Transcript 30-year old female with refractory bleeding

Blood Coagulation
- Screening assays and single factor assessment -
Jørn Dalsgaard Nielsen
Thrombosis Centre
Gentofte Hospital
Copenhagen, Denmark
ADP
Ca++
Serotonin
Inhibition
PF3
IL-1
TNF
Tromboxane A2
Activated platelet
PF4
GP Ib-IX
Activation
Aktivation of
of monocytic
monocytic
cells,
cells, induced
induced by
by
endotoxin,
endotoxin, cytokines,
cytokines, etc.
etc.
GP IIb-IIIa
Tissue factor
F VIIa
F VII
Inactivates F VIIa ¤ F X ¤
tissue factor -kompleks
F IX
FX
PF3, Ca++, F VIIIa
F XIa
F XIa
F Xa
PC
PCa
F VIIIi
PS
F Vi
F XIIa
F VIII
FV
PF3, Ca++, F Va
Prothrombin
F XI ¤ HMWK----
Kallikrein
F XII---PK ¤ HMWK----
t-PA
Thrombin
PAI-1
Antithrombin
F XIII
Heparin Cofactor II
Inhibits thrombin
Plasminogen
Plasmin
Inhibits serine proteases
Fibrinogen
Polymerizing
fibrin
F XIIIa
Crosslinked
fibrin
α2-antiplasmin
Fibrinogen/Fibrin
degradation
products
Is testing of single factors necessary
in patients with suspected
haemostatic dysfunction ?
• It depends on who you are addressing
• A surgeon:
– Will the patient bleed ?
– Can I stop bleeding with fresh-frossen plasma ?
• A haematologist:
– Single factor assessment is often necessary to
establish a correct diagnosis
Indications for evaluation of
haemostatic function
• Clinical problem  Biochemical defect?
Coagulation
Screening Fibrinolysis
Bleeding
Thromboembolism
Platelets
Endothelium
Single
factor
assay
Further
screening
• Biochemical defect  Clinical problem?
Abnormal test
result
Further
screening
Single
factor
assay
Prophylaxis/
treatment
indicated?
or not?
The challenge of evaluation of
clotting abnormalities
• In vitro assessment of haemostasis is difficult
because the important interaction between the
endothelium and blood components cannot be
evaluated in a single assay.
• So-called ”global tests” can be used to test the
haemostatic capacity of blood components (plasma
and blood cells) but not the influence of
antithrombotic properties of the endothelium.
• Thrombelastography may give a clue of a clotting
defect, platelet dysfunction, or hyperfibrinolysis but
will not give the final diagnosis.
Thrombelastography
Increased in
patients
with
clotting
defects
Decreased in patients
with platelet
dysfunction or defect
fibrin formation
Reaction
time
Amplitude
TEG
Start
Minutes
Thrombelastography
TEG
• LA and HIT-2 are associated
with a high risk of thrombosis
• A shortened reaction time
might, therefore, be expected
but is not seen because the
thrombotic predisposition is
provoked by endothelial
dysfunction
The challenge of evaluation of
clotting abnormalities
• As global tests of haemostasis neither give a consice
diagnosis nor results that reliably reflect the clinical
problem, more specific assays are often needed for
the evaluation of thrombotic and haemorrhagic
disturbances of the haemostatic system.
However, separation of the
complex network of reactions
may result in a number of
other pitfalls and impede a
comprehensive view.
The challenge of evaluation of
clotting abnormalities
• Among laboratory testing, coagulation assays are the
most influenced by the inaccurate standardization of
the pre-analytical phase.
• Clotting times are influenced by:
–
–
–
–
–
–
–
–
time of tourniquet placement (<60 sec recommended)
needle size (19-22 gauge recommended)
citrate concentration (105-109 mM recommended)
incomplete filling of tubes (PT<80%, APTT<90%)
platelet count (<10*109/l recommended)
haemolysis and lipaemia
temperature and G-force during centrifugation
temperature and duration of storage until testing
The challenge of evaluation of
clotting abnormalities
• Screening methods of coagulation should
optimally be sensitive to all coagulation
defects.
• This is not the case but by combination of
simple procedures we can get close to the
final diagnosis.
Exploring coagulation
• The present theory of the function of the
coagulation system is based on numerous
studies performed in the 20th century.
• The history of the discovery of clotting factors
and development of assays may help
understanding the use of screening assays of
coagulation.
The theory of blood coagulation
Year 1900: the ’four factor’ theory
Known factors:
•
•
•
•
Fibrinogen
Prothrombin
Thromboplastin
Calcium
Cellular
damage
Prothrombin + Ca
Fibrinogen
Thromboplastin
Thrombin
Fibrin
• Hammerstein. Hoppe-Seylers Zeitschrift für physiologische Chemie 1899; 28: 98.
• Morawitz, P. Ergebnisse der Physiologie biologischen Chemie und Experimental
Pharmakologie 1905; 4: 307.
The theory of blood coagulation
Year 1935: the ’Quick’ test
Determination of the clotting
time of citrated plasma after
addition of thromboplastin
and calcium chloride
Prothrombin + Ca
Fibrinogen
Thromboplastin
Thrombin
Fibrin
• Quick AJ. J Biol Chem 1935; 109: LXXIII
The theory of blood coagulation
Year 1947: Factor V
Cellular
damage
Prothrombin + Ca
Fibrinogen
Factor V
Thromboplastin
Thrombin
Fibrin
• Owren PA. Acta Med Scand 1947; Suppl: 194
The theory of blood coagulation
Year 1947: Factor V
Cellular
damage
Prothrombin + Ca
Fibrinogen
Factor V
Thromboplastin
Factor V deficiency showed to be a rare disease,
and the discovery of FV did not explain the puzzle
that the standard coagulation test: the Quick test,
was normal in most patients with congenital
bleeding tendency.
• Owren PA. Acta Med Scand 1947; Suppl: 194
Thrombin
Fibrin
Mixing assays
• Whole blood clotting time and plasma
clotting time are prolonged in haemophiliac
patients and can be normalized by mixing
patient blood/plasma with equal parts of
normal blood/plasma.
• Both tests, however, have high CV%.
First description of APTT
The theory of blood coagulation
Year 1953: APTT
Prothrombin + Ca
Fibrinogen
Ca++
PT
Factor V
Thromboplastin
”Partial
thromboplastin”
APTT
Unknown factors
Thrombin
Incubation
Ca++
Kaolin
• Langdell et al. J Lab Clin Med 1953;41:637-47.
Fibrin
The theory of blood coagulation
Year 1959: the Roman numerical nomenclature
Factor
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
Synonyms
Fibrinogen
Prothrombin
Thromboplastin
Calcium
Accelerator globulin; proaccelerin; labile factor
Factor V derivative (not used now)
Proconvertin; stable factor; autoprothrombin I
Antihaemophilic factor A; platelet cofactor 1
Plasma thromboplastin component (PTC); Christmas factor; antihaemophilic
factor B; autoprothrombin II; platelet cofactor 2
Stuart-Prower factor
Plasmathromboplastin antecedent (PTA)
Hageman factor
Fibrin stabilizing factor
• suggested by an international committee under the chairmanship of Dr. IS Wright
The theory of blood coagulation
Year 1964: the cascade scheme
Surface contact
XII
Problems
XIIa
XI
XIa
IX
IXa
VIII
?
VII ?
VIIIa
X
Xa
V
?
Va
II
IIa
I
• Macfarlane, RG. Nature 1964; 202: 498
Ia (fibrin)
The theory of blood coagulation
Year 1975: the classic coagulation system
Internal
Surface contact
pathway
XII
External
Tissue pathway
factor
XIIa
XI
XIa
IX
IXa
VIIa
VII
Ca++
Phospholipid, Ca++, VIII
X
Xa
X
Phospholipid, Ca++, V
II
IIa
I
Ia (fibrin)
• Austen DEG & Rhymes. A laboratory manual of blood coagulation. 1975.
The theory of blood coagulation
discoveries of the last decades
• The major in vivo importance of the
external pathway
• Acceleration of coagulation by positive
feed-back mechanisms
• Inibitory mechanisms of blood
coagulation
The theory of blood coagulation today
Tissue factor
VII
VIIa
EXPRESSION OF TISSUE FACTOR
CONSTITUTIVE
INDUCED
PROHIBITED
e.g.:
epithelial cells
glial cells
e.g.:
monocytic cells
endothelial cells
e.g.:
lymphocytes
erythrocytes
IL-1
TNF-
C5a
The theory of blood coagulation today
Activation by a serine
protease, e.g. hepsin
Tissue factor
VIIa
VII
XI
XIa
IX
VIII
IXa
VIIIa
X
V
XIII
Xa
Va
IIa
XIIIa
XL-Fibrin
Fibrin
II
Fibrinogen
The theory of blood coagulation today
Activation by a serine
protease, e.g. hepsin
XII ?
Tissue factor
VIIa
VII
XI
XIa
IX
VIII
IXa
VIIIa
X
V
XIII
Xa
Va
IIa
XIIIa
XL-Fibrin
Fibrin
II
Fibrinogen
Activated
platelet
Zn2+
F XIa
F XI ¤ HMWK---F XIIa
Kallikrein
F XII---PK ¤ HMWK---urokinase
prourokinase
t-PA
PAI-1
Plasmin
Plasminogen
Natural inhibitors of blood coagulation
Tissue factor
XI
XIa
AT
IX
VIII
IXa
HC-II
VIIIa
X
V
XIII
Va
Fibrin
PCa
PC
IIa
XIIIa
XL-Fibrin
PS
Xa
II
Fibrinogen
Endothelial cell
VIIa
VII
The classic coagulation system
Surface
APTT
contact
XII
Prothrombin
Tissue factor
time
XIIa
XI
XIa
IX
IXa
VIIa
VII
Ca++
Phospholipid, Ca++, VIII
X
Xa
X
Phospholipid, Ca++, V
II
IIa
I
Thrombin time
Ia (fibrin)
• Austen DEG & Rhymes. A laboratory manual of blood coagulation. 1975.
Clotting defects and bleeding
• Coagulation factor deficiencies seldom cause
bleeding if the level of the deficient factor is
>40%.
• APTT is normal when the level of coagulation
factors is >40%.
• Therefore, APTT determined in a mixture of equal
parts of normal plasma and plasma from a
haemophiliac patient will be normal.
• Unless an inhibitor is present.
Antibodies against coagulation
factors
• Two types:
– Alloantibodies:
Patients with hereditary coagulopathy may develope
antibodies against the deficient factor when treated with
plasma-derived or recombinant factor concentrates
– Autoantibodies:
Aquired antibodies, most often against factor VIII and
typically in patients with autoimmune diseases, malignancy
and in women during pregnancy and post partum. In half of
the cases no underlying disease can be found. Incidence: 1-5
per 1.000.000.
APTT-based inhibitor test
Add APTT reagents
Determine
APTT
mix
Patient
plasma
Normal
plasma
APTT-based inhibitor test
Haemophilia
APTT: normal
mix
Patient plasma Normal plasma
Factor: <1%
100%
>50%
Patient with inhibitor
mix
Patient plasma Normal plasma
Factor: <1%
100%
APTT: prolonged
<50% due to excess of antibody
Treatment of bleeding in patients with
antibodies against coagulation factors
– In some patients (low responders) the neutralizing
effect of the antibody can be overcome by increasing
the dose of factor concentrate
– In patients with high titers of antibody recombinant
factor VIIa can be used to obtain haemostasis
The ”by-passing effect” of factor VIIa
FIX
TF
FXIa
FXI
FVIIa
FIXa
FVIIIa
FX
FXa
FVa
FV
Thrombin
Prothrombin
Cross-linked
fibrin
FVIII
AMPLIFICATION
INITIATION
FVII
Fibrin
FXIIIa
FIBRIN FORMATION
Fibrinogen
FXIII
Platelet
activati
on
30-year old female with
refractory bleeding
APTT Immediate
Plasma
(sec)
Normal pool (NP)
26,5
Patient (Pt)
91,3
Pt:NP = 1:1
38,5
2 h incub
(sec)
26,7
100,8
63,2
Aquired factor VIII deficiency with a progressive
inhibitor to factor VIII. Bethesda titer: 5.5 BU.
Algoritm for evaluation of
prolonged APTT
• Exclude preanalytical factors causing spuriously
prolonged APTT
– Underfilled tubes, delayed testing, venipuncture above heparin lock etc.
• Is the patient receiving antithrombotic treatment?
– E.g. heparin, thrombin inhibitors, vitamin K antagonists, fibrinolytics
• Defect fibrin formation?
– Determine fibrinogen concentation thrombin time
• If not – then test for inhibitors
– Lupusinhibitor (Thrombophilia)
– Antibodies against a coagulation factor (Haemophilia, aquired/cong.))
• If neg. inhibitor test: Coagulation factor deficiency
– Contact factor deficiency (No bleeding)
– Deficiency of other clotting factors (Haemophilia)
Algorithm for evaluation of
prolonged APTT
Explore possible AC treatment:
Heparin: Thrombin time
Vitamin K antagonist: INR
Fibrinogen < 3 M
Yes
Explore
hypofibrinogenaemia
No
Corrects APTT
Pt:NP 1:1 mix
Incub 2h  APTT
Corrects
APTT
Pt:NP 1:1 mix
immediate APTT
Fails to
correct
APTT
Inhibitor
present
Factor deficiency
Pt:APTT-reagent
Incub 10 minutes
Fails to
correct
APTT
Fails to
correct
APTT
Corrects
APTT
Phospholipid
dependent
Lupus
inhibitor
Procoagulant
factor deficiency
Contact
factor deficiency
Phospholipid
independent
Specific
inhibitor
Prolonged preincubation with
APTT reagent
PK-deficient plasma
Asmis et al. Thromb Res 2002;105:463-70
Algorithm for evaluation of
prolonged APTT
Explore Thrombin time: Heparin?
Fibrinogen > 3 M
INR: Vitamin K antagonist?
No
Explore
hypofibrinogenaemia
Yes
Corrects APTT
Pt:NP 1:1 mix
Incub 2h  APTT
Corrects
APTT
Pt:NP 1:1 mix
immediate APTT
Fails to
correct
APTT
Inhibitor
present
Factor deficiency
Pt:APTT-reagent
Incub 10 minutes
Fails to
correct
APTT
Fails to
correct
APTT
Corrects
APTT
Symptoms
Phospholipid
dependent
Lupus
inhibitor
Procoagulant
factor deficiency
Contact
factor deficiency
Phospholipid
independent
Specific
inhibitor
Evaluation of 177 consecutive cases of
prolonged APTT
Results
Chng et al. 2005
Evaluation of 177 consecutive
cases of prolonged APTT
100%
80%
No obvious
cause
Factor deficiencies: 15 %
60%
40%
20%
LA
0%
LA
FXII def
FXI def
FVIII def
Acq FVIII inh
vWD
FXI+XII def
FXII+vWD
No obvious cause
Chng et al. 2005
Factor XIII deficiency
• In FXIII deficiency the APTT, PT and
thrombin time are normal.
• Moderate to severe FXIII deficiency can be
diagnosed by the clot solution test.
• A fibrin clot prepared from patient plasma is
placed in 8 M urea.
• Dissolution of the clot within 24 hours is
suggestive of FXIII deficiency.
Blood coagulation
Screening assays and single factor assessment
Jørn Dalsgaard Nielsen
Thrombosis Centre
Gentofte Hospital
Copenhagen, Denmark
E-mail: [email protected]