CLIA. BinaxNOW Influenza A & B CLIA Waived. Training Packet. Inverness Medical Point of Care Diagnostic Products Better Results Mean Better Medicine

Transcription

1 CLIA Training Packet BinaxNOW Influenza A & B CLIA Waived Inverness Medical Point of Care Diagnostic Products Better Results Mean Better Medicine

2 Inverness Medical CLIA Packet The following materials are provided to all Inverness Medical Customers. Laboratories performing waived tests are expected to follow the manufacturer s guidelines and good laboratory practice. Included in this packet are the following: CLIA Quality Control Information Letter for Waived Tests. Quality Control and Patient Record Logs. List of Important Procedure Steps. Training Certificate. Proficiency Test Information. Test Procedure specifically written according to the standards of CLSI Guidelines. Competency Assessment Forms. Appendix: Material Safety Data Sheets (MSDS) to satisfy OSHA requirements.

3 Dear Customer, Thank you for your interest in the BinaxNOW Influenza A & B Test Kit. The BinaxNOW Influenza A & B Test has been categorized by CLIA as a waived complexity test. It is good laboratory practice to use Quality Control Material when performing waived tests to ensure that the test results are accurate. The BinaxNOW Influenza A & B Test Package Inserts specifies how often to use Quality Control Material. Per the BinaxNOW Influenza A & B Test Package Inserts, The BinaxNOW Test has built-in (internal) procedural controls. For daily quality control, Inverness Medical suggests that you record results of these controls for each test run. An untested strip has a blue line at the control position. If the test flows correctly and the reagents work, this blue line will always turn pink on the strip. The clearing of background color from the result window is a negative background control. The background color in the window should change from light pink to white within 15 minutes. Background color should not interfere with the reading of the test. BinaxNOW test kits contain Positive and Negative Control Swabs. These swabs will verify the entire assay. Test these swabs once with each new shipment received. Other controls may be tested in order to conform with local, state and/or federal regulations; accrediting groups, and/or your lab s standard QC procedures. In summary, good laboratory practice should be used when performing waived tests to ensure accurate results. If you should need assistance, please do not hesitate to call Technical Service at Inverness Medical Technical Services Office

4 BinaxNOW Influenza A & B Quality Control and Patient Record Lot Number Exp. Date Inverness Medical recommends that external positive and negative controls be run with each new shipment received Record the Date, Patient s Name, Patient Test Result, Internal Control Results and the performer s initials. Positive Internal Control = the pink/purple Control Line; Negative Background Control = the clear background in the test window. 1. Date Patient Name Patient ID Number Patient Results Internal Controls + - Comments Tech Reviewed By: Date:

5 BinaxNOW Influenza A & B Quality Control and Patient Record (Page 2 for Waived BinaxNOW FLU 22 Kit only) Record the Date, Patient s Name, Patient Test Result, Internal Control Results and the performer s initials. Positive Internal Control = the pink/purple Control Line; Negative Background Control = the clear background in the test window Date Patient Name Patient ID Number Patient Results Internal Controls + - Comments Tech Reviewed By: Date:

6 BinaxNOW Influenza A & B Test Important Procedure Steps 1. Leave test device sealed in its foil pouch until just before use. 2. Elute swab samples in ml of elution solution, saline or transport media within 1 hour of collection. 3. Testing should be performed as soon as possible. a. Eluted swab samples may be stored refrigerated for up to 24 hours. b. Nasal wash samples may be stored refrigerated for up to 24 hours. 4. Sample should be at room temperature for testing. Swirl gently to mix before testing. 5. INVALID RESULTS can occur when too little sample is added to the test. Be sure that the lower part of the transfer pipette is full and does not have any air spaces before you add the sample to the Sample Pad. 6. Add sample slowly (drop-by-drop) to the middle of the white sample pad so that all of the sample volume absorbs into the pad. DO NOT add sample to the pink/purple colored pad. 7. When testing nasal wash/aspirate samples, avoid viscous areas of the sample when drawing specimen into the transfer pipette. 8. Read result in window 15 minutes after closing the device. 9. A test is invalid if the Control Line remains blue or is not present at all, whether a Sample Line (s) is present or not. Repeat Invalid tests with a new test device. If the problem persists call Inverness Medical Technical Service at

7 Certification of Training This is to verify that the office staff and personnel responsible for running the BinaxNOW Influenza A & B Test at have been thoroughly in-serviced on the test and the test procedure. This has included: Review of the package insert Demonstration of the product assay Successful performance of the BinaxNOW Influenza A & B Test and interpretation of results Names of the personnel who have been trained with the BinaxNOW Influenza A & B Test and are responsible for reporting patient results: PRINT NAME SIGNATURE DATE Signature of Medical Director(s) responsible for personnel and testing: Signature Signature Date Date Inverness Medical Trainer Date

8 ...A Word about Proficiency Testing... Proficiency Testing (PT) is a type of external quality control. The practice of testing unknown specimens from an outside source provides an additional means to assure quality laboratory testing results. External quality control provides important comparisons to determine the accuracy of your testing procedures. Some states require PT testing for analytes categorized as waived tests or methods by the federal government. Because your state may have its own laboratory regulations, check to see if you must perform PT for the federallywaived tests. Whether required or not, PT for all tests performed in your laboratory can provide documentation regarding the accuracy of waived and non-regulated tests in the event of an inspection. Most importantly, it helps to ensure that all patient testing is accurate. Proficiency Testing Information Page 1 of 3

9 Tips for Successful PT Performance Strictly follow the PT provider's storage or handling requirement before testing PT specimens. Analyze PT specimens within the time frame provided by the PT provider. Contact the PT provider promptly when specimens are received damaged. You may be able to receive a replacement immediately. Avoid clerical error when filling out PT answer sheets. Be sure to enter the correct result next to the correct analyte on the answer form. Remember to identify the instrument or method you are using to perform your PT so that you are graded among your peer group. Make copies of all answer forms before submitting them to your PT provider. Proficiency Testing Information Page 2 of 3

10 Proficiency Test Providers American Association of Bioanalysts(AAB) Proficiency Testing Service 205 West Levee Street Brownsville, TX American Academy of Family Physicians (AAFP) Tomahawk Creek Pkwy Leawood, Ks College of American Pathologists (CAP) Surveys Program 325 Waukegan Road Northfield, IL College of American Pathologists (CAP) EXCEL Program 325 Waukegan Road Northfield, IL American Proficiency Institute (API) 1159 Business Park Drive Traverse City, MI Medical Laboratory Evaluation (MLE) ACP-ASIM Services 2011 Pennsylvania Avenue, NW Suite 800 Washington, DC Accutest P.O. Box 999 Westford, MA Some states provide their own in-state proficiency testing programs. Please contact your state CLIA office for more information. Proficiency Testing Information Page 3 of 3

11 Laboratory: Date Implemented Address: BinaxNOW Influenza A & B Laboratory Procedure I. Test Principle The BinaxNOW Influenza A & B test is an immunochromatographic membrane assay that uses highly sensitive monoclonal antibodies to detect influenza type A and B nucleoprotein antigens in respiratory specimens. These antibodies and a control antibody are immobilized onto a membrane support as three distinct lines and combined with other reagents/pads to construct a test strip. This test strip is mounted inside a cardboard, book-shaped hinged test device. Swab specimens require a sample preparation step, in which the sample is eluted or washed off the swab into elution solution, saline or transport media. Nasal wash/aspirate samples require no preparation. Sample is added to the top of the test strip and the test device is closed. Test results are interpreted at 15 minutes based on the presence or absence of pinkto-purple colored Sample Lines. The blue Control Line turns pink in a valid assay. II. Specimen Collection/Treatment A. Specimen: Acceptable: Nasal wash/aspirate; Nasopharyngeal or Nasal swab. Unacceptable: Specimens collected from other sources. B. Swabs: Acceptable: Cotton, rayon, foam, or polyester flexible-shaft NP swabs. Use cotton, rayon, foam, or polyester solid shaft swabs to collect nasal swab samples. Unacceptable: Calcium alginate swabs. C. Transport Media: Acceptable: Amies Media, Binax Elution Solution, Hank s Balanced Salt Solution, M4, M4-RT, M5, Stuart s Media, Saline, Brain Heart Infusion Broth, Dulbecco Medium, Phosphate Buffer, Stuart s Media, Tryptose Phosphate Broth, UTM-RT Media, Veal Infusion Broth. Unacceptable: Any other type of media. It has been determined that Sucrose-Phosphate Buffer may not be suitable for use with this test. D. Specimen Transport: Transport samples in a leak proof container. E. Specimen Storage: Process samples as soon as possible after collection. Swabs: Elute samples within 1 hr of collection. Eluted swab samples may be stored refrigerated up to 24 hrs. Nasal wash/aspirate: May be stored refrigerated up to 24 hrs. F. Handling Precautions: Patient samples, controls and test devices should be handled as though they could transmit disease. Observe established precautions against microbial hazards. CLSI Procedure Page 1 of 15

12 III Reagents and Equipment A. Reagents and Materials Provided Component Content Test Device Transfer Pipettes Positive Control Swab Negative Control Swab Elution Solution Vials for Control Swabs/ Swab Specimens A cardboard, book-like, hinged test device containing the test strip. A/Texas/1/77 was the master influenza virus strain used to develop the monoclonal antibodies incorporated into the test device. Fixed Volume (100ul) transfer pipettes used to transfer sample to the test device. Use only pipettes provided by Binax. Inactivated influenza A/ Beijing or influenza A/Texas 1/77 (H3N2) and inactivated influenza B/ Harbin or influenza B/ Hong Kong 5/72 dried onto swab. The influenza viruses are originally grown in embryonic eggs and are Formalin or gamma radiation inactivated. Formalin treated viruses are tested for inactivation and non-infectiousness by re-growing virus in embryonic eggs. Viruses are considered inactivated when no viral propagation is seen in eggs. Inactivated Streptococcus Group A dried onto swab. Organism used to inoculate the swab is heat inactivated, and then tested for inactivation and non-infectiousness by standard culture. The organisms are determined to be inactivated when no growth is present on the plate. Vials containing elution solution used to prepare the Control Swabs/Swab Specimens for testing. NP Swabs Sterile, foam swabs for use in the BinaxNOW Influenza A & B Test Package Insert B. Materials not Provided A timer, watch, or clock Nasal wash/aspirate collection containers C. Storage and Stability Store Test Kit at room temperature o C (59-86 o F). The BinaxNOW Influenza A & B Test kit and reagents are stable until the expiration dates marked on their outer packaging and containers. D. Quality Control Internal Procedural Controls The BinaxNOW Influenza A & B test has built-in (internal) procedural controls. For daily quality control, Inverness Medical suggests that you record results of these controls for each test run. An untested strip has a blue line at the Control position. If the test flows correctly and the reagents work, this blue line will always turn pink on the strip. The clearing of background color from the result window is a negative background control. The background color in the window should change from light pink to white within 15 minutes. Background color should not interfere with the reading of the test. CLSI Procedure Page 2 of 15

13 External Quality Control Testing Good laboratory practice suggests the use of positive and negative controls to guarantee that test reagents are working and the test is correctly performed. The BinaxNOW Test Kits contain Positive and Negative Control Swabs. These swabs will verify the entire assay. Test these swabs once with each new shipment received. Other controls may be tested in order to conform with local, state and/or federal regulations, accrediting groups, and/or your lab s standard Quality Control procedures. Remedial Actions When correct control results are not obtained, do not report patient results. Contact Inverness Medical Technical Services at E. Precautions For in vitro diagnostic use. Leave test sealed in its foil pouch until just before use. Do not use kit past its expiration date Do not mix components from different kit lots. The white sample pad at the top of the test strip contains reagents that extract the target antigen from the virus. To ensure best performance, add the sample SLOWLY (drop by drop) to the MIDDLE of this pad, without touching with the pipette, such that all of the sample absorbs into the pad. DO NOT add sample to the pink/purple colored pad. Solutions used to make the control swabs are inactivated using standard methods. However, patient samples, controls, and tests should be handled as though they could transmit disease. Observe established precautions against microbial hazards. The use of lab coats, gloves, and safety eye glasses is recommended. All transfer pipettes and test vials are single use items - do not use with more than one specimen. The ability of this test to detect avian influenza was determined using cultured avian influenza viruses; the performance characteristics of this test with specimens collected from humans infected with H5N1 or other avian influenzas is unknown. 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 BSL 3 + facility is available to receive culture specimens. 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. IV. Test Procedure Sample Preparation Procedure Nasal Wash/Aspirate: Nasal wash/aspirate samples do not need preparation. Go to Test Procedure. Precaution: When testing nasal wash/aspirate samples, avoid thick areas of the sample when drawing it into the transfer pipette. If the pipette becomes clogged, and the lower part of the pipette is not full, put the sample back into container by squeezing the top bulb. Redraw the sample into the pipette. Use a new pipette if needed CLSI Procedure Page 3 of 15

14 Swab (Control & Patient) Elution using Binax Elution Solution: 1. Twist off the test vial cap. 2. Put the swab to be tested into test vial. Rotate the swab vigorously (without making a lot of bubbles) three (3) times in the liquid. 3. Press the swab against the side of the vial and turn as you remove it from the vial. This removes sample from the swab. 4. Discard the swab into a container intended for contagious material. 5. Test the liquid sample (from the test vial) in the BinaxNOW Test as soon as possible. Go to Test Procedure. Swab Elution Using Transport Media: Remove sample from swab in 0.5 to 3.0 ml of saline or transport media/fluid by vigorously rotating the swab in the liquid. Refer to Specimen Collection and Handling section for acceptable transport media. Go to Test Procedure. Test Procedure: WARNING: INVALID RESULTS can occur when too little sample is added to the test. Be sure that the lower part of the transfer pipette is full and does not have any air spaces before you add the sample to the Sample Pad. If there are air spaces, put the sample back into the container by squeezing the top bulb. Redraw the sample from the bottom of the container into the pipette. Use a new pipette if needed. 1. Remove device from the pouch just prior to testing and lay flat on work bench. 2. Fill pipette by firmly squeezing the top bulb and then placing pipette tip into sample. Slowly release bulb while tip is still in sample. This will pull liquid into the pipette. Make sure there are no air spaces in the lower part of the pipette. 3. See arrow on test to find WHITE Sample Pad at the top of the test strip. SLOWLY (drop by drop) add entire contents of pipette (100uL) to the MIDDLE of this pad by squeezing the top bulb, such that all of the sample volume absorbs into this pad. DO NOT add sample to the pink/purple colored pad. 4. Immediately peel off brown adhesive liner from the test device. Close and securely seal the device. Read result in window 15 minutes after closing the device. NOTE: When reading test results, tilt the device to reduce glare on the result window if necessary V. Interpretation of Test Results NOTE: Do not read test result before or after 15 minutes as they may not be correct.. Individuals with color impaired vision may not be able to adequately interpret test results. Negative Result: The BLUE Control Line in the BOTTOM THIRD of the window turns a pink to purple color. No other line appears. Flu A Positive Result: The BLUE Control Line turns a pink to purple color and a SECOND pink to purple Sample Line appears above it in the MIDDLE THIRD of the window. Any shade of a pink to purple Sample Line, even when very faint, indicates a positive result. Flu B Positive Result: The BLUE Control Line turns a pink to purple color and a SECOND pink to purple Sample Line appears above it in the TOP THIRD of the window. Any shade of a pink to purple Sample Line, even when very faint, indicates a positive result. CLSI Procedure Page 4 of 15

15 Flu A and Flu B Positive Result: The BLUE Control Line turns a pink to purple color, AND two pink to purple Sample Lines appear above it in the MIDDLE and TOP thirds of the window. Any shade of pink to purple Sample Lines indicates positive results. Invalid: A test is invalid if the Control Line remains BLUE or is not present at all, whether a Sample Line(s) is present or not. Remedial Actions Repeat Invalid tests with a new test device. Call Inverness Medical Technical Services at , if problem persists. VI. Interfering Substances The following substances, naturally present in respiratory specimens or that may be artificially introduced into the nasal cavity or nasopharynx, were evaluated in the BinaxNOW Influenza A & B Test at the concentrations listed and were found not to affect test performance. Whole blood (1%) did not interfere with the interpretation of negative BinaxNOW Test results, but did interfere with the interpretation of Flu A LOD positive samples. Therefore, visibly bloody samples may not be appropriate for use in this test. Substance Concentration 1 OTC mouthwashes 20% 3 OTC nasal sprays 15% 3 OTC throat drops 15% 2 OTC throat sprays 20% 4-acetamidophenol 10 mg/ml Acetylsalicylic acid 15 mg/ml Albuterol 20 mg/ml Chlorpheniramine 5 mg/ml Dextromethorphan 10 mg/ml Diphenhydramine 5 mg/ml Guaiacol glycerol ether 20 mg/ml Oxymetazoline 0.05% Phenylephrine 50 mg/ml Phenylpropanolamine 20 mg/ml Rebetol 500 ng/ml Relenza 20 mg/ml Rimantadine 500 ng/ml Synagis 0.1 mg/ml Tamiflu 50 mg/ml CLSI Procedure Page 5 of 15

16 Transport Media: The following transport media were tested in the BinaxNOW Influenza A and/or B Test as negative samples (no virus present) and after inoculation with the LOD levels of Influenza A & B. Media did not impact BinaxNOW test performance, with the media alone testing negative in the BinaxNOW Test and media inoculated with LOD Influenza A & B testing positive on the appropriate test line in BinaxNOW test. Amies Media Hank s Balanced Salt Solution M4 Media M4-RT Media M5 Media Stuart s Media Saline Brain Heart Infusion Broth Dulbecco Medium Phosphate Buffer Solution UTM-RT Media Tryptose Phosphate Broth Veal Infusion Broth It has been determined that Sucrose-Phosphate Buffer may not be suitable for use with this test. VII. Limitations A negative test result does not exclude infection with influenza A and/or B. Therefore, the results obtained with the BinaxNOW Influenza A & B Test should be used in conjunction with clinical findings to make an accurate diagnosis. Additional testing is required to differentiate any specific influenza A and B subtypes of strains, in consultation with state or local public health departments. The BinaxNOW Influenza A & B Test detects both viable and non-viable influenza A and B. Test performance depends on the amount of virus (antigen) in the specimen and may or may not compare with cell culture results performed on the same specimen. Monoclonal antibodies may fail to detect, or detect with less sensitivity, influenza A and B viruses that have undergone minor amino acid changes in the target epitope region. Inadequate specimen collection or improper sample handling/transport may yield a false-negative result. Performance of the BinaxNOW Influenza A & B Test has not been established for monitoring antiviral treatment of influenza. Positive and negative predictive values of in vitro diagnostic tests are highly dependent on prevalence. False negative test results are more likely during peak activity when prevalence of disease is high. False positive results are more likely during periods of low influenza activity when prevalence is moderate to low. Individuals who have received nasally administered influenza A vaccine may test positive in commercially available influenza rapid diagnostic tests for up to three days after vaccination. Children tend to shed virus more abundantly and for longer periods of time than adults. Therefore, in vitro diagnostic tests for influenza may have lower sensitivity in adults than in children. Use of visibly bloody samples is not recommended with the BinaxNOW Influenza A & B Test. VIII. Expected Results The prevalence of influenza varies from year to year, with outbreaks typically occurring during the fall and winter months. 1 The rate of positivity found in influenza testing is dependent on many factors including the method of specimen collection, the test method used, geographic location, and the disease prevalence in specific localities. Type A viruses are typically CLSI Procedure Page 6 of 15

17 associated with most serious influenza epidemics, while Type B are typically milder. In multicenter clinical studies conducted by Binax outside the U.S. during the 2004 respiratory season and in the U.S. during the respiratory season, the average prevalence of influenza A (as determined by viral cell culture) was 18%. The average prevalence of influenza B was 3%. IX. Analytical Specificity and Cross Reactivity To determine the analytical specificity of the BinaxNOW Influenza A & B Test, 36 commensal and pathogenic microorganisms (27 bacteria, 8 viruses and 1 yeast) that may be present in the nasal cavity or nasopharynx were tested. All of the following microorganisms were negative when tested at concentrations ranging from 10 4 to 10 8 TCID 50 /ml (viruses), 10 7 to 10 8 organisms/ml (bacteria) and 10 6 organisms/ml (yeast). Bacteria Acinetobacter Bordetella pertussis Enterococcus faecalis Escherichia coli Gardnerella vaginalis Haemophilus influenzae Klebsiella pneumoniae Lactobacillus casei Legionella pneumophila Listeria monocytogenes Moraxella catarrhalis Neisseria gonorrhoeae Neisseria meningitidis Neisseria sicca Neisseria subflava Proteus vulgaris Pseudomonas aeruginosa Serratia marcescens Staphylococcus aureus Staphylococcus aureus (Cowan protein A producing strain) Staphylococcus epidermidis Streptococcus, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group F Streptococcus mutans Streptococcus pneumoniae Virus Adenovirus Coronavirus Coxsackie B4 Cytomegalovirus (CMV) Parainfluenza 1 Parainfluenza 2 Parainfluenza 3 Respiratory Syncytial Virus (RSV) Yeast Candida albicans CLSI Procedure Page 7 of 15

18 X. Analytical Reactivity The Influenza A and B strains listed tested positive in the BinaxNOW Influenza A & B Test at concentrations specified. Although the specific influenza strains causing infection in humans can vary year to year, all contain the conserved nucleoproteins targeted by the BinaxNOW test. 2 Performance characteristics of the BinaxNOW Influenza A & B Test for detecting influenza A virus from human specimens was established when H1 and H3 subtypes were prevalent. Performance characteristics of the test when other influenza A virus subtypes are emerging as human pathogens have not been established. Influenza Strain ATCC# Concentration Flu A/WS/33 (H1N1) VR CEID 50 /ml Flu A/NWS/33 (H1N1) VR CEID 50 /ml Flu A/Hong Kong/8/68 (H3N2) VR CEID 50 /ml Flu A/Aichi/2/68 (H3N2) VR CEID 50 /ml Flu A /New Jersey/8/76 (Hsw1N1) VR CEID 50 /ml Flu A/ Mal/302/54 (H1N1) VR CEID 50 /ml Flu A/Port Chalmers/1/73 (H3N2) VR CEID 50 /ml Flu A/ Hong Kong/156/97 (H5N1) x 10 2 TCID 50 /ml Flu A/ Vietnam/1194/04 (H5N1) x 10 4 TCID 50 /ml Flu A / Chicken/NY/ /01 (H5N2) x 10 4 EID 50 /ml Flu A/ Turkey/VA SEP-66/02 (H7N2) x 10 5 EID 50 /ml Flu B/Lee/40 VR CEID 50 /ml Flu B/Brigit VR CEID 50 /ml Flu B/Russia/69 VR CEID 50 /ml Flu B/Hong Kong/5/72 VR CEID 50 /ml Flu B/R75 VR CEID 50 /ml XI. Performance Characteristics The clinical performance of the BinaxNOW Influenza A & B Test was established in multicenter, prospective, clinical studies conducted at a central testing laboratory outside the US during the 2004 respiratory season and at three US trial sites during the respiratory season. Additional performance testing was conducted on retrospective frozen clinical samples collected from symptomatic patients at multiple physician offices, clinics and hospitals located in the Southern, Northeastern and Midwestern regions of the United States and from one hospital in Sweden. Clinical Studies: BinaxNOW Influenza A & B Test Performance vs. Cell Culture/ DFA- Prospective Study A total of 846 prospective specimens collected from children (less than 18 years of age) and adults (18 years or older) were evaluated in the BinaxNOW Influenza A & B Test and compared to culture/ DFA. Evaluated specimens include nasopharyngeal and nasal swabs collected from patients presenting with influenza-like symptoms. Forty-four percent (44%) of the population tested was male, 56% female, 54% pediatric (< 18 years), and 46% adult ( 18 years). No differences in test performance were observed based on patient age or gender. A/H3 and A/H1 were the predominant influenza subtypes observed during this time. BinaxNOW Influenza A & B Test performance by sample type versus cell culture/dfa, including 95% confidence intervals, is listed below. CLSI Procedure Page 8 of 15

19 BinaxNOW Influenza A & B Test Performance vs. Cell Culture/DFA for Detection of Flu A Test Sensitivity Test Specificity Sample +/+ - /+ %Sens 95% CI - /- +/- %Spec 95% CI NP Swab % 65-86% % % Nasal Swab % 74-90% % 93-98% Overall % 74-86% % 96-98% BinaxNOW Influenza A & B Test Performance vs. Cell Culture/DFA for Detection of Flu B Test Sensitivity Test Specificity Sample +/+ - /+ %Sens 95% CI - /- +/- %Spec 95% CI NP Swab % 9-91% % % Nasal Swab % 39-90% % % Overall % 39-85% % % BinaxNOW Influenza A & B Test Performance vs. Cell Culture/ DFA- Retrospective Study A total of 293 retrospective frozen clinical samples were evaluated in the BinaxNOW Influenza A & B Test and compared to culture/ DFA. All clinical samples were collected from symptomatic patients at multiple physician offices, clinics and hospitals located in the Southern, Northeastern and Midwestern regions of the United States and from one hospital in Sweden. Fifty-three percent (53%) of the population tested was male, 47% female, 62% pediatric (<18 years) and 38% adult ( 18 years). Nasal wash/aspirate specimens comprised approximately 61% of the samples tested, while NP swabs represented 39%. No differences in test performance were observed based on patient age and gender or based on sample type tested. BinaxNOW A & B Test performance by sample type versus cell culture/ DFA, including 95% confidence intervals, is listed below. BinaxNOW Influenza A & B Test Performance vs. Cell Culture/ DFA for Detection of Flu A Test Sensitivity Test Specificity Sample +/+ - /+ %Sens 95% CI - /- +/- %Spec 95% CI NP Swab % 50-86% % 81-95% Wash/Aspirate % 78-96% % 89-98% Overall % 73-90% % 88-96% CLSI Procedure Page 9 of 15

20 BinaxNOW Influenza A & B Test Performance vs. Cell Culture/ DFA for Detection of Flu B Test Sensitivity Test Specificity Sample +/+ - /+ %Sens 95% CI - /- +/- %Spec 95% CI NP Swab 0 0 N/A N/A % % Wash/Aspirate % 27-78% % 89-97% Overall % 27-78% % 92-98% Analytical Sensitivity The BinaxNOW Test limit of detection (LOD), defined as the concentration of influenza virus that produces positive BinaxNOW test results approximately 95% of the time, was identified by evaluating different concentrations of inactivated Flu A/Beijing and inactivated Flu B/Harbin in the BinaxNOW Test. Twelve (12) different operators each interpreted 2 tests run at each concentration for a total of 24 determinations per level. The following results identify a concentration of 1.03 x 10 2 ng/ml as the LOD for Flu A/Beijing and 6.05 x 10 1 ng/ml for Flu B/Harbin. Concentration (ng/ml) Flu A/Beijing # Detected % Detected 1.03 x 10 2 (LOD) 23/ x 10 1 (Cutoff) * x 10 1 (High Neg) 4/24 17 True Negative 0/24 0 Concentration (ng/ml) Flu B/Harbin # Detected % Detected 6.05 x 10 1 (LOD) 23/ x 10 1 (Cutoff) 11/ x 10 1 (High Neg) 6/24 25 True Negative 0/24 0 *Linear regression was used to calculate a line equation, which was then used to project the cutoff concentration of Flu A/Beijing. XII. Reproducibility A blind study of the BinaxNOW Influenza A & B Test was conducted at 3 separate sites using panels of blind coded specimens containing negative, low positive, and moderate positive samples. Participants tested each sample multiple times on 3 different days. There was 97% (242/250) agreement with expected test results, with no significant differences within run (replicates tested by one operator), between run (3 different days), between sites (3 sites), or between operators (6 operators). CLSI Procedure Page 10 of 15

21 XIII. Consumer Precision Studies: Swab Specimen The BinaxNOW Influenza A & B Test was evaluated by twelve (12) adults with no professional laboratory experience (intended user) at three (3) intended use sites. Each operator at each site tested thirty-five (35) samples from a randomly coded panel of true positive, true negative, limit of detection (LOD) and below LOD for both influenza A and influenza B. In order to demonstrate equivalent performance among intended users and trained laboratorians, twenty-four (24) trained laboratorians, ran blind coded panels of Flu A and Flu B samples containing the same true negative, true positive, below LOD, and LOD samples described above. As indicated by the overlapping 95% confidence intervals in the tables below, no significant differences were observed between the intended users and the expected results established by the trained laboratorians. These results demonstrate that users with no formal laboratory training can read the package insert and perform the Binax test with the same precision as trained laboratorians. Influenza A and Influenza B Sample Testing Intended Users vs. Trained Laboratorians Overall Results Participant Type Negative - % Negative Below LOD - % Detection LOD - % Detection True Positive % Detection % Invalid Tests Flu A Samples Intended User Trained Laboratorian 97% (57/59) (88-99) 99%(179/180) (97-100) 79% (46/58) (67-88) 84%(102/121) (77-90) 95% (59/62) (87-98) 99%(179/180) (97-100) 100% (60/60) (94-100) 100%(120/120) (97-100) 1.6% (4/243) 0% (0/601) Flu B Samples Intended User Trained Laboratorian 72% (43/60) (59-81) 78% (93/120) (69-84) 97% (59/61) (89-99) 99% (179/180) (97-100) 98% (58/59) (91-100) 100%(120/120) (97-100).6% (1/181) 0% (0/420) CLSI Procedure Page 11 of 15

22 Influenza A Sample Testing by Site Intended Users (IU) and Trained Laboratorians (TL) Site# Negative % Negative Below LOD % Detection LOD % Detection True Positive % Detection % Invalid Tests 1 IU 89% (17/19) (68-97) 63% (12/19) (41-84) 100% (20/20) (84-100) 100% (20/20) (84-100) 2.5% (2/80) Intended User (IU) Results 2 IU 100% (20/20) (84-100) 79% (15/19) (56-91) 90% (19/21) (71-97) 100% (20/20) (84-100) 2.4% (2/82) 3 IU 100% (20/20) ( ) 95%(19/20) (76-99) 95% (20/21) (77-99) 100% (20/20) (84-100) 0% ( 0/81) 1 TL 100% (25/25) (87-100) 64% (16/25) (44-80) 100% (25/25) (87-100) 100% (25/25) (87-100) 0% (0/100) Trained Laboratorian (TL) Results 2 TL 3 TL 100% (15/15) (79-100) 100% (20/20) (84-100) 87% (13/15) (62-96) 75% (15/20) (53-89) 100% (15/15) (79-100) 100% (20/20) (84-100) 100% (15/15) (79-100) 100% (20/20) (84-100) 0% (0/60) 0% ( 0/80) 4 TL 99% (119/120) (95-100) 95% (58/61) (87-98) 99% ( ) (95-100) 100% (60/60) (94-100) 0% (0/361) Influenza B Sample Testing by Site Intended Users (IU) and Trained Laboratorians (TL) Site# Below LOD % Detection LOD %Detection True Positive % Detection % Invalid Tests 1 IU 70% (14/20) (48-85) 95% (19/20) (76-99) 100% (19/19) (83-100) 1.7% (1/60) Intended User (IU) Results 2 IU 65% (13/20) (43-82) 95% (20/21) (77-99) 100% (20/20) (84-100) 0% (0/61) 3 IU 80% (16/20) (58-92) 100% (20/20) (84-100) 95% (19/20) (76-99) 0% ( 0/60) 1 TL 60% (15/25) (41-77) 100% (25/25) (87-100) 100% (25/25) (87-100) 0% (0/75) Trained Laboratorian (TL) Results 2 TL 3 TL 73% (11/15) (48-89) 65% (13/20) (43-82) 100% (15/15) (79-100) 100% (20/20) (84-100) 100% (15/15) (79-100) 100% (20/20) (84-100) 0% (0/45) 0% ( 0/60) 4 TL 90% (54/60) (80-95) 99% (119/120) (95-100) 100% (60/60) (94-100) 0% (0/240) CLSI Procedure Page 12 of 15

23 Liquid Specimen According to the 1995 CLIA Rule, Inverness Medical conducted their Consumer Precision testing of the BinaxNOW Influenza A & B Test with a total of 120 lay users at 6 sites. Participants tested proficiency panels consisting of high negative, assay cutoff, and limit of detection (LOD) samples for both influenza A and influenza B, as well as true negative samples. Expected results for each sample type were generated by trained laboratorians. Six percent (6%) of the total tests run by the lay users and 0.4% of the test run by the trained laboratorians resulted in invalid tests. The tables below detail the number of invalid tests generated by each site. As indicated by the overlapping 95% confidence intervals in the tables below, no significant differences were observed between the lay users and the expected results established by the trained laboratorians. These results demonstrate that users with no formal laboratory training can read the package insert and perform the Inverness Medical tests with a relatively high level of precision. Influenza A and Influenza B Sample Testing Lay Users vs. Trained Laboratorians Overall Results Participant Type Negative: % Negative High Negative*: % Detection Assay Cutoff*: % Detection LOD: % Detection % Invalid Tests Flu A Samples Lay User Trained Laboratorian 96% (54/56) (88-99) 100% (8/8) (66-100) 32% (18/57) (21-44) 22%(8/36) (12-38) 78% (46/59) (66-87) 64% (23/36) (47-77) 95% (57/60) (86-98) 94% (34/36) (82-98) 3.3% (8/240) 0.9% (1/117) Flu B Samples Lay User Trained Laboratorian 96% (53/55) (88-99) 100% (9/9) (69-100) 4% (2/52) (1-13) 11% (4/36) (4-25) 27% (15/56) (17-40) 49% (17/35) (33-64) 82% (45/55) (70-90) 92% (33/36) (78-97) 9.2% (22/240) 0.0% (0/116) * These levels are below the detection limit of the test. Warning: Invalid test results can occur when an insufficient volume of sample is added to the test device due to misuse of the transfer pipette. Please see the Test Procedure section for detailed instructions on the proper use of the transfer pipette. Influenza A Sample Testing by Site Lay Users (LU) and Trained Laboratorians (TL) Site# Negative: % Negative High Negative: % Detection Assay Cutoff: % Detection LOD: % Detection % Invalid Tests Lay User (LU) Results 2 LU 4 LU 100% (18/18) (82-100) 90% (18/20) (70/97) 33% (6/18) (16-57) 45% (9/20) (26-66) 70% (14/20) (48-85) 85% (17/20) (64-94) 90% (18/20) (70-97) 100% (20/20) (84-100) 5.0% (4/80) 0% (0/80) 6 LU 100% (18/18) (82-100) 16%(3/19) (6-38) 79% (15/19) (56-91) 95% (19/20) (76-99) 5.0% (4/80) Trained Laboratorian (TL) Results 1 TL 2 TL 100% (3/3) (40-99) 100% (2/2) (29-99) 0% (0/12) (0-25) 33% (4/12) (14-61) 25% (3/12) (9-54) 92% (11/12) (64-98) 100% (12/12) (75-100) 83% (10/12) (54-95) 0% (0/39) 3% (1/39) 3 TL 100% (3/3) (40-99) 33% (4/12) (14-61) 75% (9/12) (46-90) 100% (12/12) (75-100) 0% (0/39) CLSI Procedure Page 13 of 15

24 Influenza B Sample Testing by Site Lay Users (LU) and Trained Laboratorians (TL) Site# Negative: % Negative 1 LU 100% (20/20) ( Lay User 3 LU 100% (19/19) (LU) (83/100) Results 5 LU 87% (14/16) (63-96) High Negative: % Detection 5% (1/19) (1-25) 0% (0/18) (0-18) 7%(1/15) (2-30) Assay Cutoff: % Detection 25% (5/20) (11-47) 25% (5/20) (11-47) 31% (5/16) (14-56) LOD: % Detection 85% (17/20) (64-94) 79% (15/19) (56-91) 81% (13/16) (57-93) % Invalid Tests 1% (1/80) 5% (4/80) 21% (17/80) Trained Laboratoria n (TL) 1 TL 100% (3/3) (40-99) 2 TL 100% (3/3) (40-99) Results 3 TL 100% (3/3) (40-99) 0% (0/12) (0-25) 33% (4/12) (14-61) 0% (0/12) (0-25) 9% (1/11) (21-38) 67% (8/12) (39-86) 67% (8/12) (39-86) 75% (9/12) (46-91) 100% (12/12) (75-100) 100% (12/12) (75-100) 0% (0/38) 0% (0/39) 0% (0/39) XIV. References 1. Williams, KM, Jackson MA, Hamilton M. (2002) Rapid Diagnostic Testing for URIs in Children: Impact on Physician Decision Making and Cost. Infect. Med. 19(3): Dowdle, W.R, Kendal, A.P., and Noble, G.R Influenza Virus, p Manual of Clinical Microbiology, 3 rd edition, in Lennette, et. al (ed.). American Society for Microbiology, Washington, D.C. 3. Key facts about Avian Influenza (Bird Flu) and Avian Influenza A (H5NI) virus CDC Publication, May 24, Avian Influenza Infection in Humans CDC Publication. May 24, Updated Interim Guidance for Laboratory Testing of Persons with Suspected Infection with Avian Influenza A (H5N1) Virus in the United States CDC Health Alert, June 7, BinaxNOW Influenza A & B Package Insert CLIA Waived All trademarks referenced are trademarks of their respective owners. All rights reserved. CLSI Procedure Page 14 of 15

25 Test Procedure Review Supervisor Date Reviewed Supervisor Date Reviewed CLSI Procedure Page 15 of 15

26 BinaxNOW Influenza A & B Competency Assessment A Word about Personnel Competency Assessment According to the CLIA 88 Rules and Regulations, laboratories possessing a certificate of waiver are only required to: Enroll in the CLIA program. Pay applicable certificate fees biennially. Perform only tests that have been classified as waived by the FDA or CDC. Follow manufacturers test instructions. Currently, there are no personnel requirements that must be met by certificate of waiver laboratories. However, certificate of waiver laboratories are expected to follow good laboratory practices. Good Laboratory Practice Good laboratory practices are a set of procedures that span all phases of testing (preanalytical, analytical and post-analytical) and are employed by a laboratory for the purpose of achieving accurate and timely test results. All laboratories, regardless of complexity, are recommended to follow these practices. At a minimum, laboratories are recommended to have: 1. A procedure manual that contains procedures for all tests performed in the laboratory. 2. A system to train all employees and ensure their competence to perform laboratory tests. 3. A system to ensure that patient preparation and specimen collection, identification, handling, and storage policies and procedures are in place and adequately followed. 4. A system to ensure that all paperwork associated with a patient s test is handled in a manner to facilitate the timely reporting of results, including panic values, confidentiality of results, and storage of the results. 5. General policies and procedures written and followed regarding laboratory safety including the use of Universal Precautions and the prohibition of eating, drinking, or storage of food in the laboratory. 6. A system designed to oversee the storage of kits, reagents, and controls as required by the manufacturer and to ensure that when outdated they are properly disposed. 7. A system developed to monitor the use of, and interpret the results of, either the manufacturer s built-in controls or external quality control material to ensure that the test system is operating correctly. 1 Competency Assessment Although not a requirement per CLIA law, evaluation of competency of testing personnel is an important part of good laboratory practice. Providing regular training, appropriate education, and evaluating and maintaining competency of testing personnel is essential to achieving accurate test results. Evaluating and documenting the performance of the individuals responsible for testing can be semi-annually during the first year the individual tests patient specimens. Thereafter, annual evaluations are sufficient unless test methodology or instrumentation changes, in which case, prior to reporting patient test results, the individual's performance can be re-evaluated to include the use of the new test methodology or instrumentation. Competency Assessment Page 1 of 5

27 The procedures for evaluating the competency of the staff can include but are not limited to: 1. Direct observations of routine patient test performance, including patient preparation, if applicable, specimen handling, processing and testing. 2. Monitoring the recording and reporting of test results. 3. Review of intermediate test results or worksheets, quality control records, proficiency testing results, and preventive maintenance records. 4. Direct observation of performance of instrument maintenance and function checks. 5. Assessment of test performance through testing previously analyzed specimens, internal blind testing samples or external proficiency testing samples. 6. Assessment of problem solving skills. In addition, evaluation of competency may also include a written quiz. Competency Assessment Tools As an Inverness Medical customer you are provided with the following to help maintain and evaluate testing personnel competence: The Training Certificate in the CLIA packet can be used as documentation that appropriate training has been provided to all testing personnel for the BinaxNOW Influenza A and B test. We have provided you with a written quiz that can be administered to all testing personnel as part of their competency assessment. A Competency Assessment Checklist has been created that can be used to verify and document that all areas of competency for the BinaxNOW Influenza A and B test have been evaluated. 1. COLA LabGuide, General Information on Waived Tests, 11-1, 4/9/97. Attachments: Competency Assessment Answer Key Competency Assessment Personnel Checklist Competency Assessment Quiz Competency Assessment Page 2 of 5

28 BinaxNOW Influenza A & B Quiz Answer Key 1. F The BinaxNOW Influenza A & B kit may be stored at room temperature. 2. T The BinaxNOW Influenza A & B test device should be left in the foil pouch until just before use. 3. F Nasal Wash/Aspirate and Nasopharyngeal or Nasal swabs are the acceptable sample types for the Influenza A & B BinaxNOW kit. Throat swabs are unacceptable. 4. F Nasal wash samples should be run as soon as possible or may be refrigerated for up to 24 hours. 5. T Transport media may be used to elute a nasopharyngeal swab or a nasal swab. Refer to the Specimen Collection and Handling Section for acceptable media. 6. F Nasopharyngeal swab must be eluted within 1 hour of collection. 7. F Sample should be added SLOWLY to the MIDDLE of the White Sample Pad such that all of the sample volume absorbs into the pad. 8. T BinaxNOW Influenza A & B test results should be read at 15 minutes. Results read before or after 15 minutes may be inaccurate. 9. F The Control Line must change from a blue color to a pink-to-purple color in order for the test result to be valid. 10. T A positive BinaxNOW Influenza A & B test will have a pink-to-purple control line and a second pink-to-purple sample line appears above it. Competency Assessment Page 3 of 5

29 Testing Personnel Competency Assessment Employee: Date: Test Method: BinaxNOW Influenza A & B Test Procedure Satisfactory Unsatisfactory Observation of Test Performance: Patient Preparation (if applicable) Specimen Handling/Processing Testing Recording/Reporting Results Assessment of Test Performance Using Known Samples Review of Records: Patient/Quality Control Log Sheet Records Proficiency Testing Records Assessment of Problem Solving Skills (Attach all supporting documents) Not Applicable Comments/Corrective Actions Evaluator: Date: Employee: Date: Competency Assessment Page 5 of 5

30 BinaxNOW Influenza A & B Quiz Name: Date: Circle T (True) or F (False) for each Question: 1. The BinaxNOW Influenza A& B test kit requires refrigeration at 2 to 8 C. T F 2. The BinaxNOW Influenza A & B test device should not be removed from the foil pouch until just before use. T F 3. Throat swabs are an acceptable sample type for testing on the BinaxNOW Influenza A & B test. T F 4. Nasal Wash samples may be stored at room temperature for 24 hours. T F 5. Transport media may be used with the BinaxNOW Influenza A & B test. T F 6. NP swabs may be stored at room temperature for 24 hours before elution. T F 7. Sample should be added quickly to the top of the white sample pad. T F 8. Test Results should be read at 15 minutes. T F 9. If the Control Line remains BLUE in color the test results are valid. T F 10. The appearance of a pink-to-purple Control Line and a pink-to-purple Sample Line is a positive result. T F Reviewed By: Date: Competency Assessment Page 5 of 5

31 Material Safety Data Sheet BinaxNOW Influenza A&B Test Kit Cat. # , , , 41603J, J Nasopharyngeal Swab Specimen Accessory Pack Cat. # Control Swab Accessory Pack Cat. # THE FOLLOWING INFORMATION PROVIDED IS BELIEVED TO BE CORRECT AND ACCURATE. IT DOES NOT PURPORT TO BE ALL-INCLUSIVE AND SHALL BE USED AS A GUIDE. BINAX, INC. SHALL NOT BE HELD LIABLE FOR ANY DAMAGE RESULTING FROM HANDLING, CONTACT WITH, OR MIS-USE OF THIS PRODUCT. Section I. General Information Chemical Name & Synonyms: NOT APPLICABLE Trade Name & Catalogue Number: BinaxNOW Influenza A&B Test Catalogue Numbers , , BinaxNOW Nasopharyngeal Swab Specimen Accessory Pack Catalogue Number BinaxNOW Control Swab Accessory Pack Catalogue Number Chemical Family: In Vitro Diagnostic Test Kits Formula: NOT APPLICABLE Proper DOT Shipping Name: NOT APPLICABLE DOT Hazard Classification: NOT APPLICABLE Contact: Binax, Inc. Phone: US: Southgate Road Outside US: Scarborough, Maine Fax: Poison Control Center: Section II Hazardous Ingredients/Identity Information Item Percent (optional) Exposure Limits Elution Solution Detergent NIOSH REL: none OSHA PEL: none Saline Solution Preservative Positive Control Swabs Inactivated Flu A Inactivated Flu B Negative Control Swabs Inactivated Streptococcus Group A NIOSH REL: none OSHA PEL: none NIOSH REL: none OSHA PEL: none NOT APPLICABLE NOT APPLICABLE NOT APPLICABLE Section III Physical Data Boiling Point ( C): Detergent: undetermined Saline Solution (NaCl): 1413C Preservative: 189C Vapor Pressure (mm Hg): Detergent: N/A Saline Solution (NaCl): N/A Preservative: 0.06 Vapor Density (Air = 1.0): N/A Appearance: Packaged kit with ampoules, test swabs, and test device. Specific Gravity (water = 1.0): Detergent: Saline Solution (NaCl): 2.16 Preservative: 1.03 Percent volatile by Volume (%):N/A Detergent: N/A Saline Solution (NaCl): 0 Preservative: <95 Evaporation Rate: N/A Odor: None known M-013, Rev 2, 4/24/06 Author: Lisa Irish 1 of 3

32 Section IV. Health Hazard Data Detergent Route(s) of entry: skin? Yes Inhalation? Yes Ingestion? Yes Health hazards: Acute: Irritating to eyes, respiratory system and skin. Chronic: None known Symptoms of exposure: Irritating to eyes, respiratory system and skin Carcinogenicity: NTP? Not listed IARC listed? Yes OSHA Regulated? Not listed Medical conditions aggravated by exposure: None known Emergency and first aid procedures: ALWAYS CALL A PHYSICIAN OR POISON CONTROL CENTER Skin: flush with water for at least 15 minutes. Eyes: flush with copious amounts of water for at least 15 minutes, if symptoms persist, consult physician. Ingestion: Call physician immediately. Inhalation: remove to fresh air. If not breathing give artificial respiration. If breathing is difficult, give oxygen. Saline Solution Route(s) of entry: skin? Yes Inhalation? Yes Ingestion? Yes Health hazards: Acute: May irritate damaged skin, absorption can occur with effects similar to those via ingestion. May cause mild irritation to the respiratory tract. Chronic: None known. Symptoms of exposure: Very large doses can cause vomiting, diarrhea, and prostration. Dehydration and congestion occur in most internal organs. Hypertonic salt solutions can produce violent inflammatory reactions in the gastrointestinal tract. Carcinogenicity: NTP? Not listed IARC listed? Yes OSHA Regulated? Not listed Medical conditions aggravated by ACGIH Carcinogen? Rating A4 exposure: Emergency and first aid procedures: ALWAYS CALL A PHYSICIAN OR POISON CONTROL CENTER Eye: Immediately flush eyes with plenty of water for at least 15 minutes, lifting upper and lower eyelids occasionally. Get medical attention if irritation persists. Skin: Wash exposed area with soap and water. Get medical advice if irritation develops. Inhalation: Remove to fresh air. Get medical attention for any breathing difficulty. Ingestion: If large amounts were swallowed, give water to drink and get medical advice. Preservative Route(s) of entry: skin? Yes Inhalation? Yes Ingestion? Yes Health hazards: Acute: Causes burns, corrosive. May be harmful if inhaled. Material is extremely destructive to the tissue of the mucous membranes and upper Chronic: None known. Symptoms of exposure: May include burning sensation, coughing, wheezing, laryngitis, shortness of breath, headache, nausea and vomiting respiratory tract. Carcinogenicity: NTP? Not listed IARC Monograph? Not Listed OSHA Regulated? Yes. Medical conditions aggravated by exposure: None known. Emergency and first aid procedures: ALWAYS CALL A PHYSICIAN OR POISON CONTROL CENTER Skin: Flush with copious amounts of water for at least 15 minutes. Remove contaminated clothing. Eyes: Immediately flush with copious amounts of water for at least 15 minutes. Inhalation: Remove to fresh air. If not breathing give artificial respiration. If breathing is difficult give oxygen. Ingestion: Wash out mouth with water provided person is conscious. M-013, Rev 2, 4/24/06 Author: Lisa Irish 2 of 3