Guidance for Industry

Revised Recommendations Regarding

Invalidation of Test Results of Licensed

and 510(k) Cleared Bloodborne

Pathogen Assays Used to Test Donors

Additional copies are available from:

Office of Communication, Training and Manufacturers Assistance (HFM-40)

1401 Rockville Pike, Rockville, MD 20852-1448

(Tel) 1-800-835-4709 or 301-827-1800

http://www.fda.gov/cber/guidelines.htm

U.S. Department of Health and Human Services

Food and Drug Administration

Center for Biologics Evaluation and Research (CBER)

July 2001
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TABLE OF CONTENTS

I. INTRODUCTION ................................................................................................................. 1

II. BACKGROUND.................................................................................................................... 1

III. SPECIFIC RECOMMENDATIONS................................................................................... 3

A. CLIA REQUIREMENTS FOR CONTROL REAGENTS................................................................... 3

B. INVALIDATION OF TEST RESULTS BASED ON TEST KIT PACKAGE INSERT................................. 3

C. INVALIDATION OF TEST RESULTS BASED ON CLIA REGULATIONS .......................................... 4

IV. REFERENCES ...................................................................................................................... 5
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GUIDANCE FOR INDUSTRY

Revised Recommendations Regarding Invalidation of Test Results of

Licensed and 510(k) Cleared Bloodborne Pathogen Assays Used to

Test Donors

This guidance document represents FDA’s current thinking on invalidating test results based on

the Clinical Laboratory Improvement Act of 1988 (CLIA) required control reagents. It does not

create or confer any rights for or on any person and does not operate to bind FDA or the public.

 An alternative approach may be used if such approach satisfies the requirements of the

applicable statutes and regulations.

I. INTRODUCTION

To reduce infectious disease transmission by blood and blood products, donor samples from blood

donations are tested for markers of pathogenic bloodborne infections, including antibodies, antigens,

and nucleic acids that may indicate the presence of etiologic agents such as human immunodeficiency

virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell lymphotropic virus

(HTLV), cytomegalovirus (CMV), syphilis, and others. The validity of screening and supplemental

(confirmatory) test assay results is determined by the performance of test kit manufacturer supplied

reagents labeled as Acontrols@, used in accordance with the test kit instructions. Historically, FDA has

not required additional assay-specific quality control procedures beyond those documented in the test

kit package inserts. FDA now recommends implementation of quality assurance and quality control

procedures that involve use of external control reagents beyond those provided by the test kit

manufacturer in order to contribute to overall testing accuracy and therefore to blood safety. This

guidance finalizes the draft guidance entitled AGuidance for Industry-Revised Recommendations for the

Invalidation of Test Results When Using Licensed and 510(k) Cleared Bloodborne Pathogen Assays to

Test Donors@ that was announced in the Federal Register of September 1, 1999 (64 FR 47847). This

guidance also supercedes the January 3, 1994, guidance document on this topic (Ref. 1). These

invalidation procedures should be part of blood establishments= comprehensive quality assurance

programs (Ref. 2) and documented in blood establishments= Standard Operating Procedures (SOPs).

II. BACKGROUND

On May 28, 1992, September 25, 1992, September 26, 1996, and December 13, 1996, the Blood

Products Advisory Committee (BPAC) discussed in open public meetings circumstances under which

test results may be invalidated, including actions based on the use of additional quality control reagents

in donor testing. These discussions included general aspects of quality assurance related to bloodborne

pathogen testing, invalidation procedures based on test kit product insert requirements, and additional
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procedures to invalidate test results based on criteria developed by the user. FDA issued guidance on

this subject on January 3, 1994 (Ref. 1). In an effort to further clarify FDA=s guidance and to help

ensure compliance with the Clinical Laboratory Improvement Act of 1988 (CLIA), at the December

13, 1996, BPAC meeting, the FDA, the Centers for Disease Control and Prevention (CDC), and the

Health Care Financing Administration (HCFA) discussed elements of CLIA. CDC described the

differences between a calibrating reagent and a reagent serving as an assay control. Based on CLIA

regulations, CDC stated that if a test kit supplied Acontrol@ reagent is used to calculate the assay cutoff,

regardless of its container label title, the reagent serves the function of a calibrator and may not serve

simultaneously as a control reagent. In such instances, an additional reagent should be included in the

assay run to meet CLIA control reagent requirements.

For example, if a test kit includes two reagents labeled as negative control and positive control

respectively, and the negative control reagent is used to calculate the assay cutoff, the negative control

serves as a calibrator and may not also serve as an assay control reagent. Since CLIA regulations

require use of a minimum of two controls (one negative and one positive) each day of testing, a negative

control from an external source should be included in the run to meet CLIA requirements. Such

external controls may be developed in-house or procured, and the blood establishment must develop

acceptance/rejection criteria for their use. For further information, blood establishments should refer to

CLIA regulations under Title 42, Code of Federal Regulations (CFR), Part 493, or contact the State

Survey Agency in the state where the laboratory is located or the accrediting organization with which the

laboratory is affiliated.

It is important to note that the test kit package insert instructions must continue to be followed in order

for runs to be considered valid for donor testing. 21CFR 606.65(e). Accordingly, externally supplied

reagents should not be substituted for test kit supplied reagents to calculate the assay cutoff, but may be

used additionally to satisfy CLIA requirements.

As a result of discussions held during BPAC meetings, and additional discussions among CDC, HCFA,

and FDA, FDA has developed revised recommendations for blood establishments, integrating current

CLIA provisions for invalidating test results based on CLIA designated control reagents. 

Because blood safety relies significantly on the accuracy of testing, user-developed acceptance/rejection

rules should be designed only with the objective of enhancing blood safety. Accordingly, rules

developed by blood establishments to meet either CLIA requirements or other in-house developed

requirements should only be used to invalidate nonreactive results. Reactive results obtained from

a screening test or confirmatory/supplemental test run that satisfies the test kit=s criteria for

acceptance should not be invalidated. Such reactive results remain as the initial test of record, and,

in the case of a screening test, these specimens should be retested in duplicate as specified in the test kit

manufacturer=s instructions. The duplicate retest of a reactive specimen (duplicate rescreen) is designed

to reduce the probability of obtaining a false positive result for a truly negative, but non-specifically

reactive specimen. A donor sample with an invalidated nonreactive result may be retested singly.
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III. SPECIFIC RECOMMENDATIONS

These recommendations for procedures to invalidate test results, based on: 1) test kit package insert

rejection criteria; and 2) CLIA provisions, are applicable to all blood establishments and should be

included in their SOPs.

A. CLIA Requirements for Control Reagents

CLIA regulations require control reagents to be used according to 42 CFR 493. If a

bloodborne pathogen test kit uses any of its manufacturer supplied reagents to serve as a

calibrator function, i.e., either or both of the test kit controls (negative or positive) are used to

calculate the assay cutoff, then CLIA regulations require that (an) additional Acontrol@ reagent(s)

be included in each run (see table below). Such reagents may be procured or developed inhouse. In any case, prior to placing the additional controls in routine use, each lot of such

reagents should have: 1) a known dating period, i.e., established stability (supplied by a control

reagent manufacturer or established by the user on in-house developed control reagent); and 2)

known performance parameters, i.e., specifications for acceptance. Prior to implementation,

additional control reagents should be qualified, i.e., evaluated for suitability by the user applying

generally accepted laboratory quality control procedures to establish acceptance/rejection

criteria, to minimize possible incompatibilities that may exist with particular test kits before

putting control reagents into use.

TEST KIT

REAGENT(S)

USED IN CALCULATION OF

THE CUTOFF?

ADDITIONAL CONTROL

REAGENT REQUIRED

Negative Control Yes Yes (Negative Control)

Negative Control No No

Positive Control Yes Yes (Positive Control)

Positive Control No No

Positive and Negative

Control

Yes Yes (Positive and Negative Controls)

B. Invalidation of Test Results Based on Test Kit Package Insert

All results, both reactive and non-reactive, must be considered invalid when it has been

determined that the assay, or part of the assay, has not been performed according to the test kit

package insert instructions, (e.g., improper procedure, compromised reagent, faulty equipment)

or that the test kit package insert defined run acceptance criteria have not been met, (e.g.,

controls out of specified range) [21 CFR 606.65(e)]. Whenever such situations occur, test

results must be invalidated and the subsequent valid assay becomes the initial test of record. 

The determination of nonadherence to the test kit package insert requirements may be a result
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of an investigation into an unexplained discrepancy, a routine review of the assay data, or

concerns raised by a blood establishment's quality control procedures. For example, an

investigation of procedures may be performed in response to unexpected external control

reagent values or failure to meet the blood establishment's additional statistical acceptance

criteria for rate of reactivity. Documentation of each incident of invalidation should include the

basis for invalidation, the details of an investigation, including records of supervisory oversight,

the outcome of the investigation, and, if indicated, any corrective action taken. All of these

actions should be taken prior to repeat testing of donor samples.

C. Invalidation of Test Results Based on CLIA Regulations

When the test kit package insert instructions have been met, but CLIA control requirements

have not been met: 1) any reactive results must not be considered invalid and 2) nonreactive results should be invalidated. All specimens giving initially reactive results should

be re-tested in duplicate, or as the package insert directs [21 CFR 606.65(e)]. If either of the

repeated duplicates is reactive, the unit should be classified as repeatedly reactive and no further

repeat testing should be performed. Specimens giving repeatedly reactive test results then

should be further assayed and reported as recommended in FDA Recommendations to Blood

Establishments (Ref. 3, 4, 5, 6, 7, 8, 9) regarding viral marker testing of donations. Invalidated

non-reactive specimens should be re-tested singly and those results, if valid, should become the

test of record.

Invalidation criteria pursuant to CLIA requirements should be based on site-user developed

rules, e.g., population reactive prevalence data, or standard deviation based limits, which may

be derived from generally accepted clinical laboratory quality control algorithms. 

Documentation for all incidents of invalidation should include the basis for invalidation, the

details of an investigation, including records of supervisory oversight, the outcome of the

investigation, and, if indicated, any corrective action taken. All of these actions should be taken

prior to repeat testing of donor samples.

Reactive results should only be invalidated when an assay run either fails to meet test kit

package insert acceptance criteria or the assay was not performed in accordance with the test

kit package insert. Accordingly, invalidation of reactive test results should not be based solely

on either CLIA required quality control reagent performance or site supplemental quality control

rules. Examples of circumstances when reactive results should not be invalidated include CLIA

required control reagent values exceeding site established quality control acceptance limits, or

reactive rates exceeding site defined upper limits.
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IV. REFERENCES

1. FDA Recommendations for the Invalidation of Test Results When Using Licensed Viral Marker

Assays to Screen Donors, 3 January 1994, Center for Biologics Evaluation and Research,

FDA.

2. Guideline for Quality Assurance in Blood Establishments, 11 July 1995, Center for Biologics

Evaluation and Research, FDA.

3. Revised Recommendations for Testing Whole Blood, Blood Components, Source Plasma and

Source Leukocytes for Antibody to Hepatitis C Virus Encoded Antigen (Anti-HCV), 23 April

1992, Center for Biologics Evaluation and Research, FDA.

4. Revised Recommendations for Testing Whole Blood, Blood Components, Source Plasma and

Source Leukocytes for Antibody to Hepatitis C Virus Encoded Antigen (Anti-HCV), 5 August

1993, Center for Biologics Evaluation and Research, FDA.

5. Additional Recommendations for Testing Whole Blood, Blood Components, Source Plasma

and Source Leukocytes for Antibody to Hepatitis C Virus Encoded Antigen (Anti-HCV), 16

May 1996, Center for Biologics Evaluation and Research, FDA.

6. Recommendations for the Management of Donors and Units that are Initially Reactive for

Hepatitis B Surface Antigen (HBsAg), 2 December 1987, Center for Biologics Evaluation and

Research, FDA.

7. Revised Recommendations for the Prevention of Human Immunodeficiency Virus (HIV)

Transmission by Blood and Blood Products, 23 April 1992, Center for Biologics Evaluation

and Research, FDA.

8. FDA Recommendations Concerning Testing for Antibody to Hepatitis B Core Antigen (AntiHBc), 10 September 1992, Center for Biologics Evaluation and Research, FDA.

9. HTLV-I Antibody Testing, 29 September 1989, Center for Biologics Evaluation and Research,

FDA.