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 

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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
Contains Nonbinding Recommendations 

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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 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 

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. 
Contains Nonbinding Recommendations 

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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. 
Contains Nonbinding Recommendations 

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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 
Contains Nonbinding Recommendations 

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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 donor’s 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, 
Contains Nonbinding Recommendations 

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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 
Contains Nonbinding Recommendations 

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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 manufacturer’s instructions (see Table 1). 
Contains Nonbinding Recommendations 

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Table 1. Implementation of a Licensed NAT Kit 

If you... And... Then we recommend that you: 

you are already FDA 

approved to perform 

infectious disease testing for 

your Whole Blood and blood 

components 

report in your next annual report (AR) 

(§ 601.12(d)). Submit an instruction 

circular and revised labeling, as 

described in section VI. (§ 601.12(f)) 

will perform 

licensed NAT 

in your facility 

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.) 

you have already 

supplemented your BLA to 

use this laboratory for 

infectious disease testing for 

your Whole Blood and blood 

components 

report in your next AR (§ 601.12(d)). 

Submit an instruction circular and 

revised labeling, as described in section 

VI. 

you have not used the 

laboratory before and they 

already perform infectious 

disease testing for Whole 

Blood and blood components 

submit a changes being effected in 30 

days (CBE-30) supplement to your 

BLA (§ 601.12(c)). Submit an 

instruction circular and revised 

labeling, as described in section VI. 

will use an FDA 

registered 

contract 

laboratory to 

perform 

licensed NAT 

the laboratory has not 

previously performed 

infectious disease testing for 

Whole Blood and blood 

components 

submit a PAS to your BLA (§ 

601.12(b)). 

Include an instruction circular and 

revised labeling, with your supplement. 

(see section VI.) 

will 

concurrently 

discontinue 

testing for HIV1 p24 antigen 

 include this information in the 

submission described above, and 

submit revised labeling as described in 

section VI. 

 

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. 
Contains Nonbinding Recommendations 

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Table 2. Implementation of NAT using a Licensed NAT Assay Test Facility 

If you... and... Then we recommend that you: 

you already perform 

infectious disease 

testing on your Source 

Plasma 

submit a CBE-30 supplement to your BLA 

(§601.12(c)), including information on the 

procedure/algorithm for pooling and the 

number of samples in the master pool. This 

information may be included in the CBE-30 

supplement for implementing NAT. 

will perform 

sample pooling 

at your facility 

infectious disease 

testing is new at your 

facility 

submit a PAS to your BLA (§ 601.12(b)), 

including SOPs, validation data on pooling, 

storage, sample shipment, and information 

on software and data management systems. 

the contractor is 

already registered to 

perform infectious 

disease testing for 

a. Whole Blood and 

blood components or

b. pooling of Source 

Plasma donor samples 

for NAT 

submit a CBE-30 supplement to your BLA (§ 

601.12 (c)) including information on the 

procedure/algorithm for pooling and the 

number of samples in the master pool. This 

information may be included in your CBE-30 

supplement for implementing NAT. 

will use a 

registered 

contractor to 

perform 

pooling in 

support of 

licensed NAT 

the contractor is 

already registered but 

has not previously 

performed infectious 

disease testing for 

a. Whole Blood and 

blood components or 

b. pooling of Source 

Plasma donor samples 

for NAT testing 

submit a PAS to your BLA (§601.12) and 

additional information described above for 

PAS submissions. 

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 
Contains Nonbinding Recommendations 

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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
Contains Nonbinding Recommendations 

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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. 
Contains Nonbinding Recommendations 

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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. Bailliere’s 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).