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The Diagnostic Hybrids, Inc. device, D3 DFA Cytomegalovirus Immediate Early Antigen Identification Kit, is intended for use in the qualitative detection and identification of human Cytomegalovirus (CMV) immediate early antigen (IEA) in cell cultures by immunofluorescence using fluoresceinated monoclonal antibodies (MAbs).

This product is not intended for use in testing blood or plasma donors and is not intended for use in direct detection of cytomegalovirus in clinical specimens.

Two murine derived monoclonal antibodies (MAbs) are used in the Diagnostic Hybrids, Inc. (DHI) device, D3 DFA Cytomegalovirus Immediate Early Antigen Identification Kit (CMV-IEA ID Kit), and are directed against CMV immediate early antigen (pp 72). The MAbs used in the Kit have been shown to be highly specific, with no cross-reactivity to other cultured viruses. The MAbs have been labeled by DHI using Fluorescein Isothiocyanate (FITC).

Kit Components:

1. CMV-IEA DFA Reagent, 10-mL. One dropper bottle containing a mixture of two murine MAbs directed against CMV immediate early antigen (pp 72). The MAbs are both IgG1 (k) isotype. The buffered, stabilized, aqueous solution contains Evans Blue as a counter-stain and 0.1% sodium azide as preservative.
2. CMV Antigen Control Slides, 5-slides. Individually packaged control slides containing wells with cell culture derived positive and negative control cells. Each slide contains one Negative well of uninfected cells and one Positive well of CMV infected cells. Each slide is intended to be stained only one time.
3. Mounting Fluid, 7-mL. One dropper bottle of an aqueous, buffered, stabilized solution of glycerol (ph 8.2 ± 0.2) and 0.1% sodium azide.
4. 40X PBS Concentrate, 25-mL. One bottle containing a 40X concentrate consisting of 4% sodium azide (0.1% sodium azide after dilution to 1X using de-mineralized water) in a phosphate buffered saline (PBS) solution.

Patient samples are inoculated onto susceptible cell monolayers and cultured. After a defined incubation period, the cells to be tested for the presence of CMV-IEA are fixed in acetone. The CMV-IEA DFA Reagent is added to the cells which are then incubated for 15 to 30 minutes at 35° to 37°C, the stained cells are washed with the supplied PBS Solution (diluted), and a drop of the supplied Mounting Fluid is placed on the prepared cells. The cells are examined using a fluorescence microscope. The cells infected with CMV and expressing the CMV-IEA will have apple-green fluorescent nuclei while uninfected cells will contain no fluorescence but will be stained red by the Evans Blue counter-stain.

If no fluorescent cells are found, report result as "No cytomegalovirus detected". If fluorescent cells are found in the CMV-IEA DFA Reagent stained monolayer, report result as "Cytomegalovirus isolated by cell culture".

The provided 510(k) summary describes the D3 DFA Cytomegalovirus Immediate Early Antigen Identification Kit and its performance in comparison to predicate devices. Here's a breakdown of the requested information:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state "acceptance criteria" in terms of specific numerical thresholds for positive or negative percent agreement that the sponsor had to meet to claim substantial equivalence. Instead, the study aims to show that the D3 DFA CMV-IEA ID Kit performs comparably to legally marketed predicate devices. The performance is reported as percent agreement with the comparator devices, which implicitly serves as the standard for acceptance.

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

• Total Clinical Specimens: 1060 specimens were cultured initially, with 1026 included in the final analysis after exclusions. This represents the sample size for the test set.
• Data Provenance: The clinical study involved three external clinical laboratory sites and the DHI internal laboratory.
  • Study Site 1: Collected and cultured 314 fresh specimens during August 2006.
  • Study Site 2: Cultured 300 specimens (72 fresh and 228 archival). Archival specimens were collected from June 2005 through September 2006 and stored at -70°C.
  • Study Site 3: Tested 146 fresh specimens from February 2007 through May 2007.
  • Study Site 4: Cultured 300 frozen prospectively collected archival specimens from a clinical reference laboratory in the Eastern U.S. in February 2007. These were stored at -70°C.
• Retrospective/Prospective: The study included both prospective (fresh specimens) and retrospective (archival/frozen specimens) data. Most sites included fresh specimens, while Study Site 2 and 4 specifically mention archival/frozen specimens. "Prospectively collected" for archival specimens refers to how they were collected for the archive, not necessarily how they were collected for this specific study.

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

The document does not specify the number or qualifications of experts used to establish the ground truth for the test set. The ground truth for clinical performance was established by comparison to legally marketed predicate devices (Bartels Cytomegalovirus Immediate Early Antigen Kit and Light Diagnostics CMV Direct Immunofluorescence Assay), meaning the results from these predicate devices were used as the reference standard. The interpretation of these predicate device results would have typically been done by trained laboratory personnel, but no specific expert qualifications are provided.

4. Adjudication Method for the Test Set

The document does not describe any specific adjudication method (e.g., 2+1, 3+1) for resolving discrepancies or establishing the ground truth when comparing the D3 DFA Cytomegalovirus Immediate Early Antigen Identification Kit to the comparator devices. The comparison is mainly presented as percentage agreement.

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

No multi-reader multi-case (MRMC) comparative effectiveness study, evaluating the effect size of human readers improving with AI vs. without AI assistance, was explicitly reported. This device is a diagnostic kit (fluorescent antibody test), not an AI algorithm assisting human readers. The performance is assessed by comparing its direct output to predicate devices.

6. Standalone (Algorithm Only Without Human-in-the-Loop Performance) Study

This device is a standalone diagnostic kit. Its performance, as described by the analytical and clinical studies, refers to the kit's ability to qualitatively detect and identify CMV IEA in cell cultures. The results are interpreted by trained laboratory personnel examining the stained cells under a fluorescence microscope, but the "performance" described is of the kit itself, rather than an AI or algorithm in a human-in-the-loop context.

7. Type of Ground Truth Used

The ground truth used for the clinical performance evaluation was the results obtained from legally marketed predicate devices (Bartels Cytomegalovirus Immediate Early Antigen Kit and Light Diagnostics CMV Direct Immunofluorescence Assay) when applied to cultured specimens. In the context of the analytical studies, ground truth was based on known concentrations of CMV (TCID50) in cell cultures.

8. Sample Size for the Training Set

The document does not describe a "training set" in the context of machine learning or AI models. This device is a traditional laboratory diagnostic kit utilizing monoclonal antibodies, not an AI-driven system. Therefore, there is no training set in the typical sense. The "training" for such a kit would involve optimization and validation during its development phase, but these details are not provided as a distinct "training set size."

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

Since there is no training set mentioned in the context of an AI/ML device, the concept of establishing ground truth for a training set does not apply here. The "development" of the kit would be based on well-established immunological principles and laboratory validation procedures to ensure the monoclonal antibodies are specific and effective, rather than training an algorithm on a dataset with ground truth.

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The NucliSens® CMV pp67 assay is a nucleic acid amplification-based qualitative assay to be used in conjunction with the NucliSens® Reader for the detection of human cytomegalovirus (HCMV) pp67 mRNA in EDTA anticoagulated whole blood from adult transplant donors and HIV infected individuals. It is intended as an aid in the diagnosis of active (acute or reactivated) HCMV infection. This product is not intended for use in screening of blood or plasma donors.

The NucliSens® CMV pp67 assay is a nucleic acid amplification-based qualitative assay to be used in conjunction with the NucliSens®™ Reader for the detection of human cytomegalovirus (HCMV) pp67 mRNA in EDTA anticoagulated whole blood from adult transplant donors and HIV infected individuals. It is intended as an aid in the diagnosis of active (acute or reactivated) HCMV infection. This product is not intended for use in screening of blood or plasma donors.

The NucliSens® CMV pp67 assay is comprised of four separate stages:

• Nucleic acid release
• Nucleic acid isolation
• Nucleic acid amplification
• Nucleic acid detection

The provided text describes the NucliSens® CMV pp67 assay and its performance, primarily in comparison to existing methods (CMV pp65 antigenemia assay and cell culture) for diagnosing active HCMV infection in transplant and HIV-infected populations. However, it does not explicitly state "acceptance criteria" in the format of defined thresholds that the device had to meet. Instead, the study provides performance metrics (sensitivity, specificity, agreement) and concludes that the device performed "as well as" the predicate device.

Let's extract the relevant information based on your request, inferring "acceptance criteria" from the comparative performance.

1. Table of Acceptance Criteria and Reported Device Performance

As explicit acceptance criteria are not stated, the table below will summarize the comparative effectiveness results, which are analogous to demonstrating the device meets performance expectations for substantial equivalence. The predicate device's performance (or a combined reference standard) implicitly sets the bar.

Note: The document states "The conclusions drawn from the clinical tests demonstrate that the NucliSens™ CMV pp67 assay is as safe, as effective, and performed as well as the legally marketed CMV Brite™ CMV Antigenemia Detection Test Kit and typical cell culture in the diagnosis of active HCMV infection." This implies that performance comparable to these reference methods serves as the de facto acceptance criteria.

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

• Interfering Substances: 60 samples (10 unspiked negative, 10 spiked positive for each of 6 categories).
• Analytical Specificity (CMV Strains/Cross-reactivity):
  • HCMV Strains: Number not specified, but 5 laboratory strains (Towne, C87, Davis, ESP, AD169) were tested.
  • Cross-reactivity (other infections/diseases): 86 samples (various infections/diseases, tested unspiked and spiked with HCMV RNA; e.g., 10 HIV-1, 8 HTLV-I/II, 10 HSV-I, etc.)
  • Cross-reactivity (related Herpes viruses): 4 cell lines (HHV-6A, HHV-6B, HHV-7, HHV-8 infected).
  • Cross-reactivity (bacterial flora): 4 types of bacterial flora, each with 2 aliquots (unspiked and spiked).
• Analytical Specificity (Whole Blood Donors): 100 donors (50 HCMV seronegative, 50 HCMV seropositive).
• Analytical Sensitivity (CMV Infected Population): 51 clinical specimens.
• Reproducibility: 4 samples tested multiple times (repeated 8 times per lot/site/tech combination for some, 4 or 6 times for others). This involved 3 sites, with 1-3 technicians per site, and 3 lots of reagents. Total tests: (37+32+29) + (44+31+33) + (30+35+34) totals 325-334 specific tests for each specimen type (1-4).
• Limit of Detection: Based on the reproducibility data (4 samples with known RNA input, tested repeatedly).
• Clinical Performance (Transplant Population): 50 patients; multiple specimens collected over an extended period. Total valid tests considered: 368.
• Clinical Performance (HIV-1 Infected Population): 50 individuals; multiple specimens collected over an extended period. Total valid tests considered: 450.

Data Provenance:

• Analytical Specificity (CMV Strains/Cross-reactivity): North America and Europe for clinical/analytical studies; laboratory strains AD169, Towne, C87, Davis, ESP.
• Analytical Specificity (Whole Blood Donors): Not specified, but generally implies a US-based population if the study was for FDA clearance.
• Analytical Sensitivity (CMV Infected Population): United States (New England, Northeast, and West Coast regions).
• Reproducibility: 3 sites, likely in different geographical locations (internal to the company or contracted third parties).
• Clinical Performance (Transplant Population): Italy (longitudinal study).
• Clinical Performance (HIV-1 Infected Population): The Netherlands (longitudinal study).
• Overall: The data is primarily prospective (longitudinal studies) and retrospective (existing positive/negative panels) from multiple countries including the USA, Italy, and The Netherlands.

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

The document does not explicitly mention the number or specific qualifications of experts (e.g., radiologists with 10 years of experience) used to establish ground truth.

Instead, the "ground truth" (referred to as "true state of nature") for the clinical studies was established by conventional laboratory methods:

• For the analytical sensitivity and clinical performance studies, ground truth was determined by cell culture and/or an FDA-cleared CMV pp65 antigenemia assay.
• These are established diagnostic laboratory tests, and their interpretation would typically be performed by trained clinical laboratory professionals or physicians overseeing patient diagnosis.

4. Adjudication Method for the Test Set

The document describes the "ground truth" being established by either antigenemia OR culture being positive for a "CMV positive" designation, and BOTH antigenemia AND culture being negative for a "CMV negative" designation. This serves as the adjudication method for the reference standard.

• "If either antigenemia or culture were found positive for a specimen, that particular specimen was regarded as CMV positive. If both antigenemia and culture were negative, the state of nature for that specimen was considered CMV negative."

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

No. This device is a diagnostic assay (in vitro diagnostic device), not an imaging device or AI-assisted diagnostic tool that would involve human "readers" in the typical sense of an MRMC study assessing image interpretation. The NucliSens® CMV pp67 assay is an automated nucleic acid amplification test. It does not involve human readers interpreting AI output.

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

Yes, the studies presented are for the standalone (algorithm only) performance of the NucliSens® CMV pp67 assay. It's an automated molecular diagnostic test; the "algorithm" is the entire test procedure from sample processing to result interpretation by the NucliSens® Reader. Its performance is compared directly against the established laboratory methods (antigenemia and cell culture).

7. The Type of Ground Truth Used (expert consensus, pathology, outcomes data, etc.)

The primary ground truth used in the clinical performance studies was a composite reference standard of:

• Traditional laboratory methods: Cell culture and an FDA-cleared CMV pp65 antigenemia assay results.
• For the analytical specificity and sensitivity studies, the ground truth involved known CMV strains, known negative panels, and clinical panels confirmed by existing diagnostic tests.

8. The Sample Size for the Training Set

The document does not explicitly describe a separate "training set" for the NucliSens® CMV pp67 assay in the context of machine learning or algorithm development. This is typical for a traditional in vitro diagnostic (IVD) assay which follows a pre-defined chemical and molecular reaction protocol rather than being "trained" iteratively on data. The validation studies (analytical and clinical) serve to demonstrate the performance of the fixed assay.

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

Since there is no explicitly defined "training set" in the context of an algorithm that learns from data, this question is not directly applicable. If one were to consider any preliminary data used during the assay's development (e.g., to optimize primer design, reaction conditions), the ground truth for such internal development would typically come from well-characterized positive and negative samples, often using established methods like viral culture or PCR. However, this is not detailed in the 510(k) summary.

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The CMV Brite™ Turbo Kit is intended for the qualitative detection of Cytomegalovirus (CMV) lower matrix protein pp65 by indirect immunofluorescence using microscopy in isolated peripheral blood leukocytes obtained from ethylenediaminetetraacetic acid (EDTA) or heparin anticoagulated human peripheral blood. The detection of CMV pp65 in human perpheral blood cells aids in the diagnosis of acute or reactivated CMV infection. This product is not FDA cleared (approved) for use in testing (i.e., screening) of blood or plasma donors.

The CMV antigenemia assay has been developed using a cocktail of two monoclonal antibodies (C10/C11) directed against CMV lower matrix protein pp65(6). The assay uses the C10/C11 cocktail in an indirect immunofluorescence staining of cytospin preparations of peripheral blood leukocytes. The CMV Brite™ Turbo antigenemia assay is completed within two hours of blood collection which saves time and means a rapid answer for the clinician. The CMV Brite™ Turbo method consists of:

• Direct lysis of peripheral blood erythrocytes a.
• Preparation of cytospin slides b.
• Fixation and permeabilization C.
• Indirect immunofluorescence staining using monoclonal antibodies directed d. against CMV pp65 protein
• Reading and evaluation of results e.
  The first step in the CMV Brite™ Turbo method involves direct lysis of the peripheral blood erythrocytes(22). Following lysis the leukocytes are cytocentrifuged onto a slide, fixed and permeabilized to allow subsequent detection of CMV pp65 antigen. The presence of the CMV pp65 antigen is detected by the C10/C11 antibody cocktail and visualized by means of a specific secondary FITC-labeled antibody. CMV antigenpositive leukocytes exhibit homogeneous yellow-green polylobate nuclear staining when observed using a fluorescence microscope. The number of CMV antigen-positive cells are counted per duplicate stain.
  The whole procedure can be performed in approximately 2 hours. The total analysis time has been shortened by performing direct erythrocyte lysis on whole blood and avoiding dextran sedimentation. Further time has been saved by shortening individual steps in the protocol so that the whole CMV antigenemia procedure has been reduced in time by more than 50%.

Here's a breakdown of the acceptance criteria and study information for the CMV Brite™ Turbo Kit, based on the provided text:

Acceptance Criteria and Reported Device Performance

The provided document specifically compares the CMV Brite™ Turbo Kit to its predicate device, the CMV Brite™ Test Kit, in terms of performance characteristics. The "acceptance criteria" are implied by the performance of the predicate device, which the new device aims to be "as safe, effective, and performs as well as."

Note: The document states, "The performance characteristics of the CMV Brite™ Turbo Kit are the same as those established for the CMV Brite™ Test Kit." However, the comparative table (Table 1) then lists slightly different numerical values for sensitivity, specificity, and predictive values. This might indicate that "the same" refers to generally comparable performance rather than identical numerical results, or that the table itself is the primary source of the "established" performance for the new device based on its own testing. For the purpose of this response, I'm listing the specific numbers presented in the table for the CMV Brite™ Turbo.

Study Details

1. Sample size used for the test set and the data provenance:

   • Test Set Sample Size: The document does not explicitly state the sample size used for the test set.
   • Data Provenance: The document does not specify the country of origin of the data or whether it was retrospective or prospective.

2. Number of experts used to establish the ground truth for the test set and the qualifications of those experts:

   • The document does not specify the number of experts or their qualifications for establishing ground truth for the test set. The nature of the device (immunofluorescence staining read by microscopy) implies a human reader, but details about independent review or consensus are missing.

3. Adjudication method for the test set:

   • The document does not describe any specific adjudication method (e.g., 2+1, 3+1, none) for the test set.

4. 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:

   • No, an MRMC comparative effectiveness study was not done. This device is a test kit for laboratory use, involving manual interpretation via fluorescence microscopy, not an AI-assisted diagnostic tool. Therefore, the concept of human readers improving with AI assistance is not applicable here.

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

   • No, a standalone algorithm-only performance study was not done. This device is a manual laboratory test kit that requires human interpretation (microscopy of stained cells) to detect CMV pp65 antigen. It is not an algorithm or AI-driven system.

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

   • The type of ground truth is not explicitly stated. Given the nature of CMV antigenemia testing, the ground truth would typically be established by clinical diagnosis of CMV infection, often supported by other laboratory methods (e.g., PCR, viral culture, or clinical outcomes), or by expert interpretation of the gold standard for CMV antigenemia. The document describes the new device being compared to a "legally marketed device" (CMV Brite™ Test Kit), implying the predicate device serves as a reference, but the ultimate ground truth for both is not detailed.

7. The sample size for the training set:

   • The document does not mention a "training set" as this is not an machine learning/AI device. The studies described are for validating the performance of a wet laboratory assay.

8. How the ground truth for the training set was established:

   • Not applicable, as there is no "training set" for this type of device.

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The Light Diagnostics Cytomegalovirus Direct Immunofluorescence Assay is intended for use in centrifugation enhanced shell vials in the qualitative detection and identification of immediate early antigen- of human CMV.

Light Diagnostics Cytomegalovirus Direct Immunofluorescence Assay (CMV DFA) uses the standard laboratory direct immunofluorescence technique for the culture confirmation of cytomegalovirus. The DFA is based on the principle of antigen identification using a detector monoclonal antibody conjugated to fluorescein isothiocyanate. The substrate consists of a slide prepared from the tissue cultured cells from a clinical specimen inoculum. Anti CMV FITC laheled antibody is applied to the substrate. The antibody will bind to specific antigen, if present, in the substrate. The fluorescein conjugated monoclonal antibody allows for visualization of the antigen / antibody complex by flucrescence microscopy. Mouse monoclonal antibody is used as the detector antibody in Light Diagnostics Cytomegalovirus Direct Immunofluorescence Assay. The use of monoclonal antibody ensures increased specificity and reduced non-specific interference. The monoclonal antibody is specific for a 68-72 kDa non-structural protein designated as immediate early (IE) antigen of human CMV.

Here's a summary of the acceptance criteria and study details for the Light Diagnostics Cytomegalovirus Direct Immunofluorescence Assay, based on the provided text:

Acceptance Criteria and Device Performance

Study Details

1. Sample Size used for the test set and data provenance:

   • Sample Size: 516 specimens.
   • Data Provenance: Not explicitly stated, but clinical evaluation was performed. It's likely retrospective given the comparison to existing methods, but this is an inference. No country of origin is mentioned.

2. Number of experts used to establish the ground truth for the test set and qualifications of those experts:

   • This information is not provided in the summary. The ground truth was established by comparison to "indirect immunofluorescence tests," which are themselves diagnostic methods, not necessarily expert review.

3. Adjudication method for the test set:

   • This information is not provided in the summary. The comparison was made against "indirect immunofluorescence tests."

4. 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:

   • An MRMC study was not performed, nor is AI involved. This is a diagnostic assay, not an AI-assisted interpretation tool.

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

   • Yes, this is a standalone diagnostic assay. Its performance (sensitivity and specificity) was evaluated independently against another diagnostic method.

6. The type of ground truth used:

   • The ground truth was established by comparison to indirect immunofluorescence tests for the detection and identification of CMV. This indicates using a previously established and accepted diagnostic method as the reference standard.

7. The sample size for the training set:

   • This information is not provided. This device is a diagnostic assay, not a machine learning model, so the concept of a "training set" for model development isn't directly applicable in the same way. The development involved characterizing the monoclonal antibody and testing it with reference virus strains and clinical isolates, but a specific "training set size" is not mentioned.

8. How the ground truth for the training set was established:

   • As mentioned above, the concept of a "training set" for model development isn't directly applicable. For the development/characterization of the assay components, the ground truth was established by:
     • Reacting the anti-CMV antibody with reference virus strains and clinical isolates (implying known CMV status for these samples).
     • Evaluating cross-reactivity with "a variety of viral pathogens and host cell controls" and "a variety of microorganisms" (implying known non-CMV status for these samples).

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