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Coagulation Assays – Part 2 
Larry Smith, PhD 
Director, Coagulation/Hemostasis 
Assistant Attending
Objectives 
• Types of assays in the laboratory 
• Monitoring LMWH 
• Thrombophilia work-ups 
• Lupus Anticoagulant Testing 
• HIT 
• Factor Assays 
2
Types of Assays 
• Functional Assays 
 Clot-based assays 
 Good screening assays 
 Based on a functioning cascade 
 Affected by preanalytical variables 
▫ May require additional testing 
 Chromogenic assays 
 Measure the activity of a specific enzyme rather than 
general biologic function 
 Not affected by most preanalytical variables 
3 
Enzyme of interest 
Peptide pNA Peptide pNA 
Yellow color 
develops, absorbance 
reading at 405 nm 
(Substrate) 
+
Types of Assays 
 Immunologic-based assays 
 LIA- or ELISA-based technologies 
 Most measure the amount of protein present rather than function 
 Some measure a functional component 
• Confirmatory assays 
▫ DNA-based assays 
4
Types of Deficiencies 
• Type I Deficiency 
 Parallel reduction in both FUNCTION and AMOUNT of 
protein present 
 True deficiency 
 Functional assay (clot-based assay) 
• Type II Deficiency 
 Represents dysproteinemia 
 Total amount of protein > than the activity 
 Functional + Antigenic assays 
5
Anti-Xa Heparin Assay 
• Specifically determines anticoagulant 
activity of LMWH and UFH by 
measuring ability of heparin-bound 
antithrombin to inhibit a single 
enzyme 
• More specific than aPTT since it 
measures inhibition of a single 
enzyme 
• Major advantage is lack of biologic 
factors that affect its result 
• Limitations of Heparin Assay 
▫ Clinical data examining outcomes 
is limited 
Eikelboom JW. Thromb Haemost 2006;96:547-52. 
Francis JL. Pharmacotherapy 2004;24:108S-19S. 
Plasma [heparin] + Antithrombin 
Excess 
FXa 
[AT-Heparin-Xa] + Residual FXa 
Chromogenic 
substrate 
pNA 
Inversely proportional to the anticoagulant activity 
concentration in the plasma sample
Antithrombin Deficiency 
• Single chain glycoprotein 
▫ Synthesized in the liver 
▫ SERPIN 
• Inherited deficiencies 
▫ Associated with increased risk of VTE 
▫ 0.02 – 0.2% in general 
▫ 1-2% of patients with VTE 
• Acquired deficiencies 
▫ Decreased synthesis 
 LD 
▫ Drug-induced decreased synthesis 
 L-asparaginase 
 Heparin 
▫ Increased clearance 
 Active thrombosis 
 DIC 
 Nephropathies 
7
Antithrombin Assays (functional) 
• The assay relies on the AT in the patient’s plasma*** 
▫ Reflects of the functional AT status of the patient 
8 
AT + Heparin [AT*Heparin] 
[AT*Heparin] + FXa (excess) [AT-FXa- Heparin] + FXa (residual) 
Chromogenic substrate 
FXa (residual) 
Peptide + pNA 
Inversely proportional to the AT activity concentration in the plasma sample
Classification of Antithrombin Deficiency 
9 
Type Interpretation 
Type I Quantitative 
• Parallel reduction in functional and immunologic AT – 
50% of normal 
• Antigen = Activity 
Type II Qualitative 
• Greater reduction in functional assay in comparison to 
immunologic assay 
• Antigen > Activity 
T2-HBS  mutation in heparin binding domain 
T2-RS  mutation in reactive site of AT 
T2-PL  mutation in both heparin binding domain and reactive site of AT
Protein C Deficiency 
• 1960 – Seeger described it 
anticoagulant role 
• 1980 – Griffin et al associated PC 
deficiency with VTE 
• VKD serine protease that is 
activated by IIa to aPC 
▫ Inhibits Va and VIIIa  shuts off 
thrombin generation 
▫ Exhibits 
 Anti-inflammatory activities 
 Anti-apoptotic activities 
▫ Inhibits PAI-1  enhanced 
fibrinolysis 
10 
http://practical-haemostasis.com/Thrombophilia%20Tests/pc_assays.html
Protein C Deficiency 
• Congenital Deficiency 
• Heterozygous PC deficiency: 
• ~0.2% of the general population 
• ~3% of patients with VTE 
• ~85% of PC mutations are type 1 
• Acquired Deficiency 
▫ Decreased synthesis 
 LD 
 VKD/VKA 
▫ Drug-induced decreased synthesis 
 L-asparaginase 
▫ Increased clearance 
 Acute thrombosis 
 Acute medical illness 
 DIC 
 SCD 
 Trauma 
11
Protein C Assays 
• Functional assays 
▫ No single test for PC is 100% sensitive and specific for abnormalities 
▫ Antigenic assay 
12 
Clot-based Assay (functional) Chromogenic Assay (functional) 
aPTT assay to measures the anticoagulant 
effect of aPC  due to its ability to inhibit 
FV and FVIII 
aPC cleaves a substrate  release of 
chromophore generating a color change 
Subject to a number of preanalytical 
variables 
 FVIII 
 FVL 
 Hyperlipidemia 
 DTI 
 Heparin 
 Lupus Anticoagulant 
 OAT 
Subject to fewer preanalytical variables 
 Detects most functional defects but 
not all 
 PF3 binding defect 
 May be affected by OAT 
Misleadingly low 
levels 
False normal results
PC Assays 
• Dilute patient’s plasma (1:10) in PC-deficient plasma 
▫ Clot-based assay 
▫ Chromogenic-based assay 
13 
Plasma + Venom  incubate 
aPC + substrate-pNA  release of pNA 
Hydrolysis of aPC on a specific chromogenic substrate
Protein S Deficiency 
• Described in 1984, Comp 
• TOTAL PS circulates in 2 forms: 
▫ Bound PS—60% 
 C4B-BP—nonfunctional 
▫ Free PS—40%-functional 
• Serves as a cofactor for PC 
▫ Binds aPC to the 
phospholipid surface 
▫ VKD glycoprotein synthesized in 
the liver 
Free 
PS 
14 
C4B-BP 
Total Protein S 
Bound 
PS
Protein S Deficiency 
• Congenital Deficiency 
• Heterozygous PS deficiency: 
▫ ~2% in general population 
▫ ~3-6% in recurrent thrombosis or family history 
• Acquired Deficiency 
▫ Decreased synthesis 
 LD 
 VKD/VKA 
▫ Drug-induced decreased synthesis 
 L-asparaginase 
 OCT 
 HRT 
▫ Increased clearance 
 Acute thrombosis 
 DIC 
 Acute medical illness 
 Trauma 
15
Protein S Assays 
Three types of assays 
1. Clot-based functional PS assay—”activity” assay 
 Based on aPC inactivation of FVa and FVIIIa 
 Plasma + PNP(PS free)+ aPC + Add CaCL2 Clot 
1. Antigenic-based Free PS assay 
 Free PS is adsorbed on the C4BP latex particle  triggers an agglutination reaction with 
the second latex reagent which is sensitized with a monoclonal antibody directed against 
human Protein S 
 The degree of agglutination is directly proportional to the free PS concentration 
 Immunologic assay  measures “free” (functional) portion of PS 
3. Antigenic-based Total PS assay 
 Immunologic assay that measures PS bound to C4BBP + free PS 
16 
Type PS (Activity) PS (Free) PS Total C4B-BP 
I Decreased Decreased Decreased Normal 
II Decreased Normal Normal Normal 
III Decreased Decreased Normal Elevated
Preanalytical Variables Affecting PS Deficiency 
• Activity assay may be misleading low 
▫ FVL 
▫ Elevated FVIIII 
▫ VKA/VKD 
▫ LD – not universal (there are extra-hepatic sites of PS synthesis) 
▫ SCD 
▫ Type II deficiencies are very rare 
 May be helpful if there is a high index of suspicion 
▫ Pregnancy and some women on OCT 
▫ HIV infection 
▫ Acute phase response 
• Marlar et al. (2012) AJCP. 137; 173-175 
▫ Found increased number of falsely low PS activity when PS activity is used as first assay 
• Marlar et al. (2011) Am J Hem. 86;418-421 
▫ Free PS assay  considered by many to be more reliable than activity 
▫ Diagnoses 95-99% of PS deficiencies 
1717 
Type III
18 
aPC-Resistance—Screening assay 
• aPC-resistance 
▫ Dahlbäck et al in 1993 
 Noted a blunted response in 
aPTT’s of a group of 
patients with thrombophilia 
when aPC was added 
▫ Shuts-off thrombin 
generation 
 Via inhibition FVa and 
FVIIIa 
▫ Ratio of 2 aPTT’s 
__(aPTT plus aPC)__ 
(aPTT minus aPC) 
• http://www.wardelab.com/arc_2.html 
• Normal : adding aPC prolongs aPTT 
• FVL: adding aPC does NOT prolong aPTT
aPC-Resistance—Screening assay 
• “Screening assay” for FVL mutation 
▫ ~95% of aPC-resistance is caused by a defect in the Factor V molecule 
• Sensitivity and specificity approach 100% with modified assay 
▫ Uses FV-deficient normal plasma + patient plasma 
▫ Requires a normal aPTT in the patient 
▫ Should not be used in patient on OAC or with LA 
• Mutation later described in 1994 by Bertina et al 
▫ Caused by single point mutation in the FV gene 
▫ Substitution of adenine for guanine at 1691 – G1961A 
▫ Changes arginine to glutamine at 506 – R506Q 
19
PG20210 Mutation 
 Poort et al, 1996 
 Single nucleotide substitution 
G20210A in the 3’ UT regions of the 
prothrombin gene 
 G ® A substitution at nucleotide 
20210 in prothrombin gene 
 Results in elevated levels of 
prothrombin (~30% increase) 
 No screening test available 
 Occurs primarily in Caucasians--~3% 
in general population 
 2-5-fold increased risk of VTE 
20
ISTH Criteria for Lupus Anticoagulant Testing 
The ISTH has defined the minimum diagnostic criteria for lupus 
anticoagulants to include 
1. A prolonged clotting time in a screening assay such as the aPTT 
2. Mixing studies indicating the presence of an inhibitor 
3. Confirmatory studies demonstrating phospholipid dependence of the 
inhibitor 
a. Screen – decreased amount of phospholipids  prolonged 
clotting time 
b. Confirm—increased amount of phospholipids  shortened 
clotting time 
4. No evidence of other inhibitor-based coagulopathies 
 Specific factor assays if the confirmatory step is negative or there is 
evidence of a specific factor inhibitor 
21
ISTH Criteria for Lupus Anticoagulant Testing 
• Updated ISTH guidelines (2009) ISTH 
▫ Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, de Groot PG. Update of 
the guidelines for lupus anticoagulant detection. J Thromb Haemost 2009; 7: 
1737–40 
▫ Choice of tests 
1. Two tests based on different principles 
2. dRVVT should be the first test considered 
3. Seconds test should be a sensitive aPTT (low 
phospholipids and silica as activator) 
4. LA should be considered as positive if one of the two tests 
gives a positive result 
22
Detection of LA 
• Assays 
dRVVT* 
SCT* 
 HEX 
 Kaolin CT 
 dPT 
23 
dPT 
Why do we see so few LA’s 
on the extrinsic side??? 
SCT 
DRVVT 
Clot-based assays
dRVVT Screen (Normal plasma) 
24 
X 
Xa 
Va 
Xa 
Prothrombin 
dRVVT 
Thrombin 
Ca2+ 
Fibrinogen Fibrin 
Phospholipid 
(PF3)
dRVVT Screen (Lupus Anticoagulant) 
25 
X 
Xa 
Va 
Xa 
Prothrombin 
dRVVT 
Thrombin 
Ca2+ 
Fibrinogen Fibrin 
Low 
Phospholipid 
Content
dRVVT Confirm (LA) 
26 
X 
Xa 
Va 
Xa 
Prothrombin 
dRVVT 
Thrombin 
Ca2+ 
Fibrinogen Fibrin 
High 
Phospholipid 
Content
Laboratory Tests for HIT 
27 
FFuunnccttiioonnaall ((AAccttiivvaattiioonn)) 
Washed 
Platelets 
Washed 
Platelets 
 Serotonin release 
 HIPA 
 ATP release 
 Flow cytometry 
 Serotonin release 
 HIPA 
 ATP release 
 Flow cytometry 
• Primarily detect IgG antibody complex 
• Higher specificity for detecting clinically 
relevant pathogenic antibodies 
AAnnttiiggeenniicc 
Serum or 
Plasma 
Serum or 
Plasma 
 ELISA-based 
 PGI-based 
 ELISA-based 
 PGI-based 
IgG-specific 
IgG/M/A
Serotonin Release Assay (SRA) 
28 
14C-Serotonin 
Y 
Y 
Y 
Y Y 
% 14C-Serotonin Release 
0 0.1 1.0 10 
Heparin Concentration (U/mL) 
100 
80 
60 
40 
20 
0 
Heparin = 
Antibody = 
PF4 = 
Y
Heparin-Induced Platelet Aggregation (HIPA) 
29 
0 U/ml 
Heparin 
0.5 U/mL 
Heparin 
100 U/mL 
Heparin
ELISA-based Assay 
Y 
Patient 
Plasma 
Sample 
PF4 
coated 
ELISA 
plate 
Wash 
Tagged goat anti-human Ig+ 
alkaline phosphatase 
Detect 
absorbance 
substrate 
 Anti-human IgG + AP 
 Heparin Antibody 
 Heparin:PF4 complex 
Y 
Y 
YYYYY 
Y Y 
30
Factor Assays 
• Used to establish the % activity of a 
circulating factor 
• Procedure 
▫ Patient’s plasma deficient in a 
specific factor 
▫ Dilute 1/10, 1/20/ 1/40 with a 
normal plasma deficient in the 
specific factor above 
▫ Perform PT or aPTT and compare 
the seconds to a standard curve 
 1/10 = 100% 
 1/20 = 50% 
 1/40 = 25% 
▫ Patient is run in multiple dilutions 
to check for the presence of an 
inhibitor 
31 
Std curve 
Patient
Summary 
• Broad menu of assays that can potentially be performed as part of a 
hypercoagulable workup 
• These assays help to identify risk factors that may contribute to thrombosis 
• Clot-based assays should be interpreted with caution 
• All of the assays have their advantages and disadvantages 
▫ Chromogenic PC and Free PS assays are the assays of choice to screen for these 
deficiencies 
▫ ISTH Subcommittee on Thrombosis and WHO recommend Free PS to screen for 
PS deficiency 
Ballard RB, Marques, MB. Pathology Consultation on the Laboratory Evaluation of Thrombophilia: When, 
How, and Why. Am J Clin Pathol 2012;137-553-560 
32
The End 
33

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Coagulation assays

  • 1. Coagulation Assays – Part 2 Larry Smith, PhD Director, Coagulation/Hemostasis Assistant Attending
  • 2. Objectives • Types of assays in the laboratory • Monitoring LMWH • Thrombophilia work-ups • Lupus Anticoagulant Testing • HIT • Factor Assays 2
  • 3. Types of Assays • Functional Assays  Clot-based assays  Good screening assays  Based on a functioning cascade  Affected by preanalytical variables ▫ May require additional testing  Chromogenic assays  Measure the activity of a specific enzyme rather than general biologic function  Not affected by most preanalytical variables 3 Enzyme of interest Peptide pNA Peptide pNA Yellow color develops, absorbance reading at 405 nm (Substrate) +
  • 4. Types of Assays  Immunologic-based assays  LIA- or ELISA-based technologies  Most measure the amount of protein present rather than function  Some measure a functional component • Confirmatory assays ▫ DNA-based assays 4
  • 5. Types of Deficiencies • Type I Deficiency  Parallel reduction in both FUNCTION and AMOUNT of protein present  True deficiency  Functional assay (clot-based assay) • Type II Deficiency  Represents dysproteinemia  Total amount of protein > than the activity  Functional + Antigenic assays 5
  • 6. Anti-Xa Heparin Assay • Specifically determines anticoagulant activity of LMWH and UFH by measuring ability of heparin-bound antithrombin to inhibit a single enzyme • More specific than aPTT since it measures inhibition of a single enzyme • Major advantage is lack of biologic factors that affect its result • Limitations of Heparin Assay ▫ Clinical data examining outcomes is limited Eikelboom JW. Thromb Haemost 2006;96:547-52. Francis JL. Pharmacotherapy 2004;24:108S-19S. Plasma [heparin] + Antithrombin Excess FXa [AT-Heparin-Xa] + Residual FXa Chromogenic substrate pNA Inversely proportional to the anticoagulant activity concentration in the plasma sample
  • 7. Antithrombin Deficiency • Single chain glycoprotein ▫ Synthesized in the liver ▫ SERPIN • Inherited deficiencies ▫ Associated with increased risk of VTE ▫ 0.02 – 0.2% in general ▫ 1-2% of patients with VTE • Acquired deficiencies ▫ Decreased synthesis  LD ▫ Drug-induced decreased synthesis  L-asparaginase  Heparin ▫ Increased clearance  Active thrombosis  DIC  Nephropathies 7
  • 8. Antithrombin Assays (functional) • The assay relies on the AT in the patient’s plasma*** ▫ Reflects of the functional AT status of the patient 8 AT + Heparin [AT*Heparin] [AT*Heparin] + FXa (excess) [AT-FXa- Heparin] + FXa (residual) Chromogenic substrate FXa (residual) Peptide + pNA Inversely proportional to the AT activity concentration in the plasma sample
  • 9. Classification of Antithrombin Deficiency 9 Type Interpretation Type I Quantitative • Parallel reduction in functional and immunologic AT – 50% of normal • Antigen = Activity Type II Qualitative • Greater reduction in functional assay in comparison to immunologic assay • Antigen > Activity T2-HBS  mutation in heparin binding domain T2-RS  mutation in reactive site of AT T2-PL  mutation in both heparin binding domain and reactive site of AT
  • 10. Protein C Deficiency • 1960 – Seeger described it anticoagulant role • 1980 – Griffin et al associated PC deficiency with VTE • VKD serine protease that is activated by IIa to aPC ▫ Inhibits Va and VIIIa  shuts off thrombin generation ▫ Exhibits  Anti-inflammatory activities  Anti-apoptotic activities ▫ Inhibits PAI-1  enhanced fibrinolysis 10 http://practical-haemostasis.com/Thrombophilia%20Tests/pc_assays.html
  • 11. Protein C Deficiency • Congenital Deficiency • Heterozygous PC deficiency: • ~0.2% of the general population • ~3% of patients with VTE • ~85% of PC mutations are type 1 • Acquired Deficiency ▫ Decreased synthesis  LD  VKD/VKA ▫ Drug-induced decreased synthesis  L-asparaginase ▫ Increased clearance  Acute thrombosis  Acute medical illness  DIC  SCD  Trauma 11
  • 12. Protein C Assays • Functional assays ▫ No single test for PC is 100% sensitive and specific for abnormalities ▫ Antigenic assay 12 Clot-based Assay (functional) Chromogenic Assay (functional) aPTT assay to measures the anticoagulant effect of aPC  due to its ability to inhibit FV and FVIII aPC cleaves a substrate  release of chromophore generating a color change Subject to a number of preanalytical variables  FVIII  FVL  Hyperlipidemia  DTI  Heparin  Lupus Anticoagulant  OAT Subject to fewer preanalytical variables  Detects most functional defects but not all  PF3 binding defect  May be affected by OAT Misleadingly low levels False normal results
  • 13. PC Assays • Dilute patient’s plasma (1:10) in PC-deficient plasma ▫ Clot-based assay ▫ Chromogenic-based assay 13 Plasma + Venom  incubate aPC + substrate-pNA  release of pNA Hydrolysis of aPC on a specific chromogenic substrate
  • 14. Protein S Deficiency • Described in 1984, Comp • TOTAL PS circulates in 2 forms: ▫ Bound PS—60%  C4B-BP—nonfunctional ▫ Free PS—40%-functional • Serves as a cofactor for PC ▫ Binds aPC to the phospholipid surface ▫ VKD glycoprotein synthesized in the liver Free PS 14 C4B-BP Total Protein S Bound PS
  • 15. Protein S Deficiency • Congenital Deficiency • Heterozygous PS deficiency: ▫ ~2% in general population ▫ ~3-6% in recurrent thrombosis or family history • Acquired Deficiency ▫ Decreased synthesis  LD  VKD/VKA ▫ Drug-induced decreased synthesis  L-asparaginase  OCT  HRT ▫ Increased clearance  Acute thrombosis  DIC  Acute medical illness  Trauma 15
  • 16. Protein S Assays Three types of assays 1. Clot-based functional PS assay—”activity” assay  Based on aPC inactivation of FVa and FVIIIa  Plasma + PNP(PS free)+ aPC + Add CaCL2 Clot 1. Antigenic-based Free PS assay  Free PS is adsorbed on the C4BP latex particle  triggers an agglutination reaction with the second latex reagent which is sensitized with a monoclonal antibody directed against human Protein S  The degree of agglutination is directly proportional to the free PS concentration  Immunologic assay  measures “free” (functional) portion of PS 3. Antigenic-based Total PS assay  Immunologic assay that measures PS bound to C4BBP + free PS 16 Type PS (Activity) PS (Free) PS Total C4B-BP I Decreased Decreased Decreased Normal II Decreased Normal Normal Normal III Decreased Decreased Normal Elevated
  • 17. Preanalytical Variables Affecting PS Deficiency • Activity assay may be misleading low ▫ FVL ▫ Elevated FVIIII ▫ VKA/VKD ▫ LD – not universal (there are extra-hepatic sites of PS synthesis) ▫ SCD ▫ Type II deficiencies are very rare  May be helpful if there is a high index of suspicion ▫ Pregnancy and some women on OCT ▫ HIV infection ▫ Acute phase response • Marlar et al. (2012) AJCP. 137; 173-175 ▫ Found increased number of falsely low PS activity when PS activity is used as first assay • Marlar et al. (2011) Am J Hem. 86;418-421 ▫ Free PS assay  considered by many to be more reliable than activity ▫ Diagnoses 95-99% of PS deficiencies 1717 Type III
  • 18. 18 aPC-Resistance—Screening assay • aPC-resistance ▫ Dahlbäck et al in 1993  Noted a blunted response in aPTT’s of a group of patients with thrombophilia when aPC was added ▫ Shuts-off thrombin generation  Via inhibition FVa and FVIIIa ▫ Ratio of 2 aPTT’s __(aPTT plus aPC)__ (aPTT minus aPC) • http://www.wardelab.com/arc_2.html • Normal : adding aPC prolongs aPTT • FVL: adding aPC does NOT prolong aPTT
  • 19. aPC-Resistance—Screening assay • “Screening assay” for FVL mutation ▫ ~95% of aPC-resistance is caused by a defect in the Factor V molecule • Sensitivity and specificity approach 100% with modified assay ▫ Uses FV-deficient normal plasma + patient plasma ▫ Requires a normal aPTT in the patient ▫ Should not be used in patient on OAC or with LA • Mutation later described in 1994 by Bertina et al ▫ Caused by single point mutation in the FV gene ▫ Substitution of adenine for guanine at 1691 – G1961A ▫ Changes arginine to glutamine at 506 – R506Q 19
  • 20. PG20210 Mutation  Poort et al, 1996  Single nucleotide substitution G20210A in the 3’ UT regions of the prothrombin gene  G ® A substitution at nucleotide 20210 in prothrombin gene  Results in elevated levels of prothrombin (~30% increase)  No screening test available  Occurs primarily in Caucasians--~3% in general population  2-5-fold increased risk of VTE 20
  • 21. ISTH Criteria for Lupus Anticoagulant Testing The ISTH has defined the minimum diagnostic criteria for lupus anticoagulants to include 1. A prolonged clotting time in a screening assay such as the aPTT 2. Mixing studies indicating the presence of an inhibitor 3. Confirmatory studies demonstrating phospholipid dependence of the inhibitor a. Screen – decreased amount of phospholipids  prolonged clotting time b. Confirm—increased amount of phospholipids  shortened clotting time 4. No evidence of other inhibitor-based coagulopathies  Specific factor assays if the confirmatory step is negative or there is evidence of a specific factor inhibitor 21
  • 22. ISTH Criteria for Lupus Anticoagulant Testing • Updated ISTH guidelines (2009) ISTH ▫ Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, de Groot PG. Update of the guidelines for lupus anticoagulant detection. J Thromb Haemost 2009; 7: 1737–40 ▫ Choice of tests 1. Two tests based on different principles 2. dRVVT should be the first test considered 3. Seconds test should be a sensitive aPTT (low phospholipids and silica as activator) 4. LA should be considered as positive if one of the two tests gives a positive result 22
  • 23. Detection of LA • Assays dRVVT* SCT*  HEX  Kaolin CT  dPT 23 dPT Why do we see so few LA’s on the extrinsic side??? SCT DRVVT Clot-based assays
  • 24. dRVVT Screen (Normal plasma) 24 X Xa Va Xa Prothrombin dRVVT Thrombin Ca2+ Fibrinogen Fibrin Phospholipid (PF3)
  • 25. dRVVT Screen (Lupus Anticoagulant) 25 X Xa Va Xa Prothrombin dRVVT Thrombin Ca2+ Fibrinogen Fibrin Low Phospholipid Content
  • 26. dRVVT Confirm (LA) 26 X Xa Va Xa Prothrombin dRVVT Thrombin Ca2+ Fibrinogen Fibrin High Phospholipid Content
  • 27. Laboratory Tests for HIT 27 FFuunnccttiioonnaall ((AAccttiivvaattiioonn)) Washed Platelets Washed Platelets  Serotonin release  HIPA  ATP release  Flow cytometry  Serotonin release  HIPA  ATP release  Flow cytometry • Primarily detect IgG antibody complex • Higher specificity for detecting clinically relevant pathogenic antibodies AAnnttiiggeenniicc Serum or Plasma Serum or Plasma  ELISA-based  PGI-based  ELISA-based  PGI-based IgG-specific IgG/M/A
  • 28. Serotonin Release Assay (SRA) 28 14C-Serotonin Y Y Y Y Y % 14C-Serotonin Release 0 0.1 1.0 10 Heparin Concentration (U/mL) 100 80 60 40 20 0 Heparin = Antibody = PF4 = Y
  • 29. Heparin-Induced Platelet Aggregation (HIPA) 29 0 U/ml Heparin 0.5 U/mL Heparin 100 U/mL Heparin
  • 30. ELISA-based Assay Y Patient Plasma Sample PF4 coated ELISA plate Wash Tagged goat anti-human Ig+ alkaline phosphatase Detect absorbance substrate  Anti-human IgG + AP  Heparin Antibody  Heparin:PF4 complex Y Y YYYYY Y Y 30
  • 31. Factor Assays • Used to establish the % activity of a circulating factor • Procedure ▫ Patient’s plasma deficient in a specific factor ▫ Dilute 1/10, 1/20/ 1/40 with a normal plasma deficient in the specific factor above ▫ Perform PT or aPTT and compare the seconds to a standard curve  1/10 = 100%  1/20 = 50%  1/40 = 25% ▫ Patient is run in multiple dilutions to check for the presence of an inhibitor 31 Std curve Patient
  • 32. Summary • Broad menu of assays that can potentially be performed as part of a hypercoagulable workup • These assays help to identify risk factors that may contribute to thrombosis • Clot-based assays should be interpreted with caution • All of the assays have their advantages and disadvantages ▫ Chromogenic PC and Free PS assays are the assays of choice to screen for these deficiencies ▫ ISTH Subcommittee on Thrombosis and WHO recommend Free PS to screen for PS deficiency Ballard RB, Marques, MB. Pathology Consultation on the Laboratory Evaluation of Thrombophilia: When, How, and Why. Am J Clin Pathol 2012;137-553-560 32

Notes de l'éditeur

  1. This lecture is a continuation of a previous lecture on coagulation assays. Here we will discuss assays involved in thrombophilia testing. Objectives: 1) review of thrombophilia and risk factors; 2) discuss assays that identify risk factors; 3) review preanalytical variables that interfere with these assays
  2. What types of assays are there for identifying patients at risk and how good are they? Functional assays for type I deficiencies Antigenic assays for type II deficiencies DNA-based assays for confirmation The general principle of a chromogenic assay, in its simplest form: A protein is converted to its active formThe cleaved peptide releases the chromophore, in this example pNA, which gives off a color. In this example, PNA is used. PNA = para-nitroaniline, which appears as a yellow color.
  3. What types of assays are there for identifying patients at risk and how good are they? Functional assays for type I deficiencies Antigenic assays for type II deficiencies DNA-based assays for confirmation The general principle of a chromogenic assay, in its simplest form: A protein is converted to its active form. the. The cleaved peptide releases the chromophore, in this example pNA, which gives off a color. In this example, PNA is used. PNA = para-nitroaniline, which appears as a yellow color.
  4. For most clinical coagulation evaluations, the main goal is to test for function (preferred method) and if decreased then confirm by antigenic assay to determine the type of deficiency (quantitative or qualitative). PS may be the exception, since the PS activity assay is infulenced by a viariety of conditions.
  5. Described by Eggberg in coworkers in 1963 Single-chain glycoprotein, 58,200 Daltons, plasma ½-life ~ 3 days Chromosome 1 (SERPINC1) gene – wide variety of mutations have been identified
  6. Plasma is incubated with an excess of bovine Xa in the presence of heparin (in the patient). Heparin binds to AT  inactivation of Xa. Residual Xa left is measured by its ability to hydrolyze a chromogenic substrate (S-2765) added to the test tube. Absorbance is measured at 405nm and is inversely proportional to the AT activity concentration in the plasma sample. Some methods use Factor Xa instead of IIa in reagent. This theoretically decreases the contribution from other proteins such as heparin cofactor II. IV-heparin therapy can cause lowering of the AT level in plasma by ~25%. Not affected by HCII
  7. VKD serine protease Single chain glycoprotein, 62KDa, produced in the liver and converted to its active form by thrombin  becomes aPC Protein C gene (PROC) located on the long arm of chromosome 2 (2q13-q14)
  8. VKD serine protease Single chain glycoprotein, 62KDa, produced in the liver and converted to its active form by thrombin  becomes aPC Protein C gene (PROC) located on the long arm of chromosome 2 (2q13-q14)
  9. Clot-based assay: Clotting time of the aPTT (or PT) will be influenced by the amount of Va and VIIIa present in the reaction mixture and in turn this will be influenced by the activity of aPC. aPC is generated from the conversion of PC to aPC by Protac. So, if there is a reduction in circulating PC levels, then less aPC will be generated, less Va and VIIIa will be inactivated and the clotting times will be shorter. Chromogenic assay: Protac is added to PPP and incubated. A chromogenic substrate for aPC is added. aPC cleaves the substrate releasing pNA and the change in optical density is measured and compared to a standard reference curve. (Calcium, PF3 or coagulation activator is necessary since the test plasma serves as the only source of PC—clot formation is not necessary for this test). A note about chromogenic protein C assays: they may overestimate the true level of protein C in patients treated with oral anticoagulants like warfarin. This is because the non-carboxylated forms of protein C formed in warfarin-treated patients are also activated by Protac and can then cleave the chromogenic substrate. The chromogenic assay detects only abnormalities of PC activation (that is when protein C is converted to APC) and abnormalities of the enzymatic active site. In rare cases the patient may have defects in the part of the molecule that binds FVa and FVIIIa or the part that binds protein S or phospholipid. These defects are not detected by the chromogenic assay. Measures anticoagulant activity of APC exerted against natural substrates – FVa & FVIIIa. Argument for clotting based assays – measures natural substrate. Chromogenic based – measures amidolytic activity against synthetic substrate. Argument against clotting based assays – many interfering substances. Argument for chromogenic – fewer interfering substances, but not natural substrate. Rare reported mutations that affect catalytic site which can not be detected by chromogenic assays. Venom is Protac.
  10. Protein C assays can be clotting or chromogenic. The clotting assay is widely used, though the chromogenic assay is the method of choice. A note about chromogenic protein C assays: they may overestimate the true level of protein C in patients treated with oral anticoagulants like warfarin. This is because the non-carboxylated forms of protein C formed in warfarin-treated patients are also activated by Protac and can then cleave the chromogenic substrate. The chromogenic assay detects only abnormalities of PC activation (that is when protein C is converted to APC) and abnormalities of the enzymatic active site. In rare cases the patient may have defects in the part of the molecule that binds FVa and FVIIIa or the part that binds protein S or phospholipid. These defects are not detected by the chromogenic assay. Measures anticoagulant activity of APC exerted against natural substrates – FVa & FVIIIa. Argument for clotting based assays – measures natural substrate. Chromogenic based – measures amidolytic activity against synthetic substrate. Argument against clotting based assays – many interfering substances. Argument for chromogenic – fewer interfering substances, but not natural substrate. Rare reported mutations that affect catalytic site which can not be detected by chromogenic assays. Venom is Protac.
  11. Protein S, APC’s cofactor, circulates in the plasma free or bound to the complement protein C4b binding protein. 40% of protein S is in the free, functionally active form, which is what APC binds to. Binding affinity of C4BP high, therefore all C4BP molecules will be bound to PS. C4BP is an acute phase reactant. PEG precipitation takes out form bound to C4bBP. Free form in the supernatant. The gene (PROS1) located in chromosome 3 (3p11.1-3p11.2)
  12. VKD serine protease Single chain glycoprotein, 62KDa, produced in the liver and converted to its active form by thrombin  becomes aPC Protein C gene (PROC) located on the long arm of chromosome 2 (2q13-q14)
  13. Functional assay – measures on the FREE (physiologically-active) PS. Free PS – monoclonal ab directed against PS epitopes that are not accessible in the bound form and these are usually the residues that are involved in the binding to C4BBP. Second assay involves used PEG to separate the bound PS from C4BBP and then measures the free PS. Total PS – measures both PS bound to C4BBP and the Free form of PS There is a good correlation between FPS antigen (LIA) assay and functional PS activity so many labs choose only to measure FPS. HOWEVER, this may miss some rare cases in which the immunological PS level is normal but there is a functional abnormality (i.e. a true Type 2 deficiency).
  14. In SCD PS may bind to sickle cells, however, PS is not decreased during sickle cell crisis.
  15. Approximately 90% of APC Resistance is caused by a defect in the Factor V molecule, known as the Factor V Leiden gene mutation. This is explained by a point mutation in the coagulation factor V gene, changing arginine 506 in the factor V molecule to glutamine. So now APC can’t recognize the cleavage site at position 506, which allows for longer duration of thrombin generation and may lead to a hypercoagulable state. In other words, the FVa molecule isn’t allowing APC to do its job of inactivating FVa and ultimately inhibiting thrombin generation. In normal patients, adding APC to the aPTT prolongs the aPTT In patients with the FVL mutation, adding APC shortens the aPTT because they are resistant to cleavage by the APC complex Dilute in FV deficient plasma to normalize all other plasma proteins. Theoretically could test samples from patients on OAC. Better discrimination between heterozygotes and homozygotes. Limitations – With modified test (diluting in FV deficient plasma) – many limitations eliminated. No significant difference between fresh and frozen plasma. Lot to lot changes should be evaluated to verify cut-off range. High FVIII levels (associated with pregnancy & acute phase response) should not be an issue with modified test (unless really high!). With classic APTT test – the baseline APTT must be within 25-40 seconds. If results from different laboratories are to be compared, it may be beneficial to normalize the APC ratio against the APTC ratio of a normal plasma pool. Note: only 90% of APCr is due to FV Leiden mutation, so if using the modified assay alone may not pick up the 10% of people who are APCr for other reasons. High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states). Prothrombin and FX concentrations below 50% tend to produce higher APC ratios High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states). Prothrombin and FX concentrations below 50% tend to produce higher APC ratios High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states). Prothrombin and FX concentrations below 50% tend to produce higher APC ratios High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states). Prothrombin and FX concentrations below 50% tend to produce higher APC ratios
  16. Approximately 90% of APC Resistance is caused by a defect in the Factor V molecule, known as the Factor V Leiden gene mutation. This is explained by a point mutation in the coagulation factor V gene, changing arginine 506 in the factor V molecule to glutamine. So now APC can’t recognize the cleavage site at position 506, which allows for longer duration of thrombin generation and may lead to a hypercoagulable state. In other words, the FVa molecule isn’t allowing APC to do its job of inactivating FVa and ultimately inhibiting thrombin generation. In normal patients, adding APC to the aPTT prolongs the aPTT In patients with the FVL mutation, adding APC shortens the aPTT because they are resistant to cleavage by the APC complex Dilute in FV deficient plasma to normalize all other plasma proteins. Theoretically could test samples from patients on OAC. Better discrimination between heterozygotes and homozygotes. Limitations – With modified test (diluting in FV deficient plasma) – many limitations eliminated. No significant difference between fresh and frozen plasma. Lot to lot changes should be evaluated to verify cut-off range. High FVIII levels (associated with pregnancy & acute phase response) should not be an issue with modified test (unless really high!). With classic APTT test – the baseline APTT must be within 25-40 seconds. If results from different laboratories are to be compared, it may be beneficial to normalize the APC ratio against the APTC ratio of a normal plasma pool. Note: only 90% of APCr is due to FV Leiden mutation, so if using the modified assay alone may not pick up the 10% of people who are APCr for other reasons. High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states). Prothrombin and FX concentrations below 50% tend to produce higher APC ratios High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states). Prothrombin and FX concentrations below 50% tend to produce higher APC ratios High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states). Prothrombin and FX concentrations below 50% tend to produce higher APC ratios High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states). Prothrombin and FX concentrations below 50% tend to produce higher APC ratios The mutation (a 1691G→A substitution) removes a cleavage site of the restriction endonuclease MnII. Molecular Cause: Mutation of an APC cleavage site in Factor V Mutation Site: Amino acid #506 is Arg and mutated to Gln DNA and protein sequence: Amino Acid:Arg506Gln506 Nucleotide:CGACAA Molecular Function: APC cleaves Arg-Gly bond. If mutated to Gln, then bond not cleaved. FVa remains active generating more fibrin clot. High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states). Prothrombin and FX concentrations below 50% tend to produce higher APC ratios APC resistance due to the presence of the FV:Q506 allele is inherited as an autosomal dominant trait and has a prevalence of 2-13% in the general population. Frequencies of APC resistance among patients with venous thrombosis range from 20-60%. APC resistance is highly prevalent. Individuals who are heterozygous for Factor V Leiden have a 2 – 5 fold increased thrombotic risk, while homozygotes have up to an 80-fold increase. Most heterozygotes are asymptomatic unless other risk factors are present. Simple blood tests are available to detect APC Resistance and it is part of the routine thrombophilia screen
  17. A single nucleotide polymorphism at position 20210 in the 3’-untranslated region of the prothrombin gene (FII*20210GA) is associated with elevated plasma prothrombin levels and an increased risk of venous thrombosis. The FII*20210GA nucleotide substitution did not change the restriction map of the prothrombin gene. Poort et al (1996) designed a mutant reverse oligonucleotide that introduces a Hind III restriction site (A↓AGCTT) at nucleotide position 20214 in the mutant sequence but not in the normal sequence. The G-to-A transition at position 20210 in the 3’-untranslated region of the prothrombin gene is detected by PCR amplification and Hind III digestion. After Hind III digestion of the amplified DNA from a person homozygous for FII*20210G (the more frequent allele), the size of the band is unchanged (lane 1). By contrast, after Hind III digestion of the amplified DNA fragment obtained from a person heterozygous for FII*202010G/A, bands of 345 bp and 322 bp are detected (lane 2). The 23 bp band is not visible in the figure. After Hind III digestion from the amplified DNA from a person homozygous for FII*20210A, the fragment is fully digested (lane 3). Lane M: size marker ФX174 DNA-Hae III digest, Lane U: undigested PCR product). The prothrombin gene mutation is found in approximately 3% of the population and leads to an increase in prothrombin levels, which can generate excess thrombin, leading to an increased risk of VTE. It is the second most common genetic risk factor for VTE, with a relative risk of 2.8. substitution of adenine for guanine at position 20210 of the prothrombin gene. Although this falls outside the reading frame for the protein, it leads to high levels of prothrombin and a possibly increased risk of thrombosis (Poort et al 1996 Polymorphism at bp 20210 in 3’ UT Increases Prothrombin Levels (?) Mechanism: longer surviving mRNA Genetic Abnormality Heterozygote and Homozygote Assay: DNA based only Relative Risk:2.0
  18. There are two types of laboratory tests available to confirm a diagnosis of HIT: a functional assay and an antigen assay. Functional assays detect HIT-IgG on the basis of their ability to activate platelets. Activation can be measured in a number of different ways, including SRA, platelet aggregation, ATP release, and flow cytometry. A washed platelet system is recommended. Of the available assays, the serotonin release assay has the most advantages. The citrated plasma assay is widely used, specifically, the platelet aggregation. This assay measures the aggregation of normal donor platelets by patient serum or plasma in the presence of heparin. However, it is not recommended because of its low sensitivity and low specificity. Antigen assays detect the binding of antibodies to immobilized PF4-heparin complexes. The most typical of the direct antigen assay performed on serum or plasma is the heparin-PF4 ELISA. Functional assays detect HIT-IgG on the basis of their ability to activate platelets. Activation can be measured in a number of different ways, including SRA, platelet aggregation, ATP release, and flow cytometry. A washed platelet system is recommended. Of the available assays, the serotonin release assay has the most advantages. The citrated plasma assay is widely used, specifically, the platelet aggregation. This assay measures the aggregation of normal donor platelets by patient serum or plasma in the presence of heparin. However, it is not recommended because of its low sensitivity and low specificity.
  19. Radio-labeled, washed platelets from reactive donors are incubated with heat-treated PATIENT serum in the presence of heparin. The test is positive if 14C-serotonin release occurs at therapeutic (0.1 u/mL) but not at high (100 u/mL) heparin concentrations. Laboratory assays for HIT antibodies. Top: the SRA. Washed platelets loaded with radiolabeled 14C-serotonin are incubated with patient serum and pharmacologic concentrations of heparin. Visentin et al,5 PF4-heparin complexes formed at a heparin:PF4 molar ratio of approximately 1:2 are optimal for HIT antibody detection, and reactions of antibodies with this target are inhibited when high doses of heparin are added. A significant fraction of serum samples from patients suspected of having HIT react with the unmodified complexes, sometimes quite strongly, but fail to inhibit with excess heparin. What these antibodies recognize is uncertain, but they are not likely indicative of HIT. Findings showed that fewer than 10% of patients judged to have HIT on clinical grounds failed the confirmatory test, whereas approximately one-third of those who did not appear to have HIT did so. Blood, 9 September 2010, Vol. 116, No. 10, pp. 1761-1766.
  20. Aggregation at the low concentration of heparin must by greater than 20% compared to normal control and 0% at the higher concentration of heparin. Measures platelet aggregation induced by IgG in serum or plasma of HIT patients treated with heparin. Donor platelets are incubated with the patient’s plasma contain IgG antibodies. The presence of HIT antibodies may cause platelet aggregation. Advantages Easily performed in most laboratories -- Specificity greater than 90%. Disadvantages --Low sensitivity: 35%–81%, Heparin can activate platelets in the absence of HIT antibodies, Reactivity varies among donor platelets.
  21. The PF4/heparin EIA. The assay shown utilizes PF4 and heparin bound in optimal stoichiometric concentrations to detect HIT antibodies. Alternatively, PF4 can be bound to certain other polyanions, such as polyvinyl sulfonate (not shown).