COAGULATION TESTS AND  Sample becomes turbid once the

clot is being formed so the optical

FIBRINOLYSIS
FOUR GENERAL TECHNIQUES
MANUAL
THE TILT TUBE METHOD REQUIRES
GENTLE TILTING OF THE TUBE BACK
AND FORTH AT THE RATE OF ABOUT
ONCE PER SECOND UNTIL A FIBRIN
WEB IS FORMED.
 Encountered when doing manual
APTT and prothrombin time testing
 Gentle tilting of the tube, back and
forth, until a fibrin is formed and that
is how you measure (example)
coagulation testing time which
involves APTT and prothrombin
time.
THE NICHROME WIRE LOOP
TECHNIQUE EMPLOYS THE USE OF A
WIRE LOOP THAT IS PASSED THROUGH
THE MIXTURE AT THE RATE OF TWO
SWEEPS PER SECOND UNTIL A
FORMED CLOT ADHERES TO THE
LOOP.
 Technique employed when we do
clot retraction time
AUTOMATED
density is the one being measured in
order to detect how fast the clot is
being formed.
CLOTTING TIME
 Time when the blood clots
LEE AND WHITE METHOD
 Uses test tubes, put blood taken
from venipuncture, wait until –
incubate it at 37C, wait blood to form
clot
• SCREEN SECONDARY
HEMOSTASIS
(INTRINSIC AND COMMON PATHWAY)
 Function of this test – screening for
secondary hemostasis particularly in
intrinsic and common pathway
 Intrinsic pathway is activated by
negatively charge surface such as
collagen
PRINCIPLE:
EXPOSE BLOOD TO A NEGATIVELY
CHARGED SURFACE
 Such as glass – which is the main
material of the test tube

: A CLOT IS DETECTED EITHER BY USE NV: 5-15 MIN

OF A MOVING PROBE IMMERSED IN
THE MIXTURE THAT IS TRIGGERED BY
CLOT FORMATION OR BY THE CHANGE
IN OPTICAL DENSITY OF THE MIXTURE
WHEN A CLOT FORMS.

 Most common technique in

automated device for coagulation
testing time – optical density.
 LABORATORY SCREENING
Clotting time – slide method
(Intrinsic)
 The surface of the glass tube
initiates the clotting process. This
test sensitive to the factors involved
in the intrinsic pathway.
 The expected range for clotting time
is 4-10 mins.
 A new innovation
 Done by puncturing (source of the
blood)
 Then put blood in a glass slide,
which is negatively charge
 this test is sensitive to the factors
involve in the intrinsic pathway
 Wait, and swirl with something sharp
until it agitates, pull it up, and a fibrin
string is formed, an indication of clot
formation
 If u have a problem with intrinsic
pathway, or problem with extrinsic
pathway or both, which corresponds
to a problem of the common
pathway
• PROTHROMBIN TIME
 Measurement of extrinsic pathway
• ACTIVATED PARTIAL
THROMBOPLASTIN TIME (APTT)
 For intrinsic pathway
 When two of these test is abnormal
– indicative of a problem in the
common pathway
 Blue – intrinsic, for APTT (for
monitoring heparin therapy)
 Pink – extrinsic, for PT (monitoring
of courmadin therapy, such as
warfarin for instance)
 When these two are abnormal, it is
an indication of deficiency/disorder
that corresponds to the factors
involved in common pathway
PROTHROMBIN TIME
IS A USEFUL SCREENING PROCEDURE
FOR EXTRINSIC AND COMMON
PATHWAY.
SAMPLE: CITRATED PLASMA
 Citrate - Usually blue colored and
the second tube in the series of
blood collection
 Sodium citrate is the one that binds
with calcium to form insoluble
complexes – to inhibit calcium
 Why inhibit calcium? If calcium is
being inhibited, therefore no clotting
to be done. (it is an anticoagulant
choice for coagulation test)
 Platelet pore plasma is also use,
that’s why in coagulation test, the
centrifugation is much more longer
since you need to ensure that the
sample is platelet pore
 Platelet is required for coagulation –
do not use platelets
 Platelet pore plasma, that is being
anticoagulated by sodium citrate
REAGENT: TISSUE THROMBOPLASTIN,
0.025 MOL/L SOLUTION OF CALCIUM
CHLORIDE.
 Tissue thromboplastin is a substitute
for factor III
 Calcium chloride is a substitute for
calcium that is being inhibited
 Once these two is being mix with the
citrated platelet pore plasma, they’re
going to activate the extrinsic
pathway and the common pathway
 Then observe for clot formation
 If there is no deficiency, then there is
normal PT time
• NORMAL RANGE: 10-15 SEC
SENSITIVE TO: FACTORS VII, V, X, I AND
II
ACTIVATED PARTIAL
THROMBOPLASTIN TIME (APTT)
 Principle is the same with PT
 Reagent + citrated pore plasma but
the significance to measure the
intrinsic pathway and common
pathway
 Does not measure platelets and
factor XIII
 Factor XIII “fibrin stabilizing factor”
MEASURES ALL FACTORS IN THE
INTRINSIC AND COMMON PATHWAY
EXCEPT FACTOR XIII AND PLATELETS.
SAMPLE: CITRATED PLASMA
REAGENT: PARTIAL THROMBOPLASTIN,
CALCIUM CHLORIDE, ACTIVATORS
 Partial thromboplastin is a substitute
for platelet
 Calcium chloride substitute for
calcium
 Activators used as substitute for
collagen – which is negatively
charge surface
ACTIVATORS: SILICA, KAOLIN, ELLAGIC
ACID, CELITE
 No need activators in intrinsic
because tissue thromboplastin is
only needed to make calcium
chloride react with the plasma and
coagulation cascade will continue
 For intrinsic to be activated, it needs
a negatively charge surface
 Why activators are needed? You
need to use a non-contact surface
NORMAL RANGE: 28-46 SEC
OTHER COAGULATION CONVERT FIBRINOGEN O FIBRIN. TIME
IS RECORDED AS CLOT IS FORMED
TESTS
AFTER REPTILASE IS ADDED TO
1. THROMBIN TIME (TT)- PLASMA.
MEASURES THE AVAILABILITY
NV: 10-15 SEC
OF FUNCTIONAL FIBRINOGEN OR
DECREASED AMOUNT (75-100
MG/DL). AFFECTED BY HEPARIN.

 Thrombin time test is the

specific test for fibrinogen
which is factor I
 If there is fibrinogemia –
decrease amount it will result
to abnormal thrombin time
 Thrombin is the activated
form of prothrombin
 Substrate for factor II?
Factor I.
 This test is specific for factor
I 3. STYPVEN TIME (RUSSELL'S VIPER
 But the drawback – if the VENOM TIME)
patient is undergoing heparin
therapy, this test will be - USED TO DETECT DEFICIENCIES OF
prolonged THE COMMON PATHWAY (FACTORS X,
 Since heparin inhibits V, II AND I)
thrombin (factor II inhibitor) PRINCIPLE:
PRINCIPLE: MEASURED THROMBIN IS STYPVEN IS A THROMBOPLASTIN LIKE
ADDED TO PLASMA (activated factor II) SUBSTANCE THAT ACTIVATES FACTOR
AND TIME FOR FIBRIN CLOT X.
FORMATION IS RECORDED.
 Any deficiency in factors X, V, II, and
NV: 10-14 SEC (ACCORDING TO I, will result to impaired clot
BROWN) formation that will result the Stypven
2. REPTILASE TIME- SAME AS time to be abnormally prolonged
THROMBIN TIME BUT NOT AFFECTED WHEN THIS SUBSTANCE IS ADDED TO
BY HEPARIN. PLASMA TOGETHER WITH PLATELIN
 Measures factor I (PLATELET SUBSTITUTE) AND CACL2.
 Reptilase Vs TT – reptilase is not COAGULATION WILL ACTIVATE AND
affected by heparin TIME IS RECORDED UNTIL CLOT IS
PRINCIPLE: REPTILASE (ATROXIN) IS AN FORMED.
ENZYME FOUND IN THE VENOM OF THE
BOTHROPS ATROX SNAKE THAT CAN
  Platelet phospholipid is needed for
conversion of factor II to its activated
form, platelin.
NV: 6-10 SEC
4. DUCKERT'S TEST/ 5M UREA
SOLUBILITY TEST
 Test for factor XIII
- DETECTS FACTOR XIII DEFICIENCY
CLOT FORMED AFTER COAGULATION IS
ADDED WITH 5 M UREA.
NV: CLOT IS INSOLUBLE AFTER 24
HOURS
 This is Normal, this is a qualitative
test – not quantitative
NOTE: SUBSTITUTE FOR 5M UREA
 1% MONOCHLORACETIC ACID
 2% ACETIC ACID
 If there is deficiency in the factor
XIII, fibrin clot is unstable, that’s why
the clot will be soluble for 24hrs after
adding urea reagent
FIBRINOLYSIS
 After coagulation cascade, fibrin is
formed.
 Thrombolytics will lyse the clot and
for hemostatic it will form a clot
 To regulate this, plasmin, a protein,
is present which will breakdown the
clot
FIBRINOLYSIS
• IS THE FINAL STAGE OF
COAGULATION
• IT IS THE SYSTEMIC,
ACCELERATING HYDROLYSIS OF
FIBRIN BY PLASMIN
 Plasminogen is activated to
being a plasmin
 It is being activated by tissue
plasminogen activator and
also urokinase
 Tissue plasminogen activator
inhibitor 1 is the one that
makes it balance – so not all
plasminogen will be
converted to plasmin
  Why? Because plasmin is
the one that breaks down the
clot, it should not be too
activated
 Function of plasmin?
It degrades the fibrin – if ever
the clot is not needed, it
FIBRINOLYSIS PROCESS
should be dissolved, this
should also facilitates
remodeling extracellular
matrix, this also promotes
cell migration – this actually
helps promote healing in the
injured blood vessel
PROTEINS NECESSARY IN
THE FIBRINOLYSIS
PROCESS
• PLASMINOGEN- SERINE
PROTEASE ZYMOGEN; ACTIVE
FORM IS PLASMIN WHICH
DIGESTS FIBRIN AD FIBRINOGEN
 The main protein, when
activated it will be plasmin
• TISSUE PLASMINOGEN
ACTIVATOR (TPA)- SECRETED
BY ENDOTHELIUM AND
ACTIVATES PLASMINOGEN
 One of the promoter of
fibrinolysis
• UROKINASE- SECRETED BY
KIDNEYS AND ACTIVATES
PLASMINOGEN
• PLASMNIOGEN ACTIVATOR
INHIBITOR 1 (PAI-1)- FROM
ENDOTHELIUM, INHIBITS TPA
• ALPHA 2-ANTIPLASMIN- INHIBITS
PLASMIN
• THROMBIN ACTIVATABLE
FIBRINOLYSIS INHIBITOR-
SUPPRESSES FIBRINOLYSIS
 These 3 proteins are the
ones inhibits fibrinolysis
1. THE FIBRIN FORMED IS
COMPOSED OF TWO DOMAINS: D
AND E
2. PLASMINOGEN IN PLASMA IS
ACTIVATED BY TPA (from
endothelium) AND UROKINASE
(from kidney) INTO ACTIVE
ENZYME PLASMIN
3. PLASMIN, THEN CLEAVES THE
PEPTIDE BONDS ON THE
ARGININE AND LYSINE AMINO
ACID THAT CONNECTS THE TWO
DOMAINS.
 Vit K dependent amino acid?
Glutamic acid
4. THE FIBRIN DIGESTED WILL
THEN FORM THE FIBRIN
DEGRADATION PRODUCTS
(FDPS) NAMELY:
 FRAGMENT X (D-E-D),
 FRAGMENT Y (D-E),
 FRAGMENT D, (E) ,
 FRAGMENT D-D (D-
DIMER).
 D dimer is being measured in
test that corresponds to
active fibrinolysis such as in
disseminated intravascular
coagulation wherein the D-
dimer would elevated or
becomes positive
FORMATION OF D-DIMER
(ARROWS ARE ACTED BY PLASMIN)

 Different arrows corresponds

to the plasmin
 (refer to the picture)
In fibrinogen, it can promote
formation of fragment X, D, Y
and E.
 But if it is cross linked fibrin,
wherein fibrinogen is being
converted by thrombin,
wherein fibrin – degradation
products would be formed –
but u can form here it’s a
complex wherein the two D-
domain connected to each
other, that’s what u called- D-
Dimer.
 If it’s coming from fibrin – you
can detect D-dimers ,
because the D-domain is
being cross linked to each
other