D-dimer is a fibrin fragment that results when plasmin acts on cross-linked fibrin in the presence of factor XIII.
50 of piasma
100 |l of PT reagent
3 min incubation
Figure 20.14 Prothrombin time procedure.
50 of plasma
50 |l of aPTT reagent
Figure 20.15 Activated partial thromboplastin time procedure.
3 min incubation
3 min incubation
Therefore, D-dimers are formed from an insoluble fibrin clot. This semiquantitative assay, available since the 1990s, provides evidence of normal or abnormal levels of D-dimer. Latex particles are coated with mouse anti-D-dimer monoclonal antibodies. When mixed with plasma containing D-dimers, agglutination will occur. The test plays an important role in detecting and monitoring patients suspected of thrombotic disorders. Its clinical uses are for detecting deep vein thrombosis (DVT), pulmonary embolism (PE), and, in patients with disseminated intravascular coagulation (DIC), postoperative complications or septicemia. Quantitative d-dimer procedures are available, using latex-enhanced turbidimetric methods. The qualitative test, however, has widespread use in most coagulation laboratories as a screening test for D-dimers.
Reagents and Equipment
1. D-dimer kit containing reagents (stored at 2 to 8°C), good until expiration date on the kit.
a. Test reagent solution containing red cell anti-XL-FDP antibody conjugate b. Negative control solution containing 0.9% saline solution c. Positive control solution containing purified D-dimer fragment
2. Plastic agglutination trays
3. White plastic stirrers
5. Pipette 10 pL with disposable tips
Specimen Collection and Storage
1. Collect venous whole blood into a vacuum tube with 3.2% sodium citrate. There needs to be a 9:1 dilution of blood to anticoagulant.
a. 4.5 mL of blood with 0.5 mL of anticoagulant OR
b. 2.7 mL of blood with 0.3 mL of anticoagulant
2. Collection of venous blood into heparin is acceptable.
3. Store specimens at 18 to 24°C. Specimens should be tested within 4 hours from the time of specimen collection. If testing will take place after 4 hours, specimens must be refrigerated at 2 to 8°C and are good up to 24 hours.
1. Quality control is performed under several conditions a. Daily b. When opening a new kit c. When receiving a new shipment d. When a new lot number is put into use
2. A whole blood sample that has a negative D-dimer result is used for the quality control.
3. Quality control method a. Follow directions in the procedure to do the quality control, steps 1 through 5.
b. Now add 1 drop of positive control to the test well, and proceed with steps 6 through 8b.
1. Allow reagents to come to room temperature for at least 20 minutes before use.
2. Specimen should be thoroughly mixed; do not allow cells to settle out.
3. For each sample, pipette 10 pL of whole blood into each reaction well; the first labeled (nega-
320 PartV • Laboratory Procedures tive control well) and (test well) on a plastic agglutination tray.
4. Add 1 drop of the negative control to the negative control well.
6. With a plastic stirrer, mix the contents of each well thoroughly for 3 to 5 seconds, using a different stirrer for each well and spreading the reagent across the entire well surface.
7. To promote agglutination, mix by gentle rocking of the plastic agglutination tray for 2 minutes.
8. At the end of the 2 minutes, observe for the presence of agglutination.
a. Positive results: agglutination is present in the test well compared to no agglutination in the negative well.
b. If the negative control well agglutinates, the test is invalid.
c. If the test result is negative, add 1 drop of positive control to the test well and rock the plastic tray. Agglutination should occur within 15 seconds. If agglutination does not occur with the addition of the positive control, the test is invalid.
1. Positive: Agglutination seen in the test well and no agglutination seen in the negative control well.
2. Negative: No agglutination seen in the test well and the negative control well. This would be confirmed by adding the positive control to the test well and agglutination occurs.
3. Invalid a. Agglutination occurs in the negative control well.
b. No agglutination occurs with the positive control.
Negative: No agglutination seen in negative agglutination well (<0.5 mg/L) Positive: Agglutination seen in undiluted sample (0.5 to 4.0 mg/L) Positive samples can be diluted 1:8 or 1:64 to provide more specific data on the amount of D-dimer present.
The presence of cold agglutinins in patient samples can cause agglutinations in patient's blood. This may cause agglutination of the negative control, thereby invalidating the test results.
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