nal 252017N-05 NADAL Mononucleosis Test Instruction Manual

nal 252017N-05 NADAL Mononucleosis Test

Intended Use

The NADAL® Mononucleosis Test is a rapid, chromatographic immunoassay for the qualitative detection of heterophile antibodies to Epstein-Barr virus (EBV) in human whole blood, serum or plasma. The test is intended for use as an aid in the diagnosis of infectious mononucleosis (IM) and designed for professional use only.

Introduction and Clinical Significance

Infectious Mononucleosis (IM) is caused by the Epstein-Barr virus (EBV), which is a member of the herpesvirus family. The symptoms of IM are: fever, sore throat and swollen lymph glands. In very rare cases, heart or central nervous system problems may occur. Diagnosis of IM is made based on the presence of heterophile anti-EBV antibodies. Heterophile anti- EBV antibodies belong to the IgM class. They are present in 80-90% of acute IM cases and can be detected in 60-70% of patients during the first week of clinical illness.1,2,3,4 The NADAL® Mononucleosis Test is a simple test that utilises an extract of bovine erythrocytes to qualitatively and selectively detect heterophile anti EBV antibodies in whole blood, serum or plasma in just minutes.

Test Principle
The NADAL® Mononucleosis Test is a lateral flow immunoassay for the qualitative detection of heterophile antibodies to Epstein-Barr virus (EBV) in whole blood, serum or plasma.
In this test, antigens extracted from bovine erythrocytes are immobilised in the test line region of the test cassette. During testing, the specimen reacts with further antigens extracted from bovine erythrocytes and precoated onto particles which have been applied to the conjugate pad. The mixture migrates chromatographically along the membrane and interacts with the immobilised antigens. If the specimen contains heterophile anti-EBV antibodies, a coloured line will appear in the test line region, indicating a positive result. If the specimen contains no heterophile anti-EBV antibodies, no coloured line will appear in the test line region, indicating a negative result.

To serve as a procedural control, a coloured line should always appear in the control line region (C), indicating that a proper volume of specimen has been added and membrane wicking has occurred.

Reagents and Materials Supplied

• 5 NADAL® Mononucleosis test cassettes incl. disposable pipettes
• 1 buffer
• 1 positive control (diluted human plasma containing  heterophile anti-EBV antibodies, 0.09% sodium azide)
• 1 negative control (diluted human plasma, 0.09% sodium azide)
• 1 package insert

Additional Materials Required

• Specimen collection containers
• Centrifuge (for serum/plasma)
• Timer
• Lancet (for fingerstick whole blood only)
• If necessary, heparinised capillary tubes and dispensing bulb (for fingerstick whole blood only)

Storage & Stability
Test kits should be stored at 2-30°C and used before the expiry date printed on the packaging. Test cassettes are stable until the expiration date printed on foil pouches. Test cassettes should remain in sealed pouches until use. Do not freeze tests.
Do not use tests beyond the expiration date.

Warnings and Precautions

• For professional in-vitro diagnostic use only.

• Carefully read through the test procedure prior to testing.

• Do not use the test beyond the expiration date indicated on the package.

• Do not use the test if the foil pouch is damaged.

• Do not reuse tests.

• Do not add samples to the reaction area (result area).

• In order to avoid contamination, do not touch the reaction area (result area).

• Avoid cross-contamination of specimens by using a new specimen collection container for each specimen obtained.

• Do not eat, drink or smoke in the area where specimens and test kits are handled.

• Wear protective clothing such as laboratory coats, disposable gloves and eye protection when specimens are being assayed.

• Handle all specimens as if they contain infectious agents.
  Observe established precautions for microbiological risks throughout all procedures and standard guidelines for the appropriate disposal of specimens.

• The test kit contains products of animal origin. Certified knowledge of the origin and/or sanitary state of the animals does not completely guarantee the absence of transmissible pathogenic agents. It is therefore recommended that these products be treated as potentially infectious, and handled in accordance with usual safety precautions (e.g., do not ingest or inhale).

• Humidity and temperature can adversely affect test results.

• Used testing materials should be discarded according to local regulations.

Specimen Collection and Preparation

The NADAL® Mononucleosis Test can be performed using whole blood (from venipuncture or fingerstick), serum or plasma.
To collect venipuncture whole blood specimens:
Collect blood specimens with anticoagulants (sodium or lithium heparin, potassium or sodium EDTA, sodium oxalate, sodium citrate) following standard laboratory procedures.

To collect fingerstick whole blood specimens:

• Wash the patient’s hand with soap and warm water or clean it with an alcohol swab. Allow it to dry.
• Massage the hand without touching the intended puncture site by rubbing down the hand towards the fingertip of themiddle or ring finger.
• Puncture the skin with a sterile lancet. Wipe away the first  sign of blood.
• Gently rub the hand from wrist to palm to finger to form a rounded drop of blood over the puncture site.

Separate serum or plasma from blood as soon as possible to avoid haemolysis. Use only clear, non-haemolysed specimens.
Testing should be performed immediately after specimen collection. Do not leave specimens at room temperature for prolonged periods of time. Serum and plasma specimens can be stored at 2-8°C for up to 3 days. For long-term storage, specimens should be kept below -20°C. Whole blood collected by venipuncture should be stored at 2-8°C if the test is to be run within 2 days of collection. Do not freeze whole blood specimens. Fingerstick whole blood should be tested immediately. Bring specimens to room temperature prior to testing. Frozen specimens should be completely thawed and mixed well prior to testing. Specimens should not be frozen and thawed repeatedly. If specimens are to be shipped, they should be packed in compliance with federal regulations for transportation of etiologic agents.

Test Procedure

Bring tests, buffer, specimens and/or controls to room temperature (15-30°C) prior to testing.

1. Remove the test cassette from the sealed foil pouch and place it on a clean and level surface. The test cassette should be used immediately after opening the foil pouch.
   Label the test cassette with the patient or control identification.

2.  a) For serum or plasma specimens:
   Holding the dropper vertically, add 1 drop (approximately 25 μL) of serum or plasma to the sample well (S) of the test cassette.
   b) For venipuncture whole blood specimen:
   Holding the dropper vertically, add 2 drops (approximately 50 μL) of whole blood to the sample well (S) of the test cassette.
   c) For fingerstick whole blood specimen:
   Apply the end of the capillary tube to the blood until it is filled with approximately 50 μL. Avoid air bubbles. Place the bulb onto the top end of the capillary tube and squeeze the bulb to dispense the whole blood specimen into the sample well (S) of the test cassette.

3. Holding the buffer bottle vertically, add 1 drop (approximately 55 μL) of buffer to the sample well (S).

4. Start the timer.

5. Wait for the coloured line(s) to appear. Read the result after 5 minutes. Do not interpret the result after more than 10 minutes.

Result Interpretation

Positive:
Two coloured lines appear on the membrane. One line appears in the control line region (C) and the other line appears in the test line region (T).

Note:
The colour intensity in the test line region (T) may vary depending on the concentration of anti-EBV antibodies present in the specimen. Therefore, any shade of colour in the test line region (T) should be considered positive.

Negative:
One coloured line appears in the control line region (C). No apparent coloured line appears in the test line region (T).

Invalid:
The control line (C) fails to appear.
Results from any test which has not produced a control line at the specified reading time must be discarded. Please review the procedure and repeat the test with a new test cassette. If the problem persists, discontinue using the test kit immediately and contact your distributor.

Insufficient specimen volume, incorrect operating procedure or expired tests are the most likely reasons for the control line failure.

Quality Control
An internal procedural control is included in the test cassette:
A coloured line appearing in the control line region (C) is considered an internal procedural control. It confirms sufficient specimen volume, adequate membrane wicking and correct procedural technique.
Good laboratory practice (GLP) recommends the use of control materials to ensure proper test kit performance.

Procedure for External Quality Control Testing

1. Holding each bottle vertically, add 1 drop (approximately 40 μL) of positive or negative control solution to the sample well (S) of the test cassette. Then, holding the bottle vertically, add 1 drop (approximately 55 μL) of buffer to the sample well (S).
2. Continue with step 4 of ‘Test Procedure’.
3. If the controls do not yield the expected results, do not use the obtained results. Repeat the test or contact your distributor.

Limitations

• The NADAL® Mononucleosis Test is for in-vitro diagnostic use only. The test should only be used for the detection of anti-EBV antibodies in whole blood, serum or plasma specimens. Neither the quantitative value nor the rate of increase in concentration of anti-EBV antibodies can be determined by this qualitative test.

• The NADAL® Mononucleosis Test only detects the presence of anti-EBV antibodies in specimen and should not be used as the sole criterion for diagnosis of infectious
  mononucleosis infection.

• As with all diagnostic tests, all results obtained with this test should be interpreted in conjunction with other clinical information available to the physician.

• If the test result is negative and clinical symptoms persist, additional testing using other clinical methods is recommended. A negative result does not at any time preclude the possibility of IM infection.

Expected Results
Infection with Epstein-Barr virus (EBV) during adolescence or young adulthood causes infectious mononucleosis (IM) in 35% to 50% of reported cases.1,5 The incidence of EBV-associated IM in the USA has been estimated at 45 per 100,000 and is the highest in adolescent and young adults – roughly 2 out of 1,000. No seasonal pattern of EBV infection exists. The incubation period is 10 to 60 days, though the incubation period of 7 to 14 days is common amongst children and adolescents.

Performance Characteristics

Sensitivity
The NADAL® Mononucleosis Test has been evaluated with specimens confirmed positive or negative by a leading, commercially-available slide agglutination test. The slide agglutination test served as a reference method for the NADAL® Mononucleosis Test. The result shows that the sensitivity of the NADAL® Mononucleosis Test is >99.9% relative to the slide agglutination test.

Specificity
The NADAL® Mononucleosis Test uses antigens that are highly specific for anti- EBV antibodies in whole blood, serum or plasma. The results show that the specificity of the NADAL®
Mononucleosis Test is 98.9% relative to the slide agglutination test.

NADAL® Mononucleosis Test vs. slide agglutination test

| Slide agglutination test
---|---

NADAL®

Mononucleosis Test

| Positive          Negative          Total
Positive

Negative Total

| 60| 1| 61
0| 89| 89
60| 90| 150

• Relative sensitivity: >99.9% (95.1% – 100%)*
• Relative specificity: 98.9% (94% – 99.9%)*
• Overall agreement: 99.3% (96.3% – 99.9%)*
• 95% Confidence interval

Interfering substances
Negative and middle-positive plasma and serum specimens (ELISA confirmed) were spiked with the analytes at the concentrations listed below and tested in triplicate.

The analytes showed no interference with the NADAL®
Mononucleosis Test. There were no obvious differences among the results obtained with three lots of tests.

Precision
Inter- and intra-lot variability
Three independent lots of the NADAL® Mononucleosis Tests were tested with negative as well as low, medium and high positive serum and plasma specimens in 10-fold
determinations. All results matched the expectations. No deviating results were obtained within the 10-fold determinations or between the different lots.

Cross-reactivity
Samples, positive for the following parameters were tested with the NADAL® Mononucleosis Test:

No cross-reactivity was observed indicating that the NADAL®
The mononucleosis Test has a high degree of specificity for human anti-EBV antibodies.

References

1. Hickey SM, Strasburger VC. What Every Pediatrician Should Know About Infectious Mononucleosis In Adolescents. Pediatr Clin North Am. 1997;44(6):1541-56.
2. Omori M. Mononucleosis http://www.emedicine.com/EMERG/topic309.htm
3. Linde A. Diagnosis of Epstein-Barr-Virus-related diseases. Scand J Infect Dis Suppl. 1996; 100:83-8.
4. Papesch M, Watkins R. Epstein-Barr-Virus infectious mononucleosis. Clin Otolaryngol. 2001; 26(1):3-8.
5. CDC National Center for Infectious Diseases. EBV & IM: http://www.cdc.gov/ncidod/diseases/ebv.htm

Symbol

CE marking of conformity

Consult instructions for use

In-vitro diagnostic medical device

Temperature limitation

Batch code

Do not reuse

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References

• nal von minden GmbH - Spezialist für hochwertige in-vitro Diagnostik
• Meningitis: Practice Essentials, Background, Pathophysiology
• nal von minden GmbH - Spezialist für hochwertige in-vitro Diagnostik

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