This guidance was written prior to the February 27, 1997 implementation of FDA's 
Good Guidance Practices, GGP's. It does not create or confer 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 statute, regulations, or both. 
This guidance will be up dated in the next revision to include the standard elemnt s of GGP 's .  DRAFT  Original vers., May 15, 1992 
REVIEW CRITERIA FOR PREMARKET APPROVAL OF IN VITRO DIAGNOSTIC 
DEVICES -FOR- D-ET ECTIONO F ANTIBODIES -TO  PARVOVT-EKB ~~ 
This is a flexible document representing the current major 
concerns and suggestions regarding -in -vit ro diagnostic devices 
employing immunochemical or other methodologies for detection of 
antibodies to Parvovirus B19 (B19) in human serum or plasma 
specimens.  It is based on 1) current basic science, 2) clinical 
experience, and 3) the Safe Medical Devices Act of 1990 (SMDA) 
and FDA regulations in the Code of Federal Regulations (CFR). 
As advances are made in science and medicine, these review 
criteria will be re-evaluated and revised as necessary to 
accommodate new knowledge. 
PURPOSE:  The purpose of this document is to provide guidance on 
information to present to the Food and Drug Administration (FDA) 
before a device to detect antibodies to B19 in serum or plasma 
specimens may be approved for marketing.  This document is an 
adjunct to the CFR and the FDA 87-4214 Premarket Approval (PMA) 
Manual. 
DEFINITION:  This generic type device is intended for use in 
clinical laboratories as an -in -vit ro diagnostic test for 
qualitative or semi-quantitative measurement of antibodies to 
B19 in human serum or plasma by immunochemical and other 
methodologies.  A PMA submission must show the device has 
clinical utility and that there is reasonable assurance of its 
safety and efficacy. 
In addition to this guidance, refer to the National Committee 
for Clinical Laboratory Standards (NCCLS), "Specifications for 
Immunological Testing for Infectious DiseasesM1.  The NCCLS 
document may be used for definitions of terms used in this 
guidance document. 
PRODUCT CODE(S):  To be assigned 
CLASSIFICATION:  I11 
PANEL:  MICROBIOLOGY (83) 
REVIEW REQUIRED:  Premarket Approval (PMA) 
I.  CLINICAL INDICATION/SIGNIFICANCE/INTENDED  -USE  
Provide a concise discussion to include the following as 
appropriate.  Support the discussion with key literature 
citations. 
A.  Background description of the virus and infectious 
process(es). 
B.  Description of etiology fo;  the disease syndromes for which 
this device was developed. 
C.  Salient concerns of the medical community including relevant  DRAFT  Original vers., May 15, 1992 
medical/societal  issues that may impact the review process 
or possibly the development of public policy. 
D.  Significance of false positive and false negative results. 
E.  Historical summary of all test methodologies used to detect 
antibodies to B19. 
11.  DEVICE DESCRIPTION 
Key issues in the PMA review of a new device are the specific 
intended use (the analyte detected, the clinical utility, and the 
indications for use), the type of specimen tested, and the 
technology utilized.  The following descriptive information must 
be included to adequately characterize the new -in -vit ro device. 
Appropriate literature references that have been subjected to peer 
review should be attached. 
A.  Intended -Use.  
Describe the intended use based on the technology/methodology 
employed in the device.  The following questions should be 
addressed: 
1.  what patient populations should be tested? 
2.  What are the conditions and limitations for use of the 
device when used to diagnose or manage a specific 
syndrome? 
3.  What is the clinical utility of the device in specific 
patient populations? 
B.  Detailed Principle -of -the- Te st Methodology. 
Discuss the principle of the test methodology(ies).  For new 
types of technologies, provide information to substantiate 
application of the methodology to the detection of specific 
antibodies.  Cite literature references where appropriate. 
If available, furnish copies of appropriate scientific 
references for any recombinant or synthetic protein utilized. 
~f scientific references are not available, discuss how the 
antigen was determined to be representative of the native 
antigen.  Include a complete description of the following 
components if appropriate: 
1.  Any pretreatment procedure. 
2.  Antigen utilized in the assay. 
a.  If a native antigen, from what source was the 
antigen obtained. 
b.  If a recombinant antigen or synthetic peptide 
(oligonucleotide), from what source was the native  DRAFT  Original vers., May 15, 1992 
antigen derived and what nucleic acid or protein 
sequence was used to prepare the recombinant. 
3.  Define antisera used in the assay. 
a.  Specify the species in which antisera was produced. 
b.  If a native antigen was used as the immunogen, 
identify the source from which the antigen was 
obtained. 
c.  If a recombinant antigen was used as the immunogen, 
furnish the source of the native nucleic acid and 
provide the sequence for the derived recombinant. 
d.  Explain how the specificity of the antisera  was 
determined. 
4.  Enzymatic, fluorescent, or other substrate used to 
detect the antigen-antibody complexes. 
5.  Determination of the cut-off value(s) or endpoint(s) for 
the assay. Provide validation data as described in 
I11 .A.l. 
6.  Controls/calibrators  included in the assay kit and what 
aspects of the procedure are verified. 
Quality control material should be representative of and 
correlate with the intended use and clinical utility of 
the device.  In addition to the specific requirements 
listed below, the manufacturer should refer to the 
current FDA CLIA '88 Quality Control Guidance. 
Qualitative assay: 
a.  At a minimum, include in the device or make 
available two controls (positive and negative) in 
the same matrix as test specimens indicated for use 
with the assay.  Controls should be within a 
statistically significant range of the cut-off. 
This range should be appropriate for the 
statistical method used to determine the cut-off. 
Provide documentation and justification for the 
value selected for the control. 
b.  Make recommendations and justify the frequency 
of testing control material. 
Semi-quantitative assay: 
In addition to the controls and recommendations listed 
for a qualitative assay, include a control at the upper 
end of the linear range with a known expected value. 
7.  Any additional reagents or methods which contribute to  DRAFT  Original vers., May 15, 1992 
the effectiveness of the device. 
8.  Collection and transport materials provided in the kit 
or recommended for use. 
9.  Software elements and dedicated instrumentation that are 
responsible for specimen handling and/or  that are used 
to calculate assay results.  See requirements for Minor 
- 
Level of concern in Reviewer Guidance for Computer 
Controlled Medical Devices Undergoing 510(k) Review 
(available from the Division of Small Manufacturers 
Assistance).  Furnish the following for dedicated 
instrumentation and software elements: 
a.  Reference premarket notification [510(k)] 
submission  for any dedicated instrument. 
b.  Algorithms used to calculate results in either 
dedicated or non-dedicated instruments. 
c.  Mathematical curve-fitting method(s) used when 
results are calculated non-manually. 
C.  Merits -and  Limitations -of -the  Methodology(s). 
Discuss the merits and limitations/advantages/disadvantages 
of the test methodology(ies) of the new device. 
D.  Specimen Type(s). 
List all specimen types (matrices) indicated for use with the 
device. 
111. SPECIFIC PERFORMANCE CHARACTERISTICS 
The FDA requests different types and amounts of data and 
statistical analyses to market in-vitro diagnostic devices.  The 
amount and types of data requested depend on the intended use 
and the technological characteristics of the new device.  The 
data and statistical evaluation should be sufficient to 
determine if the device is safe and effective for all claimed 
specimen type(s).  Additional data may be necessary to 
' 
substantiate certain claims of intended use or clinical 
significance, and to validate use of a new technology. 
Clearly document all protocols for -in -vit ro testing.  Present 
test data with analyses and conclusions.  Summarize results and 
include explanations for unexpected results and any additional 
testing performed.  When appropriate, charts (scattergrams, 
histograms, ROC curves, etc.) may be used as part of analyses 
and conclusions.  Furnish all raw laboratory data. 
Submission of the following data is required to determine the 
device's  ability to detect B19-specific antibodies: 
A.  Analytical Laboratory Studies.  DRAFT  Original vers., May 15, 1992 
1.  Validation of Cut-off and/or  Calibration Curve. 
Describe the rationale for determination of the assay 
cut-off(s).  Furnish descriptive information and 
laboratory data to show how the cut-off (CO) 
(distinction between positivity and negativity) was 
determined for the assay. 
a.  Define the population used, including the following 
information: 
(1). geographical area(s) from which the population 
was derived; 
(2). number of samples comprising the population, 
with samples summarized according to gender 
and age groups in decades. 
(3)  the month of the year during which sample was 
collected. 
(4). graphical (e.g., histogram, scattergram, etc.) 
representation of population characteristics. 
b.  Define the statistical method used to determine the 
. 
cut-of f 
c.  Present ROC analysis of cut-off selection and other 
graphical representations as appropriate. 
d.  Define the basis for the equivocal zone. 
e.  If semi-quantitative, perform appropriate clinical 
studies to show the relationship of results to the 
diagnostic stage (e.g., early, acute, waning 
infection) for which value ranges have been 
established.  For each diagnostic stage, present 
results from a minimum of 10 patients. 
f.  For devices which determine interpolated values 
(e.g., ELISAS),  verify the accuracy and working 
range of the calibration curve used by serially 
diluting patient samples. 
2.  Establish the prevalence of the analyte in two diverse 
asymptomatic populations using the specified CO. 
a.  Assay a statistically significant number of 
specimens which are representative of the intended 
use, clinical utility, and matrix of the specimens. 
b.  Summarize the distribution of males and females 
according to age groups in decades, geographical 
area, month of year specimens were collected, and 
the number of positive, negative, and equivocal 
results.  DRAFT  Original vers., May 15, 1992 
3.  Assay Specificity 
a.  Perform cross-reactivity studies with sera 
containing relatively high titers against rubella, 
rubeola (measles), mumps, influenza A, influenza B, 
parainfluenza, HSV1, HSV2, CMV, EBV, VZV, and 
Mycoplasma pneumoniae.  Test sera from five (5) 
patients with each disease; the predominate 
antibody class should be the class measured in the 
device.  Furnish the antibody "titer1'o f the 
potential cross-reactant samples and the method 
used to determine the "titer". 
b.  If the antigen utilized in the device is a 
recombinant, test sera containing antibodies 
against the organism in which the vectors were 
induced for cross-reactivity with the organism. 
c.  If the antisera utilized in the device were 
produced by using a recombinant as the immunogen, 
test sera containing antibodies against the 
organism in which the vectors were induced for 
cross-reactivity with the antisera. 
d.  For immunoglobulin class-specific devices, test ten 
sera positive in moderate to high levels for 
specific antibody class other than what is detected 
in the device for type specificity (i.e., if the 
assay is for IgG antibody then test ten sera which 
are positive for IgM antibody to show the device 
will not detect specific IgM antibody).  Retest 
following removal of IgM or IgG. 
NOTE:  Serum samples may be artificially produced; 
describe method of preparation. 
e.  If an absorbent is used to remove interference of 
RF, ANA, and specific IgG, document the amount of 
IgG removed in mg/dL  or mg/sample  volume.  State 
this value in the LIMITATIONS section of the 
package insert. 
f.  If device is for the detection of IgM antibodies 
the following studies are required: 
(1). Test five sera containing high levels of RF 
and ANA with virus-specific IgG.  Provide 
levels of RF and ANA tested. 
(2). Describe the methods used to determine the 
amounts of virus-specific IgG, RF, and ANA 
present.  Devices used should be legally 
marketed devices. 
4.  Interference Studies.  DRAFT  Original vers., May 15, 1992 
Any potentially cross-reacting or interfering substances 
potentially encountered in specific specimen types or 
conditions should be tested using the assay system, 
e.g.,  storage conditions, hemolysis, lipemia, 
freeze-thawing, etc. 
a.  Verify that recommended storage conditions are 
compatible with the assay.  State the optimal 
conditions based on specimen storage stability 
studies.  Both false positivity and false 
negativity should be evaluated. 
b.  If the use of plasma is claimed, a study with each 
anticoagulant must be performed to show the 
anticoagulant does not interfere with the assay. 
(1). For each anticoagulant, test 10 concurrent 
serum and plasma specimens which have 
reactivity near the CO.  These specimens may 
be artificially produced as long as the matrix 
is maintained and it is noted in the 
submission that the samples were artificially 
created. 
(2). For each anticoagulant, test 10 concurrent 
non-reactive serum and plasma specimens. 
c.  If heating of the specimen is claimed not to 
interfere with the assay: 
(1). Test 10 specimens with reactivity near the CO, 
for each matrix type claimed, heated and not 
heated.  These specimens may be artificially 
produced as long as the matrix is maintained 
and it is noted in the submission that the 
samples were artificially created. 
(2). Test 10 specimens which are non-reactive for 
each matrix claimed, heated and not heated. 
5.  Reproducibility 
The National Committee for Clinical Laboratory Standards 
(NCCLS) recommends an analysis of variance experiment 
that permits estimatian of within-run and total standard 
deviations (SD).2  See the NCCLS Guideline for 
recommended data collection formats and calculations. 
Perform separate calculations for each specimen tested 
for within-run and total precision. 
Test six to ten patient blinded sera with varying 
degrees of reactivity plus controls supplied with device 
in triplicate on three different days at three 
laboratory sites (6-10 sera tested X  3 X 3 days X 3 
sites).  One testing site may be in-house.  DRAFT  Original vers., May 15, 1992 
For calculated endpoint tests (e.g., ELISAs), present 
coefficients of variation for each set of values for 
with-in run and total precision, using absorbance values 
and reporting units defined in the test procedure. 
For single endpoint assays, provide percentage of 
results that are negative, borderline/equivocal,  or 
positive for each set of tests. 
If dedicated instrumentation is used in specimen 
handling, or reading and interpreting results, use a 
different instrument at each site.  If non-dedicated 
instruments are used, state specifications of 
instrument(s) used at each site. 
6.  Stability 
Document stability from three different manufactured 
lots that represent real time studies.  Accelerated 
stability studies are acceptable as interim data only. 
Include testing to show that the reagents are stable 
under variable shipping temperatures. 
B.  Clinical Studies. 
Clinical studies provide data on the ability of the system to 
accurately detect antibodies to B19.  It should be 
demonstrated that the performance of the device is safe and 
effective when used as an aid in the diagnosis of specific 
B19 infections. 
Provide the names and telephone numbers of principal 
investigators and sites at which testing was performed. 
Clinical testing should be performed by at least three 
independent investigators at separate independent locations 
not affiliated with the manufacturer.  Identify the clinical 
laboratory sites by institutional name and address; include 
the name, title, and phone number of the responsible 
investigators at each site. 
The tests should be performed on an adequate number of 
positive and negative clinical specimens (following 
collection, storage, and testing instructions recommended in 
the package insert) from a population consistent with the 
intended use of the device.- The sensitivity and specificity 
calculated from this population should be stated in the 
PERFORMANCE CHARACTERISTICS section of the package insert. 
1.  Test samples submitted to the clinical laboratory for 
the rule out of illness associated with rash or other 
symptoms of B19 infection. 
2.  Clinical confirmation of infection may be done by 
clinical diagnosis, immune electron microscopy, or 
other standard methods.  DRAFT  Original vers., May 15, 1992 
a.  Clinical diagnosis may be based on the following 
criteria: 
(1). documented contact with infected individuals 
during a community outbreak (usually April 
through May). 
(2). typical "slapped-cheek" rash which during 
clearing may take on a lacelike appearance on 
the trunk of the body. 
(3). symmetric polyarthropathy. 
(4). absence of increased markers to other 
illnesses associated with rash (measles, 
rubella, etc.). 
(5). reticulocytopenia and a fall in hemoglobin 
concentration 
(6). bone marrow cytology. 
For pediatric patients (1) and/or  (2) is 
considered diagnostic.  For immunocompetent 
adults, (I), (2) (with or without trunk rash), and 
(3) is diagnostic.  When the symptomatology is not 
diagnostic, at least two of the criteria (1-6) 
must be documented for establishing the diagnosis. 
b.  If other standard testing methods are used, the 
antigen should be different than the antigen 
present in the device.  This additional testing 
may be performed at the manufacturervs or other 
site(s).  Note in the submission where this 
testing was performed.  Provide a description of 
the antigen used!  criteria for positivity and 
negativity, and quality control performed. 
Furnish copies of any pertinent scientific 
references for the testing method with the 
submission. 
3.  As an option, results obtained from a well-documented 
panel of  sera such as the CDC panel or a panel 
established by the manufacturer may be included with 
the submission.  If a manufacturerrs sera panel is 
used, furnish the criteria for defining the diagnosis 
and/or  disease stage of the patients from whom samples 
were obtained.  This study may be conducted at the 
manufacturer's  facility or at a clinical laboratory 
site. 
4.  Test sera from documented illnesses which produce 
symptoms similar to B19 illness.  Provide the assay 
results, final diagnosis, and how the diagnosis was 
determined with the data.  Examples of diseases to 
include are:  DRAFT  Original vers., May 15, 1992 
a.  Kawasaki disease 
b.  Aplastic anemia caused by other than B19 infection 
c.  Thyroiditis 
d.  Hemolytic uremic syndrome 
e.  Rheumatoid arthritis 
f.  Lupus erythematosus 
g.  Contact dermatitis and other diseases which may 
include a rash (e.g., Lyme disease, Rocky Mountain 
spotted fever, syphilis, meningococcal disease, 
streptococcal infections, toxoplasmosis, etc.) 
5.  Additional testing of sera from a small group of 
chronically B-19-infeyted patients may be appropriate 
depending on the intended use. 
IV.  LABELING CONSIDERATIONS 
The following are additional details for some of the points in the 
statute [502(f)(l)]  and regulations [21 CFR S 809.10(b)]. 
A.  -The  Intended -Use  Statement 
The intended use statement should be a concise description of 
the essential information about the product.  It should 
communicate the following information: 
1.  Test methodology. 
2.  Whether the assay detects a specific antibody class. 
3.  Indications for use. 
These conditions for use may be addressed further in 
either the Summary and Explanation, Limitations, or 
Performance Characteristics section of the package 
insert. 
f 
4.  What specimen source(s) may be tested. 
5.  If the assay is to be used only with special 
instrumentation. 
C.  Specimen Collection -and  Handlinq 
1.  State the type of specimen to be collected, and the 
types of collection devices which may be used. 
2.  State the conditions for patient preparation, e.g., 
page 10  DRAFT  Original vers., May 15, 1992 
timing of collection, order of collection, etc. 
3.  Provide adequate directions for sample collection 
and/or  references for appropriate collection 
procedures, e.g.,  textbooks, journals, etc. 
4.  Identify interfering substances or conditions. 
5.  State the specimen storage conditions and stability 
periods. 
D.  Quality Control 
Information provided in a Quality Control section should 
include the following information: 
1.  Provide recommendations for frequency of quality 
control. 
2.  Provide directions for interpretation of the results of 
quality control samples. 
3.  The Quality Control section should conclude with a 
statement similar to the following: "If controls do not 
behave as expected, results are invalid and patient 
results should not be reported". 
4.  Refer to the current CLIA '88  FDA Quality Control 
Guidance for additional information. 
E.  ExpectedResults 
1.  Reference expected prevalence of antibodies to B19 in 
different populations. 
2.  Indicate that prevalence may vary depending on 
geographical location, age, gender of population 
studied, type of test employed, specimen collection and 
handling procedures, clinical and epidemiological 
history of individual patients, etc. 
F.  Limitations -of -the- Te st. 
' 
List important test limitations and all known 
contraindications, with references when appropriate.  The 
following are examples of statements which may apply: 
1.  Anti target-specific viral IgG antibodies may compete 
with the less avid target specific viral IgM antibodies, 
which would decrease the sensitivity of the 
assay.3  Test only indicated specimen types.  Testing of 
other specimen types may result in false negative or 
positive results. 
2.  The predictive value of a positive test decreases when 
prevalence decreases.  Interpretation of positive 
page 11  DRAFT  Original vers., May 15, 1992 
results in a low risk patient population should be made 
with caution.  Usefulness of this test has only been 
established in testing sera from a specific population 
(e.g., adult patients with arthropathies). 
Performance Characteristics: 
Summarize the data upon which the performance characteristics 
are based, e.g.,  clinical sensitivity and specificity 
compared to clinical diagnosis; also include summary of 
reproducibility studies.  Positive and negative predictive 
values should be based on specific populations sampled for 
each disease syndrome.  State the prevalence at each testing 
site.  Also show the effect of prevalence on positive and 
negative predictive values in different test populations. 
1.  Present cross-reactivity studies in a tabular form, 
indicating negative, positive, and 
borderline/equivocal/indeterminate  results for each 
condition/disease. 
2.  Summarize within-run and total reproducibility. 
3.  Present data from clinical studies, using separate 
categories for different patient categories.  All 
borderline/equivocal/indeterminate  results should be 
clearly displayed.  Discrepancies between test and 
clinical diagnosis may be discussed and presented as 
footnotes. 
BIBLIOGRAPHY 
4 
National Committee for Clinical Laboratory Standards. 
Specifications for immunological testing for infectious 
diseases, proposed guideline. 1991. Order code I/LA  18-P. 
National Committee for Clinical Laboratory Standards. 
Evaluation of precision performance of clinical chemistry 
devices, tentative guideline. 1991. Order Code EP5-T2. 
Schmidt NJ, Emmons RW. General principles of laboratory 
diagnostic methods for viral, rickettsia1 and chlamydial 
infections. In: Schmidt NJ, Emmons, RW, eds, Diagnostic 
procedures for viral, rickettsial, and chlamydial 
infections, 6th edition. Washington, DC: American Public 
Health Assoc. 1989:l-35. 
. 
VI  REFERENCES 
1.  Anderson LJ. Role of parvovirus B19 in human disease. 
Pediatric Infectious Disease Journal 1987;6:711-718. 
2.  Anderson MJ, Lewis E, Kidd'IM, et al. An outbreak of 
erythema infeciosum associ?ted with human parvovirus 
infection. J Hyg (Camb) 1984;93:85-93. 
page 12  DRAFT  Original vers., May 15, 1992 
Anderson MJ, Higgins PG, Davis LR, et al. Experimental 
parvoviral infection in humans. J Infect Dis 
1985;152:257-265. 
Kajigaya S, Shimada TI Fujita S, Young NS. A genetically 
engineered cell line that produces empty capsids of B19 
(human) parvovirus. Proc Natl Acad Sci USA 
1989;86:7601-7605. 
Kutzman GJ, Cohen BJ, Field AM,  et al. Immune response to 
B19 parvovirus and an antibody defect in persisten viral 
infection. Journal of Clinical Investigations 
1989;84:1114-1123. 
Pattison JR, Jones SE, Hodgson J, et al. Parvovirus 
infections and aplastic crisis in sickle-cell anaemia. 
Lancet 1984;1:664-665. 
9 
Paver WK, Clarke SKR. Comparison of human fecal and serum 
parvo-like viruses. J Clin Micro 1976;4:67-60 
Siegl GI Bates RC, Berns KI, Carter BJ, et al. 
Characteristics and taxonomy of Parvoviridae. Intervirology 
1985;23:61-73. 
Simpson RW, McGinty L, Simon L, et al. Association of 
parvoviruses with rheumatoid arthritis of humans. Science 
1984;223:1425-1428. 
Torok TJ, Wang QY, Gary GW, et al.  Prenatal diagnosis of 
intrauterine infection with parvovirus B19 by the polymerase 
chain reaction technique. Clin Inf Dis 1992; 14:149-155. 
Wos SM, Cukor G.  Parvoviruses, Chp 30 in:  Schmidt NJ and 
Emmons RW, editors. Diagnostic Procedures for Viral, 
Rickettsia1 and Chlamydia1 Infections, sixth edition, 
Washington: American Public Health Association, Inc. 
1989:1103-1111. 
page 13