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) Clot Detection
37oC
Activated partial thromboplastin times (aPTT)
PP Plasma Activators CaCl Clot Detection
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. Immediate and incubated @37oC for 1 hour
Mixing Studies
Patient sample + Pooled normal plasma
Immediate and 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 2 12 25 50 75
100
% 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
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

16. 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.