Prothrombin Time (PT/ Protime)

PT is prolonged in congenital single-factor deficiencies of factor X, Vll or V,

profound prothrombin deficiency, and fibrinogen deficiency when the
fibrinogen level is 100 mg/dl or less. When the PT is prolonged, but the PTT/
APTT and Thrombin Clotting Time (TCT) results are normal, factor VII activity
may be deficient. Any suspected single-factor deficiency is confirmed with a
factor assay.
The PT is not affected by factor VIII or IX deficiency, because the
concentration of tissue factor in the reagent is high, and those factors are
bypassed in thrombin generation.
The lab should select thromboplastin reagents that are maximally sensitive to
Coumadin and relatively insensitive to heparin. Many manufacturers
incorporate polybrene in their thromboplastin reagent to neutralize heparin.
The MT may detect unexpected heparin by using the TCT test.
LACs may prolong some thromboplastin reagents. They are anti-PPL.
Coumadin is often prescribed to prevent thrombosis in patients with LACs, but
the PT may be an unreliable monitor of therapy in such cases. Patients who
have LAC and are taking Coumadin should be monitored using an alternative
system, such as chromogenic factor X assay.
The PT is most sensitive to factor VII deficiency( prolonged result), moderately
sensitive to factor V and X deficiencies, sensitive to severe fibrinogen and
prothrombin deficiencies, and insensitive to deficiencies of factors VIII, IX
and XIII.
The PT is prolonged in multiple factor deficiency disorders that include
deficiencies of factors VII and X.
Laboratory Evaluation:
Test of Phase II of Coagulation
H. Serum Prothrombin Time or Prothrombin
Consumption Test (PCT)
- best considered as a test of platelet phospholipid
activity. If the prothrombin time and the APTT are
normal, a short PCT indicates a deficiency of PF3 due to
thrombocytopenia or thrombopathia/
thrombocytopathy. If there is deficiency of any of the
factors required for coagulation of blood or plasma in
glass, Factor II will be incompletely consumed & more
than normal will be present in the serum after 1 hour.
The PCT is prolonged if any of the essential factors are
below 2% or3% of the normal.
Reference interval= 26-37 seconds

I. Thromboplastin Generation Test

1. Bigg’s and Macfarlane Method
2. Hick’s-Pitney Kaolin Modification Method
Thromboplastin Generation Time (TGT)
Principle; For normal thromboplastin activity to
develop in blood, HF,platelets,PTC,PTA,Factor V,
Stuart-Prower factor and ionized calcium are all
[Link] 4 rgts when added together supply
all the ingredients [Link] then, can give
way to the intrinsic thromboplastin generating
mechanism.
Al(OH)3 normal platelets
Normal plasma Calcium (M/40 CaCl2)
PPP + mixture( above).
Note the time for Pt’s plasma to clot. This is a measure
of conversion of prothrombin to thrombin and the
conversion of fibrinogen to fibrin. If result is
prolonged eventhough coagulation factors are normal,
inhibitors may be present.
Laboratory Evaluation:

G. Substitution Tests/ Mixing Studies

- this can be adopted if primary tests like PT or APTT are
abnormally prolonged and the indicate a factor
deficiency. The patient’s deficient plasma is diluted 1:1
with a plasma or serum substitute and the APTT or PT is
repeated. A correction if the original prolonged APTT or
PT indicates that the deficient factor has been added to
the patient’s plasma by substitution solutions as follow:
1. Aged plasma- lacks labile factors V & VIII but
retains normal activity of all factors. Contains Factors I,II,
VII,IX,X,XI,XII
2. Fresh adsorbed plasma- lacks vitamin K
dependent factors (II, VII, IX, X) but retains activity of all
other coagulation factors. Contains Factors V,VIII, XI, XII
3. Aged serum- lack factors I, II, V, VIII but retains
normal activity of all coagulation factors. Contains
Factors VII,IX,X,XI,XII.
Laboratory Evaluation
FACTOR DEFICIENT PLASMA OR SERUM
Extrinsic Intrinsic Normal Adsorbed Aged Serum
Pathway Pathway Plasma Plasma
(PT) (APTT)
I I + + (-)
II II + (-) (-)
V V + + (-)
VII + (-) +
VIII + + (-)
IX + (-) +
X X + (-) +
XI + + +
XII + + +
Laboratory Evaluation:
Test of Phase IV of Coagulation
1. Thrombin Time (TT) or Thrombin Clotting Time (TCT)
- test for the deficiency of fibrinogen or its inhibitors
Principle: Pt’s plasma + thrombin reagent
Result: time of clot formation
Reference interval.= 15-20 seconds.
A prolonged TT can be seen in patients with therapeutic
heparin, patients with high levels of antithrombin or FSPs and with
any disorder associated with hypofibrinogenemia( less than
100mg/dL) or in the presence of antithrombotic agent such as FDPs,
paraproteins, or UFH. Afibrinogenemia/ dysfibrinogenemia also
prolong TCT. TCT is part of the PTT mixing study protocol. Used to
determine if UFH is present whenever the PTT is prolonged. TCT
not sensitive to factor XIII deficiency. May assess the presence of
dabigatran( oral direct thrombin inhibitor).A normal TCT rules
out dabigatran.A TCT modification, the plasma-diluted TCT,provides
a quantitative measure of dabigatran when used with calibrators
of specific drug concentrations.

2. Reptilase test- variation of TT. Reptilase rgt is used instead of

thrombin to convert fibrinogen to fibrin.
Reptilase test
Principle: Atroxin (reptilase rgt trademark)+ pt’s
plasma.
The rgt release fibrinopeptides A from the
fibrinogen [Link] resulting polymers
polymeryze end-to-end forming a clot( fibrin
polymer)
Reference interval: 10-15 secs.
Indications:
[Link] can test for functional fibrinogen when the
TT is prolonged because of heparin(therapy) as
this test is not inhibited by heparin unlike TT.
[Link] test demonstrates only minimum inhibition
by FSP.
Both Thrombin Time & reptilase-R test are
prolonged in:
a. Factor I deficiency
b. Dysfibrinogenemia
c. Streptokinase therapy
d. Presence of FDPs /FSPs
3. Fibrindex Test- plasma added with thrombin
will result in clotting. Fibrindex is the trademark
for thrombin reagent.
Reference interval- starts to clot in 5-10 secs. In
30-60 seconds , a firm clot is formed.
Clot- based Coagulation Screening Tests
The PT, PTT/APTT, fibrinogen assays, TCT all use
the clotting time principle of Lee and White
method.
Many specialized tests, such as coagulation
factor assays, tests of fibrinolysis, inhibitor
assays, reptilase time,Russell viper venom time,
and dilute Russell viper venom time, are also
based on the relationship between time to clot
formation and coagulation system function.
Other tests
Factor XIII Screening Test
Factor XIII exists in the plasma in an inactive
state and is activated by thrombin during
Fibrinogen-Fibrin conversion. Activated Factor
XIII causes the formation of covalent bonds
between the fibrin monomers, thus stabilizing
the fibrin polymer. When Factor XIIIa is present,
the fibrin clot formed is insoluble in 5M urea &
1% monochloroacetic acid when left standing
for 24 hrs. A deficiency in this factor is rare.
Generally, a 1% level of this factor is sufficient to
make a clot insoluble in 5M urea.
Laboratory Evaluation:
Test of Phase IV of Coagulation
4. Fi-test (Immunological Test)
- a rapid slide test based on the agglutination of
fibrinogen-coated red blood cells by the latex
anti-human fibrinogen reagent. Normally,
presence of fibrinogen is indicated by
agglutination.

5. Fibrinogen Titer Method

- serial dilutions of plasma are diluted with
thrombin. The titer is the highest dilution in
which a fibrin clot can be seen, and is related to
the fibrinogen concentration and indirectly to
the presence of circulating anticoagulants.
Laboratory Evaluation:
Test of Phase IV of Coagulation
6. Assay of Plasma Fibrinogen
- several accurate methods are now available
for the quantitative assay of plasma fibrinogen.
Fibrinogen is usually converted into fibrin
which is quantified by gravimetric,
nephelometric, chemical, immunologic and
precipitation methods.
Methods
a. Ellis and Stransky Method
b. Stirland’s Method
c. Turbidimetric Method of Partfantjev [Link]
d. Ratnoff and Menzie Method
e. Fibrin Clot Method
Assays for Fibrinogen
7. Clauss procedure for measuring clottable
fibrinogen, a modification of the TCT, the
recommended method.
This test is based on the fact that changes in the
concentration of fibrinogen are very sensitive to high
concentrations of thrombin, when the fibrinogen level
is low (e.g. 5-50 mg/dL). The clotting time of this test is
sensitive to small changes in the fibrinogen
concentration but relatively unaffected by changes in
the thrombin concentration. There is an inverse
relationship between the interval to clot formation
and the concentration of functional fibrinogen. When
the thrombin rgt is concentrated and the PPP is
diluted, the relationship is linear provided the
fibrinogen concentration is 100-500 mg/dL
Reference Range: 150-400 mg/dL but each laboratory
Assays for Fibrinogen
8. PT-derived fibrinogen assay– alternative to
Clauss method. Optical coagulometers estimate
fibrinogen by assessing reaction mixture turbidity
while performing the [Link] method gives
fibrinogen result with each PT.
Tests for Inhibitors of Coagulation
1. Dilute Russell viper venom time test screening test for lupus
anticoagulants.
[Link] Mixing studies- use a moderate or a high Phospholipid-
PTT reagent to detect LAC (a non-specific inhibitor- has a wide
variety of target antigens)
LAC are found in 1-2% of randomly selected individuals.
[Link] for Specific Factor Inhibitors- Ig G immunoglobulins
directed against coagulation factors.
Usual Cause:
factor concentrate treatment
The most common specific inhibitor is anti-Factor VIII, is directed
against 10%-20% of patients with severe hemophilia and anti-
factor IX is detected in 1%-3% of factor IX-deficient patients.
Autoantibodies to factor VIII occasionally arise to those without
hemophilia, usually in young women, where they are associated
in post-partum bleeding syndrome or in patients over 60 y/o
with autoimmune disorders. The presence of these acquired
antibodies is called acquired hemophilia. Alloantibodies and
autoantibodies to factor VIII is associated with severe
hemorrhage.
Tests for Inhibitors of Coagulation
4. Nijmegen- Bethesda Assay for Anti-Factor
VIII Inhibitor- confirms and quantify anti-factor
VIII inhibitors which are typically IgG4- class
immunoglobulins
Result : The percentage of factor VIII activity
neutralized is proportional to the level of
inhibitor [Link] incubation, residual factor
VIII in the patient PPP-PNP mixture is measured
using the factor VIII factor activity assay.
Automated Instruments/Equipment
Performs clot-based , immunoassays, and
chromogenic assays.
Each instrument offers unique advantage/s.
a. high throughput
b. reduced reagent volume
c. Integral bar code reader
d. Cap piercing
e. Automatic sample dilution
Many offer user-programmable methods & pre-
programmed methods
Principles in Automated Machines
1. Electromechanical methods
Principle: Measurement of the conduction or
impedance of an electrical current by the
formation of fibrin.e.g. Fibrometer.
2. Photo-Optical methods
Principle: A change in light transmission
measured as optical density (absorbance) versus
time can be used to quantitatively determine the
activity of various coagulation stages or factors.
Machines in this type can be used to detect APTT,
PT,Fibrinogen levels & Thrombin Time.
Quantitative factors assays based on the
Automated Machines
APTT (factors XIII, IX,XI,XII) and
PT (factors V,VII, & X) are examples of available
machines. These microprocessor –controlled
instruments have separate detector cells w/
their own light emitting diode (LED) light source,
w/c is driven by a constant current regulator to
give each a noise-free light beam. The light beam
passes through a cuvette, w/c is altered by fibrin
clot formation.. The light beam then passes
through a diffuser and falls on the sensor, w/c
instantly converts the transmitted light into an
electrical signal (amplitude signal) w/c is
converted to a digital value for processing. The
computer –processed data is sent to visual
display monitor and printer.
 Thrombotic Hemostasis Panel Assays:
Antithrombin
Factor VIII:C
Heparin
Lupus anticoagulant
Protein C
Protein S & free Protein S
The routine screening procedures used to detect
coagulopathy are:
platelet count
bleeding time
prothrombin time(PT)
APTT
thrombin time (TT)
tourniquet test