D3 FASTPOINT L-DFA PARAINFLUENZA VIRUS/ADENOVIRUS IDENTIFICATION KIT

K093415 · Diagnostic Hybrids, Inc. · GQS · Dec 23, 2009 · Microbiology

Device Facts

• Record ID: K093415
• Device Name: D3 FASTPOINT L-DFA PARAINFLUENZA VIRUS/ADENOVIRUS IDENTIFICATION KIT
• Applicant: Diagnostic Hybrids, Inc.
• Product Code: GQS · Microbiology
• Decision Date: Dec 23, 2009
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3400
• Device Class: Class 1

Intended Use

The Diagnostic Hybrids, Inc. device, D³ FastPoint L-DFA Parainfluenza Virus/Adenovirus Identification Kit is intended for the qualitative identification of adenovirus and to screen for the presence of parainfluenza virus types 1, 2, and 3 in nasal and nasopharyngeal swabs and aspirates/washes specimens from patients with signs and symptoms of respiratory infection by direct detection of immunofluorescence using monoclonal antibodies (MAbs). It is recommended that specimens found to be negative for parainfluenza virus and adenovirus after examination of the direct specimen result be confirmed by cell culture. Negative results do not preclude parainfluenza virus and adenovirus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.

Device Story

The D3 FastPoint L-DFA PIV/ADV Kit is an in vitro diagnostic reagent for respiratory specimen analysis. It utilizes a blend of murine monoclonal antibodies (MAbs) directly labeled with R-phycoerythrin (PE) for parainfluenza virus types 1, 2, and 3, and fluorescein isothiocyanate (FITC) for adenovirus. Respiratory specimens are permeabilized and stained in a liquid suspension format, incubated for 5 minutes, rinsed, centrifuged, and re-suspended. The resulting cell suspension is loaded onto a slide and examined via fluorescence microscopy. Infected cells exhibit specific fluorescence (golden-yellow for parainfluenza, apple-green for adenovirus); non-infected cells appear red due to Evans Blue counter-stain, with nuclei appearing orange-red from propidium iodide. The device is used in clinical laboratories by trained personnel to assist in the diagnosis of respiratory infections. Results are intended to be used in conjunction with clinical findings and, if negative, confirmed by cell culture.

Clinical Evidence

Prospective clinical study at 4 U.S. sites (n=1519 specimens). Compared to FDA-cleared DSFA devices with culture confirmation. For nasal wash/aspirate: Adenovirus sensitivity 92.3%, specificity 100%; HPIV sensitivity 92.0%, specificity 99.3%. For nasal/nasopharyngeal swab: Adenovirus sensitivity 100%, specificity 100%; HPIV sensitivity 92.9%, specificity 100%.

Technological Characteristics

In vitro diagnostic reagent kit using direct immunofluorescence. Components: PE-labeled MAbs (PIV 1-3), FITC-labeled MAbs (Adenovirus), proprietary non-acetone fixative, Evans Blue/propidium iodide counter-stains, PBS, and re-suspension buffer. Detection via fluorescence microscopy. Manual liquid suspension staining protocol. Preservative: 0.1% sodium azide.

Indications for Use

Indicated for qualitative identification of adenovirus and screening for parainfluenza virus types 1, 2, and 3 in nasal/nasopharyngeal swabs and aspirates/washes from symptomatic patients with respiratory infection. Prescription use only.

Regulatory Classification

Identification

Parainfluenza virus serological reagents are devices that consist of antigens and antisera used in serological tests to identify antibodies to parainfluenza virus in serum. The identification aids in the diagnosis of parainfluenza virus infections and provides epidemiological information on diseases caused by these viruses. Parainfluenza viruses cause a variety of respiratory illnesses ranging from the common cold to pneumonia.

Predicate Devices

• D³ Ultra DFA Respiratory Virus Screening & ID Kit (K061101)
• D³ Duet DFA RSV/Respiratory Virus Screening Kit (K081928)

Submission Summary (Full Text)

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Diagnostic Hybrids, Inc.

K093415

## D3 FastPoint L-DFA Parainfluenza Virus/Adenovirus Identification Kit

10/30/2009 DEC 2 3 2009 Page 1 of 13

# Section 05, 510(k) Summary

#### Applicant:

DIAGNOSTIC HYBRIDS, INC. 1055 East State Street Suite 100 Athens, OHIO 45701

#### Contact Information:

Ronald H. Lollar, Senior Director Product Realization, Management, and Marketing 1055 East State Street Suite 100 Athens, Ohio 45701 740-589-3300 - Corporate number 740-589-3373 - Desk phone [phone] - Fax [e-mail]

#### Date of preparation of 510(k) summary:

October 30, 2009

#### Device Name:

Trade name - D2 FastPoint L-DFA Parainfluenza Virus/Adenovirus Identification Kit Common name - D3 FastPoint Parainfluenza Virus/Adenovirus Identification Kit Classification name - Parainfluenza virus serological reagents, Adenovirus serological reagents Product Code - GQS, GNY Regulation - 21 CFR 866.3400, 21 CFR 866.3020 Regulatory Class - Class I Panel Microbiology (83)

### Legally marketed devices to which equivalence is claimed:

### D3 Ultra DFA Respiratory Virus Screening & ID Kit (k061101)

Intended Use: The Diagnostic Hybrids, Inc. D3 Ultra DFA (direct fluorescent antibody) Respiratory Virus Screening & ID Kit (D3 Ultra) is intended for the qualitative detection and identification of the influenza A, influenza B, respiratory syncytial virus (RSV), adenovirus, parainfluenza 1, parainfluenza 2 and parainfluenza 3 virus in respiratory specimens, by either direct detection or cell culture method, by immunofluorescence using fluoresceinated monoclonal antibodies (MAbs). It is recommended that

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specimens found to be negative after examination of the direct specimen result be confirmed by cell culture. Negative results do not preclude respiratory virus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.

- Performance characteristics for influenza A were established when . influenza A/H3 and A/H1 were the predominant influenza A viruses in circulation. When other influenza A viruses are emerging, performance characteristics may vary.
- If infection with a novel influenza A virus is suspected based on current . clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL3+ facility is available to receive and culture specimens.

### D3 Duet DFA RSV/Respiratory Virus Screening Kit (k081928)

Intended Use: The Diagnostic Hybrids, Inc. device, D3 Duet DFA RSV/Respiratory Virus Screening Kit (D5 Duet RSV Kit), is intended for the qualitative detection and identification of respiratory syncytial virus, while screening for influenza A virus, influenza B virus, adenovirus, and parainfluenza virus types 1, 2 and 3 viral antigens, in nasal and nasopharyngeal swabs and aspirates or in cell culture. The assay detects viral antigens by immunofluorescence using monoclonal antibodies (MAbs), from patients with signs and symptoms of respiratory infection.

It is recommended that specimens found to be negative after examination of the direct specimen result be confirmed by cell culture. Negative results do not preclude influenza virus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.

Performance characteristics for influenza A virus detection and identification were established when influenza A (H3N2) and influenza A (H1N1) were the predominant influenza A strains circulating in the United States. Performance characteristics for influenza A virus detection and identification were established when influenza A H3N2 and influenza A H1N1 were the predominant influenza A strains circulating in the United States. When other influenza A viruses are emerging, performance characteristics may vary. If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to a state or local health department for testing. Viral

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culture should not be attempted in these cases unless a BSL3+ facility is available to receive and culture specimens.

### Device Description:

The D3 FastPoint L-DFA Parainfluenza Virus/Adenovirus Identification Kit (D3 FastPoint PIV/ADV Kit) uses a blend (called an "L-DFA Reagent") of viral antigenspecific murine monoclonal antibodies that are directly labeled with either Rphycoerythin (PE) (parainfluenza virus types 1, 2 and 3) or fluorescein isothiocyanate (FITC) (adenovirus) for the qualitative identification of adenovirus and to screen for the presence of parainfluenza virus types 1, 2, and 3.

#### Kit Components:

- D3 FastPoint L-DFA PIV/Adenovirus Reagent, 4.0-mL. One dropper bottle 1. containing a mixture of PE-labeled murine monoclonal antibodies directed against parainfluenza virus types 1, 2, or 3 antigens and FITC-labeled murine monoclonal antibodies directed against adenovirus antigens. The buffered, stabilized, aqueous solution contains Evans Blue and propidium iodide as counter-stains and 0.1% sodium azide as preservative.
- 40X PBS Concentrate, 25-mL. One bottle of 40X PBS concentrate containing 2. 4% sodium azide (0.1% sodium azide after dilution to 1X using de-mineralized water).
- Re-suspension Buffer, 6.0-mL. One bottle of a buffered glycerol solution and 3. 0.1% sodium azide.
- D3 FastPoint L-DFA Parainfluenza Virus/Adenovirus Antigen Control Slides, 4. 5-slides. Five individually packaged control slides containing 2 wells with cell culture-derived positive and negative control cells. Each positive well contains cells infected with either parainfluenza virus type 3 or adenovirus. The negative wells contain non-infected cells. Each slide is intended to be stained only one . time.
- D3 FastPoint L-DFA Specimen Slides and Coverslips, 50-slides with 5. coverslips. Fifty pack of 3-well specimen slides.

The cells to be tested are derived from respiratory specimens from patients with signs and symptoms of respiratory infection. The cells are permeabilized and stained concurrently in a liquid suspension format with the L-DFA Reagent. After incubating at 35℃ to 37℃ for 5-minutes, the stained cell suspensions are rinsed with 1X PBS. The rinsed cells are pelleted by centrifugation and then re-suspended with the Resuspension Buffer and loaded onto a specimen slide well. The cells are examined using a fluorescence microscope. Cells infected with parainfluenza virus types 1, 2 and 3 will exhibit golden-yellow fluorescence. Cells infected with adenovirus will exhibit apple

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green fluorescence due to the FITC. Non-infected cells will exhibit red fluorescence due to the Evans Blue counter-stain. Nuclei of intact cells will exhibit orange-red fluorescence due to the propidium iodide.

#### Intended Use:

The Diagnostic Hybrids, Inc. device, D3 FastPoint L-DFA Parainfluenza Virus/Adenovirus Identification Kit is intended for the qualitative identification of adenovirus and to screen for the presence of parainfluenza virus types 1, 2, and 3 in nasal and nasopharyngeal swabs and aspirates/washes specimens from patients with signs and symptoms of respiratory infection by direct detection of immunofluorescence using monoclonal antibodies (MAbs).

It is recommended that specimens found to be negative for parainfluenza virus and adenovirus after examination of the direct specimen result be confirmed by cell culture. Negative results do not preclude parainfluenza virus and adenovirus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.

| TABLE 5.1: Characteristics of the D³ FastPoint PIV/ADV Kit are compared to those of the<br>following Diagnostic Hybrids (DHI) predicate devices | | | | |
|-------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Characteristics | D³ FastPoint PIV/ADV Kit<br>(Subject Device) | D³ Ultra Kit<br>510(k) #k061101 | D³ Duet RSV Kit<br>510(k) # k081928 | |
| Intended Use | The Diagnostic Hybrids, Inc. device, D³ FastPoint L-DFA<br>Parainfluenza Virus/<br>Adenovirus Identification<br>Kit is intended for the<br>qualitative identification of<br>adenovirus and to screen for<br>the presence of parainfluenza<br>virus types 1, 2, and 3 in<br>nasal and nasopharyngeal<br>swabs and aspirates/washes<br>specimens from patients with<br>signs and symptoms of<br>respiratory infection by<br>direct detection of<br>immunofluorescence using<br>monoclonal antibodies<br>(MAbs).<br>It is recommended that<br>specimens found to be<br>negative for parainfluenza<br>virus and adenovirus after<br>examination of the direct<br>specimen result be confirmed | The Diagnostic<br>Hybrids, Inc. D³<br>Ultra™ DFA (direct<br>fluorescent<br>antibody)<br>Respiratory Virus<br>Screening & ID Kit<br>is intended for the<br>qualitative detection<br>and identification of<br>the influenza A,<br>influenza B,<br>respiratory syncytial<br>virus (RSV),<br>adenovirus,<br>parainfluenza 1,<br>parainfluenza 2 and<br>parainfluenza 3<br>virus in respiratory<br>specimens, by either<br>direct detection or<br>cell culture method,<br>by<br>immunofluorescence<br>using monoclonal<br>antibodies (MAbs). | The Diagnostic<br>Hybrids, Inc. device<br>D³ Duet DFA<br>RSV/Respiratory<br>Virus Screening Kit,<br>is intended for the<br>qualitative detection<br>and identification of<br>respiratory syncytial<br>virus, while<br>screening for<br>influenza A virus,<br>influenza B virus,<br>adenovirus, and<br>parainfluenza virus<br>types 1, 2 and 3 viral<br>antigens, in nasal<br>and nasopharyngeal<br>swabs and aspirates<br>or in cell culture.<br>The assay detects<br>viral antigens by<br>immunofluorescence<br>using monoclonal<br>antibodies (MAbs). | |
| TABLE 5.1: | Characteristics of the D³ FastPoint PIV/ADV Kit are compared to those of the following Diagnostic Hybrids (DHI) predicate devices | | | |
| Characteristics | D³ FastPoint PIV/ADV Kit<br>(Subject Device) | D³ Ultra Kit<br>510(k) #k061101 | D³ Duet RSV Kit<br>510(k) # k081928 | |
| | by cell culture. Negative<br>results do not preclude<br>parainfluenza virus and<br>adenovirus infection and<br>should not be used as the<br>sole basis for diagnosis,<br>treatment or other<br>management decisions. | antibodies (MAbs).<br>It is recommended<br>that specimens<br>found to be negative<br>after examination of<br>the direct specimen<br>result be confirmed<br>by cell culture.<br>Negative results do<br>not preclude<br>respiratory virus<br>infection and should<br>not be used as the<br>sole basis for<br>diagnosis, treatment<br>or other<br>management<br>decisions. | from patients with<br>signs and symptoms<br>of respiratory<br>infection.<br>It is recommended<br>that specimens<br>found to be negative<br>after examination of<br>the direct specimen<br>result be confirmed<br>by cell culture.<br>Negative results do<br>not preclude<br>influenza virus<br>infection and should<br>not be used as the<br>sole basis for<br>diagnosis, treatment<br>or other<br>management<br>decisions. | |
| Target Viruses | adenovirus, parainfluenza<br>virus type 1, parainfluenza<br>virus type 2, parainfluenza<br>virus type 3 | influenza A virus,<br>influenza B virus,<br>respiratory<br>syncytial virus,<br>adenovirus,<br>parainfluenza virus<br>type 1,<br>parainfluenza virus<br>type 2,<br>parainfluenza virus<br>type 3 | influenza A virus,<br>influenza B virus,<br>respiratory<br>syncytial virus,<br>adenovirus,<br>parainfluenza virus<br>type 1,<br>parainfluenza virus<br>type 2,<br>parainfluenza virus<br>type 3 | |
| Monoclonal antibodies<br>(MAbs) | The D³ FastPoint L-DFA<br>PIV/Adenovirus Reagent<br>contains 9 MAbs to<br>adenovirus (3) and<br>parainfluenza virus (6) | The Respiratory<br>Virus DFA<br>Screening Reagent<br>contains 15 MAbs to<br>7 different<br>respiratory viruses<br>(influenza A virus,<br>influenza B virus,<br>respiratory syncytial<br>virus, adenovirus,<br>parainfluenza virus<br>type 1,<br>parainfluenza virus<br>type 2,<br>parainfluenza virus<br>type 3) | The<br>RSV/Respiratory<br>Virus DFA<br>Screening Reagent<br>contains 15 MAbs to<br>7 different<br>respiratory viruses<br>(influenza A virus,<br>influenza B virus,<br>adenovirus,<br>parainfluenza virus<br>type 1, parainfluenza<br>virus type 2,<br>parainfluenza virus<br>type 3), plus 2 MAbs | |
| TABLE 5.1: Characteristics of the D³ FastPoint PIV/ADV Kit are compared to those of the<br>following Diagnostic Hybrids (DHI) predicate devices | | | | |
| Characteristics | D³ FastPoint PIV/ADV Kit<br>(Subject Device) | D³ Ultra Kit<br>510(k) #k061101 | D³ Duet RSV Kit<br>510(k) # k081928<br>syncytial virus. | |
| Labeling method | Direct labeling<br><br>- using R-Phycoerythrin (R-<br>PE) to label the MAbs to<br>parainfluenza virus types 1,<br>2, and 3.<br><br>- using fluorescein<br>isothiocyanate (FITC) to<br>label the MAbs to<br>adenovirus. | Direct labeling<br><br>- using fluorescein<br>isothiocyanate<br>(FITC) to label all<br>MAbs with<br>fluorescein. | Direct labeling<br><br>- using R-<br>Phycoerythrin (R-<br>PE) to label the<br>MAbs to respiratory<br>syncytial virus.<br><br>- using fluorescein<br>isothiocyanate<br>(FITC) to label all<br>other MAbs with<br>fluorescein. | |
| R-Phycoerythrin-labeled<br>MAbs | parainfluenza virus types 1,<br>2, and 3 | None | respiratory syncytial<br>virus | |
| Fluorescein-labeled MAbs | adenovirus | influenza A virus,<br>influenza B virus,<br>respiratory syncytial<br>virus, adenovirus,<br>parainfluenza virus<br>type 1,<br>parainfluenza virus<br>type 2,<br>parainfluenza virus<br>type 3 | influenza A virus,<br>influenza B virus,<br>adenovirus,<br>parainfluenza virus<br>type 1, parainfluenza<br>virus type 2,<br>parainfluenza virus<br>type 3 | |
| Cell Fixative | Proprietary Non-Acetone<br>based system | Acetone | Acetone | |
| Cell Counter-stain | Propidium Iodide, Evans<br>Blue | Evans Blue | Evans Blue | |
| Performance characteristics | | | | |
| Staining patterns | Parainfluenza 1, 2, 3: The<br>fluorescence is cytoplasmic.<br>Cells appear round.<br>Adenovirus: The<br>fluorescence is cytoplasmic<br>or bright nuclear or both.<br>Cells appear round.<br>Negative: Cells fluoresce<br>red due to the Evans Blue<br>counter-stain.<br>Nuclei: Cell Nuclei<br>fluoresce orange-red due to<br>the Propidium Iodide<br>counter-stain. | Influenza A and B:<br>The fluorescence is<br>cytoplasmic, nuclear<br>or both.<br>Cytoplasmic<br>staining is often<br>punctate with large<br>inclusions while<br>nuclear staining is<br>uniformly bright.<br>Respiratory<br>Syncytial Virus:<br>The fluorescence is<br>cytoplasmic and<br>punctate with small<br>inclusions in the<br>syncytia. | Influenza A and<br>B: The<br>fluorescence is<br>cytoplasmic,<br>nuclear or both.<br>Cytoplasmic<br>staining is often<br>punctate with large<br>inclusions while<br>nuclear staining is<br>uniformly bright.<br>Respiratory<br>Syncytial Virus:<br>The fluorescence<br>is cytoplasmic and<br>punctate with<br>small inclusions in<br>the syncytia. | |
| TABLE 5.1: Characteristics of the D³ FastPoint PIV/ADV Kit are compared to those of the<br>following Diagnostic Hybrids (DHI) predicate devices | | | | |
| Characteristics | | D³ FastPoint PIV/ADV Kit<br>(Subject Device) | D³ Ultra Kit<br>510(k) #k061101 | D³ Duet RSV Kit<br>510(k) # k081928 |
| | | | 3: The fluorescence<br>is cytoplasmic and<br>punctate with<br>irregular inclusions.<br>Types 2 and 3 cause<br>the formation of<br>syncytia.<br>Adenovirus: The<br>fluorescence is<br>cytoplasmic and<br>punctate or bright<br>nuclear or both.<br>Negative: Cells<br>fluoresce red due to<br>the Evans Blue<br>counter-stain. | Parainfluenza 1,<br>2, 3: The<br>fluorescence is<br>cytoplasmic and<br>punctate with<br>irregular<br>inclusions. Types<br>2 and 3 cause the<br>formation of<br>syncytia.<br>Adenovirus: The<br>fluorescence is<br>cytoplasmic and<br>punctate or bright<br>nuclear or both.<br>Negative: Cells<br>fluoresce red due<br>to the Evans Blue<br>counter-stain. |
| Analytical<br>specificity<br>(cross-<br>reactivity<br>studies; various<br>strains of<br>microorganism<br>s and cell lines) | Viruses | Device Reagents are not reactive with these numbers of microorganisms | | |
| | Viruses | 59 | 31 | 32 |
| | Bacteria | 22 | 18 | 25 |
| | <i>Chlamydia</i><br>spp. | 1 | 1 | 3 |
| | Yeast | 1 | 0 | 1 |
| | Protozoan | 0 | 0 | 1 |
| | Cell lines | N/A | 17 | 17 |

### Technological Characteristics, Compared to Predicate Device:

Sec05 FastPoint PIV-ADV 09OCT30

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#### " Diagnostic Hybrids, Inc.

.

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Sec05_FastPoint_PIV-ADV_09OCT30

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#### Analytical Performance:

#### Precision/Reproducibility:

Assay precision, intra-assay variability and inter assay variability were assessed with a reproducibility panel consisting of 5 randomized panel members.

The hPIV/adenovirus panel consisted of the following

- a. Low level parainfluenza virus type 1 (C-35 strain) infected cells.
- b. Low level adenovirus (ATCC type 1) infected cells.
- Low level parainfluenza virus type 1 (C-35 strain) infected cells mixed c. with mid level adenovirus (ATCC type 1) infected cells.
- d. Low adenovirus (ATCC type 1) infected cells mixed with mid level parainfluenza virus type 1 (C-35 strain) infected cells.
- Mid level non-infected (negative) cells. e.

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The low level is estimated to contain between 4 to 10% infected cells in the sample. The mid level is estimated to contain between 20 to 25% infected cells in the sample. Each sample contains 2.5 x 102 to 3.5 x 102 total cells.

Each panel was tested daily in two separate runs for 5-days by four different laboratories (40 total runs). The following results were recorded:

- a. Presence or absence of golden-yellow fluorescence.
- b. Percent of cells exhibiting golden-vellow fluorescence.
- c. Presence or absence of apple-green fluorescence.
- d. Percent of cells exhibiting apple-green fluorescence.

For the D3 FastPoint L-DFA Parainfluenza Virus/Adenovirus Identification Kit, the combined data from the four Study Sites demonstrated reproducible detection of parainfluenza virus type 1 (hPIV-1) by the R-PE labeled MAbs and reproducible detection of adenovirus by the FITC-labeled MAbs. The presence of hPIV-1 infected cells was reported in 100% (120/120) of the wells in which the infected cells were expected. The presence of adenovirus infected cells was reported in 100% (120/120) of the wells in which the infected cells were expected. The absence of infected cells was reported in 100% (40/40) of the wells in which infected cells were not present. The total percent agreement for the D3 FastPoint L-DFA Parainfluenza Virus/Adenovirus Identification Kit was 100% (280/280):

| TABLE 5.2: Reproducibility Study Results using the L-DFA Reagent | | | | | | | | | |
|------------------------------------------------------------------|--------------------------------------------|-----------------|-------------------------|-------------------|-----------------|-------------------------|-------------------------------|-------------------------------|-------------------------|
| Site | Panel<br>Member | Negative | Adenovirus<br>Low Level | hPIV Low<br>Level | Mixed Infection | | Mixed Infection | | |
| | | | | | Concentration | No<br>infected<br>cells | 4 to 10%<br>infected<br>cells | 4 to 10%<br>infected<br>cells | Adenovirus<br>Mid Level |
| Site<br>1 | Agreement<br>with<br>Expected result | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 70/70<br>(100%) |
| Site<br>2 | Agreement<br>with<br>Expected result | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 70/70<br>(100%) |
| Site<br>3 | Agreement<br>with<br>Expected result | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 70/70<br>(100%) |
| Site<br>4 | Agreement<br>with<br>Expected result | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 10/10<br>(100%) | 70/70<br>(100%) |
| | Total Agreement with<br>Expected<br>result | 40/40<br>(100%) | 40/40<br>(100%) | 40/40<br>(100%) | 40/40<br>(100%) | 40/40<br>(100%) | 40/40<br>(100%) | 40/40<br>(100%) | 280/280<br>(100%) |
| | 95% CI | 91.2 -<br>100% | 91.2 -<br>100% | 91.2 -<br>100% | 91.2 -<br>100% | 91.2 -<br>100% | 91.2 -<br>100% | 91.2 -<br>100% | 98.7 -<br>100% |

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### Limit of Detection:

Analytical Limit of Detection (LoD) of the L-DFA Reagent was addressed using dilution series of infected model cells. Model cells for parainfluenza virus types 1, 2, and 3 (ATCC strains C-35, Greer, and C243) and adenovirus (ATCC type 1) were diluted with non-infected cells to produce a suspension equivalent to 1,000 infected cells per milliliter. This level theoretically yields approximately 25 infected cells per 25-uL of suspension. This suspension was then serially diluted to a theoretical level of less than 1 cell per milliliter. (NOTE: This level was the target to begin with a low positive level. Actual starting levels vary, however, and are within 1 dilution of the 25 infected cell target level). 25-uL aliquots from each dilution level were spotted onto 10 replicate microscope slides, and then stained according to the instructions for use described in this product insert. Each cell spot was examined at 200X magnification. Results were reported as numbers of positive replicates for each set of 10. Analytical detection limits for each of the 4 analytes were defined as the lowest dilutions at which at least 9 out of 10 replicates were detected. LoD study results are summarized in Table 5.3 below:

| TABLE 5.3: | Limit of Detections of the D³ FastPoint L-DFA PIV/Adenovirus Reagent | | |
|-------------------------------|----------------------------------------------------------------------|------------------------------------------|-----------------------|
| Virus Strain | Infected cells/mL | Number of replicates with positive cells | LOD determination |
| Adenovirus<br>(ATCC type 1) | 1000 | 10/10 | 100 infected cells/mL |
| | 200 | 10/10 | |
| | 100 | 9/10 | |
| | 50 | 5/10 | |
| | 25 | 1/10 | |
| | 12.5 | 0/10 | |
| | 6 | 0/10 | |
| | 3 | 0/10 | |
| | 1.5 | 0/10 | |
| | 0.8 | 0/10 | |
| hPIV-1<br>(ATCC strain C-35) | 500 | 10/10 | 100 infected cells/mL |
| | 100 | 10/10 | |
| | 50…