Predicate Devices

Not Found

Reference Devices

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes a PCR and capillary electrophoresis-based in vitro diagnostic device for genetic testing. There is no mention of AI or ML in the intended use, device description, performance studies, or key metrics. The analysis is based on established molecular biology techniques.

Therapeutic

No
The device is an in vitro diagnostic (IVD) device used to detect and identify CGG repeats in the FMR1 gene, aiding in the diagnosis and carrier screening of Fragile X related disorders. It is not intended for treatment or direct therapy.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the device is "an in vitro diagnostic device" and is "intended as an aid in the post-natal diagnosis of fragile X syndrome, and fragile Xassociated disorders... and for carrier testing in adults of reproductive age." This directly indicates its diagnostic purpose.

SaMD (Software as a Medical Device)

No

The device is an in vitro diagnostic kit that includes reagents and is used with a specific hardware analyzer (Applied Biosystems® 3500 Dx Series Genetic Analyzer). While it mentions "AmplideX reporter software," this software is part of a larger system that includes physical components and chemical reagents, making it a hardware/software system, not a software-only medical device.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

The document explicitly states in the "Intended Use / Indications for Use" section:

"The AmplideX® Fragile X Dx & Carrier Screen Kit is an in vitro diagnostic device that uses polymerase chain reaction (PCR) and capillary electrophoresis to detect and identify the number of cytosine-guanine (CGG) repeats in the fragile X mental retardation-1 (FMR1) gene using genomic DNA isolated from peripheral whole blood specimens."

This statement directly identifies the device as an in vitro diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The AmplideX® Fragile X Dx & Carrier Screen Kit is an in vitro diagnostic device that uses polymerase chain reaction (PCR) and capillary electrophoresis to detect and identify the number of cytosine-guanine (CGG) repeats in the fragile X mental retardation-1 (FMR1) gene using genomic DNA isolated from peripheral whole blood specimens. It is solely intended as an aid in the post-natal diagnosis of fragile X syndrome, and fragile Xassociated disorders [i.e., fragile X-associated tremor/ataxia syndrome (FXTAS) or fragile Xassociated primary ovarian insufficiency (FXPOI)], and for carrier testing in adults of reproductive age. Assay results are solely intended to be interpreted by healthcare professionals who are board certified in molecular genetics and to be used in conjunction. with other clinical and diagnostic findings, consistent with professional standards of practice. Reflex testing, clinical genetic evaluation, and genetic counseling should be offered as appropriate. The test is for use on the 3500Dx Series Genetic Analyzer.

This test is not indicated for use for fetal diagnostic testing, newborn screening or for standalone diagnostic purposes.

Product codes (comma separated list FDA assigned to the subject device)

OYV

Device Description

The AmplideX Fragile X Dx & Carrier Screen Kit (hereafter referred to as the AmplideX Kit) includes reagents sufficient for 100 reactions and are for use on the Applied Biosystems® 3500 Dx Series Genetic Analyzer (8 and 24 capillary) with AmplideX reporter software. A description of the reagents and the storage conditions are shown in Table 1 below.

Item	Storage Temperature
GC-Rich Amp Buffer	-15 to -30°C
GC-Rich Polymerase Mix	-15 to -30°C
ROX 1000 Size Ladder	-15 to -30°C
Diluent	-15 to -30°C
Fragile X Positive Control	-15 to -30°C
Fragile X Primer Mix	-15 to -30°C

Additional reagents required but not provided with the kit:

Reagents for isolation and purification of genomic DNA (gDNA)
POP-7 ™ Polymer
Hi-Di™ Formamide
DS-30 Matrix Standard Kit (Dye Set D)

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

Post-natal diagnosis, adults of reproductive age.

Intended User / Care Setting

Healthcare professionals who are board certified in molecular genetics.

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Diagnostic Performance Study:

Sample Size: 207 leftover specimens (111 female, 96 male).
Data Source: Patient samples submitted for routine FMR1 5' UTR mutation testing across clinical sites. Specimens were selected using a specific protocol to minimize bias.
Annotation Protocol: Specimens were categorized into three groups (full mutation, premutation, and normal/intermediate) based on available Southern Blot results.

Carrier Screening Performance Study:

Sample Size: 207 specimens from females (years of age). 80 evaluable subjects.
Data Source: Specimens from clinical sites.
Annotation Protocol: Categorization into Full Mutation/Premutation, Intermediate, or Normal genotypes using an alternate orthogonal independently validated PCR assay (FMR1 Dual-PCR Test) as the comparator. The FMR1 Dual-PCR Reference Method result is the average of the longest CGG repeat counts from each of the two sets of primers.

Accuracy of Mosaic Alleles:

Sample Size: 49 specimens from the two clinical studies (27 from diagnostic, 22 from carrier screening) where mosaicism was detected by AmplideX. 35 of these had no mosaic results reported by orthogonal PCR method. 40 randomly selected samples were further evaluated for unbiased resolution of mosaic results, leading to 34 samples for detailed analysis (31 no evidence of mosaicism, 3 yielded mosaic results by both methods). Total of 83 independent specimens, 4 discordant after manual evaluation.
Data Source: Specimens from the clinical validation studies.
Annotation Protocol: Manual evaluation of orthogonal PCR method results for mosaicism.

Testing of International WHO Standards:

Sample Size: 5 samples from the WHO International Standard Fragile X Syndrome Reference Panel (NIBSC code: 08/158).
Data Source: WHO International Standard Fragile X Syndrome Reference Panel.
Annotation Protocol: A single operator performed each reaction in triplicate across three different kit lots. 135 total sample measurements collected, 134 passed QC.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

1. Analytical Performance:

Precision (Repeatability and Reproducibility - 3 site reproducibility):
- Study Type: Multi-site reproducibility study.
- Sample Size: Panel of 11 samples (7 clinical whole blood, 4 contrived) representing all FXS genotype categories.
- Key Results: 1980 total sample measurements (excluding controls), 1.1% invalid rate. For 10 of 11 samples, >97% genotype agreement was observed. Sample ASGN-018 showed 54.7% agreement for Full Mutation due to mosaicism not being found in 81/179 replicates. Reproducibility of allele size (number of repeats) for expected allele lengths (excluding those identified as "mosaic") showed >98% reproducibility based on prespecified precision criteria.
Precision (lot-to-lot reproducibility):
- Study Type: Within-laboratory precision study.
- Sample Size: Same 11-sample panel from the 3-site reproducibility study.
- Key Results: 2376 total sample measurements (excluding controls), 2.5% invalid rate. Greater than >99% categorical genotype agreement observed for all samples (excluding invalids). ASGN-018 showed 52.8% agreement for Full Mutation but full agreement if bordering categories (pre- or full mutation) are allowed. Reproducibility of all alleles (excluding "mosaic" alleles) showed 100% reproducibility with specifications.
Precision (Mosaicism):
- Study Type: Reproducibility/LoD study for mosaicism at minimal DNA input.
- Sample Size: A panel of samples representing different mosaic/major allele combinations generated by mixing DNA from 7 clinical specimens. Each panel member tested with 3 lots in duplicates by 2 operators across 3 days (36 replicate measurements per specimen).
- Key Results: Greater than 95% hit rate demonstrated for claimed LoD for various mosaic allele combinations (e.g., Intermediate-Normal 2.0% MAF, Premutation-Normal 2.0% MAF, Full Mutation-Normal 6.1% MAF, etc.).
DNA extraction reproducibility/equivalence:
- Study Type: Methods comparison.
- Sample Size: 5-member sample panel (all FMR1 genotype categories).
- Key Results: Three common commercial DNA extraction methods assessed. All three yielded sufficient DNA. 100% categorical genotype agreement and 100% of measured alleles within target precision range.
Thermal Cycler equivalence:
- Study Type: Thermal cycler comparison.
- Sample Size: 5-member sample panel (all FMR1 genotype categories).
- Key Results: Tested on three separate thermal cyclers. 100% genotype category agreement and 100% of identified alleles within respective target precision ranges.
Detection Limit (Limit of Detection for mosaic alleles):
- Study Type: LoD confirmation.
- Sample Size: Panel of 30 samples (different mosaic/major allele combinations). Each panel member tested with 3 lots in duplicates by 2 operators across 3 days (36 replicate measurements per specimen).
- Key Results: Greater than 95% hit rate demonstrated for claimed LoD for mosaic alleles (Table 10).
DNA Input:
- Study Type: Robustness study.
- Sample Size: Panel of 8 samples (6 clinical, 2 contrived). 960 total sample measurements.
- Key Results: 100% categorical genotype agreement for all valid samples and input levels (20-80ng is recommended range). QC failure rate was low within the recommended range.
Interfering Substances (Endogenous Interference):
- Study Type: Interference testing.
- Sample Size: Panel of samples.
- Key Results: Hemoglobin and EDTA affected DNA yield. For processed samples, genotype category agreement > 95% and 100% of alleles within precision range.
Stability (Specimen Handling Stability):
- Study Type: Specimen storage stability.
- Sample Size: 14 clinical specimens.
- Key Results: 100% genotype category agreement and all allele peaks within target precision for samples stored up to 2 weeks at 2-8 °C.
Stability (DNA freeze/thaw Stability):
- Study Type: Freeze/thaw cycle stability.
- Sample Size: A contrived sample panel.
- Key Results: 100% genotype category agreement and all allele peaks within target precision for up to (b)(4) freeze-thaw cycles.
Stability (PCR & Capillary Electrophoresis (CE) Reagent Product Stability):
- Study Type: PCR product and CE product stability.
- Sample Size: 5-member sample panel (4 clinical + 1 contrived).
- Key Results: PCR products stable for up to 3 days at 2-8 °C (100% categorical genotype agreement, >95% alleles within range). CE samples stable at ambient temperature for up to 24 hours (100% agreement, 100% alleles within range).
Stability (Real Time Kit Stability):
- Study Type: Real-time kit storage stability.
- Sample Size: 5-member sample panel.
- Key Results: 100% categorical genotype agreement and 100% alleles within precision range for at least 1 year storage at -30°C to -15°C. Ongoing for 25-month to support 24-month stability.
Stability (Freeze/Thaw Kit Stability):
- Study Type: Kit freeze/thaw stability.
- Sample Size: 5-member sample panel.
- Key Results: 100% categorical genotype agreement and 100% alleles within precision range for up to eight freeze/thaw cycles.
Stability (Kit Shipping Stability):
- Study Type: Shipping simulation.
- Sample Size: 5-member sample panel.
- Key Results: 100% of measured alleles within target precision range and 100% genotype category agreement after ISTA 7D, 24-hour summer temperature profile.

2. Clinical Performance:

Diagnostic Performance (Full Mutation):
- Study Type: Clinical validation study comparing AmplideX to Southern Blot.
- Sample Size: 207 specimens.
- Key Results:
  - Positive Percent Agreement (PPA): 95.71%
  - Negative Percent Agreement (NPA): 99.3%
  - Overall Percent Agreement (OPA): 97.6%
  - One of 3 discordant samples was borderline (194-repeat by AmplideX, >200 by Southern Blot).
Diagnostic Performance (Premutation vs. Normal or Intermediate assessment):
- Study Type: Clinical validation study comparing AmplideX to Southern Blot.
- Sample Size: Not explicitly stated, inferred from 138 in contingency table (69 positive, 69 negative).
- Key Results:
  - PPA: 100.0%
  - NPA: 97.1%
  - OPA: 98.6%
Carrier Screening Performance:
- Study Type: Clinical validation study comparing AmplideX to FMR1 Dual-PCR.
- Sample Size: 207 specimens from females.
- Key Results:
  - Agreement for Premutation/Full Mutation: 100.0%
  - Agreement for Intermediate: 85.7% (97.1% when accounting for assay precision with 8 borderline samples)
  - Agreement for Normal: 98.6%
  - Method comparison via Passing-Bablok regression analysis for CGG repeat length: slope of 1.0 (95% CI: 1.0-1.0), intercept of 0 (95% CI: 0.0-0.0), indicating no measurable deviation from identity.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

**Diagnostic Performance (Full Mutation vs.

Regulation Number and Section

§ 866.5970 Inherited nucleotide repeat disorder DNA test.

(a)
Identification. An inherited nucleotide repeat disorder DNA test is a prescription in vitro diagnostic device that is intended to detect and identify the number of nucleotide repeats in a gene using genomic DNA isolated from post-natal patient specimens. It is solely intended as an aid for carrier testing and as an aid for the diagnosis of inherited nucleotide repeat-associated disorders. Assay results are solely intended to be used in conjunction with other clinical and diagnostic findings. These tests do not include those indicated for use for fetal diagnostic testing or newborn screening.(b)
Classification. Class II (special controls). The special controls for this device are:(1) The intended use on the device's label required under § 809.10(a)(2) of this chapter and device's labeling required under § 809.10(b)(2) of this chapter must include a statement that assay results are solely intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, and that reflex testing, clinical genetic evaluation, and genetic counseling should be offered as appropriate.
(2) The labeling required under § 809.10(b) of this chapter must include:
(i) A warning that mosaicism detected in one tissue may not reflect mosaicism in other tissues and that the significance of mosaicism should be interpreted with caution in conjunction with other laboratory and clinical information (
e.g., sex of patient, diagnostic testing or carrier screening, patient symptoms) and should include appropriate genetic counseling.(ii) A prominent statement that this test is not indicated for use for fetal diagnostic testing, newborn screening or for stand-alone diagnostic purposes.
(iii) Information that addresses how to interpret different result outputs specific to the technology, such as (peaks) in the electropherograms.
(3) Design verification and validation must include the following:
(i) Appropriate design features and control elements incorporated into the testing procedure that mitigate the risk of incorrect clinical results. These include controls as determined acceptable by FDA that:
(A) Enable the user to determine when the amplification may yield incorrect results,
(B) Enable the user to determine when cross contamination may have occurred;
(C) Software risk control measures that address device system hazards;
(D) Provide software traceability that ensures all hazards are adequately controlled and that all controls have been validated in the final device design; and
(E) Ensure the instructions for use and test reports appropriately inform the user about the limitations of the assay.
(ii) Validated and acceptable, as determined by FDA, criteria for test result interpretation and reporting, including result outputs.
(iii) Acceptable, as determined by FDA, evidence demonstrating the clinical validity of the device which supports each indicated diagnostic use, including for each genotype and associated phenotype used in providing a clinical determination for the target population.
(iv) Evidence demonstrating acceptable, as determined by FDA, analytical device performance. Patient specimens must represent the full spectrum of expected clinical results and be obtained through unbiased collection. Specimens must be representative of all categories of results and across the range of repeat sizes (
e.g., categories and repeat sizes for Fragile X syndrome are: normal 1-44 repeats; intermediate 45-54 repeats; premutation 55-200 repeats, full mutation greater than 200 repeats), across a range of allelic combinations, be near decision points, and be from both male and female subjects. The number of specimens tested must be sufficient to obtain unbiased estimates of device performance. Analytical validation must include data demonstrating acceptable, as determined by FDA:(A) Agreement with a comparator method(s) determined to be acceptable by FDA. This evidence must demonstrate the accuracy for detecting the size of the nucleotide repeats and the diagnostic categorical calls in DNA in the indicated specimen type(s) from patients that are representative of the intended use population. Accuracy must be assessed for both diagnostic and carrier subsets independently.
(B) Device precision including repeatability and reproducibility, using clinical samples. The study must evaluate all possible sources of variability including, as appropriate, between-site and between operator at a minimum of three sites of which two must be external with a minimum of two operators per site, between-day on a minimum of 3 non-consecutive days, between-run, within-run, between-lot in a minimum of three lots, and between instrument on a minimum of three instruments. Precision must be demonstrated per specimen and determine for both categorical call and by the size of the repeat (
i.e., the percentage of replicates for which the allele fell within the target precision size range). Precision data must be calculated and presented with and without results determined to be invalid.(C) Device performance at the limit of detection of each allele across the range of sizes and as a function of the indicated DNA input for the assay.
(D) Specificity of the reagents for their targets, absence of cross-reactivity, evaluation of sources of interference relevant to the specimen type, and a demonstration of the absence of cross contamination.
(E) Performance of the pre-analytical methods, including DNA extraction methods.
(F) Performance of the device across the range of indicated DNA input concentrations for the assay.
(G) Specimen stability throughout indicated specimen storage ranges, including under expected storage and transport conditions.
(v) Robust evidence demonstrating that the number and frequency of incorrect results due to mosaicism are clinically acceptable, as determined by FDA.
(vi) An appropriate traceability plan to minimize the risk of incorrect results over time, including a description of the molecular size standards and other reagents that may be required for result interpretation, as applicable, that demonstrate the reliable interpretation of the size of the fragments.
(vii) Acceptable, as determined by FDA, device stability protocols and acceptance criteria, that are sufficient to ensure indicated analytical and clinical performance throughout the indicated device stability period. The protocols and acceptance criteria must be adequate to demonstrate that there is no degradation in signal intensity of full mutations when testing a specimen at the latest indicated time point within the indicated device stability that is comprised of the lowest indicated DNA input that can be used.

Summary

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EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR AmplideX® Fragile X Dx & Carrier Screen Kit DECISION SUMMARY

A. DEN Number:

DEN190023

B. Purpose for Submission:

De Novo request for evaluation of automatic class III designation for the AmplideX® Fragile X Dx & Carrier Screen Kit

C. Measurands:

Trinucleotide (CGG) repeats in the FMR1 gene

D. Type of Test:

Polymerase chain reaction (PCR)-based nucleic acid amplification followed by size resolution with capillary electrophoresis

E. Applicant:

Asuragen, Inc.

F. Proprietary and Established Names:

AmplideX® Fragile X Dx & Carrier Screen Kit

G. Regulatory Information:

1. Regulation section:
  21 CFR 866.5970
1. Classification:
  Class II
1. Product code(s):
  OYV
1. Panel:
  Immunology (82)

1

H. Indications for use:

1. Indications for use:

The AmplideX® Fragile X Dx & Carrier Screen Kit is an in vitro diagnostic device that uses polymerase chain reaction (PCR) and capillary electrophoresis to detect and identify the number of cytosine-guanine (CGG) repeats in the fragile X mental retardation-1 (FMR1) gene using genomic DNA isolated from peripheral whole blood specimens. It is solely intended as an aid in the post-natal diagnosis of fragile X syndrome, and fragile Xassociated disorders [i.e., fragile X-associated tremor/ataxia syndrome (FXTAS) or fragile Xassociated primary ovarian insufficiency (FXPOI)], and for carrier testing in adults of reproductive age. Assay results are solely intended to be interpreted by healthcare professionals who are board certified in molecular genetics and to be used in conjunction. with other clinical and diagnostic findings, consistent with professional standards of practice. Reflex testing, clinical genetic evaluation, and genetic counseling should be offered as appropriate. The test is for use on the 3500Dx Series Genetic Analyzer.

This test is not indicated for use for fetal diagnostic testing, newborn screening or for standalone diagnostic purposes.

1. Special conditions for use statement(s):
  For prescription use

For in vitro diagnostic use

1. Special instrument requirements:
  Applied Biosystems® 3500 Dx Series Genetic Analyzer (K191030) using the AmplideX Fragile X Reporter Software v 1.0.

I. Device Description:

The AmplideX Fragile X Dx & Carrier Screen Kit (hereafter referred to as the AmplideX Kit) includes reagents sufficient for 100 reactions and are for use on the Applied Biosystems® 3500 Dx Series Genetic Analyzer (8 and 24 capillary) with AmplideX reporter software. A description of the reagents and the storage conditions are shown in Table 1 below.

Item	Storage Temperature
GC-Rich Amp Buffer	-15 to -30°C
GC-Rich Polymerase Mix	-15 to -30°C
ROX 1000 Size Ladder	-15 to -30°C
Diluent	-15 to -30°C
Fragile X Positive Control	-15 to -30°C
Fragile X Primer Mix	-15 to -30°C

Table 1. Components of the AmplideX Kit

2

Additional reagents required but not provided with the kit:

Reagents for isolation and purification of genomic DNA (gDNA)
POP-7 ™ Polymer ●
Hi-Di™ Formamide ●
DS-30 Matrix Standard Kit (Dye Set D) ●

J. Standard/Guidance Document Referenced (if applicable):

Guidance for Industry and FDA Staff: Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices; May 11, 2005

General Principles of Software Validation; Final Guidance for Industry and FDA Staff; January 11,2002

Guidance for Industry - Cybersecurity for Networked Medical Devices Containing Off-the- Shelf (OTS) Software; January 14, 2005

Guidance for Industry. FDA Reviewers and Compliance on Off-the-Shelf Software Use in Medical Devices: September 9, 1999

CLSI Standard EP05-A3, Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline - Second Edition.

CLSI Standard EP07-A2, Interference Testing in Clinical Chemistry, Approved Guideline -Second Edition.

CLSI Standard EP17-A2, Protocols for the Determination of Limits of Detection and Limits of Quantification, Approved Guideline - Second Edition.

CLSI Standard EP25-A, Evaluation of Stability on In Vitro Diagnostic Reagents, Approved Guideline.

K. Test Principle:

Fragile X syndrome (FXS) and Fragile X associated disorders are inherited genetic conditions characterized by expanded cytosine-guanine (CGG) nucleotide repeats in the 5' untranslated region of the FMR1 gene. Expansion of the repeats is associated with hypermethylation and inactivation of gene expression and subsequent loss of protein expression. This expansion can lead to a variety of consequences depending on the length of the CGG expansion. The AmplideX Fragile X Dx & Carrier Screen Kit quantifies the number of CGG repeats in the alleles of a given sample of purified gDNA using polymerase chain reaction (PCR) followed by size resolution with capillary electrophoresis (CE).

Human gDNA is isolated from EDTA-treated whole human blood (fresh or stored at 2 to 8 °C for up to 14 days) using a DNA extraction kit capable of gDNA purity A260280 range of 1.6-2.0. A total DNA input of 20-80ng is needed to run the reaction. Purified gDNA is first added to a PCR reaction well containing a Master Mix with the GC-Rich Amp Buffer, GC-Rich Polymerase Mix, Fragile X Primer Mix, and either a specimen or control (Fragile X Diluent or Fragile X

3

Positive Control) in a total of 13 uL. After thermal cycling, unpurified PCR products are directly mixed with Hi-Di™ Formamide and ROX 1000 Size Ladder. Following denaturation, amplicons are resolved on an Applied Biosystems® 3500 Dx Genetic Analyzer running POP-7™ polymer and analyzed using the AmplideX Fragile X Reporter.

Calibration

The ROX 1000 Size Ladder included in the kit is added to each sample to allow for proper fragment sizing analysis by the AmplideX® Fragile X Reporter. Spectral calibration of the 3500 Dx instrument with DS-30 Matrix Standard Kit is required in order to properly detect and analyze samples containing ROX dye and is performed prior to the assay run.

Quality Control

The AmplideX Fragile X Reporter performs a series of OC checks on the batch controls No Template control (NTC) and Positive Control (PC) as well as on the individual samples. The software assesses three general modalities of failure: Ladder Error (LD), Low Signal (LS), and Contamination (CT). If one or more of these failures occurs in the positive control, it invalidates the run as a Batch QC failure. The NTC is expected to give a Low Signal (LS) error.

Controls

Fragile X Positive Control and Diluent (for use as an NTC) are included in the kit. A single positive and no template control must be included in every run for the analysis software to interpret sample results correctly. The NTC should not generate any peak calls from the Reporter output (Figure 5). The Positive Control includes alleles at 18 ±1, 30 ±1, 32 ±1, 56 ±1, 85 ±3, 114 ±3, and > 200 CGG repeats, and yields a full mutation genotype (Figure 1).

Image /page/3/Figure/7 description: The image shows a graph with Fragment Size (bp) on the x-axis and RFU on the y-axis. There are several peaks on the graph, labeled with their corresponding fragment sizes: 18, 30, 32, 56, 85, 114, and >200. The peaks represent the relative fluorescence units (RFU) at different fragment sizes. The graph spans from approximately 150 bp to 1200 bp.

Figure 1. An electropherogram of a positive control (PC)

For a valid run, the AmplideX Fragile X Reporter output will identify each of these alleles within the expected precision range (Table 2) for their respective CGG repeat length.

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CGG Repeat Range	Precision
1-70	$\pm$ 1
71-120	$\pm$ 3
121-199	$\pm$ 5%
≥ 200	N/A

Table 2. CGG Repeat Size Precision

Interpretation of Results

AmplideX Fragile X Reporter software provides automated analysis of the resulting electropherogram to identify and size FMR1 alleles. It then categorizes the genotype of the sample into one of the following categories based on the size of the repeats as a Normal (NOR), Intermediate (INT), Premutation (PM), or Full mutation (FM) as shown in Table 3:

Genotype	Size (CGG) repeats
Normal	1-44
Intermediate	45-54
Premutation	55-199
Full Mutation	$\ge$ 200

Table 3. FMR1 CGG repeat length ranges and their corresponding genotypes

Individuals with normal (5'-UTR FMR1

Figure 2. FMR1 CGG repeat length ranges and their corresponding genotypes and outcomes.

Mosaicism

Mosaicism can result when a new genetic alteration arise after an embryo is formed. Both size mosaicism and methylation mosaicism occur in fragile X syndrome. The kit may also detect low levels of genetic mosaicism in CGG repeat length. The AmplideX® Fragile X Dx & Carrier Screen Kit reports all detected FMR1 alleles for each sample and determines the associated genotype category based on the longest detected allele. The assay does not distinguish between mosaic and non-mosaic alleles. The kit is not designed to detect or report methylation status.

Due to the challenges associated with size mosaicism, (e.g., due to both accurate or false results) when the assay detects more than one (1) peak for a male or more than two (2) peaks for a female, users are informed in the instructions for use to carefully review the electropherogram; and recommended to confirm the presence of size mosaicism and considered in conjunction with other laboratory and clinical information (e.g., sex of patient, diagnostic testing or carrier screening, patient symptoms) and should include consultation with a genetic professional..

L. Performance Characteristics:

1. Analytical performance:

Precision (Repeatability and Reproducibility) a.
The precision of the AmplideX Fragile X Kit was assessed based on both agreement for the categorical call (i.e., normal, intermediate, premutation, and full mutation) and for precision of the size of the CGG repeats (i.e., measurements of fragile X allele lengths). Within the precision studies, specific target criteria around CGG repeat length were applied, as shown in Table 2 above.

i. Precision (3 site reproducibility)

Between-laboratory precision, or reproducibility, was assessed using a panel of 11 samples consisting of 7 clinical whole blood specimens and 4 contrived samples that collectively represent allele sizes spanning all FXS genotype categories, including those at or near category boundaries. Contrived samples were genomic DNA extracted from cell lines with known gender, genotype, and expected allele sizes, spiked into leukocyte-depleted whole human blood. Table 4

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describes the panel members.

| Sample ID | Sex | Source | Expected Number
CGG Repeats | Genotype Category |
|-----------|--------|--------|--------------------------------|-------------------|
| ASGN-109 | (b)(4) | | | Normal |
| ASGN-112 | | | | Normal |
| ASGN-005 | | | | Intermediate |
| ASGN-111 | | | | Intermediate |
| ASGN-103 | | | | Premutation |
| ASGN-113 | | | | Premutation |
| ASGN-101 | | | | Premutation |
| ASGN-016 | | | | Premutation |
| ASGN-018* | | | | Full Mutation* |
| ASGN-023 | | | | Full Mutation |
| ASGN-104 | | | | Full Mutation |

Table 4. Sample Panel used in precision and reproducibility study

ASGN-018 has a full-mutation mosaic allele at >200 repeats and therefore this is the call made by the software, a normal allele at 20 repeats, and a premutation allele at 197 repeats. Mosaic results that do not lead to categorical change not listed above.

The study was executed at three laboratories over 5 non-consecutive days utilizing a single kit lot, two operators per site, and a different 3500Dx instrument at each site. For each run, there were 6 PCR replicates per specimen. The study yielded 1980 total sample measurements excluding controls (3 sites x 2 operators x 6 replicates x 5 days = 180 replicates per sample). Of these, 30 replicates failed QC metrics (1.1% invalid rate) and were further evaluated for genotype category agreement and allele peak precision. The agreement was determined based upon the majority call.

The reproducibility of the assay for categorical call is shown in Table 5. For 10 of 11 samples, >97% genotype agreement was observed. Sample ASGN-005, ASGN-111 and ASGN-113 were not 100% due to the appearance of an extra allele which resulted in different categorical calls for 11 replicates because the assay software is designed to yield an interpretation based on the largest repeat fragment observed. These results were not observed for these samples in the lotto-lot precision study. ASGN-018 has a full-mutation mosaic allele at >200 repeats, a normal allele at 20 repeats, and a premutation allele at 197 repeats. The mosaic allele was not found in 81/179 replicates. A limitation statement in the instructions for use alerts users to carefully review and confirm mosaic results.

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| Sample | Genotype
Category | Total
number of
Replicates
(of 180) | Number of
Replicates
in
Agreement | Agreement
(%) | CI (95%) |
|----------|----------------------|----------------------------------------------|--------------------------------------------|------------------|------------|
| ASGN-109 | Normal | 178 | 178 | 100 | 97.9, 100 |
| ASGN-112 | Normal | 178 | 178 | 100 | 97.9, 100 |
| ASGN-005 | Intermediate | 179 | 177 | 98.9 | 96, 99.7 |
| ASGN-111 | Intermediate | 175 | 170 | 97.1 | 93.5, 98.8 |
| ASGN-103 | Premutation | 178 | 178 | 100 | 97.9, 100 |
| ASGN-113 | Premutation | 179 | 177 | 98.9 | 96, 99.7 |
| ASGN-101 | Premutation | 174 | 174 | 100 | 97.8, 100 |
| ASGN-016 | Premutation | 178 | 178 | 100 | 97.9, 100 |
| ASGN-018 | Full Mutation | 179 | 98 | 54.7 | 47.4, 61.9 |
| ASGN-023 | Full Mutation | 177 | 177 | 100 | 97.9, 100 |
| ASGN-104 | Full Mutation | 175 | 175 | 100 | 97.9, 100 |

Table 5. Three sites reproducibility-Categorical Genotype Agreement by Sample Excluding Invalids.

The reproducibility of the assay for allele size (number of repeats) is shown in Table 6 for each allele in the sample. Reproducibility was based on identifying the mode for each repeat. Replicates results were considered to agree if they fell within the precision specifications described in Table 2. As noted in the reproducibility for categorical calls, 11 replicates had artifacts and are shown below. The reproducibility of the expected allele lengths with the exception of the alleles identified as "mosaic" showed >98% reproducibility based on the prespecified precision criteria. The samples with contaminants/artifacts are listed for information purposes.

Table 6. Three sites Reproducibility- Percent of Allele Replicates within Target Precision Range Excluding Invalids.

| Sample ID | Mode Expected
CGG Length
(± Target
Precision) | Measured
CGG
Length | No of
replicates
at that
size | Total
Number of
replicates | % within
Target
Precision
Range |
|-----------|-------------------------------------------------------------|---------------------------|----------------------------------------|----------------------------------|------------------------------------------|
| ASGN -109 | $30 \pm 1$ | 29 | 1 | 178 | 100.0 |
| | | 30 | 177 | | |
| ASGN-112 | $30 \pm 1$ | 30 | 177 | 178 | 100.0 |
| | | 31 | 1 | | |
| | $29 \pm 1$ | 29 | 177 | 178 | 100.0 |
| Sample ID | Mode Expected
CGG Length
( $\pm$ Target
Precision) | Measured
CGG
Length | No of
replicates
at that
size | Total
Number of
replicates | % within
Target
Precision
Range |
| | | 30 | 1 | | |
| | Contamination/
artifact | 34 | 1 | 1 | 0.6% |
| ASGN-005 | 29 $\pm$ 1 | 29 | 179 | 179 | 100.0 |
| | 45 $\pm$ 1 | 45 | 141 | | 100.0 |
| | | 46 | 38 | | |
| | Contamination/
artifact | > 200 | 2 | 2 | 1.1 |
| ASGN-111 | 29 $\pm$ 1 | 29 | 173 | 175 | 100.0 |
| | | 30 | 2 | | |
| | 50 $\pm$ 1 | 50 | 87 | 175 | 98.9 |
| | | 51 | 86 | | |
| | | 52 | 1 | | |
| | | 53 | 1 | | |
| | Contamination/
artifact | 74

200 | 1
4 | 5 | 2.9 |
| ASGN-103 | 30 $\pm$ 1 | 30 | 177 | 178 | 100.0 |
| | | 31 | 1 | | |
| | 56 $\pm$ 1 | 55 | 81 | 178 | 100.0 |
| | | 56 | 96 | | |
| | | 57 | 1 | | |
| ASGN-113 | 30 $\pm$ 1 | 30 | 179 | 179 | 100.0 |
| | 103 $\pm$ 3 | 102
103
104
105 | 23
82
73
1 | 179 | 100.0 |
| | Mosaic Result* | 84
88
89 | 1
7
7 | 15 | 8.4 |
| | Contamination/
artifact | >200 | 2 | 2 | 1.1 |
| | | | | | |
| ASGN-101 | 35 $\pm$ 1 | 35 | 174 | 174 | 100 |
| | 93 $\pm$ 3 | 92 | 30 | 174 | 100.0 |
| | | 93 | 132 | | |
| Sample ID | Mode Expected
CGG Length
(± Target
Precision) | Measured
CGG
Length | No of
replicates
at that
size | Total
Number of
replicates | % within
Target
Precision
Range |
| | | 94 | 12 | | |
| | Mosaic result* | 97 | 2 | 9 | 5.2 |
| | | 98 | 7 | | |
| | 18 ± 1 | 18 | 178 | 178 | 100.0 |
| | | 113 | 37 | | |
| | | 114 | 115 | | |
| ASGN-016 | 114 ± 3 | 115 | 23 | 178 | 99.4 |
| | | 116 | 2 | | |
| | | 119 | 1 | | |
| | | | | | |
| | 20 ± 1 | 20 | 179 | 179 | 100.0 |
| | 198 ± 5%
(10 CGG) | 195 | 12 | 179 | 100.0 |
| | | 196 | 20 | | |
| | | 197 | 57 | | |
| | | 198 | 65 | | |
| | | 199 | 22 | | |
| ASGN-018 | Mosaic result | > 200 | 98 | 109 | 60.1 |
| | | 184 | 1 | | |
| | | 183 | 1 | | |
| | | 182 | 2 | | |
| | | 181 | 6 | | |
| | | 180 | 1 | | |
| | Contamination/ | 30 | 1 | 2 | 1.1 |
| | artifact | 103 | 1 | | |
| | | | | | |
| ASGN-023 | > 200 | > 200 | 177 | 177 | 100.0 |
| | | | | | |
| ASGN-104 | 24 ± 1 | 24 | 175 | 175 | 100.0 |
| | > 200 | > 200 | 175 | 175 | 100.0 |

8

9

Represents potential mosaic allele. Mosaic results may be below the limit of detection for mosaicism.

ii. Precision (lot-to-lot reproducibility)_

Within-laboratory precision was assessed running the same panel in the 3-site reproducibility study and described in Table 4. Each sample was tested in duplicate with 3 kit lots, 3 operators, and for over 12 non-consecutive days. The study was executed on 3 instruments and yielded 2376 total sample measurements excluding controls (2 replicates X 3 lots X 3 operators X 12 days = 216 per sample). Of the 2376 total sample measurements, 60 were invalid (Invalid rate 2.5%). Samples were evaluated for genotype category agreement and allele peak precision. The agreement was determined based upon the majority call. Excluding invalids, greater than >99%

10

categorical genotype agreement was observed for all samples as shown in Table 7.). Samples ASGN-112 and ASGN-016 were not 100% due to the appearance of third alleles (3 replicates) which resulted in different categorical calls. These results were not observed in the 3-site reproducibility result ASGN-018 has a full-mutation mosaic allele at >200 repeats, a normal allele at 20 repeats, and a premutation allele at 197 repeats. The mosaic allele was not found in 100/212 replicates. Allowing this sample to have a categorical call of a pre or full mutation results in 100% (212/212) agreement between replicates (95% CI: 98.2%-100%) and overall agreement of 99.9% (2313/2316, 95% CI: 99.6%-100%).In addition, as shown in Table 8, the reproducibility of the all alleles with the exception of the alleles identified as "mosaic" showed 100% reproducibility with the specifications). Precision across 3 lots was acceptable. Specimens with artifacts are listed for information purposes and calculated in the categorical section.

| Sample | Genotype
Category | Total
Number of
Replicates
from 216 | Replicates
in
Agreement | Agreement
(%) | CI
(95%) |
|----------|----------------------|----------------------------------------------|-------------------------------|------------------|-------------|
| ASGN-109 | Normal | 200 | 200 | 100 | 98.1, 100 |
| ASGN-112 | Normal | 214 | 213 | 99.5 | 97.4, 100 |
| ASGN-005 | Intermediate | 209 | 209 | 100 | 98.2, 100 |
| ASGN-111 | Intermediate | 213 | 213 | 100 | 98.2, 100 |
| ASGN-103 | Premutation | 213 | 213 | 100 | 98.2, 100 |
| ASGN-101 | Premutation | 212 | 212 | 100 | 98.2, 100 |
| ASGN-113 | Premutation | 208 | 208 | 100 | 98.2, 100 |
| ASGN-016 | Premutation | 213 | 211 | 99.1 | 96.6, 99.7 |
| ASGN-018 | Full Mutation | 212 | 112 | 52.8 | 46.1, 59.4 |
| ASGN-023 | Full Mutation | 212 | 212 | 100 | 98.2, 100 |
| ASGN-104 | Full Mutation | 210 | 210 | 100 | 98.2, 100 |

Table 7. Lot to lot reproducibility - Genotype Category Agreement by Sample Excluding Invalids.

Table 8: Lot to lot reproducibility - Percent of Allele Replicates within Target Precision Range Excluding Invalids.

| Sample ID | Mode Expected
CGG Length
( $\pm$ Target Precision) | Reported
CGG
Length | No of
replicates
at that size | Total
Number of
Replicates | % within
Target
Precision
Range |
|-----------|----------------------------------------------------------|---------------------------|-------------------------------------|----------------------------------|------------------------------------------|
| ASGN-109 | 30 $\pm$ 1 | 29
30 | 4
196 | 200 | 100.0 |
| ASGN-112 | 29 $\pm$ 1 | 28 | 2 | 214 | 100.0 |
| Sample ID | Mode Expected
CGG Length
(± Target Precision) | Reported
CGG
Length | No of replicates
at that size | Total
Number of
Replicates | % within
Target
Precision
Range |
| ASGN-101 | 30 ± 1 | 29 | 212 | 214 | 100.0 |
| | | 29 | 2 | | |
| | | 30 | 212 | | |
| | Contamination/
artifact | 114 | 1 | 1 | 0.05 |
| | 35 ± 1 | 35 | 212 | 212 | 100.0 |
| | 92 ± 3 | 92 | 187 | 212 | 100 |
| | | 93 | 24 | | |
| | | 94 | 1 | | |
| | Mosaic result* | 96 | 10 | 22 | 10.4 |
| | | 97 | 12 | | |
| | Contamination/
artifact | 30 | 1 | 3 | 1.4 |
| | | 55 | 1 | | |
| | | 79 | 1 | | |
| ASGN-005 | 29 ± 1 | 29 | 209 | 209 | 100.0 |
| | 45 ± 1 | 45 | 209 | | |
| ASGN-111 | 29 ± 1 | 29 | 213 | 213 | 100.0 |
| | 50 ± 1 | 50 | 208 | | |
| | | 51 | 5 | | |
| ASGN-103 | 30 ± 1 | 30 | 213 | 213 | 100.0 |
| | 55 ± 1 | 55 | 207 | | |
| | | 56 | 6 | | |
| ASGN-101 | 92 ± 3 | 92 | 187 | 212 | 100.0 |
| | | 93 | 24 | | |
| | | 94 | 1 | | |
| | 30 ± 1 | 30 | 208 | 208 | 100.0 |
| | 102 ± 3 | 101 | 5 | 208 | 100.0 |
| | | 102 | 123 | | |
| | | 103 | 70 | | |
| ASGN-113 | | 104 | 9 | | |
| | | 105 | 1 | | |
| | Mosaic result* | 87 | 25 | 45 | 21.6 |
| | | 88 | 20 | | |
| | 18 ± 1 | 17 | 21 | 213 | 100.0 |
| | | 18 | 192 | | |
| ASGN-016 | 113 ± 3 | 113 | 176 | 213 | 100.0 |
| | | 114 | 32 | | |
| | | 115 | 4 | | |
| | | 116 | 1 | | |
| | Contamination/
artifact | > 200 | 2 | 2 | 9.4 |
| ASGN-018 | 20 ± 1 | 19 | 3 | 212 | 100.0 |
| Sample ID | Mode Expected
CGG Length
(± Target Precision) | Reported
CGG
Length | No of
replicates
at that size | Total
Number of
Replicates | % within
Target
Precision
Range |
| | 196 ± 5% (10 CGG) | 20 | 209 | 212 | 100.0 |
| | | 195 | 19 | | |
| | | 196 | 112 | | |
| | | 197 | 60 | | |
| | | 198 | 17 | | |
| | | 199 | 3 | | |
| | | > 200 | 1 | | |
| | Mosaic allele | 178 | 2 | 131 | 52.8 |
| | | 179 | 6 | | |
| | | 180 | 5 | | |
| | | 181 | 5 | | |
| | | 182 | 1 | | |
| | | >200 | 112 | | |
| ASGN-023 | > 200 | > 200 | 212 | 212 | 100.0 |
| ASGN-104 | 24 ± 1 | 23 | 1 | 210 | |
| | | 24 | 209 | | 100.0 |
| | > 200 | > 200 | 210 | 210 | 100.0 |

11

12

Represents potential mosaic allele. Mosaic results may be below the limit of detection for mosaicism.

iii. Precision (Mosaicism)

Mosaicism is well known for Fragile X and was observed in 6 panel members out of 11 in the precision study. These results were not present in 100% of the replicates. To assess the reproducibility at the claimed limit of detection for mosaicism, a mosaicism reproducibility/LOD study was conducted with minimal DNA input (see below in the Detection of Limit Section).

A sample panel representing different mosaic/major allele combinations was generated by mixing DNA from 7 clinical specimens of different repeat sizes. The targeted MAFs are at or higher than the estimated LoD. The samples were tested at the lowest total DNA input level (20 ng). Note, one clinical specimen used to create the panel was later determined to be an outlier due to reproducibly unusual peak pattern. Subsequently, panel members involving this specimen were removed from data analysis. Each panel member was tested by 3 lots of AmplideX Fragile X Dx & Carrier Screen Kit in duplicates by 2 operators across 3 days. The study was executed on 2 instruments (one 8-capillary and one 24capillary) and vielded a total of 36 replicate measurements per specimen (2 replicates x 2 operators x 3 lots x 3 days = 36 replicates per sample). Greater than 95% hit rate were demonstrated for the claimed LoD listed in Table 9.

13

| Allele Combination | | LOD
(%MAF) | Valid
Replicates
(n) | %
Detected | 95% CI |
|-----------------------|-----------------------|---------------|----------------------------|---------------|------------|
| Mosaic | Major | | | | |
| Intermediate | Normal | 2.0 | 34 | 100.0 | 90.0-100.0 |
| Premutation | Normal | 2.0 | 36 | 94.4 | 82.0-98.0 |
| Full Mutation | Normal | 6.1 | 36 | 100.0 | 90.0-100.0 |
| Full Mutation | Premutation
(Low) | 7.0 | 36 | 100.0 | 90.0-100.0 |
| | Premutation
(High) | 7.0 | 35 | 100.0 | 90.0-100.0 |
| Premutation
(Low) | Premutation
(High) | 7.0 | 36 | 100 | 90.0-100.0 |
| Premutation
(High) | Full Mutation | 10.0 | 36 | 100 | 90.0-100.0 |
| Premutation
(Low) | Premutation
(High) | 5.0 | 34 | 97.1 | 85.0-99.0 |
| | | | 36 | 100.0 | 90.0-100.0 |
| Premutation
(High) | Premutation
(Low) | 5.0 | 36 | 100.0 | 90.0-100.0 |

Table 9. Reproducibility of Mosaic Samples

iv. DNA extraction reproducibility/equivalence

Three common commercial DNA extraction method for extracting genomic DNA from whole human blood were assessed using a 5-member sample panel representing all FMR1 genotype categories. The methods evaluated included: (D) (4) solution precipitation, manual silica spin column, and manual magnetic bead. Each extraction method was performed times per panel member. The extracted DNA was amplified in duplicate and analyzed on a single instrument.

All three methods yielded sufficient concentrations of DNA (on average) to meet the recommended input (b)(4) for the test kit. Categorical genotype agreement was 100% across all samples and 100% of all measured alleles were within target precision range. These results support the claim that the test can be used on gDNA generated using generally available DNA extraction methods capable of yielding at least 20ng/uL and A260-280 1.6-2.0.

Thermal Cycler equivalence V.
A 5-member sample panel representing all FMR1 genotype categories was amplified on three separate thermal cyclers and then sized on a single CE instrument. Four PCR runs were tested on each thermal cycler. In each PCR run

14

four replicates of each sample were tested.

For all sample replicates, across and within all thermal cvcler types, genotype category calls were in 100% agreement. 100% of all identified independent alleles were within their respective target precision ranges. The study results support the use of commonly available commercial thermal cyclers that can run the specified protocol for the test kit.

b. Linearity/assay Reportable Range:
Not applicable.
c. Detection Limit
- i. Limit of Detection

The FMR1 gene is present on the X chromosome with one copy in males and two copies in females. The detection of the repeats is influenced by the DNA concentration and the size of the repeats. The "Limit of Detection" for detecting minor or mosaic alleles was assessed in combination with the reproducibility study.

The limit of detection (LoD) for mosaic alleles in different mosaic/major allele combinations were first estimated by testing mixed cell line DNA with known genotypes at 6 mosaic allele frequencies (MAF 10, 5, 2, 1, 0.1, and 0.01% genotypes at 6 mosaic allele frequencies (MAF 10, 5, 2, 1, 0.1, and 0.01%). The
allele frequency demonstrating >000 correct categorical call rate within befor
replicates were replicates were identified. The estimated LoD were then confirmed by testing a panel of 30 samples representing different mosaic/major allele combinations. The panel was generated by mixing DNA from clinical specimens of different repeat sizes with targeted MAFs at or higher than the estimated LoD. The samples were tested at the lowest total DNA input level (20 ng). Note, one clinical specimen used to create the panel was later determined to be an outlier due to an unusual peak pattern. Subsequently 6 panel members involving this specimen were removed from data analysis. Each panel member was tested by 3 lots of AmplideX Fragile X Dx & Carrier Screen Kit in duplicates by 2 operators across 3 days. The study was executed on 2 instruments (one 8-capillary and one 24capillary) and yielded a total of 36 replicate measurements per specimen (2 replicates X 2 operators x 3 lots x 3 days = 36 replicates per sample). Greater than 95% hit rate were demonstrated for the claimed LoD listed in Table 10.

Table 10. Limit of Detection for mosaic alleles in different major allele backgrounds

Mosaic Allele	Major Allele	LoD for Mosaic Allele (%MAF)
Intermediate	Normal	2.0
Premutation	Normal	2.0
Full Mutation	Normal	6.1

15

Normal	Premutation	2.0
Premutation	Premutation	5.0
Full Mutation	Premutation	7.0
Normal	Full Mutation	2.0
Premutation	Full Mutation	10.0

ii. DNA Input

The DNA input range for the assay is 20-80ng total (or 10-40 ng/uL). A study was conducted to assess a range of DNA input to span (6) fold below and " fold higher than this range to determine the robustness of the assay to imprecision of DNA measurements that might occur in a laboratory. DNA concentration and purity were quantified using standard spectrophotometric methods. Input levels were established using a panel of 8 samples (6 clinical samples and 2 contrived), representing all FMR1 genotype categories, with DNA mass inputs corresponding to 1, 10, 20, 40, 80, and 160 ng per reaction. Each reaction was performed in duplicate. The study was executed by a single operator using two kit lots, and generated 20 replicates per dilution level per sample, resulting in 160 replicates per dilution level, and a total of 960 sample measurements excluding controls.

Categorical genotype agreement was 100% for all valid samples and all input levels. All expected allele peaks were within the target precision range. The OC failure rate at 1 ng, 10 ng, 20 ng, 40 ng, 80 ng, and 160 ng were 6.25%, 1.88%, 0.6%, 1.3%, 0%, and 0.63% respectively. These results support the recommended input range of 20-80 ng of gDNA for the assay as determined by standard absorbance or spectrophotometric methods.

d. Traceability

The assay uses molecular standards for use with fragment analysis on the instrument, and positive control that includes a range of CGG repeats. The assay is not traceable to any international standard, however an assessment of the accuracy of the molecular ladder used in the assay was conducted by testing the international standards. Refer to Clinical Section below.

e. Interfering Substances

Endogenous Interference

Endogenous interference was established by testing the interferents in accordance with CLSI EP07-A2 guidelines on a panel of "" samples ("" clinical and(b)(4) contrived). Interferents including conjugated bilirubin, unconjugated bilirubin, triglycerides, hemoglobin, and EDTA were tested. Each interferent, at its appropriate concentration, was added directly to an aliquot of a blood sample. Sodium hydroxide (diluent for hemoglobin and EDTA) as well as water were also tested to observe any solvent effects. An aliquot of untreated blood (no interferents added) was also extracted. Each condition was tested in triplicate.

16

Effects on DNA concentration yield were observed with hemoglobin (reduced yield) and EDTA (enhanced yield). Technical issues with the interference included low volume, low yield, sample loss, high screening QC failure rate, and instrument deviations, leading to some samples not completing processing. A limitation regarding the impact of excess hemoglobin and EDTA on the assay failure rate is included in the instructions for use. For samples that could be processed, the genotype category agreement was > 95% and the number of alleles within the expected precision range was 100%.

rv-Over and Cross- Contamination

g. Stability

i. Specimen Handling Stability
The ability of the assay to utilize DNA extracted from whole human blood stored at 2 to 8 °C for up to 2 weeks was determined by testing a panel of 14 clinical specimens representing all FMR1 genotype categories. For all samples, triplicate extractions were performed using independent aliquots on the day each sample was received to generate a T0 measurement. For subsequent timepoints, duplicate extractions were done. Extracted samples were stored at 2 to 8 ℃ for the duration of the study. A single operator using a single kit lot executed the study.

Genotype category agreement was 100% across all timepoints for each sample and all expected allele peaks were within target precision for all timepoints across all samples. These results support the use of sample DNA extracted from whole human blood that has been stored for up to 2 weeks at 2 to 8 ℃.

ii. DNA freeze/thaw Stability
DNA freeze/thaw stability was tested using a " contrived sample panel across (6)(4) freeze/thaw cycles. (b) (4) (D)(4) (4) This study was

executed by a single operator using a single lot of kit reagents and a single instrument.

Genotype category agreement was 100% across all timepoints for each sample

17

and all expected allele peaks were within target precision for all timepoints across all samples. These results support that extracted DNA is useable for at least (b)(4) freeze-thaw cycles to support (b) (4)

PCR & Capillary Electrophoresis (CE) Reagent Product Stability iii. PCR &CE product stability was assessed in two phases using a 5-member sample panel (4 clinical + 1 contrived) representing all FMR1 genotype categories. The time first phase evaluated post-amplification DNA stability at 4 ℃ across points for up to "" days ((b)(4) hours). With " replicates per sample at each time point, a total of (b)(4) measurements were collected.
The second phase evaluated the on-instrument, ambient temperature stability of samples prepared for CE analysis. CE deck time stability was established by preparing a (b) (4)

(b) (4

For PCR product stability, categorical genotype agreement was 100% for all timepoints across all samples, and > 95% of all measured alleles were within target precision range. The study results demonstrated that PCR products generated by the assay were stable for up to 3 days when stored at 2 to 8 ℃.

For CE product study, categorical genotype agreement was 100% for all timepoints across all samples and 100% of all measured alleles were within target precision range. The study results further demonstrated that samples prepared for CE analysis were stable at ambient temperature for up to 24 hours.

iv. Real Time Kit Stability

The real time kit stability was assessed using a 5-member sample panel representing all FMR1 genotype categories. Three lots of reagents were stored at -30°C to -15°C(b) (4)

(b) (4)

(b) (4) The study is still ongoing for (b) (4) timepoints.

Categorical genotype agreement was 100% for all timepoints across all samples and 100% of all measured alleles were within target precision range. The study results support at least a 1-year storage stability at this time. The study will carry on until 25-month to support 24-month stability.

Freeze/Thaw Kit Stability V.

The freeze/thaw kit stability was assessed using the same 5-member sample panel oresenting all FMR1 genotype categories as in real time kit stability study.

18

Categorical genotype agreement was 100% for all timepoints across all samples and 100% of all measured alleles were within target precision range. The study results support the stability of the kit for up to eight freeze/thaw cycles.

vi. Kit Shipping Stability
The kit shipping stability was assessed using the same 5-member sample panel representing all FMR1 genotype categories as in real time kit stability study. The temperature profile tested was based on the International Safe Transit Association (ISTA) 7D, 24-hour summer temperature profile. This profile (Table 11) was repeated for(b) (4) days to create the domestic and international shipping simulations, respectively.

Table 11. Shipping simulation based on the ISTA 7D 24-hour "Summer Profile".
	CHILL THE COLL COLL

Image /page/18/Figure/5 description: The image shows the title "Summer Profile [24-hr cycle]" and the text "All the kits tested across all the different shipping configurations showed 100% of their". The text (b)(4) is present twice in the image. The image appears to be a snippet of a larger document.

tested across all the different shipping configurations showed 100% of their measured alleles within the target precision range. In addition, all sample replicates showed 100% genotype category agreement.

2. Clinical Performance

Two multi-center clinical validation studies were conducted to validate a diagnostic claim and a carrier testing claim for the AmplideX Fragile X Dx & Carrier Screen Kit on the Applied Biosystems 3500 Dx series of capillary electrophoresis instruments.

a. Diagnostic Performance

i. Full Mutation
Diagnostic performance was assessed for Full Mutation classification using Southern

19

Blot as the reference method. Leftover specimens were obtained from patient samples representing the intended use population submitted for routine FMR1 5 UTR mutation testing across ? linical sites. Specimens were selected using a specific
protocol so as to minim ''e bias. Sixty-nine (%(4) specimens were enrolled into each of the three categories (full mutation, premutation, and normal/intermediate) based on available Southern Blot result, and then tested on the AmplideX Fragile X Dx & Carrier Screen Kit. A total of 207 specimens were evaluated, including 111 specimens from female patients and 96 from male patients. The results demonstrated agreement for full mutation positive percent agreement (PPA), 95.71%, negative percent agreement (NPA) 99.3%, and overall percent agreement (OPA) 97.6%. One of the 3 discordant samples was borderline sample, 194-repeat by AmplideX and >200 peak by Southern Blot.

The agreement between the AmplideX and Southern blot for full mutation are shown in Table 12.

	Reference Method (Southern Blot)
		Positive (≥

| Negative (200 peak by Southern Blot.

ii. Premutation vs. Normal or Intermediate assessment Agreement between Southern blot analysis for premutation and normal/intermediate were also compared to the Amplide X. The results demonstrated PPA 100%, NPA 97.1% and OPA 98.6%, all exceeded 95% and all two-sided Wilson score 95% confidence intervals were at or above 90%. The results are shown in Table 13.

20

	Reference Method (Southern Blot)
	Positive (55-199)	Negative (FMR1 Dual-PCR)
---------------------------------------------	-------------------------	-----------------	-------
Premutation
/ Full
mutation (>

| Intermediate
(45-54) | Normal
( 200 | 2 | 2 | | |
| 07/168 | 33-41,
300-401† | 39 | 26 | 26 | 52 | 100.0 |
| | | > 200 | 26 | 26 | | |
| 07/170 | 353-960† | > 200 | 27 | 27 | 27 | 100.0 |
| 07/174 | 97-127 | 110 | 24 | 27 | 54 | 100.0 |
| | | 111 | 2 | | | |
| | | 112 | 1 | | | |
| | | 117 | 12 | | | |
| | | 118 | 11 | | | |
| | | 119 | 4 | | | |

Table 18. Summary of Results for the WHO Sample Panel

TExpected allele size range is greater than 200 CGG; AmplideX Fragile X Reporter calls any peak larger than 200 CGG as ">200".

The test correctly classifies and sizes the international Fragile X reference standards from the World Health Organization (WHO) and the National Institute for Biological Standards and Control (NIBSC).

e. Fragile X-associated tremor/ataxia syndrome (FXTAS) or fragile X-associated primary ovarian insufficiency (FXPOI)

Literature was used to support the clinical validity of the claims associated with fragile X-associated tremor/ataxia syndrome (FXTAS) or fragile X-associated primary ovarian insufficiency (FXPOI) (Wheeler, A et al. 2017, Gallagher, A et al 2012, and Hantash, F et al. 2011).

25

M. Instrument Name

Programmable thermal cycler capable of executing the assay-specific PCR protocol ●
. Applied Biosystems 3500 Dx Series Genetic Analyzer (8-capillary or 24-capillary)

N. System Descriptions

1. Modes of Operation:
  Applied Biosystems 3500 Dx Series Genetic Analyzer (8-capillary or 24-capillary) with the following reagents:
POP-7™ Polymer ●
Hi-Di Formamide ●
Dye Set D (DS-30) Matrix Standard Kit

AmplideX Fragile X Reporter Software

1. Software:
  FDA has reviewed applicant's Hazard Analysis and software development processes for AmplideX Fragile X Reporter Software:

Yes X

1. Specimen Identification:
  Specimen identification is entered manually.
1. Specimen Sampling and Handling:
  Blood may be stored for up to 14 days at 2-8 ℃ prior to extraction. Extracted DNA is stable for at least (b freeze-thaw cycles.
1. Calibration:
  The ROX fluorescent dye is part of the ROX 1000 Size Ladder included in the kit. This ladder must be added to each sample to allow for proper fragment sizing analysis by the AmplideX Fragile X Reporter. Spectral calibration of the 3500 Dx instrument with DS-30 Matrix Standard Kit (Dye Set D) (P/N TBD) is required in order to properly detect and analyze samples containing ROX dye, and should be performed prior to any assay runs.

26

6. Quality Control:

The AmplideX Fragile X Reporter performs a series of QC checks on the batch controls (NTC and PC) as well as on the individual samples. The software assesses three general modalities of failure: Ladder Error (LD), Low Signal (LS), and Contamination (CT), If one or more of these failures occurs in the positive control, it invalidates the run as a Batch QC failure. The NTC is expected to give a Low Signal (LS) error. Rerun the flagged sample if one or more of these failures occurs in a sample.

Refer to the AmplideX Fragile X Reporter Software User Guide for more detailed description of the QC check parameters and error codes.

O. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:

Not applicable

P. Patient Perspective

This submission did not include specific information on patient perspectives for this device.

Q. Identified Risks to Health and Identified Mitigations

Identified Risks to Health and Identified Mitigations:

Identified Risks to Health	Mitigation Measures
Incorrect test results	Certain design verification and validation
Certain labeling information
Incorrect interpretation of test results	Certain design verification and validation
Certain labeling information

R. Benefit/Risk Summary:

SUMMARY
	The AmplideX® Fragile X Dx & Carrier Screen Kit is an in vitro
diagnostic device that uses polymerase chain reaction (PCR) and
capillary electrophoresis to detect and identify the number of
cytosine-guanine-guanine (CGG) repeats in the fragile X mental
retardation-1 (FMR1) gene using genomic DNA isolated from
peripheral whole blood specimens. The probable benefit of the
device is significant as it can aid in the diagnosis of Fragile X
Syndrome and can be used for carrier testing in adults of
reproductive age, for reproductive and genetic consultation. In
Summary of
Benefits	addition, there is also significant benefit in that it can aid in the
diagnosis of Fragile X-Associated Tremor/Ataxia Syndrome
(FXTAS) and Fragile X-Associated Primary Ovarian Insufficiency
(FXPOI), along with other clinical-pathological factors.
	There is risk associated with the use of this device mainly due to
incorrect test results (false positives, false negatives, and failure to
provide a result) and incorrect interpretation of test results by the
user, in the context of an aid in diagnosis for Fragile X Syndrome,
FXTAS and FXPOI, and for carrier testing.
Summary of Risks	The probable risk associated with the use of this device is that false
positives, false negatives, and failure to provide a result or incorrect
interpretation of test results by the user, can result in significant
negative clinical consequences. Erroneous device results could
adversely influence clinical consultation when used an aid in
diagnosis for Fragile X Syndrome, FXTAS or FXPOI and
reproductive consultation for carrier testing.
	In addition, it is important to note that this test can report out
mosaic results. Mosaicism detected in blood may not reflect
mosaicism in other tissues. The significance of reported mosaicism
should be interpreted with caution in conjunction with other
laboratory and clinical information (e.g., sex of patient, diagnostic
testing or carrier screening, patient symptoms) and should include
consultation with genetic professionals.
	In summary, there are potential risks associated with erroneous
results or incorrect interpretation by the user of this test due to the
analytical and clinical evidence provided and due to the test
reporting out mosaic results, which need to be interpreted with
caution.
Summary of Other
Factors	The risks of false positive and false negative results are mitigated
by the supportive analytical validation for this device. In addition,
the risk is partially attenuated because the assay results are intended
to be interpreted by a qualified molecular geneticist and the results
of this device are to be used with other clinical and diagnostic
findings, consistent with professional standards of practice. In
addition, an important mitigation is that this test is intended to be
used with reflex testing, clinical genetic evaluation, and genetic
counseling, which should be offered as appropriate. In addition, the
risks of this test are mitigated by the indication that states that this
test excludes its use for fetal diagnostic testing, newborn screening
or for stand-alone diagnostic purposes.

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S. Benefit/Risk Conclusion

Given the device's indications for use, required general controls and special controls established for this device, the probable benefits outweigh the probable risks.

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T. Conclusion

The De Novo request for the Amplide X Fragile X Dx and Carrier Screen Kit device is granted, and the device is classified under the following and subject to the special controls identified in the letter granting the De Novo request:

Product Code: Device Type: Class: Regulation:

OYV AmplideX Fragile X Dx & Carrier Screen Kit II (special controls) 21 CFR 866.5970