← Product Code [PSZ](/productcode/PSZ) · K071591

# RAMP INFLUENZA  A/B ASSAY (K071591)

_Response Biomedical Corp. · PSZ · Apr 16, 2008 · Microbiology · SESE_

**Canonical URL:** https://fda-staging.innolitics.com/device/K071591

## Device Facts

- **Applicant:** Response Biomedical Corp.
- **Product Code:** [PSZ](/productcode/PSZ.md)
- **Decision Date:** Apr 16, 2008
- **Decision:** SESE
- **Submission Type:** Traditional
- **Regulation:** 21 CFR 866.3328
- **Device Class:** Class 2
- **Review Panel:** Microbiology
- **Attributes:** Real-World Evidence, Pediatric

## Real-World Evidence

| Submission | Device | Sponsor | RWD Sources | RWE Use Summary | Key Tags |
| --- | --- | --- | --- | --- | --- |
| K071591 · Apr 16, 2008 | RAMP INFLUENZA A/B ASSAY | Response Biomedical Corp. | Retrospective frozen clinical nasopharyngeal swab samples; Retrospective frozen clinical nasal wash/aspirate samples | Retrospective clinical samples were used to supplement the prospective clinical trial performance data for the RAMP Influenza A/B Assay. | Retrospective study; Frozen clinical samples; Clinical performance |

### Clinical Evidence

| Study Design | Population | Comparator | Key Endpoints |
| --- | --- | --- | --- |
| Retrospective evaluation of frozen clinical samples | Patients with respiratory symptoms; Sample Size: 75 nasopharyngeal swabs and 130 nasal wash/aspirate samples; Number of Sites: 2 | Cell culture | Positive and negative percent agreement |

## Indications for Use

The RAMP® Influenza A/B Assay is a qualitative immunochromatographic assay used to identify the presence of Influenza A and Influenza B nucleoprotein antigens in nasal wash, nasal aspirate, nasopharyngeal aspirate, and nasopharyngeal swab specimens from symptomatic patients. It is an in vitro diagnostic assay that aids in the rapid differential diagnosis of influenza viral infections in symptomatic patients. A negative test is presumptive and it is recommended these results be confirmed by cell culture. Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other management decisions. The test performance characteristics for Influenza B were established primarily with retrospective, frozen specimens. Users may wish to further evaluate the sensitivity performance of this test for Influenza B using fresh samples.

## Device Story

RAMP Influenza A/B Assay is a qualitative immunochromatographic test for detecting Influenza A and B nucleoprotein antigens. Input: nasal wash, nasal aspirate, nasopharyngeal aspirate, or nasopharyngeal swab samples. Process: sample mixed with buffer and fluorescent-dyed antibody-conjugated particles; mixture applied to test cartridge; sample migrates along strip; antigens bind to specific antibodies; RAMP 200 instrument measures fluorescence at detection and internal standard zones. Output: RAMP Ratio calculated by instrument; compared to pre-defined thresholds for positive/negative result. Used in central labs, stat-labs, and point-of-care facilities by trained operators. Provides rapid results (~15 mins) to aid clinical diagnosis; negative results require cell culture confirmation. Benefits: rapid differential diagnosis compared to traditional culture methods.

## Clinical Evidence

Prospective multi-center trial (n=844) and retrospective study (n=205) compared RAMP Influenza A/B Assay to cell culture. Prospective study included pediatric and adult patients. Sensitivity for Influenza A ranged from 69.6% to 91.7% and for Influenza B from 50.0% to 100% across age groups and sample types. Specificity was high, ranging from 89.1% to 100%. Retrospective testing showed positive percent agreement of 76.7-84.6% for Flu A and 77.8-84.6% for Flu B.

## Technological Characteristics

Qualitative immunochromatographic assay using fluorescent-dyed particles. Requires RAMP 200 instrument for fluorescence measurement and ratio calculation. Compatible with various transport media and swab materials (foam, polyester, rayon, nylon). Not for use with calcium alginate swabs. Standalone instrument-based system.

## Regulatory Identification

An influenza virus antigen detection test system is a device intended for the qualitative detection of influenza viral antigens directly from clinical specimens in patients with signs and symptoms of respiratory infection. The test aids in the diagnosis of influenza infection and provides epidemiological information on influenza. Due to the propensity of the virus to mutate, new strains emerge over time which may potentially affect the performance of these devices. Because influenza is highly contagious and may lead to an acute respiratory tract infection causing severe illness and even death, the accuracy of these devices has serious public health implications.

## Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) The device's sensitivity and specificity performance characteristics or positive percent agreement and negative percent agreement, for each specimen type claimed in the intended use of the device, must meet one of the following two minimum clinical performance criteria:
(i) For devices evaluated as compared to an FDA-cleared nucleic acid based-test or other currently appropriate and FDA accepted comparator method other than correctly performed viral culture method:
(A) The positive percent agreement estimate for the device when testing for influenza A and influenza B must be at the point estimate of at least 80 percent with a lower bound of the 95 percent confidence interval that is greater than or equal to 70 percent.
(B) The negative percent agreement estimate for the device when testing for influenza A and influenza B must be at the point estimate of at least 95 percent with a lower bound of the 95 percent confidence interval that is greater than or equal to 90 percent.
(ii) For devices evaluated as compared to correctly performed viral culture method as the comparator method:
(A) The sensitivity estimate for the device when testing for influenza A must be at the point estimate of at least 90 percent with a lower bound of the 95 percent confidence interval that is greater than or equal to 80 percent. The sensitivity estimate for the device when testing for influenza B must be at the point estimate of at least 80 percent with a lower bound of the 95 percent confidence interval that is greater than or equal to 70 percent.
(B) The specificity estimate for the device when testing for influenza A and influenza B must be at the point estimate of at least 95 percent with a lower bound of the 95 percent confidence interval that is greater than or equal to 90 percent.
(2) When performing testing to demonstrate the device meets the requirements in paragraph (b)(1) of this section, a currently appropriate and FDA accepted comparator method must be used to establish assay performance in clinical studies.
(3) Annual analytical reactivity testing of the device must be performed with contemporary influenza strains. This annual analytical reactivity testing must meet the following criteria:
(i) The appropriate strains to be tested will be identified by FDA in consultation with the Centers for Disease Control and Prevention (CDC) and sourced from CDC or an FDA-designated source. If the annual strains are not available from CDC, FDA will identify an alternative source for obtaining the requisite strains.
(ii) The testing must be conducted according to a standardized protocol considered and determined by FDA to be acceptable and appropriate.
(iii) By July 31 of each calendar year, the results of the last 3 years of annual analytical reactivity testing must be included as part of the device's labeling. If a device has not been on the market long enough for 3 years of annual analytical reactivity testing to have been conducted since the device received marketing authorization from FDA, then the results of every annual analytical reactivity testing since the device received marketing authorization from FDA must be included. The results must be presented as part of the device's labeling in a tabular format, which includes the detailed information for each virus tested as described in the certificate of authentication, either by:
(A) Placing the results directly in the device's § 809.10(b) of this chapter compliant labeling that physically accompanies the device in a separate section of the labeling where the analytical reactivity testing data can be found; or
(B) In the device's label or in other labeling that physically accompanies the device, prominently providing a hyperlink to the manufacturer's public Web site where the analytical reactivity testing data can be found. The manufacturer's home page, as well as the primary part of the manufacturer's Web site that discusses the device, must provide a prominently placed hyperlink to the Web page containing this information and must allow unrestricted viewing access.
(4) If one of the actions listed at section 564(b)(1)(A)-(D) of the Federal Food, Drug, and Cosmetic Act occurs with respect to an influenza viral strain, or if the Secretary of Health and Human Services (HHS) determines, under section 319(a) of the Public Health Service Act, that a disease or disorder presents a public health emergency, or that a public health emergency otherwise exists, with respect to an influenza viral strain:
(i) Within 30 days from the date that FDA notifies manufacturers that characterized viral samples are available for test evaluation, the manufacturer must have testing performed on the device with those viral samples in accordance with a standardized protocol considered and determined by FDA to be acceptable and appropriate. The procedure and location of testing may depend on the nature of the emerging virus.
(ii) Within 60 days from the date that FDA notifies manufacturers that characterized viral samples are available for test evaluation and continuing until 3 years from that date, the results of the influenza emergency analytical reactivity testing, including the detailed information for the virus tested as described in the certificate of authentication, must be included as part of the device's labeling in a tabular format, either by:
(A) Placing the results directly in the device's § 809.10(b) of this chapter compliant labeling that physically accompanies the device in a separate section of the labeling where analytical reactivity testing data can be found, but separate from the annual analytical reactivity testing results; or
(B) In a section of the device's label or in other labeling that physically accompanies the device, prominently providing a hyperlink to the manufacturer's public Web site where the analytical reactivity testing data can be found. The manufacturer's home page, as well as the primary part of the manufacturer's Web site that discusses the device, must provide a prominently placed hyperlink to the Web page containing this information and must allow unrestricted viewing access.

## Predicate Devices

- BinaxNOW® Influenza A & B Test ([K062109](/device/K062109.md))

## Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
K071591

B. Purpose for Submission:
This is an application for a qualitative immunochromatographic assay used with the RAMP® 200 reader for the qualitative detection of Influenza A and Influenza B nucleoprotein antigens in human nasal wash/aspirate, nasopharyngeal aspirate, and nasopharyngeal swab specimens.

C. Measurand:
Influenza A and Influenza B nucleoprotein antigens

D. Type of Test:
Qualitative immunochromatographic test that utilizes the RAMP® 200 reader for the differential determination of Influenza A and Influenza B in nasal wash/aspirate, nasopharyngeal aspirate, and nasopharyngeal swab samples.

E. Applicant:
Response Biomedical Corp.

F. Proprietary and Established Names:
RAMP Influenza A/B Assay

G. Regulatory Information:
1. Regulation section:
21 CFR section 866.3330, Influenza virus serological reagents.
2. Classification:
Class I
3. Product code:
GNX

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4. Panel:

Microbiology (83)

H. Intended Use:

1. Intended use(s):

The RAMP® Influenza A/B Assay is a qualitative immunochromatographic assay used to identify the presence of Influenza A and Influenza B nucleoprotein antigens in nasal wash, nasal aspirate, nasopharyngeal aspirate, and nasopharyngeal swab specimens from symptomatic patients. It is an *in vitro* diagnostic assay aids in the rapid differential diagnosis of influenza viral infections in symptomatic patients. A negative test is presumptive and it is recommended these results be confirmed by cell culture. Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other management decisions.

The test performance characteristics for Influenza B were established primarily with retrospective, frozen specimens. Users may wish to further evaluate the sensitivity performance of this test for Influenza B using fresh samples.

2. Indication(s) for use:

Same as intended use.

3. Special conditions for use statement(s):

For prescription use only.

4. Special instrument requirements:

To be used only with the RAMP® 200 reader.

I. Device Description:

The RAMP Influenza A/B Assay is a qualitative immunochromatographic test that utilizes the RAMP® 200 reader for the differential determination of Influenza A and Influenza B in nasal wash/aspirate, nasopharyngeal aspirate, and nasopharyngeal swab samples. A wash/aspirate or swab sample is mixed with Sample Buffer using Assay Tip containing fluorescent-dyed latex particles coated with monoclonal antibodies targeting the Influenza A and B nucleoproteins, and applied into the sample well of the Test Cartridge. The sample migrates along the strip. Fluorescent-dyed latex (test) particles, coated with anti-Influenza A and anti-Influenza B nucleoprotein monoclonal antibodies

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bind to Influenza A or B nucleoprotein antigens, respectively, if present in the sample. As the sample migrates along the strip, Influenza-bound particles are captured at either the Influenza A or the Influenza B detection zone which is coated with either anti-Influenza A or anti-Influenza B nucleoprotein monoclonal antibodies, respectively. Excess fluorescent- dyed particles are captured at the internal standard zone. RAMP® 200 reader then measures the amount of fluorescence emitted by the complexes bound at the two detection zones (Influenza A and Influenza B) and at the internal standard zone. The RAMP® 200 reader calculates a ratio (RAMP Ratio) of each influenza zone (A or B) fluorescence reading to the internal standard zone fluorescence reading. The reader compares these ratios to pre-defined threshold limits to determine a positive or negative result for Influenza A and Influenza B in the tested sample.

# J. Substantial Equivalence Information:

1. Predicate device name(s):

BinaxNOW Influenza A & B Test

2. Predicate K number(s):

(K062109)

3. Comparison with predicate:

|  Similarities  |   |   |
| --- | --- | --- |
|  Item | Device | Predicate  |
|   | RAMP Influenza A/B Assay | BinaxNOW Influenza A & B Test  |
|  Features/Technical Information |  |   |
|  Intended Use | The RAMP® Influenza A/B Assay is a qualitative immunochromatographic assay used with the RAMP® 200 reader to identify the presence of Influenza A and Influenza B nucleoprotein antigens in nasal wash, nasal aspirate, nasopharyngeal aspirate, and nasopharyngeal swab specimens from symptomatic patients. It is an in vitro diagnostic assay aids in the rapid differential diagnosis of influenza viral infections in symptomatic patients. A negative test is presumptive and it is recommended these results be confirmed by cell culture. Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other management decisions. | The BinaxNOW Influenza A & B Test is an in vitro immunochromatographic assay for the qualitative detection of Influenza A and B nucleoprotein antigens in nasopharyngeal (NP) Swab, nasal swab, and nasal wash/aspirate specimens. It is intended to aid in the rapid differential diagnosis of Influenza A and B viral infections. Negative test results should be confirmed by cell culture.  |

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|  Qualitative/Quantitative | Qualitative | Qualitative  |
| --- | --- | --- |
|  Test Principle | Immunochromatographic fluorescence immunoassay | Immunochromatographic colloidal gold immunoassay  |
|  Antibodies Used | Two mouse monoclonal antibodies recognizing the nucleoprotein of the Influenza A virus and two mouse monoclonal antibodies recognizing the nucleoprotein of the Influenza B virus. | Unknown antibodies recognizing the nucleoprotein of the Influenza A and influenza B viruses.  |
|  Specimen Type | Nasal wash, nasal aspirate, nasopharyngeal aspirate, and nasopharyngeal swab specimens | Nasal wash, nasal swab, nasopharyngeal aspirate, and nasopharyngeal swab specimens  |
|  Reagent Stability | In sealed pouch: Up to stated expiration date stored at 15-30°C. Do not remove from pouch until ready to use. | In sealed pouch: Up to stated expiration date stored at 15-30°C. Do not remove from pouch until ready to use.  |
|  Test Time | 15 minutes | 15 minutes  |
|  Waste Handling | Dispose of Test Cartridge as per correct institutional biohazard procedure. | Dispose of Test Cartridge as per correct institutional biohazard procedure.  |
|  Self Contained | Yes | Yes  |
|  Portable | Yes | Yes  |
|  Patient Usage | No | No  |
|  Standard Curve | Not Applicable | Not Applicable  |
|  External Controls | RAMP Influenza A/B Test kit contains Positive and Negative Control swabs. These swabs will monitor the performance of the entire system. | BinaxNOW Influenza A&B Test kit contains Positive and Negative Control swabs. These swabs will monitor the performance of the entire assay.  |
|  Analytes | Influenza A and Influenza B | Influenza A and Influenza B  |
|  Product Type | In Vitro Diagnostic | In Vitro Diagnostic  |
|  Differences  |   |   |
| --- | --- | --- |
|  Item | Device | Predicate  |
|   | RAMP Influenza A/B Assay | BinaxNOW Influenza A & B Test  |
|  Features/Technical Information |   |   |
|  Sample Preparation | Use provided disposable dropper to mix the sample in provided pre-measured sample buffer vial. | Elution of swab in sample buffer. None for aspirate.  |
|  Test Procedure | Mix sample with provided transfer device and add sample to Test Cartridge. | Add sample to Test Device in a drop-wise manner expelling all material from the Binax supplied pipette.  |

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|  Automated Processing | Instrument transport of Test Cartridge within reader is the only moving step. No internal liquid handling. One-step immunochromatographic assay requiring no additional washes. | Added sample develops on Test Device providing visual result.  |
| --- | --- | --- |
|  Read Results | Read results on instrument screen, or print with optional printer. | Visual read for presence or absence of control and test lines.  |
|  Instrument | RAMP 200 | None  |
|  Automated Analysis | Yes | No. Operator must read result precisely 15 minutes after sample application. Results read before or after 15 minutes may not be accurate.  |
|  Quality Controls | Provided in every Test Cartridge. Built in performance controls for routine QC requirements. A comparison of the internal standard and the assay result indicates that sufficient properly mixed sample was applied to the test device and that unbound fluorescent label washed sufficiently from the detection zone, and the device was inserted and read properly by the instrument. Background fluorescence measurement serves as a negative control. Reader controls also prevent a used or expired cartridge from being read by the reader. Antibody quality, system function and assay timing are checked on each assay run. An unacceptable result from the control displays a warning message without a test result on the instrument indicating that the test should be repeated. | Provided in every Test Device. Built-in procedural controls. An untested device has a blue line at the “Control” position. If the test flows and the reagents work, this blue line will always turn pink in a tested device. The clearing of background color from the result window is a negative background control. The background color in the window should be light pink to white within 15 minutes. Background color should not hinder reading of the test.  |
|  System Quality Control | A built-in Internal Quality Control (IQC) function performs an automated test of all Reader analytical systems on a scheduled or operator-determined basis to provide a record of proper Reader functionality. | Not Applicable  |

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|  Calibrator | Lot card contains calibration and expiration information and is supplied with every kit. System will not process cartridge if lot card information is not stored in instrument. | Not Applicable  |
| --- | --- | --- |
|  Result Interpretation | Negative or Positive for Influenza A and B; automated analysis, result displayed on LCD screen of instrument | Negative or Positive for Influenza A or B; result visually read by presence or absence of test lines at 15 minutes following sample application. Results read before or after 15 minutes may be inaccurate.  |

# K. Standard/Guidance Document Referenced (if applicable):

Not applicable.

# L. Test Principle:

The RAMP Influenza A/B Assay is a qualitative immunochromatographic test that utilizes the  $\mathrm{RAMP}^{\text{®}}$  200 reader for the differential determination of Influenza A and Influenza B in nasal wash/aspirate, nasopharyngeal aspirate, and nasopharyngeal swab samples. A wash/aspirate or swab sample is mixed with Sample Buffer using Assay Tip containing fluorescent-dyed latex particles coated with monoclonal antibodies targeting the Influenza A and B nucleoproteins, and applied into the sample well of the Test Cartridge. The sample migrates along the strip. Fluorescent-dyed latex (test) particles, coated with anti-Influenza A and anti-Influenza B nucleoprotein monoclonal antibodies bind to Influenza A or B nucleoprotein antigens, respectively, if present in the sample. As the sample migrates along the strip, Influenza-bound particles are captured at either the Influenza A or the Influenza B detection zone which is coated with either anti-Influenza A or anti-Influenza B nucleoprotein monoclonal antibodies, respectively. Excess fluorescent-dyed particles are captured at the internal standard zone.  $\mathrm{RAMP}^{\text{®}}$  200 reader then measures the amount of fluorescence emitted by the complexes bound at the two detection zones (Influenza A and Influenza B) and at the internal standard zone. The  $\mathrm{RAMP}^{\text{®}}$  200 reader calculates a ratio (RAMP Ratio) of each influenza zone (A or B) fluorescence reading to the internal standard zone fluorescence reading. The reader compares these ratios to pre-defined threshold limits to determine a positive or negative result for Influenza A and Influenza B in the tested sample.

The  $\mathrm{RAMP}^{\text{®}}$  200 is a general use fluorometer that analyzes results produced by immunoassays that use a fluorophore having an excitation wavelength of  $560~\mathrm{nm}$  and an emission wavelength of  $610~\mathrm{nm}$ .

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![img-0.jpeg](img-0.jpeg)

## M. Performance Characteristics (if/when applicable):

### 1. Analytical performance:

#### a. Precision/Reproducibility:

The reproducibility of the RAMP Influenza A/B Assay was evaluated using a panel of 6 simulated samples that included high negative, low positive (the assay LoD) and moderate positive (2 X the assay LoD) Influenza A or Influenza B samples. The influenza strains used to prepare the simulated samples were Influenza A/Hong Kong/8/68 and Influenza B/Lee/40. The influenza strains were re-titered prior to testing. Panel samples were tested in triplicates at each of the 3 testing sites by 2 operators for 5 days using 1 RAMP Influenza A/B kit lot (6 samples X 3 replicates X 2 operators X 5 days X 3 sites = 540). The overall percent agreement for the RAMP Influenza A/B Assay was 100%, with no significant differences within run (same operator on same day), between run, operators, and sites. The RAMP Flu A/B assay is a qualitative assay based on numerical RAMP Ratio values. The overall RAMP Ratio %CV across all sites ranged from 9.6% to 15.1% depending upon analyte type and concentration tested.

|   | Panel Member ID | Influenza A High Negative | Influenza A Low Positive | Influenza A Medium Positive | Influenza B High Negative | Influenza B Low Positive | Influenza B Medium Positive | Total Agreement All (%)  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  Viral Titer (EID50/ml) | 50 | 300 | 600 | 60 | 316 | 632  |   |
|  Site 1 | Agreement with Expected result | 30/30 | 30/30 | 30/30 | 30/30 | 30/30 | 30/30 | 180/180 (100%)  |
|   |  Mean RAMP Ratio Value | 692 | 2224 | 4252 | 414 | 1192 | 2045  |   |
|   |  % CV | 11.6% | 6.3% | 7.9% | 16.4% | 8.9% | 7.9%  |   |
|  Site 2 | Agreement with Expected result | 30/30 | 30/30 | 30/30 | 30/30 | 30/30 | 30/30 | 180/180 (100%)  |
|   |  Mean RAMP Ratio Value | 661 | 1993 | 3859 | 384 | 1076 | 1952  |   |
|   |  % CV | 7.7% | 8.1% | 9.3% | 15.6% | 9.5% | 7.0%  |   |

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|  Site 3 | Agreement with Expected result | 30/30 | 30/30 | 30/30 | 30/30 | 30/30 | 30/30 | 180/180 (100%)  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  Mean RAMP Ratio Value | 704 | 2326 | 4545 | 431 | 1212 | 2164  |   |
|   |  % CV | 14.5% | 9.9% | 13.5% | 11.3% | 9.5% | 10.5%  |   |
|   | Total Agreement with Expected result | 90/90 (100%) | 90/90 (100%) | 90/90 (100%) | 90/90 (100%) | 90/90 (100%) | 90/90 (100%) | 540/540 (100%)  |
|   |  95% CI | 96%-100% | 96%-100% | 96%-100% | 96%-100% | 96%-100% | 96%-100% | 99%-100%  |
|   |  Overall Mean RAMP Ratio | 685 | 2181 | 4219 | 410 | 1160 | 2054 |   |
|   |  Overall % CV | 11.9% | 10.4% | 12.6% | 15.1% | 10.6% | 9.6% |   |

b. Linearity/assay reportable range:
Not applicable.

c. Traceability, Stability, Expected values (controls, calibrators, or methods):
Not applicable.

d. Detection limit:

The analytical sensitivity (limit of detection or LoD) of the RAMP Influenza A/B Assay was determined using quantified (EID₅₀/mL) cultures of 2 Influenza A (1 H1N1 and 1 H3N2) and 2 Influenza B strains serially diluted in either viral transfer media (VTM) to simulate a swab sample type or phosphate buffered saline (PBS) solution to simulate a wash/aspirate sample type. The influenza strains were re-titered prior to testing. Each viral strain was tested in replicates of 20 per concentration of virus.

Analytical sensitivity (LoD) as defined as the lowest concentration, at which ≥ 95% of all replicates tested positive, ranged from 3.0×10² to 6.4×10² EID₅₀/mL for the Influenza A strains and 2.8×10² to 7.1×10⁴ EID₅₀/mL for the Influenza B strains in VTM and PBS.

|  Viral Strain | LoD Concentration  |
| --- | --- |
|  Influenza A/Hong Kong/8/68 (H3N2), ATCC VR-544 in VTM | 3.0 X 10² EID₅₀/ml  |
|  Influenza A/Hong Kong/8/68 (H3N2), ATCC VR-544 in PBS | 5.0 X 10² EID₅₀/ml  |
|  Influenza A/PR/8/34 (H1N1), ATCC VR-95 in VTM | 2.0 X 10² EID₅₀/ml  |
|  Influenza A/PR/8/34 (H1N1), ATCC VR-95 in PBS | 6.4 X 10² EID₅₀/ml  |

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e. Analytical reactivity:

The analytical reactivity of the RAMP Influenza A/B Assay was evaluated against multiple strains of Influenza A (H1N1 and H3N2 subtypes) and Influenza B. The panel consisted of 3 Influenza A subtype H1N1, 2 Influenza A subtype H3N2, and 3 Influenza B strains. 5 replicates were tested for each viral strain. The concentrations for analytical reactivity testing ranged from 3.0x10^{2} to 3.0x10^{3} EID_{50}/mL for the Influenza A strains and 1.6x10^{1} to 9.5x10^{3} EID_{50}/mL for the Influenza B strains. The influenza strains were re-titered prior to testing.

|  Viral Strain | Analytical Reactivity Concentration | RAMP Assay Result  |
| --- | --- | --- |
|  Influenza A/FM/1/47 (H1N1) ATCC VR-97 | 3.0 X 10^{3} EID_{50}/ml | 5/5 Flu A Positive  |
|  Influenza A/NWS/33 (H1N1) ATCC VR-219 | 3.0 X 10^{2} EID_{50}/ml | 5/5 Flu A Positive  |
|  Influenza A/New Jersey/8/76 (H1N1), ATCC VR-897 | 3.0 X 10^{2} EID_{50}/ml | 5/5 Flu A Positive  |
|  Influenza A/Aichi/2/68 (H3N2) ATCC VR-547 | 9.0 X 10^{2} EID_{50}/ml | 5/5 Flu A Positive  |
|  Influenza A/Victoria/3/75 (H3N2), ATCC VR-822 | 9.0 X 10^{2} EID_{50}/ml | 5/5 Flu A Positive  |
|  Influenza B/GL/1739/54 ATCC VR-103 | 9.5 X 10^{3} EID_{50}/ml | 5/5 Flu B Positive  |
|  Influenza B/Taiwan/2/62 ATCC VR-295 | 1.6 X 10^{1} EID_{50}/ml | 5/5 Flu B Positive  |
|  Influenza B/Hong Kong/5/72 ATCC VR-823 | 6.3 X 10^{3} EID_{50}/ml | 5/5 Flu B Positive  |

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# f. Analytical specificity:

The analytical specificity of the RAMP Influenza A/B Assay was evaluated by testing a panel consisting of 17 bacteria and 15 viruses that may be present in the nasal cavity or nasopharynx. Bacterial isolates were tested at  $10^{6}$  cfu/mL and viral isolates were tested at approximately  $10^{5}$  TCID $_{50}$ /mL at  $n = 3$  replicates each. The bacterial and viral organisms were re-grown and re-titered prior to cross-reactivity testing. Analytical specificity of the RAMP Influenza A/B Assay was  $100\%$ .

|  Strain/Isolate | Concentration | RAMP Flu A result | RAMP Flu B result  |
| --- | --- | --- | --- |
|  Adenovirus, Type 1 | 105 TCID50/mL | 3/3 Neg | 3/3 Neg  |
|  Adenovirus, Type 7a | 105.15 TCID50/mL | 3/3 Neg | 3/3 Neg  |
|  Respiratory Syncytial Virus (RSV) | 105 TCID50/mL | 3/3 Neg | 3/3 Neg  |
|  Human coronavirus, strain OC43 | 105 TCID50/mL | 3/3 Neg | 3/3 Neg  |
|  Human coronavirus Strain 229E | 105.23 TCID50/mL | 3/3 Neg | 3/3 Neg  |
|  Cytomegalovirus | 105.15 TCID50/mL | 3/3 Neg | 3/3 Neg  |
|  Enterovirus, Type 68 | 105.15 TCID50/mL | 3/3 Neg | 3/3 Neg  |
|  Epstein Barr Virus | 105 TCID50/mL | 3/3 Neg | 3/3 Neg  |
|  Measles | 105 TCID50/mL | 3/3 Neg | 3/3 Neg  |
|  Mumps virus | 105 TCID50/mL | 3/3 Neg | 3/3 Neg  |
|  Human Parainfluenza, Type 1 | 104.75 TCID50/mL | 3/3 Neg | 3/3 Neg  |
|  Human Parainfluenza, Type 2 | 105 TCID50/mL | 3/3 Neg | 3/3 Neg  |
|  Human Parainfluenza, Type 3 | 105 TCID50/mL | 3/3 Neg | 3/3 Neg  |
|  Human metapneumovirus | 105 TCID50/mL | 3/3 Neg | 3/3 Neg  |
|  Human Rhinovirus, Strain 1A | 105 TCID50/mL | 3/3 Neg | 3/3 Neg  |
|  Bordetella pertussis | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |
|  Corynebacterium Sp. | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |
|  Escherichia coli | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |
|  Haemophilus influenzae | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |
|  Lactobacillus casei | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |
|  Legionella pneumophila | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |
|  Moraxella catarrhalis | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |
|  Mycobacterium tuberculosis avirulent | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |
|  Mycoplasma pneumoniae | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |
|  Neisseria menigitidis | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |
|  Neisseria sicca | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |
|  Psudomonas aeruginosa | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |
|  Streptococcus pneumoniae | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |
|  Streptococcus pyogenes (Group A) | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |
|  Streptococcus salivarius | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |
|  Staphylococcus epidermidis | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |
|  Staphylococcus aureus (Protein A producer) | 106 cfu/mL | 3/3 Neg | 3/3 Neg  |

Note: RAMP Influenza A/B Assay potential cross-reactivity with Chlamydophilia pneumoniae has not been determined.

{10}

g. Assay cut-off:

The lot-specific Threshold 1 (Normal cut-off) values and the fixed Threshold 2 (Extreme cut-off) values are programmed into the lot-specific Lot Cards and thus the RAMP Ratio values are invisible to the end user. Only negative or positive results are displayed. The Lot Card provides the reader with information specific to the lot, including lot number, expiration date, thresholds and standard curve information.

A. Threshold 1 (Normal cut-off):

1) Each RAMP Influenza A/B kit has lot-specific Threshold 1 levels. The Threshold 1 values (separate values for Influenza A and Influenza B) for each lot are based on QC testing data of the specific combination of component lots (Sample Buffer, Test Cartridge, and Assay Tips) used in the Influenza A/B kit lot. QC testing is performed on each kit lot to ensure that the lot meets release specifications as well as to provide the data to set the Threshold 1 values.

The algorithm used to determine the lot-specific Threshold 1 values is the same for each lot and uses defined calculations based on the results of the QC testing data of that lot, and the Threshold Factors that were based on the clinical trial results to optimize sensitivity and specificity of the RAMP Influenza A/B test (ROC Optimization).

B. Threshold 2 (Extreme cut-off):

1) During development of the RAMP Influenza A/B Assay, it was observed that when testing clinical samples with extremely high concentrations of Influenza A, there appeared to be interference in the Influenza B Assay potentially resulting in a False Positive Influenza B result in addition to the true positive Influenza A result. Testing was performed on the four Influenza A/B kit lots manufactured for the clinical trials to characterize this interference between multiple lots of the Influenza A/B Assays. Based on this testing, it was observed that the interference was very reproducible between kit lots. Threshold 2 values of 20,000 for Influenza A and 10,000 were arbitrarily set. The pre-determined "extreme" Threshold 2 values are fixed values and are applied to all test kit lots.

2) Based on this characterization of the interference of high levels of Influenza A antigen in the Influenza B Assay, an algorithm was derived to provide Threshold 1 for clinical samples that are negative or have low to moderate levels of Influenza A, but to switch to Threshold 2 if a double positive (Influenza A and Influenza B) was determined and the level of Influenza A was extremely high.

Because a simultaneous double infection of Influenza A and Influenza B in the same patient is exceedingly rare, the algorithm and Threshold 2 values

11

{11}

were biased towards minimizing the possibility of displaying a double Influenza A positive and Influenza B positive result.

h. Interfering Substances:

Whole blood and a number of other potentially interfering substances (medications and over the counter (OTC) products) that may be present naturally or artificially introduced in the nasal cavity or nasopharynx were evaluated in the RAMP Influenza A/B Assay. The substances were added to a negative sample (viral transport media), an Influenza A LoD positive sample (300 EID₅₀/mL), an Influenza A 2x LoD positive sample (600 EID₅₀/mL), an Influenza B LoD positive sample (316 EID₅₀/mL) and an Influenza B 2x LoD positive sample (632 EID₅₀/mL) and tested at n=3 replicates each in the RAMP Influenza A/B Assay. The influenza strains used to prepare the samples were Influenza A/Hong Kong/8/68 and Influenza B/Lee/40. These influenza strains were re-titered prior to interference testing.

None of the substances tested at the concentrations indicated interfere with the test results of negative or positive Influenza A and Influenza B samples in the RAMP Influenza A/B Assay. The RAMP Flu A/B assay is a qualitative assay based on numerical RAMP Ratio values. The Percent of Mean Control Ratio values were calculated using these RAMP Ratios.

12

{12}

|  Substance Tested | Conc. Tested | RAMP Results  |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |  Negative |   | Influenza A LoD |   | Influenza A 2x LoD |   | Influenza B LoD |   | Influenza B 2x LoD  |   |
|   |  | RAMP Flu A/B results | Percent of Mean Control Ratio FluA/FluB | RAMP Flu A/B results | Percent of Mean Control Ratio | RAMP Flu A/B results | Percent of Mean Control Ratio | RAMP Flu A/B results | Percent of Mean Control Ratio | RAMP Flu A/B results | Percent of Mean Control Ratio  |
|  Control (No interfering substance) | N/A | 3/3 Neg | 100% / 100% | 3/3 Flu A Pos | 100% | 3/3 Flu A Pos | 100% | 3/3 Flu B Pos | 100% | 3/3 Flu B Pos | 100%  |
|  Halls Throat Drop | 15% w/v | 3/3 Neg | 120% / 105% | 3/3 Flu A Pos | 101% | 3/3 Flu A Pos | 103% | 3/3 Flu B Pos | 99% | 3/3 Flu B Pos | 120%  |
|  Cepacol Throat Drop | 15% w/v | 3/3 Neg | 91% / 101% | 3/3 Flu A Pos | 98% | 3/3 Flu A Pos | 101% | 3/3 Flu B Pos | 105% | 3/3 Flu B Pos | 118%  |
|  Fisherman’s Friend Throat Drop | 15% w/v | 3/3 Neg | 120% / 105% | 3/3 Flu A Pos | 101% | 3/3 Flu A Pos | 103% | 3/3 Flu B Pos | 99% | 3/3 Flu B Pos | 120%  |
|  4-Acetamidophenol | 10 mg/mL | 3/3 Neg | 110% / 117% | 3/3 Flu A Pos | 99% | 3/3 Flu A Pos | 100% | 3/3 Flu B Pos | 104% | 3/3 Flu B Pos | 117%  |
|  Acetylsalicylic Acid | 15 mg/mL | 3/3 Neg | 89% / 106% | 3/3 Flu A Pos | 98% | 3/3 Flu A Pos | 97% | 3/3 Flu B Pos | 110% | 3/3 Flu B Pos | 118%  |
|  Chlorphiramine | 5 mg/mL | 3/3 Neg | 107% / 110% | 3/3 Flu A Pos | 101% | 3/3 Flu A Pos | 95% | 3/3 Flu B Pos | 112% | 3/3 Flu B Pos | 118%  |
|  Diphenylhydramine | 5 mg/mL | 3/3 Neg | 109% / 126% | 3/3 Flu A Pos | 99% | 3/3 Flu A Pos | 100% | 3/3 Flu B Pos | 115% | 3/3 Flu B Pos | 116%  |
|  Phenylpropanol-amine HCl | 20 mg/mL | 3/3 Neg | 126% / 100% | 3/3 Flu A Pos | 91% | 3/3 Flu A Pos | 96% | 3/3 Flu B Pos | 113% | 3/3 Flu B Pos | 113%  |
|  Oseltamivir Phosphate (Tamiflu) | 50 mg/mL | 3/3 Neg | 106% / 101% | 3/3 Flu A Pos | 105% | 3/3 Flu A Pos | 105% | 3/3 Flu B Pos | 106% | 3/3 Flu B Pos | 111%  |
|  Rimantadine HCl | 500 ng/mL | 3/3 Neg | 103% / 103% | 3/3 Flu A Pos | 105% | 3/3 Flu A Pos | 94% | 3/3 Flu B Pos | 111% | 3/3 Flu B Pos | 113%  |
|  Ribavirin (Rebetol) | 100 mg/mL | 3/3 Neg | 137% / 136% | 3/3 Flu A Pos | 111% | 3/3 Flu A Pos | 106% | 3/3 Flu B Pos | 118% | 3/3 Flu B Pos | 120%  |
|  Scope Mouthwash | 20% v/v | 3/3 Neg | 88% / 99% | 3/3 Flu A Pos | 96% | 3/3 Flu A Pos | 94% | 3/3 Flu B Pos | 98% | 3/3 Flu B Pos | 112%  |
|  Good and Kind Mouthwash | 20% v/v | 3/3 Neg | 97% / 118% | 3/3 Flu A Pos | 97% | 3/3 Flu A Pos | 106% | 3/3 Flu B Pos | 99% | 3/3 Flu B Pos | 125%  |

{13}

|  Cepacol Mouth Wash | 20% v/v | 3/3 Neg | 88% / 85% | 3/3 Flu A Pos | 103% | 3/3 Flu A Pos | 104% | 3/3 Flu B Pos | 98% | 3/3 Flu B Pos | 118%  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Flonase Nasal Spray | 15% v/v | 3/3 Neg | 103% / 104% | 3/3 Flu A Pos | 109% | 3/3 Flu A Pos | 100% | 3/3 Flu B Pos | 95% | 3/3 Flu B Pos | 113%  |
|  Rhinocort Nasal Spray | 15% v/v | 3/3 Neg | 85% / 88% | 3/3 Flu A Pos | 109% | 3/3 Flu A Pos | 101% | 3/3 Flu B Pos | 90% | 3/3 Flu B Pos | 122%  |
|  Nasonex Nasal Spray | 15% v/v | 3/3 Neg | 104% / 90% | 3/3 Flu A Pos | 103% | 3/3 Flu A Pos | 106% | 3/3 Flu B Pos | 92% | 3/3 Flu B Pos | 113%  |
|  Oxymetazoline HCl | 0.05% v/v | 3/3 Neg | 116% / 95% | 3/3 Flu A Pos | 101% | 3/3 Flu A Pos | 103% | 3/3 Flu B Pos | 97% | 3/3 Flu B Pos | 108%  |
|  Phenylephrine HCl | 10 mg/mL | 3/3 Neg | 126% / 119% | 3/3 Flu A Pos | 101% | 3/3 Flu A Pos | 99% | 3/3 Flu B Pos | 106% | 3/3 Flu B Pos | 117%  |
|  Guaiacol Glycerol Ether (Benylin) | 20 mg/mL | 3/3 Neg | 166% / 153% | 3/3 Flu A Pos | 118% | 3/3 Flu A Pos | 113% | 3/3 Flu B Pos | 118% | 3/3 Flu B Pos | 124%  |
|  Dextromethorphan | 2 mg/mL | 3/3 Neg | 95% / 101% | 3/3 Flu A Pos | 115% | 3/3 Flu A Pos | 115% | 3/3 Flu B Pos | 126% | 3/3 Flu B Pos | 134%  |
|  Salbutamol Sulfate | 400 ng/mL | 3/3 Neg | 102% / 92% | 3/3 Flu A Pos | 98% | 3/3 Flu A Pos | 99% | 3/3 Flu B Pos | 111% | 3/3 Flu B Pos | 116%  |
|  Whole Blood* | 2% v/v | 3/3 Neg | 126% / 91% | 3/3 Flu A Pos | 75% | 3/3 Flu A Pos | 90% | 3/3 Flu B Pos | 106% | 3/3 Flu B Pos | 98%  |

* A warning is added to the package insert to reflect that samples contaminated with whole blood in greater concentrations (visibly bloody samples) may interfere in the interpretation of the assay.

i. Transport Media Compatibility:

Eight transport media (six commercial transport media and two clinical site prepared transport media) were evaluated for compatibility in the RAMP Influenza A/B Assay by testing a negative sample (transport media only), an Influenza A LoD positive sample (300 EID50/mL), an Influenza A 2x LoD positive sample (600 EID50/mL), an Influenza B LoD positive sample (316 EID50/ml), and an Influenza B 2x LoD positive sample (632 EID50/ml) at n=3 replicates each in the RAMP Influenza A/B Assay. The influenza strains used to prepare the samples were Influenza A/Hong Kong/8/68 and Influenza B/Lee/40. These influenza strains were re-titered prior to media testing.

All negative samples (media only) tested negative. All inoculated samples (media with Influenza A or Influenza B) tested positive for the appropriate virus. The RAMP Flu A/B assay is a qualitative assay based on numerical RAMP Ratio values. The %CVs were also calculated for the RAMP Ratios. None of the tested transport media interfered with the performance of the RAMP Influenza A/B Assay.

{14}

|  Transport Media Tested | RAMP Results  |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  Negative |   | Influenza A LoD |   | Influenza A 2x LoD |   | Influenza B LoD |   | Influenza B 2x LoD  |   |
|   |  Results | Mean RAMP Ratio FluA/FluB | Results | Mean RAMP Ratio | Results | Mean RAMP Ratio | Results | Mean RAMP Ratio | Results | Mean RAMP Ratio  |
|  Copan Universal Transport Media (UTM) | Neg (3/3) | 388 / 295 | Flu A Pos (3/3) | 1559 | Flu A Pos (3/3) | 2630 | Flu B Pos (3/3) | 1143 | Flu B Pos (3/3) | 1928  |
|  Remel M4 Media | Neg (3/3) | 495 / 307 | Flu A Pos (3/3) | 1629 | Flu A Pos (3/3) | 3068 | Flu B Pos (3/3) | 1254 | Flu B Pos (3/3) | 2339  |
|  Remel M4-RT Media | Neg (3/3) | 530 / 364 | Flu A Pos (3/3) | 1711 | Flu A Pos (3/3) | 2984 | Flu B Pos (3/3) | 1218 | Flu B Pos (3/3) | 2349  |
|  Remel M5 Media | Neg (3/3) | 409 / 242 | Flu A Pos (3/3) | 1548 | Flu A Pos (3/3) | 2684 | Flu B Pos (3/3) | 1189 | Flu B Pos (3/3) | 1759  |
|  Starplex Transport Media | Neg (3/3) | 366 / 269 | Flu A Pos (3/3) | 1529 | Flu A Pos (3/3) | 2673 | Flu B Pos (3/3) | 1110 | Flu B Pos (3/3) | 1844  |
|  Phosphate Buffered Saline (PBS) Solution | Neg (3/3) | 345 / 272 | Flu A Pos (3/3) | 1632 | Flu A Pos (3/3) | 2939 | Flu B Pos (3/3) | 1132 | Flu B Pos (3/3) | 2055  |
|  St. Louis Children's Hospital In-house Media | Neg (3/3) | 319 / 306 | Flu A Pos (3/3) | 1684 | Flu A Pos (3/3) | 3297 | Flu B Pos (3/3) | 1371 | Flu B Pos (3/3) | 2309  |
|  Texas Children's Hospital In-house Media | Neg (3/3) | 332 / 263 | Flu A Pos (3/3) | 1669 | Flu A Pos (3/3) | 2559 | Flu B Pos (3/3) | 1202 | Flu B Pos (3/3) | 2019  |
|  Total Mean RAMP Ratio |  | 398 / 290 |  | 1620 |  | 2854 |  | 1203 |  | 2075  |
|  RAMP Ratio SD |  | 77 / 37 |  | 68 |  | 258 |  | 83 |  | 232  |
|  RAMP Ratio % CV |  | Flu A 19% Flu B 13% |  | 4% |  | 9% |  | 7% |  | 11%  |

j. Sample Collection Swabs Compatibility:

Four swab materials were evaluated for compatibility in the RAMP Influenza A/B Assay by testing a negative sample (swab alone with no virus present), an Influenza A LoD positive sample (300 EID50/mL after extraction), an Influenza A 2x LoD positive sample (600 EID50/mL after extraction), an Influenza B LoD positive sample (316 EID50/mL after extraction), and an Influenza B 2x LoD positive sample (632 EID50/mL after extraction) at  $n = 3$  replicates each in the RAMP Influenza A/B Assay. The influenza strains used to prepare the samples

{15}

were Influenza A/Hong Kong/8/68 and Influenza B/Lee/40. These influenza strains were re-titered prior to swab testing. Each swab was dosed with 20 µL of the appropriate sample and extracted into 3 mL Copan Universal Transport media by vortexing for 30 seconds to get the target concentration for each sample prior to testing in the RAMP Influenza A/B Assay. Each replicate tested was prepared from a separate swab. Swabs made of sterile foam, polyester, nylon, or rayon were evaluated. The RAMP Flu A/B assay is a qualitative assay based on numerical RAMP Ratio values. The %CVs were also calculated based on the RAMP Ratios for the testing

The negative sample (swab alone) tested negative. At least two of three of each swab type inoculated with Influenza A or Influenza B at the LoD tested positive for the virus.

|  Swab Material Tested | RAMP Results
Influenza A Threshold 1= 1023
Influenza B Threshold 2= 649  |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  Negative |   | Influenza A LoD |   | Influenza A 2x LoD |   | Influenza B LoD |   | Influenza B 2x LoD  |   |
|   |  Results | Mean RAMP Ratio FluA/FluB | Results | Mean RAMP Ratio | Results | Mean RAMP Ratio | Results | Mean RAMP Ratio | Results | Mean RAMP Ratio  |
|  Foam | Neg (3/3) | 441 / 231 | Flu A Pos (3/3) | 1392 | Flu A Pos (3/3) | 2354 | Flu B Pos (3/3) | 1171 | Flu B Pos (3/3) | 2194  |
|  Polyester | Neg (3/3) | 370 / 228 | Flu A Pos (2/3) | 1116 | Flu A Pos (3/3) | 2342 | Flu B Pos (2/3) | 735 | Flu B Pos (3/3) | 1431  |
|  Rayon | Neg (3/3) | 406 / 252 | Flu A Pos (3/3) | 1267 | Flu A Pos (3/3) | 2169 | Flu B Pos (2/3) | 661 | Flu B Pos (3/3) | 1342  |
|  Nylon | Neg (3/3) | 543 / 353 | Flu A Pos (3/3) | 1581 | Flu A Pos (3/3) | 2629 | Flu B Pos (3/3) | 1147 | Flu B Pos (3/3) | 2002  |
|  Total Mean RAMP Ratio |  | 440 / 266 |  | 1339 |  | 2374 |  | 928 |  | 1742  |
|  RAMP Ratio SD |  | 75 / 59 |  | 197 |  | 190 |  | 268 |  | 420  |
|  RAMP Ratio % CV |  | Flu A 17% Flu B 22% |  | 15% |  | 8% |  | 29% |  | 24%  |

{16}

# k. Sample Stability Testing:

The RAMP Influenza A/B Assay was evaluated to determine the effect of time of storage of the sample at  $2 - 8^{\circ}\mathrm{C}$  to assess stability of detection of the antigen in the sample. Clinical samples from the British Columbia Centre for Disease Control (BCCDC) equivalent to those tested in the clinical trial were thawed and diluted in viral transport media to simulate samples that were close to the Threshold 1 ratio values. The clinical samples were then tested immediately and after 72 hours storage at  $2^{\circ}\mathrm{C}$  and  $8^{\circ}\mathrm{C}$ .

The results indicate that samples tested in the RAMP Influenza A/B Assay are stable for at least 72 hours when stored at  $2 - 8^{\circ}\mathrm{C}$ .

|  Influenza A RAMP Ratio (Results)  |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- |
|   | Time 0 | 72 hrs at 2°C |   | 72 hrs at 8°C  |   |
|  Sample ID (dilution) (BCCDC) | Ratio (Result) | Ratio (Result) | Percent of Time 0 Ratio | Ratio (Result) | Percent of Time 0 Ratio  |
|  A5VI 27170-A (1/20) | 1556 (Flu A Pos) | 1816 (Flu A Pos) | 117 | 1486 (Flu A Pos) | 96  |
|  A5VI 27597-A (1/6) | 2466 (Flu A Pos) | 2883 (Flu A Pos) | 117 | 2480 (Flu A Pos) | 101  |
|  A6VI 01262-B (1/6) | 364 (Negative) | 406 (Negative) | 112 | 339 (Negative) | 93  |
|  A5VI 27163-A (1/10) | 1016 (Flu A Pos) | 948 (Flu A Pos) | 93 | 1094 (Flu A Pos) | 108  |
|  A5VI 27308-A (1/10) | 1289 (Flu A Pos) | 1224 (Flu A Pos) | 95 | 1216 (Flu A Pos) | 94  |
|  A5VI 27606-A (1/40) | 1943 (Flu A Pos) | 2241 (Flu A Pos) | 115 | 2016 (Flu A Pos) | 104  |
|  A5VI 27559-A (1/20) | 2576 (Flu A Pos) | 2873 (Flu A Pos) | 112 | 3033 (Flu A Pos) | 118  |
|  A5VI 27562-A (1/20) | 1710 (Flu A Pos) | 1720 (Flu A Pos) | 101 | 1777 (Flu A Pos) | 104  |
|  A5VI 27213-A (1/10) | 706 (Negative) | 980 (Flu A Pos) | 139 | 976 (Flu A Pos) | 138  |
|  A5VI 27349-A (1/10) | 446 (Negative) | 437 (Negative) | 98 | 413 (Negative) | 93  |
|  10008549 (1/20) | 374 (Negative) | 460 (Negative) | 123 | 400 (Negative) | 107  |
|  10008549 (1/40) | 320 (Negative) | 394 (Negative) | 123 | 416 (Negative) | 130  |
|  10017303 (1/20) | 443 (Negative) | 490 (Negative) | 111 | 375 (Negative) | 85  |
|  10041464 (1/6) | 411 (Negative) | 378 (Negative) | 92 | 382 (Negative) | 93  |
|  10045523 (1/6) | 438 (Negative) | 440 (Negative) | 101 | 331 (Negative) | 76  |
|  10008549 (1/80) | 385 (Negative) | 293 (Negative) | 76 | 473 (Negative) | 123  |
|  Mean % Recovery |  |  | 108 |  | 104  |

{17}

|  Influenza B RAMP Ratio (Results)  |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- |
|   | Time 0 | 72 hrs at 2°C |   | 72 hrs at 8°C  |   |
|  Sample ID (dilution) (BCCDC) | Ratio (Result) | Ratio (Result) | Percent of Time 0 Ratio | Ratio (Result) | Percent of Time 0 Ratio  |
|  A5VI 27170-A (1/20) | 186 (Negative) | 108 (Negative) | 58 | 181 (Negative) | 97  |
|  A5VI 27597-A (1/6) | 250 (Negative) | 246 (Negative) | 98 | 129 (Negative) | 52  |
|  A6VI 01262-B (1/6) | 668 (Flu B Pos) | 516 (Flu B Pos) | 77 | 713 (Flu B Pos) | 107  |
|  A5VI 27163-A (1/10) | 209 (Negative) | 143 (Negative) | 68 | 137 (Negative) | 66  |
|  A5VI 27308-A (1/10) | 259 (Negative) | 136 (Negative) | 53 | 228 (Negative) | 88  |
|  A5VI 27606-A (1/40) | 155 (Negative) | 168 (Negative) | 108 | 164 (Negative) | 106  |
|  A5VI 27559-A (1/20) | 129 (Negative) | 233 (Negative) | 181 | 236 (Negative) | 183  |
|  A5VI 27562-A (1/20) | 264 (Negative) | 248 (Negative) | 94 | 150 (Negative) | 57  |
|  A5VI 27213-A (1/10) | 119 (Negative) | 148 (Negative) | 124 | 193 (Negative) | 162  |
|  A5VI 27349-A (1/10) | 182 (Negative) | 93 (Negative) | 51 | 223 (Negative) | 123  |
|  10008549 (1/20) | 2657 (Flu B Pos) | 2825 (Flu B Pos) | 106 | 2945 (Flu B Pos) | 111  |
|  10008549 (1/40) | 1215 (Flu B Pos) | 1468 (Flu B Pos) | 121 | 1433 (Flu B Pos) | 118  |
|  10017303 (1/20) | 443 (Negative) | 518 (Flu B Pos) | 117 | 474 (Flu B Pos) | 107  |
|  10041464 (1/6) | 110 (Negative) | 137 (Negative) | 125 | 176 (Negative) | 160  |
|  10045523 (1/6) | 150 (Negative) | 111 (Negative) | 74 | 223 (Negative) | 149  |
|  10008549 (1/80) | 684 (Flu B Pos) | 700 (Flu B Pos) | 102 | 861 (Flu B Pos) | 126  |
|  Mean % Recovery |  |  | 97 |  | 113  |

# 2. Comparison studies:

a. Method comparison with predicate device:

Not applicable.

b. Matrix comparison:

Not applicable.

# 3. Clinical studies:

{18}

a. Prospective Clinical studies

The performance of the RAMP Influenza A/B Assay was compared to cell culture in a prospective study conducted as part of a multi-center trial in North America during the 2006-2007 influenza season when influenza A/H3 (36.5%) and A/H1 (63.5%) were the predominant Influenza A viruses in circulation¹. Four independent laboratories (located in distinct geographic regions) evaluated the RAMP Influenza A/B Assay in parallel with cell culture. Eight hundred Forty-four (844) fresh specimens (nasal wash/aspirate, nasopharyngeal aspirate, or nasopharyngeal swab samples) prospectively collected during the 2006-2007 influenza season were tested. Across the sites these samples were drawn from an approximately equal mix of pediatric (0 – 21 years) and adult patients (21+ years) with approximately equal numbers of male and female patients. The mean (standard deviation) age of the patients was 28.6 (30.7) years. The results of the prospective clinical trials based on sample type are given in the following tables:

|  Prospective Fresh Nasopharyngeal Swab | RAMP Influenza A |   |   | RAMP Influenza B  |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  Tissue Culture | Positive | Negative | Total | Positive | Negative | Total  |
|  Positive | 53 | 13 | 66 | 7 | 5 | 12  |
|  Negative | 19 | 541 | 560 | 12 | 602 | 614  |
|  Total | 72 | 554 | 626 | 19 | 607 | 626  |
|   |  |  | 95% CI |  |  | 95% CI  |
|  Sensitivity | 53/66 | 80.3% | 68.7-89.1% | 7/12 | 58.3% | 27.7-84.8%  |
|  Specificity | 541/560 | 96.6% | 94.8-97.9% | 602/614 | 98.0% | 96.6-99.0%  |
|  Prospective Fresh Nasal Wash/Aspirate | RAMP Influenza A |   |   | RAMP Influenza B  |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  Tissue Culture | Positive | Negative | Total | Positive | Negative | Total  |
|  Positive | 36 | 9 | 45 | 9 | 0 | 9  |
|  Negative | 11 | 162 | 173 | 2 | 207 | 209  |
|  Total | 47 | 171 | 218 | 11 | 207 | 218  |
|   |  |  | 95% CI |  |  | 95% CI  |
|  Sensitivity | 36/45 | 80.0% | 65.4-90.4% | 9/9 | 100% | 66.4-100%  |
|  Specificity | 162/173 | 93.6% | 88.9-96.8% | 207/209 | 99.0% | 96.6-99.9%  |

{19}

The results of the prospective clinical trials based on sample type and stratified by patient age group are given in the following tables:

|  Prospective Fresh Nasopharyngeal Swab (Age 0-5) | RAMP Influenza A |   |   | RAMP Influenza B  |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  Tissue Culture | Positive | Negative | Total | Positive | Negative | Total  |
|  Positive | 11 | 1 | 12 | 1 | 0 | 1  |
|  Negative | 12 | 187 | 199 | 6 | 204 | 210  |
|  Total | 23 | 188 | 211 | 7 | 204 | 211  |
|   |  |  | 95% CI |  |  | 95% CI  |
|  Sensitivity | 11/12 | 91.7% | 61.5-99.8% | 1/1 | 100% | NA  |
|  Specificity | 187/199 | 94.0% | 89.7%-96.8% | 204/210 | 97.1% | 93.9-98.9%  |
|  Prospective Fresh Nasopharyngeal Swab (Age 6-21) | RAMP Influenza A |   |   | RAMP Influenza B  |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  Tissue Culture | Positive | Negative | Total | Positive | Negative | Total  |
|  Positive | 12 | 3 | 15 | 2 | 2 | 4  |
|  Negative | 4 | 66 | 70 | 4 | 77 | 81  |
|  Total | 16 | 69 | 85 | 6 | 79 | 85  |
|   |  |  | 95% CI |  |  | 95% CI  |
|  Sensitivity | 12/15 | 80.0% | 51.9-95.7% | 2/4 | 50.0% | 6.8-93.2%  |
|  Specificity | 66/70 | 94.3% | 86.0-98.4% | 77/81 | 95.1% | 87.8-98.6%  |
|  Prospective Fresh Nasopharyngeal Swab (Age 22-59) | RAMP Influenza A |   |   | RAMP Influenza B  |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  Tissue Culture | Positive | Negative | Total | Positive | Negative | Total  |
|  Positive | 16 | 7 | 23 | 2 | 2 | 4  |
|  Negative | 1 | 136 | 137 | 1 | 155 | 156  |
|  Total | 17 | 143 | 160 | 3 | 157 | 160  |
|   |  |  | 95% CI |  |  | 95% CI  |
|  Sensitivity | 16/23 | 69.6% | 47.1-86.8% | 2/4 | 50.0% | 6.8-93.2%  |
|  Specificity | 136/137 | 99.3% | 96.0-100% | 155/156 | 99.4% | 96.5-100%  |
|  Prospective Fresh Nasopharyngeal Swab (Age >=60) | RAMP Influenza A |   |   | RAMP Influenza B  |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  Tissue Culture | Positive | Negative | Total | Positive | Negative | Total  |
|  Positive | 14 | 2 | 16 | 2 | 1 | 3  |
|  Negative | 2 | 152 | 154 | 1 | 166 | 167  |
|  Total | 16 | 154 | 170 | 3 | 167 | 170  |
|   |  |  | 95% CI |  |  | 95% CI  |
|  Sensitivity | 14/16 | 87.5% | 61.7-98.4% | 2/3 | 66.7% | 9.4-99.2%  |
|  Specificity | 152/154 | 98.7% | 95.4-99.8% | 166/167 | 99.4% | 96.7-100%  |

{20}

|  Prospective Fresh Nasal Wash/Aspirate (Age 0-5) | RAMP Influenza A |   |   | RAMP Influenza B  |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  Tissue Culture | Positive | Negative | Total | Positive | Negative | Total  |
|  Positive | 24 | 4 | 28 | 6 | 0 | 6  |
|  Negative | 6 | 117 | 123 | 1 | 144 | 145  |
|  Total | 30 | 121 | 151 | 7 | 144 | 151  |
|   |  |  | 95% CI |  |  | 95% CI  |
|  Sensitivity | 24/28 | 85.7% | 67.3-96.0% | 6/6 | 100% | 54.1-100%  |
|  Specificity | 117/123 | 95.1% | 89.7-98.2% | 144/145 | 99.3% | 96.2-100%  |
|  Prospective Fresh Nasal Wash/Aspirate (Age 6-21) | RAMP Influenza A |   |   | RAMP Influenza B  |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  Tissue Culture | Positive | Negative | Total | Positive | Negative | Total  |
|  Positive | 12 | 5 | 17 | 3 | 0 | 3  |
|  Negative | 5 | 41 | 46 | 1 | 59 | 60  |
|  Total | 17 | 46 | 63 | 4 | 59 | 63  |
|   |  |  | 95% CI |  |  | 95% CI  |
|  Sensitivity | 12/17 | 70.6% | 44.0-89.7% | 3/3 | 100% | 29.2-100%  |
|  Specificity | 41/46 | 89.1% | 76.4-96.4% | 59/60 | 98.3% | 91.1-100%  |
|  Prospective Fresh Nasal Wash/Aspirate (Age 22-59) | RAMP Influenza A |   |   | RAMP Influenza B  |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  Tissue Culture | Positive | Negative | Total | Positive | Negative | Total  |
|  Positive | 0 | 0 | 0 | 0 | 0 | 0  |
|  Negative | 0 | 3 | 3 | 0 | 3 | 3  |
|  Total | 0 | 3 | 3 | 0 | 3 | 3  |
|   |  |  | 95% CI |  |  | 95% CI  |
|  Sensitivity | 0/0 | NA | NA | 0/0 | NA | NA  |
|  Specificity | 3/3 | 100% | 29.2-100% | 3/3 | 100% | 29.2-100%  |
|  Prospective Fresh Nasal Wash/Aspirate (Age >=60) | RAMP Influenza A |   |   | RAMP Influenza B  |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  Tissue Culture | Positive | Negative | Total | Positive | Negative | Total  |
|  Positive | 0 | 0 | 0 | 0 | 0 | 0  |
|  Negative | 0 | 1 | 1 | 0 | 1 | 1  |
|  Total | 0 | 1 | 1 | 0 | 1 | 1  |
|   |  |  | 95% CI |  |  | 95% CI  |
|  Sensitivity | 0/0 | NA | NA | 0/0 | NA | NA  |
|  Specificity | 1/1 | 100% | NA | 1/1 | 100% | NA  |

{21}

b. Retrospective Clinical studies

In order to supplement the prospective study, performance of the RAMP Influenza A/B Test was also evaluated by two independent laboratories comparing to the original cell culture testing results (cell culture testings were performed originally using fresh specimens) on 75 retrospective frozen clinical nasopharyngeal swab samples and 130 retrospective frozen clinical wash/aspirate samples. The results of the retrospective clinical trials based on sample type are given in the following tables:

|  Retrospective Frozen Nasopharyngeal Swab | RAMP Influenza A |   |   | RAMP Influenza B  |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  Tissue Culture | Positive | Negative | Total | Positive | Negative | Total  |
|  Positive | 11 | 2 | 13 | 23 | 7 | 30  |
|  Negative | 0 | 62 | 62 | 0 | 45 | 45  |
|  Total | 11 | 64 | 75 | 23 | 52 | 75  |
|   |  |  | 95% CI |  |  | 95% CI  |
|  Positive Percent Agreement | 11/13 | 84.6% | 54.5-98.1 % | 23/30 | 76.7% | 57.7-90.1%  |
|  Negative Percent Agreement | 62/62 | 100% | 94.2-100% | 45/45 | 100% | 92.1-100%  |
|  Retrospective Frozen Nasal Wash/Aspirate | RAMP Influenza A |   |   | RAMP Influenza B  |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  Tissue Culture | Positive | Negative | Total | Positive | Negative | Total  |
|  Positive | 35 | 10 | 45 | 33 | 6 | 39  |
|  Negative | 2 | 83 | 85 | 2 | 89 | 91  |
|  Total | 37 | 93 | 130 | 35 | 95 | 130  |
|   |  |  | 95% CI |  |  | 95% CI  |
|  Positive Percent Agreement | 35/45 | 77.8% | 62.9-88.8% | 33/39 | 84.6% | 69.5-94.1%  |
|  Negative Percent Agreement | 83/85 | 97.6% | 91.8-99.7% | 89/91 | 97.8% | 92.3%-99.7%  |

c. Other clinical supportive data (when a. and b. are not applicable):

Not applicable.

4. Clinical cut-off:

Not applicable.

{22}

5. Expected values/Reference range:

The prevalence of Influenza varies each year with epidemics occurring during the fall and winter months in the US. Variables that affect the rate of positivity observed in respiratory testing include: the efficiency and timing of specimen collection, handling and transport of the specimen, the time of year, age of the patient, and local disease prevalence. During the 2006-2007 US respiratory season, the combined prevalence of Influenza A and Influenza B was 13.2%¹. In the 2007 RAMP Influenza A/B multi-center clinical study (samples collected between January and April), the prevalence as observed with culture of Influenza A was 13.2% (111/844), Influenza B was 2.5% (21/844).

N. Instrument Name:

RAMP® 200 reader

O. System Descriptions:

1. Modes of Operation:

The RAMP® 200 measures the amount of fluorescence emitted by antibody/antigen complexes bound at two detection zones (Influenza A and Influenza B) and at the internal standard zone. The RAMP® 200 calculates a ratio (RAMP Ratio) using the fluorescence reading of each detection zone (A and B) and the internal zone. The reader compares these ratios to predefined threshold limits to determine a positive or negative result for Influenza A and Influenza B in the tested sample.

2. Software:

FDA has reviewed applicant’s Hazard Analysis and software development processes for this line of product types:

Yes ☐ X ☐ or No ☐

3. Specimen Identification:

User enters Patient ID/Sample ID using either the touch screen of the Control Module or optional barcode scanner.

4. Specimen Sampling and Handling:

Not applicable

5. Calibration:

¹ Centers for Disease Control and Prevention. 2007. Update: Influenza activity--United States and worldwide, 2006-07 season, and composition of the 2007-08 influenza vaccine. MMWR 56(31); 789-794.

{23}

Each box of test strips is supplied with a Lot Card containing the calibration information for each specific manufacturing lot. The Reader determines the lot information by reading the barcode on the bottom of each Test Cartridge and reports the result based on the fluorescence ratio calculated for the Cartridge and comparing it to the specific lot information loaded into the memory from the associated Lot Card.

6. Quality Control:

When the RAMP® 200 is turned on, it performs a Self-Test which verifies the system calibration, the cartridge transport mechanism and the Reader optics. During the self-test the system also determines remaining battery power, verifies the system clock and verifies the instrument calibration. In addition, the RAMP® 200 has an Internal Quality Control (IQC) function. IQC can be scheduled to be performed at a pre-specified interval (1-24 hours) and takes less than 1 minute to complete. The IQC function repeats all the start-up checks and ensures appropriate instrument operation.

The RAMP® 200 scans the entire length of the Assay strip within the Cartridge. There are 2 areas on the strip to which the fluorescently labeled antibodies bind, the "detection zone" and the "internal standard zone". The Reader monitors the sample flow across the length of the Assay Strip. Should an operator delay for too long between loading the sample and inserting the Cartridge into the Reader, insert a Cartridge that has already been run, apply too little sample to the Cartridge, or not use the supplied Assay Tip from the kit Pouch, the flow detected will not adhere to expected patterns and an error will be reported. In this way each Cartridge assures appropriate operator technique. Prior to reporting a result, the Reader software checks that the background signal (measured from areas outside of the detection zone and internal standard zones) is not higher than a predetermined value and that the total amount of fluorescence is above a predefined value. If either of these parameters is not met, an error is reported.

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

Not applicable

Q. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

R. Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

24

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**Source:** [https://fda-staging.innolitics.com/device/K071591](https://fda-staging.innolitics.com/device/K071591)

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