Guidance for Industry 
 
Use of Nucleic Acid Tests on Pooled and 

Individual Samples from Donors of 

Whole Blood and Blood Components 

(including Source Plasma and Source 

Leukocytes) to Adequately and 

Appropriately Reduce the Risk of 

Transmission of HIV-1 and HCV 

 
 
Additional copies of this guidance are available from the Office of Communication, Training and 
Manufacturers Assistance (HFM-40), 1401 Rockville Pike, Rockville, MD  20852-1448, or by 
calling 1-800-835-4709 or 301-827-1800, or from the Internet at 
http://www.fda.gov/cber/guidelines.htm. 
 
For questions concerning labeling, reporting or licensing issues, contact the Division of Blood 
Applications at 301-827-3543. For questions regarding testing issues, contact the Division of 
Emerging Transfusion Transmitted Diseases at 301-827-3008. 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Biologics Evaluation and Research 
October 2004   Contains Nonbinding Recommendations 
Table of Contents 

 
 
I. 	  INTRODUCTION...........................................................................................................1 
 
II. 	  BACKGROUND .............................................................................................................2 
 
III. 	  RECOMMENDATIONS FOR DONOR SCREENING..............................................3 
 
IV. 	  ADDITIONAL TESTING CONSIDERATIONS ........................................................5 
 
V. 	  REQUIREMENTS FOR REPORTING IMPLEMENTATION OF HIV-1 and HCV 

NAT FOR LICENSED BLOOD AND PLASMA ESTABLISHMENTS (21 CFR 

601.12) ..............................................................................................................................5 
 
A. 	  Implementation of a licensed NAT kit for Whole Blood and blood 

components including Source Plasma and Source Leukocytes for which 

instruments and software for pooling of samples are referenced in the 

product insert and are validated as part of the test procedure ......................6 
 
B. 	  Implementation of NAT using a licensed NAT assay test facility approved for 

use on pools of samples from Source Plasma donors.......................................7 
 
VI. 	  PRODUCT LABELING AND DISPOSITION............................................................8
  
A.   Whole Blood and blood components for transfusion.......................................8 
 
B.   Blood components intended for further manufacture.....................................9 
 
C.   Reactive units and product disposition .............................................................9 
 
VII.	   IMPLEMENTATION ..................................................................................................10
  
VIII.	   REFERENCES..............................................................................................................11 
 
 
i 
  Contains Nonbinding Recommendations 
 
Guidance For Industry 

 
Use of Nucleic Acid Tests on Pooled and Individual Samples from 

Donors of Whole Blood and Blood Components (including Source 

Plasma and Source Leukocytes) to Adequately and Appropriately 

Reduce the Risk of Transmission of HIV-1 and HCV 

 
 
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 
 
FDA's blood testing rule requires you, an establishment that collects blood and blood 
components (e.g. Whole Blood and blood components including Source Plasma and Source 
Leukocytes), to test each donation of human blood or blood component intended for use in 
preparing a product, including donations intended as a component of, or used to prepare a 
medical device, for evidence of infection due to specific communicable disease agents(21 CFR 
610.40(a)). This rule also requires you to use one or more approved screening tests as necessary 
to reduce adequately and appropriately the risk of transmission of communicable disease.  (21 
CFR 610.40(b)) In the preamble to this final rule, we discussed the approved donor screening 
tests that we believed were necessary to reduce adequately and appropriately the risk of 
transmission of Human Immunodeficiency Virus type 1 (HIV-1) and hepatitis C virus (HCV).  
We also stated that as technology advances, we would issue guidance describing those tests that 
we believe would adequately and appropriately reduce the risk of transmission of communicable 
disease agents. (66 FR 31146, 31162 June 11, 2001). 
 
The purpose of this guidance is to inform you that:   
 
  1)  FDA has licensed nucleic acid tests (NAT) as tests to screen blood donors for 
HIV-1 ribonucleic acid (RNA), and HCV RNA; and 
 
  2)  these licensed tests can detect evidence of infection at a significantly earlier stage 
than is possible under previously approved tests using antibody or antigen 
detection technology; including the HIV-1 p24 antigen test. 
 
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We believe that these newly licensed tests are now widely available, and that these tests meet the 
criteria for screening tests   that are necessary to reduce adequately and appropriately the risk of  
transmission of communicable disease through blood products. 
 
The biologics regulations require licensed blood and Source Plasma manufacturers to report 
manufacturing changes, such as a change in donor testing, to FDA ( 601.12).  We further 
explain those reporting requirements in Section V of this guidance. 
 
This guidance combines and finalizes the draft guidance Use of Nucleic Acid Tests on Pooled 
Samples from Source Plasma Donors to Adequately and Appropriately Reduce the Risk of 
Transmission of HIV-1 and HCV dated December 2001 (January 31, 2002, 67 FR 4719) and 
the draft guidance Use of Nucleic Acid Tests on Pooled and Individual Samples from Donors of 
Whole Blood and Blood Components for Transfusion to Adequately and Appropriately Reduce 
the Risk of Transmission of HIV-1 and HCV dated March 2002 (April 9, 2002, 67 FR 17077). 
 
II.  BACKGROUND 
 
Transmission of HIV and HCV by blood and blood components has been dramatically reduced 
as a result of implementation of sensitive tests for viral antibody and antigen.  The major sources 
of remaining risk of transmission of HIV and HCV by blood and blood components include 
window period donations (donations that occur when the donor is infectious, but before the 
infection can be detected using approved tests), viral variants (which may not be detectable using 
tests designed to identify the common forms of  a virus), atypical seroconversion as indicated by 
an unusually prolonged window period or lack of seroconversion, and laboratory testing error 
(Ref. 1, 2). In addition, initiatives by the Source Plasma industry, including the study of NAT 
performed under an investigational new drug application (IND), have greatly reduced the 
frequency of infectious units entering plasma pools for further manufacturing (Ref. 3). Although 
effective viral clearance methods are in place for almost all plasma derivatives, measures to 
reduce the occurrence of window period donations are expected to further reduce the residual 
risk of HIV or HCV infectious units entering plasma pools, and enhance the overall safety of 
blood products. 
 
In 1994, we held a workshop to explore the potential application of nucleic acid based methods 
to screen samples from blood donors for HIV.  At that time, it was thought that although these 
methods were clearly sensitive, they were not ready for implementation on a large scale.  
However, the workshop fueled interest in developing systems for implementing nucleic acid 
methodology for testing blood and plasma donations, since the methodology might be used to 
identify earlier stages of infection, and therefore reduce the window period for donations. 
 
Subsequently, test kit manufacturers and blood organizations, in collaboration with the 
government (The National Institutes of Health (NIH) and FDA), actively pursued development 
of NAT assays for HIV-1 RNA and HCV RNA.  The sensitivity of NAT on pooled samples and 
labor intensiveness of testing individual donations made testing pooled samples more efficient 
than testing individual donations. 
 
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Clinical studies to evaluate NAT were initiated in 1997 under INDs.  Large-scale studies were 
necessary to demonstrate the efficacy of NAT as a donor screening method, primarily because 
the frequency of window period donations is low.  We have worked with manufacturers to 
validate NAT assays for donor screening through review of their Biologics License Applications 
(BLAs) (Ref. 4). In December 1999, we provided industry with guidance on manufacturing and 
assay validation for licensure of NAT. 
 
By 1997, some manufacturers in Europe had voluntarily instituted NAT on pooled samples of 
plasma.  At about that time, the European Union issued a directive requiring HCV RNA testing 
for all plasma fractionated in Europe by July 1, 1999, and stated that HIV-1 RNA testing would 
be required at a later date. The European directive, which applied to both Source Plasma and 
recovered plasma, provided further impetus to the rapid development of NAT for blood and 
plasma donations in the United States. 
 
Since September 2001, we have licensed several NAT assays for the detection of HIV-1 RNA 
and HCV RNA in Whole Blood and blood components including Source Plasma.  Validation 
data submitted by manufacturers support replacement of currently licensed assays for HIV-1 p24 
antigen by both pooled and individual NAT. 
 
FDA has previously issued guidance on Revised Recommendations for Testing Whole Blood, 
Blood Components, Source Plasma and Source Leukocytes for Antibody to Hepatitis C Virus 
Encoded Antigen (Anti-HCV) (Memorandum  to All Blood Establishments, April 23, 1992, 
http://www.fda.gov/cber/bldmem/hcv042392.pdf) and Revised Recommendations for the Prevention 
of Human Immunodeficiency Virus (HIV) Transmission by Blood and Blood Products 
(Memorandum to All Blood Establishments, April 23, 1992, 
http://www.fda.gov/cber/bldmem/hiv042392.pdf). 
 
III.   RECOMMENDATIONS FOR DONOR SCREENING 
 
Under 21 CFR 610.40(b), you must perform one or more screening tests approved by the FDA as 
necessary to reduce adequately and appropriately the risk of transmission of communicable 
disease agents, including HIV-1 and HIV-2, and, HCV.  Note that the regulation requires testing 
for all of these agents, and does not provide an exemption from the requirement to test for all 
named agents if a donation will be discarded on the basis of reactive tests for a single disease 
agent such as HIV-1. 
 
We recommend that you meet the requirement for conducting adequate and appropriate testing 
for HIV and HCV, as required by 21 CFR  610.40(b), in accord with points 1 and 2 below.  
Note that, although we recommend the use of HIV-1 NAT and HCV NAT on units that are not 
reactive on a donor screening test for the detection of antibodies to HIV or HCV, respectively, 
we do not believe that HIV-1 NAT and HCV NAT are necessary in all instances.  We do not 
believe that a licensed HIV-1 NAT is part of the adequate and appropriate testing required under 
610.40(b) for donations that are reactive on a test for the detection of antibodies to HIV-1 and are 
to be discarded or used in the manufacture of non-injectable products.  Similarly, we do not 
believe that a licensed HCV NAT is part of the adequate and appropriate testing required under 
610.40(a) for donations that are reactive on a donor screening test for the detection of antibodies 
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to HCV and are to be discarded or used in the manufacture of non-inject able products.  
Nevertheless, you may decide to perform HIV-1 and HCV NAT in order to obtain useful 
information regarding the donors infection status.  This information may be useful as part of 
donor notification. 
 
  1) 	 For HIV-1: To meet the requirement to use one or more approved screening tests, 
as necessary to adequately and appropriately reduce the risk of transmission of 
communicable disease, we recommend that you use an FDA licensed donor 
screening test for the detection of antibodies to HIV-1 (either a stand alone HIV-1 
test or a combination HIV-1/HIV-2 test).  If the FDA licensed test for detection of 
antibodies to HIV-1 is negative or non-reactive, we recommend that, as part of 
adequate and appropriate screening, you test the donation further using an FDA 
licensed HIV-1 NAT.  Testing using the tests for the antibodies to HIV-1 and 
HIV-1 NAT may be performed concurrently. 
 
If that FDA licensed test for the detection of antibodies to HIV-1 is reactive and 
the donation will be discarded or used in the manufacture of non-injectable 
products, we believe that you have met the standard for adequate and appropriate 
screening for HIV-1. Although you may choose to test such a reactive donation 
by using an FDA licensed HIV-1 NAT, and although additional testing may 
provide information that would be useful to the donor, we do not believe that it is 
necessary to perform this additional test.  However, you must proceed to 
supplemental testing for HIV-1 using an additional, more specific test, as required 
by 21 CFR  610.40(e). 
 
Conversely, if a donation that is reactive on a test for the detection of antibodies 
to HIV-1 will be used for autologous transfusion or for further manufacturing into 
injectable products, we recommend that you test the donation using an FDA 
licensed HIV-1 NAT. 
 
We believe that, when HIV-1 NAT is used in accordance with these 
recommendations, the use of the HIV-1 p24 test (previously recommended by 
FDA) is not necessary to reduce adequately and appropriately the risk of 
transmission of HIV-1. 
 
2) 	 For HCV:  To meet the requirement to use one or more approved screening tests, 
as necessary to adequately and appropriately reduce the risk of transmission of 
communicable disease, we recommend that you use an FDA licensed donor 
screening test for the detection of antibodies to HCV.  If the FDA licensed test for 
detection of antibodies to HCV is negative or non reactive, we recommend that, 
as part of adequate and appropriate screening, you test the donation further using 
an FDA licensed HCV NAT.  Testing using a test for antibodies to HCV and the 
HCV NAT test may be performed concurrently. 
 
If that FDA licensed test for the detection of antibodies to HCV is reactive and the 
donation will be discarded or used in the manufacture of non-injectable products, 
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  Contains Nonbinding Recommendations 
we believe that you have met the standard for adequate and appropriate screening 
for HCV.  Although you may choose to test such a reactive donation by using an 
FDA licensed HCV NAT, and although additional testing may provide 
information that would be useful to the donor, we do not believe that it is 
necessary to perform this additional test.  However, you must proceed to 
supplemental testing for HCV using an additional, more specific test, as required 
by 21 CFR  610.40(e). 
   
Conversely, if a donation that is reactive on a test for the detection of antibodies 
to HCV will be used for autologous transfusion or for further manufacturing into 
injectable products, we recommend that you test the donation using an FDA 
licensed HCV NAT. 
 
IV. 	 ADDITIONAL TESTING CONSIDERATIONS 
 
You must defer a donor who tests reactive by a donor-screening test ( 610.41), you must 
perform a supplemental (additional, more specific) test on donations that test reactive on an 
initial screening test ( 610.40(e)), and you must make reasonable attempts to notify a donor who 
has been deferred based on the results of tests for communicable diseases ( 630.6). 
 
V. 	 REQUIREMENTS FOR REPORTING IMPLEMENTATION OF HIV-1 AND 
HCV NAT FOR LICENSED BLOOD AND PLASMA ESTABLISHMENTS (21 
CFR 601.12) 
 
Under 21 CFR 601.12, biologics license applicants are required to inform FDA about each 
change in the product, production process, quality controls, equipment, facilities, responsible 
personnel, or labeling established in the approved application(s).  Before distributing a product 
made using a change (including instrumentation), an applicant must demonstrate, through 
appropriate validation and/or studies, the lack of adverse effect of the change on the identity, 
strength, quality, purity, or potency of the product as they may relate to the safety or 
effectiveness of the product (21 CFR 601.12(a)).  The type of notification to FDA required under 
this section varies according to the potential of the change to have an adverse effect on the 
identity, strength, quality, purity, or potency of the product, as they may relate to the safety or 
effectiveness of the product.  Notification may be by annual report (when there is a minimal 
potential for adverse effect, 21 CFR 601.12(d)), changes being effective supplement (when there 
is a moderate potential for adverse effect and particular assurances are provided to FDA, 21 CFR 
601.12(c)(5)), changes being effective in 30 days supplement (when there is a moderate potential 
for adverse effect, 21 CFR 601.12(c)), or prior approval supplement (PAS) (when there is a 
substantial potential for adverse effect, 21 CFR 601.12(b)).  Labeling changes are addressed in 
21 CFR 601.12(f). 
 
In section V.A. below, we describe our recommendations for reporting your implementation of a 
licensed NAT kit. 
 
In section V.B., below, we describe our recommendations for reporting implementation of NAT 
using a licensed NAT assay test facility approved for use on pools of samples from Source 
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  Contains Nonbinding Recommendations 
Plasma donors.  In addition, if you intend to use a licensed NAT test facility, and you or your 
contract laboratory intend to use instruments and software for sample pooling that have not 
previously been validated using these assays, we believe this manufacturing change would 
present a substantial potential for adverse effect.  We recommend that you submit the change in a 
PAS, and include validation data, information on the instruments and software used, the 
procedure/algorithm for pooling and the number of samples in the master pool 
 
A. 	 Implementation of a licensed NAT kit for Whole Blood and blood 
components including Source Plasma and Source Leukocytes for which 
instruments and software for pooling of samples are referenced in the 
product insert and are validated as part of the test procedure 
 
The following recommendations for reporting manufacturing changes under 601.12 apply when 
implementing a licensed NAT (with the particular test to be used identified in your submission) 
according to the manufacturers instructions (see Table 1).   
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  Contains Nonbinding Recommendations 
 
Table 1. Implementation of a Licensed NAT Kit  
 
If you...  And...  Then we recommend that you: 
will perform  you are already FDA  report in your next annual report (AR) 
licensed NAT  approved to perform   ( 601.12(d)). Submit an instruction 
in your facility  infectious disease testing for  circular and revised labeling, as 
your Whole Blood and blood  described in section VI. ( 601.12(f)) 
components 
testing is new at your facility  submit a prior approval supplement 
(PAS) to your BLA ( 601.12(b)). 
Include an instruction circular and 
revised labeling, with your supplement 
(see section VI.) 
 
will use an FDA  you have already  report in your next AR ( 601.12(d)). 
registered  supplemented your BLA to  Submit an instruction circular and 
contract  use this laboratory for  revised labeling, as described in section 
laboratory to  infectious disease testing for  VI. 
perform   your Whole Blood and blood 
licensed NAT  components 
you have not used the  submit a changes being effected in 30 
laboratory before and they  days (CBE-30) supplement to your 
already perform infectious  BLA ( 601.12(c)). Submit an 
disease testing for Whole  instruction circular and revised 
Blood and blood components  labeling, as described in section VI.  
the laboratory has not  submit a PAS to your BLA ( 
previously performed  601.12(b)). 
infectious disease testing for  Include an instruction circular and 
Whole Blood and blood  revised labeling, with your supplement. 
components  (see section VI.) 
will    include this information in the 
concurrently  submission described above, and 
discontinue  submit revised labeling as described in 
testing for HIV- section VI. 
1 p24 antigen 
   
B. 	 Implementation of NAT using a licensed NAT assay test facility approved for 
use on pools of samples from Source Plasma donors 
 
If a licensed NAT assay test facility will perform testing or testing and pooling of the 
samples, we recommend that you submit a CBE-30 to supplement your BLA ( 
601.12(c), and revised labeling to supplement your BLA as described in section VI.  If 
you intend to discontinue testing for HIV-p24 antigen, we recommend that you include 
this information in your CBE-30 supplement. 
 
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  Contains Nonbinding Recommendations 
Table 2. Implementation of NAT using a Licensed NAT Assay Test Facility 
 
If you...  and...  Then we recommend that you: 
will perform  you already perform   submit a CBE-30 supplement to your BLA 
sample pooling  infectious disease  (601.12(c)), including information on the 
at your facility  testing on your Source  procedure/algorithm for pooling and the 
Plasma   number of samples in the master pool.  This 
information may be included in the CBE-30 
supplement for implementing NAT.  
infectious disease  submit a PAS to your BLA ( 601.12(b)), 
testing is new at your  including SOPs, validation data on pooling, 
facility   storage, sample shipment, and information 
on software and data management systems.   
will use a  the contractor is  submit a CBE-30 supplement to your BLA ( 
registered  already registered to  601.12 (c)) including information on the 
contractor to  perform infectious  procedure/algorithm for pooling and the 
perform   disease testing for  number of samples in the master pool.  This 
pooling in  a. Whole Blood and  information may be included in your CBE-30 
 
support of  blood components or   supplement for implementing NAT.   
licensed NAT  b. pooling of Source 
Plasma donor samples 
for NAT 
the contractor is  submit a PAS to your BLA (601.12) and 
already registered but  additional information described above for 
has not previously  PAS submissions.  
performed infectious 
disease testing for 
a. Whole Blood and 
blood components or  
b. pooling of Source 
Plasma donor samples 
for NAT testing 
 
VI.  PRODUCT LABELING AND DISPOSITION 
 
A.  Whole Blood and blood components for transfusion 
 
Consistent with the labeling for other infectious disease markers, nonreactive NAT 
results need not appear on the container label.  We recommend that you include these test 
results in the instruction circular.  Upon implementation of a licensed NAT, we 
recommend that you use an instruction circular that reflects the results of NAT, and the 
discontinuation of HIV-1 p24 antigen testing, if applicable, and that you submit a revised 
instruction circular incorporating the results of NAT for HIV-1 and HCV, as well as 
discontinuation of HIV-1 p24 antigen testing as a changes being effected supplement 
under  601.12(f)(2)(i)(E).  In a Final Guidance for Industry, An Acceptable Circular of 
Information for the Use of Human Blood and Blood Components, December 2003 
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  Contains Nonbinding Recommendations 
http://www.fda.gov/cber/gdlns/circbld.htm, FDA recognized as acceptable for use by 
manufacturers of blood and blood components intended for transfusion subject to United 
States statutes and regulations, the instruction circular entitled Circular of Information 
for the Use of Human Blood and Blood Components (Circular) dated July 2002, 
http://www.fda.gov/cber/gdlns/crclr.pdf. This circular has incorporated the results of 
NAT and the discontinuation of HIV-1 p24 antigen testing. 
 
B.  Blood components intended for further manufacture 
 
Upon implementation of a licensed NAT, we recommend that you include NAT results 
on the labeling for blood components intended for further manufacture into injectable or 
non-injectable products. We recommend that you use the following statement on the 
labeling for donations that test nonreactive: 
 
  Nonreactive for HIV-1 RNA and HCV RNA. 
 
Alternatively, you may report NAT results in combination with the results of other 
infectious disease testing.  
 
If you wish to use a statement regarding NAT results that is consistent with the 
recommendations above, we recommend that you submit this labeling change as a 
changes being effected supplement under  601.12(f)(2)(i)(E). 
 
NOTE: For section B (above), we do not recommend that you submit an additional 
labeling supplement if you have already received approval for an alternate test statement. 
 
C.  Reactive units and product disposition 
 
NAT reactive units must not be shipped or used, except as provided in  610.40(h)(2).  If 
released for these uses, the units must be relabeled consistent with the labeling 
requirements in  606.120, 610.40, and 640.70. 
 
Additionally, under  610.40(h)(2)(ii)(B), (C), and (E), you must label the reactive unit 
with the Biohazard legend and with the following cautionary statements as applicable: 
 
Reactive for HIV-1 RNA, 
 
or  
 
Reactive for HCV RNA,  
 
or  
 
Reactive for HIV-1 RNA and HCV RNA, 
 
and either  
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Caution: For Further Manufacturing into In Vitro Diagnostic Reagents for Which There 
Are No Alternative Sources, 
 
or  
 
Caution: For Laboratory Research Use Only. 
 
VII.  IMPLEMENTATION  
HIV-1 and HCV NAT for testing samples from donors of Whole Blood and blood components 
(including Source Plasma and Source Leukocytes) may involve the use of complex pooling and 
testing systems.  We recognize that it may require time to implement these systems.   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. 
 
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VIII.  	REFERENCES 
 
1. 	 M.P. Busch and S. H. Kleinman, Nucleic Acid Amplification Testing of Blood Donors 
for Transfusion- Transmitted Infectious Diseases, Transfusion 2000; 40:143-159. 
2. 	 S.H. Kleinman and M.P. Busch, The Risks of Transfusion-Transmitted Infection: Direct 
Estimation and Mathematical Modeling. Baillieres Clinical Hematology 2000; 13:631- 
649. 
3. 	 General Accounting Office, Blood Plasma Safety:  Plasma Product Risks Are Low If 
Good Manufacturing Practices Are Followed, GAO/HEHS-98-205. 
4. 	 Guidance for Industry: In the Manufacture and Clinical Evaluation of In Vitro Tests to 
Detect Nucleic Acid Sequences of Human Immunodeficiency Viruses Type 1 and 2. 
December 1999 (December 14, 1999; 64 FR 71147) 
(http://www.fda.gov/cber/gdlns/hivnas.htm). 
 
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