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Review Criteria for Assessment of Cytogenetic Analysis Using Automated and Semi-Automated Chromosome Analyzers. |
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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 updated in the next revision to include the standard elements of GGP’s.
DRAFT DOCUMENT
FOR MANUFACTURERS
VERSION: Original
DATE: 7/15/91
REVIEW CRITERIA FOR ASSESSMENT OF CYTOGENETIC ANALYSIS
USING AUTOMATED AND SEMI-AUTOMATED CHROMOSOME ANALYZERS.
This is a flexible draft document representing the current major
concerns and suggestions regarding in vitro automated and
semi-automated chromosome analyzers for cytogenetic analysis. It
is based on 1) current basic science, 2) clinical experience, 3)
current standard laboratory practice, and 4) previous submissions
by manufacturers to the FDA. 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 for chromosome analysis may be cleared
for marketing. This information enables FDA
to make better informed decisions based on a
uniform data base.
DEFINITION: The generic type device is intended for use
in the cytogenetics laboratory to aid
laboratory personnel in performing certain
procedures used in karyotyping human
metaphase/prometaphase cells for in vitro
cytogenetic analysis.
PRODUCT CODE: LNJ - 88
REGULATION
NUMBER: CFR ~ 864.5260 Automated cell-locating
device.
PANEL: PATHOLOGY
CLASS: II
REVIEW REQUIRED: 510(k)
REGULATORY ISSUES:
Manufacturers are encouraged to develop devices that will be
compatible with guidelines of laboratory regulatory
organizations.
Most cytogenetics laboratories subscribe to certain proficiency
testing and accreditation agencies or are state regulated. The
College oœ American Pathologist (CAP) and the Council of Regional
Genetics Networks (CORN) offer voluntary proficiency testing. In
New York State, the Department of Health is responsible for
laboratory testing and certification; the State of Oregon
licenses cytogenetics laboratories.
In New York State, cytogenetics laboratories that use image
analysis computer systems must develop a system for record
retention and retrieval that meets laboratory licensure quality
control standards.
I. Background
A. Cytogenetics in Clinical Practice
Cytogenetic analysis is an in vitro clinical laboratory
procedure that evaluates the chromosomes of a cell.
Clinical cytogenetics is the study of chromosomes and
their correlation to the phenotype (observable clinical
characteristics). Certain clinical characteristics
occur consistently in association with a particular
chromosome abnormality. This phenotype-karyotype
correlation is useful to the clinician in making a
clinical genetic diagnosis and prognosis.
There are two basic classes of chromosome aberrations:
numeric and structural. Common abnormalities of
chromosome number include aneuploidy, mosaicism, and
polyploidy. Structural abnormalities include
duplications, deletions, inversions, shifts, fragile
sites, etc.
Chromosome aberrations are found in association with
many anomalies of sexual development such as the Turner
syndrome (XO) and the Kleinfelter syndrome (XXY);
mental retardation (Trisomy 21 in the Down syndrome and
fragile X in the Fragile X syndrome); complex
malformation syndromes (Trisomy 13); spontaneous
abortions; and malignant disorders (chromosome
translocation between chromosome 9 and 22 in chronic
myeloid leukemia).
In addition to studies for detection of classic
chromosome abnormalities, more recent biotechnologies
add a new dimension to traditional cytogenetic
analysis. Cytogenetic procedures may be used to study
cell cycle associated phenomenon, gene amplification
(homogeneous staining regions and double minutes),
clastogen challenge, chromosome breakage syndromes,
chromosome fragile sites, and polymorphisms to monitor
organ/tissue transplantation. DNA probes that are site
specific are used for identification of chromosome
abnormalities by in situ hybridization using chromosome
specific DNA probes and for purposes of gene mapping.
Clinicians request cytogenetic analysis for individuals
with clinical findings or a medical or family history
indicative of a chromosome abnormality. Requests for
cytogenetic studies have increased in the last decade
due to the demand for prenatal chromosome testing and
because of the widespread use of chromosome analysis in
cancel diagnosis and monitoring.
An accurate cytogenetics analysis is essential for the
clinician to make an accurate clinical genetic
diagnosis and prognosis for patient management,
pregnancy planning and prenatal diagnosis.
An incorrect diagnosis, both false positive and false
negative, will have far reaching medical and legal
implications. Therefore it is imperative that any
device used for purposes of cytogenetic analysis is
sensitive, specific, safe, and effective.
B. Historical Background
The correct chromosome number in humans, 46, was determined
in 1956 by Tjio and Levan2. Prior to this date the normal
human chromosome number was considered to be 48. With this
new knowledge came the recognition that certain chromosome
abnormalities were associated with specific congenital
defects.
Improved techniques for handling mitotic chromosomes
awakened interest in human cytogenetics. In 1959 Lejeune,
et al., described the first chromosome abnormality
associated with a clinical syndrome, trisomy 21 in the Down
syndrome3. With the advent of chromosome banding techniques
in the 1970s, it was possible to identify with certainty all
chromosome pairs and to characterize more accurately
abnormalities of chromosome number and structure. For a
given banding technique, each chromosome pair exhibits a
unique pattern of differential staining along the length of
the chromosome.
The traditional method for analyzing chromosomes is labor
intensive. By the early 1960s, development of instruments
for 4 automated analysis of metaphase chromosomes was well
under way. The primary objective was to develop automated
systems which perform as well as a cytogeneticist using
conventional techniques and are faster and more cost
effective.
Because of the rapid increase in the work load of the
cytogenetics laboratory and the improved computer
capabilities for image processing, these devices are being
used with increased frequency to automate parts of the
manual procedure.5,6 Various computer hardware and software
features are available which are designed to assist in one
or more steps of the process outlined in II.C.
C. Basic Steps in Routine Cytogenetic Analysis7,8,9
1. Specimen Types and Cell Preparation. (Automated
chromosome analyzers are not involved in this step.)
a. Obtain a specimen with a large population of
cycling (dividing) cells
Specimens that contain rapidly dividing cells (e g ,
bone marrow, solid tumors and chorionic villus) may be
harvested without culturing (direct method). Other
types of issue (e g., peripheral blood lymphocytes,
skin fibroblasts and amniotic fluid cells) must be
cultured in a nutritive media and controlled
environment before harvesting.
b. Harvest the cells
Arrest cell division at the metaphase stage of the cell
cycle by adding colcemid or a similar agent that
inhibits spindle fiber formation. Treat the cells with
a hypotonic agent to swell the cells and to facilitate
a better spread of the metaphase chromosomes when they
are dropped onto a microscope slide or are grown on
coverslips. Treat them with a fixative to kill the
cells, clarify the chromosome morphology, and enhance
the basophilic property of the chromosomes.
c. Prepare the slides and stain the cells
2. Selection and Analysis of cell with Metaphase
Chromosomes (Automated Chromosome Analyzers may modify
or aid in one or more of these processes.)
a. Select a predetermined number of metaphase spreads
of suitable quality for study and count the
chromosomes to determine the modal chromosome
number.
b. Select several representative spreads for detailed
analysis. The number of cells selected varies
depending on individual laboratory practices and
the clinical indication for testing.
c. Photograph the representative metaphase spreads
and make an appropriate number of photographic
prints.
d. Cut individual chromosomes from the prints. Pair
and arrange them in a standard format following
the International System for Human Cytogenetic
Nomenclature (ISCN), 1985 guidelines10.
e. Prepare a final report using standard nomenclature
that includes a summary and interpretation of the
reservations and the number of cells counted and
analyzed. Send the report to the referring
clinician. Keep permanent records including a
copy of the final report, the original metaphase
images and karyotypes, and the microscope slides
on file for a period of time determined by the
individual institution or other regulatory agency.
II. Device Description
A. General Principles and Features
Key issues in the review of these devices center on
specific intended use statements and claims dependent
on the type of device manufactured.
The following features/capabilities are representative
of devices currently marketed or being developed in the
United States for clinical use. Describe fully these
and any other features for which claims are made in the
labeling section of the 51OK submission.
Work Station
Hardware/Software
Electronic camera with automatic focusing
Monitor (color, monochrome)
Word Processor
Special Decision Making Features
Automatic karyotyping
Metaphase finding
Chromosome finding
Satellite analysis
in situ hybridization analysis
Sister chromatin exchange (SCE) analysis
Automatic cutting and/or separation of
chromosomes
Image manipulation
Positioning
Rotating
Moving
Eliminating artifact (dodging)
Enlarging selected metaphases, chromosome
pairs, or individual chromosomes
Enhancing
Contrasting
Image capturing process by a TV camera
Resolving gray scale (levels of grayness)
Printer interface
Microscope interface
Capacity for networking
Data storage mechanism and capacity
Ability to recognize and analyze prophase
chromosomes
Staining methods the instrument can utilize
Chromosome recognition capability
Training feature
B. Description of Specific Features:
1. Metaphase Finders/Scanners aid the
cytotechnologist to locate rapidly suitable
metaphases for analysis. These instruments
automatically scan the microscope slide to locate
likely metaphase spreads. The instrument may rank
the metaphase cells according to quality and store
their microscope slide coordinates in the
computer's data base. Metaphase finding/scanning
instruments are not always accurate. In some
cases their use is limited to specific types of
staining and they may not be appropriate for use
with certain types of disorders.
2. Chromosome Counters determine the chromosome
number by automatically counting the number of
chromosomes in a given metaphase spread.
3. Photomicroscopy and Photographic Dark Room
Processes eliminate the need for photomicroscopy,
photographic dark room work and cutting and
pasting chromosomes when performing karyotyping.
The process uses digital image processing to
digitize the metaphase images by dividing the
picture into a grid of pixels. The resolution and
detail is determined by the number of pixels in
the image and the range in the level of contrast
(grayness). The level of grayness may
theoretically range from 0 to 256. Optical
information about each pixel as well as its
location may be processed and stored in the
computer data base. The metaphase chromosomes are
manipulated (cut) and arranged (pasted) in pairs
on the karyotype card (projected onto the computer
monitor). Some instruments are designed to do the
"cutting and pasting" automatically (see section,
B.4.b., below) - others require the operator to
manipulate the images (see section B.4.a., below).
4. Interactive and Automatic Karyotyping
a. Interactive systems have no decision making
ability and depend on the operator to
classify the chromosomes from the computer
screen and arrange them on the computerized
karyotype sheet.
b. Automatic karyotyping systems exist with
varying amounts of decision making ability.
Chromosomes are classified on the basis of
chromosome dimension (e.g., ratio of short
arm to long arm) and banding pattern profile.
5. Enhancement, Alteration and Manipulative Features.
Some instruments have features that enhance or
contrast chromosome images in metaphases and/or
individual chromosomes to improve the banding
characteristics of the chromosome(s). Other
features alter or allow the operator to alter
chromosome morphology and/or other cellular
characteristics. These features include:
straightening, enlarging, trimming,
"mirror-image", "enhancement" (selectively
altering staining pattern within a metaphase
spread or within a chromosome region), and
dodging/lifting the cytoplasmic background.
6. Specialized Analysis (in development) A few
instruments have the ability to perform analysis
of specialized studies such as chromosome
satellites, in situ hybridization and sister
chromatid exchange (SCE). Some chromosome
analyzers automatically count the number of
satellites or SCEs per metaphase cell or the
number of hybridized probe sites per interphase or
metaphase cell. Although this feature is currently
being used for research purposes, it has not been
cleared by the FDA for clinical use.
7. Hard Copy Prints. Most systems are capable of
producing near photographic quality, printer
generated hard copy of the metaphase images and
karyotypes.
8. Generating Reports. Many instruments have
capabilities of generating (preparation &
printing) a final summary for the referring
clinician and of handling the billing process and
other bookkeeping.
9. Computerized Patient Data Storage, Retrieval and
Archival Systems. A data base may include
metaphase images and karyotypes, patient
identifying information, and final reports.
10. Training Feature (in development). This feature
permits the operator to teach the system to
recognize the chromosomes preparations particular
to a given laboratory. It allows the operator to
"train" the instrument to recognize different
staining preparations. Although this feature is
currently being used for research purposes, it has
not been cleared by the FDA for clinical use.
11. Telecommunication Features allows for site-to-site
image transmission.
12. Networking features provide networking between
workstations and local areas.
III. Specific Performance Characteristics
Support specific parameters of importance to the operation
of the instrument by data generated with the device.
Demonstrate that the device is substantially equivalent to a
legally marketed predicate device. Conduct performance
studies to demonstrate that the device is safe and effective
by comparing the device's performance to the manual
reference method of chromosome analysis. See section II.B.,
"Device Description" for details of performance
characteristics required for specific feature.
Address all aspects of performance characteristics as stated
in section III.C. in the Performance Characteristics section
of the Labeling. Provide the following specific information
on reproducibility/precision and accuracy for instruments
with features that warrant such studies. Include a detailed
study protocol, generated data and statistical analysis of
the data in any submission to the include a summary of the
performance data in the Performance Characteristics section
of the Labeling
A. Analytical/Laboratory/in vitro Studies
1. Reproducibility Studies
Study a sufficient number of control specimens and
test specimens with the types of chromosome
abnormalities or characteristics for which claims
are made (e.g., normal, aneuploidies, structural
rearrangements, fragile sites, sister chromatin
exchange, etc.) to demonstrate:
a. Within Sample Reproducibility
Does the device give the same results on
repeated trials (analysis) of a given
procedure? e.g., does it locate the same
cells on a given slide, rank cells in the
same manner, give the same chromosome count
on a given cell, generate the same karyotype
for a given cell, etc.
b. Between Instrument Reproducibility
Do different devices give the same results
for a given procedure? e.g. do they locate
the same cells on a given slide, rank the
cells in the same manner, give the same
chromosome count on a given cell, generate
the same karyotype for a given cell, etc.
2. Comparison Studies
Comparison studies provide data on the ability of
the instrument to determine accurately specific
results as compared to the manual method for
chromosome analysis. (If a method other than the
manual method is used justify the choice of the
method and include pertinent references.)
Perform the test on a sufficient number of
specimens with and without chromosome aberrations
and calculate the following parameters:
a. Relative Diagnostic Sensitivity: the
probability that the instrument will
correctly identify an abnormality determined
to be abnormal by the reference method.
b. Relative Diagnostic Specificity: the
probability that the instrument will
correctly identify as normal a specimen
determined to be normal by the reference
method.
3. Specifications
Describe the relative quality: Does the
instrument achieve the same (or better band
resolution compared to the reference method
according to the ISCM (1985) guidelines10?
B. Software Documentation
All computer software should comply with the FDA's
Policy for Regulation of Computer Products. For
general information contact the FDA Division of Small
Manufacturers (phone, 800-638-2041). For specific
information contact the Division of Product
Surveillance (phone, 301-427-8156).
C. Special Considerations for Specific Features Described
in II.B.
All devices that have any decision making features
should also have a feature which allows the operator to
interact, edit and override the work generated by the
device.
Several features of imaging analysis computer systems
need special consideration which are addressed below.
For each of the following features, provide
reproducibility and comparison studies, specifications,
and software documentation unless otherwise specified.
Incorporate the following considerations as appropriate
in the Labeling (e.g., Intended Use, Methods,
Limitations, etc.).
1. Metaphase Finders
a. State which staining methods may be used with
the device. State what types of preparations
may be analyzed (e.g., air dried, grown on
coverslips, primary colonies, etc.)
b. Provide data to demonstrate that the device
does not introduce bias in selection of
metaphases with respect to chromosome number
(e.g., aneuploidy, polyploidy),
endoreduplication, poor chromosome morphology
as in some malignant cells, structural
chromosome abnormality (e.g., translocations,
dicentrics, fragments, etc.), or chromosome
staining factors.
c. Provide data to demonstrate the performance
of the metaphase ranking feature. State in
the Labeling whether metaphase finders will
or will not detect certain cellular
abnormalities such as micronuclei, nucleolar
organizing region (NOR) alterations and other
abnormalities of the cytoplasm or nucleus
which would be noted by an astute observer.
d. Since these instruments are not always
accurate for finding and ranking metaphases,
place the following or (similar) statement in
the Limitations Section of the Labeling.
"The cytogenetic technologist/
cytogeneticist should always review
slides independently of the metaphase
scanner/finders."
e. State whether or not it is appropriate to use
the metaphase finder for studies such as drug
sensitivity, tumor hard tissue, etc. State
any limitations in the Limitations section of
the Labeling.
2. Chromosome Counters
a. State which staining methods may be used with
the device.
b. State limitations imposed by how well the
chromosomes are spread (e.g., overlapping
chromosomes, too much spread, broken cells,
too many metaphases in one location, etc.).
c. Provide data to demonstrate that bias is not
introduced into chromosome counts by
aneuploidy, polyploidy, endoreduplication,
radial formations, chromosome pulverization,
poor chromosome morphology as in some
malignant cells, and structural chromosome
abnormality (dicentrics, fragments, etc.).
If any incorrect chromosome counts result,
this should be declared in the limitations
section of the package insert.
d. Include the following or similar statement in
the Limitations Section of the Labeling:
"In general there will be one or more
errors in determining the correct
chromosome number for the population of
cells studied. It is the responsibility
of the operator to determine the correct
modal chromosome number."
3. Interactive Karyotyping Systems (no decision
making ability)
Reproducibility and comparison studies are not
required since this feature has no decision making
ability.
4. Automated Karyotyping
Reproducibility and comparison studies are
required only for the decision making features of
the device.
Include the following or similar statement in the
Limitations section of the Labeling:
"In general, there will be one or more errors
in the computer-generated karyotype
Therefore, it must always be examined and
edited as a final interactive manual
operation by a qualified cytogeneticist or
cytogenetic technologist."
5. Enhancement, Alteration and Manipulation Features
Altering chromosome morphology in any way (aside
from improving culturing and staining techniques)
is not an accepted standard of professional
practice in cytogenetic analysis.
a. Comparison data are not required for
enhancement, alteration or manipulation
features.
b. Any device with features that in any way
alter chromosome morphology should also have
a built-in feature that automatically and
permanently marks/designates these
alterations in the karyotype.
c. Digitized straightening of chromosomes will
often artificially induce extra bands and
thus make it impossible to determine,
unequivocally, whether the straightened
chromosome is, indeed, normal. Designate any
straightened chromosomes as stat.
d. Assure that the device does not have the
capacity to induce artifacts.
e. Features that "lift" cellular background
should not be automatic and should have a
built-in feature that requires operator
activation. Such a feature should not be
used unless the operator has already examined
the cell to assure that the material to be
lifted is true artifact and not chromatin
material such as double minutes.
f. Include a statement about the potential for
misuse of each feature under the Limitations
section of the Labeling and elsewhere in the
operators manual whenever use of these
features are described. Use the following or
a similar statement about features listed in
this section:
"It is the responsibility of the
cytogenetics technician and/or the
cytogeneticist to utilize all features
in compliance with standard laboratory
practice and regulatory guidelines."
6. Photomicroscopy and Dark Room Process
a. Reproducibility and comparison data are not
required.
b. Compare the resolution of the digitized
images to standard microscopic resolution in
terms of the resolution standards (400, 500
and 850 bands) of the ISCN, 1985 guidelines10.
c. For devices that have no decision making
ability, include the following or similar
statement in the Intended Use section of the
Labeling:
"The device does not locate metaphase
spreads; it does not rank the given
cells according to quality; it does not
automatically classify chromosomes; it
does require and relies completely on
the operator to manipulate the digitized
microscope images."
7. Hard Copy Prints
a. Reproducibility and comparison data are not
required.
b. Describe the quality and resolution of the
hard copy print in the Principle of the
Procedure section of the Labeling. State
whether the quality/resolution of the
computer generated print is equivalent to (as
good as) standard photomicroscopy (levels of
gray) and whether the device meets the
minimum resolution standard set by the ISCN,
1985 guidelines.
c. Describe the composition, quality and
durability of the photo image paper and
whether contrast deteriorates with age.
i. A precaution/warning statement in the
Precautions section of the Labeling will
be required for paper containing mercury
or other toxic substances.
ii. State how long the prints will remain of
archival quality. The durability of
these hard copy prints is important
since some proficiency/licensing
agencies require long term storage of
metaphase images (up to 25 years for New
York State).
8. Computerized Patient Data Storage, Retrieval and
Archival System.
a. Reproducibility and comparison data are not
required.
b. This feature should comply with section
III.B. of this document. The following
issues are of special concern and should be
addressed in the Principles of Procedure
section of the labeling:
i. adequate security control which may
require several levels of "password"
security to assure protection and
confidentially of patient information;
ii. adequate safe guards to protect against
accidental or virus generated deletion
of the data
iii. an explanation of where and how the data
are stored; and
iv. a recommendation of multiple identifying
codes (e.g., for patient identifying
data, metaphases, karyotypes, final
report, etc.) to assure correct and
usable storage and retrieval of
information from the data base and
archival system.
IV. Labeling Considerations
The Labeling (Operator's Manual or Package Insert) should
include all information listed in the in vitro diagnostic
Labeling regulations 21 CFR ~ 809.10(b)(6) plus additional
pertinent headings found under 21 CFR ~ 809.10(b):
A. Intended Use Statement
Describe concisely the functions/features of the
device. State clearly that a qualified cytogenetic
technologist and/or cytogeneticist must edit and/or
confirm all computer-generated data/results and make
the final judgment/decision.
For devices that have no decision making ability,
include the following or similar statement in this
section:
"The device does not locate metaphase spreads; it
does not rank the given cells according to
quality; it does not automatically classify
chromosomes; it does require and relies completely
on the operator to manipulate the digitized
microscope images."
B. Limitations of the Device
Include the following:
1. a statement that decision making capabilities of
the instrument do not relieve the
cytogeneticist/cytogenetics technologist of the
responsibility to review and edit all work
generated by the device and that the final
decision must be made by a qualified
cytogeneticist;
2. appropriate statements of precaution outlined for
each feature in the Performance Characteristics
section (III-C). In some cases, limitation
statements for more than one features may be
combined; and
3. these general statements for all devices that have
features with decision making ability:
"In general, there will be one or more errors
in the computer-generated data/karyotypes.
Therefore, it must always be examined and
edited as a final interactive manual
operation by qualified cytogenetic
personnel."
"The final clinical diagnosis must be made by
qualified medical personnel."
C. Performance Characteristics
Provide a summary of all reproducibility and comparison
studies (sensitivity and specificity) when performed as
requested by Section III.
Also address other performance characteristics that relate
to specific features (as described in section III-C).
V. Bibliography
1. Letter. September 1989 Cytogenetic Proficiency Test,
General Comments. New York State Department of Health.
2. Tjio, JH and Levan, A: The chromosome number in man.
Hereditas, 42:1, 1956.
3. Lejeune, J, Gautier, M, and Turpin, MR: Etude dais
chromosomes somatiques de neut enfants mongoliens.
C.R. Acad. Sci. (Paris) 248, 1721-1722, 1959.
4. Lubs, HA and Ledley, RS: Automated analysis of
differentially stained human chromosomes. Nobel.
23:61-76, 1973.]
5. Lifshitz, MS and DeCresce, RP: Genetiscan Digital
Karyotype System. Lab Med, 18(6):402-403, 1987.
6. Bender MK: Karyotyping System with Special Reference to
the KARYOTEC 100. Presentation: 34th Annual Scientific
Meeting of The Royal College of Pathologists of
Australia. Hong Kong, 1989.
7. Rooney DE and Czepulkowski BH: Human Cytogenetics: A
Practical Approach. Oxford, England: ILR Press; 1986.
8. Verma RS and Buba A. Chromosomes: A manual of Basic
Techniques. New York, NY: Pergamon; 1989.
9. Priest JH: Medical Cytogenetics and Cell Culture. 2nd
ed. Philadelphia, PA: Lea and Fabiger; 1977.
10. Harden DG and Klinger HP: An International System for
Human Cytogenetic Nomenclature (1985). Basel,
Switzerland: S Karger; 1985.
This document was still considered current as of July 1997.
It will be reviewed again in July 1998.
Updated September 5, 1997
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