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) PP Plasma + Activator (+ CaCl2) Clot Detection 37oC Activated partial thromboplastin times (aPTT) PP Plasma + Activators CaCl2 Clot Detection Optical methods: change in turbidity Mechnical methods: change in motion

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

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

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

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

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

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

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

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

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 2 12 25 50 75 100 % activity

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

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

% Residual Activity Bethesda units 100 80 0.3 75 0.4 70 0.5 65 0.6 60 0.7 55 0.9 50 1.0 45 1.2 40 1.3 35 1.5 30 1.7 25 2.0 20 2.3 15 2.7 10 3.3 A patient sample producing a residual factor activity of 50% is said to contain 1 Bethesda unit of inhibitor

Average : 4.5 B.U. Patient sample results % Residual Activity Bethesda units 100 80 0.3 75 0.4 70 0.5 65 0.6 60 0.7 55 0.9 50 1.0 45 1.2 40 1.3 35 1.5 30 1.7 25 2.0 20 2.3 15 2.7 10 3.3 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.