FOLLOWING MANUFACTURER S INSTRUCTIONS COURSE INSTRUCTIONS

Transcription

1 STATE OF WEST VIRGINIA DEPARTMENT OF HEALTH AND HUMAN RESOURCES Joe Manchin III Governor Patsy A. Hardy, FACHE, MSN, MBA Cabinet Secretary FOLLOWING MANUFACTURER S INSTRUCTIONS COURSE INSTRUCTIONS 1. If you have not already registered and paid for the course, you must complete the registration form at the end of the chapter exams. Submit the form with your payment and the completed exams. 2. Write your name in the designated area of each chapter exam. Unidentified exams will not be graded. 3. The exams are generally made up of four sections. Follow the instructions given in each section: A. Multiple Choice C. Matching B. True or False D. Short Answer 4. Complete all questions of the examination. Participants are reminded that this is an individual self-study course. Taking the examination is not meant to be a group effort. Credits are based on individual merit. Therefore, participants will be on the honor system when completing this examination. Group study can be done after the exams are graded and returned. 5. The Training and Evaluation Section has no regulated deadline. To avoid urgent requests and to allow you to meet any program deadlines for which you may be planning to use the CEUs, we ask that you return completed exams in plenty of time to allow for grading. Return completed exams to: West Virginia Office of Laboratory Services Training & Evaluation Section th Avenue South Charleston WV The exams for this course are in two sections: exam one will cover material in the course text; exam two will cover material in the manufacturer s insert. Thank you for your cooperation. BUREAU FOR PUBLIC HEALTH OFFICE OF LABORATORY SERVICES th Avenue South Charleston, West Virginia Phone: (304) FAX: (304)

2 FOLLOWING MANUFACTURER S INSTRUCTIONS COURSE OUTLINE Section Topic Page 1 Introduction and Definitions 1 2 Sections of a Manufacturers Insert 8 3 Course References 32 4 P-Strips Package Insert 33 Exams 35 Any product names used in this course does not constitute an endorsement from this agency. All text and illustrations are copyright of their respective owners and have been adapted to fit this course. The information is used for educational purposes only.

3 The Importance of FOLLOWING MANUFACTURER S INSTRUCTIONS One of the most frequently cited laboratory deficiencies of physician office and point-of-care testing is failure to follow manufacturer s instructions. Requirements such as quality control, specimen collection, reagent storage temperatures, along with many other important testing details are contained within the manufacturer s instructions. Regardless of the test complexity under the Clinical Laboratory Improvement Amendments (CLIA-88) regulations, a test system is approved by the Food and Drug Administration (FDA) for its test category with the assumption that the test user is following the manufacturer s instructions. Any deviations could lead to erroneous patient test results. This is why CLIA-88 regulations will reclassify a waived or moderate complexity test to the high complexity category if manufacturer s instructions are deviated in any way. Manufacturers provide written instructions with every reagent lot or control in the form of a package insert. Often, package inserts are discarded without being read; quality control is not being followed as written and testing personnel do not know the location of the instrument manuals. This section will focus on the importance of manufacturer s instructions. Package inserts will be reviewed and explained in detail. Manufacturer package inserts and instrument manuals follow the same general format as specified by the FDA. In most cases, they are written so well that CLIA-88 regulations will permit the laboratory to use these instructions as the test procedure. Be aware that if the manufacturer s manuals and package inserts are used in lieu of a formally written procedure manual, they must be signed and dated as approved by the director. If any changes occur from the original, the changes must be signed and dated as well. Some supplemental material pertaining to the specific policies of the work site must be added to each manufacturer s instructions to make a complete procedure manual. WVOLS T&E 2010 FMI Page 1

4 Written, site-specific information to be used in addition to the manufacturer s instructions include: o o o o o Specific instructions for reporting test results Procedure for notification of erroneous results to the physician or referring health facility Assigning panic values for all tests Written protocols for a backup system in the event a test system becomes inoperable or quality control fails Name and location of the testing laboratory should appear at the top of each procedure Definitions of terms used in this course: acceptable range control accuracy analysis analyte analytic antibody anticoagulant antigen antiserum assayed values The range of results that indicate adequate performance when analyzing a control sample. The range is shown in the package insert accompanying the control. Correctness; freedom from error. The extent to which measurements agree with the true value of the quantity being measured. The laboratory procedure that enables you to measure the amount of an analyte in a specimen. The substance or constituent being measured, e.g. glucose. The phase of testing which is the actual step-by-step performance of the test and quality control. A body of substance, usually in serum and other body fluids, that specifically counteracts the effects of a foreign substance or organism (as a disease-producing microorganism introduced into the body). Agent which prevents blood coagulation. A substance (includes proteins, carbohydrates, and microorganisms) that when introduced into the body stimulates the production of an antibody, specific to that antigen. Serum containing antibodies. The measurement of the amount of a constituent in a specimen; a test. WVOLS T&E 2010 FMI Page 2

5 calibration CLIA, CLIA-67, or CLIA-88 control corrective action critical limits (panic values) cross reaction The process by which readings obtained from an instrument or other measuring device is related to known concentrations of an analyte. Clinical Laboratory Improvement Amendments. Regulations established by the federal government in 1967 and 1988 to improve quality of laboratory testing. A material, solution, or lyophilized preparation having known value or content used in quality control. Action taken when quality control tests or instrument calibration are beyond an acceptable range. Test results of a patient s specimen that are dangerously low or high; use for emergency notification of clinicians. Every laboratory should establish its own panic values. The interaction of an antigen with an antibody formed against a different antigen with which the first antigen shares identical or closely related antigenic determinants. Tests with low specificity and high sensitivity are subject to more cross reactivity. deficiency Noncompliance to a standard. (CLIA or other regulatory agency) diagnostic laboratory tests diluent distilled water expiration date false positive (result) false negative (result) FDA Laboratory tests which are used for the basis of diagnosing disease (along with symptoms). Diagnostic tests are specific for the disease state and usually do not require further confirmatory tests. Substance used to dilute a concentrated or freeze-dried reagent or control - usually distilled water or saline. The condensate collected when water has been boiled (distilled) to remove impurities. The last day a reagent, kit, control, etc., can be used; the expiration date of the unopened container is printed on the product by the manufacturer. If the expiration date changes when the product is opened, the laboratorian must record the new expiration date on the product. If this date is given as month/year, the product expires on the last day of that month. A positive test result for a patient who is negative for the constituent or condition being tested for. A negative test result for a patient who is positive for the constituent or condition being tested for. Food and Drug Administration. The branch of the federal government which regulates laboratory test systems and kits, and classifies tests in CLIA-88 complexity categories. WVOLS T&E 2010 FMI Page 3

6 hemolysis high complexity laboratory immunoassay in-control in-vitro incubation kit The destruction of red blood cells resulting in the liberation of hemoglobin from the cells. The serum or plasma appears pink to red in color. The category of CLIA regulated laboratories which performs tests of the highest technical difficulty as defined by the FDA. High complexity laboratories are subject to inspection and the most stringent regulatory requirements. A quantitative determination of antigenic substances (proteins, microorganisms, hormones, drugs) or antibody by serological means. Term used to describe the testing procedure when the results from a control sample or series of control samples are within the acceptable control range. Testing conducted outside of the human body in a test tube, test kit, cartridge, or on a slide. Placing in an optimal test environment such as proper temperature or humidity for growth of microorganisms or a chemical or biological reaction to occur. All materials packaged together, needed to perform a test. laboratory A facility for the virological, microbiological, serological, chemical. immunochematological, hematological, biophysical, cytological, pathological, or other examination providing information for the diagnosis, prevention, or treatment of human beings. These examinations also include procedures to determine, measure, or otherwise describe the presence or absence of various substances or organisms in the body. Facilities only collecting or preparing specimens (or both) or only serving as a mailing service and not performing testing are not considered laboratories. laboratory (CLIA-88 definition) lipemia A facility for the biological and chemical examination of materials derived from the human body for purposes of providing information for the diagnosis, prevention, or treatment of disease or the assessment of health of human beings. Condition of excess fat in the blood manifested by a milky appearance of blood serum or plasma. lot number (control number) The number given to a batch of a product by the manufacturer. mean The average of the numerical results obtained from a series of analyses. WVOLS T&E 2010 FMI Page 4

7 method Analytical procedure; the instructions including procedures, material, equipment, and everything else needed for an analyst to perform an analysis. moderate complexity laboratory The category of CLIA regulated laboratories which are subject to inspection, testing, and personnel requirements more stringent than for waived laboratories. NBS thermometer National Bureau of Standards thermometer; readings of thermometers used in laboratory instruments must be compared to reading with NBS thermometer. NCCLS National Committee for Clinical Laboratory Standards; committee of experts that develops and publishes standards for laboratory procedures. NIST Certified Thermometer Laboratory grade thermometers which meet certain specifications for accuracy, design, and performance. They are calibrated against NIST traceable equipment. A certificate is provided with each thermometer indicating traceability to NIST standards and serial number. This type of thermometer should be used for monitoring temperatures in a clinical laboratory. normal values (expected values, reference values) A range of values established for each analyte that includes the results expected when performing a test on a healthy person. OSHA Occupational Safety and Health Administration. The branch of the Federal Department of Labor which regulates employee workplace safety. Bloodborne Pathogens and Chemical Hygiene are OSHA regulations particularly affecting laboratories. out-of-control Term used to describe the testing procedure when the result from a control sample is outside the acceptable control range. package insert or product insert Information material that comes with instruments, reagents, and other laboratory products giving instructions for the use of the product and other information required of the manufacturer by the US Food and Drug Administration. panic values Test results that fall outside of the low or high critical limit for the particular test in question requiring URGENT notification of the appropriate medical personnel. WVOLS T&E 2010 FMI Page 5

8 physician performed microscopy procedures (PPMP) A subcategory of moderate complexity testing in which certain microscopic tests are performed by the clinicians only. This category of laboratory is not subject to a CLIA inspection but must follow moderate complexity requirements. 1. The liquid part of the blood in which the cellular elements are suspended. plasma point-of-care tests post-analytic 2. The clear liquid that separates from whole blood when the blood is collected in a tube containing anticoagulant (purple top tube). This portion contains particulate matter, clotting factors, and fibrinogen. Laboratory testing performed in a nontraditional clinical laboratory setting such as at the patient s hospital unit. Laboratory testing for direct patient care in a physician office is often included with the definition. The phase of laboratory testing which involves reporting and interpretation of the test result. pre-analytic Phase of laboratory testing which includes test selection, specimen collection, transport, and handling. precision (reproducibility) predictive value procedure manual qualitative quality assurance The measure of the closeness of the results obtained when analyzing the same sample more than once; the measure of agreement between replicate measurements. The smaller the variation between results, the greater the precision. The probability of the presence or absence of disease given the results of a test. A laboratory manual that contains the methods, materials, and other information needed to do a test. A term applied to tests that detect whether a particular analyte, constituent, or condition is present. A comprehensive set of policies, procedures, and practices necessary to make sure that the laboratory s results are reliable. QA includes recordkeeping, calibration and maintenance of equipment, quality control, proficiency testing, and training. quality control The set of laboratory procedures designed to ensure that the test method is working properly and that the results meet the diagnosis needs of the physician. QC includes testing control samples, charting results, and analyzing them statistically. WVOLS T&E 2010 FMI Page 6

9 quantitative reagent reagent strip reconstitute reliability result sample A term applied to tests that give results expressing the numerical amount of an analyte in a specimen. A substance that produces a chemical reaction in a sample, allowing an analyte to be detected and measured. A plastic strip to which are affixed test reagents. Also referred to as dipsticks. Reagent strips are immersed briefly in blood or other body fluids for analysis. To add a diluent to a freeze-dried calibrator, control, or reagent. The method s capacity to maintain both accuracy and precision. The value obtained by analysis for a particular analyte in a particular sample. The part of a specimen that is used for an analysis. screening laboratory tests sensitivity serological test serum Standard (CLIA) titer waived complexity laboratory A laboratory test which is used for mass examination of the population to detect the existence of a particular disease or condition. Screening tests are highly sensitive but require more specific follow up confirmatory or diagnostic tests. The ability of a test to give a positive result for patients that have the disease or condition they are tested for; measured as the ratio of positive tests to the total number of tests in those that have the disease, expressed as a percentage. Laboratory testing which requires antigen-antibody reactions in-vitro. The cell-free portion of the blood from which the fibrinogen has been separated in the process of clotting. Specific requirements of regulations. Each requirement is assigned a D-Tag (Deficiency) number. The highest dilution of a serum which causes clumping of microorganisms or antigens, or otherwise gives a positive reaction. The lesser of the three categories of laboratories under the CLIA-88 regulations. Waived laboratories employ test methodologies that are simple to perform so as to render the likelihood of erroneous results negligible or are methodologies approved for home use. Except for complaints or random monitoring, a waived laboratory is not subject to inspection. There are no formal education requirements for personnel. WVOLS T&E 2010 FMI Page 7

10 SECTIONS OF A MANUFACTURERS PACKAGE INSERT Manufacturers provide written instructions with every reagent lot or control that is produced. These package inserts follow the same general format: 1. Title (Heading) 2. Summary, Explanation, and Intended Use 3. Principles of the Test 4. Reagents and Materials 5. Safety Precautions 6. Reagent Storage, Handling, and Preparation 7. Specimen Collection and Handling 8. Test Procedure 9. Quality Control 10. Interpretation of Results (Expected Values) 11. Limitations of Procedure 12. Specific Performance Characteristics 13. References or Bibliography 14. Revision Date The information contained within each section of a package insert will be reviewed and explained in detail. Do not discard the literature without reading it. Remember from the introduction: CLIA regulations reclassify a waived or moderate complexity test to the high complexity category if the manufacturer s instructions are deviated in any way. WVOLS T&E 2010 FMI Page 8

11 1. Title (Heading) This section may include any or all of the following: Exact name of the test US license number Manufacturer s name (possibly address and telephone number) Number of the tests per kit Catalog number for ordering CPT code Instrument or test system for which the product is used Examples of information from various manufacturers: 1. Affirm VPII 24 test kits Microbial Identification Test In-Vitro Diagnostic use Catalog No.: CPT code: Glucometer Elite Blood glucose test strips and code system for use with all Glucometer Elite blood glucose meters. 3. Bayer Reagent Strips Bayer package insert for the following products: Multistix 10SG, Multistix 9, Multistix 9SG, Multistix 8SG, Multistix 7, etc. Test for glucose, bilirubin, ketone, specific gravity, blood, ph, protein, urobilinogen, nitrite, and leukocytes in urine. WVOLS T&E 2010 FMI Page 9

12 2. Summary, Explanation, and Intended Use The Summary, Explanation, and Intended Use sections are combined into one section by some manufacturers and other manufacturers separate each section individually. For purposes of this course, the information contained in these sections is combined. The Summary section of the package insert provides a brief overall description of the test system or product. This section serves as the opening paragraph to the manufacturer s literature. Some people may not read beyond this information so it is important to contain important facts that catch the reader s attention. More of the same information is repeated in greater detail throughout the package insert. The Explanation of the Test section information is a detailed description of the method and the reason for performing the test. The Intended Use information outlines correct use(s) for the test system. For example: the intended use of blood glucose meters is to monitor the glucose level of patients for point-of-care testing, personal use, or to screen a large group of people such as in a mall screening. If a glucose monitor is used in a diagnostic test procedure such as a glucose tolerance, it is used beyond its intended use. This places the instrument in a high complexity category under the CLIA-88 regulations. These sections contain any or all of the following information: The name and manufacturer of the test will be repeated along with the method of testing. Words such as screening, monitoring, or diagnostic test. Often, waived test systems have the terms screening or monitoring attached to them. If a test is a screening or monitoring test, then confirmatory or diagnostic tests must follow. A brief description of the test method. Physical description of the test material(s). The type(s) of specimen used in the test. Whether the test is qualitative or quantitative. The disease state, symptoms, and clinical information which support the use of the test. Prevalence of the disease. Comparison of the test method to other methods for detection of the same analyte or organism. WVOLS T&E 2010 FMI Page 10

13 Examples of information from various manufacturers: 1. Urine reagent strips (Summary, Explanation, and Intended Use) Bayer Urinalysis Reagent Strips are firm plastic strips to which are affixed several separate reagent areas. Depending on the product being used, Bayer strips provide tests for glucose, bilirubin, ketone, specific gravity, blood, ph, protein, urobilinogen, nitrite, and leukocytes in urine. Test results may provide information regarding the status of carbohydrate metabolism, kidney and liver function, acid-base balance, and urinary tract infection. The reagent test areas on test strips are ready to use upon removal from the bottle, and the entire reagent strip is disposable. The strips may be read visually, requiring no additional laboratory equipment for testing. Certain configurations of strips may also be read instrumentally, using the Clinitek. 2. Blood Glucose Test Strips (Summary, Explanation, and Intended Use) The Glucometer Elite Blood Glucose Test Strips are used by persons with diabetes and by health care professionals to monitor glucose concentration in blood and are intended for use with all the Glucose Elite systems. The glucose test strips may also be used by health care professionals to test neonatal and arterial blood specimens. The strips are specific for glucose and have been calibrated to give plasma/serum equivalent glucose results. When the tip of the test strip touches a drop of blood, the strip fills automatically through simple capillary action. The blood is drawn into a reaction chamber and a reading is displayed on the meter. Only a small amount of blood is needed and no wiping or blotting is required. The rest provides a quantitative measurement of glucose from mg/dl. 3. SUDS HIV-1 Test (NOTE: in this example the Intended Use, and Summary & Explanation Sections are separate sections.) A. Intended Use The Murex single use diagnostic system HIV-1 test is an in-vitro qualitative microfiltration enzyme immunoassay intended for use by properly trained personnel as a screening test for the rapid detection of antibodies to Human Immunodeficiency Virus Type-1 (HIV- 1) in plasma or serum. The SUDS HIV-1 test can be used as a screening test in hospitals, laboratories, medical clinics, physician offices, and emergency care situations. WVOLS T&E 2010 FMI Page 11

14 B. Summary and Explanation of the Test Acquired Immunodeficiency Syndrome (AIDS) can be caused by two etiologic agents, which are designated as Human Immunodeficiency Virus Type-1 and Human Immunodeficiency Virus Type-2. Serological data reveal those ADIS patients and individuals infected by HIV-1 produce antibodies against HIV-1 proteins. Enzyme-linked immunosorbent assay (ELISA) is the test method used in the Murex test system for the detection of antibodies to HIV-1. Using this and other immunoassay systems, a high correlation of seropositivity for HIV-1 has been found. The presence of antibodies to HIV-1 is indicative of previous exposure to HIV-1 but not necessarily a diagnosis of AIDS. Samples which are repeatedly reactive for HIV-1 antibodies should be tested using additional, more specific tests such as Western Immunoblot and IFA. It is inappropriate to use the results of the SUDS HIV-1 test as the sole criterion for the diagnosis of AIDS or to exclude the possibility of having been exposed to or infected with HIV-1. In neonates, the presence of antibodies to HIV-1 is indicative of exposure to HIV- 1 but not necessarily of HIV-1 infection due to the acquisition of maternal antibodies which persist for up to six months. WVOLS T&E 2010 FMI Page 12

15 3. PRINCIPLES OF THE TEST This portion of the package insert is a detailed explanation of the chemical or biological principles of the test method. This section includes terminology which is highly technical in nature. This information may not seem to be important in the day-to-day operation of the laboratory, but, if problems are encountered, knowing how the system works can help in troubleshooting. Test systems are not as simple as they appear to be. For example: a complex series of reactions is occurring every time a reagent strip is immersed in a urine specimen. The following are examples or information from various manufacturers: 1. Urine Reagent Strip (Principle of the Test) Glucose: this test is based on a double sequential enzyme reaction. One enzyme, glucose oxidase, catalyzes the formation of gluconic acid and hydrogen peroxide from the oxidation of glucose. A second enzyme, peroxidase, catalyzes the reaction of hydrogen peroxide with a potassium iodide chromogen to oxidize the chromogen to colors ranging from green to brown. 2. Affirm VPII Microbial Identification Test (Principles of the Test) The Affirm VPII Microbial Identification test is based on the principles of nucleic acid hydridization. In nucleic acid hydridization tests, also known as DNA probe tests, complementary nucleic acid strands align to form specific, double-stranded complexes called hybrids. The test uses two distinct single-stranded nucleic acid probes for each organism, a capture probe and a color development probe, that are complementary to unique genetic sequences of the target organisms. The capture probes are immobilized on a bead embedded in a Probe Analysis Card (PAC) which contains a separate bead for each target organism. The color development probes are contained in a multi-well reagent cassette. WVOLS T&E 2010 FMI Page 13

16 4. Reagents and Materials The Reagents and Materials section of the package insert outlines the reagent within a test system and the chemical properties of each. This section lists additional reagents and equipment required to perform the test. Often, test kits will include dry chemicals (lyophilized) which require the addition of a liquid or diluent to make a solution. A description of the type of liquid (e.g. distilled water or kit solution) and the volume required for rehydration of the dry chemical us provided. The information included in the Reagent and Materials Section is as follows: The chemical constituents of all reagents (e.g. ph - 0.2% w/w methyl red; 2.8% w/w bromthymol blue; 97% w/w nonreactive ingredients, sodium azide). Material provided to perform the test (e.g. analysis cards, filter tips). Reagent quantities (e.g. 24 reagent cassettes, Latex antigen suspension ml vial). Positive and negative controls if included within a kit. Materials required but not provided - latex gloves, specific volume pipette, thermometer, processor, etc. Examples of information from various manufacturers: 1. Glucometer Elite (Reagents) 29.1% w/w glucose oxidase 32.0% w/w potassium ferricyanide 38.9% w/w nonreactive ingredients 2. SUDS HIV-1 Kit (Reagents and/or Kit Components) A. Latex-antigen suspension ml vial One dropper vial containing latex particles with bound HIV-1 virus antigens in a buffered protein solution. WVOLS T&E 2010 FMI Page 14

17 B. Enzyme-antibody conjugate ml vial One dropper vial containing goat anti-human IgC and anti-igm conjugated to alkaline phosphatase. C. Diluent - 15 ml One bottle containing buffered protein solution. Preservative: 0.1% sodium azide. D. Wash Reagent - 30 ml One bottle containing buffered solution. E. Substrate ml One dropper vial containing buffered substrate. F. Stop solution - 6 ml One dropper vial containing 0.3 M citrate. G. Positive control ml One vial containing heat inactivated and detergent treated plasma or serum, positive for antibodies to HIV-1, diluted in a buffered protein solution. Negative for hepatitis B surface antigen; hepatitis C antibody status is unknown. H. Negative control ml One vial containing human plasma or serum negative for antibodies to HIV-1. I. Micropipettes - 30 each J. Calibrated dropper K. Color key WVOLS T&E 2010 FMI Page 15

18 5. Safety Precautions The Precautions section of a package insert provides very important information related to workplace safety. It describes the chemical hazards or biohazards associated with the test and personal protective equipment required to perform the test safety. The Precautions section serves as a document reference for compiling work place safety manuals and policies. It may be referred to as documentation during an Occupation Safety and Health Administration (OSHA) or hazardous waste inspection. The information in the section includes: General bloodborne pathogen precautions for handling specimens. Safety precautions in handling of reagents and controls. Examples of information from various manufacturers: 1. Handle the positive and negative controls as if they are capable of transmitting infection. 2. Substrate solution contents are combustible and must be kept away from heat or flame. Specific safety practices associated with the test method. Examples of information from various manufacturers: 1. Do not pipette by mouth. 2. Wear a lab coat and latex gloves while handling kit reagents and specimens. 3. Wipe spills thoroughly with a 10% solution of bleach. Hazardous waste disposal requirements. Examples of information from various manufacturers: 1. Dispose of all test specimens and materials used in the test method in the biohazardous waste container. 2. All reagents contain sodium azide as a preservative. Sodium azide can react with lead and copper plumbing to form potentially explosive metal azides. If reagents are flushed in the plumbing system, always flush with large quantities of water. WVOLS T&E 2010 FMI Page 16

19 6. Reagent Storage, Handling, and Preparation This section describes the storage, handling, and preparation requirements for all reagents and controls within a test system. Reagents stored improperly will lead to erroneous testing results. Failure to store and handle reagents according to manufacturer s instruction is a frequent CLIA deficiency. The information within this section includes: Temperature requirements for storage of all reagents within a test system. This includes monitoring and recording of room, refrigerator, and freezer temperatures (if applicable) with a NIST traceable thermometer. Reagents must be stored within the specified temperature range. Examples: 1. Store test strips between C. 2. Store wash reagent between 3-8 C. Atmospheric conditions. Examples: 1. Store test strips away from moisture. 2. Store test cartridges away from light. 3. Do not remove desiccant from the bottle. Material preparation. Examples: 1. Allow all reagents and specimens to reach room temperature before use. 2. The latex-antigen suspension should be resuspended before use by shaking vigorously. 3. Use a new micropipette for each specimen or control. 4. Reagent must be reconstituted with 3 ml of distilled water. WVOLS T&E 2010 FMI Page 17

20 Handling of reagents to prevent errors. Examples of information from various manufacturers: 1. Do not remove strip from the bottle until immediately before it is to be used for testing. Replace cap immediately and tightly after removing reagent strip. 2. Work areas and specimen containers should be free of detergents and other contaminating substances. 3. Avoid touching the chemical test areas of the reagent strips. 4. Do not use reagents after expiration date. 5. Do not mix materials from kits with different lot numbers. 6. Do not interchange vial or bottle caps to avoid cross-contamination. WVOLS T&E 2010 FMI Page 18

21 7. Specimen Collection and Handling The Specimen Collection and Handling section is one of the most important reference sections of the package inserts. Regardless of how meticulously the test is performed, the test result is only as good as the integrity of the specimen. This section outlines the type of specimen to be used and how the specimen should be handled to preserve its integrity. It also refers to the pre-analytic factors which could affect test performance. Errors associated with preanalytic and post-analytic factors far exceed errors in the analytic phase of testing. This section includes the following information: Type of specimen required Examples: Serum, plasma, whole blood, urine, vaginal fluid, finger stick Specific instructions for collection of the specimen Examples of information from various manufacturers: 1. All samples must be collected using the swabs and tubes provided in the kit. 2. Written instructions for the collection of a clean-catch urine specimen should be provided to patients for the collection of a clean catch urine specimen. 3. Place swab against the back wall of the throat and move up and down in the uvula area. White patches in the tonsillar area are particularly productive for streptococcal isolation. 4. Collect blood specimen by venipuncture in an EDTA vacutainer tube. Patient preparation Examples of information from various manufacturers: 1. Fasting instructions for blood glucose and other clinical chemistry tests. 2. Recommendations for first morning urine specimen. WVOLS T&E 2010 FMI Page 19

22 Specimen storage requirements Example: Serum or plasma may be stored refrigerated at 2-8 C for up to 24 hours. If longer storage is required the serum or plasma should be stored frozen at -70 C. Preservation to be used if testing is prolonged Examples: 1. Refrigeration 2. Chemical additives Factors that interfere with the specimen integrity Examples: 1. Specimens that have been frozen should be centrifuged at 15,000 RPM before testing with the SUDS kit to remove particulate matter. 2. Contamination of the urine specimen with skin cleansers which contain chlorhexidine may affect protein and bilirubin in test results. Testing time limitations Examples: 1. Urine stored at room temperature should be tested within one hour of voiding. 2. The total time between placing the swab into the sample collection tube and proceeding with the sample preparation should be no longer than one (1) hour if the sample is stored at room temperature, or four (4) hours if the sample is stored at 2-8 C. WVOLS T&E 2010 FMI Page 20

23 8. Test Procedure The Test Procedure section is the actual step-by-step instructions on performing the test. It is written in cookbook fashion with every detail outlined. This section features the analytical phase of laboratory testing. Often this is the only part of the manufacturer s instructions that is read by testing personnel. The importance of this section must not be underplayed but it is not the only part of manufacturer s instructions. It is a good idea to copy this portion and place it near the testing area for the laboratory personnel as a reference in the day-to-day operation of the laboratory. Information that may be found in the Test Procedure section includes: Proper mixing of the specimen (if applicable) Reaction time, read time, or incubation time Step-by-step instructions on the mechanics of performing the test Signs of errors occurring Identification of equipment, such as pipette size, test cartridges Sequential use and quantity of reagents to perform the test Incubation temperature How to recognize the test result (e.g. color change, line, numerical value) Visual illustrations of the steps in the procedure Automated processing Example: Urine Reagent Strip (Test Procedure) 1. Collect fresh urine specimen in a clean, dry, container. Mix well immediately before testing. 2. Remove one strip from the bottle and replace cap. Completely immerse reagent areas of the strip in the urine and remove immediately to avoid dissolving out reagents. WVOLS T&E 2010 FMI Page 21

24 3. While removing, run the edge of the entire length of the strip against the rim of the urine container to remove excess urine. Hold the strip in a horizontal position to prevent possible mixing of chemicals from the adjacent reagent areas and/or contaminating the hands with urine. 4. Compare the reagent areas to corresponding color chart on the bottle label at the reaction time specified on the bottle. Hold the strip close to the color blocks and match carefully. Avoid laying the strip directly on the color chart. 5. Read the glucose and bilirubin tests at 30 seconds after dipping. Read the ketone test at 40 seconds; the specific gravity test at 45 seconds; ph, protein, urobilinogen, blood, and nitrite at 60 seconds. WVOLS T&E 2010 FMI Page 22

25 9. Quality Control The Quality Control (QC) section of the package insert describes the specific quality control requirements for the test method. Good laboratory practice must always include quality control to validate the testing process. Regardless of the test complexity (waived, moderate, or high) quality control must be performed at a frequency specified by the manufacturer. Examples of the differences in quality control instructions include: o o o Two levels of control are run each day of patient testing; this is the general CLIA-88 QC rule. For qualitative tests, the two levels of controls are positive and negative (reactive or nonreactive). For quantitative tests, the controls are in the normal and abnormal numerical levels. Test kits may have internal and external controls. The internal controls test must be run and documented daily while the external controls are run when a new kit is opened and if problems are suspected. Some Strep antigen detection kits in the waived category will require that the external control be tested by each operator with a 25 size test kit when the kit is first opened. o The hematology specialty requires two levels of controls to be performed every eight (8) hours of testing. The Quality Control section of the package insert includes the following information: Description of the type of control specimens Control levels that must be run to validate test Frequency of running controls Controls recommended for use General troubleshooting guidelines in the event the controls fail Description of how to interpret control results Instructions on how to run controls If control preparation is required, the instructions on how to reconstitute the controls Calibration instructions, if applicable WVOLS T&E 2010 FMI Page 23

26 Example: Blood Glucose Test Strips (Quality Control) Why? To check the performance of the meter, test strip, and testing technique. The meter test strip and testing technique are acceptable if the control test results fall within the specified control range listed on the end flap of the test strip carton. Because control ranges can change for each new lot of test strips, always use the control ranges given on the end flap of the carton of test strips. How? Run a control test by following the illustrated control test directions in the Glucometer Elite user s guide. When? Any time you want to check the performance of your meter, test strips, or testing technique. Performance is only assured when the control test result falls within the specific range listed on the flap of the Glucometer Elite test strip carton. The manufacturer recommends that individuals run a control test and consult with their physician or health care professional before adjusting medication based on the glucose meter test results. When test results are questionable: 1. Be sure the drop of sample completely fills the tip of the test strip. 2. Confirm that the strips are within the expiration date. 3. Do not use test strips from a torn or damages foil packet. 4. Be sure you have calibrated the meter by setting the correct function number. 5. Check meter performance using the check strip. 6. Check meter and strip performance using the normal and abnormal controls. WVOLS T&E 2010 FMI Page 24

27 10. Interpretation of Results (Expected Values) The Interpretation of Results section is also referred to as the Expected Values or Results Section - depending on the manufacturer. This section provides basic instruction on how to relate the results of a test as clinically meaningful information. It may be a description of a color change or an appearance of a line as a positive or negative result. Illustrations and color keys corresponding to concentrations of a substance are often used to aid in the interpretation of test results. In quantitative tests, the meanings of the corresponding numerical values are described. A description of the expected values for the typical normal healthy population is also included in this section. The Interpretation of Results section may include any or all of the following information: Describe what reaction is considered normal or abnormal. Recommend follow-up confirmatory test(s). Recommend when a test must be repeated before reporting. Define normal patient population reference ranges (should be validated by the director to the norms of the patient, i.e. pediatric, gender, location, geriatric). Describe whether a test method is a screening test (which requires further testing) or a diagnostic test. Provide flow chart of the steps to follow depending on the test observation. Describe visual control zone or endpoint of reaction(s) and procedure to follow should the control zone fail. Example: SUDS HIV-1 package insert (Interpretation of Results) A color key is provided to assist in the determination of color. Whether or not a blue color is produced in the center circle, the outer circles should remain white and serve as control zones to indicate that the washing steps were properly performed. If one or both of the outer circles develops blue color, regardless of the presence of the blue color in the center circle, the test result is inconclusive and the specimen should be retested. Specimens which do not produce a blue color are considered non-reactive by the test method and may be considered negative for HIV-1 antibodies. The presence of any blue color in the center circle, as viewed from the bottom of the SUDS test cartridge, indicates that the specimen is initially reactive for the HIV-1 test. Any specimen producing a blue color on initial testing should be retested in duplicate. A repeatedly reactive result should be tested using an additional, more specific test (e.g. Western Blot or IFA). WVOLS T&E 2010 FMI Page 25

28 11. Limitation of the Procedure The Limitation of the Procedure section details the specific factors or variables which interfere with test results. This section may also include statements on correlation of laboratory results with patient clinical signs and symptoms. Knowing interfering factors helps to avoid potentially erroneous test results. For example: if a manufacturer states that hemolysis (breakdown of RBCs that produce a red serum or plasma) may interfere with test results, then a hemolyzed specimen should not be used in the test method. This section supplies information that is an important part of troubleshooting. The Limitation of the Procedure section may include any or all of the following information: Substances which cause an abnormal color to a specimen. Specimens kept beyond the recommended storage time and temperature. Most common procedural errors to avoid. Correlation of patient symptoms with test results. Instrument reportable range in which a substance or an organism can be detected by the test method. The minimal concentration of a substance which will affect test results. Mechanical, physical, and chemical causes of false-positive and false-negative test results. Examples of information from various manufacturers: 1. Affirm VPII Microbial Identification Test (Limitations of Procedure) Specimens held longer than one (1) hour at room temperature or four (4) hours at 2-8 C prior to preparation may cause false results. Prepared specimens held longer than 24 hours at room temperature prior to processing may give inaccurate results. A positive result for Candida, Gardnerella, and/or Trichomonas means nucleic acid for Candida species, G. vaginalis, and/or T. vaginalis is present in the sample and indicates the patient has candidiasis, bacterial vaginosis, and/or trichomoniasis when consistent with clinical signs and symptoms. The performance of this test on patient specimens collected during or immediately after antimicrobial therapy is unknown. The presence or absence of Candida species, G. vaginalis, or T. vaginalis cannot be used as a criterion for therapeutic success or failure. 2. Urinalysis (Limitations of Procedure) Substances that cause abnormal urine color such as drugs containing azo dyes (e.g. pyridium) may affect the readability of the reagent areas on urinalysis reagent strips. The color development on the reagent pad may be masked, or a color reaction may be produced on the pad that could be interpreted visually and/or instrumentally as a false-positive. WVOLS T&E 2010 FMI Page 26

29 12. Specific Performance Characteristics The Specific Performance Characteristics section of the package insert describes how well the test has performed in clinical and analytical studies. Independent investigators evaluate the test system using test specimens from patients exhibiting and not exhibiting the symptoms to which the test pertains. Graphs and charts are sometimes included depicting how this method compares to other test methods which detect the same substance or organism. The FDA requires these studies to be conducted before the test method is placed on the market for use. This information is used mostly by persons in a supervisory capacity who decides whether a test meets their needs. Terms that are often used in this section are sensitivity, specificity, predictive value, and detectable levels. Explanations of these terms are needed to explain adequately the information in the specific performance characteristics section of the package insert. Sensitivity - This term refers to the proportion of people with the disease who have a positive test. A sensitive test will rarely miss people who truly have the disease and produce very few false-negatives. A test with no false-negative errors would be considered the most sensitive test (100%) for the disease under investigation. There are circumstances when it is important to find all of the individuals with a particular problem. An example would be a screening for a disease with a significant morbidity in which early treatment greatly improves the outcome. Finding everyone with the disorder can be done by subjecting high-risk individuals who have been identified by a highly sensitive, but less specific test (screening test), to a second test of very high specificity (confirmatory test) to confirm the diagnosis. The formula used to calculate sensitivity is: Sensitivity = True-positive test results (TP) True-positive test results (TP) + False-negative test results (FN) x 100 Specificity - This term refers to the proportion of people without the disease who have a negative test. A specific test will rarely misclassify people free of disease and produce few falsepositives. A test with no false-positive errors would qualify as 100% specific for the disease under investigation. As stated above, highly specific tests are used to confirm a diagnosis. However, it is important to have a fairly high specificity of all tests. The formula used to calculate specificity is: Specificity = True-negative test results (TN) True-negative test results (TN) + False-positive test results (FP) x 100 Predictive Value - This term refers to the probability of the presence or absence of disease given the results of a test. In CLIA, this might be called Relationship of Patient Information to Patient Test Results. It is common sense laboratory testing. For example, if a rapid flu kit detects a number of positive patients at a time of year when flu is not normally seen, the laboratory director may scrutinize the results more closely for false-positives. Predictive value is determined by the sensitivity and the specificity of the test and the prevalence of disease in the population being tested. Predictive value positive (PVP) is the probability of diseases in a patient with a positive test result. Predictive value negative (PVN) is the probability of not having disease when the test result is negative. WVOLS T&E 2010 FMI Page 27

30 The formula for investigative studies to calculate predictive value are: Predictive Value Positive (PVP) = True-positive test results (TP) True-positive test results (TP) + False-positive test results (FP) x 100 Predictive Value Negative (PVN) = True-negative test results (TN) True-negative test results (TN) + False-negative test results (FN) x 100 Example of how the manufacturer determines the sensitivity and specificity of a test kit: A manufacturer evaluates a new pregnancy test for sensitivity and specificity. They enlist 800 female study subjects, 300 pregnant and 500 non-pregnant. After testing all subjects, the manufacturer constructs the following table: Test Results Pregnant Subjects Non-pregnant Subjects Total Positive 250 (TP) 4 (FP) 254 Negative 50 (FN) 496 (TN) 546 Total 300 (TP+FN) 500 (TN+FP) 800 Sensitivity = (TP) (TP) + (FN) x 100 Sensitivity = x 100 = 83% Specificity = (TP) (TP) + (FN) x 100 Specificity = x 100 = 99% WVOLS T&E 2010 FMI Page 28

31 Detectable levels - The lowest concentration of an analyte in a specimen which the test method can detect. This relates to the sensitivity of the test. Example: Glucose (reagent strip) Leukocytes (reagent strip) mg/dl 5-15 cells/hpf Any concentration under these values will not be detected by the reagent strip method. Other information that is included in the Specific Performance Characteristic section includes: Variable factors which influence test results such as color perception, the presence of inhibitory factors found in a specimen, lighting conditions, temperature, etc. Correlations with other test methods. For example: Reagent strip specific gravity - This method correlates within with values obtained with the refractometer method. For increased accuracy, may be added to readings from urine with ph equal to or greater than 6.5. Strips read instrumentally are automatically adjusted for ph by the instrument. Disease states that may cross react with the test method and produce false-positive test results. Causes of false-negative test results. WVOLS T&E 2010 FMI Page 29