**Guidance for Industry**

**and FDA Staff**

**Class II Special Controls Guidance Document: Sirolimus Test Systems**

**Document issued on: \[[Insert release date of FR Notice\]]{.mark}**

For questions regarding this document contact Avis Danishefsky at
301-594-1243 ext.161 or by email at atd@cdrh.fda.gov.

![](media/image1.png)

**U.S. Department of Health and Human Services**

**Food and Drug Administration**

**Center for Devices and Radiological Health**

**Division of Chemistry and Toxicology Devices**

**Office of In Vitro Diagnostic Device Evaluation and Safety**

**Preface**

**Public Comment**

Written comments and suggestions may be submitted at any time for Agency
consideration to the Division of Dockets Management, Food and Drug
Administration, 5630 Fishers Lane, Room 1061, (HFA-305), Rockville, MD,
20852. Alternatively, electronic comments may be submitted to
<http://www.fda.gov/dockets.ecomments>. When submitting comments, please
refer to Docket No. 2004D-0412. 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 at:
[http://www.fda.gov/cdrh/oivd/guidance/1300.pdf]{.underline}, or to
receive this document by fax, call the CDRH Facts-On-Demand system at
800-899-0381 or 301-827-0111 from a touch-tone telephone. Press 1 to
enter the system. At the second voice prompt, press 1 to order a
document. Enter the document number 1300 followed by the pound sign (#).
Follow the remaining voice prompts to complete your request.

> **Table of Contents**

[1. Introduction 4](#introduction)

[2. Background 5](#background)

[3. The Content and Format of an Abbreviated 510(k) Submission
6](#the-content-and-format-of-an-abbreviated-510k-submission)

[4. Scope 8](#scope)

[5. Risks to Health 8](#risks-to-health)

[6. Performance Characteristics 9](#performance-characteristics)

[General Study Recommendations 9](#general-study-recommendations)

[Specific Performance Characteristics
10](#specific-performance-characteristics)

[*Precision* 10](#precision)

[*Recovery* 11](#recovery)

[*Linearity* 11](#linearity)

[*Sensitivity* 12](#sensitivity)

[*Specificity for parent compound* 13](#specificity-for-parent-compound)

[*Interference* 14](#interference)

[*Specimen collection and handling conditions*
16](#specimen-collection-and-handling-conditions)

[*Method comparison* 16](#method-comparison)

[*Studies at external sites* 18](#studies-at-external-sites)

[*Calibrator and Control Material* 18](#calibrator-and-control-material)

[7. Labeling 19](#labeling)

[8. New Instrument Applications 21](#new-instrument-applications)

[9. References 22](#references)

**Guidance for Industry and FDA Staff**

**Class II Special Controls Guidance Document:**

**Sirolimus Test Systems**

***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.***

# 1. Introduction

This guidance was developed as a special controls guidance to support
the classification of sirolimus (rapamycin) test systems into class II.
Sirolimus test systems are intended to quantitatively determine
sirolimus concentrations in whole blood as an aid in the management of
transplant patients receiving therapy with sirolimus. Many aspects of
this document, especially those concerning performance characteristics
and risks to health, were developed using information FDA obtained from
the Therapeutic Drug Management (TDM) Roundtable. This working group was
composed of representatives from laboratory medicine as well as device
manufacturers.

This guidance document addresses instrument-based chromatographic assays
or immunoassays used in central clinical laboratories. It does not
address assays that use other methodologies or point of care assays.

This guidance is issued in conjunction with a Federal Register notice
announcing the classification of sirolimus test systems. Any firm
submitting a premarket notification (510(k)) for a sirolimus test system
will need to address the issues covered in this special controls
guidance document. However, the firm need only show that its device
meets the recommendations of the guidance or in some other way provides
equivalent assurances of safety and effectiveness.

FDA\'s guidance documents, including this guidance, do not establish
legally enforceable responsibilities. Instead, guidance documents
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 guidance
documents means that something is suggested or recommended, but not
required.

**The Least Burdensome Approach**

The issues identified in this guidance document represent those that we
believe need to be addressed before your device can be marketed. In
developing the guidance, we carefully considered the relevant statutory
criteria for Agency decision-making. We also considered the burden that
may be incurred in your attempt to follow the statutory and regulatory
criteria in the manner suggested by the guidance and in your attempt to
address the issues we have identified. We believe that we have
considered the least burdensome approach to resolving the issues
presented in the guidance document. If, however, you believe that there
is a less burdensome way to address the issues, you should follow the
procedures outlined in the document, "A Suggested Approach to Resolving
Least Burdensome Issues." It is available on our Center web page at:
<http://www.fda.gov/cdrh/modact/leastburdensome.html>.

# 2. Background

FDA believes that special controls, when combined with the general
controls, will be sufficient to provide reasonable assurance of the
safety and effectiveness of a sirolimus test system. A manufacturer who
intends to market a device of this generic type should (1) conform to
the general controls of the Federal Food, Drug, and Cosmetic Act (the
Act), including the premarket notification requirements described in 21
CFR 807 Subpart E, (2) address the specific risks to health associated
with sirolimus test systems identified in this guidance, and (3) obtain
a substantial equivalence determination from FDA prior to marketing the
device.

This guidance document identifies the classification regulation and
product code for sirolimus test systems (Refer to Section 4 --
**Scope**). In addition, other sections of this special control guidance
document list the risks to health identified by FDA and describe
measures that, if followed by manufacturers and combined with the
general controls, will generally address the risks associated with a
sirolimus test system and lead to a timely premarket notification
(510(k)) review and clearance. This document supplements other FDA
documents regarding the specific content requirements of a premarket
notification submission. You should also refer to 21 CFR 807.87 and
other FDA documents on this topic, such as the **510(k) Manual -
Premarket Notification: 510(k) - Regulatory Requirements for Medical
Devices**, <http://www.fda.gov/cdrh/manual/510kprt1.html>.

As explained in "**The New 510(k) Paradigm - Alternate Approaches to
Demonstrating Substantial Equivalence in Premarket Notifications; Final
Guidance**[^1]," a manufacturer may submit either a Traditional 510(k)
or an Abbreviated 510(k). FDA believes an Abbreviated 510(k) provides
the least burdensome means of demonstrating substantial equivalence for
a new device, particularly when FDA has issued a guidance document that
provides recommendations on what should be addressed in a submission for
the device. Alternatively, manufacturers considering modifications to
their own cleared devices may lessen the regulatory burden by submitting
a Special 510(k).

# 3. The Content and Format of an Abbreviated 510(k) Submission

An Abbreviated 510(k) submission must include the required elements
identified in 21 CFR 807.87, including the proposed labeling for the
device sufficient to describe the device, its intended use, and the
directions for its use. In an Abbreviated 510(k), FDA may consider the
contents of a summary report to be appropriate supporting data within
the meaning of 21 CFR 807.87(f) or (g); therefore, we recommend that you
include a summary report. The report should describe how this guidance
document was used during the device development and testing and the
methods or tests used. The report should also include a summary of the
test data or description of the acceptance criteria applied to address
the risks identified in this document, as well as any additional risks
specific to your device. This section suggests information to fulfill
some of the requirements of 21 CFR 807.87 as well as some other items
that we recommend you include in an Abbreviated 510(k).

> **Coversheet**
>
> The coversheet should prominently identify the submission as an
> Abbreviated 510(k) and cite the title of this class II special
> controls guidance document.
>
> **Proposed labeling**
>
> Proposed labeling should be sufficient to describe the device, its
> intended use, and the directions for its use. (Refer to Section 7 for
> specific information that should be included in the labeling for
> devices of the types covered by this document.)
>
> **Summary report**
>
> We recommend that the summary report contain the following:

-   A description of the device and its intended use. We recommend that
    the description include a complete discussion of the performance
    specifications and, when appropriate, detailed, labeled drawings of
    the device. You should also submit an \"indications for use\"
    enclosure.[^2]

-   A description of the device design.

-   An identification of the Risk Analysis method(s) used to assess the
    risk profile in general as well as the specific device's design and
    the results of this analysis. (Refer to Section 5 for the risks to
    health generally associated with the use of this device that FDA has
    identified.)

-   A discussion of the device characteristics that address the risks
    identified in this class II guidance document, as well as any
    additional risks identified in your risk analysis.

-   A description of the test method(s) you have used or intend to use
    to address each performance aspect identified in Section 6 of this
    class II special controls guidance document. If you follow a
    suggested test method, you may cite the method rather than
    describing it. If you modify a suggested test method, you may cite
    the method but should provide sufficient information to explain the
    nature of and reason for the modification. For each test, you may
    either (1) briefly present the data resulting from the test in clear
    and concise form, such as a table, **or** (2) describe the
    acceptance criteria that you will apply to your test results.[^3]
    (See also 21 CFR 820.30, Subpart C - Design Controls for the Quality
    System Regulation.)

-   If you choose to rely on a recognized standard for any part of the
    device design or testing, you may include either: (1) a statement
    that testing will be conducted and meet specified acceptance
    criteria before the product is marketed, or (2) a declaration of
    conformity to the standard.[^4] Because a declaration of conformity
    is based on results from testing, we believe you cannot properly
    submit a declaration of conformity until you have completed the
    testing the standard describes. For more information, please refer
    to section 514(c)(1)(B) of the Act and the FDA guidance, **Use of
    Standards in Substantial Equivalence Determinations; Final Guidance
    for Industry and FDA**,
    <http://www.fda.gov/cdrh/ode/guidance/1131.html>.

If it is not clear how you have addressed the risks identified by FDA or
additional risks identified through your risk analysis, we may request
additional information about aspects of the device's performance
characteristics. We may also request additional information if we need
it to assess the adequacy of your acceptance criteria. (Under 21 CFR
807.87(l), we may request any additional information that is necessary
to reach a determination regarding substantial equivalence.)

As an alternative to submitting an Abbreviated 510(k), you can submit a
Traditional 510(k) that provides all of the information and data
required under 21 CFR 807.87 and described in this guidance. A
Traditional 510(k) should include all of your methods, data, acceptance
criteria, and conclusions. Manufacturers considering modifications to
their own cleared devices should consider submitting Special 510(k)s.

The general discussion above applies to any device subject to a special
controls guidance document. The following is a specific discussion of
how you should apply this special controls guidance document to a
premarket notification for a sirolimus test system.

# 4. Scope

The scope of this guidance is limited to the following devices as
described in 21 CFR 862.3840 (product code NRP):

> [21 CFR 862.3840 Sirolimus Test System]{.underline}
>
> A sirolimus test system is a device intended to quantitatively
> determine sirolimus concentrations in whole blood. Measurements are
> used as an aid in the management of transplant patients receiving
> therapy with sirolimus.

# 5. Risks to Health 

There are no known *direct* risks to patient health. However, an
indirect risk is that failure of the test to perform as indicated or
error in interpretation of results may lead to improper patient
management. For example, a falsely low sirolimus measurement could
contribute to a decision to raise the dose of sirolimus above that which
is necessary for therapeutic benefit. This could result in increased
risk in the form of thrombocytopenia, leukopenia, anemia, or
hyperlipidemia (Meier-Kriesche, 2000). A falsely high sirolimus
measurement could contribute to a decision to decrease the dose below
that which is necessary for immunosuppression. This could result in
increased risk of rejection of the transplanted organ.

Optimal ranges for sirolimus are expected to depend on the specific
assay used because of variations in metabolite cross-reactivity among
immunoassays. For example, assay biases ranging from 8-21% and up to 40%
have been observed for immunoassays relative to chromatographic methods
(Salm, 2000; Jones, 2000). Therefore, use of a sirolimus assay to adjust
a treatment regimen without knowledge of performance of the assay used
or its specific optimal ranges could lead to improper patient management
due to error in interpretation. Optimal ranges also depend on other
clinical factors, including patient drug tolerance, immunosuppressive
regimen, and time post-transplant. To address these issues, the
Therapeutic Drug Management Roundtable has recommended that each
institution establish optimal ranges for sirolimus, based on the
specific assay used at that institution, and other factors relevant to
their patient population. This is similar to the recommendations, for
other immunosuppressant drugs, in the National Academy of Clinical
Biochemistry's, "Guidelines for Therapeutic Drug Monitoring Services".
The manufacturer should also clearly portray performance observed for a
new assay relative to a gold standard (e.g., measures of bias,
variability, cross-reactivity) in the labeling.

For chromatographic methods, optimal ranges for whole blood trough
sirolimus concentrations following kidney transplantation have been
suggested as 5-15 ng/ml (Mahalati, 2001) when given in combination with
cyclosporine and 12-24 ng/ml following cyclosporine withdrawal
(Rapamune® package insert).

In the table below, we have identified the risk to health generally
associated with the use of sirolimus test systems. The measures we
recommend to mitigate this identified risk are given in this guidance
document. You should also conduct a risk analysis to identify any other
risks specific to your device and describe the risk analysis method. If
you elect to use an alternative approach to address a particular risk
identified in this guidance document, or have identified risks
additional to those in the guidance, you should provide sufficient
detail to support the approach you have used to address that risk. It
would also be helpful to consult with FDA concerning your studies in
such cases.

  ----------------------------------- -----------------------------------
  **Identified Risk**                 **Recommended Mitigation Measures**

  Improper patient management (due to Documented test system performance
  failure of the test to perform as   throughout the measurement range
  indicated or error in               and appropriate labeling. See
  interpretation).                    sections 6 and 7 (performance and
                                      labeling).
  ----------------------------------- -----------------------------------

# 6. Performance Characteristics 

## General Study Recommendations 

We recommend that you use samples or sample pools derived from patients
taking sirolimus in precision and linearity studies, as well as method
comparison studies. This is important because patient samples reflect
the relevant proportions of free and bound drug, metabolites, and other
drugs commonly co-administered to transplant patients and therefore help
demonstrate robustness of the assay. Spiked samples or control or
calibrator material may be appropriate to supplement the analytical
studies; however, we do not recommend using these types of samples as
the only matrix in the evaluations because they may not provide an
accurate assessment of the performance characteristics.

We recommend that you perform all of your analytical protocols in
accordance with the procedures you plan to recommend to users in the
labeling, in order to reflect performance expected by the user. We
recommend that you ensure that all steps (e.g., cell lysis, extraction,
and centrifugation) are included in the various analytical studies and
that manufacturer recommended quality control and calibration procedures
are followed. We recognize that evaluations that require freezing of
samples (for example, between- run precision studies) may necessitate
use of hemolyzed samples.

So that results can be correctly interpreted, you should provide
appropriate specifics concerning protocols. For example, when referring
to NCCLS evaluation protocols or guidelines, we recommend that you
indicate which specific aspects of the guidelines you followed and which
you modified.

In studies using spiked samples, we recommend that you provide
information about the purity of drugs, metabolites, or potential
interferents you used.

Whole blood is the matrix recommended in consensus statements from major
scientific groups associated with organ transplantation (Holt, 2002;
Yatscoff, 1995). For assays intended for use with matrices other than
venous whole blood, it would be necessary to demonstrate a strong
correlation between matrices using specimens from patients on drug
therapy. We recommend that you contact FDA to discuss your protocol
before initiating a study of this type.

## Specific Performance Characteristics 

### *Precision* 

> We recommend that you characterize within-run and total precision of
> your test system. The document "Evaluation of Precision Performance of
> Clinical Chemistry Devices; Approved Guideline" (1999) NCCLS, Document
> EP05-A includes appropriate guidelines for experimental design,
> computations, and format for statement of claims.
>
> We recommend that you evaluate precision for at least three
> concentrations spanning most of the range of the test system.
> Typically these concentrations are chosen to represent the (a)
> sub-therapeutic range or near low end of the reportable range, (b)
> concentrations considered to be within the therapeutic ranges, and (c)
> toxic range.
>
> In order to demonstrate robustness of the test system, you should
> include samples from patients taking sirolimus in your evaluation of
> total precision at the various concentration levels. If it is not
> feasible to conduct the entire precision evaluation using patient
> samples, then we recommend that you supplement precision evaluation of
> patient samples with spiked whole blood samples, pools, or control
> material. If patient samples at sub-therapeutic levels or the low end
> of the assay range are not available, it may be appropriate to dilute
> patient samples of higher concentration.
>
> In order to validate precision of the entire assay procedure, as it
> will be performed by the user, you should include evaluation of the
> effect of pre-treatment steps (such as extraction procedures).
> Therefore, we recommend performing pre-treatment steps separately for
> individual replicates in your evaluation of within-run and total
> precision.
>
> We recommend that you include the following in the description of your
> precision evaluation, as relevant:

-   Sample types (e.g., pooled samples from patients taking sirolimus,
    spiked whole blood samples or pools, control samples).

-   Point estimates of the concentrations evaluated.

-   Description of how you evaluated the effect of pre-treatment steps
    on precision (e.g., by individually extracting the replicate
    samples).

-   Sites at which the precision protocol was run.

-   Number of days, runs, and observations.

-   Identification of factors that were held constant and those that
    were varied during the evaluation (e.g., instruments, calibration,
    reagent lots, and operators).

-   Description of your computational methods (including equations, if
    they were modified from those described in NCCLS EP05-A).

-   Results, e.g., coefficients of variation and standard deviations for
    within-run and total precision at each level.

### *Recovery* 

> As a measure of accuracy, we recommend that you characterize the
> percent recovery (bias) of sirolimus with your assay. To address this,
> we recommend evaluating whole blood samples or pools spiked with known
> concentrations of sirolimus. You should determine the target
> concentrations of the samples using a reliable method that is
> independent of your assay. Gravimetric methods using drug material of
> defined high purity or well-validated reference chromatography methods
> are appropriate. We recommend that you evaluate samples with
> concentrations that span a significant part of the reportable range
> and include potential medical decision levels. We recommend that you
> include sufficient replicates at each level so that you can
> meaningfully evaluate your results. You should assess the effect of
> any pre-treatment steps on recovery in your assay. To address this, we
> recommend performing the pre-treatment steps separately for each
> target concentration.
>
> We recommend that you include the following in the description of your
> recovery evaluation:

-   Sample types (e.g., spiked whole blood) and preparation.

-   Target concentrations of the samples and the method by which these
    were independently determined.

-   Description of material used for spiking.

-   Description of how you accounted for the effect of pretreatment
    steps on recovery (e.g., by individually pre-treating samples at the
    various levels you evaluated).

-   Definition or method of calculating recovery, including number of
    replicates evaluated.

-   Results, e.g., recoveries observed.

> We recommend that you indicate the range of recoveries or mean and
> standard deviation for each concentration level when you report
> results, since this approach may be more informative than describing
> only average recoveries at each concentration level.

### *Linearity* 

> You should characterize the linear range of the test system. We
> recommend serially diluting positive whole blood samples or pools from
> patients taking sirolimus with sirolimus-free whole blood, to generate
> samples evenly distributed across the entire assay range. We recommend
> that you evaluate a minimum of 5 sirolimus-positive levels within the
> claimed linear range and include multiple samples at each level. In
> order to evaluate whether there is any effect of pre-treatment steps
> on assay linearity, we recommend that you perform the pre-treatment
> steps separately for samples at each of the various target levels.
>
> In cases where samples from patients taking sirolimus are not
> available for the high end of the assay range, we recommend that you
> evaluate linearity by dilution of patient samples at the highest
> concentration that is available. In such cases, if evaluation of
> patient samples does not span the assay range, results can be
> supplemented with data from spiked samples.
>
> The document "Evaluation of the Linearity of Quantitative Analytical
> Methods; Approved Guideline" (2003) NCCLS Document EP06-A describes a
> protocol for sample preparation, value assignment, and a format for
> statement of claims, as well as one approach to statistical design and
> analysis methods for evaluation of the linear range of an assay.
>
> Some immunoassays may exhibit a \"high dose hook effect,\" in which
> there is a fall in response of the assay at high concentrations.
> Whenever appropriate (e.g., for two-site or sandwich immunoassays),
> you should extend linearity studies beyond the reportable range to the
> highest concentrations that may be encountered in clinical settings in
> order to evaluate whether your device exhibits a high dose hook
> effect.
>
> We recommend that you include the following in the description of your
> linearity evaluation:

-   Sample types (e.g., whole blood samples from patients taking
    sirolimus) and preparation.

-   Target concentrations and the methods or calculations you used to
    determine these concentrations.

-   Description of how you evaluated the effect of pre-treatment steps
    on linearity (e.g., by individually pre-treating samples at each of
    the levels evaluated).

-   Number of samples and replicates evaluated.

-   Statistical methods you used to evaluate linearity.

-   Results.

> We recommend that results include a table of the target concentration
> versus the observed concentrations, in addition to the assay range of
> linearity. We also recommend that you include the acceptable maximum
> differences from linearity, if you used the approach described in
> NCCLS EP06-A. If applicable, you should also include data from your
> high-dose hook evaluation.
>
> If you recommend to users that they should dilute samples that are
> above the reportable range, you should provide a specific protocol for
> dilution and include your results for validation of that protocol. The
> validation description should include the concentration range tested
> and the recoveries observed.

### *Sensitivity* 

> You should evaluate bias and precision at the claimed sensitivity
> level of the test system and demonstrate that results meet your
> acceptance criteria. Therefore, we recommend that you include samples
> at your claimed sensitivity level within the evaluations of recovery
> and total precision described above.
>
> It may also be appropriate to report the limit of quantitation (LoQ).
> We define this as the lowest drug concentration that can be reliably
> detected and for which assay bias and precision meet your stated
> acceptance criteria. The document "Protocols for Determination of
> Limits of Detection and Limits of Quantitation; Proposed Guideline"
> (2004) NCCLS Document EP17-P describes a method for establishing the
> limit of quantitation. This document proposes a minimum of 40
> replicates, from 3-5 different samples and determined from 5 or more
> runs. In order to assess whether your acceptance criteria for
> precision and accuracy at the sensitivity level can be met over time,
> we recommend that you perform multiple runs on separate days
> (preferably, non-consecutive days). If possible, we recommend use of
> multiple instruments to capture variability. Bias can be estimated by
> comparing the average concentration based on your assay to the value
> that you determined based on reference material or a reference method
> independent of your assay (and specific for sirolimus parent
> compound). Precision can be estimated by the total standard deviation
> of the samples evaluated with your assay.
>
> You should include evaluation of the effect that pre-treatment steps
> might have on bias and precision at the assay sensitivity level. In
> order to address this, we recommend performing the pre-treatment steps
> separately for each of the individual replicates and levels in your
> evaluations, to simulate conditions for patient samples.
>
> In the description of your evaluation of the limit of quantitation, we
> recommend that you include the (bulleted) points listed in the
> precision and recovery sections above, as applicable. You should state
> your acceptance criteria for bias and precision at the assay
> sensitivity level and provide results to demonstrate these criteria
> were met.
>
> In some cases, you may find it useful to provide additional measures
> of sensitivity, such as the limit of blank or limit of detection (for
> proposed definitions, see NCCLS EP-17P).

### *Specificity for parent compound* 

> As a measure of assay specificity, you should characterize
> cross-reactivity with sirolimus metabolites. Primary known metabolites
> appropriate for sirolimus specificity studies include: 41-O-demethyl-,
> 7-O-demethyl, 12-hydroxy-, 16-O-demethyl, 39-O-demethyl, 27,
> 39-O-di-demethy-, and dihydroxy-sirolimus (Mahalati, 2001). We
> recommend that you spike sirolimus-free whole blood with the
> metabolites to a final concentration consistent with the highest
> concentration expected for the intended use population. When
> metabolites are not available, you may be able to estimate the effect
> of specific metabolites by measuring the metabolites present in
> multiple patient specimens using an appropriate chromatographic method
> and comparing results to your assay. We recommend that you include
> specimens from patients with elevated creatinine concentration when
> available because such patients typically show higher than average
> metabolite concentrations. We recommend that you consult with FDA
> prior to undertaking this alternative type of study.
>
> We recommend that you include the following in the description of your
> evaluation:

-   Types of samples used for spiking.

-   Concentration and purity of metabolite (and parent compound, if
    present) in samples.

-   Computational methods for determining cross-reactivity, including
    number of replicates evaluated.

-   Results (e.g., percent cross-reactivity) for each metabolite.

### *Interference* 

> You should characterize the effects of potentially interfering
> compounds on assay performance. We recommend that you test the
> compounds listed below. If other potentially interfering compounds
> become known during widespread use of the assay, you should test these
> as well.
>
> \(1\) endogenous compounds, such as the following (examples of upper
> limit concentrations are given in parentheses):

-   bilirubin (60 mg/dL)

-   triglycerides (1500 mg/dL)

-   cholesterol (500 mg/dL)

-   uric acid (20 mg/dL)

-   rheumatoid factor (500 IU/ml)

-   hematocrit (15-60%)

-   albumin (12 g/dL)

-   gamma globulin (12 g/dL)

-   human anti-mouse antibodies (HAMA)

> \(2\) commonly co-administered drugs, including drugs listed below. If
> other relevant drugs become known, you should also evaluate these.

-   cyclosporine

-   mycophenolic acid and its metabolite, MPAG

-   acyclovir

-   amphotericin B

-   ciprofloxacin

-   erythromycin

-   fluconazole

-   flucytosine

-   gentamicin

-   itraconazole

-   ketoconazole

-   gancyclovir (and pro-drugs)

-   rifampin

-   tacrolimus

-   tobramycin

-   vancomycin

-   common over-the-counter drugs

> \(3\) anticoagulants or preservatives with which the sample is likely
> to come in contact, such as EDTA.
>
> Interference studies typically involve adjusting sirolimus
> concentrations in whole blood samples to near high and low medical
> decision levels, adding the potential interferent to these samples and
> determining any bias in recovery relative to control sample(s).
> Interference studies using samples naturally high in the endogenous
> compound being tested can be informative and we recommend that you
> consider this approach when such samples are available. Guidelines for
> interference testing are described in detail in "Interference Testing
> in Clinical Chemistry; Approved Guideline" (2002) NCCLS Document
> EP07-A. That document includes guidelines for setting decision
> criteria as well as for protocol designs and statistical methods for
> evaluating interference and establishing validating and verifying
> interference claims. We recommend that you consider the following
> guidelines from that document when planning interference studies.

-   For endogenous substances, test at the highest concentration
    expected based on experience with the intended use population.

-   For drug levels, test to levels 3 times the highest acute peak
    concentration reported following therapeutic dosage.

-   For specimen additives, test up to levels five times the recommended
    concentration.

> If you observe interference at the concentration levels tested, you
> should test lower levels in order to determine the lowest
> concentration that could cause interference. We recommend that you
> test replicate samples in these protocols.

We recommend that you include the following in the description of your
evaluation:

-   Specific compounds and concentrations tested for interference.

-   Sample types and preparation (e.g., spiked whole blood pools,
    samples naturally high in endogenous compounds).

-   Concentrations of sirolimus in the sample.

-   Percent bias (relative to a control sample) for the compounds
    tested, and the definition/calculations you used to determine this.

> When reporting results, we recommend that you identify any observed
> trends in bias (i.e., negative or positive) across the concentration
> range of the compound tested for interference. We recommend that you
> include the standard deviation or range of the observed recoveries at
> the interferent concentrations you evaluate. This approach may be more
> informative than listing only average recoveries.
>
> For substances that you characterize as non-interfering in your
> labeling, you should state the criteria on which this is based, e.g.,
> "inaccuracies due to these substances are less than X% at the
> sirolimus concentrations tested." If any compounds are known from the
> literature or other sources to interfere with the test system, you
> should also include this information.

### *Specimen collection and handling conditions* 

> You should substantiate the recommendations in your package insert for
> specimen storage and transport, by assessing whether the device can
> maintain acceptable performance (e.g., precision, accuracy) over the
> storage times and temperatures, including freeze/thaw cycles, that you
> recommend to users. We recommend that you evaluate sample aliquots
> stored under the conditions of time, temperature, or allowed number of
> freeze/thaw cycles recommended in the package insert. You should state
> the criteria for acceptable range of recoveries under your recommended
> storage and handling conditions. You should also identify any other
> sources of pre-analytical error, such as binding to a specimen
> container or gel.

### *Method comparison* 

> Sirolimus assays vary significantly in terms of cross-reactivity
> patterns with metabolites whose therapeutic and toxic effects are not
> well-defined (Gallant-Haidner, 2000). Therefore, you should compare
> the new assay to a candidate reference method, specific for the parent
> compound. We recommend that you compare your assay to a carefully
> validated high performance liquid chromatography method that measures
> parent drug specifically, such as methods described as reference
> procedures (e.g., Salm, 2000; Streit, 2002).
>
> We recommend that you follow the guidelines provided in the document,
> "Method Comparison and Bias Estimation Using Patient Samples; Approved
> Guideline" (2002), NCCLS Document EP09-A2, concerning experimental
> guidelines and statement of claims. Sirolimus (Rapamune^®^) is
> currently indicated for the prophylaxis of organ rejection in patients
> receiving renal transplants. Therefore, you should evaluate renal
> transplant patient samples with drug concentrations distributed across
> the reportable range of the assay. (If, in the future, the drug and
> assay are indicated for additional transplant populations, these
> should also be included in the evaluation.) Banked (retrospective)
> samples are appropriate for these studies provided that appropriate
> information concerning sample characterization (listed below) is
> available. We recommend including samples from multiple geographic
> sites or clinical centers to enhance the chance that samples will
> represent a broad range of individuals and treatment regimens.
>
> Appropriate sample size depends on factors such as precision,
> interference, assay range, and other performance characteristics of
> the test. The number of patients should also be large enough so that
> inter-individual variation can be observed. We recommend that you
> provide a statistical justification to support the study sample size.
> We recommend that the sample size target, however supported, include
> 100 or more samples distributed fairly evenly over 50 or more
> individual patients.
>
> If you choose to include multiple measurements from individual
> patients, we recommend that you summarize your results of appropriate
> statistical analyses such as Analysis of Variance, Generalized
> Estimating Equations, or Bootstrapping, to account for correlation of
> repeat measurements within patients in the study. If you choose to
> include multiple measurements from individuals, it would be beneficial
> if they range over time post-transplant.
>
> For your results to be properly interpreted, you should provide
> relevant information on the samples tested. We recommend that you
> include the following information:

-   The number of individual patients represented by the samples.

-   The number of data points.

-   The number of clinical sites.

-   Characterization regarding the time of last dose, e.g., trough
    samples. (We currently consider evaluation of trough samples
    sufficient, as long as the sample concentrations span the assay
    range.)

-   Selection (inclusion/exclusion) criteria for samples.

-   Other known sample characteristics relevant to interpretation of
    results.

> Factors such as other co-administered immunosuppressant drugs (e.g.,
> cyclosporine), age range (e.g., adults), and time post-transplant
> (e.g., chronic, acute) can affect drug metabolism and consequently,
> assay bias (Gallant-Haidner, 2000; Lampen, 1998; Kaplan, 1998; Kelly,
> 2002). Therefore, we recommend that you describe these features of the
> general sample population whenever possible. You should also indicate
> if samples were collected from patients with specific clinical
> outcomes, or from centers using atypical or novel drug regimens.
>
> You should clarify the comparator reference method used, and include a
> summary of the validation of that method and references from the
> literature describing the method.
>
> We recommend that you include the following in the results of your
> method comparison:

-   Methods you used to statistically analyze the data.

-   Scatterplots of the new assay versus the reference (e.g., LC-MS)
    method, including all data points, the estimated regression line,
    and the line of identity. Data points in the plot should represent
    individual measurements (unless you are instructing users to report
    an average of multiple measurements).

-   A description of the method used to fit the regression line and
    results of regression analysis, including the slope and intercept
    with their 95% confidence limits, the standard error of the estimate
    (calculated in the y direction), and correlation coefficient. If
    both the comparator and the new assay are subject to measurement
    error, a regression method such as the Deming method may be
    appropriate, rather than Least Squares.

-   Graphs of difference in measurements (i.e., new device minus
    reference method) versus the reference method, to illustrate
    variability. Appropriate representations could include a bias plot
    of difference in measurements (y - x) versus the reference method
    (x), as recommended in NCCLS Document EP9-A , or versus the mean of
    y and x, as recommended by Bland and Altman (Bland, 1995).

> We recommend that you stratify analyses for samples representing
> different patient groups for whom differences in assay bias might be
> expected, if you included such samples in your study. Some examples of
> such groups include samples drawn at different time points with
> respect to dose (e.g., trough samples versus other time points) or
> samples representing patients at various times post-transplant (e.g.,
> acute or chronic).
>
> If the bias in your method comparison exceeds 25% relative to the
> reference procedure, or if the variability in results among patient
> samples is unusually large, you should address the reasons for the
> discordance and describe steps to be taken to minimize risk of patient
> mismanagement that is based on the results of such tests.

### *Studies at external sites* 

> You should demonstrate performance (bias and precision) in at least
> two external sites, in addition to that of the manufacturer's site. We
> recommend that you include this as part of the method comparison study
> described above. You should initially analyze data from individual
> sites separately to evaluate any inter-site variation. Method
> comparison results from the individual sites can be pooled in the
> package insert, if you demonstrate that there are no significant
> differences in results among sites.

### *Calibrator and Control Material*

> We recommend that you provide the following information concerning
> assay calibrators and controls:

-   Protocol description and acceptance criteria for real-time stability
    studies of opened and unopened calibrators and controls. This should
    include the methods or analyses you used and your criteria for
    recovery at the expiration date.

-   Protocol description for value assignment and validation of the
    various calibrator and control levels. This should include the
    methods or analyses used and your results or acceptance criteria for
    recovery.

-   Identification of traceability to a domestic or international
    standard reference material.

-   Protocol and acceptance criteria for the transfer of performance of
    a primary calibrator to a secondary calibrator.

> For information about calibrators marketed separately as class II
> devices under 862.1150, see the guidance \"Abbreviated 510k
> Submissions for *In Vitro* Diagnostic Calibrators,\"
> <http://www.fda.gov/cdrh/ode/calibrator.html>.

# 7. Labeling

The premarket notification should include labeling in sufficient detail
to satisfy the requirements of 21 CFR 807.87(e).[^5] The following
suggestions are aimed at assisting you in preparing labeling that
satisfies these requirements.

> **Specimens**
>
> We recommend that you include the following:

-   Discussion of the importance of consistency and accurate recording
    of time of blood draw with respect to the last dose of sirolimus, if
    relevant for interpretation of results.

-   Discussion of any limitations or instructions related to the
    specimen type, such as appropriate matrices or anticoagulants (in
    most cases, EDTA).

-   Instructions concerning preserving integrity of the specimen, such
    as required temperatures or materials for collection, transport,
    storage (short and long term), and assay procedural steps. Storage
    conditions that you recommend to the user should be based on the
    conditions you have validated for your test system. You should
    clearly define any acceptance criteria that you apply in determining
    the recommended storage conditions. Additional information on
    storage conditions based on literature can be cited if it is
    applicable to your test system.

> **Assay procedure**
>
> We recommend that you include the following:

-   Time limits and temperature requirements for the procedural steps.
    Whenever applicable, you should describe expected appearance of the
    specimen through various procedural steps and advise users of any
    signs that may indicate whether the assay is proceeding correctly.

-   A validated procedure for dilution, if you instruct users to dilute
    samples with values above the highest calibrator.

-   Steps that users can take to minimize the effect of carryover, or
    other causes of bias or irreproducibility, based on procedures you
    have validated for your test system.

> **Quality control**
>
> We recommend that you advise users of the specifics of calibration and
> quality control procedures necessary to ensure performance claims. You
> should include recommendations for appropriate quality control
> specimens. Consensus documents recommend that whole blood assays
> should employ whole blood controls with well-characterized drug
> preparations.
>
> **Limitations**
>
> We recommend that you include limitations such as the following, when
> appropriate for your device type:
>
> Various immunoassays may yield results that differ from each other and
> from chromatographic assays on the same clinical sample. Therefore, it
> is important that the same analytical method be used consistently for
> monitoring immunosuppressant concentrations for an individual patient.
> Laboratories should identify the method used, when reporting results.
>
> Patients with impaired drug metabolism or clearance may show the most
> variation in measured values for immunoassays. For such patients, use
> of this assay may be supported with a chromatographic method more
> specific for the parent compound.
>
> Clinical trials have shown large intrapatient variability observed in
> trough sirolimus concentrations (MacDonald, 2000), indicating that
> optimal dose adjustment should be based on more than a single trough
> sample.
>
> You should identify any exogenous or endogenous factors known to
> affect results and describe the effect on results (e.g., highly
> lipemic samples may cause falsely low results).
>
> We recommend that you cite references that list drugs currently known
> to alter metabolism of sirolimus and to affect blood concentrations of
> the parent or metabolites in an appropriate section of the package
> insert.
>
> **Optimal Concentration Range**
>
> Since the optimal concentration ranges may vary depending on the
> methodology used, as well as the clinical state of the individual,
> stating one specific therapeutic range is usually not appropriate for
> current sirolimus immunoassays**.** You should include cautionary
> explanations concerning the lack of firm optimal concentration ranges
> to the user and discuss both patient variability and test variability.
> For example:
>
> **The optimal concentration range for sirolimus in whole blood using
> this assay has not been established. Optimal concentration ranges vary
> according to the specific assay used, and therefore should be
> established for each specific assay. Values obtained with different
> assay methods should not be used interchangeably due to differences in
> cross-reactivity with metabolites, nor should correction factors be
> applied. Laboratories should include identification of the assay used
> in order to aid in interpretation of results. Each institution should
> establish the optimal ranges based on the specific assay used and
> other factors relevant to their patient population.**
>
> **Optimal ranges depend upon the patient's clinical state, individual
> differences in sensitivity to immunosuppressive and nephrotoxic
> effects of sirolimus, co-administration of other immunosuppressants,
> time post-transplant and a number of other factors. Therefore,
> individual sirolimus values cannot be used as the sole indicator for
> making changes in treatment regimen and each patient should be
> thoroughly evaluated clinically before changes in treatment regimens
> are made.**
>
> **Performance Characteristics**
>
> We recommend that you describe the protocol and results for each
> performance characteristic discussed in Section 6. Your representation
> of protocol and results in the package insert should include
> information cited in Section 6 that would be relevant to aid the user
> in understanding test performance. Results should include scatterplots
> of the new assay versus the reference (e.g., LCMS) method. In some
> cases, graphs or tables of inter-individual variation or equivalent
> information may also be appropriate in order to clearly represent
> results of the method comparison for the user. See also applicable
> sections in the NCCLS guidelines cited in Section 6 concerning
> statements of claims.

# 8. New Instrument Applications

For information concerning application of cleared test systems to
additional analyzers, see the guidance entitled \"Guidance for Industry
and FDA Staff; Replacement Reagent and Instrument Family Policy,\"
available at
[[http://www.fda.gov/cdrh/oivd/guidance/950.html]{.underline}](http://www.fda.gov/cdrh/oivd/guidance/950.html ).
The approach described in that guidance is appropriate in cases when
performance characteristics with a new analyzer meet your pre-defined
acceptance criteria using a proper validation protocol. If performance
characteristics do not meet your pre-determined acceptance criteria, a
Special 510(k) is appropriate.

When the new analyzer does not involve any changes in reagents, sample
treatment, or assay procedure that could affect cross-reactivity or
partitioning of metabolites, you might determine that it is sufficient
to compare samples using the new instrument to the previously cleared
instrument. In this case, we recommend that you still include results of
the original method comparison for the test system versus the LCMS
reference procedure in the package insert, so that users can properly
interpret results. When application to a new analyzer also includes
changes that could affect cross-reactivity, we recommend that you
compare the new assay to a reference method, in order to validate that
performance is not affected.

# 9. References 

Bland JM, Altman DG. Comparing methods of measurement: Why plotting
difference against standard method is misleading. *Lancet*
1995;346:1085-1087.

Gallant-Haidner HL, Trepanier DJ, Freitag DG, Yatscoff RW.
Pharmacokinetics and metabolism of sirolimus. *Ther Drug Monit*
2000;22:31-5.

Holt DW, Lee T, Jones K, Johnston A. Validation of an assay for routine
monitoring of sirolimus using HPLC with mass spectrometric detection.
*Clinical Chemistry* 2000;46:1179-1183.

Jones K, Saadat-Lajevardi S, Lee T, Horwatt R, Hicks D, Johnston A, Holt
DW. An immunoassay for the measurement of sirolimus, *Clinical
Therapeutics* 2000;22 (Suppl B):B49-B61.

Kaplan B, Meier-Kriesche H, Napoli KL, Kahan BD. The effects of relative
timing of sirolimus and cyclosporine microemulsion formulation
co-administration on the pharmacokinetics of each agent. *Clin Pharmacol
Ther* 1998;63:48-53.

Kelly P, Kahan BD. Review: metabolism of immunosuppressant drugs. *Curr
Drug Metab* 2002; (3):275-87.

Lampen A, Zhang Y, Hackbarth I, Benet LZ, Sewing KF, Christians U.
Metabolism and transport of the macrolide immunosuppressant sirolimus in
the small intestine. *J Pharmacol Exp Ther* 1998;285:1104-12.

MacDonald A, Scarola J, Burke JT, Zimmerman JJ. Clinical
pharmacokinetics and therapeutic drug monitoring of sirolimus, in Shaw,
LM (ed.), Advances in Therapeutic Drug Monitoring for
Immunosuppressants: A review of Sirolimus. *Clinical Therapeutics*
2000;22 (Suppl B): B101-B121.

Mahalati K, Kahan BD. Clinical pharmacokinetics of sirolimus. *Clinical
Pharmacokinetics* 2001;40:573-585.

Meier-Kriesche H, Kaplan B. Toxicity and efficacy of sirolimus:
relationship to whole blood concentrations. *Clinical Therapeutics*
2000;22 (Suppl B):B93-B100.

Salm P, Taylor PJ, Pillans PI. The quantification of sirolimus by
high-performance liquid chromatography-tandem mass spectrometry and
microparticle enzyme immunoassay in renal transplant recipients.
*Clinical Therapeutics* 2000;22 (Suppl B):B71-B85.

Streit G, Armstrong VW, Oellerich M. Rapid liquid chromatography-tandem
mass spectrometry routine method for simultaneous determination of
sirolimus, everolimus, tacrolimus, and cyclosporin A in whole blood.
*Clinical Chemistry* 2002;48:955-958.

Yatscoff RW, Boeckx R, Holt DW, Kahan BD, LeGatt DF, Sehgal S, Soldin
SJ, Napoli K, Stiller C. Consensus guidelines for therapeutic drug
monitoring of rapamycin: report of the consensus panel. *Ther Drug
Monit* 1995;17(6):676-80.

[^1]: <http://www.fda.gov/cdrh/ode/parad510.html>

[^2]: Refer to
    [http://www.fda.gov/cdrh/ode/indicate.html](http://www.fda.gov/cdrh/ode/indicate.html )
    for the recommended format.

[^3]: If FDA makes a substantial equivalence determination based on
    acceptance criteria, the subject device should be tested and shown
    to meet these acceptance criteria before being introduced into
    interstate commerce. If the finished device does not meet the
    acceptance criteria and, thus, differs from the device described in
    the cleared 510(k), FDA recommends that submitters apply the same
    criteria used to assess modifications to legally marketed devices
    (21 CFR 807.81(a)(3)) to determine whether marketing of the finished
    device requires clearance of a new 510(k).

[^4]: See Required Elements for a Declaration of Conformity to a
    Recognized Standard (Screening Checklist for All Premarket
    Notification \[510(K)\] Submissions),
    <http://www.fda.gov/cdrh/ode/reqrecstand.html>.

[^5]: Although final labeling is not required for 510(k) clearance,
    final labeling must also comply with the requirements of 21 CFR 801
    and 21 CFR 809.10 before a medical device is introduced into
    interstate commerce. Labeling recommendations in this guidance are
    consistent with the requirements of part 801 and section 809.10.