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APPENDIX III
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intrinsic pathway. To standardize the activation of contact factors, the plasma is pre-incubated with the kaolin platelet substitute mixture. During this phase, factor XIIa is produced which cleaves factor XI to factor XIa but coagulation does not proceed beyond this in the absence of calcium. When calcium is added, factor XIa activates factor IX and coagulation commences. The test depends on contact factors, factors VIII and IX but also on the reactions with factors X,V, prothrombin and fibrinogen. It is sensitive to the presence of anticoagulants and heparin in plasma.
Reagents
Platelet poor plasma
Diagen Kaolin Platelet Substitute Mixture Lot No.
Procedure
Warm up the coatron M2 coagulation analyzer for 2-3 minutes. Using a
micropipette, dispense 0.2ml of kaolin platelet substitute into the micro-cuvette and incubate in coagulometer at 370C. 0.1ml of test plasma is added and left in cuvette for 2 minutes at same temperature. 0.1ml of 0.025M Cacl2 (pre-warmed at 370C) is added and this automatically commences the test. The time taken for the change in optical density from plasma to clot formed is then reported. Each test is done in duplicate and results are expressed in mean of the duplicate reading. Normal range is between 36-50 seconds.
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Principle of antithrombin assay
The USCN human antithrombin kit is a sandwich enzyme immunoassay for in vitro measurement of antithrombin in human plasma. Standards or samples are added to the appropriate microtitre plate wells with a biotin conjugated antibody specific to antithrombin. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microtitre plate well and incubated. After TMB
substrate solution is added, only those wells that contain antithrombin, biotin-conjugated antibody and enzyme – biotin-conjugated Avidin will exhibit change in colour. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the colour change is measured spectrophotometrically at a wavelength of 450nm±10nm. The concentration of antithrombin is then determined by comparing the optical density of the samples to the standard curve.
Assay procedure (Summary)
1. Prepare all reagents, samples and standards as instructed. Determine 7 wells for standard, 1 well for blank and samples.
2. Add 100µl standard or sample to each well. Incubate for 2 hours at 370C.
3. Aspirate but don’t wash. Add 100 µl prepared Detection Reagent A into each well. Incubate for 1 hour at 370C after covering it with plate sealer.
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4. Aspirate and wash 3 times with 350 µl of Wash Solution. After the each wash, remove the liquid by aspirating or decanting then snap plate against absorbent paper.
5. Add 100µl prepared Detection Reagent B into each well. Incubate for 30 minutes at 370C after covering it with the plate sealer.
6. Aspirate and wash 5 times
7. Add 90 µl Substrate Solution. Incubate 15-25 minutes at 370C while protecting from light. The liquid will turn blue by the addition of Substrate Solution.
8. Add 50 µl Stop Solution. The liquid will turn yellow by the addition of Stop Solution. Mix the liquid by tapping side of the plate to ensure a uniform colour change
9. Remove any water or fingerprint at the bottom of the plate and confirm there is no bubble on the surface of the liquid. Run the microplate reader and conduct measurement at 450nm immediately.
Principle of PAI-1 assay
The USCN human PAI-1 kit is a sandwich enzyme immunoassay for in vitro measurement of PAI-1 in human plasma. Standards or samples are added to the appropriate microtitre plate wells with a biotin conjugated antibody specific to
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PAI-1. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microtitre plate well and incubated. After TMB substrate solution is added, only those wells that contain PAI-1, biotin-conjugated antibody and enzyme – conjugated Avidin will exhibit change in colour. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the colour change is measured spectrophotometrically at a wavelength of 450nm±10nm. The
concentration of PAI-1 is then determined by comparing the optical density of the samples to the standard curve.
Assay procedure (Summary)
1. Prepare all reagents, samples and standards as instructed. Determine 7 wells for standard, 1 well for blank and samples.
2. Add 100µl standard or sample to each well. Incubate for 2 hours at 370C.
3. Aspirate but don’t wash. Add 100 µl prepared Detection Reagent A into each well. Incubate for 1 hour at 370C after covering it with plate sealer.
4. Aspirate and wash 3 times with 350 µl of Wash Solution. After the each wash, remove the liquid by aspirating or decanting then snap plate against absorbent paper.
5. Add 100µl prepared Detection Reagent B into each well. Incubate for 30 minutes at 370C after covering it with the plate sealer.
6. Aspirate and wash 5 times
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7. Add 90 µl Substrate Solution. Incubate 15-25 minutes at 370C while protecting from light. The liquid will turn blue by the addition of Substrate Solution.
8. Add 50 µl Stop Solution. The liquid will turn yellow by the addition of Stop Solution. Mix the liquid by tapping side of the plate to ensure a uniform colour change.
9. Remove any water or fingerprint at the bottom of the plate and confirm there is no bubble on the surface of the liquid. Run the microplate reader and conduct measurement at 450nm immediately.
Principle of D-dimer assay
The USCN D-dimer assay employs the competitive enzyme immunoassay technique. A monoclonal antibody specific to D-dimer is precoated onto the microplate. A competitive inhibition reaction is launched between biotin labeled D-dimer and unlabelled D-dimer (Standard or Sample) with the pre-coated antibody specific to D-dimer. After incubation, the unbound conjugate is washed off. Next, Avidin conjugated to HRP is added to each microplate well and conjugated. The amount of bound HRP conjugate is reverse proportional to the concentration of D-dimer in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of D-dimer in the sample.
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Assay procedure (Summary)
1. Prepare all reagents, samples and standards as instructed. Determine 5 wells for standard, 1 well for blank and samples.
2. Add 50µl of dilutions of standard, blank or sample to each well. Incubate for 2 hours at 370C.
3. Add 50 µl prepared Detection Reagent A into each well and shake gently. Incubate for 1 hour at 370C after covering it with plate sealer.
4. Aspirate and wash 3 times with 350 µl of Wash Solution. After the each wash, remove the liquid by aspirating or decanting then snap plate against absorbent paper.
5. Add 100µl prepared Detection Reagent B into each well. Incubate for 30 minutes at 370C after covering it with the plate sealer.
6. Aspirate and wash 5 times
7. Add 90 µl Substrate Solution. Incubate 15-25 minutes at 370C while protecting from light. The liquid will turn blue by the addition of Substrate Solution.
8. Add 50 µl Stop Solution. The liquid will turn yellow by the addition of Stop Solution. Mix the liquid by tapping side of the plate to ensure a uniform colour change.
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9. Remove any water or fingerprint at the bottom of the plate and confirm there is no bubble on the surface of the liquid. Run the microplate reader and conduct measurement at 450nm immediately.
Principle of fibrinopeptide A (FPA)assay
The USCN fibrinopeptide A assay employs the competitive enzyme
immunoassay technique. A monoclonal antibody specific to FPA is precoated onto the microplate. A competitive inhibition reaction is launched between biotin labeled FPA and unlabelled FPA (Standard or Sample) with the pre-coated antibody specific to FPA. After incubation, the unbound conjugate is washed off. Next, Avidin conjugated to HRP is added to each microplate well and conjugated. The amount of bound HRP conjugate is reverse proportional to the concentration of FPA in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of FPA in the sample.
Assay procedure (Summary)
1. Prepare all reagents, samples and standards as instructed. Determine 5 wells for standard, 1 well for blank and samples.
2. Add 50µl of dilutions of standard, blank or sample to each well. Incubate for 1 hour at 370C.
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3. Add 50 µl prepared Detection Reagent A into each well and shake gently. Incubate for 1 hour at 370C after covering it with plate sealer.
4. Aspirate and wash 3 times with 350 µl of Wash Solution. After the each wash, remove the liquid by aspirating or decanting then snap plate against absorbent paper.
5. Add 100µl prepared Detection Reagent B into each well. Incubate for 30 minutes at 370C after covering it with the plate sealer.
6. Aspirate and wash 5 times
7. Add 90 µl Substrate Solution. Incubate 15-25 minutes at 370C while protecting from light. The liquid will turn blue by the addition of Substrate Solution.
8. Add 50 µl Stop Solution. The liquid will turn yellow by the addition of Stop Solution. Mix the liquid by tapping side of the plate to ensure a uniform colour change.
9. Remove any water or fingerprint at the bottom of the plate and confirm there is no bubble on the surface of the liquid. Run the microplate reader and conduct measurement at 450nm immediately.
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Principle of fibrinopeptide B (FPB)assay
The USCN fibrinopeptide B assay employs the competitive enzyme
immunoassay technique. A monoclonal antibody specific to FPB is precoated onto the microplate. A competitive inhibition reaction is launched between biotin labeled FPB and unlabelled FPB (Standard or Sample) with the pre-coated antibody specific to FPB. After incubation, the unbound conjugate is washed off. Next, Avidin conjugated to HRP is added to each microplate well and conjugated. The amount of bound HRP conjugate is reverse proportional to the concentration of FPB in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of FPB in the sample.
Assay procedure (Summary)
1. Prepare all reagents, samples and standards as instructed. Determine 5 wells for standard, 1 well for blank and samples.
2. Add 50µl of dilutions of standard, blank or sample to each well. Incubate for 1 hour at 370C.
3. Add 50 µl prepared Detection Reagent A into each well and shake gently. Incubate for 1 hour at 370C after covering it with plate sealer.