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The BOND MMR Antibody Panel is intended to be used for the qualitative identification by light microscopy of human mismatch repair (MMR) proteins MLH1, MSH2, MSH6 and PMS2 in formalin-fixed, paraffin-embedded (FFPE) colorectal cancer (CRC) tissue sections by immunohistochemical staining. The BOND MMR Antibody Panel includes BOND Ready-to-Use Primary Antibody MLH1 (Mismatch Repair Protein) (ES05), BOND Ready-to-Use Primary Antibody MSH2 (Mismatch Repair Protein) (79H11), BOND Ready-to-Use Primary Antibody MSH6 (Mismatch Repair Protein) (EP49) and BOND Ready-to-Use Primary Antibody PMS2 (Mismatch Repair Protein) (EP51). The BOND MMR Antibody Panel is intended for use on the BOND-III or BOND-MAX fully automated systems with BOND Polymer Refine Detection.

The BOND MMR Antibody Panel is indicated for the detection of MMR protein deficiency as an aid in the identification of potential hereditary nonpolyposis colorectal cancer (HNPCC)/Lynch Syndrome in patients diagnosed with CRC. Patients with "MMR Loss" results should receive additional diagnostic testing consistent with clinical practice guidelines for diagnosis of Lynch syndrome. The BOND MMR Antibody Panel is not intended for use in indications other than CRC. This test should not be used for diagnosis of CRC.

The clinical interpretation of any staining or its absence when using the BOND MMR Antibody Panel should be complemented by morphological studies and proper controls and should be evaluated within the context of the patient's clinical history and other diagnostic tests by a qualified pathologist.

The clinical performance of this device to guide treatment of MMR deficient patients has not been established.

The BOND MMR Antibody Panel [subject device] consists of the following BOND Ready-to-Use (RTU) Primary Antibody (PA) products:

• MLH1 (Mismatch Repair Protein) (ES05) (PA0988-U) .
• MSH2 (Mismatch Repair Protein) (79H11) (PA0989-U) .
• . MSH6 (Mismatch Repair Protein) (EP49) (PA0990-U)
• . PMS2 (Mismatch Repair Protein) (EP51) (PA0991-U)

The BOND MMR Antibody Panel is intended for use on the BOND-III or BOND-MAX fully automated systems with BOND Polymer Refine Detection (DS9800). The BOND MMR Antibody Panel is indicated for the detection of mismatch repair protein deficiency as an aid in the identification of potential Hereditary Non-Polyposis Colorectal Cancer (HPNCC)/Lynch Syndrome in patients diagnosed with CRC.

MLH1 (Mismatch Repair Protein) (ES05) is a mouse anti-human monoclonal antibody produced as a tissue culture supernatant, and supplied in Tris buffered saline with carrier protein, containing 0.35 % ProClin™ 950 as a preservative and in a total volume of 7 mL. The antibody is optimally diluted for use on the automated BOND-MAX or BOND-III instrument staining platforms in combination with BOND Polymer Refine Detection (DS9800).

MSH2 (Mismatch Repair Protein) (79H11) is a mouse anti-human monoclonal antibody produced as a tissue culture supernatant, and supplied in Tris buffered saline with carrier protein, containing 0.35 % ProClin™ 950 as a preservative and in a total volume of 7ml. The antibody is optimally diluted for use on the automated BOND-MAX or BOND-III instrument staining platforms in combination with BOND Polymer Refine Detection (DS9800).

MSH6 (Mismatch Repair Protein) (EP49) is a rabbit anti-human monoclonal antibody produced as an affinity-purified tissue culture supernatant, and supplied in Tris buffered saline with carrier protein, containing 0.35 % ProClin™ 950 as a preservative and in a total volume of 7ml. The antibody is optimally diluted for use on the automated BOND-MAX or BOND-III instrument staining platforms in combination with BOND Polymer Refine Detection (DS9800).

PMS2 (Mismatch Repair Protein) (EP51) is a rabbit anti-human monoclonal antibody produced as an affinity purified tissue culture supernatant, and supplied in Tris buffered saline with carrier protein, containing 0.35 % ProClin™ 950 as a preservative and in a total volume of 7ml. The antibody is optimally diluted for use on the automated BOND-MAX or BOND-III instrument staining platforms in combination with BOND Polymer Refine Detection (DS9800).

Instrument and Software: The BOND-MAX and BOND-III instruments are fully automated slide stainers that perform automated deparaffinization (dewaxing), antigen retrieval, immunohistochemistry (IHC) staining/in situ hybridization (ISH) staining, and counterstaining. The major components of the BOND staining platforms are the processing module, computer (BOND controller), handheld ID scanner, and slide label printer. The BOND staining platforms are composed of a number of discrete software components including the BOND application software, BOND instrument/processing module software, BOND service software, and Laboratory interface system - integration package (LIS-IP).

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

Device: BOND MMR Antibody Panel

Intended Use: Qualitative identification by light microscopy of human mismatch repair (MMR) proteins (MLH1, MSH2, MSH6, PMS2) in formalin-fixed, paraffin-embedded (FFPE) colorectal cancer (CRC) tissue sections by immunohistochemical staining. It aids in identifying potential hereditary nonpolyposis colorectal cancer (HNPCC)/Lynch Syndrome in CRC patients.

1. Table of Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state "acceptance criteria" in a single, consolidated table. However, the precision studies reference "pre-specified acceptance criteria of ≥85% lower bound confidence interval." The clinical performance section states the point estimates of agreement, which implicitly serve as performance benchmarks.

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

Test Set Sample Size:

• Precision Studies (Intra-run, Between-day, Between-lot):
  • 40 FFPE CRC tissue cases (10 per MMR protein: 5 protein deficient, 5 intact) - used for intra-run, between-day, between-lot precision.
  • One MSH2 case was excluded in some analyses due to insufficient tumor.
• Reproducibility Studies (Pathologist & Laboratory):
  • 30 FFPE CRC tissue cases (specific breakdown for each MMR protein provided in Table 4, e.g., MLH1: 25 intact, 5 loss).
• Inter-day and Inter-site Reproducibility (BOND-III):
  • 24 FFPE CRC tissue cases (3 intact and 3 loss cases for each of the 4 MMR proteins).
• Clinical Performance Study:
  • Initially, 155 cases procured.
  • 143 cases were eligible and tested by both methods (BOND MMR Antibody Panel and DNA sequencing panel).
  • 133 cases had valid results by both methods and were evaluable for agreement analysis. These comprised:
    • Sequential cohort: 94 cases (unknown MMR status, sequentially obtained from a single US site).
    • Enrichment cohort: 49 cases (known MMR protein deficiencies from multiple sites).

Data Provenance:

• Clinical Performance Study: Specimens for the sequential cohort were obtained from a single US site. The enrichment cohort specimens were from multiple sites. The text indicates "Eligible remnant FFPE CRC tissues ("cases") were procured." This suggests the data are retrospective, using banked FFPE tissue samples.

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

Precision & Reproducibility Studies:

• Intra-run, Between-day, Between-lot Precision: A single pathologist read and scored all stained slides. No specific qualifications are provided for this pathologist, other than being "a single pathologist."
• Intra- and Inter-Pathologist Reproducibility: 3 pathologists read and scored the 30 cases. No specific qualifications beyond "pathologist" are provided.
• Inter-Instrument Reproducibility: One pathologist evaluated the cases. No specific qualifications are provided.
• Inter-Laboratory Reproducibility: One pathologist at each of the 3 sites independently evaluated each case. No specific qualifications beyond "pathologist" are provided.
• Inter-day and Inter-site Reproducibility (BOND-III): One pathologist at each of the 3 sites read and scored the stained slides. No specific qualifications beyond "pathologist" are provided.

Clinical Performance Study:

• "One pathologist at the testing site read and scored BOND MMR Antibody Panel stained slides in accordance with the scoring guidance." No specific qualifications are provided for this pathologist.
• For the ground truth (DNA sequencing panel results): The ground truth was established by assessing "pathogenic mutation(s) likely to affect MMR protein expression in CRC" using a DNA sequencing panel. This is an objective molecular test, not dependent on expert visual interpretation.

4. Adjudication Method for the Test Set

Precision & Reproducibility Studies:

• For precision studies where a single pathologist scored, "majority score" was used as the reference where multiple replicates were performed. For reproducibility studies involving multiple pathologists or sites, concordance between pathologists/sites was evaluated. No explicit adjudication process like "2+1" or "3+1" is described for resolving discrepancies to establish a single ground truth from pathologist reads for performance metrics. Instead, the studies assess agreement between readers and sites. The "majority score" used in the precision studies implied that if there were discordant reads, the majority would determine the "true" result for that specific replicate.

Clinical Performance Study:

• The ground truth for the clinical performance study was the DNA sequencing panel result. The pathologist's interpretation of the BOND MMR Antibody Panel staining was compared against this molecular ground truth. Therefore, no pathologist adjudication of the test device results was used to establish the ground truth for this comparison.

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

• Was an MRMC study done? Yes, aspects of MRMC designs are present in the reproducibility studies, particularly the "Intra- and Inter-Pathologist Reproducibility" and "Inter-Laboratory Reproducibility" sections. These studies involved multiple pathologists reading multiple cases to assess the reproducibility of the device's output.
• Effect size of human reader improvement with AI vs. without AI assistance: Not applicable. This device is an immunohistochemistry (IHC) panel, interpreted by pathologists using light microscopy. It is not an AI-assisted diagnostic device, nor does the study evaluate human reader performance with or without AI assistance. The study focuses on the reproducibility and clinical validity of the IHC panel itself.

6. Standalone Performance Study (Algorithm Only)

• This question is not applicable as the device is an Immunohistochemistry (IHC) panel, not an algorithm or AI system. Its performance is intrinsically tied to human interpretation (by a pathologist). The studies evaluate the performance of the IHC panel as interpreted by pathologists.

7. Type of Ground Truth Used

• Precision/Reproducibility Studies: The "ground truth" for evaluating these studies was largely based on the expected protein expression status of the selected CRC tissue cases (e.g., "5 cases being protein deficient and 5 cases expressing intact protein"). For calculating agreements over repeat measurements, often a "majority score" from multiple reads or comparison to a baseline read was used as the reference.
• Clinical Performance Study: The ground truth was established by a molecular test: a DNA sequencing panel validated for detecting pathogenic mutations likely to affect MMR protein expression in CRC. This provides an objective measure of MMR gene status, which is then correlated with the protein expression detected by the IHC panel.

8. Sample Size for the Training Set

The document describes pre-market testing and performance characterization, not the development or training of an AI algorithm. Therefore, there is no mention of a "training set" sample size for an algorithm. The "Immunoreactivity" section (Table 15-17) shows the testing of the antibodies across a wide variety of normal, neoplastic, and colorectal cancer tissues (dozens to hundreds of cases across various tissue types) to characterize their expected staining patterns, which could be considered part of the development/characterization phase, but not an algorithmic "training set."

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

As there is no training set for an AI algorithm mentioned in the document (the device is an IHC panel), this question is not applicable. The "Immunoreactivity" section's characterization of normal and tumor tissue staining patterns served as the basis for understanding expected reactivity, likely through known biological knowledge and expert pathological evaluation.

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Ready-to-Use Format

For in vitro diagnostic use.

BOND Ready-to-Use Primary Antibody Progesterone Receptor (16) is a monoclonal antibody intended to the qualitative identification by light microscopy of human progesterone receptor in formalin-fixed, paraffin-embedded tissue by immunohistochemical staining using the automated BOND-III systems. Progesterone Receptor Clone (16) specifically binds to the progesterone receptor antigen located in the nucleus of progesterone receptor positive normal and neoplastic cells.

Progesterone Receptor Clone (16) is indicated as an aid in the management, prognosis and prediction of therapy outcome of breast cancer. The clinical interpretation of any staining or its absence should be complemented by morphological studies using proper controls and should be evaluated within the context of the patient's clinical history and other diagnostic tests by a qualified pathologist.

Progesterone Receptor Clone (16) is optimized for use on the Leica Biosystems automated BOND-MAX or BOND-III systems using the BOND Polymer Refine Detection kit.

Concentrated Liquid Antibody Format

For in vitro diagnostic use.

Novocastra Liquid Mouse Monoclonal Antibody Progesterone Receptor Clone (16) (Concentrated Liquid Antibody Format) is intended to be used for the qualitative identification by light microscopy of human progesterone receptor in formalin-fixed, paraffin-embedded tissue by immunohistochemical staining. Progesterone Receptor Clone (10) specifically binds to the progesterone receptor antigen located in the nucleus of progesterone receptor positive normal and neoplastic cells.

Progesterone Receptor Clone (16) Monoclonal Antibody (Concentrated Liquid Antibody Format) is indicated as an aid in the management, prognosis and prediction of therapy outcome of breast cancer. The clinical interpretation of any staining or its absence should be complemented by morphological studies using proper controls and should be evaluated within the context of the patient's clinical history and other diagnostic tests by a qualified pathologist.

Progesterone Receptor (16) is a mouse anti-human monoclonal antibody produced as a tissue culture supernatant. This antibody is utilized to perform a qualitative immunohistochemical (IHC) assay to identify Progesterone Receptor expression in human breast cancer tissue routinely processed and paraffin-embedded for histological examination.

Progesterone Receptor (PGR) Clone 16 Primary Antibody is provided in a Ready-to-Use (RTU) and a concentrated liquid format. The RTU format is supplied in Tris buffered saline with carrier protein, containing 0.35% ProClin™ 950 as a preservative and is provided in two volumes (7 mL and 30 mL). The total protein concentration is approximately 10 mg/mL and the total antibody concentration is greater than or equal to 1 mg/L as determined by ELISA. The RTU format is optimally diluted for use on the automated BOND-MAX and BOND-III instrument staining platforms in combination with BOND Polymer Refine Detection (DS9800). The concentrated liquid format is provided so that customers may utilize manual staining protocols. The concentrated liquid format is a liquid tissue culture supernatant containing 15 mM sodium azide as a preservative. The total protein concentration is determined on a per batch basis and is described on the vial label, and the antibody concentration is greater than or equal to 324.0 mg/L as determined by ELISA.

The BOND-MAX and BOND-III instruments are fully automated slide stainers that perform automated deparaffinization (dewaxing), antigen retrieval, immunohistochemistry (IHC) staining/in situ hybridization (ISH) staining, and counterstaining. The major components of the BOND staining platforms are the processing module, computer (BOND controller), handheld ID scanner, and slide label printer. The BOND staining platforms are composed of a number of discrete software components including the BOND application software, BOND instrument/processing module software, BOND service software, and Laboratory interface system - integration package (LIS-IP).

Here's a breakdown of the acceptance criteria and study details based on the provided text:

1. A table of acceptance criteria and the reported device performance

The document does not explicitly state numerical acceptance criteria for immunoreactivity or stability beyond "met acceptance criteria". However, the repeatability and reproducibility sections do provide numerical agreements and state that they met criteria. The method comparison also provides numerical agreements and states it met acceptance criteria.

2. Sample size used for the test set and the data provenance (e.g. country of origin of the data, retrospective or prospective)

• Intra-run Repeatability: 6 unique breast tumor tissue cases.
• Inter-day, Inter-instrument, Inter-lot Repeatability: 27 unique breast tumor tissue cases.
• Reproducibility (Inter-laboratory & Inter-pathologist): 135 unique FFPE breast tumor tissue cases.
• Method Comparison: A total of 455 cases (implied from the table total).
• Data Provenance: The document does not explicitly state the country of origin or whether the data was retrospective or prospective. It only mentions "human progesterone receptor in formalin-fixed, paraffin-embedded tissue" and "breast tumor tissue cases."

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g. radiologist with 10 years of experience)

• Reproducibility (Inter-laboratory & Inter-pathologist): 3 pathologists were involved in scoring the slides, one per site. Their qualifications are not specified beyond "pathologist."
• Other studies (Repeatability, Method Comparison): The document does not explicitly state the number of experts used for establishing ground truth or their qualifications for these studies. The scoring for repeatability likely involved expert assessment, and method comparison relied on comparing the subject device to a predicate device, which would also implicitly rely on established expert assessment standards.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set

The document does not describe an explicit adjudication method (like 2+1 or 3+1) for resolving discrepancies. For the reproducibility studies, agreements are calculated between observers, suggesting individual pathologist assessments were compared rather than being subjected to a specific adjudication process to establish a single "ground truth" per case.

5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

This document describes the performance of an immunohistochemistry reagent/antibody, not an AI-assisted diagnostic device. Therefore, an MRMC comparative effectiveness study involving human readers improving with AI assistance is not applicable and was not performed. The studies focus on the analytical performance and reproducibility of the staining process and the antibody's interpretation by pathologists.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done

This is an immunohistochemistry reagent, not an algorithm. Therefore, a standalone algorithm-only performance study is not applicable and was not performed. The performance is intrinsically linked to its use in a laboratory setting by human interpretation.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc.)

The ground truth for the evaluations appears to be based on pathology assessments by qualified pathologists, specifically following ASCO/CAP guidelines (≥1% cut-off) for determining PR status in breast cancer tissue. This is indicated by phrases like "scored according to ASCO/CAP guidelines" and "clinical interpretation of any staining or its absence should be complemented by morphological studies... by a qualified pathologist."

8. The sample size for the training set

The document describes performance studies, not the development or training of an AI model. Therefore, there is no mention of a "training set" or its sample size.

9. How the ground truth for the training set was established

As this is not an AI model, there is no training set and thus no ground truth establishment process described for one. The "training" of the antibody is inherent to its development and optimization for specific antigen binding, which is evaluated through immunoreactivity and analytical performance studies.

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Estrogen Receptor Clone 6F11 (ER 6F11) Mouse Monoclonal antibody is intended for laboratory use to qualitatively identify estrogen receptor (ER) antigen in sections of formalin fixed, paraffin embedded breast cancer tissue by immunohistochemistry methods. Estrogen Receptor Clone 6F11 specifically binds to the ER antigen located in the nucleus of ER positive normal and neoplastic cells.

Estrogen Receptor Clone 6F1 I is indicated as an aid in the management, prognosis and predication of therapy outcome of breast cancer. The clinical interpretation of any staining or its absence should be complemented by morphological studies using proper controls and should be evaluated within the context of the patient's clinical history and other diagnostic tests by qualified pathologist.

Estrogen Receptor Clone 6F11 Ready-to-Use Primary Antibody for Bond™ and the Estrogen Receptor Clone 6F11 Liquid Concentrate Primary Antibody, Novocastra™ are optimized for use on the Leica Biosystems Bond III staining platform using the Bond Polymer Refine Detection Kit.

Estrogen Receptor Clone 6F11 is a mouse anti-human monoclonal antibody produced as a tissue culture supernatant. This antibody is utilized to perform a semi-quantitative immunohistochemical (IHC) assay to identify estrogen receptor (ER) expression in human breast cancer tissue routinely processed and paraffin-embedded for histological examination.

Estrogen Receptor Clone 6F11 primary antibody is provided in two formats, a Bond™ Ready-to-Use format (product code PA0151 (7mL) and PA0009 (30 mL)) and a concentrated liquid format (product code NCL-L-ER-6F11 (1mL) and is optimally diluted for use on the automated Bond System (Bond III ) in combination with Bond Polymer Refine Detection kit.

Total protein concentration for Estrogen Receptor clone 6F11 is approximately 3.8 g/L. The immunoglobulin concentration is approximately 75 mg/L.

The Estrogen Receptor Clone 6F11 Liquid Concentrate Primary Antibody, Novocastra" is recommended for use at a dilution of 1 in 50 when diluted in Bond Antibody Diluent (AR9352).

The Estrogen Receptor Clone 6F11 Ready-to-Use Primary Antibody for Bond™ is a tissue culture supernatant prepared at a working immunoglobulin concentration of 0.88 µg/mL. It is supplied in Tris buffered saline with carrier protein, containing 0.35% ProClin™ 950 as a preservative.

Acceptance Criteria and Device Performance for Estrogen Receptor Clone 6F11

The Leica Biosystems Estrogen Receptor Clone 6F11 (ER 6F11) primary antibody, in both Ready-to-Use (PA0151) and Liquid Concentrate (NCL-L-ER-6F11) formats, demonstrated performance across several studies to meet acceptance criteria for its intended use in immunohistochemistry for ER antigen identification in breast cancer tissue.

1. Table of Acceptance Criteria and Reported Device Performance

The provided document details various performance studies rather than explicit, pre-defined acceptance criteria with specific thresholds for each metric. However, the data presented strongly implies what would be considered acceptable performance for a device seeking governmental approval, particularly regarding reproducibility and correlation with existing methods and clinical outcomes.

Based on the studies, the implicit acceptance criteria for the device revolve around demonstrating high agreement in various precision and reproducibility studies, and acceptable clinical performance (sensitivity, specificity, PPV, NPV) when compared to a "gold standard."

Summary Table of Reported Device Performance against Implicit Acceptance Criteria:

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

The document describes several distinct test sets:

• Clinical Outcome Study (Calgary Cohort):
  • Sample Size: n=532 breast cancer patients (retrospectively analyzed); n=473 for univariate analysis; n=363 for multivariate analysis.
  • Data Provenance: Retrospective, Calgary-based patient cohort (Canada), diagnosed between 1985 and 2000.
• Precision and Reproducibility Studies (TMA and Whole Tissue Sections): These studies used unspecified "breast cancer cases."
  • Within-Run Precision (PA0151): 108 test data points (presumably cases or samples).
  • Within-Instrument Precision (PA0151): 321 test data points.
  • Between-Run Precision (PA0151): 180 test data points.
  • Between Laboratory Precision (PA0151): 101 test data points from 3 investigational sites.
  • Lot to Lot Precision (PA0151): 100 test data points using 3 reagent lots.
  • Between Observer Precision (PA0151 & NCL-L-ER-6F11): 20 whole section breast cancer cases for each format (total 40 cases if both studies were distinct).
  • Inter-Site Reproducibility (PA0151 & NCL-L-ER-6F11): 18 cases evaluated at 3 sites over 5 days (9 replicates per case).
  • Lot to Lot Reproducibility (PA0151 & NCL-L-ER-6F11): 18 cases using 3 reagent lots.
• Inter-Platform Comparison Study:
  • Sample Size: 306 invasive breast cancer specimens. An additional 452 cases were assessed to enrich the cohort, but the final reported results are for the 306 cases.
  • Data Provenance: Clinical archives, from three independent US-based testing facilities. Specimens were formalin-fixed, paraffin-embedded.

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

• Clinical Outcome Study (Calgary Cohort):
  • Number of Experts: 3 observers scored slides (likely pathologists, though specific qualifications were not given beyond "observers").
  • Qualifications: Not explicitly stated, but clinical outcome studies involving immunohistochemistry interpretation typically involve experienced pathologists.
• Precision, Reproducibility, and Inter-Platform Comparison Studies:
  • Number of Experts: 3 observers/investigational sites are mentioned for between-observer and inter-site studies.
  • Qualifications: Not explicitly stated beyond "observers" and "investigational sites." It's implied these are expert professionals (e.g., pathologists) involved in diagnostic interpretation.

4. Adjudication Method for the Test Set

• Clinical Outcome Study (Calgary Cohort): The scoring method was the "Allred scoring method." Cohen's Kappa statistic was used to quantify inter- and intra-observer reproducibility. While individual observer scores were compared, there is no explicit mention of an adjudication process (e.g., 2+1, 3+1) to establish a consensus ground truth solely among the experts for this specific test set. The ground truth for comparative effectiveness was primarily based on ligand-binding assay (LBA) and patient progression on tamoxifen.
• Precision, Reproducibility, and Inter-Platform Comparison Studies: For these studies, the "ground truth" often refers to a reference standard against which the test device is compared. For observer agreement studies, the "adjudication method" is the direct comparison of observer scores without necessarily establishing a final adjudicated truth.
  • In the inter-platform comparison study, the "reference standard device" (Estrogen Receptor Clone 6F11 Ready-to-Use Primary Antibody for Bond™ on the Bond III) effectively served as the de facto ground truth against which the Liquid Concentrate format was compared.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, And the Effect Size of How Much Human Readers Improve with AI vs. Without AI Assistance

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done in the context of human readers with vs. without AI assistance.

This document describes the validation of an immunohistochemistry (IHC) assay and its accompanying primary antibody, not an AI or digital pathology device meant to assist human readers. The studies focus on the performance and reproducibility of the IHC staining method itself.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

No, a standalone performance study in the context of an algorithm only was not done.

As noted above, this submission is for an IHC primary antibody and its associated staining platform, which is interpreted by human observers (pathologists). There is no mention of an algorithm or AI component in this device.

7. The Type of Ground Truth Used

The ground truth varied depending on the specific study:

• Clinical Outcome Study (Calgary Cohort):
  • Ligand-binding assay (LBA): Used as a "gold standard" for calculating sensitivity, specificity, PPV, and NPV of the device.
  • Patient progression on tamoxifen: Used as another "gold standard" for calculating diagnostic performance metrics, indicating real clinical outcome.
• Precision and Reproducibility Studies: For these studies, the "ground truth" was often the consensus or established score from a reference method or a designated expert, against which other measurements/observers were compared. For the between-observer studies, direct comparison of observer agreement served as the assessment.
• Inter-Platform Comparison Study: The Estrogen Receptor Clone 6F11 Ready-to-Use Primary Antibody for Bond™ on the Bond III (the existing, approved format) served as the "reference standard test" or ground truth against which the Liquid Concentrate format was evaluated.
• Stability Studies: A "control score" for Intensity Score and Proportion Score was used as the ground truth.

8. The Sample Size for the Training Set

The document does not describe a "training set" in the context of a machine learning model, as the submission concerns an IHC assay and antibody, not an AI device.

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

Not applicable, as no training set for an AI model is described.

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