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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 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.

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

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 90%.
5. 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.
6. Not explicitly reported as a percentage, but implied by the overall conclusions.
7. 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.

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