Further Testing of Donations that are 
Reactive on a Licensed Donor 
Screening Test for Antibodies to 
Hepatitis C Virus  
 
 
Guidance for Industry
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Additional copies of this guidance are available from the Office of Communication, Outreach 
and Development (OCOD), 10903 New Hampshire Ave., Bldg. 71, Rm. 3128, Silver Spring, 
MD 20993-0002, or by calling 1-800-835-4709 or 240-402-8010, or email ocod@fda.hhs.gov, or 
from the Internet at  
https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-regulatory-information-
biologics/biologics-guidances 
 
For questions on the content of this guidance, contact OCOD at the phone numbers or email 
address listed above. 
 
 
 
 
 
 
U.S. Department of Health and Human Services  
Food and Drug Administration  
Center for Biologics Evaluation and Research  
October 2019  Contains Nonbinding Recommendations 
 
Table of Contents 
 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  BACKGROUND ............................................................................................................... 2 
III.  RECOMMENDATIONS FOR FURTHER TESTING OF DONATIONS THAT 
ARE REPEATEDLY REACTIVE ON A LICENSED SCREENING TEST FOR 
ANTI-HCV ........................................................................................................................ 4 
IV.  IMPLEMENTATION AND REPORTING ................................................................... 5 
V.  REFERENCES .................................................................................................................. 6 
i  Contains Nonbinding Recommendations 
 
Further Testing of Donations that are Reactive on a Licensed Donor 
Screening Test for Antibodies to Hepatitis C Virus  
 
 
Guidance for Industry 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or 
Agency) on this topic.  It does not establish any rights for any person and is not binding on FDA 
or the public.  You can use an alternative approach if it satisfies the requirements of the 
applicable statutes and regulations.  To discuss an alternative approach, contact the FDA staff 
responsible for this guidance as listed on the title page. 
 
 
I.  INTRODUCTION 
 
We, the Food and Drug Administration (FDA), are providing you, blood establishments that 
collect Whole Blood and blood components, including Source Plasma, with recommendations 
for further testing of donations that are reactive on a licensed donor screening test for antibodies 
to hepatitis C virus (anti-HCV), as required under 21 CFR 610.40(e).  We are also providing 
guidance to blood establishments on how to report the implementation of these 
recommendations.   
 
In accordance with 21 CFR 610.40(e), each donation, including autologous donations1, found to 
be reactive by a donor screening test must be further tested using a licensed, approved or cleared 
supplemental test, when available.  If no such supplemental test is available, blood 
establishments must perform one or more licensed, approved, or cleared tests as adequate and 
appropriate to provide additional information concerning the reactive donors infection status  
(21 CFR 610.40(e)).  
 
Currently, certain hepatitis C virus (HCV) nucleic acid tests (NAT) licensed for donor screening 
are also licensed for use as a supplemental test to confirm HCV infection for specimens that are 
repeatedly reactive on a licensed donor screening test for anti-HCV.  We have referred to this 
indication as a limited supplemental claim because a reactive HCV NAT result on a donation 
that is repeatedly reactive for anti-HCV confirms HCV infection and fulfills the further testing 
requirement in 21 CFR 610.40(e) (Ref. 1).  In contrast, a nonreactive HCV NAT result on a 
donation that is repeatedly reactive for anti-HCV in a screening test does not provide adequate 
information about the donors infection status.  This guidance provides recommendations for 
adequate and appropriate testing of donors with repeatedly-reactive anti-HCV screening results 
under 21 CFR 610.40(e), using the licensed HCV NAT and licensed anti-HCV donor screening 
tests or approved or cleared anti-HCV diagnostic tests that are currently available, to provide 
additional information about the donors infection status.  
 
                                                 
1 Exceptions to this requirement for autologous donations are found in 21 CFR 610.40(e)(1)(i)  (ii). 
1  Contains Nonbinding Recommendations 
 
This guidance does not address the requirements to test each donation for evidence of HCV  
(21 CFR 610.40(a) and (b)).  You should refer to previously published guidance documents for 
recommendations regarding testing donations, donor deferral, product disposition, lookback and 
donor reentry (Refs. 1-3).  This guidance finalizes the draft guidance of the same title dated 
September 2018 and updates the recommendations related to the use of an appropriate 
multiantigen supplemental test2 contained in Guidance for Industry: Lookback for Hepatitis C 
Virus (HCV):  Product Quarantine, Consignee Notification, Further Testing, Product 
Disposition, and Notification of Transfusion Recipients Based on Donor Test Results Indicating 
Infection with HCV, dated December 2010 (December 2010 Guidance) (Ref. 1).   
 
FDA guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidance documents describe the FDAs current thinking on a topic and 
should be viewed only as recommendations, unless specific regulatory or statutory requirements 
are cited.  The use of word should in FDA guidance documents means that something is 
suggested or recommended, but not required. 
 
 
II.  BACKGROUND 
 
Hepatitis C is a blood borne disease caused by acute or chronic infection with HCV. 
Approximately 75-85% of newly infected adults and adolescents develop chronic HCV infection. 
A survey of the general United States (U.S.) population during 2003-2010 estimated that 1.3% or 
about 3.6 million persons had antibodies to HCV, indicating past or present infection; of whom, 
about 1% or 2.7 million persons are HCV RNA-positive, indicating chronic infection (Ref. 4).  
As a group, persons born during 1945-1965 demonstrate a 3.25% prevalence of anti-HCV and 
account for approximately three fourths of all chronic HCV infections among adults in the U.S. 
(Ref. 5).  Many individuals living with HCV are unaware of their infection status (Ref. 5).  NAT 
is necessary to identify active HCV infection among persons who have tested anti-HCV positive 
(Ref. 5).  Current public health initiatives aim to identify actively infected individuals to prevent 
and control HCV infection and associated chronic disease, cirrhosis, and hepatocellular 
carcinoma.   
 
The prevalence of HCV infection is substantially lower among blood donors than in the general 
population.  Based on the federally-funded Transfusion-Transmissible Infections Monitoring 
System (TTIMS) that collated infectious disease test results on about 14 million blood donations 
in 2015 to 2017, the prevalence of HCV in blood donations was 0.02% overall and 0.1% among 
first-time donors (Ref. 6).  Blood donation testing allows for the appropriate identification of 
donors infected with the virus so that they can be deferred from donation, notified, and counseled 
of their results.  Studies have demonstrated the utility of HCV NAT in an HCV supplemental 
testing algorithm for the purposes of donor notification and counseling.  Among anti-HCV 
repeatedly reactive donations, about 40% were reactive by individual donation HCV NAT 
demonstrating acute or chronic HCV infection in these donors (Ref. 7).  In this scenario, 
supplemental testing for anti-HCV is not necessary.  The counseling message to donors with 
these results is that they have ongoing HCV infection, are potentially infectious to others, and 
                                                 
2 The Chiron RIBATM HCV 3.0 assay (HCV RIBA) was used as the supplemental test for donations found to be 
reactive on a licensed screening test for anti-HCV before it was discontinued in February 2013. 
2  Contains Nonbinding Recommendations 
 
need further medical evaluation for liver disease, monitoring and possible treatment (Refs. 7, 8).  
In contrast, donors with a repeatedly reactive anti-HCV screening test and nonreactive HCV 
NAT may have a false-reactive screening test and be uninfected, may have a chronic persistent 
infection with transient or intermittent low-level viremia, or may have a resolved infection (Refs. 
7, 9).  Further testing is necessary to clarify the donors HCV infection status.  
 
The Chiron RIBATM HCV 3.0 assay (HCV RIBA) had been used as the supplemental test for 
donations found to be repeatedly reactive on a licensed screening test for anti-HCV before it was 
discontinued in February 2013.  Upon discontinuation of the HCV RIBA, FDA posted 
information on its website to assist blood establishments in managing donations that are 
repeatedly reactive on an anti-HCV screening test.  In the web posting, FDA advised blood 
establishments that they could use licensed HCV NAT tests labeled with a limited supplemental 
claim, because a reactive NAT acts as a positive supplemental test and confirms HCV infection. 
FDA did not provide recommendations for further testing when donations are repeatedly reactive 
on an anti-HCV screening test and nonreactive on HCV NAT.    
 
In 2015, FDA issued a final rule that revised the requirement for further testing under 21 CFR 
610.40(e).3  Consistent with the previous regulation, under the final rule, establishments must 
perform further testing using an approved supplemental test when one is available.  When a 
supplemental test is not available, the rule now requires the use of other tests as adequate and 
appropriate to provide additional information concerning the reactive donors infection status.    
 
In this guidance, we are providing recommendations for further testing under 21 CFR 610.40(e) 
of donations that are repeatedly reactive on a licensed screening test for anti-HCV.  Blood 
establishments must use the results of this further testing performed under 21 CFR 610.40(e) to 
notify donors who have been deferred based on the testing results (21 CFR 630.40), and to take 
the actions based on further testing described in the lookback requirements, including notifying 
consignees (21 CFR 610.47(a)(3)), notifying transfusion recipients (21 CFR 610.47(b)(3)) and 
determining disposition of quarantined, in-date blood, and blood components (21 CFR 
610.47(a)(4) and (b)(2).   
 
 
                                                 
3 See Requirements for Blood and Blood Components Intended for Transfusion or for Further Manufacturing Use; 
Final Rule (80 FR 29842, May 22, 2015).  The rule became effective May 23, 2016. 
3  Contains Nonbinding Recommendations 
 
III.  RECOMMENDATIONS FOR FURTHER TESTING OF DONATIONS THAT 
ARE REPEATEDLY REACTIVE ON A LICENSED SCREENING TEST FOR 
ANTI-HCV 
 
You must further test each donation, including each autologous donation, that is repeatedly 
reactive for anti-HCV on a licensed donor screening test, using a licensed HCV NAT that is 
labeled with the supplemental indication, according to the manufacturers instructions in the 
package insert [see 21 CFR 610.40(e) and 606.65(e)].  Consistent with the manufacturers 
instructions, you must obtain the supplemental results on an individual donation, either on the 
index donation or a follow-up sample (21 CFR 606.65(e)). We recommend the following:  
 
1.  If NAT with a supplemental indication is reactive on an individual donation for HCV, the 
result confirms HCV infection in the donor and no additional testing is necessary.  The 
donor should be counseled as HCV infected.  The test results for the donation should be 
regarded as positive for the actions based on further testing under the lookback 
requirements found in 21 CFR 610.47(a)(3) and (4) and (b)(2) and (3). 
 
2.  If NAT with a supplemental indication is nonreactive on an individual donation for HCV, 
further testing is necessary.  You should perform a second, different licensed donor 
screening test or an approved or cleared diagnostic test for anti-HCV.  
 
a.  If the result is negative, the donor should be counseled that HCV infection was not 
confirmed and is unlikely.  The test results for the donation may be regarded as 
negative for the actions based on further testing under the lookback requirements 
found in 21 CFR 610.47(a)(3) and (4) and (b)(2) and (3). 
 
b.  If the result is repeatedly-reactive for anti-HCV, no further testing is necessary, and 
the donor should be counseled appropriately.  The test results for the donation are 
considered positive for the actions based on further testing under the lookback 
requirements found in 21 CFR 610.47(a)(3) and (4) and (b)(2) and (3).  
 
3.  You must make reasonable attempts to notify the donor within 8 weeks after determining 
that the donor is deferred, and you must attempt to obtain the results of further testing 
prior to notifying the donor of the deferral (21 CFR 630.40).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  
 
A list of tests licensed for use in blood donor screening and supplemental testing is available on 
the FDA website, available at   
https://www.fda.gov/vaccines-blood-biologics/blood-donor-screening/complete-list-donor-
screening-assays-infectious-agents-and-hiv-diagnostic-assays 
 
If additional supplemental test(s) for anti-HCV are licensed, approved, or cleared in the future, 
FDA may update these recommendations as warranted. 
 
 
 
4  Contains Nonbinding Recommendations 
 
IV.  IMPLEMENTATION AND REPORTING 
 
Blood establishments should report the implementation of the recommendations in this guidance 
as follows:   
 
1.  Blood establishments that implement the recommendations in this guidance without 
modification and in their entirety:  
 
a.  We consider the implementation of the recommendations in this guidance without 
modification and in their entirety to be a minor change, because FDA has determined 
that implementation in this manner has a minimal potential 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.  Licensed blood establishments must 
report this change to FDA in the annual report under 21 CFR 601.12(d), noting the 
date the method or process was implemented.   
 
b.  Unlicensed blood establishments are not required to report this change to FDA.  
 
2.  Blood establishments that wish to implement an alternative algorithm for further testing 
donations that are repeatedly reactive on a licensed screening test for anti-HCV: 
 
a.  We consider the implementation of an alternative testing algorithm to be a major 
change with a substantial potential 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.  Therefore, licensed blood establishments must submit a 
Prior Approval Supplement (PAS) to FDA under 21 CFR 601.12(b).  We recommend 
the supplement include the following:  
 
i.  Form FDA 356h Application to Market a New or Abbreviated New Drug or 
Biologic for Human Use which may be obtained at  
https://www.fda.gov/about-fda/reports-manuals-forms/forms 
ii.  A cover letter describing the request and the contents of the submission.  
iii. A written SOP describing the testing algorithm or process.  
 
b.  If an unlicensed blood establishment implements an alternative testing algorithm, it 
must be adequate and appropriate (21 CFR 610.40(e)).  Although the unlicensed 
blood establishment is not required to report this change to FDA, we recommend the 
blood establishment submit the contents of the algorithm to FDA for review and 
comment before implementing.    
5  Contains Nonbinding Recommendations 
 
V.  REFERENCES 
 
1.  Guidance for Industry:  Lookback for Hepatitis C Virus (HCV):  Product Quarantine, 
Consignee Notification, Further Testing, Product Disposition, and Notification of 
Transfusion Recipients Based on Donor Test Results Indicating Infection with HCV, updated 
December 2010.  
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/lookback-
hepatitis-c-virus-hcv-product-quarantine-consignee-notification-further-testing-product 
2.  Nucleic Acid Testing (NAT) for Human Immunodeficiency Virus Type 1 (HIV-1) and 
Hepatitis C Virus (HCV):  Testing, Product Disposition, and Donor Deferral and Reentry; 
Guidance for Industry, updated December 2017. 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/nucleic-acid-
testing-nat-human-immunodeficiency-virus-type-1-hiv-1-and-hepatitis-c-virus-hcv-testing 
3.  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, October 2004. 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/use-nucleic-
acid-tests-pooled-and-individual-samples-donors-whole-blood-and-blood-components-0 
4.  Denniston MM, Jiles RB, Drobeniuc J, Klevens RM, et al. Chronic hepatitis C virus infection 
in the United States, National Health and Nutrition Examination Survey, Ann Intern Med 
2014;160:293-300.  
5.  Smith BD, Morgan RL, Beckett GA, Falck-Ytter Y, et al. Recommendations for the 
identification of chronic hepatitis C virus infections among persons born during 1945-1965, 
MMWR 2012;61(RR04):1-8. https://www.cdc.gov/mmwr/preview/mmwrhtml/rr6104a1.htm.  
6.  Steele WR, Notari EP, Nelson D, Anderson S, Glynn S, et al. Prevalence of human 
immunodeficiency virus, hepatitis B virus and hepatitis C virus in more than fifty-percent of 
the United States blood supply:  The Transfusion-Transmissible Infections Monitoring 
System (TTIMS); Transfusion 2017;57(Suppl. 3):46A (abstract). 
7.  Kleinman SH, Stramer SL, Brodsky JP, Cagilioti S, Busch MP.  Integration of nucleic acid 
amplification test results into hepatitis C supplemental serologic testing algorithms: 
implications for donor counseling and revision of existing algorithms.  Transfusion 2006; 
46:695-702.  
8.  Centers for Disease Control and Prevention (CDC).  Testing for HCV infection: an update of 
guidance for clinicians and laboratorians.  MMWR Morb Mortal Wkly Rep.  2013; 62:362-
365. 
9.  Busch MP, Glynn SA, Stramer SL, Orland J, et al. Correlates of hepatitis C virus (HCV) 
RNA negativity among HCV-seropositive blood donors.  Transfusion 2006; 46:469-474. 
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