GFI West Nile Virus

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 208521448, 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.

III.

Whole Blood and Blood Components ................................................................. 2

RECOMMENDATIONS FOR DONATIONS OF WHOLE BLOOD AND BLOOD
COMPONENTS................................................................................................................ 4
A.
B.
C.
D.

Testing, Unit Management, and Donor Management ....................................... 4
Switching from MP-NAT to ID-NAT.................................................................. 5
Reporting Test Implementation .......................................................................... 6
Labeling of Whole Blood and Blood Components Intended for Transfusion. 6

IV.

IMPLEMENTATION ...................................................................................................... 9

V.

REFERENCES.................................................................................................................. 9

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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 Administration’s (FDA’s) 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 1 with recommendations for testing donations
of Whole Blood and blood components for West Nile Virus (WNV) using an FDA-licensed
donor screening assay 2 . 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
transfusion 3 .
FDA’s guidance documents, including this guidance, do not establish legally enforceable
responsibilities. Instead, guidances describe FDA’s 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 FDA’s 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.

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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 FDA’s 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 IDNAT 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

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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 MPNAT 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 nonreactive 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-

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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 semiautomated 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

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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.
B.

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.

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 donor’s residential zip code or county, or other
well-specified region of comparable size that includes the donor’s 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.

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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.

D.

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 manufacturer’s 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).

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 nonreactive 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

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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.

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Figure 1. Recommendations on Testing, Unit Management, and Donor Management for
Whole Blood and Blood Components
Test blood donations using a
licensed MP-NAT for WNV
MP-NAT
non-reactive

Test blood donations
using a licensed ID-NAT
for WNV

MP-NAT reactive

Test each specimen in the pool by ID-NAT

If suitable, release
unit(s) for
transfusion.

ID-NAT
non-reactive

If suitable, release unit
for transfusion.

ID-NAT
non-reactive
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.

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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)
Non-Reactive Not needed

If suitable, release units for transfusion.
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).
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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 EnzymeLinked 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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