Affymetrix® Gene Profiling Reagents are intended for the preparation of labeled complementary RNA target from purified total RNA from fresh or frozen clinical tissue specimens for hybridization to Affymetrix® GeneChip® microarrays and the measurement of fluorescence signals of labeled RNA target using the Affymetrix GeneChip microarray instrumentation system.

Intended for use with separately FDA-cleared Affymetrix GeneChip microarray assays specifying the use of the Affymetrix Gene Profiling Reagents.

Device Description

The Affymetrix® Gene Profiling Reagents were designed for in vitro diagnostic use as an accessory to the GeneChip® MicroArray Instrumentation System. Affymetrix® Gene Profiling Reagents are intended for the preparation of labeled complementary RNA target from purified total RNA from fresh or frozen clinical tissue specimens for hybridization to Affymetrix® GeneChip® microarrays and the measurement of fluorescence signals of labeled RNA target using the Affymetrix GeneChip microarray instrumentation system. Intended for use with separately FDA-cleared Affymetrix GeneChip microarray assays specifying the use of the Affymetrix Gene Profiling Reagents.

The Affymetrix Gene Profiling Reagents consist of three kits.

Kit 1 is the RNA Control Kit consisting of Poly-A Control and Dilution Buffer. These reagents are designed to provide exogenous positive controls to monitor the entire target labeling process. The Poly-A Control and Dilution Buffer are provided with the kit to prepare the appropriate serial dilutions. After the appropriate dilution of the Poly-A Control, they are added to the total RNA and then amplified and labeled together. Examining the hybridization intensities of these controls on the array helps to monitor the amplification and labeling processes.

Kit 2 is comprised of the Transcript Synthesis and Labeling Kits A and B and includes enzyme mixes, labeling reagent, reaction buffers and purification reagents for the preparation of the labeled cRNA target. Kit 2 is optimized specifically for producing amplified and biotinylated cRNA targets to hybridize to arrays for expression analysis.

Kit 3 is comprised of Transcript Detection Kits A, B and C and includes all of the reagents to perform fragmentation of the labeled cRNA target, prepare the hybridization cocktail (including Oligo B2 and hybridization controls) and process the arrays in the Affymetrix Fluidics Station 450. The arrays are then ready to be scanned by the Affymetrix GeneChip Scanner.

AI/ML Overview

Here's an analysis of the acceptance criteria and the study that proves the device meets them, based on the provided text:

Device: Affymetrix® Gene Profiling Reagents (K103112)
Intended Use: Preparation of labeled complementary RNA target from purified total RNA from fresh or frozen clinical tissue specimens for hybridization to Affymetrix GeneChip® microarrays and the measurement of fluorescence signals of labeled RNA target using the Affymetrix GeneChip microarray instrumentation system. Intended for use with separately FDA-cleared Affymetrix GeneChip microarray assays specifying the use of the Affymetrix Gene Profiling Reagents.

1. Table of Acceptance Criteria and Reported Device Performance

Performance Characteristic	Acceptance Criteria	Reported Device Performance
Precision/Reproducibility	Median probeset signal %CV from detected probesets < 20% (between lots and between days within a lot).	Met for all combinations. - Between days (MAQC A): One-Cycle: 7.97%; Gene Profiling (1000ng): 7.17%; Gene Profiling (100ng): 7.03% - Between days (MAQC B): One-Cycle: 7.68%; Gene Profiling (1000ng): 7.24%; Gene Profiling (100ng): 9.33% - Between lots (MAQC A): One-Cycle: 8.70%; Gene Profiling (1000ng): 9.54%; Gene Profiling (100ng): 8.74% - Between lots (MAQC B): One-Cycle: 9.52%; Gene Profiling (1000ng): 9.93%; Gene Profiling (100ng): 11.72%
Repeatability	Median probeset signal %CV from detected probesets < 10% for replicates.	Met for all conditions, internal and external sites. Examples from internal testing (MAQC A, 100ng): 5.31%, 5.81%, 5.41%, 4.24%, 4.04%. External sites also met the criteria.
Input Total RNA	Reagents perform as expected with 100 ng and 1000 ng of input total RNA, and produced sufficient cRNA for hybridization (≥ 20 µg or ≥ 15 µg for Pathchip).	Testing demonstrated reagents performed as expected with 100 ng and 1000 ng. When tested with 200 ng (clinical study amount), sufficient cRNA was produced (≥ 15 µg).
cRNA Yield	Internal: ≥ 90% of samples yield ≥ 20 µg cRNA and cRNA concentration ≥ 0.625 µg/µL. External: ≥ 96% of samples yield > 20 µg cRNA and cRNA concentration ≥ 0.625 µg/µL.	Internal: 100% (30/30) of samples yielded ≥ 20 µg cRNA and concentration ≥ 0.625 µg/µL. External: 100% (32/32) of samples at both sites achieved yields of ≥ 20 µg cRNA and concentration ≥ 0.625 µg/µL.
Performance of Controls	1. .CEL files generated by successful automatic gridding for all samples with Oligo B2. 2. 3' AFFX-r2-Bs probe sets for lys, phe, dap present, with signal intensities and r-squared values ≥ 0.900 for correlation of signal intensities with relative ratio. 3. 3' AFFX-r2 probeset for bioB called present. 4. Hybridization controls (bioB, bioC, bioD, cre) followed expected relative concentration (bioB < bioC < bioD < cre).	All criteria met. Internal study: 100% (80/80) of samples passed. External study: 100% (64/64) of samples passed. Clinical studies: 100% (135/135 from 3 sites, and 32/32 from 1 site) passed.
Clinical Method Comparison	1. Specimen processing specifications met. 2. Overall success rate for generating Tissue of Origin Test - Frozen results > 90%. 3. Observed percent correct when using Gene Profiling Reagents no different from expected percent correct when using One-Cycle Reagent Kit (α = 0.05).	1. Not explicitly stated "met," but implied by successful study completion. 2. Not explicitly reported as a percentage, but implied by the overall conclusions. 3. The observed percent correct with Gene Profiling Reagents was "no different" (α = 0.05) from the One-Cycle Kit (95% bootstrap confidence limits: [-5.9%, 3.4%]). Acceptance criteria achieved.

2. Sample Size Used for the Test Set and Data Provenance

Precision/Reproducibility:
- Sample Size: MAQC A and B total RNAs were used. Specific numbers: 3 lots of Gene Profiling Reagents and 3 lots of Pathwork® Expression 3'-Amplification One-Cycle Target Reagents were each tested using 100 ng and 1000 ng of MAQC A and B total RNAs in quadruplicate. For internal consistency, one lot of each reagent was tested with 100 ng/1000 ng MAQC A/B total RNAs in quadruplicate, repeated on three different days.
- Data Provenance: Not explicitly stated (e.g., country of origin). The use of "MAQC A and B total RNAs" suggests standardized reference materials. This was an analytical, non-clinical study.
Repeatability:
- Sample Size: Same as Precision/Reproducibility for internal testing. Additionally, two external sites tested 8 replicates each using 100 ng and 1000 ng of MAQC A and B total RNAs in batches of a minimum of 8 samples.
- Data Provenance: Internal Affymetrix testing, and two external sites. Analytical, non-clinical.
cRNA Yield:
- Sample Size: Internal: 100 ng of total RNA from ten commercially available human tissues (run in triplicate). External: Eight replicates using 100 ng and 1000 ng of MAQC A and B total RNAs in batches of a minimum of 8 samples at two external sites.
- Data Provenance: Internal Affymetrix testing, and two external sites. Analytical, non-clinical.
Performance of Controls:
- Sample Size: Internal: Three lots of Gene Profiling Reagents tested using 100 ng and 1000 ng of MAQC A and B total RNAs in quadruplicate. External: Eight replicates using 100 ng and 1000 ng of MAQC A and B total RNAs in batches of a minimum of 8 samples at two external sites. Clinical studies: 45 total RNA samples tested at each of 3 clinical sites (total 135 samples) and 16 total RNA samples from 16 frozen tissues (tested twice, so 32 data points) at one site.
- Data Provenance: Internal Affymetrix testing, two external sites, and multiple prospective clinical sites.
Clinical Method Comparison (Tissue of Origin Study):
- Sample Size:
  - Part 1 (Tissue Study): 16 frozen tissues (representing 15 tumor types), each tested twice with Gene Profiling Reagents and twice with One-Cycle Reagent Kit (total 32 reactions per reagent type, 64 total).
  - Part 2 (Total RNA Study): 45 total RNA samples processed using Gene Profiling Reagents (45 reactions per site at 3 sites, total 135 reactions). For comparison, 39 of these samples had prior One-Cycle Kit results available from two sites, and 6 additional samples had prior One-Cycle Kit results from one site.
  - Combined Analysis: 16 tissues from Part 1, and 45 total RNA samples from Part 2.
- Data Provenance: Prospective clinical studies conducted at 1 external site (Part 1) and 3 clinical sites (Part 2). Tissue sources and resulting .CEL files were blinded to the analysis labs (Pathwork Diagnostics). Not specified if the data was from the US or international.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications

Not applicable directly for these studies. The ground truth for the clinical comparison was the available diagnosis of each tissue specimen (implied from clinical pathology, but not explicitly stated how many experts or their qualifications). The study design blinded the testing sites and the analysis center to this diagnosis, suggesting it was established independently.
For the analytical studies (precision, repeatability, yield, controls), the "ground truth" is based on the inherent properties of the RNA samples (MAQC A/B, commercially available human tissues) and the expected performance of the control spike-ins, not expert interpretation.

4. Adjudication Method for the Test Set

Not applicable. The clinical study compared the performance of the Gene Profiling Reagents to a predicate (One-Cycle Reagent Kit) in generating results for the Pathwork® Tissue of Origin-Frozen Test. The "truth" in this context was the existing diagnosis of the tissue, and the comparison was on the output "percent correct" of the assay, not on expert consensus readings of an image or signal. The study notes that the sites and Pathwork Diagnostics were blinded to the available diagnosis.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, an MRMC study was not done. This device is a reagent kit for gene profiling, not an imaging or diagnostic algorithm requiring human interpretation. The clinical study was a method comparison to evaluate if the new reagents performed equivalently to predicate reagents within an existing diagnostic assay (Pathwork® Tissue of Origin-Frozen Test), which involves an algorithm for analysis, not direct human reading of raw data. Therefore, an effect size of human readers improving with AI vs. without AI assistance is not relevant here.

6. Standalone (i.e. algorithm only without human-in-the-loop performance) Study

Yes, a standalone study was done in essence. The analytical performance studies (Precision, Repeatability, cRNA Yield, Performance of Controls) are all measuring the performance of the reagents and their output without human interpretation of the final diagnostic result.
The clinical method comparison study evaluates the reagents' contribution to the Pathwork® Tissue of Origin-Frozen algorithm's performance. The algorithm itself (from Pathwork Diagnostics) provides the interpretation, and it was "blinded to the available diagnosis for each specimen." The comparison was between the output of the Pathwork algorithm when using RNA prepared with Affymetrix Gene Profiling Reagents versus when using RNA prepared with the One-Cycle Reagent Kit. The "observed percent correct" was comparing the algorithm's output to the known diagnosis. This represents the performance of the system (reagents + instrumentation + algorithm) in a standalone fashion compared to known ground truth.

7. Type of Ground Truth Used

Analytical Studies: Chemical/biological properties of standardized RNA samples (MAQC A and B, commercially available human tissues), and expected signals from internal controls (spike-ins).
Clinical Method Comparison Study: "Available diagnosis of each tissue specimen." This would typically be established by pathology (histopathology, immunohistochemistry) to confirm the tissue of origin and tumor type, but the specific method or number of pathologists is not detailed. It represents an established clinical diagnosis.

8. Sample Size for the Training Set

Not explicitly stated in the provided text. The document describes a premarket notification for a reagent kit, not a machine learning algorithm that typically requires a training set. The reagents are used to prepare samples for an existing FDA-cleared microarray assay (Pathwork® Tissue of Origin-Frozen Test, K080896) and instrumentation (Affymetrix GeneChip® MicroArray Instrumentation System, K080995), which presumably had their own training and validation data (if applicable, for the Pathwork algorithm). The current submission focuses on demonstrating that the new Gene Profiling Reagents perform equivalently to the reagents used in the predicate device for this assay.

9. How the Ground Truth for the Training Set Was Established

Not applicable as no training set for the Gene Profiling Reagents themselves is described. If the Pathwork® Tissue of Origin-Frozen Test's algorithm involved a training set, the ground truth for that would have been established during its own development and clearance process (K080896), but this information is not provided in the current document.

Summary

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K103112

Image /page/0/Picture/1 description: The image shows the Affymetrix logo, which includes the company name in a stylized font and a graphic element above it. Below the logo, the text "Gene Profiling Reagents Premarket Notification" is displayed. The text is in a smaller font size compared to the company name.

April 28, 2011

Summary of Safety and Effectiveness

MAY - 4 2011

l. Submitter Information - 21 CFR 807.92(a)(1):

Submitter: Affymetrix, Inc. 3420 Central Expressway Santa Clara, CA 95051

Establishment Registration No: 3003314809 '

Contact:

Phone: Fax:

E-mail:

Chief Medical Officer, Affymetrix

Phone: 408-731-5967

Fax: 408-731-5755

E-mail: Rick_Hockett@Affymetrix.com

Rick Hockett

Regulatory Contact: Maureen Mende

MyRAQA, Inc. 3 Lagoon Drive, Suite 280 Redwood Shores, CA 94065 650-730-5020 650-730-5005 Maureen@myraqa.com

April 17, 2011 Date Prepared:

II. Name of Device and Classification - 21 CFR 807.92(a)(2):

Affymetrix Gene Profiling Reagents Name:

Class II Classification:

OVA Product Code:

AFFYMETRIX, INC.

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Image /page/1/Picture/0 description: The image shows the Affymetrix logo, which includes a stylized DNA double helix and the company name in bold, sans-serif font. Below the logo, the text "Gene Profiling Reagents Premarket Notification" is displayed in a smaller, regular font. The text indicates that the image is related to the regulatory submission of gene profiling reagents by Affymetrix.

III. Predicate Device - 21 CFR 807.92(a)(3):

Affymetrix GeneChip® Microarray Predicate Device:

Instrumentation System

IV. Device Description - 21 CFR 807.92(a)(4):

The Affymetrix Gene Profiling Reagents consist of three kits.

Kit 2 is comprised of the Transcript Synthesis and Labeling Kits A and B and includes enzyme mixes, labeling reagent, reaction buffers and purification reagents for the preparation of the

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labeled cRNA target. Kit 2 is optimized specifically for producing amplified and biotinylated cRNA targets to hybridize to arrays for expression analysis.

V. Intended Use/Indications for Use - 21 CFR 807.92(a)(5):

Special Conditions For Use Statement(s):

Intended for use with separately FDA-cleared Affymetrix GeneChip microarray assays specifying the use of the Affymetrix Gene Profiling Reagents.

VI. Performance Data - 21 CFR 807.92(b):

A. Non-Clinical Test Summaries 21 CFR 807.92(b)(1):
- i. Analytical Performance:
  - 1. Precision/Reproducibility
    - To demonstrate reproducibility studies were conducted in which three lots of Gene Profiling Reagents were tested

AFFYMETRIX, INC.

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using 100 ng and 1000 ng of MAQC A and B total RNAs in quadruplicate by three different operators. Three lots of Pathwork® Expression 3'-Amplification One-Cycle Target Reagents were tested using 1000 ng of MAQC A and B total RNAs in quadruplicate by three different operators for comparison.

In addition, one lot of the Gene Profiling Reagents was tested using 100 ng and 1000 ng of MAQC A and B total RNAs in quadruplicate. One lot of the One-Cycle Reagent Kit was tested using 1000 ng of MAQC A and B total RNAs in quadruplicate for comparison. The testing was repeated on three different days.

The median probeset signal %CV from detected probesets was calculated both between lots and between days within a lot and is reported in Table 6-1 (a - d), The acceptance criterion of %CV less than 20% was met for all combinations.

Table 6-1

a - %CV between days within a lot for MAQC A
----------------------------------------------	--	--	--

Data Sets	One-CycleDays 1, 2 and 3MAQC A	Gene Profiling1000 ngDays 1, 2 and 3MAOC A	Gene Profiling100 ngDays 1, 2 and 3MAQC A
Median CV%	7.97 %	7.17 %	7.03 %

b - %CV between days within a lot for MAQC B

Data Sets	One-CycleDays 1, 2 and 3MAQC B	Gene Profiling1000 ngDays 1, 2 and 3MAQC B	Gene Profiling100 ngDays 1, 2 and 3MAQC B
Median CV%	7.68 %	7.24%	9.33 %

c - %CV between lots for MAQC A

Data Sets	Gene Profiling	Gene Profiling
One-Cycle Lots1, 2 and 3MAQC A	1000 ngLots 1, 2 and 3MAQC A	100 ngLots 1, 2 and 3MAQC A
Median CV%8.70 %	9.54 %	8.74 %

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d - %CV between lots for MAQC B
Data Sets	One-CycleLots 1, 2 and 3MAQC B	Gene Profiling1000 ngLots 1, 2 and 3MAQC B	Gene Profiling -100 ngLots 1,2 and 3MAQC B
Median CV%	9.52 %	9.93 %	11.72 %

2. Repeatability

To demonstrate repeatability, the samples described in the Reproducibility section of this summary were used to calculate the median probeset signal %CV from detected probesets for the replicates. Table 6-2 (a - f) shows the results for all sets of replicates using the Gene Profiling Reagents and the One-Cycle Reagent Kits. The acceptance criterion for %CV less than 10% for Repeatability was met for all conditions.

Table 6-2

a - %CV for the replicates of 3 lots of the One-Cycle Reagent Kit, and one lot tested on 3 different days using 1000 ng of MAQC A.

Data Sets	Lot1 Day1One-CycleMAQC A	Lot1 Day2One-CycleMAQC A	Lot1 Day3One-CycleMAQC A	Lot2 Day1One-CycleMAQC A	Lot3 Day1One-CycleMAQC A
Median CV%	5.06%	4.55%	4.85%	3.67%	4.30%

b - %CV for the replicates of 3 lots of the Gene Profiling Reagents, and one lot tested on 3 different days using 100 ng of MAQC A.

Data Sets	Lot1	Lot2	Lot3	Lot3	Lot3
	100ng	100ng	100ng	100ng	100ng
	Day1	Day1	Day1	Day2	Day3 -
	MAOC A	MAQC A	MAQC A	MAQC A	MAOC A
Median CV%	5.31%	5.81%	5.41%	4.24%	4.04%

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Image /page/5/Picture/0 description: The image shows the Affymetrix logo, which includes the company name and a symbol resembling a DNA strand made of squares. Below the logo, the text "Gene Profiling Reagents Premarket Notification" is displayed. The text indicates that the image is related to the premarket notification of gene profiling reagents produced by Affymetrix.

c - %CV for the replicates of 3 lots of the Gene Profiling Reagents, and one lot tested on 3 different days using 1000 ng of MAQC A.

Data Sets	Lot1	Lot2	. Lot3	Lot3	Lot3
	1000ng	1000ng	1000ng	1000ng	1000ng
	Day1 -	Day1 -	Day1 -	Day2 -	Day3 -
	MAQCA	MAQCA	MAQCA	MAQCA	MAQCA
Median CV%	5.5%	4.38%	5.88%	4.54%	5.44%

d - %CV for the replicates of 3 lots of the One-Cycle Reagent Kit, and one lot tested on 3 different days using 1000 ng of MAQC B.

Data Sets	Lot1 Day1One-CycleMAQC B	Lot1 Day2One-CycleMAQCB	Lot1 Day3One-CycleMAQC B	Lot2 Day1One-CycleMAQC B	Lot3 Day1One-CycleMAQC B
Median CV%	4.57%	4.11%	4.35%	4.88%	4.64%

e - %CV for the replicates of 3 lots of the Gene Profiling Reagents, and one lot tested on 3 different days using 100 ng of MAQC B.

Data Sets	Lot1100ngDay1MAQC B	Lot2100ngDay1MAQC B	Lot3100ngDay1MAQC B	Lot3100ngDay2MAQC B	Lot3100ngDay3MAQC B
Median CV%	8.77%	4.42%	5.15%	5.46%	4.79%

f - %CV for the replicates of 3 lots of the Gene Profiling Reagents, and one lot tested on 3 different days using 1000 ng of MAQC B.

Data Sets	Lot11000ngDay1MAQCB	Lot21000ngDay1MAQCB	Lot31000ngDay1MAQCB	Lot31000ngDay2MAQCB	Lot31000ngDay3MAQCB
Median CV%	5.74%	5.33%	4.23%	4.37%	5.64%

Repeatability was also demonstrated in the testing conducted at two external sites where eight replicates using 100 ng and 1000 ng of MAQC A and B total RNAs in batches of a minimum of 8 samples were tested. The median probeset signal %CV from detected probesets was calculated for the replicates and the acceptance criteria of % CV of < 10% was met for both sites.

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3. Input Total RNA

The range for the amount of starting material to be used with the Gene Profiling Reagents was determined to be between 100 ng to 1000 ng (1ug) of total RNA. The reagents were tested in both internal and external studies using total RNA at the minimum and maximum amount of the range of input total RNA. The testing demonstrated the reagents performed as expected using 100 ng and 1000 ng of input total RNA and the amount of cRNA produced was sufficient for the hybridization of one microarray (≥ 20 µg).

The clinical study which utilized the Pathwork Tissue of Origin Test - Frozen used 200 ng of total RNA as the starting material. When the reagents were tested with 200 ng, the amount of cRNA produced was sufficient for the hybridization of one Pathchip™ microarray (≥ 15μg).

4. cRNA Yield

To demonstrate cRNA yield of the Gene Profiling Reagents testing was conducted internally by Affymetrix and at two external sites as part of the Design Validation.

The internal testing included 100 ng of total RNA from ten commercially available human tissues run in triplicate using the Transcript Synthesis and Labeling Kit (Kit 2). The test assessed the ability of the kit to vield ≥ 20 µg cRNA for greater than 90% of the total RNA samples tested and a cRNA concentration of ≥0.625 µg/µL. 100% (30/30) of the samples tested yielded greater than ≥ 20 µg cRNA and a cRNA concentration ≥0.625 µg/µL. Data is provided in Table 6-3.

The testing conducted at the two external sites included eight replicates using 100 ng and 1000 ng of MAQC A and MAQC B total RNAs in batches of a minimum of 8 samples.

AFFYMETRIX, INC.

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The acceptance criterion for this study was cRNA yield of ≥96 % of samples prepared must produce > 20 µg cRNA and cRNA concentration ≥0.625 µg/µL. 'All 32 samples (100%) at both sites achieved yields of ≥ 20 µg cRNA and cRNA concentration of ≥0.625 µg/µL.

	cRNAConcentration	AdjustedcRNA yields	AveragecRNA yields
Sample ID	μg/μL	(μg)	(μg)
Kidney Total RNA_R1	2.250	69.6
Kidney Total RNA_R2	2.112	65.4	67.1
Kidney Total RNA_R3	2.141	66.3
Pancreas Total RNA_R1	1.796	55.6
Pancreas Total RNA_R2	1.792	55.5	54.2
Pancreas Total RNA_R3	1.664	51.5
Heart Total RNA_R1	2.241	69.4
Heart Total RNA_R2	2.338	72.4	69.2
Heart Total RNA_R3	2.126	65.8
MAQCB Total RNA_R1	2.443	75.6
MAQCB Total RNA_R2	2.441	75.6	74.4
MAQCB Total RNA_R3	3.009	72.1
Liver Total RNA_R1	1.985	61.4
Liver Total RNA_R2	1.843	57.0	57.1
Liver Total RNA_R3	1.705	52.8
Breast Total RNA_R1	2.161	66.9
Breast Total RNA_R2	1.844	58.9	62.2
Breast Total RNA_R3	1.965	60.8
Testicle Total RNA_R1	2.207	68.3
Testicle Total RNA_R2	2.268	70.2	69.5
Testicle Total RNA_R3	2.261	70.0
HeLa Total RNA_R1	2.401	74.3
HeLa Total RNA_R2	2.277	72.8	73.7
HeLa Total RNA_R3	2.390	74.0
Thyroid Total RNA_R1	1.689	52.2
Thyroid Total RNA_R2	1.562	48.3	50.5
Thyroid Total RNA_R3	1.644	50.8
Skeletal Muscle Total RNA			62.3
Sample ID	cRNAConcentrationμg/μL	AdjustedcRNA yields(μg)	AveragecRNA yields(μg)
Skeletal Muscle Total RNAR2	2.043	63.2
Skeletal Muscle Total RNAR3	1.931	59.8

Table 6-3

AFFYMETRIX, INC.

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Image /page/8/Picture/0 description: The image shows the logo for Affymetrix. The logo consists of the word "Affymetrix" in a sans-serif font, with a stylized double helix symbol above the letters "ff" and a series of black squares to the right of the helix. The squares are arranged in a pattern that resembles a grid or a barcode. The logo is simple and modern, and it is likely used to represent the company's focus on genomics and biotechnology.

Gene Profiling Reagents Premarket Notification

April 28, 2011

5. Linearity/Assay Reportable Range

Not Applicable.

Traceability, Stability Expected Values (controls, 6. calibrators or methods)

a. Performance of the Controls

Performance of the Poly-A Control, Oligo B2 and hybridization controls were evaluated by testing conducted internally at Affymetrix and at two external sites as part of Design Validation. The internal testing to evaluate the performance of the Poly-A Control, Oligo B2 and hybridization controls included three lots of Gene Profiling Reagents that were tested using 100 ng and 1000 ng of MAQC A and B total RNAs and tested in quadruplicate. The testing conducted at the two external sites included eight replicates using 100 ng and 1000 ng of MAQC A and B total RNAs in batches of a minimum of 8 samples.

In both studies .CEL files were generated by successful automatic gridding for all samples tested demonstrating that the Oligo B2 performed as expected. The analysis of the Poly-A Control showed the 3' AFFX-r2-Bs probe sets

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Image /page/9/Picture/1 description: The image shows the Affymetrix logo and the text "Gene Profiling Reagents Premarket Notification". The Affymetrix logo is a stylized double helix with a series of squares above it. The text is in a simple, sans-serif font, with the company name in a larger size than the rest of the text.

for all three spikes (lys, phe and dap) were present. Signal intensities and r-squared values for the correlation of the 3' AFFX-r2-Bs signal intensities with the relative ratio for each spike, followed the relative concentration in the Poly-A Control mixture: lys < phe< dap and the rsquared values met the acceptance criteria of ≥ 0.900 for both studies. The 3' AFFX-r2 probeset for bioB was called present for all samples in both studies. The signal intensity for the hybridization controls (bioB, bioC, bioD and cre) followed the relative concentration in the mixture: bioB < bioC < bioD < cre for all samples tested in both studies. 100 % (80/80) of the samples passed the acceptance criteria for the performance of the controls in the internal study and 100% (64/64) in the external study.

In addition the performance of the controls was evaluated in the clinical studies. 45 total RNA samples were tested at each of the 3 clinical sites and 100% (135/135) passed the acceptance criteria for the Poly-A Control, Oligo B2 and hybridization controls. 16 total RNA samples from 16 frozen tissues were tested at one site and 100 % (32/32) passed the acceptance criteria for performance of the controls.

b. Stability Studies

Real-time stability studies are being conducted on the RNA Control Kit, the Transcript Synthesis and Labeling Kit and the Transcript Detection Kit. Based on the results of these studies, a shelf life from the date of manufacturing has been established for each of the Gene

AFFYMETRIX, INC.

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Image /page/10/Picture/1 description: The image shows the Affymetrix logo, which includes the company name in a stylized font and a DNA-like graphic. Below the logo, the text "Gene Profiling Reagents Premarket Notification" is displayed. The text is left-aligned and appears to be part of a document or presentation header. The overall image is clean and professional, likely representing a formal announcement or document related to gene profiling reagents.

Profiling Reagents. On-going stability studies are being conducted according to established procedures.

Open-vial stability studies have been completed for all reagent kits. Freeze-thaw stability studies have also been completed for the applicable reagents. The results of these studies support the freeze-thaw and open-vial stability recommended in the package inserts for the Gene Profiling Reagents.

c. Shipping Studies

Shipping studies under actual and simulated conditions have been conducted to confirm that the products have been designed, manufactured and packaged in such a way that they maintain transport storage conditions and packaging integrity. Additional shipping studies are being conducted to confirm that product performance is not affected under recommended storage and transportation conditions.

7. Detection Limit

Not applicable.

8. Assay Cut-Off

Not Applicable.

VII. Clinical Study Summary - 21 CFR 807.92(b)(2):

A. Comparison Studies:

i. Method Comparison:

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The performance of the Affymetrix Gene Profiling Reagents was demonstrated utilizing a previously cleared expression assay on the Affymetrix GeneChip Microarray Instrumentation System. A two part prospective clinical study was conducted demonstrating performance of the Affymetrix Gene Profiling Reagents using the Pathwork® Tissue of Origin-Frozen Test (K080896) and the Affymetrix GeneChip® MicroArray Instrumentation System cleared for RNA analysis (K080995). Both studies evaluated the performance of the Pathwork® Tissue of Origin-Frozen Test with the Gene Profiling Reagents compared to performance of the assay demonstrated in K080896 using the One-Cycle Reagent Kit.

The first part of the prospective clinical study was conducted at 1 external site and included testing of 16 frozen tissues from RNA extraction to hybridization to the Pathchip 100 microarray and scanning of the microarray on the Affymetrix GCS3000 Dx Instrument System. The 16 tissues, representing each of the 15 tumor types included in the Pathwork Tissue of Origin - Frozen cleared intended use, were tested twice using each of the Affymetrix Gene Profiling Reagents and the One-Cycle Reagent Kit included in Pathwork's K080896. The site was blinded to the available diagnosis of each tissue specimen. The resulting .CEL files were sent to Pathwork Diagnostics for analysis using proprietary Pathwork Tissue of Origin-Frozen algorithm. Pathwork Diagnostics was also blinded to the tissue sources.

The second part of the prospective clinical study was conducted at 3 clinical sites. The clinical study included the preparation of 45 total RNA samples using the Affymetrix Gene Profiling Reagents, with hybridization to the Pathchip™ microarray and scanning of the microarray on the Affymetrix GCS3000 Dx Instrument System. The resulting .CEL files were sent to Pathwork Diagnostics for analysis using proprietary

AFFYMETRIX, INC. `

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Pathwork Tissue of Origin-Frozen algorithm. The clinical sites and Pathwork Diagnostics were both blinded to the available diagnosis for each specimen. The study included 39 samples that had been tested using One-Cycle Kit reagents in the Pathwork Reproducibility study, as discussed in K080896 and 6 samples that had previously been tested with One-Cycle Kit reagents at one of the test sites. Results using One-Cycle Kit reagents were available from two sites for the 39 samples and 1 site for the 6 additional samples.

The acceptance criteria for both prospective studies were for the specimen processing specifications defined in the protocol to be met and the overall success rate for generating Tissue of Origin Test - Frozen results to be greater than 90%. Lastly, when data from these two studies were combined, the observed percent correct when using Gene Profiling Reagents could be no different from expected percent correct when using One-Cycle Reagent Kit (α = 0.05).

Table 6-4 below shows the numbers of samples used in this comparison of One-Cycle Reagents and Gene Profiling Reagents.

Protocol(unique samples)	Gene ProfilingReagents Reactions			One-Cycle KitReactions

	Site 1	Site 2	Site 3	Site 1	Site 4
Total RNA Study (n = 45)	45	45	45	45	39
Tissue Study (n = 16)	32			32
Total Reactions by Site	77	45	45	77	39
Total Reactions by Reagent	167			116

TABLE 6-4 DISTRIBUTION OF SAMPLES USED FOR ANALYSIS

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In an integrated clinical analysis the performance of the Affymetrix Gene Profiling Reagents intended for in vitro diagnostic use were compared to the One-Cycle Reagent Kit intended for research use only which were utilized by Pathwork Diagnostics in the 510(k) for the Tissue of Origin Test - Frozen (K080896). The acceptance criteria for this analysis were for the observed percent correct when using IVD reagents should be no different from expected percent correct when using One-Cycle Kit reagents (α = 0.05).

The results from this integrated analysis showed that the observed percent correct when using the Affymetrix Gene Profiling Reagents was no different (α = 0.05) from the expected percent correct when using the One-Cycle Reagent Kit (95% bootstrap confidence limits are given by (-5.9%, 3.4%)). The acceptance criteria were achieved in support of substantial equivalence for the IVD and One-Cycle Kit reagents. The combined data from these studies demonstrated the performance of the TOO assay was comparable with both sets of reagents.

ii. Matrix Comparison:

Not Applicable.

iii. Clinical Sensitivity:

Not Applicable.

iv. Clinical Specificity:

Not Applicable.

v. Clinical Cut-Off:

Not Applicable. AFFYMETRIX, INC.

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vi. Expected Values/Reference Range:

Not Applicable.

vii. Other Clinical Supportive Data: Not Applicable.

VIII. Conclusion

The submitted information in this premarket notification is complete and supports a substantial equivalence decision for the Gene Profiling Reagents.

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Image /page/15/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a circular seal with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" around the perimeter. Inside the circle is an emblem featuring a stylized depiction of an eagle or bird-like figure.

Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993

Affymetrix, Inc. c/o Maureen Mende, RAC, MBA Senior Director and Regulatory Consultant MyRAQA, Inc. 3 Lagoon Drive. Suite 280 Redwood Shores, CA 94065

MAY - 4 2011

Re: K103112

Trade/Device Name: Affymetrix® Gene Profiling Reagents Regulation Number: 21 CFR 862.2570 Regulation Name: Instrumentation for Clinical Multiplex Test Systems Regulatory Class: Class II Product Code: OVA Dated: April 22, 2011 Received: April 25, 2011

Dear Ms. Mende:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into class II (Special Controls), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); and good manufacturing practice

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Page 2 – Ms. Maureen Mende

requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820). This letter will allow you to begin marketing your device as described in your Section 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Parts 801 and 809), please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (301) 796-5450. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/cdrl/industry/support/index.html.

Sincerely yours.

Maurice M. Charn

Maria M. Chan, Ph.D. Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

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510(k) Number (if known): K103112

Device Name: Affymetrix® Gene Profiling Reagents

Indications for Use:

Affymetrix® Gene Profiling Reagents are intended for the preparation of labeled complementary RNA target from purified total RNA from fresh or frozen clinical tissue specimens for hybridization to Affymetrix GeneChip® microarrays and the measurement of fluorescence signals of labeled RNA target using the Affymetrix GeneChip® Microarray Instrumentation System.

Intended for use with separately FDA-cleared Affymetrix GeneChip microarray assays specifying the use of the Affymetrix Gene Profiling Reagents.

Prescription Use X (Part 21 CFR 801 Subpart D) AND/OR

Over-The-Counter Use _ (21 CFR 801 Subpart C)

(PLEASE DO NOT WRITE BELOW THIS LINE-CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of In Vitro Diagnostic Devices (OIVD)

Reena Philip

Division Sign-Off Office of In Vitro Diagnostic Device Evaluation and Safety

510(k) _k103112 ______________________________________________________________________________________________________________________________________________________________

Page __ of __ ]

Regulation Number and Section

§ 862.2570 Instrumentation for clinical multiplex test systems.

(a)
Identification. Instrumentation for clinical multiplex test systems is a device intended to measure and sort multiple signals generated by an assay from a clinical sample. This instrumentation is used with a specific assay to measure multiple similar analytes that establish a single indicator to aid in diagnosis. Such instrumentation may be compatible with more than one specific assay. The device includes a signal reader unit, and may also integrate reagent handling, hybridization, washing, dedicated instrument control, and other hardware components, as well as raw data storage mechanisms, data acquisition software, and software to process detected signals.(b)
Classification. Class II (special controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 862.9. The special control is FDA's guidance document entitled “Class II Special Controls Guidance Document: Instrumentation for Clinical Multiplex Test Systems.” See § 862.1(d) for the availability of this guidance document.