Guidance for Industry 
 
 
Use of Nucleic Acid Tests to Reduce the Risk of 
Transmission of West Nile Virus from Donors of 
Whole Blood and Blood Components Intended 
for Transfusion  
 
 
 
 
  
 
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/ 
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 
          November 2009 Contains Nonbinding Recommendations 
 
 
Table of Contents
 
I.  INTRODUCTION.............................................................................................................1 
II.  BACKGROUND...............................................................................................................1 
A.  Whole Blood and Blood Components.................................................................2 
III.  RECOMMENDATIONS FOR DONATIONS OF WHOLE BLOOD AND BLOOD 
COMPONENTS................................................................................................................4 
A.  Testing, Unit Management, and Donor Management.......................................4 
B.  Switching from MP-NAT to ID-NAT..................................................................5 
C.  Reporting Test Implementation..........................................................................6 
D.  Labeling of Whole Blood and Blood Components Intended for Transfusion.6 
IV.  IMPLEMENTATION......................................................................................................9 
V.  REFERENCES..................................................................................................................9 
 
i  Contains Nonbinding Recommendations 
Guidance for Industry 
 
Use of Nucleic Acid Tests to Reduce the Risk of Transmission of 
West Nile Virus from Donors of Whole Blood and Blood 
Components Intended for Transfusion   
 
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 you1 with recommendations for testing donations 
of Whole Blood and blood components for West Nile Virus (WNV) using an FDA-licensed 
donor screening assay2.  We believe that the use of a licensed nucleic acid test (NAT) will 
reduce the risk of transmission of WNV, and therefore recommend that you use a licensed NAT 
to screen donors of Whole Blood and blood components intended for transfusion for infection 
with WNV.   
 
The recommendations in section III of this guidance apply to all donations of Whole Blood (as 
defined in Title 21 Code of Federal Regulations (CFR) 640.1) and blood components for 
transfusion3.   
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe 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  
 
WNV first appeared in the United States in 1999, and has become endemic with high viral 
activity during the warm months of the year.  WNV is a mosquito-borne agent that is maintained 
                                                 
1 This guidance is intended for establishments that collect Whole Blood and blood components intended for 
transfusion. 
2 This guidance finalizes the recommendations for donations of Whole Blood and blood components in the draft 
guidance titled, Guidance for Industry:  Use of Nucleic Acid Tests to Reduce the Risk of Transmission of West Nile 
Virus from Donors of Whole Blood and Blood Components Intended for Transfusion and Donors of Human Cells, 
Tissues, and Cellular and Tissue-Based Products (HCT/Ps), dated April 2008 (April 28, 2008, 73 FR 22958). 
3 This guidance does not apply to Source Plasma or plasma derivatives. 
  1 Contains Nonbinding Recommendations 
in nature primarily between birds and mosquitoes but can also infect other animals, including 
humans.  The potential for WNV transmission by blood transfusion during the acute phase of 
infection, when infected individuals are viremic and asymptomatic, was first recognized in 2002 
(Ref. 1).  At that time, test kit manufacturers and blood organizations, with input from the Public 
Health Service (National Institutes of Health, FDA, and Centers for Disease Control and 
Prevention (CDC)), actively pursued development of NAT systems for WNV.  Retrospective 
studies have subsequently confirmed human-to-human transmission of WNV by blood 
transfusion and by organ transplantation (Refs. 2, 3).   
 
Nationwide clinical studies to evaluate a NAT for the detection of WNV were initiated in 2003, 
under FDAs Investigational New Drug Application (IND) regulations (21 CFR Part 312).  Such 
large-scale studies were necessary to help ensure blood safety and to determine the efficacy of 
investigational assays to prevent the transmission of WNV through blood transfusion, because at 
that time there was no FDA-licensed screening assay available to detect WNV infection.   
 
Since 2005, FDA has approved biologics license applications for two NAT assays for detecting 
WNV ribonucleic acid (RNA) using plasma specimens from human donors of blood.  The assays 
are intended for use in testing individual donor samples and in testing pools of human plasma 
comprised of equal aliquots of not more than either 6 or 16 individual donations (minipools) of 
whole blood and blood components, depending on the manufacturer.   
 
As explained below in section III, if the result of a licensed minipool NAT (MP-NAT) is 
reactive, and subsequent testing of the individual donation(s) (ID-NAT) comprising the tested 
minipool is reactive, then FDA would recommend treating the reactive unit(s) as though they are 
infectious.   
 
Evaluation of additional testing performed on specimens that were reactive on screening by ID-
NAT has shown that a repeat ID-NAT on index donation specimens (i.e., the same or an 
independent specimen from the index donation, which is the donation for which the test result 
was reactive), using either the same screening assay or an equally sensitive alternate NAT, 
together with a test result for antibody to WNV, has a positive predictive value of 98% (Ref. 4).   
   
Data show that up to 10% of donors who have a reactive ID-NAT that fails to be reactive on 
repeat testing by ID-NAT actually are infected, based on the presence of antibodies to WNV 
either in the index donation (ca. 8%) or on a follow-up test (ca. 2%) (Ref. 4).  Therefore, 
additional testing that would include repeat testing by ID-NAT along with testing for antibody to 
WNV may be of value in donor counseling. 
 
A.  Whole Blood and Blood Components 
 
In 2002, there were 23 confirmed cases of WNV transmission by blood or blood components 
(Ref. 3).  Only six transmissions of WNV by transfusion were documented in 2003 (Ref. 5) 
following nationwide implementation of screening for WNV by MP-NAT under an IND in July 
2003.  Retrospective studies using ID-NAT to test MP-NAT non-reactive specimens collected 
during that season identified additional reactive donations and indicated that up to 25% of 
viremic units were not detected by MP-NAT, presumably due to low viral load (Ref. 6).  Results 
  2 Contains Nonbinding Recommendations 
of these studies show that for detecting WNV, ID-NAT has greater sensitivity than MP-NAT. 
 
As a result, ID-NAT may identify reactive donations not detected by MP-NAT.  However, 
limitations in reagent availability, and personnel and logistical issues related to blood donor 
screening may not allow full implementation of ID-NAT.  During the development and 
implementation of the ID-NAT test under IND, MP-NAT of plasma samples (pools of 6 or 16 
samples), rather than ID-NAT, was the only feasible format for performing the test.  In addition, 
testing using the MP-NAT format was similar to the assay platforms being used for human 
immunodeficiency virus type 1 (HIV-1) NAT and hepatitis C virus (HCV) NAT at that time.  As 
reagent availability increases, technology advances, and personnel and logistical issues related to 
blood donor screening diminish, year-round ID-NAT testing of all donations of blood and blood 
components, using a licensed NAT, may become feasible and practical.  
 
Although year-round ID-NAT testing of all blood and blood components may not be currently 
feasible, we believe that using ID-NAT instead of MP-NAT on a limited basis during periods of 
high WNV activity to maximize the benefit to the public health is more practicable.  Statistical 
analyses were performed on the data from the retrospective studies described above to establish 
criteria for defining high WNV activity in a particular geographic region (Ref. 7).  These criteria 
were used as a trigger for ID-NAT implementation and for reversion to MP-NAT testing when 
the high WNV activity in that region subsided.  Since 2004, ID-NAT screening replaced MP-
NAT screening in those geographic regions of high WNV activity during epidemic periods 
(Refs. 7, 8) when a threshold was reached.  The threshold was usually based on the number of 
MP-NAT-reactive screening test results obtained during a one-week interval or on a cumulative 
rate for ID-NAT reactive screening test results in a particular region (Ref. 4).  
 
After selective implementation of ID-NAT during epidemic seasons, there were three additional 
transmissions of WNV by transfusion between 2004 and 2006:  one in 2004 and two in 2006.  
The WNV transmission in 2004 resulted from a donation of red blood cells which tested non-
reactive in a MP-NAT assay, but which was subsequently found to be reactive in an ID-NAT 
test.  Plasma from the donation retrospectively tested reactive by ID-NAT.  However, ID-NAT 
had not yet been implemented (Ref. 9).  The two WNV transmissions in 2006 resulted from a 
non-reactive MP-NAT donation from which red blood cells and fresh frozen plasma were 
transfused to two immunosuppressed recipients (Ref. 10).  Investigation of the 2006 cases 
showed that:  1) there were no established methods of communication linking WNV MP-NAT 
data from multiple collecting and testing facilities serving overlapping or adjacent geographic 
areas; and 2) if efficient communication mechanisms had been in place, the corresponding 
collection area would have reached the threshold for switching to ID-NAT screening, and the 
WNV-contaminated components would likely have been detected and removed from the blood 
supply (Ref. 4).  
 
At this time, there is insufficient data to support recommendation of uniform threshold criteria 
for switching from MP-NAT screening to ID-NAT screening.  Pending development of suitable 
uniform threshold criteria, we consider it appropriate for each blood establishment to define its 
own threshold criteria for switching from MP-NAT to ID-NAT screening and for reverting to 
MP-NAT screening.  Each blood establishment should follow an established standard operating 
procedure (SOP) for this decision process.  Voluntary industry practice of switching from MP-
  3 Contains Nonbinding Recommendations 
NAT to ID-NAT screening during seasonal activity has been useful in increasing the 
effectiveness of the WNV screening process.  
 
III.  RECOMMENDATIONS FOR DONATIONS OF WHOLE BLOOD AND BLOOD 
COMPONENTS 
 
Testing donations of Whole Blood and blood components for WNV using NAT involves the use 
of defined pooling and testing systems.  We recognize that licensed testing technology in a semi-
automated or fully automated format is not universally available, and that if you are currently 
performing NAT for WNV under an IND you would need time to fully implement a licensed 
system with all approved components, including the supporting software cleared as a device.  If 
you are therefore using some, but not all, of the licensed or cleared components, you should 
continue your existing IND and report the use of the licensed assay or the related cleared 
components as an amendment to your existing IND.  When you implement all licensed or cleared 
components of the test system, you may withdraw the IND in accordance with the procedures 
provided in 21 CFR 312.38. 
 
A.  Testing, Unit Management, and Donor Management 
 
1.  We recommend that you screen year-round for WNV using a licensed NAT on 
donor samples of Whole Blood and blood components intended for transfusion.  
In general, you may use either MP-NAT or ID-NAT for screening (see Figure 1 
and Table 1), except that we recommend that you use ID-NAT screening during 
high WNV activity in your region (using a previously defined geographic area).  
See section B.  
 
2.  If you perform screening using MP-NAT, you may release all units whose test 
samples comprise a non-reactive minipool, if those units are otherwise suitable for 
release. 
 
We recommend that you resolve a NAT-reactive minipool using ID-NAT to test 
each specimen in the minipool in order to identify the unit(s) that led to the 
reactivity of the minipool.  Based on the ID-NAT results, we recommend the 
following:   
 
a.  You may release all ID-NAT non-reactive units if they are otherwise suitable 
for release. 
 
b.  If one or more individual donation(s) is (are) reactive, we recommend that you 
discard the unit(s), defer the donor(s) for a period of 120 days and retrieve and 
quarantine in-date products from prior collections dating back 120 days prior 
to the donation that is ID-NAT-reactive.  We recommend that you notify the 
donor of his or her deferral and counsel the donor.  Further testing on the 
index donation using the same ID-NAT or an alternate NAT with sensitivity 
equal to or greater than that of the screening assay, in addition to testing the 
  4 Contains Nonbinding Recommendations 
specimen using a cleared test for antibodies to WNV may be of value in donor 
counseling. 
 
Note:  In the event that the NAT screening assay does not discriminate 
between WNV and other Flaviviruses that belong to the Japanese 
Encephalitis (JE) serogroup (namely, Saint Louis Encephalitis virus, 
Japanese Encephalitis virus, Murray Valley Encephalitis virus and 
Kunjin virus), the donor should be counseled that he or she tested 
positive for a JE serogroup virus, most likely WNV.  Alternatively, the 
use of a NAT assay that discriminates WNV from other members of 
the JE serogroup may be of value in donor counseling.  
 
Note:  Antibodies to viruses of the JE serogroup may cross-react on the test 
for antibodies to WNV (Refs. 11, 12).  Therefore, reactivity in a WNV 
antibody test may not be conclusive for WNV infection. 
 
3.    If you perform screening using ID-NAT, we recommend that you follow the steps 
in 2.a. and 2.b. for testing, unit management, and donor management. 
 
B.  Switching from MP-NAT to ID-NAT 
 
We recommend that you: 
 
1.  Establish and validate criteria that define high WNV activity in your 
geographic area of collection. 
2.  Define a threshold for switching from MP-NAT to ID-NAT screening 
during high WNV activity in your geographic area of collection, and for 
reverting to MP-NAT screening when the high WNV activity in your 
geographic area has subsided. 
3.  Switch from MP-NAT to ID-NAT screening as soon as feasible, but 
within 48 hours of reaching that threshold. 
4.  Establish and follow an SOP for this decision process. 
 
NOTE:  To define the geographic area for which the threshold criteria would 
apply, you may consider using the donors residential zip code or county, or other 
well-specified region of comparable size that includes the donors residence.  
Although exposure to WNV may occur in any location, it is reasonable to assume 
that exposure most likely occurred while the donor was near his or her residence, 
because mosquito activity is highest at dawn and dusk, times when many donors 
are at home.  Mechanisms for switching to ID-NAT screening that utilize defined 
geographic areas based on residential zip codes, counties, or other comparable 
well-specified regions provide a standardized method for collecting data on the 
number of NAT-reactive donations and the number of donations tested.  
 
Consideration of other epidemiological data may be useful in defining a threshold 
for switching from MP-NAT to ID-NAT screening, if such data are available.  
  5 Contains Nonbinding Recommendations 
Examples include the number of clinical cases, the number of positive birds or 
mosquito pools reported in a particular geographic area, and prior ID-NAT 
implementation history. 
 
You should switch from MP-NAT to ID-NAT screening when the WNV case 
threshold has been met or exceeded in your defined geographic area.  Blood 
establishments that share geographic collection areas should consider a 
communication plan so that data from overlapping and adjacent collection areas 
may be shared and used to assess WNV activity in a defined geographic area.  
You may use this data to determine whether your defined threshold for switching 
to ID-NAT screening has been met. 
 
C.  Reporting Test Implementation 
 
1.    If you are a licensed blood establishment and are already FDA-approved to 
perform infectious disease testing of blood products, you may use at your facility 
a licensed WNV NAT according to the manufacturers product insert, and you 
must notify us in your annual report of the testing change in accordance with 21 
CFR 601.12(d).  Also, if you have already filed a supplement to your Biologics 
License Application to use a contract laboratory to perform infectious disease 
testing of blood products, and the contract laboratory will now perform a NAT for 
WNV, you must report this change in your annual report, in accordance with 21 
CFR 601.12(d).  
 
2.    If you are a licensed blood establishment and you use a new contract laboratory to 
perform a NAT for WNV and the laboratory already performs infectious disease 
testing for blood products, then you must report this change to FDA, and may do 
so through submission of a Supplement  Changes Being Effected in 
accordance with 21 CFR 601.12(c)(1) and (5), also known as changes being 
effected immediately (CBE).  If your contract laboratory previously has not 
performed infectious disease testing for blood products, then you must submit this 
change in a prior approval supplement (PAS) in accordance with                         
21 CFR 601.12(b).   
D.  Labeling of Whole Blood and Blood Components Intended for Transfusion 
 
  Title 21 CFR 606.122(h) requires that an instruction circular, also known as the Circular 
of Information, for blood products intended for transfusion include the names and 
results of all tests performed when necessary for safe and effective use.  To comply with 
21 CFR 606.122(h), upon implementation of a licensed NAT for WNV, both licensed and 
unlicensed blood establishments must revise such instruction circular to include the non-
reactive results of a NAT for WNV.  If you are a licensed blood establishment, you may 
submit this labeling as a CBE (21 CFR 601.12(c)(1) and (5)), provided the revision is 
identical to the following statement:  
 
A Licensed Nucleic Acid Test (NAT) for West Nile Virus (WNV) RNA has 
  6 Contains Nonbinding Recommendations 
been performed and found to be non-reactive.  
 
If you are a licensed blood establishment and you wish to use a different statement, then 
you must submit the labeling change as a PAS (21 CFR 601.12(b)).  If you are an 
unlicensed blood establishment, you must revise the instruction circular under                
21 CFR 606.122(h), but you are not required to submit the revision as a supplement.  
 
  7 Contains Nonbinding Recommendations 
Figure 1.  Recommendations on Testing, Unit Management, and Donor Management for 
Whole Blood and Blood Components  
Test blood donations using a  Test blood donations 
licensed MP-NAT for WNV using a licensed ID-NAT 
for WNV  
MP-NAT  
MP-NAT reactive 
non-reactive 
ID-NAT  
non-reactive 
 Test each specimen in the pool by ID-NAT 
If suitable, release  ID-NAT  If suitable, release unit 
unit(s) for  non-reactive  for transfusion. 
transfusion.  unit(s) 
ID-NAT reactive unit(s) 
Discard unit(s). 
Defer donor(s) for 120 days. 
Notify and counsel the donor(s).* 
Retrieve and quarantine in-date products from prior collections dating 
back 120 days. 
* Additional testing on the index donation using the same ID-NAT assay or an alternate NAT 
of comparable sensitivity in addition to a cleared test for antibodies to WNV may be of value in 
donor counseling. 
Note:  In the event that the NAT screening assay does not discriminate between WNV and 
other Flaviviruses that belong to the Japanese Encephalitis (JE) serogroup (namely, 
Saint Louis Encephalitis virus, Japanese Encephalitis virus, Murray Valley 
Encephalitis virus and Kunjin virus), the donor should be counseled that he or she 
tested positive for a JE serogroup virus, most likely WNV.  Alternatively, the use of a 
NAT assay that discriminates WNV from other members of the JE serogroup may be 
of value in donor counseling.  
Note:  Antibodies to viruses of the JE serogroup may cross-react on the test for antibodies to 
WNV (Refs. 11, 12).  Therefore, reactivity in a WNV antibody test may not be 
conclusive for WNV infection. 
  8 Contains Nonbinding Recommendations 
Table 1.  Recommendations on Testing, Unit Management, and Donor Management for 
Whole Blood and Blood Components 
 
  MP- NAT   ID-NAT   Actions 
  Reactive   Reactive unit(s)   Discard the unit(s).   
      Defer the donor(s) for 120 days. 
    Notify and counsel the donor(s).* 
Retrieve and quarantine in-date products from prior 
      collections dating back 120 days. 
    Non-Reactive unit(s)   If suitable, release units for transfusion. 
  Non-Reactive   Not needed   If suitable, release units for transfusion. 
 
 * Additional testing on the index donation using the same ID-NAT assay or an alternate NAT of 
comparable sensitivity in addition to a cleared test for antibodies to WNV may be of value in 
donor counseling. 
Note:  In the event that the NAT screening assay does not discriminate between WNV and 
other Flaviviruses that belong to the Japanese Encephalitis (JE) serogroup (namely, 
Saint Louis Encephalitis virus, Japanese Encephalitis virus, Murray Valley Encephalitis 
virus and Kunjin virus), the donor should be counseled that he or she tested positive for 
a JE serogroup virus, most likely WNV.  Alternatively, the use of a NAT assay that 
discriminates WNV from other members of the JE serogroup may be of value in donor 
counseling.  
Note:  Antibodies to viruses of the JE serogroup may cross-react on the test for antibodies to 
WNV (Refs. 11, 12).  Therefore, reactivity in a WNV antibody test may not be 
conclusive for WNV infection. 
 
IV.  IMPLEMENTATION 
 
We recommend that you implement the recommendations in this guidance as soon as feasible, 
but not later than six months after the guidance issue date. 
 
 
V.  REFERENCES 
 
1.  Biggerstaff BJ, Petersen LR, Estimated Risk of West Nile Virus Transmission Through 
Blood Transfusion During an Epidemic in Queens, New York City. Transfusion. 42:1019-26 
(2002). 
2.  Pealer LN, et al., Transmission of West Nile Virus Through Blood Transfusion in the United 
States in 2002. N Engl J Med. 349:1236-45 (2003). 
  9 Contains Nonbinding Recommendations 
3.  Iwamoto M, et al., Transmission of West Nile Virus From an Organ Donor to Four 
Transplant Recipients. N Engl J Med. 348, 2196-2203 (2003). 
4.  Blood Products Advisory Committee, 89th Meeting, April 27, 2007. 
http://www.fda.gov/ohrms/dockets/ac/cber07.htm#BloodProducts. 
5.  Macedo de Oliveira A, et al., West Nile Virus Blood Transfusion-Related Infection Despite 
Nucleic Acid Testing. Transfusion. 44:1695-99 (2004). 
6.  Stramer SL, et al., West Nile Virus Among Blood Donors in the United States, 2003 and 
2004. N Engl J Med. 353:451-59 (2005). 
7.  Custer B, et al., Triggers for Switching from Minipool Testing by Nucleic Acid Technology 
to Individual-Donation Nucleic Acid Testing for West Nile Virus: Analysis of 2003 Data to 
Inform 2004 Decision Making. Transfusion. 44:1547-54 (2004). 
8.  Busch MP, et al., Screening the Blood Supply for West Nile Virus RNA by Nucleic Acid 
Amplification Testing. N Engl J Med. 353:460-67 (2005). 
9.  MMWR 2004 Centers for Disease Control and Prevention. Transfusion-Associated 
Transmission of West Nile Virus --- Arizona, 2004. MMWR. 53(36):842-44 (2004). 
10. MMWR 2007 Centers for Disease Control and Prevention. West Nile Virus Transmission  
South Dakota, 2006. MMWR. 56(04):76-79 (2007). 
11. Holmes DA, et al., Comparative Analysis of Immunoglobulin M (IgM) Capture Enzyme-
Linked Immunosorbent Assay Using Virus-Like Particles or Virus-Infected Mouse Brain 
Antigens to Detect IgM Antibody in Sera from Patients with Evident Flaviviral Infections. J 
Clin Microbiol. 43(7):3227-36 (2005). 
12. Wong SJ, et al., Immunoassay Targeting Nonstructural Protein 5 to Differentiate West Nile 
Virus Infection from Dengue and St. Louis Encephalitis Virus Infections and from Flavivirus 
Vaccination. J Clin Microbiol. 41(9):4217-23 (2003). 
 
 
 
 
 
 
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