  3 +****	****  3 +**
**	****18****	****QNS****	****N/A****	****N/A**

## Parvovirus B19 PrevalenceUsing Gen-Probe Assay

- 2,547 pools of 16 (April-May 2002 collections, N=40,752) 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 = 1:1,772 versus 1:12,800 for NGI study (7-fold higher)
- Product loss, due to discard of all members of a reactive pool of 16 = 1:111 (unacceptable)
- Based on distribution of quant. results of 23 positive pools, the addition of a 1:1,000 predilution would result in a prevalence comparable to NGI (3/40,752 = 1:13,584)

## Parvovirus B19 Titers for23 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**

## HAV PCR Test Results onManufacturing 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 1.1 x 107 copies/mL

## Slide 13

## 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
- 1:12,800 frequency using insensitive method
- 1:25,600 high-titer, acute viremic donors, IgG-negative
- 1:528 frequency using sensitive method
- 1:17,000 moderate-titer, IgG+/–, IgM+
- 1:539 low-titer, IgG+, IgM+/–

## 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
- 1:476,000 to 1:981,333 million donations

## Strategies for HAV/B19V NAT for Plasma for Further Manufacturing

**HAV UltraQual****TM****2000 RT PCR**
**and**
**Parvovirus B19****UltraQual****TM****1000 PCR****(Following a****1:1000 dilution)**

***Strategies for HAV/B19V NAT for ******Plasma for Further Manufacturing***

**Phase I – Outsource testing; process time exceeds****the dating of labile components**

**Following completion****of HIV-1/HCV NAT**

**Identify individual****NAT tubes corresponding****to recovered plasma**

**Pools of 16****(recovered****plasma only)**

**Neg**

**Plasma for Mfg.**

**Pos**

**Resolve to Rx****Pool of 16**

**Discard all in-date ****frozen products****(no FFP will exist)**

## Strategies for HAV/B19V NAT for Plasma for Further Manufacturing

- “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 (106 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

**Phase II –HAV/B19V Testing In-House (commercial kit)**