Guidance for Industry
 
 
Nucleic Acid Testing (NAT) to Reduce 
the Possible Risk of Human 
Parvovirus B19 Transmission by 
Plasma-Derived Products 
 
 
 
 
 
 
 
 
 
 
Additional copies of this guidance are available from the Office of Communication, Outreach 
and Development (OCOD) (HFM-40), 1401 Rockville Pike, Suite 200N, Rockville, MD 20852-
1448, or by calling 1-800-835-4709 or 301-827-1800, or from the Internet at 
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guida
nces/default.htm. 
 
For questions on the content of this guidance, contact OCOD at the phone numbers listed above.   
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Biologics Evaluation and Research 
July 2009  Contains Nonbinding Recommendations 
 
 
Table of Contents 
 
 
I.  INTRODUCTION.............................................................................................................1 
II.  BACKGROUND...............................................................................................................1 
III.  RECOMMENDATIONS..................................................................................................3 
IV.  REFERENCES..................................................................................................................4 
i  Contains Nonbinding Recommendations 
 
Guidance for Industry 
 
Nucleic Acid Testing (NAT) to Reduce the Possible Risk of Human 
Parvovirus B19 Transmission by Plasma-Derived Products 
 
 
This guidance represents the Food and Drug Administrations (FDAs) current thinking on this 
topic.  It does not create or confer any rights for or on any person and does not operate to bind 
FDA or the public.  You can use an alternative approach if the approach satisfies the 
requirements of the applicable statutes and regulations.  If you want to discuss an alternative 
approach, contact the appropriate FDA staff.  If you cannot identify the appropriate FDA staff, 
call the appropriate number listed on the title page of this guidance. 
 
 
I.  INTRODUCTION 
 
We, FDA, are issuing this guidance to provide you, manufacturers of plasma-derived products, 
with recommendations for performing nucleic acid testing (NAT) for human parvovirus B19 as 
an in-process test for Source Plasma and recovered plasma used in the further manufacturing of 
plasma-derived products.  Such testing will identify and help to prevent the use of plasma units 
containing high levels of parvovirus B19.  This guidance also recommends how to report to FDA 
implementation of parvovirus B19 NAT. 
 
We recognize that in the current business practice for parvovirus B19 NAT in-process testing, 
several weeks can elapse between collection of the units of Source Plasma or recovered plasma 
and identification of B19 NAT-positive pools or units.  We encourage manufacturers of plasma-
derived products to employ practices that will reduce the time between product collection and in-
process testing to allow for the meaningful notification of blood and plasma collection 
establishments of positive test results within the dating period of any blood components intended 
for use in transfusion. 
 
This guidance finalizes the draft guidance of the same title, dated July 2008. 
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe the FDAs current thinking on a topic and should be 
viewed only as recommendations, unless specific regulatory or statutory requirements are cited.  
The use of the word should in FDAs guidances means that something is suggested or 
recommended, but not required. 
 
 
II.  BACKGROUND 
 
Human parvovirus B19 is a small, non-enveloped single stranded DNA virus.  Virus clearance 
studies, using non-human parvoviruses as models for parvovirus B19, have indicated that this 
virus is highly resistant to all commonly used inactivation methods, including heat and 
1  Contains Nonbinding Recommendations 
 
solvent/detergent (S/D) treatment, and is also difficult to remove by filtration because of its small 
size.  More recent studies have demonstrated that human parvovirus B19 may be more readily 
cleared than certain model animal parvoviruses (Refs. 1, 2, 3 and 4).  The parvovirus B19 can be 
transmitted by blood components and certain plasma derivatives, and may cause morbidity to 
susceptible recipients such as pregnant women (and their fetuses exposed in utero), persons with 
underlying hemolytic disorders, and immune compromised individuals (Refs. 5 and 6).  The 
disease transmission by transfusion of blood components is rare.  However, extremely high 
levels of parvovirus B19, up to 1012 IU/mL, in plasma of acutely infected but asymptomatic 
donors may present a greater risk in plasma derivatives due to pooling of large numbers of 
plasma units in the manufacture of these products.  The virus can be detected by NAT in plasma 
pools when there are high levels of parvovirus B19 DNA in viremic donations.  For example, the 
parvovirus B19 DNA can be detected in various plasma-derived products, particularly in 
coagulation factors (Refs. 7 and 8).  There have been a few reports of parvovirus B19 infection 
associated with the administration of coagulation factors (Refs. 9 and 10) and S/D Treated 
Pooled Plasma (Refs. 5 and 11).  Parvovirus B19 DNA is less frequently detected in albumin and 
immunoglobulin products and, when detected, the levels are usually low.  There are no 
confirmed reports that albumin and immunoglobulin products have transmitted parvovirus B19 
infection. 
 
We have held or participated in several meetings to discuss the potential risk of parvovirus B19 
infection by plasma-derived products, and the strategy for reducing such risk.  The meetings 
included FDA-sponsored NAT workshops in 1999 and 2001 (Refs. 12 and 13), Blood Products 
Advisory Committee (BPAC) meetings in 1999 and 2002 (Refs. 14, 15, and 16), the National 
Heart, Lung, and Blood Institute-sponsored Parvovirus B19 workshop in 1999 (Ref. 5), and an 
ad hoc Public Health Service (PHS) panel in 2002 (discussed at the 2002 BPAC meeting (Ref. 
16)).  In these meetings, it was recognized that viral inactivation/removal steps that are routinely 
used in the manufacturing process of plasma-derived products do not alone appear to be 
sufficient to completely clear the virus if high viral load is present in the starting material.  
Therefore, in these meetings, a common recommendation for mitigating the risk of parvovirus 
B19 transmission by plasma derivatives has been to limit the virus load in the manufacturing 
plasma pool by testing the plasma donations for high titer parvovirus B19 DNA, using a 
minipool format.  This viral load reduction strategy combined with the ability of the 
manufacturing process to clear the residual virus could greatly reduce the risk of parvovirus B19 
infection by plasma-derived products. 
 
The recommended limit in this guidance for viral load of parvovirus B19 DNA in the 
manufacturing plasma pool (i.e., not to exceed 104 IU/mL) was primarily derived from studies 
that were conducted on the transmission of parvovirus B19 associated with S/D Treated Pooled 
Plasma (Refs. 5, 11, and 14).  In principle, testing in a minipool format to measure the viral load 
for parvovirus B19 DNA in a manufacturing plasma pool is acceptable in order to exclude only 
the high-titer plasma donations, thereby avoiding too great a loss of plasma for further 
manufacturing.  Furthermore, during the viremic period for parvovirus B19 infected donors, 
which can be very lengthy, low levels of parvovirus B19 coexist with parvovirus B19 antibodies 
2  Contains Nonbinding Recommendations 
 
(potentially complexing with and neutralizing the virus).  Therefore, it is undesirable to remove 
plasma units with low levels of B19 DNA, because it would diminish the parvovirus B19 
antibody levels in plasma pools and in some of the resulting plasma-derived products (Refs. 17 
and 18). 
 
 
III.  RECOMMENDATIONS 
 
We recommend that you implement the following procedures to detect the presence of 
parvovirus B19 DNA: 
 
  For all plasma-derived products, you should perform parvovirus B19 NAT as an in-
process test to ensure that the viral load of parvovirus B19 DNA in the manufacturing 
pools does not exceed 104 IU/mL. 
  Use parvovirus B19 NAT on minipool samples to screen plasma units intended for 
further manufacturing into plasma-derived products.  Primers and probes selected for 
parvovirus B19 NAT should detect all known genotypes of the virus (Ref. 19). 
  When identified, you should not use individual plasma units, intended for further 
manufacturing into plasma-derived products, when such units are found to have a titer 
of parvovirus B19 DNA that might result in plasma manufacturing pools exceeding a 
parvovirus B19 DNA titer of 104 IU/mL. 
 
You should assess validation data demonstrating the accuracy, sensitivity, specificity, 
reproducibility, and other performance characteristics of the parvovirus B19 NAT assay used for 
the detection of parvovirus B19 DNA in the Source Plasma and recovered plasma, and for 
demonstrating that the viral load of parvovirus B19 DNA in the manufacturing pool does not 
exceed 104 IU/mL.  
 
If the above recommendations are implemented, you must inform FDA, as required under 21 
CFR 601.12(a).  You may submit these changes as a Supplement-Changes Being Effected 
supplement (CBE supplement), under 21 CFR 601.12(c)(5).     
 
3  Contains Nonbinding Recommendations 
 
IV.  REFERENCES 
 
1.  Mani, B., Gerber, M., Lieby P., Boschetti, N., Kempf, C., Ros, C. Molecular mechanism 
underlying B19 virus inactivation and comparison to other parvoviruses. Transfusion (2007); 47: 
17651774.  
 
2.  Blumel J, Stuhler A, Dichtemuller H. Kinetics of inactivating human parvovirus B19 and 
porcine parvovirus by dryheat treatment. Transfusion (2008); 48:790791.  
 
3.  Berting A, Modrof J, Unger U, Gessner M, Klotz A, Poesler G, Kreil TR. Inactivation of 
parvovirus B19 during STIM4 vapor heat treatment of three coagulation factor concentrates. 
Transfusion (2008); 48:12201226  
 
4.  Boschetti N, Niederhauser I, Kempf C, Stuhler A, Lower J, Blumel J. Different susceptibility 
of B19 virus and mice minute virus to low pH treatment. Transfusion (2004); 44: 1079-1086. 
   
5.  Brown, K.E., Young N.S., Alving, B.M., Luiz, H., and Barbosa, L.H.  Parvovirus B19 
implications for transfusion medicine.  Transfusion (2001); 41:130-135. 
 
6.  Young, N.S., and Brown, K.E.  Parvovirus B19.  The New England Journal of Medicine 
(2004); 350:586-597. 
 
7.  Saldanha, J., and Minor, P.  Detection of human parvovirus B19 DNA in plasma pools and 
blood products derived from these pools: implications for efficiency and consistency of removal 
of B19 DNA during manufacture.  British Journal of Haematology (1996); 93:714-719. 
 
8.  Schmidt, I., Blumel, J., Seitz, H., Willkommen, H., and Lower, J.  Parvovirus B19 DNA in 
plasma pools and plasma derivatives.  Vox Sanguinis (2001); 81:228-235.  
 
9.  Blumel, J., et al.  Parvovirus B19 transmission by heat-treated clotting factor concentrates.  
Transfusion (2002); 42:1473-1481. 
 
10. Wu, C.G., et al. Parvovirus B19 transmission by a high-purity factor VIII concentrate.  
Transfusion (2005); 45:1003-1010. 
 
11. Koenigbauer, U.F., Eastlund, T., and Day, J.W.  Clinical illness due to parvovirus B19 
infection after infusion of solvent/detergent-treated pooled plasma.  Transfusion (2000); 
40:1203-1206. 
 
12. Transcript of Workshop on Implementation of Nucleic Acid Testing, December 14, 1999.  . 
http://www.fda.gov/downloads/BiologicsBloodVaccines/NewsEvents/WorkshopsMeetingsConfe
rences/TranscriptsMinutes/UCM055438.pdf. 
 
4  Contains Nonbinding Recommendations 
 
13. Transcript of Application of Nucleic Acid Testing To Blood Borne Pathogens and Emerging 
Technologies Workshop, December 4, 2001.  
http://www.fda.gov/downloads/BiologicsBloodVaccines/NewsEvents/WorkshopsMeetingsConfe
rences/TranscriptsMinutes/UCM055339.pdf. 
 
14.   Transcript of FDAs Blood Products Advisory Committee (BPAC) Meeting, September 
1999.  
http://www.fda.gov/AdvisoryCommittees/CommitteesMeetingMaterials/BloodVaccinesandOther
Biologics/BloodProductsAdvisoryCommittee/default.htm. 
 
15. Transcript of FDAs Blood Products Advisory Committee (BPAC) Meeting, March 14, 2002.  
http://www.fda.gov/AdvisoryCommittees/CommitteesMeetingMaterials/BloodVaccinesandOther
Biologics/BloodProductsAdvisoryCommittee/default.htm. 
 
16. Transcript of FDAs Blood Products Advisory Committee (BPAC) Meeting, December 2002.  
http://www.fda.gov/AdvisoryCommittees/CommitteesMeetingMaterials/BloodVaccinesandOther
Biologics/BloodProductsAdvisoryCommittee/default.htm. 
 
17. Lefrere, Jean-Jacques, et al. Persistent B19 infection in immunocompetent individuals: 
implications for transfusion safety.  Blood (2005); 106(8):2890-2895. 
 
18. Cassinotti, P., Siegl, G. Quantitative evidence for persistence of human parvovirus B19 DNA 
in an immunocompetent individual.  Eur J Clin Microbiol Infect Dis (2000); 19:886-895. 
 
19. Servant, A., et al. Genetic diversity within human erythroviruses: identification of three 
genotypes.  J Virol (2002); 76:9124-34. 
 
 
 
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