Diagnosis of thrombophilia

rev. C
March 2012

Diagnosis of Thrombophilia - Presentation

Diagnostica Stago - all rights reserved

Diagnosis of thrombophilia

Definition of thrombophilia
Deficiencies in inhibitors
Antithrombin
Protein C
Protein S

Definition of thrombophilia
Thrombophilia can be defined as a tendency to
thrombosis
Only weakens the coagulation response to cope with
environmental fluctuations
Does not necessarily cause clinical impairment =
RISK FACTOR
Interaction required with other components before
onset of clinical disorders
Inherited thrombophilia is a genetically determined
tendency to develop thrombosis

Inherited thrombophilia
Symptoms
Features

Clinical: venous thromboembolism

Spontaneous or out of proportion
Usually deep vein thrombosis of lower limbs, pulmonary embolism,
superficial thrombophlebitis, sometimes unusual locations

Includes deficiencies of 3 natural anticoagulant proteins

Antithrombin
Protein C
Protein S

Includes also specific mutations in the genes for factor V (factor

V Leiden) and factor II or prothrombin (prothrombin 20210G>A)
Prevalence of these deficiencies is low

Acquired thrombophilia
Symptoms
Features
No family history
Occurs in association
with a trigger event e.g. trauma, pregnancy, puerperium,
surgery, immobilisation, postoperative period ...
with other clinical disorders, e.g. malignancy, nephrotic
syndrome, HIT
with Lupus Anticoagulant (LA) and Antiphospholipid
antibodies (APA)

Acquired risk factors and interactions

with inherited thrombophilia

Varga EA. Clinical Genetics, 2012, 81(1): 7-17

Situational risk factors (1/2)

Cancer
six- to sevenfold increased risk of VTE to those without cancer
magnitude of risk varies with cancer type, extent of disease and
increases during treatment

Central venous catheters

strongest risk factor for upper extremity thrombosis
two- to threefold higher in factor V Leiden and prothrombin 20210G>A
heterozygotes

Varga EA. Clinical Genetics, 2012, 81(1): 7-17

Situational risk factors (2/2)

Immobility and hospitalization
risk of VTE increased with all types of immobility including bed rest,
lower extremity plaster casts and paralysis or weakness resulting from
neurologic disease
prolonged bed rest confers five- to sixflod increase in risk
shorter periods of bed rest and reduced use of an extremity are
associated with a two- to threefold increased thrombotic risk

Surgery
all types of surgery increase the risk of VTE

Trauma
risk of VTE is nearly 13-fold after major trauma requiring hospitalization
the risk increases with the severity of the injuries and is highest in those
with multiple limb or plevic fractures
Varga EA. Clinical Genetics, 2012, 81(1): 7-17

Inherited thrombophilias
prevalence and relative risks

Varga EA. Clinical Genetics, 2012, 81(1): 7-17

Inherited thrombophilia and pregnancyassociated VTE

Varga EA. Clinical Genetics, 2012, 81(1): 7-17

Haemostasis balance
Activation and
inhibitory pathways
RISK FACTORS
FOR THROMBOSIS

Inhibitors
Factors

Factors

+
Inhibitors

Inhibitor

Factors

Inhibitors

XIIa
TF-VIIa
Xa

AT

XIa
IXa

VIIIa

Xa
AT: Antithrombin
APC: Activated Protein C
PS: Protein S
TFPI: Tissue Factor Pathway
Inhibitor
TF: Tissue Factor

TFPI

IIa

Va

PS
APC

Heritable thrombophilia screening:

who should be screened? (1/6)
Neonates and children with purpura fulminans should be tested urgently for
protein C and S deficiency (grade 1B).
A variety of functional methods may be required to identify specific severe type 2
functional defects when levels of protein C or S are not <5% (grade 1B).
Adults who develop skin necrosis in association with oral VKAs should be tested
for protein C and S deficiency after VKA treatment is withdrawn (grade 2B).
Asymptomatic relatives with high risk thrombophilia (deficiency in AT, Protein C
and Protein S) should only be considered in selected thrombosis-prone families
(grade 1B). Risks, benefits and limitations of testing should be discussed.
All hospitalized patients should be assessed for risk of venous thrombosis
regardless of heritable thrombophilia based on a clinical risk assessment (grade
1B).
Baglin T. British Journal of Haematology, 2010, 149: 209-220

Heritable thrombophilia screening:

who should be screened? (2/6)
In pregnant women:
pregnancy-associated venous thrombosis primarily in relation to clinical
risk
previous event due to a minor provoking factor, e.g. travel, should be
tested and considered for prophylaxis if a thrombophilia is found.
asymptomatic pregnant women with a family history of venous
thrombosis be tested if an event in a first-degree relative was
unprovoked, or provoked by pregnancy, combined oral-contraceptive
exposure or a minor risk factor (grade 2C). The result will be more
informative if the first-degree relative has a known thrombophilia.

Baglin T. British Journal of Haematology, 2010, 149: 209-220

Recommendations for laboratory testing

in case of venous thrombosis (3/6)
Testing at the time of acute venous thrombosis is not
indicated.
As treatment of acute venous thrombosis is not influenced by
test results, testing can be performed later if indicated.
Initiation and intensity of anticoagulant therapy following a
diagnosis of acute thrombosis should be the same in patients
with and without heritable thrombophilia (grade 1B).
Decisions regarding duration of anticoagulation in unselected
patients should be made with reference to whether or not a
first episode of venous thrombosis was provoked or not, other
risk factors, and risk of anticoagulant therapy-related
bleeding, regardless of whether a heritable thrombophilia is
known (grade 1B). Baglin T. British Journal of Haematology, 2010, 149: 209-220

Recommendations for assays

(4/6)

PT testing should be measured to detect the effect of oral

VKAs, which will cause a reduction in protein C and S levels.
Functional assays should be used to determine antithrombin
and protein C levels.
Chromogenic assays of protein C activity are less subject to
interference than clotting assays and are preferable.
Immunoreactive assays of free protein S antigen are
preferable to functional assays. If a protein S activity assay is
used in the initial screen, low results should be further
investigated with an immunoreactive assay for free protein S.
Baglin T. British Journal of Haematology, 2010, 149: 209-220

Recommendations for assays

(5/6)

If an APC (Activated Protein C) resistance assay is performed

to detect the F5G1691A then the modified APC sensitivity test
(predilution of the test sample in factor V-deficient plasma), as
opposed to the original APC sensitivity tests should be used.
If positive the mutation should be confirmed by a direct genetic
test. An APC resistance assay is unnecessary if a direct
genetic test for F5G1691A is used initially.
Repeat testing for identification of deficiency of antithrombin,
protein C and protein S is indicated and a low level should be
confirmed on one or more separate samples.
Deficiency should not be diagnosed on a single abnormal
result.
Baglin T. British Journal of Haematology, 2010, 149: 209-220

Recommendations for interpretation

(6/6)

Rigorous internal quality control assurance and satisfactory

participation in accredited external quality assessment
schemes are mandatory.
Thrombophilia testing must be supervised by experienced
laboratory staff and the clinical significance of the results
must be interpreted by an experienced clinician who is aware
of all relevant factors that may influence individual test
results in each case.

Baglin T. British Journal of Haematology, 2010, 149: 209-220

Laboratory testing for inhibitors

Activity (functional) assays
Clotting test

Chromogenic assay

Antigenic assays
Immunological assay

Preanalytical variables
Sample:
Sampling on citrate 0.109M (1 vol for 9 blood vol
blood)
No polystyrene tube
Transportation at room temperature
Analysis 4 hours after blood sampling
Fresh or frozen plasma without platelets
Platelets < 10.109 /L (10 000 / L)
Double-centrifugation
Quick defrosting in 37C, homogenization of the plasma and
the immediate testing

Antithrombin (AT)

Antithrombin (AT)
Glycoprotein synthesised in the liver
Major inhibitor of factors IIa and Xa
Normal plasma level usually between 80 and
120 %
AT congenital deficiency: levels of 40 to 60 %

Antithrombin (AT)
Physiological variation
AT level is low until the age of 6 months
Marked decrease after the 13th week of
pregnancy and during post partum
AT level in women up to the menopause is
lower than in men
Acquired AT deficiency
Heparin therapy, severe liver disease, nephrotic
syndrome, preeclampsia, DIC,

Hereditary AT deficiency
Type I:
True deficiency
Low antigenic and functional levels

Type II:
Presence of abnormal molecules
Normal antigenic BUT low functional levels
RS, effect on Reactive Site
HBS, effect on Heparin Binding Site
PE, Pleiotropic (or multiple) effects

Prevalence in general population 1:5000

Stago reagents for AT

STA-Stachrom AT III
Chromogenic test
Detection of different types of AT deficiencies (I & II)
Fully automtaed on STA line

Liatest AT III
Immuno-turbidimetric assay
Quantification of the antigenic AT
Manual assay

Diagnosis strategy
ATIII

Quantitatif
deficiency

A: Abnormal < 80%

N : Normal > 80%

Qualitatif
deficiency
RS*

HBS**

* RS: Reactive Site

**HBS: Heparin Binding
Site

1st step

STA - Stachrom
AT

A
2nd step

Liatest AT

ProgressiveAT (same as
Stachrom AT III, but
without heparin)

Allows diagnosis of any type of deficiencies

The more oftenly used

Proposal for a diagnosis approach

Activity assay

Chromogenic assay
STA-Stachrom AT III

Normal
> 80%

Abnormal
< 80%

No AT deficiency

Antigenic
determination
+ Criteria
of
thrombophilia

Confirm abnormality
on a new sample, same test

Normal
> 80%

Abnormal
< 80%

Confirm discrepancy AT deficiency

Qualitative defect (rare)
Antigenic determination (LiatestAT III)

STA-Stachrom AT III
STA-Stachrom AT III 3 (4x3mL)
STA-Stachrom AT III 6 (4x6mL)
Functional assay
Automated test
Long stability
7 days on STA -R and STA -Compact
8 days sur le STA Satellite
21 days at 2-8C (original vial)
Clinically validated
Detection of all AT deficiencies

STA-Stachrom AT III - Principle

Give the specificity and
the sensitivity of the test
excess of
IIa
Patient

AT

heparin

excess IIa

+
Chromogenic
substrate CBS

AT

IIa

IIa

IIa

IIa

IIa

Paranitroaniline
Measure at = 405 nm

Intensity of the coloration is inversely proportional

to the AT level in the plasma

STA-Stachrom AT III - Specificity

Main features
Short time reaction (60 sec)
High concentration of heprain
Bovin thrombin (FII) + specific buffer

An anti-IIa based assay functional assay

Allows the detection of some variants not evidenced by
other activity techniques (anti-Xa)
Allows detection of HBS type II variants
Rare: incidence 1:300

Cambridge II, Stockholm AT

Not sensitive to heparin cofacteur II (HCII)

Perry et al.
Thromb Haemost 1998; 79 : 249-253

Using an anti-Xa based assay individual with the Ala384Ser mutation,

approximately 50% of cases can be missed

Ala384Ser mutation: Cambridge type II variant

Antovic et al.
Lab Hematol. 2002; 8: 37-40

Some type II deficiencies are not detected with

a factor Xa inhibition-based test

ANTITHROMBIN
STA-Stachrom AT III

Packaging

R1 Bovin Thrombin
R2 CBS 61.50
R3 Solvant with heparin

Volume

ATIII 3
ATIII 6

Nomber of tests

PE = 100 L
AT III (3 ml): 30 theorical tests , 23 real tests
AT III (6 ml): 60 theorical tests, 53 real tests

Stability

7 days on board STA Compact & STA-R

Evolution ES

Calibrator

STA-Unicalibrator

Controls

4 x 3 mL
4 x 6 mL

STA-Coag Control N+P

STA-System Control N+P

Competition
AT

Siemens versus Stago

Berichrom ATIII
Thrombin bovin (Lyo) 5/15 mL
Substrate 3/6 mL
Buffer 30/100 mL

Stability
4-48 h on-board (system dependent)
2 weeks at 2-8C

Turn around time

3 min

Tests number
50 tests

Not sensitive to Cambridge &

Stockholm variants

STA-Stachrom AT III
Bovine thrombin (Lyo) 3/6 mL
Substrate CBS 61.50
Buffer with heparin

Stability
7 days on-board
21 days at 2-8C
Validated on instrument

Turn around time

60 sec

Tests number
30 (x3 vials)
60 tests (x6 vials)

Detection of all type II

Cambridge & Stockholm variants
Not sensitive to heparin
cofacteur II (HC II)

Siemens versus Stago

Innovance AT
human FXa (liquid) 5/15 mL
Substrate 3/6 mL
Buffer 30/100 mL

Stability
4-48 h on-board (system dpt)
4 weeks at 2-8C

TAT
3 min

Tests number
50 tests

Not sensitive to Cambridge &

Stockholm & Denver variants

STA-Stachrom AT III
Bovine thrombin (Lyo) 3/6 mL
Substrate CBS 61.50
Buffer with heparin

Stability
7 days on-board
21 days at 2-8C
Validated on instrument

TAT
60 sec

Tests number
30 (x3 vials)
60 tests (x6 vials)

Detection of type II Cambridge &

Stockholm & Denver variants
Not sensitive to heparin HC II

IL versus Stago
Liquid Antithrombin
buffered bovine FXa (liquide) 4 mL
with heparin
Substrate 2 mL

Stability
48 h on-board
5 weeks at 2-8C
for optimal stability, keep the vial in the
fridge between tests

Performances
not sensitive to heparin HCII
not sensitive to Stockholm & Denver
variants

STA-Stachrom AT III
bovine thrombin (Lyo) 3/6 mL
Substrate CBS 61.50
Buffer with heparin

Stability
7 days on-board
21 days at 2-8C
validated on instrument

TAT
60 sec

Tests number
30 & 60 tests

Detection of type II Cambridge &

Stockholm & Denver variants
Not sensitive to heparin HC II

Conclusion - Antithrombin (1/3)

Chromogenic assay
Stago

Siemens

IL

Principle

Features

STA-Stachrom ATIII

bovine thrombin
functional assay

7 days on-board STA line

21 days at 2-8C
buffer with heparin

Berichrom ATIII

bovine thrombin
functional assay

4-48 hours on-board

15 days at 2-8C
buffer without heparin

Innovance AT

anti-Xa functional
assay

4-48 hours on-board

28 days at 2-8C
buffer without heparin

Liquid Antithrombin

anti-Xa functional
assay

48 hours on-board
35 days at 2-8C
buffer with heparin

Cooper PC. Int. J. Lab. Hem; 2012. 9: 1-11

Conclusion - Antithrombin (2/3)

Assays that use factor Xa may fail to detect the common variant, AT
Cambridge II, which can be detected by assays using bovine
thrombin.
AT activity assays are essential for the detection of AT deficiency
because type II defects are relatively common in patients with
heritable deficiency.
An excess of antigen over activity level suggests the presence of
functionally defective. Consequently the association of a functional
and an antigenic assay can detect all forms of AT deficiency.

Cooper PC. Int. J. Lab. Hem; 2011. 33(3): 227-237

Conclusion - Antithrombin (3/3)

An excess of antigen over activity level suggests the presence of
functionally defective. Consequently the association of a functional
and an antigenic assay can detect all forms of AT deficiency.
STA Stachrom ATIII (functional assay)

Liatest AT (antigenic assay)

Stago provides customers with the complete range of reagents for all
AT deficiencies determination.
Cooper PC. Int. J. Lab. Hem; 2011. 33(3): 227-237

Protein C

Protein C system

PC

C4bBP

PS

Total
PS

VIIIa
Va
VIIIi

TM

IIa PC

EPCR

Endothelium
PCa:
Activated Protein C
TM:
Thrombomodulin
PS:
Protein S
C4bBP: C4b Binding-Protein
EPCR: Endothelial Cell PC Receptor

Free
PS

PCa

Vi

Vi: V inactivated
VIIIi: VIII inactivated
Adapted from Esmon CT et al.,
Thromb. Haemostasis, 78 (1), 70, 1997

Protein C
Synthesised by the liver
Vitamin-K dependant glycoprotein
Normal plasma range: 70 - 130 %
Levels for heterozygous PC deficient patients
between 25 % and 70 %

Prevalence of the congenital deficiency in the

general population: 0.1 - 0.3 %

Protein C
Physiological variations
At birth, low PC levels are observed due to liver
immaturity
PC increases slightly during pregnancy and during
the puerperium
In adults, the protein C level is independent of age and sex

Acquired deficiencies
Oral Anticoagulant therapy, liver disorders, nephrotic
syndrome, DIC, ...

Hereditary protein C deficiency

Type I:
True congenital deficiency
Reduced PC antigen and PC activity

Type II :
Abnormal molecule
Normal PC antigen but reduced PC activity
Abnormal binding to other proteins (mostly PS)
Abnormal binding to PPL (severe types)

Stago reagents for Protein C

STA-Staclot Protein C
3 x 1 mL
Clotting method
Detects any type of PC deficiency (I and II)
Detects simultanously:
catalytic activity
FVa / FVIIIa interactions
PS / PL / Ca2+ interactions

STA-Stachrom Protein C
6 x 3 mL
Chromogenic method
Detects any type of PC deficiency (I and II partly)
Well standardized and less interferences

PC diagnosis strategy

*AC: Anticoagulant
**AM: Amidolytic

Proposal for a diagnosis approach

Activity assay

Activity
e.g. STA-Staclot PC or STA-Stachrom PC
assay
Normal
> 70%

Abnormal
< 70%

No PC deficiency

Antigenic
determination
+ Criteria
of
thrombophilia

Confirm abnormality
on a new sample, same test

Normal
> 70%

Abnormal
< 70%

Confirm the discrepancy

Qualitative defect (rare)
Antigenic determination
Asserachrom PC

PC deficiency

STA-Staclot Protein C
Functional assay
Snake venom-based assay
Detects any deficiencies
Golden standard, reference thrombosis centers

Automated on coagulation analysers

Specific
No interference by heparin up to 1 IU/ml, by factor
deficiencies and protein S levels

Stability:
8 hours on board

STA-Staclot Protein C
Principle

50 l

50 l

Two-step reaction
For the quantitative measurement of
the functional PC level
Anticoagulant reaction
50 l

Purified extract of
Agkistrodon contortirx venom

1/10
dil.

37C

180 sec.

50 l

CaCl2
37C

STA-Staclot Protein C
Role of reagents
Total
***
C4 BP PS
PC
PS
***Va
b

***IIa PC

TM

***
VIIIa

EPCR

Endothelium

***

Free PCa
PS

37C

Vi

activates PC > test not directly

dependent of the plasma PC

Deficient plasma which

adds all the requested factors for coagulation
CaCl2

VIIIi

Ca2+ to trigger the coagulation reaction

PC diagnosis strategy

*AC : Anticoagulant
**AM : Amidolytique

STA-Stachrom Protein C
Principle
One-step reaction
For the quantitative determination
of the functional PC level
Amidolytic reaction

Purified extract
of Agkistrodon
contortirx venom
50 l

50 l

50 l

Substr
Monitoring
Absorbance
180 sec.

37C

STA-Stachrom Protein C
Chromogenic assay principle
PC

aPC

Activated
Protein C

Specific Protein C
activator

Protein C

(Agkistrodon contortrix contortrix)

aPC

aPC

Paranitroaniline
Measure at = 405 nm

Chromogenic
substrate CBS

Intensity of the coloration is directly proportional

to the PC level in plasma

STA-Stachrom Protein C
Chromogenic functional assay
Snake venom-based assay
Detects any type of PC deficiency (I and II partly)
Quantitative determination of the functional PC level

Automated on most analysers

Specific
No interference by heparin up to 1 IU/ml, by factor deficiencies,
PS levels or Lupus Anticoagulant

Excellent stability
21 days on board STA instruments
1 month at 4C

PROTEIN C
STA-Staclot PC

STA-Stachrom PC

Principle

R1 PC deficient Plasma
R2 Agkistrodon Contortrix Contortrix
(Protac)

R1 Agkistrodon Contortrix
Controtrix (Protac)
R2 CBS 42.46

Kit content

ref 00737: 3 x 1 mL

ref 00671: 6 x 3 mL

Number of tests

PE = 50 L
3 x 20 theoretic tests
3 x 16 real tests

PE = 100 L
6 x 30 theoretic tests
6 x 23 real tests

Stability

8 hours on board

21 days on board

Calibrator

STA -Unicalibrator

STA -Unicalibrator

Calibration

4 points / series

3 points per lot

Control

STA -System Control N+P

STA -System Control N+P

Minimum patient per week

16 - 4 cal -2ct = 10 patients

23 - 3 cal - 2 ct = 18 patients

Competition
PC

Siemens versus Stago

Protein C Reagent
manual or ou automated method
PC-Activator 1x3 mL
APTT reag. for PC 1x10 mL
PC deficient plasma 4x1 mL

Stability
8 hours 15-25C (on-board)
2 days at 2-8C
7 days < -18C
Tests number (manual method)
60 tests per vial (PC Activator)
60 tests per kit

Interferences with LA et FVL

STA-Staclot Protein C
automated method
PC-Activator 3x1 mL
PC-deficient plasma
only 2 reagents

Stability
8 hours on-board
validated on instrument

Tests number
60 tests per kit
20 tests per vial

no interference
with LA or FV leiden

Siemens versus Stago

Berichrom Protein C
Manual or automated method
PC-Activator 5/10 mL
Substrate reagent 3mL
Hepes Buffer solution 30 mL

Stability
PC activator: 2 weeks (2-8C) or 8
h (37C) ; on-board ???
Substrate: 6 weeks (2-8C) ou 1
week (37C)
Calibrator: 4 hours
Controls: 4 hours

Tests nb (manual method)

10 tests per vial (10 mL)
x3 vials = 30 tests
5 tests per vial (5 mL)
x4 vials = 20 tests

STA-Stachrom Protein C
Automated method
PC-Activator (3 mL)
Substrate reagent (6 mL)
Owren on-board

Stability

PC Activator: 21 days OB
Substrat: 21days OB
Calibrator: 4 hours
Controls: 8 hours

Tests nb
30 tests per vial
180 tests per kit (6 vials)

IL versus Stago
HemosIL ProClot
Automated method
PC-Activator 4x1.5 mL
PC-deficient plasma 4x1 mL
PC control plasma 2x1 mL
(low level of Protein C)

Stability
PC-Activator: 15 days (2-8C)
or 60 days (-20C flc origine) ; OB ??
PC-deficient plasma: 4 hours (ACL Top:
15-25C) or 7 days (-20C)
PC control plasma: 4 hours (15-25C)
or 7 days (-20C)

Limitations
Bilirubin (>20 mg/dl), triglycerides (>
500 mg/dl), hemoglobin (> 150 mg/dl)
interferences with LA, high FVIII level,
FVL

STA Staclot Protein C

Automated method
PC-Activator 3x1 mL
PC-deficient plasma
no control in the kit

Stability
8 hours OB
OB validation

System controls
8 h stability OB

No limites with turbid plasma

no interference with LA or FVL
Tests number
60 tests per kit
20 tests per vial

IL versus Stago
HemosIL PC
Automated method
Diluant 1x8 mL
PC-activator 2x2.5 mL
Chromogenic susbtrate 2x2 mL

Stability
Diluant: keep at 2-8C
PC-activator: 5 days at 15C (Futura,
Advance et ACL Top) ; 3 months (2-8C,
original vial)

STA-Stachrom Protein C
Automated method
PC-Activator (3 mL)
Substrate reagent (6 mL)
Owren on-board

Stability

PC Activator: 21 days OB
Substrate: 21 days OB
Calibrator: 4 hours
Controls: 8 hours

chromogenic substrate: 5 days at

15C (Futura, Advance et ACL Top) ; 3
months (2-8C)

Tests number
60 tests (kit)

Tests number
30 tests per vial
180 tests per kit (6 vials)

Conclusion - Protein C
Clotting

Chromogenic

Immunological

Stago

STA-Staclot PC

STA-Stachrom PC

Asserachrom PC

Dade Behring
(Siemens)

Protein C Reagent

Berichrom PC

HemosIL Proclot
PC

HemosIL PC

IL

Stago is the only supplier to offer a complete range of reagents

for PC determination
identify the 3 types of deficiencies

Functional assay should be the first choice to determine

protein C levels (Cooper, 2012).
Chromogenic assay for protein C is less subject to interference
than clotting assays and are preferable (Cooper, 2012).
Cooper PC. Int. J. Lab. Hem; 2012. 9: 1-11

Protein S

Protein S
Produced by the liver
Vitamin-K dependent glycoprotein
Normal range (free PS Ag):
Men: 108% (SD 16.5%)
Women: 88% (SD 19.5%)
2 forms of protein S:
Free PS (# 40 %) and PS-C4b BP

Protein S
Plasmatic concentration: 25 mg/ml
Two forms of the protein S
Free ~ 40 %
only active form, cofactor activity on PC
Bound to C4b-BP
not active form
C4b-BP: bound the PS to PPL
C4bBP

PS
PS

PS
PS

bound PS: 60%

free PS: 40%

reversible

activate the haemostasis

Protein S
Physiological variation
At birth: low total PS, normal free PS level
Total and free PS levels are lower in woman than
in man, and increase with age
PS levels decrease during pregnancy
Acquired deficiency
Warfarin, DIC, liver disease, oral contraception,
nephrotic syndrome, AIDS, inflammatory
syndrome (C4bBP ) ...

PS diagnosis strategy

Allows diagnosis of any type of deficiencies

The more oftenly used

Proposal for a diagnosis approach

Activity assay
Activity assay

e.g. STA-Staclot Protein S

Normal
> 65 % *

* For men

No PS deficiency

Antigenic
determination
+ Criteria
of
thrombophilia

Abnormal
< 65% *

Confirm abnormality
on a new sample, same test

Normal
> 65% *
Confirm discrepancy

Abnormal
< 65% *
Protein S deficiency

Qualitative defect
Antigenic determination
e.g. Asserachrom Free PS or STA-Liatest Free PS

Protein S testing
Protein S activity
STA-Staclot Protein S
2 x 1 mL
Clotting test

Free protein S antigen

Asserachrom Free Protein S
STA-Liatest Free Protein S
6 x 2 mL et 6 x 5 mL
Immunological assay

Total protein S antigen

Asserachrom Total Protein S
Liatest Protein S
Manual assay

Protein S
STA -Staclot PS

Kit

Packaging
Number of test
Stability
Minimum test/day
Calibrator
Control

R1 PS deficient Plasma
R2 aPC
R3 bovine FVa
> Functional test

STA-Liatest Free PS
and

R1 Latex
R2 Buffer

2 x 1 ml

6 x 6 ml
3 x 2 ml

2 x 20 / kit

240 / kit, 40 / vial

39 / kit, 13 / vial

4 hours on board

5 days on board
32 hours on board

20

STA-Unicalibrator

pre-calibrated reagent

STA-System Control N+P

STA-Liatest Control N+P

STA-Staclot Protein S
Principle
Two-step reaction
Anticoagulant reaction

50 l

1/10
dil.

50 l

50 l

50 l

37C

Clotting assay based on the direct PS action

on its physiological targets (PCa, factor Va)

120 sec.

50 l

CaCl2
37C

PC

STA- Staclot Protein S

Role of reagents
***
Total
***
VIIIa
C4 BP PS
PS
***
Va
b

***IIa PC

TM
Endothelium

EPCR

***

Free PCa
PS

VIIIi

Vi

activated factor V added in excess *** > independent test from potential
patient's Fact V deficiency
deficient plasma which adds all the requested factors for coagulation ***
activated PC added in excess > test not directly dependent of the
plasma PC ***
CaCl2

Ca2+ to trigger the coagulation reaction

STA-Staclot Protein S
Advantages
Functional assay
Clotting assay
detects any type of PS deficiency (type I, II, III)

APTT based method

no risk of interference of FVIIa generated in vivo or in
vitro (frozen samples)

Protein S deficient plasma

no interference by factor deficiencies

PCa and FVa included in the reagents

optimal and standardized levels of PCa and FVa
no in vitro activation of patient PC
no interference by APC Resistance

Heparin inhibitor included in reagent

no interference by therapeutic heparin up to 1 IU/ml

Diagnostic strategy

STA-Liatest Free Protein S

Features
Quantitative and direct measurement
Latex Immuno Assays (LIA)
Based on the use of latex microparticles

2 MAbs specific of free PS

Automated on STA line
Quick turn around time (< 10 min)
Precalibrated and barcoded assay
Liquid and ready to use reagents
Measuring range: 10% -150%
Detection limit: 7%
No hook effect up to 200%

STA-Liatest Free Protein S

Good correlation with the referent assay

Blood Coagulation and Fibrinolysis, 2001

Key features of
STA-Liatest Free Protein S
Features
Automated test
Quick & simple to
measure Free PS
Liquid and ready to
use
Precalibrated

Advantages
Very easy to use
5 days OB stability
One-shot test or series
of batch
High level of
measurement up to
150% (without redilution)
Standardized
Secure

Limitations
Dosage of the inactive Free protein S:
Qualitative deficiency
VKA treatment

Risk of false diagnosis if used first

complementary test to clotting assay, which
remains the gold standard

Competition
PS

Siemens versus Stago

Protein S Ac
Protein S Ac deficient plasma
Protein S Ac APC reag. (contains
human PC, lyo)
Protein S Ac starting reag.
(contains the activator RVV
linked to PL)

Stability
8 h (15-25C)
2 months (< -18C)

Tests number
?

Interference with FV Leiden

STA-Staclot Protein S
PS deficient Plasma
PCa
Bovine FV

Stability
8 h (15-25C)

Tests number
40 tests per kit
20 tests per vial

No interference with FV
Leiden and factor
deficiencies

IL versus Stago
HemosIL ProS
PT based assay (rabbit
recombinant TF)
sensitive to factor VII
frozen sample can to FVII
activation

PS reagent
PS deficient plasma
PC control plasma

Stability
PS reagent:
16 h-2 days (2-8C)
1 h (15C - ACL Top)
4 hours (ACL 8000/9000)

PS control plasma:
4 hours on-board

STA-Staclot Protein S
APTT based assay
not sensitive to FVII

PS deficient Plasma
PCa
Bovine FV
not sensitive to factor V
Leiden
not sensitive to factor
deficiencies

Stability
8 hours on-board

Controls
8 hours on-board

IL versus Stago
HemosIL Free protein S
Immunological test in 2 steps
Latex coated with C4bBP
Latex coated with anti PS (Mab)

Kit contains
Buffer (Liq)
Latex C4bBP (Lyo)
Latex PS (Liq)
to reconstitute Latex C4bBP
stabilize 30 min
Take care to bubles !!!

Calibration
necessary

One package
3 x 25 tests

Stability
1 week on-board (ACL Top)
1 months (2-8C)

STA-Liatest Free PS
1 step
latex coated with 2 Mabs

Kit contains
Buffer (Liquid)
Liatest (Liquid)
Ready to use

Calibration
precalibration

2 packages :
3x2 mL for 20 tests
6x5 mL for 50 tests

Stability
5 days on-board (STA Liatest
Free PS 6x5 mL)

Thrombophilia screening,
in practice

Thrombophilia laboratory testing

Functional assays first
AT activity
STA-Stachrom AT III

Protein C activity
STA-Staclot Protein C
STA-Stachrom Protein C

Protein S activity
STA-Staclot Protein S

Thrombophilia laboratory testing

Antigenic assays as complementary tests
AT antigen
Liatest AT III

Protein C antigen
Asserachrom Protein C

Free PS antigen
STA-Liatest Free Protein S
Asserachrom Free Protein S

Total PS antigen
Asserachrom Total Protein S

Thrombophilia laboratory testing

Other parameters for a global testing:
FV mutation
Prothrombin mutation
DNA analysis
level of FII

High coagulation factor (FVIII, FXI)

Antiphospholipid antibodes
LA
anticardiolipin or anti-2GP1 antibodies

Factor inhibitors

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