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Factor Assays

Factor Assays. Robert Gosselin MT (ASCP), CLS. Most labs: Clot based: Modified PT or aPTT PT based: usually II, V, VII, X aPTT based: usually VIII, IX, XI, XII Chromogenic Antigenic - ELISA. Coagulation Testing in the Clinical Laboratory. Prothrombin times (PT)

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Factor Assays

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  1. Factor Assays Robert Gosselin MT (ASCP), CLS

  2. Most labs: Clot based: Modified PT or aPTT PT based: usually II, V, VII, X aPTT based: usually VIII, IX, XI, XII Chromogenic Antigenic - ELISA

  3. Coagulation Testing in the Clinical Laboratory Prothrombin times (PT) PP Plasma + Activator (+ CaCl2) ClotDetection 37oC Activated partial thromboplastin times (aPTT) PP Plasma + Activators CaCl2 Clot Detection 37oC Optical methods: change in turbidity Mechnical methods: change in motion

  4. Coagulation Testing in the Clinical Laboratory Factor VIII assay: clot based Dil PP Plasma + Factor VIII deficient plasma + Activators Clot detection CaCl2 37oC

  5. Factor Activity assays Calibrator of known value (e.g. Factor VIII 95%) 1:5: 95% = 49.0 s 1:6: 79% = 51.3 s 1:10: 47.5% = 58.4 s 1:20: 23.75% = 66.3 s 75 70 65 Seconds 60 55 50 45 2 12 25 50 75 100 % activity

  6. Factor Activity assays Patient sample: 1:5: 60.2 s 1:10: 67.2 s 1:20: 71.9 s 75 50% 24% x 2 = 48% 70 12.5% x 4 = 50% 65 Seconds 60 Average: 49% 55 50 45 2 12 25 50 75 100 % activity

  7. Chromogenic factor VIII Factor IXa, Ca, PL Factor Xa Factor X Patient’s factor VIII Factor Xa Peptide + pNA S-2765

  8. Factor Assays: when and why Prolonged PT and/or aPTT Mixing study correction vonWillebrands w/u (Factor VIII) Thrombophilia w/u Factors VIII, IX

  9. Mixing Studies Patient sample + Pooled normal plasma Immediate and incubated @37oC for 1 hour PT/aPTT corrects PT/ aPTT does correct Factor Deficiency Inhibitor

  10. Factor Activity assays Patient sample: 1:5: 60.2 s 1:10: 64.4 s 1:20: 67.5 s 75 50% 37.5% x 2 = 75% 70 25% x 4 = 100% 65 Seconds 60 Average: 75% Poor agreement 55 50 45 2 12 25 50 75 100 % activity

  11. Factor VIII Inhibitor assay Neat and pre-diluted samples with Buffer Dil PPP + PNP + Factor VIII deficient plasma + Activators Clot detection CaCl2 37oC

  12. Factor Inhibitor Assay Calibrator: PNP + Buffer 1:5:100% = 60s 1:10: 50% = 62s 1:20: 25% = 66s 1:40:12.5% = 71s 1:80: 6.25% = 75s 85 80 75 Seconds 70 65 60 55 25 50 75 100 2 12 % activity

  13. Factor Inhibitor Assay Patient sample 1:5:100% = 55 s 1:10: 50% = 61s 1:20: 25% = 66 s >100 % 85 84 x 2 = >100% 80 63 x 4 = >100% 75 Seconds 70 No inhibitor present 65 60 55 2 12 25 50 75 100 % activity

  14. Factor Inhibitor Assay- Bethesda method Patient sample Neat sample: = 80 s 1:5 pre: 50% = 61s 1:10 pre: 25% = 66 s <20 % 85 50% 80 75% 75 Seconds 70 65 60 55 2 12 25 50 75 100 % activity

  15. % Residual Activity Bethesda units A patient sample producing a residual factor activity of 50% is said to contain 1 Bethesda unit of inhibitor

  16. % Residual Activity Bethesda units Patient sample results Neat sample: <20% residual factor VIII activity 1:5 pre: 50% residual activity 1 B.U 1:10 pre: 75% residual activity 0.4 B.U. x 5 = 5 B.U x 10 = 4 B.U. Average : 4.5 B.U.

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