MODIFICATION TO D3 ULTRA DFA RESPIRATORY VIRUS SCREENING & ID KIT

K092300 · Diagnostic Hybrids, Inc. · GNW · Aug 28, 2009 · Microbiology

Device Facts

• Record ID: K092300
• Device Name: MODIFICATION TO D3 ULTRA DFA RESPIRATORY VIRUS SCREENING & ID KIT
• Applicant: Diagnostic Hybrids, Inc.
• Product Code: GNW · Microbiology
• Decision Date: Aug 28, 2009
• Decision: SESE
• Submission Type: Special
• Regulation: 21 CFR 866.3330
• Device Class: Class 1

Intended Use

The Diagnostic Hybrids, Inc. D3 Ultra DFA (direct fluorescent antibody) RESPIRATORY VIRUS SCREENING & ID KIT 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 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.

Device Story

The D3 Ultra DFA Respiratory Virus Screening & ID Kit is an in vitro diagnostic assay used in clinical laboratories to detect and identify seven common respiratory viruses. The device utilizes a panel of fluorescein-labeled murine monoclonal antibodies (MAbs) that bind to specific viral antigens in respiratory specimens (e.g., nasopharyngeal aspirates, washes). The procedure involves fixing clinical specimens or cell culture monolayers onto glass slides with acetone, followed by incubation with the DFA reagents. If viral antigens are present, they are visualized as apple-green fluorescence under a fluorescence microscope, while uninfected cells are counter-stained red with Evan's Blue. The kit includes a screening reagent for initial detection and individual virus-specific reagents for identification. Results assist healthcare providers in confirming viral etiology, which informs clinical management and antibiotic stewardship. The device is intended for professional use by trained laboratory personnel.

Clinical Evidence

Clinical performance was established through prospective and retrospective studies comparing the subject device to the predicate. Studies evaluated 849 specimens across three sites. For fresh specimens (n=326), positive percent agreement (PPA) was 95.5% and negative percent agreement (NPA) was 98.3%. Frozen specimens and clinical isolates showed 100% agreement. No clinical data for 2009 H1N1 clinical specimens was established.

Technological Characteristics

The device consists of fluorescein-labeled murine monoclonal antibodies (MAbs) in aqueous solution with Evan's Blue counter-stain and 0.1% sodium azide preservative. It utilizes direct immunofluorescence (DFA) for antigen detection. The system requires a fluorescence microscope (FITC filter: 490nm excitation, 520nm emission). Components include screening and specific reagents, antigen control slides, wash solution, and mounting fluid. The assay is manual and requires acetone fixation.

Indications for Use

Indicated for respiratory virus screening and identification.

Regulatory Classification

Identification

Influenza virus serological reagents are devices that consist of antigens and antisera used in serological tests to identify antibodies to influenza in serum. The identification aids in the diagnosis of influenza (flu) and provides epidemiological information on influenza. Influenza is an acute respiratory tract disease, which is often epidemic.

Predicate Devices

• D³ Ultra DFA Respiratory Virus Screening & ID Kit (K061101)

Submission Summary (Full Text)

{0}------------------------------------------------

# Special 510(k): Device Modification De Ultra DFA Respiratory Virus Screening & ID Kit

Image /page/0/Picture/1 description: The image shows a logo for Diagnostic Hybrids. The logo includes the company name in bold, black letters, with the tagline "Integrating Science and Humanity" underneath. To the right of the text is a stylized graphic that resembles a DNA strand intertwined with a human figure. The number K092300 is written below the logo.

### DATE OF PREPARATION OF 510(k) SUMMARY

July 23, 2009

### APPLICANT

**AUG 2 8 2009**

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

### CONTACT INFORMATION

Ronald H. Lollar Senior Director, Product Realization, Management, and Marketing E-mail: [e-mail] Telephone: [phone] Desk Extension: 740-589-3373 FAX: [phone]

### DEVICE NAME

५:

Trade name: D 3 Ultra DFA Respiratory Virus Screening & ID Kit Common name: Respiratory Virus DFA Assay Classification name: Antisera, Cf, Influenza A, B, C Product Code: GNW Regulation: 21 CFR § 866.3330, Class I, Influenza virus serological reagents, Panel Microbiology (83)

### LEGALLY MARKETED DEVICE

Do Ultra DFA Respiratory Virus Screening & ID Kit, K061101

## DESCRIPTION of DEVICE MODIFICATION

The product insert has been modified. The following has been added (see below):

Table 15 in the product insert has been updated to include reactivity data on influenza A virus Mexico/4108/2009 and California/07/2009 strains. The following language was included with the data:

"Although this test has been shown to detect the 2009 H1N1 influenza virus in two cultured isolates, the performance characteristics of this device with clinical

{1}------------------------------------------------

specimens that are positive for the 2009 H1N1 influenza virus have not been established. The D Ultra DFA Respiratory Virus Screening & ID Kit can distinguish between influenza A and B viruses, but it cannot differentiate influenza subtypes."

## INTENDED USE

The Diagnostic Hybrids, Inc. D3 Ultra DFA (direct fluorescent antibody) RESPIRATORY VIRUS SCREENING & ID KIT 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 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.

## ASSESSMENT OF NON-CLINICAL PERFORMANCE DATA FOR EQUIVALENCE

Not Applicable

## ASSESSMENT OF NON-CLINICAL PERFORMANCE DATA FOR EQUIVALENCE

The risk analysis method used to assess the impact of the modification was a Failure Modes and Effects Analysis (FMEA). The modification to device labeling poses no additional risk.

## BIOCOMPATABILITY

www.cdc.gov

² FDA Guidance Document: In Vitro Diagnostic Devices to Detect Influenza A Viruses: Labeling and Regulatory Path; Issued 4/10/2006

{2}------------------------------------------------

·

.

## Not applicable

# STERILIZATION

Not applicable

:

{3}------------------------------------------------

D3 Ultra DFA 1 Respiratory Virus Screening & ID 2 Kit 3 4 For In Vitro Diagnostic Use I. INTENDED USE 5 б The Diagnostic Hybrids, Inc. Do Ultra DFA (direct fluorescent antibody) RESPIRATORY 7 8 VIRUS SCREENING & ID KIT is intended for the qualitative detection and identification 9 of the Influenza A. Influenza B. Respiratory Syncytial Virus (RSV), Adenovirus, Parainfluenza 1, Parainfluenza 2 and Parainfluenza 3 virus in respiratory specimens, by 10 either direct detection or cell culture method, by immunofluorescence using 11 fluoresceinated monoclonal antibodies (MAbs). It is recommended that specimens found 12 13 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 14 ાં ર sole basis for diagnosis, treatment or other management decisions. 16 Performance characteristics for influenza A were established when influenza A/H3 and 17 . A/H1 were the predominant influenza A viruses in circulation. When other influenza 18 A viruses are emerging, performance characteristics may vary. 19 If infection with a novel influenza A virus is suspected based on current clinical and 20 . epidemiological screening criteria recommended by public health authorities, 21 specimens should be collected with appropriate infection control precautions for novel 22 virulent influenza viruses and sent to state or local health departments for testing. 23 Viral culture should not be attempted in these cases unless a BSL3+ facility' is 24 available to receive and culture specimens.2 ટર્ટ II. SUMMARY AND EXPLANATION OF THE TEST ટેર 27 With the addition of new antiviral drugs for the treatment of Influenza', more rapid and 28 sensitive tests for respiratory virus detection 45 and the increasing need to be more 29 discriminating in the use of antibiotics , early detection and identification of the infecting 30 viral agent has grown substantially in importance. Viral identification is becoming 31 increasingly important in ruling out bacteria as the cause of respiratory infections. Virus 32 33 identification by either direct antigen detection or cell culture using fluorescent monoclonal antibodies continues to be the standard method in virology laboratories. 34 રે રે Influenza A and B Influenza viruses (family Orthomyxoviridae) contain a single-stranded RNA genome 36 which is present in 8 separate segments of ribonucleoprotein. This segmentation of the 37 38 genome is rare among viruses and probably contributes to the rapid development of new influenza strains through interchange of gene segments if two different viruses infect the 39 same cell. There are 3 types of influenza, A, B and C. Type A has counterparts in birds 40 and pigs as well as humans, while types B and C are known only in man. Due to the 41 possibility of another pandemic caused by Influenza A, as occurred in 1918 when 25-35 42 million people worldwide died, the Centers for Disease Control (CDC) and the World 43

{4}------------------------------------------------

Health Organization (WHO) maintain surveillance of influenza strains and make 44

predictions of suitable strains for vaccine production. વર્સ

Influenza infects an estimated 120 million people in the US, Europe and Japan each year 46

- and it is estimated that in the US there are 75,000 deaths annually from pneumonia caused 47
- by influenza. Primary viral pneumonia or pneumonia from secondary bacterial infections 48
- are the primary causes of morbidity of the viral infection. Pandemics of influenza A 49
- occur about every 10 to 30 years and epidemics of either influenza A or B occur annually. ર૦
- Infections are seasonal, typically extending from November to April in the northern રી I
- hemisphere. Complications tend to occur in the young, elderly and persons with chronic 52 રે 3 cardio-pulmonary diseases.
- રવે Incubation time is 1-3 days with rapid spread by inhalation via aerial droplets and fomites. ર ર It is characterized by fever, myalgia, headache and pharyngitis. .
- Influenza A and B are most commonly isolated in A549/Mv1Lu mixtures (R-Mix™1), ર્ડ
- A549/MDCK mixtures (R-Mix Too™1), Rhesus MK, MDCK, MRC-5 and A549 cells . 57

#### ર 8 Adenovirus

- Adenoviruses (family Adenoviridae) are non-enveloped, double stranded DNA viruses. રતે
- There are 49 serotypes, further divided into 6 groups, A to F, with most associated with રેજી
- respiratory and ocular infections. Generally, adenovirus infections in adults have a low ୧।
- morbidity with the exceptions of immunocompromised patients and individuals living in 62
- cramped quarters where infections can cause atypical pneumonia. Virus spread is 63
- commonly via aerial droplets and fomites where they infect the mucous membranes of the 64 ર્ણ રહ્યું eye, respiratory tract and gut'.
- Adenovirus can be isolated in A549/Mv1Lu mixtures (R-Mix™), A549/MDCK mixtures 66
- (R-Mix Too™), HEp2, HEK, A549 and MRC-5 cells.8 67

#### Parainfluenza Viruses 1, 2 and 3 68

- Parainfluenza viruses (family Paramyxoviridae) are enveloped viruses with a single, રેતે
- negative strand RNA genome. The 4 different types, 1 to 4, cause croup and viral 70
- pneumonia in children under the age of 5 years and cause upper respiratory illness in 71
- adults. Parainfluenza is the number 2 leading cause of lower respiratory illness in children 72
- (after RSV). Outbreaks caused by parainfluenza viruses occur during alternate years in the 73
- fall (P1 and P2) or throughout the year, with increased activity in the spring (P3) 10 74
- 75 Parainfluenza viruses can be isolated in A549/Mv1Lu mixtures (R-Mix™), A549/MDCK
- mixtures (R-Mix Too™), Rhesus MK, MRC-5 and LLC-MK2 cells. Trypsin is helpful in 76
- the medium for recovery of types 1 and 2 but not type 3 8. 77
- 78 Respiratory Syncytial Virus (RSV)
- RSV (family Paramyxoviridae) is an enveloped virus with a single, negative strand RNA 79
- genome. RSV infections cause viral bronchiolitis and pneumonia in infants and the 80
- common cold in adults". RSV is usually a seasonal (winter and early spring) infection 81
- 82 with epidemics lasting up to 5 months. Peak mortality due to RSV occurs in 3-4 month
- old infants. There are two major subtypes. A and B; Subtype B is characterized as the 83
- asymptomatic strain that the majority of the population experiences. The more severe 84

Tel [phone]
FAX [phone]
Diagnostic Hybrids, Inc.
[www.dhiusa.com](http://www.dhiusa.com)
350 West State Street
Athens, OH 45701

Page 13-2 of 13-31 pages

The use of R-Mix™ and R-Mix Too™ cells is covered by U.S. Patent Number 6,168,915 with additional patents pending.

{5}------------------------------------------------

clinical illnesses involve Subtype A strains which tend to predominate in most outbreaks 12. 8 રે

૪૯ RSV is the primary viral cause of lower respiratory disease in infants and young children.

Re-infections do occur but tend to be limited to minor upper respiratory infections13. RSV 87

- is also now recognized as a significant problem in certain adult populations. These include 88 the elderly, persons with cardiopulmonary diseases, and immunocompromised hosts 4. 89
- 90

RSV is commonly detected directly in cells from the nasopharyngeal epithelium by ਰੇ I

- staining with immunofluorescent reagents' although it can be isolated in cell cultures of 92
- A549/Mv1Lu mixtures (R-Mix™), A549/MDCK mixtures (R-Mix Too™), HEp2, Vero, ਰੇਤੇ

े पै LLC-MK2 and MRC-5 cells®.

- તેરે
#### III. PRINCIPLE OF THE PROCEDURE તેરિ

- 97
The Diagnostic Hybrids, Inc. D3 Ultra DFA RESPIRATORY VIRUS SCREENING & ID 98

ਰੇਰੇ KIT uses viral antigen-specific murine monoclonal antibodies that are directly labeled

- with fluorescein for the rapid detection and identification of respiratory viruses. 100
- The kit includes a DFA Screening Reagent that contains a blend of murine monoclonal 101
- antibodies (MAbs) directed against seven respiratory viruses (Influenza A, Influenza B, 102
- 103 Respiratory Syncytial Virus, Adenovirus, Parainfluenza 1, Parainfluenza 2, and
- Parainfluenza 3) plus seven separate DFA Reagents, each consisting of MAb blends 104
- directed against a single respiratory virus. The kit can be used for direct specimen or cell 105
- 106 culture screening and final virus identification.
- The cells to be tested, either derived from a clinical specimen or cell culture, are fixed in 107
- acetone. The DFA Screening Reagent is added to the cells to determine the presence of 108
- viral antigens. After incubating at 35℃ to 37℃, the stained cells are rinsed with the 109
- diluted Wash Solution. A drop of the supplied Mounting Fluid is added and a coverslip is 110
- placed on the prepared cells. The cells are examined using a fluorescence microscope. 111
- Virus infected cells will be stained with viral specific apple-green fluorescence when 112
- stained with the DFA Screening Reagent while uninfected cells will contain no 113
- fluorescence but will be stained red by the Evan's Blue counter-stain. If the specimen 114
- contains fluorescent cells, the particular virus is identified using the separate DFA । । ર
- Reagents on new, separate cell preparations. 116

If on examination of a direct stained specimen, no fluorescent-stained cells are found and 117

- all the cells stain red from the Evan's Blue, it is recommended that the specimen be 118
- cultured and stained using the DFA Screening Reagent. If fluorescent cells are seen, the 119 120 identification of the virus is determined as described above.
- Cell preparations are fixed in acetone. The individual DFA reagents are added to the cell 121
- preparations. After incubating at 35° to 37°C, the stained cells are rinsed with the diluted 122
- Wash Solution. A drop of the supplied Mounting Fluid is added and a coverslip is placed 123
- on the stained cells. The cells are examined using a fluorescence microscope for the 124
- presence of viral specific apple-green fluorescence. The unknown respiratory virus is then 125 identified and reported. 126
- 127

#### IV. REAGENTS 128

- 129
Tel [phone] FAX [phone] Diagnostic Hybrids, Inc. www.dhiusa.com

350 West State Street Athens, OH 45701

{6}------------------------------------------------

#### A. Kit Components 130

1. Respiratory Virus DFA Screening Reagent - 10-mL. One dropper bottle containing a 131 132 mixture of fluorescein labeled murine monoclonal antibodies directed against respiratory

133 viral antigens of Influenza A, Influenza B, Respiratory Syncytial Virus (RSV),

- Adenovirus, Parainfluenza 1, Parainfluenza 2 and Parainfluenza 3. The buffered, 134
stabilized, aqueous solution contains Evan's Blue as a counter-stain and 0.1% sodium ા ૩૨

- azide as preservative. I 36
2. Influenza A DFA Reagent - 2-mL. One dropper bottle containing fluorescein labeled 137 murine monoclonal antibodies directed against antigens produced by Influenza A virus 138 I ਤੇਰੇ (strain Texas 1/77, H3N2) infected cells. The buffered, stabilized, aqueous solution contains Evan's Blue as a counter-stain and 0.1% sodium azide as preservative. 140

3. Influenza B DFA Reagent - 2-mL. One dropper bottle containing fluorescein labeled 141 murine monoclonal antibodies directed against antigens produced by Influenza B virus 142 (Hong Kong 5/72) infected cells. The buffered, stabilized, aqueous solution contains 143 Evan's Blue as a counter-stain and 0.1% sodium azide as preservative. 144

4. RSV DFA Reagent - 2-mL. One dropper bottle containing fluorescein labeled murine 145 monoclonal antibodies directed against antigens produced by RSV (Long strain) infected 146 147 cells. The buffered, stabilized, aqueous solution contains Evan's Blue as a counter-stain and 0.1% sodium azide as preservative. 148

5. Adenovirus DFA Reagent - 2-mL. One dropper bottle containing fluorescein labeled 149 murine monoclonal antibodies directed against antigens produced by Adenovirus (Type 3-1 20 GB strain and Type 6-tonsil 99 strain) infected cells. The buffered, stabilized, aqueous 151 solution contains Evan's Blue as a counter-stain and 0.1% sodium azide as preservative. 152

6. Parainfluenza 1 DFA Reagent - 2-mL. One dropper bottle containing fluorescein 153 labeled murine monoclonal antibodies directed against antigens produced by Parainfluenza 154 ાં રેર 1 (VP-1 strain) infected cells. The buffered, stabilized, aqueous solution contains Evan's Blue as a counter-stain and 0.1% sodium azide as preservative. । રહ

7. Parainfluenza 2 DFA Reagent - 2-mL. One dropper bottle containing fluorescein 157 labeled murine monoclonal antibodies directed against antigens produced by Parainfluenza I રાજ 2 (Greer strain) infected cells. The buffered, stabilized, aqueous solution contains Evan's । રેતે Blue as a counter-stain and 0.1% sodium azide as preservative. 160

8. Parainfluenza 3 DFA Reagent - 2-mL. One dropper bottle containing fluorescein 161 labeled murine monoclonal antibodies directed against antigens produced by Parainfluenza 162 3 (C243 strain) infected cells. The buffered, stabilized, aqueous solution contains Evan's 163

- 164 Blue as a counter-stain and 0.1% sodium azide as preservative.
। ୧୯ 9. Respiratory Virus Antigen Control Slides - 5-slides. Five individually packaged

control slides containing wells with cell culture derived positive and negative control cells. 166

- Each positive well is identified as to the virus infected cells present, i.e., Influenza A, 167
- Influenza B. Respiratory Syncytial Virus (RSV), Adenovirus, Parainfluenza 1, 1 68
- Parainfluenza 2 and Parainfluenza 3. The Negative well contains uninfected cells. Each । ୧୯୯
- slide is intended to be stained only one time. 170

{7}------------------------------------------------

171 10. Normal Mouse Gamma Globulin DFA Reagent - 10-mL. One dropper bottle

172 containing a mixture of fluorescein labeled murine gamma globulin that has been shown to

173 be un-reactive with any of the listed respiratory viruses. The buffered, stabilized, aqueous

174 solution contains Evan's Blue as a counter-stain and 0.1% sodium azide as preservative.

11. Wash Solution Concentrate - 25-mL. One bottle containing a 40X concentrate 175

176 consisting of Tween 20 and 4% sodium azide (after dilution to 1X in water, the

177 concentration of sodium azide in the solution is 0.1%) in Phosphate Buffered Saline.

178 12. Mounting Fluid - 15-mL. One dropper bottle containing an aqueous, buffered, stabilized solution of glycerol and 0.1% sodium azide. 179

#### B. Warnings and Precautions 180

- 181 1. For in vitro diagnostic use.
- 2. Cells may have some potential to be hazardous. Personnel working with these cultures 182 must be properly trained in safe handling techniques1516/7, and have experience with 183 tissue culture before attempting this procedure. 184
- I 85 3. All procedures must be conducted in accordance with the CDC 4" edition Biosafety in Microbiological and Biomedical Laboratories, 1999, and CLSI Approved Guideline 186 M29-A, Protection of Laboratory Workers from Instrument Biohazards and Infectious 187 Disease Transmitted by Blood, Body Fluids, and Tissue. 188
- 189 4. Acetone, a reagent that is required for the test but not provided in the kit, is a flammable, volatile organic solvent. Use it in a well-ventilated area and keep away 190 from flames and other sources of ignition. । ਰੇ।
- 192 5. Sodium azide is included in the Wash Solution Concentrate at 4%, and in the other
- solutions in this kit at 0.1%. A MSDS for sodium azide or for Diagnostic Hybrids, Inc 193 (DHI) reagents containing sodium azide is available by contacting a Diagnostic 194
- I તેર Hybrids' Technical Service Representative.
- 196 a. Reagents containing sodium azide should be considered a poison. If products 197 containing sodium azide are swallowed, seek medical advice immediately and show product container or label. [Refer to NIOSH, National Institute for 198 Occupational Safety and Health; CAS#: 2628-22-8; and also to GHS, The । ਉਹੇ 200 Globally Harmonized System of Classification and Labeling of Chemicals.]
- b. Aqueous solutions of sodium azide, when mixed with acids, may liberate toxic gas (sodium azide in water exists is ionic equilibrium with hydrazoic acid. which when mixed with acid may liberate a toxic gas).
- c. Any reagents containing sodium azide should be evaluated for proper disposal. 204 205 Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. If products containing sodium azide are discarded into 206 207 a drain, flush with a large volume of water to prevent azide build-up. Check 208 with regulatory agencies to determine at what concentration sodium azide may 209 cause a product to be regulated as hazardous waste.

210 6. Evan's Blue counter-stain is a potential carcinogen. If skin contact occurs. flush with 211 water immediately.

7. The DFA Reagents are supplied at working strength. Any dilution of the DFA 212

213 Reagents will decrease sensitivity.

201

202 203

{8}------------------------------------------------

- 8. Reagents should be used prior to their expiration date. 214 215 9. Each Respiratory Virus Antigen Control Slide should be used only once. Do not re-216 use a Control Slide. 217 10. Microbial contamination of DFA Reagents may cause a decrease in sensitivity. 218 11. Store 1X Wash Solution and PBS in a clean container to prevent contamination. 12. All specimens and materials used to process them should be considered potentially 219 infectious and handled in a manner which prevents infection of laboratory personnel. 220 221 Decontamination is most effectively accomplished using a 0.05% solution of sodium 222 hypochlorite (1:100 dilution of household bleach). 223 13. Although Antigen Control Slides have been shown to be non-infectious, the same precautions taken in handling and disposing of other infectious materials should be 224 225 employed in their use. 226 14. Never pipette reagents or clinical samples by mouth; avoid all contact of clinical 227 samples with broken skin. 228 15. Avoid splashing and the generation of aerosols with clinical samples. 229 16. Use aseptic technique and sterile equipment and materials for all tissue culture 230 procedures. 231 17. Reusable glassware must be washed and thoroughly rinsed free of all detergents. 232 18. Do not expose DFA Reagents to bright light during staining or storage. 19. Use of other reagents than those specified with the components of this kit may lead to 233 234 erroneous results. 235 236 C. Preparation of 1X Wash Solution 237 1. After storage at 2° to 8°C, some salts in the Wash Solution Concentrate may have 238 crystallized. Warm the solution to room temperature to re-dissolve the crystals and 239 mix. 240 2. Add contents of the fully dissolved 25-mL Wash Solution Concentrate to 975-mL of 241 de-mineralized water. 3. · Label the 1X Wash Solution with a sixty (60) day expiration date after reconstitution 242 243 and store at room temperature (20° to 25°C).
- 244

#### 245 D. Storage Instructions

| TABLE 1 | | |
|---------|-----------------------------------------|---------------------------------|
| 1. | Respiratory Virus DFA Screening Reagent | Store at 2° to 8°C in the dark. |
| 2. | Influenza A DFA Reagent | |
| 3. | Influenza B DFA Reagent | |
| 4. | RSV DFA Reagent | |
| 5. | Adenovirus DFA Reagent | |
| 6. | Parainfluenza 1 DFA Reagent | |
| 7. | Parainfluenza 2 DFA Reagent | |
| 8. | Parainfluenza 3 DFA Reagent | |
| 9. | Mounting Fluid | |
| 10. | Normal Mouse Gamma Globulin DFA Reagent | |

Tel [phone] FAX [phone]

Diagnostic Hybrids, Inc. www.dhiusa.com

{9}------------------------------------------------

| 11. Respiratory Virus Antigen Control Slides | Store at 2° to 8°C. |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------|
| 12. Wash Solution Concentrate<br>NOTE: The Concentrate may crystallize when<br>stored at 2° to 8°C. The crystals will<br>dissolve when the Concentrate is warmed<br>to room temperature. | Store liquid at 2° to 8°C<br>prior to dilution. |
| 13. 1X Wash Solution | Store at room temperature<br>(20° to 25°C). |

246

#### 247 E. Stability

248 Reagents and components will retain their full potency through the expiration date shown 249 on the label of each bottle when stored at recommended temperatures. Light exposure of 250. the DFA Reagents should be kept to a minimum.

Discard 1X Wash Solution if it becomes cloudy. 251

252

#### V. SPECIMEN COLLECTION AND PREPARATION 253

254

255 Proper collection and handling of the patient specimen are the most important factors in successful respiratory virus detection. Specimen collection, specimen processing, and cell 256 culture of viruses should be attempted only by personnel that have been trained in such 257 procedures. Care should be taken during all specimen collection and handling to avoid 258 259 generation of aerosols.

- 260
#### A. Specimen Collection18 261

Aspirates and Washes containing secretions from the nasopharyngeal epithelium provide 262

the best specimens for direct specimen testing since they will contain large numbers of 263 264 epithelial cells.

265 Aspirates can be collected using a sterile, soft polyethylene #8 infant feeding tube attached 266 to a disposable aspiration trap connected to a suction device.

267 Washes can be collected by instilling and aspirating 1- to 2-mL of saline in the patient's 268 nostril while the patient is in a supine position.

Aspirates and washes should be diluted with equal volumes of transport medium contained 269 in a centrifuge tube with several sterile glass beads. 270

- Swabs from nasal, throat and nasopharyngeal areas often do not contain sufficient 271
- 272 numbers of columnar epithelial cells to allow for direct specimen detection of respiratory 273 viruses.
- 274

#### 275 B. Specimen Transport and Storage

276 All potentially infectious agents should be transported according to International Air

- Transport Association (IATA), International Civil Aviation Organization, (ICAO), Titles 277
- 42 and 49 of the US Code of Federal Regulations, or other regulatory requirements, as 278
- 279 may be applicable.
- 280 Specimens should be transported on wet ice to the laboratory and processed and tested as
- 281 soon as possible and then stored at 2° to 8°C.

| Tel [phone] | Diagnostic Hybrids, Inc. | 350 West State Street |
|------------------|--------------------------|-----------------------|
| FAX [phone] | www.dhiusa.com | Athens, OH 45701 |

Page 13-7 of 13-31 pages

{10}------------------------------------------------

Specimens should be stored at 2° to 8°C for no longer than 48 hours before being tested. If 282

longer storage is required, the specimens should be frozen at -70℃ or lower. 283

284 Freezing and thawing of specimens should be avoided since this will result in a loss of

285 viability of viruses, leading to decreased sensitivity of the test.

286

#### VI. PROCEDURE 287

- 288
#### A. Materials Provided 289

- 290 1. Respiratory Virus DFA Screening Reagent
- 291 2. Influenza A DFA Reagent
- 3. Influenza B DFA Reagent 292
- 4. RSV DFA Reagent 293
- 294 5. Adenovirus DFA Reagent
- જીતેર 6. Parainfluenza 1 DFA Reagent
- 296 7. Parainfluenza 2 DFA Reagent
- 297 8. Parainfluenza 3 DFA Reagent
- 298 9. Normal Mouse Gamma Globulin DFA Reagent
- 299 10. Respiratory Virus Antigen Control Slides
- 300 11. Mounting Fluid
- 301 12. Wash Solution Concentrate
- 302

#### B. Materials Required But Not Provided 303

- 1. Fluorescence microscope with the correct filter combination for FITC (excitation peak 304 305 = 490 nm, emission peak = 520nm).
- 2. Cell culture for respiratory virus isolation. Suggested cell lines that are susceptible to 306 respiratory viruses include LLC-MK2, HEp-2, A549 cells, R-Mix™ and R-Mix TooTM 307 Mixed Cells, and primary Rhesus monkev kidnev cells, all available from DHI. 308
- 300 3. Cover slips (22 x 50mm) for Antigen Control Slides and for specimen slides.
- 310 4. Universal Transport Medium (available from DHI).
- 5. R-Mix Refeed medium (for use with R-Mix™ and R-Mix Too™ Mixed Cells) or 311 other standard Refeed medium. Available from DHI. 312
- 313 6. Reagent grade acetone (>99% pure) chilled at 29 to 8°C for fixation of direct specimen slides and shell vials. 314
- NOTE 1: Keep the reagent grade acetone container tightly sealed to avoid hygroscopic 315 absorption of water, which may cause a hazy, nonspecific, background fluorescence. 316
- NOTE 2: A mixture of 80% acetone/20% de-mineralized water is used for fixing cells 317
- 318 in plastic multi-well plates. Store at ambient temperature.
- 319 7. Sterile graduated pipettes: 10-mL, 5-mL, and 1-mL.
- 320 8. Sterile Pasteur pipettes or other "transfer"-type pipettes.
- Fine-tipped forceps. 321 9.
- 200-mL wash bottle. 322 10.
- 11. Bent-tip teasing needle (for removal of coverslip from a shell vial for the typing 323
- 324 portion of the procedure); fashion the teasing needle by bending the tip of a syringe

{11}------------------------------------------------

| 325 | | needle or similar object (i.e. mycology teasing needle) against a benchtop or with a pair of forceps taking care to avoid injury. |
|---------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 326 | | pair of forceps taking care to avoid injury. |
| 327 | 12. | Sodium hypochlorite solution, 0.05% (1:100 dilution of household bleach). |
| 328 | 13. | Humid chamber (e.g. covered Petri dish with a damp paper towel placed in the bottom). |
| 329 | | bottom). |
| 330 | 14. | Glass microscope slides. |
| 331 | 15. | Acetone-cleaned multi-well glass microscope slides (2-well and 8-well masked slides). |
| 332 | | slides). |
| 333 | 16. | Blotters for multi-well glass microscope slides: Two- and 8-well absorbent blotters, used to blot excess liquid from the mask to prevent spread of liquid or stained cells from one well to the other. |
| 334 | | used to blot excess liquid from the mask to prevent spread of liquid or stained cells |
| 335 | | from one well to the other. |
| 336 | 17. | Sterile nylon flock swab or polyester swab, non-inhibitory to respiratory viruses and tissue culture. |
| 337 | | tissue culture. |
| 338 | 18. | Incubator, 35° to 37°C (CO2 or non-CO2 , depending on the cell culture format used). |
| 339 | 19. | Centrifuge with free swinging bucket rotor. |
| 340 | 20. | De-mineralized water for dilution of Wash Concentrate Solution and for dilution of the reagent grade acetone for use in polystyrene multi-well plates (see item VI.B.5). |
| 341 | | the reagent grade acetone for use in polystyrene multi-well plates (see item VI.B.5). |
| 342 | 21. | PBS (Phosphate Buffered Saline), sterile, for use in rinsing and suspending cells. |
| 343 | 22. | Control viruses: Known strains of the 7 respiratory viruses for use in monitoring the cell culture and staining procedures. Such control virus strains can be obtained from Diagnostic Hybrids, Inc. |
| 344 | | cell culture and staining procedures. Such control virus strains can be obtained from |
| 345 | | Diagnostic Hybrids, Inc. |
| 346 | 23. | Aspirator Set-up: Vacuum aspirator with disinfectant trap containing sufficient household bleach (5%) that the concentration is not decreased by more than 100 fold as it is diluted with discarded fluids. |
| 347 | | household bleach (5%) that the concentration is not decreased by more than 100 fold |
| 348 | | as it is diluted with discarded fluids. |
| 349 | 24. | Wash Container: Beaker, wash bottle or Coplin jar for washing slides. |
| 350 | 25. | Fixing Container: Coplin jar, slide dish or polyethylene holder for slides for use in fixing the cells on the slides. |
| 351 | | fixing the cells on the slides. |
| 352 | 26. | Inverted Light Microscope: Used for examining the monolayers of cells prior to inoculation and examination for toxicity and CPE. It should have between 100X to 400X magnification capability. |
| 353 | | inoculation and examination for toxicity and CPE. It should have between 100X to |
| 354 | | 400X magnification capability. |
| 355 | | |
| 356 | | <b>C. Preliminary Comments and Precautions</b> |
| 357 | | 1. Adhere to the recommended volumes and times in the following procedure to ensure that accurate results are obtained. |
| 358 | | 2. For specimen swabs received in transport medium with glass beads, vortex vigorously for about 15 seconds to dissociate adhered cells. For swabs not received in transport medium, transfer them to a tube of transfer medium containing glass beads and vortex vigorously for about 15 seconds to dissociate adhered cells. |
| 359 | | For specimen swabs received in transport medium with glass beads, vortex |
| 360 | | vigorously for about 15 seconds to dissociate adhered cells. For swabs not received |
| 361 | | in transport medium, transfer them to a tube of transfer medium containing glass |
| 362 | | beads and vortex vigorously for about 15 seconds to dissociate adhered cells. |
| 363 | | 3. When staining with fluorescent reagents and examining cells microscopically for fluorescence, it is very important to include controls, both positive and negative, to monitor the procedure and performance of the reagents. It is recommended that such controls be run with each batch of patient specimens. |
| 364 | | fluorescence, it is very important to include controls, both positive and negative, to |
| 365 | | monitor the procedure and performance of the reagents. It is recommended that |
| 366 | | such controls be run with each batch of patient specimens. |
| 367 | | 4. The closed, humidified container for holding the slides during incubation should be kept in the incubator so it is at incubator temperature when the slides are placed in |
| 368 | | kept in the incubator so it is at incubator temperature when the slides are placed in |
| 369 | | it. By doing this, the cells and antibody solution will come up to temperature more |
| 370 | | rapidly, yielding more intense stains in shorter periods of time. |
| 371 | | REGARDING CELL CULTURE TESTING: |
| 372 | | 5. Good Laboratory Practice dictates that positive and negative virus controls be run |
| 373 | | with each new batch of cells to confirm their performance in culturing specific |
| 374 | | viruses. |
| 375 | | 6. It is good practice to retain the medium removed from the positive monolayers |
| 376 | | until after staining results have been obtained. If there is any question concerning |
| 377 | | the specimen results, the medium can be passed to another monolayer for repeat |
| 378 | | testing. |
| 379 | | 7. If using cell cultures in polystyrene multi-well plates, the acetone fixative must be |
| 380 | | diluted with water to 80% by adding 20 mL of water to 80 mL of acetone. |
| 381 | | 8. Do not allow the monolayers to dry before fixing; this can lead to high background |
| 382 | | staining and decreased sensitivity. |
| 383 | | 9. Do not allow the antibody reagents to dry on the monolayers; this can lead to high |
| 384 | | background. |
| 385 | | REGARDING IMMUNOFLUORESCENCE MICROSCOPY: |
| 386 | | 10. It is good practice to examine the positive and negative controls before examining |
| 387 | | the test specimens. If one of these fails to perform as expected, review the steps |
| 388 | | and conditions under which the test was performed to determine the cause(s). Do |
| 389 | | not report results until controls perform properly. |
| 390 | | 11. There are three aspects of the fluorescence microscope that must be functioning |
| 391 | | properly and optimally in order to achieve maximum brightness of fluorescence: |
| 392 | | i. The activation light source has a finite life and as it ages, its output decreases, |
| 393 | | resulting in lower fluorescence intensity from the DFAs. |
| 394 | | ii. The light source is focused by a number of lenses and mirror(s). For |
| 395 | | maximum intensity, these must be properly aligned. |
| 396 | | iii. The filters used in the light path must be appropriate for the particular fluor, |
| 397 | | in this case, fluorescein. |
| 398 | | 12. There are several fluorescent artifacts that may be observed in the cell monolayers |
| 399 | | being examined: |
| 400 | | i. Cell debris, lint, etc. can nonspecifically adsorb DFAs, resulting in highly |
| 401 |…