Establishing the Performance 
Characteristics of In Vitro 
Diagnostic Devices for the 
Detection of Antibodies to Borrelia 
burgdorferi 
Guidance for Industry and Food 
and Drug Administration Staff
 
 
Document issued on: March 28, 2013 
The draft of this document was issued on January 5, 2011.
 
. 
 
For questions regarding this document contact Prasad Rao, Ph.D. at 301-796-6203 or by 
email at prasad.rao@fda.hhs.gov. 
        U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Devices and Radiological Health 
Office of In Vitro Diagnostics and Radiological Health  
Division of Microbiology Devices 
 
               Contains Nonbinding Recommendations 
 
Preface 
 
Public Comment 
You may submit written comments and suggestions at any time for Agency consideration 
to the Division of Dockets Management, Food and Drug Administration, 5630 Fishers 
Lane, rm. 1061, (HFA-305), Rockville, MD, 20852.  Submit electronic comments to 
http://www.regulations.gov.  Identify all comments with the docket number listed in the 
notice of availability that publishes in the Federal Register.  Comments may not be acted 
upon by the Agency until the document is next revised or updated. 
Additional Copies 
Additional copies are available from the Internet.  You may also send an e-mail request to 
dsmica@fda.hhs.gov to receive an electronic copy of the guidance or send a fax request to 
301-827-8149 to receive a hard copy.  Please use the document number 1721 to identify 
the guidance you are requesting. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  2               Contains Nonbinding Recommendations 
 
 
Table of Contents 
 
Introduction ....................................................................................................................... 4 
Background ....................................................................................................................... 4 
Scope................................................................................................................................... 5 
Risks to Health .................................................................................................................. 6 
Establishing Performance Characteristics ..................................................................... 6 
A. Controls ..................................................................................................................... 6 
B. Performance Studies ................................................................................................ 6 
C. CLIA Waiver .......................................................................................................... 11 
References ........................................................................................................................ 12 
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 Establishing the Performance 
Characteristics of In Vitro 
Diagnostic Devices for the 
Detection of Antibodies to Borrelia 
burgdorferi 
Guidance for Industry and Food 
and Drug Administration Staff
 
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 FDA staff responsible for 
implementing this guidance.  If you cannot identify the appropriate FDA staff, call the 
appropriate number listed on the title page of this guidance.  
   Introduction 
FDA is issuing this guidance to provide industry and agency staff with recommendations 
for studies to establish the analytical and clinical performance of in vitro diagnostic 
devices (IVDs) intended for the detection of antibodies to Borrelia burgdorferi.  These 
devices are used to aid in the diagnosis of Lyme disease.   
FDA's guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe the Agency'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 Agency guidances means that 
 
something is suggested or recommended, but not required.
 Background  
This document recommends studies for establishing the performance characteristics of in 
vitro diagnostic devices for the detection of antibodies to B. burgdorferi in human serum, 
plasma, and blood.  Serological testing for antibodies to B. burgdorferi in Lyme disease 
diagnostics is a two step procedure [Ref. 1, 2].   Initial testing is done by an enzyme 
immunoassay (EIA) or immunofluorescent assay (IFA); specimens yielding positive or 
equivocal results are tested further by using a Western immunoblot assay.  Specimens 
negative by a sensitive EIA or IFA do not need further testing.  Results from Western 
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blot assays for antibodies to B. burgdorferi are supplemental rather than confirmatory.  
Two-step positive results provide supportive evidence of exposure to B. burgdorferi, 
which could support a clinical diagnosis of Lyme disease but should not be used as a sole 
criterion for diagnosis.  
A manufacturer who intends to market an in vitro diagnostic device for detection of 
antibodies to B. burgdorferi must conform to the general controls of the Federal Food, 
Drug, and Cosmetic Act (FD&C Act) and, unless exempt, obtain premarket clearance or 
approval prior to marketing the device (sections 510(k), 513, 515 of the FD&C Act; 21 
U.S.C. 360(k), 360c, 360e).  This document is intended to supplement 21 CFR 807.87 
(information required in a premarket notification) and other FDA resources such as 
Premarket Notification 510(k) at: 
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/HowtoMarketYour
Device/PremarketSubmissions/PremarketNotification510k/default.htm.  Guidance on the 
content and format for abbreviated and traditional 510(k)s is available in the guidance 
entitled Format for Traditional and Abbreviated 510(k)s found at: 
http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/Guidanc
eDocuments/ucm084396.pdf.   
Information regarding the use of standards can be found in section 514(c)(1)(B) of the 
FD&C Act (21 U.S.C. 360d(c)(1)(B)), and in the FDA guidance entitled Use of 
Standards in Substantial Equivalence Determinations, at 
http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/Guidanc
eDocuments/ucm073756.pdf.  The Special 510(k) is an option for manufacturers 
considering modifications to their own cleared devices.  Information on how to prepare a 
Special 510(k) is available at: 
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/HowtoMarketYour
Device/PremarketSubmissions/PremarketNotification510k/ucm134573.htm.   
Further information on device testing can be found in the guidance entitled In Vitro 
Diagnostic (IVD) Device Studies  Frequently Asked Questions at, 
http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/Guidanc
eDocuments/ucm071230.pdf, and the guidance entitled Guidance on Informed Consent 
for In Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not 
Individually Identifiable at, 
http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/Guidanc
eDocuments/ucm071265.pdf. 
 
Scope 
This document recommends studies for establishing the performance characteristics of 
EIA or Western blot devices for the detection of antibodies to B. burgdorferi in human 
serum, plasma, and blood.  The following is the product code for B. burgdorferi (Lyme 
disease) Class II devices cleared under 21 CFR 866.3830: 
LSR  Reagent, Borrelia Serological Reagent    
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This document does not apply to B. burgdorferi nucleic acid amplification assays.  Please 
contact the Division of Microbiology Devices in the Office of In Vitro Diagnostics and 
Radiological Health for further information on nucleic acid amplification devices.  
 
Risks to Health 
Lyme disease is a significant public health concern.  It is the most common vector-borne 
infectious disease in North America.  The infectious agent in Lyme disease is the 
spirochete B. burgdorferi.  Definitive diagnosis of B. burgdorferi infection is complicated 
by the varied nature of the clinical symptoms and the overlap of these symptoms with 
numerous other infectious and non-infectious diseases.   
Failure of devices for the detection of B. burgdorferi antibodies to perform as expected, 
or failure to correctly interpret results may lead to incorrect patient management 
decisions and inappropriate public health disease reporting.  In the context of individual 
patient management, a false negative report could lead to a delay or failure to provide 
treatment.  A false positive report could lead to unnecessary or inappropriate treatment.  
Therefore, establishing the performance of these devices and understanding the risks that 
might be associated with their use is critical to their safe and effective use.  
The studies conducted by manufacturers to establish the performance of B. burgdorferi 
antibody detection devices are the basis for determining the safety and effectiveness and 
substantial equivalence of these devices.   
Establishing Performance Characteristics  
A. Controls 
When conducting the performance validation studies described below, we recommend 
that you run appropriate controls every day of testing for the duration of the analytical 
and clinical studies. 
B. Performance Studies 
We recommend you perform the following studies:   
1.  Cross-reactivity 
Specimens known to contain potentially cross reactive antibodies to B.burgdorferi 
should be evaluated.  We recommend that a minimum of ten specimens should be 
tested for each type.  Examples of these include but are not limited to: (1) specimens 
from patients with infections such as tick-borne relapsing fever; syphilis and other 
treponemal infections; rickettsial diseases; ehrlichiosis; babesiosis; leptospirosis; 
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infections with parvovirus B19, influenza viruses, Epstein-Barr virus, 
cytomegalovirus, and H. pylori; and (2) patients with diagnoses that can be confused 
with the late manifestations of Lyme disease such as chronic fatigue syndrome, 
fibromyalgia, rheumatoid arthritis, autoimmune diseases, and multiple sclerosis.  
 
Interference  
We recommend that you characterize the effects of potential interferents on assay 
performance.  Examples of experimental designs, including guidelines for selecting 
interferents for testing, are described in CLSI documents EP7-A2 [Ref. 3].  Potential 
sources of interference can include high levels of compounds normally found in 
serum, such as triglycerides, hemoglobin (i.e., hemolyzed samples), bilirubin, protein, 
and lipids.  We recommend that you state the criteria or level at which non-
interference is determined.  
2.  Precision 
Within-Laboratory Precision/Repeatability  
We recommend that you conduct within-laboratory precision studies for devices that 
include instruments or automated components.  You may perform these studies in-
house, i.e., within your own company. 
We recommend that you test sources of variability (such as operators, days, assay 
runs, etc.) for a minimum of 12 days (not necessarily consecutive), with 2 runs per 
day, and 2 replicates of each sample per run.  These test days should span at least two 
calibration cycles.  The test panel should consist of 3-6 patient samples at three levels 
of antibodies that include:  
  A negative sample: a sample with no analyte such that results of repeated tests 
of this sample are negative 100% of the time.   
  A high negative sample (C  concentration): a sample with an analyte 
5
concentration below the clinical cut-off such that results of repeated tests of 
this sample are negative approximately 95% of the time (and results are 
positive approximately 5% of the time). 
  A low positive sample (C  concentration): a sample with a concentration of 
95
analyte just above the clinical cut-off such that results of repeated tests of this 
sample are positive approximately 95% of the time.  
   A moderate positive sample: a sample with a concentration at which one 
can anticipate positive results approximately 100% of the time (e.g., 
approximately two to three times the concentration of the clinical cut-off). 
CLSI documents EP5-A2 [Ref. 4] and EP12-A [Ref. 5] contain further information 
about designing and performing precision studies.   
 
 
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Reproducibility 
The protocol for the reproducibility study may vary slightly depending on the assay 
format.  As a general guide, we recommend the following protocol: 
  Evaluate the reproducibility of your test at 3 testing sites (for example, two 
external sites and one in-house site). 
  Use a five day testing protocol, including a minimum of two runs per day, 
(unless the assay design precludes multiple runs per day) and three replicates 
of each panel member per run. 
  Each day, have at least two operators at each facility perform the test.  We 
recommend that, for rapid testing or point-of-care (POC)1 devices, you 
include a larger number of devices in your evaluation, in order to best 
represent the settings in which the devices will be used. 
  Use the same sample panel as described in the repeatability study above.   
CLSI document, EP15-A2 [Ref. 6], contains additional information on reproducibility 
study design. 
3. Specimen Collection and Handling Conditions 
We recommend that you substantiate statements in your labeling about specimen storage 
and transport by assessing whether the device can maintain acceptable performance over 
the range of storage times and temperatures recommended to users.  For example, an 
appropriate study would include an analysis of aliquots stored under the recommended 
conditions of time, temperature, or specified number of freeze/thaw cycles.  We 
recommend that you state the criteria for an acceptable range of recoveries under the 
recommended storage and handling conditions as described in the CLSI document, H18-
A [Ref. 7]. 
4. Assay Cut-Off Point 
We suggest that data be furnished to explain how the assay cut-off point was selected and 
established.  If appropriate, information should be provided on the use of an equivocal 
zone for testing.  If data suggests that an equivocal zone is not appropriate, this should be 
explained. 
5. Clinical Performance Studies 
 Current recommendation for Lyme disease serology testing is a two step procedure [Ref. 
1, 2]:   
                                                 
1 Point-of-care tests, also known as bedside or near-patient tests, is a term that encompasses any diagnostic 
testing near the site of patient care regardless of whether the device is intended for use by a trained medical 
professional or by a lay user.  The person conducting the diagnostic testing near the point of care, whether a 
trained medical professional or a lay user, is the caregiver in that instance as that person is the person 
providing care. 
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Step I:  EIA tests for IgM and/or IgG antibodies 
Step II: Western blot tests for IgM or IgG antibodies when EIA tests are positive 
or equivocal 
Since the Borrelia burgdorferi strains from the United States (U.S.) differ markedly from 
Europe and other non-U.S. endemic regions, all antigenic reagents used in Lyme disease 
devices to be marketed in the U.S. should be manufactured from the U.S. strain sensu 
stricto B31 Borrelia burgdorferi spirochete and not from other non-U.S. strains.  This is 
particularly true when whole cell sonicate is used as the antigenic reagent to capture B. 
burgdorferi antibodies in both EIA and Western blot devices.  However, it is acceptable 
to augment antigens from the U.S. strain of B. burgdorferi with recombinant proteins of 
defined length but not total cell lysates from the European species of Borrelia.  This 
applies to first tier EIA tests only and not to Western blot tests for IgM or IgG antibodies.   
Clinical studies are necessary to establish the performance characteristics for both EIA 
and Western blot devices.  Generally, we recommend that performance be assessed in the 
testing environment in which the device will ultimately be used (i.e., clinical laboratory) 
by individuals who will use the test in clinical practice (e.g., trained technologists).  The 
following sets forth what is expected in terms of establishing the performance 
characteristics for each type of device.  
Study Protocol 
We recommend that you develop a detailed study protocol that includes specific 
patient inclusion and exclusion criteria, the type and number of specimens needed, 
directions for use, and a statistical analysis plan that accounts for variances to prevent 
data bias.  You need to provide a statistical justification to support the sample size of 
the study population.  We recommend that you include this and any other relevant 
protocol information in your premarket submission.  
We encourage sponsors to contact the Division of Microbiology Devices to request a 
review of their proposed studies and selection of specimen types prior to study 
initiation.      
Study Sites 
We recommend that you conduct your studies at a minimum of three separate sites, 
one of which may be in-house. Note, we do not believe non-U.S. clinical testing site 
data are appropriate given that different strains are found in other countries.  Clinical 
investigations of unapproved and uncleared in vitro diagnostic devices, including 
diagnostic devices for Lyme disease, are subject to the investigational device 
exemption (IDE) provisions of Section 520(g) of the Federal Food, Drug, and 
Cosmetic Act (21 U.S.C. 360j) and the implementing regulations. You should 
consider how 21 CFR part 812 (IDEs) applies to your particular study and refer to 21 
CFR part 50 (informed consent), and 21 CFR part 56 (institutional review board 
review) for other applicable requirements.  
We recommend that the performance evaluation for devices intended for point-of-
care (POC) use include, at a minimum, one site at a clinical laboratory as well as 2 or 
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more sites representative of non-laboratory settings in which the device is intended to 
be used (e.g., an emergency department).     
 
Study Design 
The following studies should be conducted and submitted with a 510(k) application 
for either a Lyme disease EIA device or a Lyme disease Western blot device.  Study 
designs are similar for both IgM and IgG tests.  
a. Sensitivity Study:  A study consisting of a minimum of 100 well characterized 
clinically or culture confirmed Lyme disease specimens should be 
conducted with the test device.  These archived specimens should contain 
samples from early, early disseminated, and late phases of the disease.  The 
sensitivity of the test device on these specimens should be compared to a 
predicate device.  A pedigree for each characterized patient contributing 
samples for the studies should be included; this pedigree should encompass 
available clinical and laboratory testing information.  
The following breakout of the patient population is suggested for sensitivity 
testing:     
a.  Initial (acute) samples from patients with documented erythema 
migrans (EM) or culture positive disease (<1 month, 1-2 months, 23 
months after symptom onset). 
b.  Convalescent samples from patients with documented EM or culture 
positive results, stratified by time of draw after initial appearance of 
symptoms (3-12 months). 
c.  Testing of known Lyme disease patients with presentations other than 
EM, e.g., neuro-, arthritic, etc., in which the time interval from 
infection to symptom onset may be unknown or more than 1 year.  
Testing of specimens from patients across all ranges is recommended.  
b.Prospective Study:  A study designed to test non-selected, prospectively 
collected consecutive specimens from a minimum of three geographically 
distinct locations within the U.S. should be conducted.  Testing of these 
samples using the test device and a comparison device, should occur at a 
minimum of 3 sites, one of which could be internal.  
i.  For EIA devices:  The results should be compared to a predicate 
device.  All positive and equivocal samples by the test device and the 
predicate device should be tested by an FDA-cleared IgM and/or IgG 
Western blot assay as applicable.     
ii.  For Western blot devices:  The prospective study samples should be 
tested initially by an FDA cleared first-step EIA.  All EIA positive and 
equivocal samples should be tested by the test device and the predicate 
Western blot.  Positive and negative % agreement between the two 
devices should be provided.       
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Interpretation of Western blot results:  The interpretation of Western blot results 
should follow the recommended criteria described by the Centers for Disease Control 
(CDC) [Ref. 1] and the Second National Conference on Serological Diagnosis of 
Lyme Disease [Ref. 2].  An IgM immunoblot is considered positive if two of the 
following three bands are present: 23 kDa (OspC), 39 kDa (BmpA), and 41 kDa 
(Fla).  An IgG immunoblot is considered positive if five of the following 10 bands are 
present: 18 kDa, 23 kDa (OspC), 28 kDa, 30 kDa, 39 kDa (BmpA), 41 kDa (Fla), 45 
kDa, 58 kDa, 66 kDa, and 93 kDa. 
Analytical Specificity Testing:  For the determination of analytical specificity, testing 
of samples from both endemic and non-endemic regions is recommended (minimum 
of 100 samples from each region).  These samples should be obtained from an 
asymptomatic population but should not include pre-screened blood donors.  The 
results, expressed as % positives and % negatives, should be presented separately as 
endemic and non-endemic subjects. 
CDC Reference Panel Testing:  A standard panel of positive and negative specimens 
provided by the CDC for testing Lyme disease detection devices should be tested by 
the new device and the results provided as % agreement with the expected results.  
The data should be stratified by disease stage; early, intermediate, and late and be 
compared to the predicate device.  
  
6. Labeling    
Proposed labeling for the device must comply with the requirements of 21 CFR 
809.10.  As part of meeting these requirements we recommend that your labeling include 
a description of quality control recommendations, which should include a clear 
explanation of what controls and calibrators are to be used with the assay and how often 
they should be used. 
C. CLIA Waiver  
If you are seeking waiver for your device under the Clinical Laboratory Improvement 
Amendments of 1988 (CLIA),2  we recommend that you consult with the Division of 
Microbiology Devices staff regarding the design of specific studies to support the CLIA 
waiver application for your device.  Guidance for Industry and FDA Staff, 
Recommendations: Clinical Laboratory Improvement Amendments of 1988 (CLIA) 
Waiver Applications for Manufacturers of In Vitro Diagnostic Devices, is available at 
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocument
s/ucm079632.htm. 
 
                                                 
2 See 42 U.S.C.  263a(d)(3). 
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References  
1.  CDC. Recommendations for test performance and interpretation from the Second 
National Conference on Serologic Diagnosis of Lyme Disease. MMWR 
1995;44:590-591.  
2.  Association of State and Territorial Public Health Laboratory Directors and the 
Centers for Disease Control and Prevention. Recommendations. In: Proceedings 
of the Second National Conference on Serologic Diagnosis of Lyme Disease 
(Dearborn, Michigan). Washington, DC: Association of State and Territorial 
Public Health Laboratory Directors 1995; 1-5.  
3.  Clinical and Laboratory Standards Institute. 2005. Interference Testing in Clinical 
Chemistry; Approved Guideline. EP7-A2. Clinical and Laboratory Standards 
Institute, Wayne PA. 
4.  Clinical and Laboratory Standards Institute. 2004. Evaluation of Precision 
Performance of Quantitative Measurement Methods; Approved Guideline. EP5-
A2. Clinical and Laboratory Standards Institute, Wayne PA. 
5.  Clinical and Laboratory Standards Institute. 2002. User Protocol for Evaluation of 
Qualitative Test Performance; Approved Guideline. EP12-A. Clinical and 
Laboratory Standards Institute, Wayne PA. 
6.  Clinical and Laboratory Standards Institute. 2006. User Verification of 
Performance for Precision and Trueness; Approved Guideline. EP15-A2. Clinical 
and Laboratory Standards Institute, Wayne PA. 
7.  Clinical and Laboratory Standards Institute. 1990.  Procedures for Handling and 
Processing of Blood Specimens; Approved Guideline. H18-A. Clinical and 
Laboratory Standards Institute, Wayne PA.  
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