SPECIAL LABORATORY EVALUATION OF

COAGULATION
TESTING METHODOLOGIES
Clot formation endpoint in coagulation studies
The development of monoclonal antibodies has made highly specific antisera available
for immunologic testing, and synthetic substrates have made it possible to view
coagulation from an enzymatic perspective. In addition, new instruments and methods
have been developed to keep pace with the expanding knowledge of hemostasis
Fibrin Endpoint
Fibrin endpoint methodologies depend on detection of a fibrin clot as it is formed
through proteolytic cleavage of fibrinogen. Methods for clot detection
Commonly used:
o Manual tilt-tube techniques
o Electromechanical (fibrin strand)
o Optical density turbidity
Optical systems measure the rate of change of optical density, and
thus the results are potentially more reproducible than those of
manual or electromechanical methods
Coagulation times will differ significantly depending on the instrument reagent, and
instrument-reagent combination being used Thus, it is imperative that each laboratory
establish the reactivity of its reagent-instrument combination or system to the various
factors and its own reference ranges for each determination
Immunologic Methods
There are hereditary variants of coagulation proteins that have normal antigenic
properties but lack functional activity
o Cross-Reactive Material (CRM)
o It is important to identify these variants in order to understand the
pathophysiology of coagulation disorders
1. Ouchterlony Double Diffusion
Antigen and antibody specific to that antigen placed in separate wells cut in
agar will diffuse toward each other in a radial manner and precipitate as
immune complexes when they meet.
The resultant precipitate can be seen as a transverse line (straight or
curved) between the two wells.
Not quantitative but will establish whether two antigens are serologically
identical or different
2. Radial Immunodiffusion
Specific antibody is incorporated into agar, and antigen is placed in wells cut in the
agar.
As the antigen diffuses, it precipitates with the antibody, forming a halo around the
well.
A linear relation exists between the square of the diameter of the ring and the
concentration of antigen in the well.
3. Laurell Rocket Electrophoresis
Agarose containing antibody specific for the antigen to be measured is poured on a
glass plate.
Antigen is placed in wells cut on one side of the plate, and direct electric current is
applied. As the antigen migrates and contacts antibody molecules, a rocketshaped precipitate is formed

The height is proportional to the antigen concentration.

4. Crossed Immunoelectrophoresis
Two-dimensional method
Plasma is electrophoresed through plain agarose to separate the antigens. Agar
containing antibody specific to the antigen being measured is then poured on the
plate, and a second electrophoresis is performed at right angles to the first
As the antigen-antibody complexes form, they create bell-shaped precipitin lines.
Abnormalities can be detected by abnormal migration patterns or abnormally
shaped precipitin areas
5. Radioimmunoassay
A known concentration of radiolabeled antigen and an unknown concentration of
antigen in a test sample compete for binding sites on a known amount of antibody.
The bound antigen is separated from the free antigen, and the ratio of
radioactivity associated with the bound antigen to the free antigen is determined.
6. Enzyme-Linked Immunosorbent Assay
Adaptation of RIA. The antigen-specific antibody in this case is linked to an
enzyme, and a substrate for that enzyme is then added
The amount of color is directly proportional to the concentration of antigen
7. Synthetic Substrates
Small peptide chains consisting of ammo acid sequences that mimic the natural
substrate of an enzyme. An indicator group
o Chromophore or a Fluorophore - attached to the synthetic substrate,
which is cleaved by the enzyme for which the substrate has specificity
o Synthetic substrate assays allow coagulation testing to be approached as a
series of enzymatic reactions m an accurate and sensitive manner. A sound
knowledge of enzyme kinetics is necessary for optimal use of synthetic
substrate assays
Endpoint or initial rate analysis may be performed, and many assays have been
adapted for use on automated instrumentation.

INDIRECT TESTS FOR COAGULATION PROTEINS 0R SYSTEMS

Early Tests for Coagulation Abnormalities
1. Two-Stage Prothrombin time - first method to measure the prothrombin
concentration indirectly in plasma
Test plasma is defibrinated by the addition of thrombin
After the clot is removed, all prothrombin in the plasma is converted to
thrombin by adding thromboplastin, calcium, and a source of factors V, VII, and X.
The thrombin formed is then measured by adding aliquots of the activation
mixture to standard fibrinogen solutions and recording the clotting time.
o Prothrombin units/ mL of plasma.
2. Prothrombin consumption test - performed on serum from blood that has been
allowed to clot for 60 minutes
o Severe deficiencies of factors V, VIII, IX, X, XI, or XII, as well as platelet
deficiencies or abnormalities, will slow the rate of prothrombin
conversion, thus leaving significant amounts of prothrombin in the serum.
3. Thromboplastin Generation Test (TGT) - barium sulfate or aluminum hydroxide
will adsorb to certain coagulation factors
o If BaSO4 or Al(OH)3 is added to normal plasma, factors II, VII, IX, and X will be
adsorbed and thus can be removed by centrifugation. Likewise if blood is

allowed to clot, the remaining serum will not contain fibrinogen, factor II, the
labile factors V and VIII, or XIII.
o Therefore, if normal adsorbed plasma, normal serum, platelets, and CaC1 2
are mixed, the mixture cannot form a clot until prothrombin (factor II) is
supplied by the addition of normal plasma.
o TGT reagents can be used in either the PT or the APTT systems
THROMBIN CLOTTING TIME
Principle
o Addition of thrombin to plasma bypasses all coagulation reactions except
polymerization of fibrinogen.
o The thrombin clotting time measures the conversion of fibrinogen to
fibrin and is not influenced by deficiencies of the other coagulation factors.
o Dysfibrinogenemia, hypofibrinogenemia, fibrin split products, immunologic
antithrombins, and the presence of abnormal globulins will prolong the
thrombin clotting time
o The thrombin clotting time also is prolonged by heparin and may be used to
monitor heparin therapy

Procedure
A standardized thrombin solution is added to citrated platelet poor plasma
Comments
Platelets contain platelet factor 4, which is a heparin-neutralizing substance
Therefore, the specimen must be obtained and handled carefully to avoid platelet
disruption. The plasma tested must be platelet free.
Thrombin times should not be performed on heparinized plasma samples that have
been frozen.
Thrombin is highly unstable.
To preserve its activity, it is diluted to 100 NIH units/ mL with a solution of equal
parts 0.15 M NaCl and glycerol. Aliquots are then frozen just prior to use, an
aliquot of the stock thrombin is thawed and diluted with 0.15 M NaCl to give a
control value established by each laboratory. Thrombin activity decreases after 20
minutes at 37C.
Protamine can be added to determine if the thrombin time is prolonged because of
heparin or other antithrombin activity, as protamine neutralizes heparin.
Control
A normal control sample must be run
Reference Ranges
Ranges are:
o 8 to 9 seconds
o 15 to 20 seconds
o less than 24 seconds
o 20 to 25 seconds
Depending on the thrombin concentration selected, reported reference

REPTILASE TIME

Principle
Thrombin-like enzyme isolated from the reptile Bothrops atrox
Cleaves fibrinopeptides from the alpha chain of fibrinogen, whereas thrombin
releases fibrinopeptides from both the alpha and the beta chains.

Not influenced by heparin or immunologic antithrombins

Fibrin (ogen) split products will prolong the Reptilase time to a lesser degree than
the thrombin time, whereas dysfibrinogenemia will more greatly affect the
Reptilase time. Therefore, if both the Reptilase and the thrombin time tests are
performed, the cause of the prolonged thrombin time can be identified more easily

Procedure
Reptilase is added to citrated platelet-poor plasma
The time necessary for clot formation is proportional to the amount and quality of
fibrinogen present.
Comments
Platelet-poor plasma
Reptilase is capable of aggregating platelets
If thrombin is present in the specimen, it may interfere with Reptilase. This
problem can be eliminated by adding heparin, which will inhibit thrombin but not
Reptilase.
After reconstitution with water, reptilase is stable for 6 hours at 37C and for 5
days at 4C
Reference Range
18 to 20 seconds.

FIBRINOGEN
1. Precipitation or denaturation methods - in which heat, salt, or another agent
is used to denature fibrinogen to an insoluble state.
The suspended fibrinogen precipitate may be measured turbidimetrically, or the
precipitated fibrinogen may be isolated and measured by colorimetric or ultraviolet
protein quantitation. These methods generally are semi-quantitative and are
subject to many technical problems.
2. Turbidimetric or fibrin clot density procedures - based on photo-optical
measurement of the change in turbidity of plasma as fibrinogen is converted to
fibrin by thrombin.
The kinetics of this conversion have been used to measure the fibrinogen
concentration. Turbidimetric procedures are the basis for some automated
fibrinogen methods that have been introduced.88'99 Modifications of the original
procedure have improved sensitivity at lower fibrinogen levels and have eliminated
heparin interference.
3. Coagulable protein assays - performed by clotting the plasma sample using
thrombin or CaC12, isolating the fibrin clot, and then measuring the fibrin by
weight, chemically, or by ultraviolet absorbance
Ancrod - a thrombin-like enzyme that is specific for fibrinogen and not
influenced by heparin, also has been used to clot plasma. The coagulable
protein assay is considered the reference procedure for fibrinogen
measurement. The principal disadvantage of this type of assay is the time
required to perform the test.
4. Immunologic assays - utilize antibodies to fibrinogen in assays such as radial
immunodiffusion, kinetic latex agglutinometry, measurement of turbidity, or rocket
immunoelectrophoresis
Immunologic assays are time consuming and also measure fibrin (ogen)
degradation products. Because immunologic assays measure dysfunctional as well

as functional fibrinogen, performing an immunologic fibrinogen assay along with a

functional fibrinogen assay is a useful tool in diagnosing dysfibrinogenemia.
5. Modified thrombin time fibrinogen determination - most widely performed
clinical fibrinogen
first described by Clauss in 1957
The clotting time of diluted plasma to which a high concentration of thrombin
has been added is inversely proportional to the fibrinogen concentration
This procedure is relatively insensitive to heparin and fibrin (ogen)
degradation products
MODIFIED THROMBIN CLOTTING TIME ASSAY FOR FIBRINOGEN
Procedure
A high concentration of thrombin is added to diluted, citrated, platelet-poor
plasma to convert fibrinogen to fibrin.
The clotting time is inversely proportional to the fibrinogen concentration
Comments
Wrong blood:anticoagulant ratio or lack of correction of citrate volume for a low or
high hematocrit may cause erroneous results.
A higher dilution of the test plasma must be made if the clotting time is too short
A lesser dilution of plasma is necessary for very long clotting times: however, less
than a 1:3 dilution should not be tested. Appropriate dilution factors must be used
in calculating results
Care must be taken to avoid obtaining specimens through heparinized lines.
If the patient is receiving fibrinolytic therapy, either epsilon aminocaproic acid
(EACA) or soybean trypsin inhibitor should be added to the collection tube to
prevent in vitro clot lysis.
Hemolysis or lipemia may interfere with photo-optical measurements.
Manual, semi-automated, or automated endpoint detection devices may be used,
but each will result in a different endpoint.
The thrombin reagent (100 NIH units/mL) is unstable
Known normal and abnormal controls should be run every 20 samples or every
shift. Duplicates should agree within 0.5 seconds
Reference Range
170 to 410 mg/ dL.

INHIBITOR STUDIES
SCREENING TESTS
Principle
Coagulation testing abnormalities may be caused by either a factor deficiency or
an inhibitor
o The inhibitors action may be confined to a specific factor (c.g., factor VIII
inhibitor); it may be nonspecific (e. g., lupus anticoagulant); or it may be
global, affecting several factors simultaneously
Correction studies with normal plasma are performed to differentiate a factor
deficiency from an inhibitor (Table 53-3).
Procedure
Patient plasma is mixed with normal plasma, which will correct the abnormality
caused by a factor deficiency.

If the abnormality is secondary to the presence of an inhibitor, the abnormality

will not be corrected.
Testing is performed both immediately on mixing and after incubation for 2 hours
at 37C
o Testing after incubation will reveal the time and temperature dependency of
the inhibitor.
Comments
Citrated blood specimen is centrifuged immediately to prevent interference from
platelets.
Normal plasma is preferably obtained from a normal plasma pool, although it may
be obtained by drawing blood from a healthy donor
o This plasma is treated in the same manner as the patient specimen.
Depending on the system to be tested, prothrombin time (PT) or activated partial
thromboplastin time (APTT) reagents will be necessary
Fresh pooled normal plasma mixed 1:1 with Owrens Veronal buffer is used as
the control and is incubated at 37C in the same manner as the patient specimen
The interpretation of this test is based on the correction, if any, of the abnormal
test time (
o If the abnormality is attributable to a factor deficiency, the addition of one
part normal plasma will correct the abnormal test time close to the normal
range. If the abnormality is secondary to an immediate acting inhibitor (e.g.,
factor IX inhibitor, heparin), the addition of normal plasma will not correct
the abnormal test time.
o If, however, the inhibitor is time or temperature dependent (e. g., factor VIII
inhibitor) an immediate correction will be noted, but on incubation, the test
time will become prolonged if the inhibitor titer is high enough. Lupus
anticoagulants generally are immediate acting but may be time dependent.
Equivocal results may be obtained in borderline or mild abnormalities.
If an inhibitor is indicated, a more sensitive and specific test should be performed
to identify and quantify it.
Reference Range
Reference range for the PT or APTT will differ

INHIBITOR IDENTIFICATION AND QUANTITATION

Lupus anticoagulant special type of circulating anticoagulant

Generally identified in the laboratory through the tissue thromboplastin
inhibition test, the platelet neutralization procedure, or the agarose
plasma gel technique.
Factor VIII inhibitor - may be quantitated by the agarose inhibitor plasma gel
technique or the Bethesda inhibitor assay.

Tissue Thromboplastin Inhibition Test

Modified clotting test
Citrated plasma is incubated at 37C with dilute thromboplastin, CaCl2 is added,
and the clotting time is measured
The results are expressed as a ratio of the clotting time (in seconds) of the patient
sample to that of the control sample
The TTI is considered normal if this ratio is 1:1 or less; borderline if 1:2 to 1:3;
and abnormal if it is greater than 1:3
Neither sensitive nor specific.
Platelet Neutralization Procedure
Freeze-thawed platelets serve as an additional source of phospholipid and
neutralize lupus anticoagulant
o are added to the test plasma, and an APTT is performed

Significant shortening of the abnormal APTT is indicative of lupus anticoagulant

Agarose Plasma Gel Technique

Agarose containing fresh normal plasma is poured on a plate. Patient and control
plasmas are placed in wells cut into the agar and allowed to diffuse into the gel.
After incubation, the plate is flooded with CaCl2 solution
The agarose will turn cloudy or opaque as fibrin is formed in the agarose
In the presence of an inhibitor, a clear area or zone of inhibition will be seen
surrounding the well containing that plasma.
Bethesda Inhibitor Assay
Factor VIII inhibitor can be quantitated by mixing test plasma with a known amount
of factor VIII and incubating the mixture for 2 hours at 37C. After incubation, the
amount of residual factor VIII is measured by specific factor assay. By comparing
the factor VIII activity in the patient incubation mixture with that in the control
mixture, the amount of inhibitor present in the former can be calculated. The result
is expressed in Bethesda units; one Bethesda unit of inhibitor is defined as the
amount that will inactivate 50% of the factor VIII activity present

FACTOR ASSAYS
Currently, a variety of methods are in use for the measurement of factor activity,
including one-stage clotting methods, two-stage clotting methods, fluorometric methods,
and colorimetric methods. The fluorometric and colorimetric methodologies are based on
the principles that govern synthetic substrates. The one-stage clotting method is widely
used because of its simplicity.
1. ONE-STAGE CLOTTING ASSAYS
Principle
One-stage factor assays are based on a simple modification of the PT or APTT.
Assays for factors II, V, VII, and X generally are based on the PT, whereas assays for
factors VIII, IX, XI, and XII are based on the APTT.
Procedure
The clotting time of a substrate plasma known to be deficient in the factor to be assayed
is shortened or corrected by adding diluted factor-containing plasma. By using
various dilutions of a known normal plasma or standard, a curve is constructed. Unknown
sample values are derived by using an appropriate dilution and converting the clotting
time to factor activity from the standard curve.

Comments
Citrated platelet-poor plasma should be kept on slush ice.
Plasma should be tested within 1 to 2 hours and dilutions within 30 minutes.
Frozen plasma may be used provided the standard liquid reference plasma has
likewise been frozen.
Normal reference plasma and factor-deficient plasma may be obtained
commercially in lyophilized form.
Known controls should be run simultaneously with the unknown samples.

Reference Range
50% to 150%

Factor VIII Immunologic Assays

Factor VIII-related antigen (factor VIII R:Ag) most widely studied

coagulation protein using immunologic methods.
o Laurell rocket technique or rocket electroimmunodiffusion (EID)
most convenient
o Immunoradiometric assay (IRMA)
o ELISA
o Crossed
immunoelectrophoresis
and
SDS-polyacrylamide
gel
methods - used to characterize qualitative abnormalities of the molecule.

RISTOCETIN COFACTOR
Originally introduces as an antibiotic
Can cause thrombocytopenia
Factor VIII protein is able to bind ristocetin and platelet membranes, causing the
platelets to agglutinate.
In cases of von Willebrands disease, which is secondary to a deficiency of this
cofactor, ristocetin will not agglutinate platelets.
Principle
Agglutination of fixed platelets in response to ristocetin depends on the presence of von
Willebrand factor in plasma.
Procedure
Plasma dilutions and ristocetin are added to normal fixed platelets, and the rate of
agglutination is observed using an aggregometer. Test plasma and normal standard
plasma are treated similarly. A standard curve of the percent of von Willebrand factor
versus either the slope or the percent of agglutination is constructed. Patient samples
are compared with the standard curve, and percentages of von Willebrand factor are
calculated.

Comments
The normal platelet reagent consists of platelets fixed in formalin or
paraformaldehyde
Citrated platelet-poor normal plasma or a plasma standard with a known value is
used in the construction of a standard curve
Reference Range
50% to 150%

FACTOR XIII
fibrin-stabilizing factor
Transglutaminase that is activated by thrombin in the presence of Calcium
Principle
In the presence of activated factor XIII, the fibrin clot that forms is insoluble in urea or
monochloroacetic acid
Procedure
Test plasma is clotted. The clot is immersed in 5 M urea and served to see if it is
dissolved or remains intact.

Comments
A normal control should be run simultaneously with the unknown sample
A control tube containing both a normal clot and the test clot in 5 M urea should be
observed
The normal clot should contain sufficient factor XIII to keep the test clot from
dissolving regardless of its factor XIII level

o If the clot dissolves in this tube, the presence of abnormal fibrinolytic activity
should be investigated
This procedure should be considered a screening test for the almost total absence
of factor XIII, because levels as low as 1% to 2% are adequate for hemostasis and
will result in a normal test
To detect individuals with less than 50% but more than 2% activity, immunologic
methods are necessary. These include immunoelectrophoresis to measure factor
XIII subunit A and S levels or SDS polyacrylamide gel electrophoresis.
Reference Range
In the presence of factor XIII, the clot should remain insoluble for 24 hours at 37C.

Fletcher Factor
Participates in the contact phase of the intrinsic system
First described by Hathaway and associates in 1965
Laboratory detection is initially made by a simple screening test in which the
prolonged APTT is corrected by additional incubation with an activator such as
Celite or kaolin
Partial thromboplastins containing ellagic acid as an activator do not usually
detect this deficiency.
Fletcher factor deficiency is confirmed by a quantitative assay using a modified
APTT technique and factor-deficient substrate or a synthetic substrate.
REGULATORY PROTEINS
Antithrombin III
Assayed by either immunologic or functional methods.
Immunologic assays : RIA, RID and Laurell rocket immunoelectrophoresis
o Do not distinguish free-circulating AT-III from that which is complexed and
inactive
o Milligrams per deciliter
o 20 - 50mg/dL

FUNCTIONAL ASSAYS - based on the fact that AT-III inhibits activated serine
proteases
o Clotting technique Employing synthetic substrates based on the fact that AT-III inhibits activated
serine proteases
Reference Range
80% to 120%

Protein C
Immunologic methods
o Laurell rocket immunoelectrophoresis technique
o RIA techniques
o ELISA methodology
3 - 4ug of protein C per milliliter.

Functional Assays
o Automated clot-based method - described by Odegard and associates.

Protein S
Immunologic methods

o Laurell rocket immunoelectrophoresis technique

1 ug/mL

Functional Assays
o Based on the ability of protein S to serve as a cofactor for the anticoagulant
effects of activated protein C