Safety Tips for Laboratorians

This section provides safety tips for conducting laboratory tests properly.

• Important Issues for Allergen-Specific IgE Testing
• Blood Glucose Meters: Getting the Most Out of Your Meter
• C-Reactive Protein (CRP): The Differences Amongst Various Assays
• Cautions in Using Rapid Tests for Detecting Influenza A Viruses
• Blood Human Chorionic Gonadotropin (hCG) Assays: What Laboratorians Should Know about False-Positive Results
• Troponin: What Laboratorians Should Know to Manage Elevated Results
• Home Pregnancy Tests – How to Use a Popular Test Wisely
• Practice Caution in Using Bio-Rad Platelia™ Aspergillus EIA
• Use of Backup Testing for Negative Rapid Group A Strep Tests
• Follow Good Laboratory Practices with Waived Laboratory Tests
• Performance and Cautions in Using Rapid Influenza Virus Diagnostic Tests
• Built-in Controls for Single Use Disposable Tests
• Common Problems with the Use of Glucose Meters
• Make Sure Glucose Meters Used in Testing Neonates are FDA Cleared for Testing in Neonates
• Useful Tips to Increase Accuracy and Reduce Errors in Test Results from Glucose Meters
• Reporting Problems with Laboratory Tests and Equipment

Troponin: What Laboratorians Should Know to Manage Elevated Results

(posted 8/26/05)

FDA, using input provided by Advanced Medical Technology Association (AdvaMed), has developed this document in response to numerous adverse event reports of falsely elevated results sent by manufacturers and users of troponin assays and many published articles related to this subject.

1. What is the Purpose and Scope of this Communication?

The purpose of this communication is to inform laboratorians about the possibility of falsely elevated troponin results when troponin is used as a cardiac marker, and to provide information as to how to identify and verify cases of suspected falsely elevated results. This communication may also be of interest to cardiologists, primary care physicians, and emergency care physicians. In this document, we use the term “false-positive” troponin test result to refer to cases when the troponin immunoassay result is positive in the absence of acute myocardial infarction (AMI) or any other clinical condition in which troponin markers may be elevated.

This document addresses issues related to false-positive troponin results only when troponin testing is used in the diagnosis of acute myocardial infarction and not when it is used to diagnose other diseases. The information applies to both cardiac troponin I and cardiac troponin T immunoassays since the issue of falsely elevated results applies to both assay systems.

Cardiac troponins are highly sensitive and specific biochemical markers of myocardial cell necrosis and are widely used for the diagnosis of acute myocardial infarction.^1,2 Troponin levels rise and fall with acute myocardial infarction and with other clinical syndromes associated with infiltrative/inflammatory causes of myocyte death.

Troponin testing is subject to analytical interfering factors and assay malfunctions; both may lead to a falsely elevated troponin result. In addition, there are clinical conditions in which the troponin level may rise in the absence of an acute myocardial infarction . Although the elevation of troponin in association with non-AMI is generally rare, it may mislead the clinician to suspect that the patient has an acute myocardial infarction when that is not the case.

Therefore, it is important that laboratorians, working in conjunction with physicians who order the test, are aware of falsely elevated results and non-AMI causes of elevated troponin results in order to assist physicians to properly utilize troponin results in patient management.

3. Can Results of Different Troponin Assay Systems Be Compared?

No. The results of different troponin assays are not generally comparable. A large variation in cardiac troponin I concentration, in terms of absolute value, may be observed for a given patient specimen with different analytic methods.³ Until recently, there was no accepted reference standard for troponins. However, a new Standard Reference Material for Human Cardiac Troponin Complex (SRM 2921)⁴ has been introduced by the National Institute of Standards and Technology (NIST) which should aid in the future standardization of troponin assays. The SRM 2921 is intended for use in evaluating the accuracy of clinical procedures for the determination of cardiac troponin I in human serum.⁴

The analytical variability among troponin assays is due to the fact that different troponin assays have wide variations in lower detection limits, upper reference limits, diagnostic cut points, assay imprecision (coefficient of variation),⁵ and specimen matrices (i.e., serum versus plasma samples). The presence of a large number of manufacturers of troponin assays in the United States market makes standardization more difficult.

4. What Are the Analytical Interfering Factors That May Lead to Falsely Elevated Troponin Results?

Some of the analytical interfering factors that may lead to falsely elevated troponin results include: ^3,6,7

• fibrin clots in serum as a result of incompletely clotted specimen, e.g. in patients with coagulopathy or on anticoagulant therapy⁸
• heterophile antibodies, human anti-animal antibodies⁹ , rheumatoid factor^10,11, and autoantibodies
• interference from other endogenous components in the blood such as bilirubin and hemoglobin¹²
• immunocomplex formation¹³
• microparticles in specimen
• high concentration of alkaline phosphatase¹⁴
• analyzer malfunction¹⁵

5. What Are Interfering Antibodies (Heterophile and Human Anti-Animal) and How Can Laboratorians Address This Issue?

A. Definition and Sources

Circulating heterophile antibodiesⁱ and anti-animal antibodiesⁱⁱ have the potential to interfere with two-site (sandwich) or competitive immunoassays, such as troponin assays, by cross-linking the capture and label antibodies in the absence of specific analyte. ^6,16 The estimated prevalence of interfering antibodies in the general population is up to 40% of normal serum samples.^17,18

Most modern immunoassays contain nonspecific blocker immunoglobulins (which originate from the same species as the analyte-specific antibodies) in order to limit the effect of the interfering antibodies.¹⁹ However, in some instances the blocking proteins can not sufficiently neutralize the interfering antibodies. Thus, analytical errors may occur. In case of troponin assays, the presence of high levels of these antibodies may lead to falsely elevated values.

An individual may acquire these antibodies from a variety of sources including the use of mouse monoclonal antibodies in diagnostic imaging and cancer therapy; exposure to microbial antigens; exposure of veterinarians, farm workers, and food preparers to foreign proteins; the presence of domestic animals in the home; or autoimmune diseases which can give rise to autoantibodies such as rheumatoid factor. ^7,16

ⁱ Heterophile antibodies are antibodies produced against poorly defined antigens (frequently foreign proteins). The general term "heterophile antibodies" is sometimes used in the literature interchangeably to refer to heterophile antibodies, human anti-animal antibodies, rheumatoid factor, and autoantibodies.

ⁱⁱHuman anti-animal antibodies are circulating human antibodies reactive with animal proteins.⁹ Circulating antibodies with specificities for a wide range of animal immunoglobulins have been reported such as mouse, rat, rabbit, and others.⁹

B. Laboratorians May Suspect or Recognize the Presence of Interfering Antibodies in a Specimen Tested for Troponin

Because of a broad range of interfering antibodies, there is not a specific test to verify or rule out their presence in an assay. Laboratorians should suspect the occurrence of interfering antibodies in a troponin assay when the test result:

• does not agree with the patient’s clinical information for an acute myocardial infarction (see items 6 & 7 for more information)
• may not be reproducible on the same or different assay system
• is not linear after serial dilutions

C. Methods to Reduce or Remove Interfering Antibodies from Troponin Assays

After ruling out the possibility of technical errors and analyzer malfunction and after repeating the assay, the following methods may be applied for reducing or removing the effect of interfering antibodies from troponin assay results.⁹

• Run the specimen on a different assay system
• Use commercially available heterophile blocking reagents
• Remove endogenous immunoglobulins by adding endogenous immunoglobulin-free serum samples to the specimen. For example, use normal mouse (animal) sera as blocking reagent

6. If a Troponin Test Result Does Not Match the Patient’s Clinical Picture for Acute Myocardial Infarction, What Should Laboratorians Consider Doing?

If the troponin test result does not match the patient’s clinical picture for acute myocardial infarction, it is possible that the test result is falsely elevated due to analytical interfering factors, and/or analyzer malfunction. The laboratory should investigate the presence of any potentially interfering factors and verify the test result by taking the following steps.

• Find out whether the patient is on anti-coagulant therapy or has coagulopathy.
• Check for bilirubin and/or hemoglobin interference.
• Visually examine the specimen tube for any particles or clots (fibrin interference). If present, centrifuge the specimen for a second time.
• Rerun the specimen after doing a serial dilution and look for interfering antibodies by examining the linearity of results.
• If sufficient amount of specimen is available, save the sample for further testing by the laboratory or the manufacturer.
• Rerun the same specimen on a different assay system, if available, to verify the results. If another assay system is not available, send out the specimen to another laboratory to be run on a different assay system.
• Examine the analyzer thoroughly to investigate the presence of any malfunction.
• Communicate to the treating physician the discrepancy between the test result and clinical information, and inform him/her of the possibility of a false-positive value.
• If the problem does not get resolved, report the issue to the manufacturer and FDA and send the specimen to the manufacturer for further testing and investigation.

7. If a Troponin Test Result Does Not Match the Patient’s Clinical Picture for Acute Myocardial Infarction, What Should Physicians Consider Doing?

In cases when a troponin test result does not match the patient’s clinical picture for acute myocardial infarction, it is important that the physician gather all the available information and reassess the patient. The physician may:

• consider the possibility that some other clinical condition may be causing an elevated troponin level in the absence of acute myocardial infarction.
• communicate with the laboratory about the test result and ask the laboratory to rule out technical errors, analytical interfering factors, and analyzer malfunction.
• consider repeating the blood draw and retesting.
• review the clinical presentation and consider additional diagnostic testing (e.g. reconsider the nature of chest pain, repeat ECG, etc.); bear in mind that the troponin test result is only one piece of the diagnostic puzzle.

8. Is It Important that Laboratorians Follow Manufacturers’ Recommended Instructions?

Yes. For troponin testing, it is critical that patient specimens be collected and processed according to the manufacturers’ recommendations included in the device product insert. Improper collection, handling, and preparation of specimens can impact the accuracy of results.

• Store unused collection tubes and blood specimens according to the manufacturers’ recommendations.
• Follow manufacturers’ instructions for using collection tubes with anticoagulants. Some may contain insufficient anticoagulant and lead to elevated or decreased results.
• Mix the content of tubes properly at the time of blood collection to prevent incomplete clot formation (serum) and platelet clumping or clotting (plasma).
• Process specimens according to the tube manufacturer’s recommendations. Different types of tubes may have different requirements.
• Use a refrigerated, horizontal centrifuge head for best results. Use the centrifuge settings recommended by the tube manufacturer.
• Inspect samples for clots, fibrin, particulate matter, and other debris prior to processing them on an analyzer. Cellular debris from grossly hemolyzed samples may elevate test results.
• Follow manufacturer’s recommendation for running proper quality control samples. At least one control should be run at the cutoff level. If the risk stratification and acute myocardial infarction cutoff are different, separate controls should be considered at those levels.
• Follow the manufacturers’ recommended calibration and/or maintenance schedules. Analyzer malfunction is one of the common assay interfering factors that leads to inaccurate results. Laboratories reporting troponin results should perform thorough and regular system maintenance to ensure peak performance of their analyzers and to reduce the possibility of inaccurate results.

9. What Are the Current Cardiology Practice Guidelines for Troponin Reference Intervals and Decision Limits?

Current practice guidelines issued by the American College of Cardiology (ACC), the National Academy of Clinical Biochemistry (NACB), and the European Society of Cardiology (ESC) make similar recommendations regarding reference intervals, decision limits, and precision. These recommendations include: ^1,2,20

• establishing an upper reference limit at the upper 99 th percentile of normal population
• using a single cutoff positioned at or slightly above the upper reference limit
• accomplishing a total imprecision (coefficient of variation) ≤10% at the decision limit (Currently, few commercially available assays can achieve this precision level .)

10. What Are Some Clinical Conditions Associated with Cardiac Troponin Elevations in the Absence of Acute Myocardial Infarction?

Myocardial cell damage (reversible or irreversible) and elevation of cardiac troponins can occur under conditions OTHER than acute myocardial infarction due to obstructive coronary artery disease. A short list of some of these clinical conditions includes but is not limited to:^21,22,23

• myocardial cell trauma
• congestive heart failure
• left ventricular hypertrophy
• myocarditis
• pulmonary embolism
• renal failure
• infiltrative disease with cardiac involvement
• acute neurological disease including cerebrovascular accidents and subarachnoid bleeds
• rhabdomyolysis with cardiac injury

REPORTING TO FDA

If there are any questions or concerns regarding the performance of troponin test method, contact the assay manufacturer. You should report all occurrences of unusual test performance to the manufacturer, and you are encouraged to also report them to FDA. To obtain more information about medical device reporting you can refer to the FDA’s web site at: http://www.fda.gov/cdrh/mdr/index.html

All reports should be sent to:

Food and Drug Administration
Center for Devices and Radiological Health
Medical Device Reporting
P.O. Box 3002
Rockville , MD 20847-3002

For any questions or concerns regarding the content of this communication contact:

Ms. Ruth Chesler
Food and Drug Administration
Center for Devices and Radiological Health
Office of In Vitro Diagnostic Device Evaluation and Safety
2098 Gaither Road , HFZ-440
Rockville , MD 20850

Phone: (240) 276-0686
Fax: (240) 276-0651
Email: ruth.chesler@fda.hhs.gov

REFERENCES:

1 Heterophile antibodies are antibodies produced against poorly defined antigens (frequently foreign proteins). The general term "heterophile antibodies" is sometimes used in the literature interchangeably to refer to heterophile antibodies, human anti-animal antibodies, rheumatoid factor, and autoantibodies.

2 Human anti-animal antibodies are circulating human antibodies reactive with animal proteins. 9 Circulating antibodies with specificities for a wide range of animal immunoglobulins have been reported such as mouse, rat, rabbit, and others. 9

3 Alpert JS, Thygesen K, Antman E, Bassand JP. Myocardial Infarction Redefined - A Consensus Document of The Joint European Society of Cardiology/American College of Cardiology Committee for the Redefinition of Myocardial Infarction. The Joint European Society of Cardiology/American College of Cardiology Committee. Journal of the American College of Cardiology 2000; 36(3):959-969.

4 Wu AHB, Apple FS, Gibler WB, Jesse RL, Warshaw MM, Valdes R Jr. National Academy of Clinical Biochemistry Standards of Laboratory Practice: Recommendations for the Use of Cardiac Markers in Coronary Artery Diseases. Clinical Chemistry 1999; 45(7):1104-1121.

5 Ng SM, Krishnaswamy P, Morrisey R, Clopton P, Fitzgerald R, Maisel AS. Mitigation of the Clinical Significance of Spurious Elevations of Cardiac Troponin I in Settings of Coronary Ischemia Using Serial Testing of Multiple Cardiac Markers. The American Journal of Cardiology 2001; 87:994-999.

6 National Institute of Standards and Technology. http://www.nist.gov/srm Certificate of Analysis Standard Reference Material 2921, Human Cardiac Troponin Complex. 2004.

7 Apple FS. Cardiac Troponin Assays Analytical Issues and Clinical Reference Range Cut Points. Cardiovascular Toxicology 2001; 1(2):93-98.

8 Ringdahl EN, Stevermer JJ. False-Positive Troponin I in a Young Healthy Woman with Chest Pain. The Journal of the American Board of Family Practice 2002; 15(3):242-245.

9 Roongsritong C, Warraich I, Bradley C. Common Causes of Troponin Elevations in the Absence of Acute Myocardial Infarction – Incidence and Clinical Significance. Chest 2004; 125(5):1877-1884.

10 Kazmierczak SC , Sekhon H, Richards C. False-Positive Troponin I Measured with the Abbott AxSYM Attributed to Fibrin Interference. International Journal of Cardiology 2005; 101(1):27-31.

11 Kricka LJ. Human Anti-Animal Antibody Interferences in Immunological Assays. Clinical Chemistry 1999; 45(7):942-956.

12 Krahn J, Parry DM, Leroux M, Dalton J. High Percentage of False Positive Cardiac Troponin I Results in Patients with Rheumatoid Factor. Clinical Biochemistry 1999; 32(6):477-480.

13 Dasgupta A, Banerjee SK, Datta P. False-Positive Troponin I in the MEIA Due to the Presence of Rheumatoid Factors in Serum - Elimination of This Interference by Using a Polyclonal Antisera Against Rheumatoid Factors. American Journal of Clinical Pathology 1999; 112:753-756.

14 Hawkins RC. Hemolysis Interference in the Ortho-Clinical Diagnostics Vitros ECi cTnI Assay. Clinical Chemistry 2003; 49(7):1226.

15 Plebani M, Mion M, Altinier S, Girotto MA, Baldo G, Zaninotto M. False-Positive Troponin I Attributed to a Macrocomplex. Clinical Chemistry 2002; 48(4):677-679.

16 Dasgupta A, Chow L, Wells A, Datta P. Effect of Elevated Concentration of Alkaline Phosphatase on Cardiac Troponin I Assays. Journal of Clinical Laboratory Analysis 2001; 15:175-177.

17 Galambos C, Brink DS, Ritter D, Chung HD, Creer MH. False-Positive Plasma Troponin I with the AxSYM Analyzer. Clinical Chemistry 2000; 46(7):1014-1015.

18 Yeo KTJ, Storm CA, Li Y, Jayne JE, Brough T, Quinn-Hall KS, Fitzmaurice TF. Performance of the Enhanced Abbott AxSYM Cardiac Troponin I Reagent in Patients with Heterophilic Antibodies. Clinica Chimica Acta 2000; 292:13-23.

19 Gibson TN, Hanchard B. False Positive Troponin I in a Case of Metastatic Small Cell Bronchogenic Carcinoma Complicated by Pulmonary Thromboembolism. West Indian Medical Journal 2001; 50(2):171-172.

20 Fleming SM, O’Byrne L, Finn J, Grimes H, Daly KM. False-Positive Cardiac Troponin I in a Routine Clinical Population. The American Journal of Cardiology 2002; 89:1212-1215.

21 Fitzmaurice TF, Brown C, Rifai N, Wu AHB, Yeo KTJ. False Increase of Cardiac Troponin I with Heterophilic Antibodies. Clinical Chemistry 1998; 44(10):2212-2214.

22 Morrow DA, Cannon CP, Rifai N, Frey MJ, Vicari R, Lakkis N, Robertson DH, Hille DA, DeLucca PT, DiBattiste PM, Demopoulos LA, Weintraub WS, Braunwald E; TACTICS-TIMI 18 Investigators. Ability of Minor Elevations of Troponins I and T to Predict Benefit from an Early Invasive Strategy in Patients with Unstable Angina and Non-ST Elevation Myocardial Infarction: Results from a Randomized Trial. Journal of the American Medical Association 2001; 286(19):2405-2412.

23 Jaffe AS. Elevations in Cardiac Troponin Measurements: False False-positives The Real Truth. Cardiovascular Toxicology 2001; 1:87-92.

24 Hamm CW, Giannitsis E, Katus HA. Cardiac Troponin Elevations in Patients without Acute Coronary Syndrome. Circulation 2002; 106:2871-2872.

25 Li D, Keffer J, Corry K, Vazquez M, Jialal I. Nonspecific Elevation of Troponin T Levels in Patients with Chronic Renal Failure. Clinical Biochemistry 1995; 28(4):474-477.

Home Pregnancy Tests – How to Use a Popular Test Wisely

(posted 4/2/04)

The first home pregnancy tests were marketed in the mid 1970’s. These tests are one of the most popular products for home diagnostic testing. It is estimated that about 33% of women have used these tests. The tests are popular because they allow women rapid access to highly sensitive and personal information. These tests can lead to earlier diagnosis and can provide pregnant women an opportunity to seek earlier health care intervention.

FDA is involved in the premarket review of these tests. Since the 1976 Medical Device Amendment, FDA assures that new pregnancy tests perform as well as those tests on the market since 1976. Premarket compares test performance between a new test and an established test. In these kinds of pregnancy devices the new test is compared to varying levels of the hormone human chorionic gonadotropin (hCG) that is the marker for pregnancy.

Recent investigators point out that the FDA review of analytical performance does not always mean a woman is pregnant. This discrepancy is because different tests have different abilities to detect low levels of hCG. Also hCG levels differ between pregnant women depending on the timing of the onset of pregnancy with regard to a menstrual period and depending on each woman’s unique biology.

The result is that pregnancy tests may be labeled up to 99% accurate when compared to other hCG tests, not to pregnancy. This may be true based on information submitted to the FDA. Therefore, labeling of these tests should clearly indicate that there is a possibility for both false positive tests and false negative tests, so patients should contact their health care provider to discuss any result.

Patients may frequently recognize incorrect results with the passage of time. False negatives may be detected by ongoing failure to have a period or the development of other obvious signs of pregnancy. False positives may be demonstrated by the unexpected onset of menses (regular vaginal bleeding associated with “periods”.) Repeat testing and/or other investigations such as ultrasound may provide corrected results.

If a patient has a negative result, it is always wise to consider this a tentative finding. Women should not use medications and should consider avoiding potentially harmful behaviors, such as smoking or drinking alcohol, until they have greater certainty that they are not pregnant.

Since September of 2003, studies for pregnancy tests have been posted on the Office of In Vitro Diagnostic Web Page (www.fda.gov/cdrh/oivd/) under new 510(k)s – decision summaries. FDA is considering what educational or regulatory tools might be available to help clarify the status, use, and interpretation of these tests.

Practice Caution in Using Bio-Rad Platelia™ Aspergillus EIA

FDA has cleared for marketing, the Platelia™ Aspergillus EIA, manufactured in France by Bio-Rad, Marnes-la-Coquette and distributed by Bio-Rad Laboratories, Redmond, WA, which detects Aspergillus galactomannan antigen in serum samples, and is an aid in the diagnosis of Invasive Aspergillus infection. Invasive Aspergillosis is a life-threatening invasive fungal infection that often occurs in leukemia patients, organ and bone marrow transplant patients, and patients whose immune systems are compromised by illness or chemotherapy.

There have been reports in the literature, in the U.S. and from Europe, of potential drug and device interaction that may occur when using the Platelia™ Aspergillus EIA galactomannan antigen assay to test sera from patients who are receiving piperacillin/tazobactam (Zosyn®), an injectable antibacterial combination product from Wyeth Pharmaceuticals, Inc. Recent reports 1,2 have indicated that positive galactomannan antigen tests results may occur in serum samples from patients treated with Zosyn®, but without Invasive Aspergillosis. Additional studies have also demonstrated positive galactomannan antigen test results in certain batches of Zosyn®. These results are highly suggestive of reactivity of the Platelia™ Aspergillus EIA with Zosyn®. Nevertheless, as Platelia™ Aspergillus EIA can detect galactomannan antigen well before clinical or radiological signs appear, the occurrence of Invasive Aspergillosis cannot be ruled out. Therefore, patients treated with Zosyn® with positive test results should be followed carefully.

Bio-Rad has notified their customers and modified the Platelia™ Aspergillus EIA package insert to include a new Limitation of Use alerting laboratory users that positive test results in patients treated with Zosyn® should be interpreted cautiously and confirmed by other diagnostic methods.

It is therefore recommended that when reporting a positive Platelia™ Aspergillus EIA galactomannan test result, laboratories should inform physicians of the limitations of the test with regard to the potential interaction with Zosyn® and that the patient’s previous drug therapy should be taken into account.

FDA reminds users that life threatening or potentially threatening adverse device failures should be reported to the agency through MEDWATCH medical device reporting system. This may be done by visiting the MEDWATCH site at http://www.fda.gov/medwatch/ or calling 1-800-FDA-1088. Less serious adverse device failures may be reported to the FDA by sending an e-mail to fdalabtests@cdrh.fda.gov.

References:

1. Sulahian, A., S. Touratier, T. LeBlanc, P. Rousselot, F. Derouin, P. Ribaud. False Positive Aspergillus antigenemia related to concomitant administration of Tazocillin. Abstract M-2062a, 43^rd ICAAC Program.
2. Viscoli, C., M. Machetti, C. Cappellano, B. Bucci, P. Bruzzi, A. Bacigalupo. False-Positive Platelia Aspergillus test in patients receiving Piperacillin/Tazobactam. Abstract M-2062b, 43rd ICAAC Program.

Cautionary Note: Use of Backup Testing for Negative Rapid Group A Strep Tests

(posted 11/5/03)

Pharyngitis (sore throat) is caused by a number of different viruses or bacteria which set up shop in the throat and tonsil area. One of the most clinically significant bacterial organisms is group A beta-hemolytic streptococcus (GAS). Infection with GAS causes local pain and redness, bad breath, tonsillar or pharyngeal exudates and systemic symptoms like fever. Alternatively, patients may present with only mild throat pain and no fever. Anecdotal experience shows that some patients may only complain of bad breath and a history of GAS in a schoolmate or family member.^1,2,3

Serious complications of GAS include rheumatic fever, rheumatic heart disease and death. Historically, treatment with intramuscular penicillin within the first several days of symptoms prevented the rheumatic complications. Because intramuscular injection of penicillin is painful this has evolved into treatment with oral penicillins.^1,2

While the local throat infection may clear on its own in many patients, these untreated patients are still thought to be at increased risk of rheumatic complications. Unfortunately, the literature is clear that physical examination is insufficient to accurately distinguish GAS from other causes of pharyngitis which do not present any risk of rheumatic complications. Therefore, if a patient presents with appropriate signs and symptoms a throat test for strep is performed.

The historic test of choice was throat culture. A variety of very useful non-culture based methods are available which offer quick results. These rapid tests are invaluable when they are positive for strep, but caution is required when they are negative for a variety of reasons. Pediatric practice guidelines recommend all negative rapid tests in the face of overt clinical symptoms need to be supported with follow up testing.^1,4,5 This is conventionally viewed as throat culture on media which is designed to augment growth of GAS, though newer methodologies may change this recommendation.⁶ Notwithstanding, there is at least one study supporting clinical superiority of a rapid method compared to culture method.⁷

The situation in adults is more complex. While 15%-30% of children who present with pharyngitis have GAS, the percentage in adults may be as low as 5%-10%.^2,3 Also, it has been reported that adults who get GAS are at a lower risk for rheumatic complications. This has been offered as a rational supporting the lack of need for confirmatory testing in adults with a negative rapid test.⁸ However, there are studies suggesting that the rate of rheumatic complications in adults who present with local suppurative disease is equal to the rate in children.³ Further, some sources quote that up to 20% of first attacks occur in middle to later life.²

Since no rapid test has been cleared, approved, or waived through the regulatory process as a stand alone test in the face of locally suppurative disease, lack of a backup method for a negative rapid GAS test result constitutes off label use.

While the practice of medicine is not the province of FDA regulation, regulatory medicine, like clinical medicine, is always in the process of change and FDA welcomes information from academia or industry which may contribute to the evolution of this opinion.

Bibliography:

1. Committee on Infectious Diseases, Red Book: 2003 Report of the Committee on Infectious Diseases, 26’th Edition, Elk Grove Village IL, American Academy of Pediatrics, 2003, p 573-584.

2. Cotran RS, Kumar V, Collins T. Robbins Pathologic Basis of Disease, 6’th Edition, Philadelphia PA, WB Saunders Company, 1999, p 572.

3. Mandell GL, Bennett JE, Dolin R. Mandell. Douglass and Bennet’s Principles and Practice of Infectious Disease, 5’th Edition, Philadelphia PA, Churchill Livingstone, 2000, p 2119.

4. Bisno AL, et al. Diagnosis and management of Group A Streptococcal pharyngitis: A practice guideline. Clinical Infectious Diseases. 1997; 25:574-583.

5. Gieseker KE, et al. Comparison of two rapid Streptococcus pyogenes diagnostic tests with a rigorous culture standard. Pediatr Infect Dis J, 2002; 21:p 922-926.

6. Gieseker KE, et al. Evaluating the American Academy of Pediatrics diagnostic standard for Streptococcus pyogenes pharyngitis: backup culture versus repeat rapid antigen testing. PEDIATRICS June 2003. 111(6): e666-e670.

7. Uhl JR, et al. Comparison of LightCycler PCR, Rapid Antigen Immunoassay, and culture for detection of Group A Streptococci from throat swabs. J Clin Microbiol Jan 2003, 41(1): p 242-249.

8. Bisno AL, et al. Practice guidelines for the diagnosis and management of Group A Streptococcal pharyngitis. CID 2002; 35: p 113-125.

Follow Good Laboratory Practices with Waived Laboratory Tests

Background:

The Department of Health and Human Services has evaluated laboratory tests for complexity since 1992 as part of the implementation of the Clinical Laboratory Amendments of 1988 (CLIA '88). The more complicated the test, the more stringent the CLIA '88 requirements. Tests designated as high or moderate complexity can be used only by clinical laboratories that comply with CLIA '88 standards for quality assurance, quality control, and patient test management, and personnel. Simple tests designated as waived can be used by laboratories that enroll in CLIA and pay the fee but are exempt from CLIA '88 standards (i.e. community clinics, physicians' offices, or other small laboratories).

By law, waived tests should be simple to use and must provide acceptable results in the hands of untrained users. However, users must follow the manufacturer's instructions as well as good laboratory practices to ensure that test results are reliable.

Recommendations for Running Waived Tests:

In order to maximize the performance and reliability of waived tests, laboratories are encouraged to consider the following:

1. Read and follow the information found in the package inserts.
   Small deviations from the written instructions about product storage or procedural steps can have a significant impact on results. Test kits and reagents must be stored under the proper conditions and used prior to their expiration dates. You should take precautions to make sure that the correct reagents are used for each test, and reagents from different test kits are not inadvertently mixed up.
    
2. Follow the manufacturer's recommendations for running quality control (QC).
   Many waived tests have built-in quality controls that only monitor certain aspects of the test. In these cases, external quality controls may be the only way to monitor the entire test system. Read the information concerning built-in controls carefully. Manufacturers list minimum frequency requirements for their products in the package insert, but they may not be adequate for all laboratories. Take into account the environment where testing is performed when deciding how often you should run external QC. There are many factors that may be unique to your laboratory that can effect this decision, such as:
    
   • what information is provided by the built-in control;
   • the extent of safe-guards that are provided with the test system (such as a temperature monitor that alerts you when the test has been stored outside of acceptable limits),
   • the ambient environmental conditions of your laboratory;
   • storage conditions (such as whether the freezer has recycling phases);
   • personnel issues (understaffing or high turnover); or
   • the danger of a false negative test result.
      
   Select concentrations of control materials to challenge the medical decision points of the test. Selecting a control that has a concentration three times the cutoff concentration of a qualitative test will not allow you to detect reagent deterioration until patient results have already been compromised.
 
3. Train staff members to perform tests correctly. Make sure your staff is familiar with product operating instructions, quality control procedures, and record keeping. Professional organizations sponsor educational and proficiency testing programs geared toward staff that performs waived tests in outpatient or clinic laboratories. Some training courses are available over the internet.

See Also:

Centers for Medicaid and Medicare Services: CLIA '88 Program

Tests Waived by FDA from January 2000 to Present

Performance and Cautions in Using Rapid Influenza Virus Diagnostic Tests

FDA has cleared for marketing 7 rapid influenza diagnostic tests that can produce results within 30 minutes. These tests directly detect influenza A or B virus associated antigens or enzyme in throat swabs, nasal swabs, or nasal washes.

For the various products that have been FDA cleared for marketing Becton Dickinson’s Directogen Flu A test can detect only antigens associated with influenza A virus. Binax’s NOW Flu A, Binax’s NOW Flu B, and Becton Dickinson’s Directogen Flu A+B can detect and distinguish between influenza A and B virus antigens. Quidel’s QuickVue Influenza and Thermo BioStar FLU OIA can detect but do not distinguish between influenza A and B antigens. ZymeTx's ZstatFlu test can detect neuraminidase the presence of which denotes a high probability that infectious virions are present; it also does not distinguish between influenza A and B.

The Quidel Quick Vue and ZymeTx’s ZstatFlu and Quidel’s QuickVue influenza tests are considered low complexity and may be used in physicians’ offices. The other five tests mentioned above are considered moderately complex and are for use in a hospital or clinical reference laboratories.

If performed on individuals with signs and symptoms consistent with influenza, rapid influenza diagnostic tests appear to be moderately to reasonably accurate for detecting influenza virus dependent on when testing is performed during the influenza season. Potentially the rapid time for results can be very useful for managing patients with suspected influenza and for detection of institutional influenza outbreaks. However, positive testing must not be used alone to determine the cause of an individual’s symptoms. Often an individual may be co-infected with another pathogen that is the underlying cause of the symptom. Use of rapid influenza virus testing and missing an underlying cause was the subject of a public health advisory from the Centers for Drugs Evaluation and Research. 1,2 The basis for this advisory were several deaths where individuals were treated with antiviral medication but died from an underlying bacterial infection.

Laboratories should make sure that physicians using these rapid test results understand the limitations of the tests, use clinical experience, further laboratory testing, and consider local surveillance data about circulating influenza viruses when interpreting test results.

1 Public Health Advisory, Food And Drug Administration Public Health Advisory Subject: Safe And Appropriate Use Of Influenza Drugs, 12 January 2000, http://www.fda.gov/cder/drug/advisory/influenza.htm

2 FDA Talk Paper, FDA Reminds Prescribers of Important Considerations Before Prescribing Flu Drugs, January 12, 2000, http://www.fda.gov/bbs/topics/ANSWERS/ANS00995.html

The accuracy of an influenza test is determined by the sensitivity and specificity of the test to detect influenza virus antigens/enzyme compared with a reference method (cell culture) and the prevalence of influenza in the population being tested. Sensitivity is the percentage of culture confirmed influenza virus isolates detected by a test. Specificity is the percentage of virus negative cell cultures with negative rapid test results. Positive predictive value (PPV) of a test is the percentage of rapid test positive patients that have influenza virus isolated from cell culture. Negative predictive value (NPV) is the percentage of rapid test negative patients that do not have influenza virus isolated from cell culture.

Although the sensitivity and specificity of a test are unaffected by the prevalence of disease, the PPV and the NPV of a test are affected by disease prevalence. An influenza test will have the highest PPV (and lowest NPV) during peak influenza activity and the lowest PPV (and highest NPV) during periods of low influenza prevalence, i.e., at the beginning and ending of the influenza season.

Diagnostic antigen/enzyme tests for influenza are more likely to produce false positive results when the prevalence of influenza virus infections is low, such as during the late spring, summer, and early fall in North America. Confirming positive test results with viral culture is particularly important during this time period. Even when a positive test result reflects true influenza virus infection, this does not rule out the possibility of other co-existing infections. Persons with influenza are at risk for bacterial superinfections, and coincidental dual infections with other pathogens. Since some bacterial infections may have a fulminant course requiring urgent intervention, a positive test for influenza should not be used as a reason for withholding antibacterial therapy if clinical evaluation suggests a need.

False negative test results will be more common during periods of high influenza activity, such as during the winter in North America. Clinical judgment and local influenza surveillance data should be utilized to guide patient management. When compared to culture, FDA has found that the majority of rapid influenza virus assays have sensitivity >= 80 and <= 90%, and specificity in the same range. Most of these studies were performed during influenza season in the U.S. or the Southern Hemisphere.

Proper sample collection is the major determinant for obtaining accurate results. It has been reported in the literature and shown during studies conducted by companies that throat swabs are the poorest specimen source with properly collected nasal swabs or washes being the best. The adequacy of samples being tested should be carefully monitored before performing rapid tests and should be factored into interpretation of results.

FDA reminds users that life threatening or potentially threatening adverse device failures should be reported to the agency through the MEDWATCH medical device reporting system. This may be done by visiting the MEDWATCH WWW site at http://www.fda.gov/medwatch/ or calling 1-800-FDA-1088. Less serious adverse device failures may be reported to the FDA by simply sending an e-mail to fdalabtests@cdrh.fda.gov.

For additional information on using rapid tests for detecting influenza A viruses go to http://www.fda.gov/cdrh/oivd/tips/rapidflu.html

References

1. Covalciuc KA, Webb KH, Carlson CA. Comparison of four clinical specimen types for detection of Influenza A and B viruses by optical immunoassay (FLU OIA Test) and cell culture methods. J Clin Micro 1999; 37:3971-3974.

1. Boivin G, Hardy I, Kress A. Evaluation of a rapid optical immunoassay for influenza viruses (FLU OIA Test) in comparison with cell culture and reverse transcription-PCR. J Clin Micro 2002; 39:730-732.

1. Schultze D, Thomas Y, Wunderli W. Evaluation of an optical immunoassay for the rapid detection of influenza A and B viral antigens. Eur J Clin Microbiol Infect Dis 2001; 20:280-382.

1. Tucker SP, Cox C, Steaffens J. A flu optical immunoassay (ThermoBioStar’s FLU OIA): a diagnostic tool for improved influenza management. Philos Trans R Soc Lond B Biol Sci 2001; 356:1915-1024.

1. Yamazaki M, Mitamura K, Kimura K, Komiyama O. et al. Clinical evaluation of an immunochromatography test for rapid diagnosis of influenza. Kansenshogaku Zasshi. 2001; 75:1047-1053.

1. Rodriguez WJ, Schwartz RH, Thorne MM. Evaluation of diagnostic tests for influenza in a pediatric practice. Pediatr Infect Dis J 2002; 21:193-196.

1. Reina J, Padilla E, Alonso F, Ruiz DGE et al. Evaluation of a new dot blot enzyme immunoassay (Directigen flu A+B) for simultaneous and differential detection of influenza A and B virus antigens from respiratory samples. J Clin Micro 2002;40:3513-3517.

1. Uyeki TM. Influenza diagnosis and treatment in children: a review of studies on clinically useful tests and antiviral treatment for influenza. Pediatric Infect. Dis. J 2003; 22:164-177.

Updated 09/21/06

Functions and Limitations of Built-in Controls for Single Use Disposable Tests

Single-use disposable tests, such as visually read tests used for pregnancy or drug testing, often contain a built-in control. These controls are sometimes referred to as process or procedural controls. The following tips will help you understand how these controls function and their limitations.

• Built-in controls may not monitor the entire test system. There is significant variability among different devices in terms of how procedural controls work and the functions they serve. It is recommended that you read the package insert carefully, and pay particular attention to the description of the chemical or immunological reaction that takes place at the control line in the device being used. Use this information to determine what aspects of the test are monitored by the control and what variables are not monitored.

• Built-in controls are sometimes not a good indicator of the viability of test reagents. This is because the reactions occurring at control lines may be more robust than reactions occurring at the test line. For some immunochromatographic tests, the presence of a control line merely indicates that an adequate volume of sample was added and that the membrane strip is intact.

• You should select external control materials that have concentrations of the targeted analyte that are near to the cutoff concentration or medical decision level of the test. This will optimize your ability to detect early stages of reagent degradation.

• External controls are often the only way to monitor the entire test procedure, including pre-analytical steps, operator technique, procedural steps, and reagent viability.

• If the control line does not appear within the read time identified in the package insert, or if the appearance of the line or the background varies from the description provided by the manufacturer, e.g., the line is speckled instead of solid, you should not report patient results.

• When determining when and how often external controls should be run all laboratories should follow local, state, and federal regulatory requirements. Decisions about control frequency and type should be made in the context of the particular operating environment for the laboratory taking into account operator proficiency, the stability of the environment, the volume of testing, and other relevant factors.

Updated 5/1/03

Common Problems with the Use of Glucose Meters

Glucose Testing Tips:

Diabetes care has come a long way since the introduction of insulin and the first oral anti-hyperglycemic medicines. Life span and quality of life have improved for majority of affected individuals. Even better, a large part of diabetic care formerly performed in hospital clinics can now be managed at home with use of well designed home based glucose meters, a telephone and a good patient-doctor relationship.

The Office of In Vitro Diagnostics (OIVD) is charged with the job of evaluating many devices, including glucose meters. OIVD helps these meters come to the public market. Another of its tasks is the continuous evaluation of the same devices for long term safety and effectiveness not just of the devices, but of how the devices are used.

OIVD is taking this opportunity to provide some friendly tips in Point of Care glucose testing inspired by some comments we have received from manufactures and users of these devices.

Causes of false results may be patient/sample based or user/device based. Some common problems and their effects on meter glucose readings are listed below.

The advantage of Point of Care testing is eliminated if proper technique is not followed. In addition to the above recommendations, laboratory professionals must remember to wash hands and change gloves between patients. Also, clean the surface of the meter if blood gets on it. This each time, every time approach helps protect both the patient and the health care worker from blood borne agents like HIV and HCV.

All operators, from patients to non-lab health care workers to medical technologists and physicians, should be thoroughly familiar with any device prior to using it. The best way to do this is to read the package insert and user manual carefully before using a device for the first time. It sounds simple, and it is. If you have any questions, ask someone who is familiar with the device. Another option is calling the customer service telephone number located on most package inserts. The people on the other end are there to help. Another good tip is to reread the package insert every few months. It is a good practice and their may be changes.

Next, watch an experienced laboratory professional, doctor, nurse or diabetic educator perform the test. Then perform the test in front of someone who has experience in using the glucose meter and instructing others on its performance. Ask for tips.

Specific problems come up from time to time including glucose readings that don’t make sense. For example you might feel fine when the glucose meter reading is obviously too high or too low. Remember, the best way to resolve any questionable result, and the best sample from any sick patient, is still a venous blood sample tested at a central lab. Even then any result that does not fit the clinical picture needs to be investigated and, at a minimum, repeated.

For more information also see FDA’s diabetes website at: http://www.fda.gov/diabetes/

Updated 5/1/03

Make sure glucose meters used in testing neonates are FDA cleared for testing in neonates

Glucose Monitoring in Neonates:

Glucose determination in the blood or plasma is used in the diagnosis, monitoring and treatment of persons with diabetes mellitus. Neonates (full term birth to 30 days old) and premature infants (gestational age in weeks at birth up to original due date) represent a mixed group of individuals in whom glucose monitoring is crucial and who have a physiology that differs significantly from older children and adults. Prevention or treatment of hypoglycemia (low blood sugar) in this group is just as important as the recognition and treatment of hyperglycemia (high blood sugar.)

Most full term neonates are born with glucose reserves that last for about 6-24 hours and if feeding or intravenous nutrition is not started in that time period, glucose levels can fall dangerously low. Since if left untreated the child can die or survive with serious impairment, it is common practice to measure the blood glucose of neonates.

When “bedside” glucose monitors were first used for neonates, it was discovered that increased hematocrit levels could interfere with the function of some glucose monitors and produce false low glucose results. The precise mechanism of this interference is debated and may be method dependant. Some people think that the blood may interfere with color based reactions by interfering with light transmittance. An analogy is trying to count the raised fingers on someone’s hand when seen through glass versus tissue paper. In the first case it is easy. In the second situation the hand with some fingers may be recognized but the precise number is undefined. For other methods the blood may be too thick for the chemical reactions to go to completion so less glucose is detected.

FDA has published guidelines to aid the makers of these devices and several have been cleared for use in the neonatal period.

Clinicians should carefully review the claims made to help determine if the device in question is appropriate for neonatal use. It is also a good idea to perform a literature search to see if there are studies published in refereed journals investigating the candidate glucose meter in the population in question (e.g.: healthy full term newborns or critically ill preterm neonates.) Even so, no matter what device is used it is crucial that the end user clinicians validate the meters they are considering for use. The clinicians should work closely with the central lab and compare candidate devices against their central laboratory’s current reference method. Given the acuity of care, it may also be a good idea to periodically reevaluate these meters against the reference standard and to develop quality assurance and quality control programs.

In closing, increased hematocrit may interfere with some blood glucose meters. If a result does not make sense the clinician needs to do two things. First, reassess the patient. Second, if it is clinically appropriate, get a new sample and verify the result using an accepted reference method.

Bibliography:

Anonymous. Points to consider for portable blood glucose monitoring devices intended for bedside use in the neonate nursery. FDA Guidance Document (http://www.fda.gov/cdrh/ode/122.pdf). February 20, 1996.

Anonymous. Review Criteria Assessment of Portable Blood Glucose Monitoring In Vitro Diagnostic Devices Using Glucose Oxidase, Dehydrogenase or Hexokinase Methodology (Draft Document). FDA Guidance Document (http://www.fda.gov/cdrh/ode/gluc.html). February 14, 1996.

Girouard J, et al. Multicenter Evaluation of the Glucometer Elite XL Meter; an Instrument Specifically Designated for Use With Neonates. Diabetes Care. 23(8); 1149-1153. August 2000.

Henry JB. Clinical Diagnosis and Management by Laboratory Methods, 19’th Ed. Philadelphia, WB Saunders, 1996. pp 197-199.

Perkins SL, et al. Laboratory and Clinical Evaluation of Two Glucose Meters for the Neonatal Intensive Care Unit. Clinical Biochemistry. 31(2); 67-71. 1998.

Soldin SJ, Devairakkam PD, Adarwalla PK. Evaluation of the Abbot PCx® Point of Care Glucose Analyzer in a Pediatric Hospital. Clinical Biochemistry. 33(4); 319-321. 2000.

St-Louis P, Ethier J. An Evaluation of three glucose meter systems and their performance in relation to criteria of acceptability for neonatal specimens. Clinical Chimica Acta. 322; 139-148. 2002.

Updated June 19, 2003

Glucose Meter Test Results: Useful Tips to Increase Accuracy and Reduce Errors

Have you ever wondered why you got a bad glucose meter test result when there is nothing obvious wrong with your meter, your test strips are new, and you’ve been running glucose tests for years? The simple answer is that glucose meters are not perfect, and neither are the people who use them! This chart lists some tips to help you get the most accurate results from your glucose meter.

Updated July 14, 2003

Reporting Problems with Laboratory Tests and Equipment

Once an IVD device goes into widespread use, unforeseen problems can arise. FDA's premarket review cannot always detect adverse events that are rare or if the problems are related to the clinical use of the device, manufacturing problems, product labeling (including instructions for use), or user technique and skill. To identify these problems, FDA and manufacturers depend on reports from the individuals and facilities that use IVD devices.

What types of problems should be reported?

• Device problems including
  • reagent or instrument failure
  • defects in product design or development
  • product instability
  • any other device problems that compromise patient health or safety
  • failure to perform according to performance characterized in package insert
  • incorrect test results that cause or contributed to an incorrect patient diagnosis and/or treatment
     
• Use-related problems including
  • inadequate and/or misleading labeling or confusing user instructions
  • inadequate packaging or poor package design
  • any other user problems that compromise patient health or safety

Who should report IVD device problems?

• User facilities, including hospitals, ambulatory surgical facilities, nursing homes, outpatient treatment facilities, or outpatient diagnostic facilities are required to report
   
  • device-related events that have caused, or may have caused or contributed to a death to both the FDA and the manufacturer.
  • device-related events that have caused, or may have caused or contributed to a serious illness or injury to the manufacturer. If the medical device manufacturer is unknown, the serious injury is reported by the facility to FDA.
     
• All other IVD device users, including doctors and patients, are encouraged to voluntarily report device problems directly to the manufacturer and/or FDA whenever it is suspected that the product caused or contributed to an adverse outcome. FDA is interested in reports of:
   
  • product problems such as erroneous results (false positive or false negative),
  • unexplained quality control (QC) failures,
  • inaccurate or unreadable instructions for use,
  • packaging or product mix-up,
  • contamination or stability problems,
  • defective devices,
  • product confusion caused by name, labeling, design, or packaging,
  • "near misses" where under slightly different circumstances, a serious injury or death might have occurred, or
  • use error

How should IVD device problems be reported?

• Mandatory Reporting by User Facilities (MDR)
• Voluntary Reporting by Consumers (MedWatch)
• Voluntary Reporting by Healthcare Professionals (MedWatch)

If you want to notify OIVD directly of a problem with a laboratory test or other equipment, contact us at fdalabtest@cdrh.fda.gov.

What happens to your report?

When FDA receives a report from a user facility, device user or an individual healthcare professional, it is entered in the medical device postmarket surveillance database. FDA continually reviews the database to detect problems, trends, and potential hazards. When FDA detects risks or potential risks associated with the use of a particular product, the agency can take corrective actions and notify the public.

Who has access to your report?

FDA is aware that IVD device users are concerned about the issue of confidentiality and public availability of reports. For all reports, FDA holds the patient's identity in strict confidence and protects it to the fullest extent of the law. FDA will not release any patient identifiers to the public. Reporters can assist in this process by not using the patient's name, initials, or other identifying information. The reporter's identity, including the identity of a self-reporter, may be shared with the manufacturer unless requested otherwise (there is a check-off box on the report form). However, FDA will not disclose the reporter's identity in response to a request from the public, pursuant to the Freedom of Information Act.

See also:

Medical Device Reporting for User Facilities

MedWatch: The FDA Safety Information and Adverse Event Reporting Program