PRAXISDIENST 351006N-05 NADAL D-Dimer Test Instruction Manual

NADAL® D-Dimer Test (test cassette)
REF 351006N-05/351006N-10/351006N-25

Intended Use

The NADAL® D-Dimer Test is used for the qualitative detection of D-Dimer in human whole blood and plasma. The test is used as an aid in the assessment and evaluation of patients with suspected disseminated intravascular coa-gulation (DIC), deep vein thrombosis (DVT) and pulmonary embolism (PE).

Introduction

During blood coagulation process, fibrinogen is converted to fibrin by the activation of thrombin. The resulting fibrin monomers polymerise to form a soluble gel of non-crosslinked fibrin. This fibrin gel is then converted to cross-linked fibrin by thrombin activated factor XIII to form an insoluble fibrin clot. Production of plasmin, the major clot-lysing enzyme, is triggered when a fibrin clot is formed. Although fibrinogen and fibrin are both cleaved by the fibrinolytic enzyme plasmin to yield degradation products, only degradation products from cross-linked fibrin contain D-Dimer and are called cross-linked fibrin degradation products. Therefore, fibrin derivatives in human blood or plasma containing D-Dimer are a specific marker of fibrinolysis. The detection limit of the NADAL® D-Dimer Test is 500 ng/mL D-Dimer.

Test Principle

The NADAL® D-Dimer Test (whole blood/ plasma) detects D-Dimer through visual interpretation of color development in the internal strip. Anti-D-Dimer antibodies are immobilized onthe test region of the membrane, and anti-mouse antibodies are immobilized on the control region. During testing, the specimen reacts with anti-D-Dimer antibodies conjugated to colored particles and precoated onto the specimen pad of the strip. The mixture then migrates through the membrane by
capillary action and interacts with reagents on the membrane.If there is sufficient D-Dimer in the specimen, a colored band will form at the test region of the membrane. The presence of this colored band indicates a positive result, while its absence indicates a negative result. The appearance of a colored band at the control region serves as a procedural control, indication that the proper volume of specimen has been added and membrane wicking has occurred.

Reagents and Materials Supplied

• 5/10/25 NADAL® D-Dimer test cassettes, incl. disposable pipettes
• 1/2/5 buffer bottle(s)
• 1 package insert

Additional Materials Required

• Specimen collection container
• Centrifuge
• Timer

Storage & Stability

• The kit should be stored at 2-30°C until the expiry date printed on the sealed pouch.
• The test must remain in the sealed pouch until use.
• Do not freeze!
• Care should be taken to protect the components of the kit from contamination. Do not use if there is evidence of microbial contamination or precipitation. Biological conta- mination of dispensing equipments, containers or reagents can lead to false results.

Warnings and Precautions

• This 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 observing usual safety precautions (e.g. do not ingest or inhale).
• Avoid cross-contamination of specimens by using a new specimen collection container for each specimen obtained.
• Read the entire procedure carefully prior to testing.
• Do not eat, drink or smoke in the area where the specimens and kits are handled. Handle all specimens as if they contain infectious agents. Observe established  recautions against microbiological hazards throughout the procedure and follow standard procedures for proper disposal of specimens. Wear protective clothing such as laboratory coats, disposable gloves and eye protection when speci-mens are assayed.
• Do not interchange or mix reagents from different lots.
•  Humidity and temperature can adversely affect results.
• Used testing materials should be discarded according to local regulations.

Specimen Collection and Preparation

Specimen Collection

• The NADAL® D-Dimer Test (whole blood/plasma) is intended for use with human whole blood or plasma specimens only.

• Only clear, non-hemolyzed specimens are recommended for use with this test. Plasma should be separated as soon as possible to avoid hemolysis.

• Containers containing anticoagulants such as EDTA, citrate, or heparin should be used for whole blood storage.

• Bring specimens to room temperature prior to testing. Frozen specimens must be completely thawed and mixed well prior to testing. Avoid repeated freezing and thawing of specimens.

• Icteric, lipemic, hemolysed, heat treated and contaminated specimens may cause erroneous results.
  Specimen Transport and Storage

• Perform testing immediately after specimen collection. Do not leave specimens at room temperature for prolonged periods. Plasma specimens may be stored at 2-8°C for up to 1 day. 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 performed within 1 day of collection. Do not freeze whole blood specimens! Whole blood collected by fingerstick should be tested immediately.

• If specimens are to be shipped, pack them in compliance with all applicable regulations for transportation of etiological agents.

Test Procedure

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

1. Remove a test cassette from the foil pouch and use it as soon as possible. The best results will be obtained if the test is performed immediately after opening the foil pouch. Label the test cassette with the patient’s name or control identification.

2. Place the test cassette on a clean and level surface.

3. a) For plasma specimens:
   Holding a pipette vertically, add 1 drop (approximately 25 μL) of the plasma specimen to the specimen well (S) of the test cassette.
   b) For venipuncture whole blood specimens:
   Holding a pipette vertically, add 2 drops (approximately 50 μL) of the whole blood specimen to the specimen well (S) of the test cassette.
   c) For fingerstick whole blood specimens:
   Position the patient’s finger so that a drop of blood is exactly above the specimen well (S) of the test cassette. Allow 1 hanging drop of fingerstick whole blood (approximately 50 μL) to fall into the centre of the specimen well (S) of the test cassette.

4. Holding the buffer bottle vertically, add 1 drop of buffer to the specimen well (S) of the test cassette.

5. Start the timer.

6. Wait for the coloured line(s) to appear. Read the test result after 10 minutes. Do not interpret the result after more than 20 minutes.

Result Interpretation

Positive result
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 the analyte present in the specimen. Therefore, any shade of colour in the test line region (T) should be considered positive. Note that this is a qualitative test only and it cannot determine the analyte concentration in the specimen.
Negative result
One coloured line appears in the control line region (C). No apparent coloured line appears in the test line region (T).
Invalid result
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.
Note:
Insufficient specimen volume, incorrect operating procedure or expired tests are the most likely reasons for control line failure.

Quality Control

 Internal procedural controls are included in the test. A colored band appearing in the control region (C) is considered an internal positive procedural control, confirming sufficient specimen volume and correct procedural technique.
 External controls are not supplied with this kit. It is recommended that positive and negative controls be tested as a good laboratory practice to confirm the test procedure
and to verify proper test performance.

Limitations

 The NADAL® D-Dimer Test (whole blood/ plasma) is for professional in-vitro diagnostic use and should only be used for the qualitative detection of D-Dimer.
 Clinical diagnosis should not be based on the result of the D-Dimer rapid test only. The full clinical context of the patient should be included when making a diagnostic
decision, taking into account the clinical signs and other relevant information such as the «Well’s pre-test probability score» or equivalent.
 Negative D-Dimer results can occur very occasionally even in the presence of a DVT due to other factors including the age or position of a clot, heparin therapy and when the D-Dimer concentration is below the sensitivity of the test.

Expected Values

Elevated levels of D-Dimer are an indication of active fibrinolysis and have been shown in patients with disseminated intravascular coagulation (DIC), deep vein thrombosis (DVT) and pulmonary embolism (PE). Elevated levels of D-Dimer have also been reported in surgery, trauma, sickle cell disease, liver disease, severe infection, sepsis, inflammation, malignancy and in the elderly. D-Dimer levels also rise during normal pregnancy but very high levels are associated with complications.

Performance Characteristics

Analytical sensitivity
The detection limit of the NADAL® D-Dimer Test is 500 ng/mL (fibrinogen equivalent units: FEU). No hook effect was observed when testing a specimen containing a D-Dimer concentration as high as 50 µg/mL.
Diagnostic sensitivity and specificity
A clinical study was performed on 149 negative plasma specimens (EIA confirmed, Roche Cobas c701) and 153 positive plasma specimens (EIA confirmed). The results are presented in the following table:

| |

EIA

---|---|---
NADAL® D-Dimer Test| | +| –| Total
+| 151| 16| 167
–| 2| 133| 135
Total| 153| 149| 302

Relative sensitivity: 151/(151+2) = 98.7% (96.91% – 100%)
Relative specificity: 133/(133+16) = 89.3% (84.34% – 94.26%)
Overall agreement: (151+133)/(151+2+133+16) = 94.0% (91.36% – 96.72%)
95% Confidence interval
Cross-reactivity
1 mg/mL fibrinogen, 25 µg/mL fragment D and 25 µg/mL fragment E do not cross- react with the NADAL® D-Dimer Test.
Elevated levels of rheumatoid factors (RF) or heterophile antibodies might interfere with test results.
Interfering substances
Negative and positive specimens spiked with the following potentially interfering substances were evaluated in triplicates using the NADAL® D-Dimer Test.

None of the substances interfered with the assay at the concentrations tested.
Precision
Repeatability and reproducibility
Repeatability was established by testing 10 replicates of 3 specimens (0 ng/mL, 500 ng/mL and 2000 ng/mL D-Dimer) with each of 3 independent NADAL® D-Dimer test lots.
Reproducibility was established by testing triplicates of 3 specimens (0 ng/mL, 500 ng/mL and 2000 ng/mL D-Dimer) with 3 independent NADAL® D-Dimer test lots.
The NADAL® D-Dimer Test demonstrated acceptable repeatability and reproducibility. The negative and positive values were correctly identified

99% of the time.

References

1. Gaffney, P.J. D-dimer History of Discovery, Characterisation and Utility of this and other Fibrin Fragments. Fibrinolysis 7 Suppl 2:2-8; 1993

2. Lane, D.A. et al. Characterisation of Serum Fibrinogen and Fibrin Fragments Produced During Disseminated Intravascular Coagulation. Haematology. 40: 609615; 1978.

3. Keeling, D.M. et al. The Haemostasis and Thrombosis Task Force of the British Committee for Standards in Haematology. The diagnosis of deep vein thrombosis in
   symptomatic outpatients and the potential for clinical assessment and D-dimer assays to reduce the need for diagnostic imaging. Br. J. Haematol. 124(1): 1525;2004.

4. Bick, R.L. et al. Diagnostic Efficacy of the D-dimer assay in Disseminated Intravascular Coagulation (DIC) Thromb. Res. 65:785-790; 1992.

5. Bick, R.L. et al. Disseminated Intravascular Coagulation: Objective Clinical and Laboratory Diagnosis, Treatment, and Assessment of Therapeutic Response. Semin.
   Thromb. Hemost. 22(1): 69-88; 1996.

6. Scarvelis, D and Wells, P.S. Diagnosis and Treatment of Deep Vein Thrombosis. Can. Med. Assoc. J. 175 (9):1087-92; 2006

7. Subramanian, R.M. et. al. Does an Immunochromatographic D-dimer exclude acute lower limb deep venous thrombosis? Emer. Med. Austral. 18: 457-463; 2006.

8. Runyon, M.S. et. al. Comparison of the Simplify D-dimer assay performed at the bedside with a laboratory based quantitative D-dimer assay for the diagnosis of pulmonary embolism in a low prevalence emergency department population. Emerg. Med. J. 25:70-75; 2008.

9. Ginsburg, J.S. et. al. Sensitivity and specificity of a rapid whole-blood assay for Ddimer in the diagnosis of pulmonary embolism. Ann. Intern. Med. 129(12), 1006-11; 1998.

10. Hunt, F.A. et al. Serum Cross-Linked Fibrin (XDP) and Fibrinogen/Fibrin Degradation Products (FDP) in Disorders Associated with Activation of the Coagulation or
    Fibrinolytic Systems. Br. J. Haematol. 60: 715-722; 1985.

11. Smith, R.T. et al. Fibrin Degradation Products in the Post-Operative PeriodEvaluation of a New Latex Agglutination Method. AJCP. 60: 644-647; 1973.

12. Nolan, T.E. et al. Maternal Plasma D-dimer Levels in Normal and Complicated Pregnancies. Obstetrics & Gynecology. 81(2): 235-238, 1993.

Rev. 1, 2021-04-08 FS/SS

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