LABORATOR

INVESTIGATIONS
Y IN
COAGULATION
DISORDERS
 CONTENT
 INTRODUCTION S
 SPECIMEN COLLECTION AND PROCESSING
 LABORATORY INVESTIGATIONS OF
COAGULATION
SYSTEM
 SCREENING TESTS
 CONFIRMATORY TESTS
 TESTS TO EVALUATE CIRCULATING INHIBITORS
 LAB INVESTIGATIONS FOR FIBRINOLYTIC SYSTEM
 AUTOMATION
 SUMMARY
 REFERENCES
 INTRODUCTION
 Hemostasis is a process by which the body prevents
loss of blood from the vascular system and maintains it
in a fluid state.

 Involves a series of complex and regulated events

linking platelets , vascular endothelial cells and
coagulation factors.

Hemostasis includes
 Primary hemostasis
 Secondary hemostasis
 THE COAGULATION
SYSTEM
Coagulation Factor XIIa, XIa
factors Kallikrein
Phospholipids ACTIVATOR T-PA
S U-PA
CA2+

CLOTTING FIBRINOLYSIS

AT
Protein C, S
TFPI PAI- 1,2,3
α2Macroglobulin INHIBITOR α2- antiplasmin
α2- macroglobulin
α1Antitrypsin S
C1 Inactivators TAFI
Heparin Cofactor
 COAGULATION
DISORDERS
CONGENITAL DISORDERS ACQUIRED DISORDERS
Hemophilia A and B Disseminated intravascular
coagulation
Von Willebrand’s disease Deficiency of vitamin K
dependent factors
Hypofibrinogenemia Liver disease

Afibrinogenemia Massive transfusion syndrome

Dysfibrinogenemia Heparin therapy

Inhibitors of coagulation-fVIII
inhibitor, APLA
 SPECIMEN COLLECTION
AND PROCESSIN
 G
Whole blood is aseptically collected by venipuncture.
 Anticoagulant of choice is 3.2% sodium citrate.
 Volume of anticoagulant necessary for a 5 ml
blood sample adjusted according to the hematocrit
value.
 One may also choose to keep the anticoagulant volume
of
0.5 ml constant and adjust the volume of added blood
according to the hematocrit.
 The volume of blood to be added (to 0.5 ml of 0.109M
citrate) is calculated from the formula:
(60 - hematocrit)/100 × 4.5
HAEMATOCRIT VOLUME OF VOLUME OF
(%) ANTICOAGULANT (ML) BLOOD (ML)

25 – 55 0.5 4.5

20 0.7 4.3

60 0.4 4.6

70 0.25 4.75

80 0.2 4.8
 SPECIMEN

PROCESSING
Citrated whole blood is centrifuged to obtain plasma
for coagulation testing.
Platelet Poor Plasma:
 Blood sample is centrifuged at a minimum of 1700rpm
for at least 10 minutes (2500rpm for 15min) preferably
at 4°C in a refrigerated centrifuge.
 Open the samples at RT, separate and store the plasma
sample at 4°C as quickly as possible.
 When RT >25°C then labile coagulation proteins may
be affected, a refrigerated (4°C) centrifuge should be
used.
 Platelet poor plasma <10× 109/l
 SPECIMEN
PROCESSING
Samples for Immediate Testing:
 Samples should be tested within 4 hours of sample
collection when possible.
 Longer storage should be avoided for screening tests
and clotting factor assays.
 Samples for factor assays should be stored at 4°C
testing.
 Samples for screening tests and assay of factor VII
should be maintained at room temperature to avoid
the possibility of cold activation.
 SPECIMEN
PROCESSING
Deep Freezing of Plasma:
 Samples can be frozen for testing at a later stage.
 Storage at -70°Cor lower is preferable (better)
but at -35°C is adequate for most tests.
 Storage at -20°C is usually inadequate.
 Double centrifugation should be used if
samples are frozen prior to analysis for lupus

anticoagulant.
Freezing and thawing best avoided before
is determinations, results obtained APTT
sincebe
can by some
affected.
 LABORATORY O
INVESTIGATIONS
COAGULATION SYSTEM F
1.SCREENING TESTS

 Prothrombin time

 Activated partial thromboplastin time

 Thrombin time

2.CONFIRMATORY TESTS FOR FACTOR

ABNORMALITIES

 Reptilase time

 Mixing tests.

 Fibrinogen assay (modified Claus Assay)

TESTS TO EVALUATE CIRCULATING
INHIBITORS
 Platelet neutralizing procedure

 Lupus anticoagulant & antiphospholipid antibodies

 LAB INVESTIGATIONS OF
FIBRINOLYTIC SYSTEM

 Detection of FDPs using latex agglutination method

 D-dimer test

 Euglobin clot lysis

SCREENING
TESTS
 PROTHROMBIN
TIME
Principle:
 Measures the time for fibrin formation.
 Reflects the overall efficiency of the extrinsic system.
 Sensitive to changes in factor V, VII and X, and less
so to factor II (prothrombin).
 It is also unsuitable for detecting minor changes
in fibrinogen level
 May be abnormal if the fibrinogen level is very low or
if there is an inhibitor present.
 The sensitivity of the test is influenced by the
reagent and technique used and it is important to
establish a reference range locally.
 REAGENT
- Sfrom the patient.
Platelet poor plasma

- Control plasma sample.

- Thromboplastin (this may contain calcium

chloride).
- Calcium chloride – 0.025 mol/lit (only required if

thromboplastin reagent does not contain

calcium).
 IN
 R
INR – International Normalized Ratio.
 Was established by WHO and the international
committee on thrombosis and hemostasis on reporting
the result of PT.
 INR = (PT of Patient/Control PT)ISI
 Anticoagulant therapy control = 2.0-3.0

ISI
 International Sensitivity
Index.
 Given by manufacturer. to different Vitamin
 Reflects reagent K
sensitivity dependent
 RESULT
S
Results can be expressed in
1. Mean of duplicate recordings in secs
2. INR = (PT of Patient/Control PT)ISI

NORMAL RANGE
 With rabbit thromboplastin : 11-16 secs
 With recombinant human thromboplastin : 10- 12
secs.
 CAUSES OF PROLONGED
PT
 Administration of oral anticoagulants drugs (vitamin
K anticoagulants).

 Liver diseases

 Vitamin K deficiency

 DIC

 Previously underdiagnosed factor VII, X, V or

prothrombin deficiency or defect.
 ACTIVATED
THROMBOPLASTIN
PARTIAL
TIME
Principle:
 Measures the clotting time of plasma after the activation
of contact factors but without added tissue
thromboplastin.
 Indicates the overall efficiency of the intrinsic pathway.
 It is also sensitive to circulating anticoagulants
(inhibitors) and heparin.
 Depends on contact factors, factors VIII, IX as well as X,
V, prothrombin and fibrinogen.
 REAGENT

S
Platelet poor plasma

 Kaolin 0.5gm in 100ml barbitone buffered saline, pH

7.4

 Other can be used like silica, celite or elagic acid can

be used.

 Phospholipid

 Calcium chloride 0.025M

 METHO
D
1.Mix equal volumes of phospholipid reagent and the
kaolin
kaolin suspension and leave in a glass tube in the water
bath at 37 degree.
2.Place 0.1ml of plasma into a new glass tube.
3.Add 0.2ml of kaolin-phospholipid solution, mix
the contents and start the stopwatch simultaneously.
4. Leave at 37 degrees for 10mins with occasional shaking.
5.At exactly 10mins, add 0.1ml of pre-warmed CaCl2 and
start a second stopwatch.
6.Record the time taken for the mixture to clot.
7.Repeat the test on both the patient and the control
plasma atleast once.
 INTERPRETATIO
N 30 – 40 seconds
Normal range:

 A prolonged APTT with a normal PT indicates a possible

deficiency of factor VIII, IX, XI, XII, high molecular weight
kininogen, prekallikrein or the presence of an inhibitor.
 CAUSES OF PROLONGED
APTT
DIC

 Liver diseases

 Massive transfusion with stored blood.

 Administration of heparin or contamination with

heparin.

 A circulating anticoagulant.

 Deficiency of coagulation factor other than factor VII.

 THROMBIN
TIME
Principle:
 The thrombin time reflects the reaction between thrombin
and fibrinogen.
Thrombin
Fibrinogen

Fibrin

 Thrombin is added to the plasma and the clotting time is

measured.
 The Thrombin time is affected by the concentration and
reaction of fibrinogen, and by the presence of inhibitory
substances, including fibrinogen/FDP and heparin.
 REAGENT
S
 Platelet poor plasma.

 Thrombin solution which induces clotting of

normal plasma in about 15 seconds.

Normal range: 15 – 19
secs
 METHOD –
1. Pipette MANUAL
0.2 ml test and control plasma into
separate glass clotting tubes.

2.Warm to 37oC.

3.Add 0.1 ml thrombin solution to each tube.

4. Start stopwatch for each tube.

5. Measure the clotting time and observe the nature

of clot.

6.Repeat procedure for patient and control plasma.

 CAUSES OF PROLONGED
TT
 Hypofibrinogenaemia as found in DIC and, more rarely,
in a congenital defect or deficiency.
 Raised concentrations of FDP, as encountered in DIC
or liver disease.
 Extreme prolongation of the TT is nearly always a result
of the presence of unfractionated heparin.
 Dysfibrinogenaemia, either inherited or acquired, in
liver disease or in neonates.
 Hypoalbuminaemia.
 Paraproteinemia.
CONFIRMATORY
TESTS
 REPTILASE OR ANCROD
 TIMEBothrops atrox
Reptilase:
 Ancrod: Agkistrodon rhodostoma
Principle
 It is a serine protease, a thrombin like
enzyme. (thrombi
 Cleaves fibrinopeptide A from n
fibrinogen cleaves both A &B).
Method
 Addition of reptilase to platelet poor plasma initiates
clot formation.
 Clot formation detected by optical/ electromechanical
methods.
CAUSES OF PROLONGED
RT/AT interval- 18-22
Reference
seconds

 Hypofibrinogenemia

 Dysfibrinogenemia

 FDPs

 Other circulating
anticoagulants
 DIFFERENTIATION OF CONDITIONS
ASSOCIATED WITH PROLONGED TT
USING RT

Condition Thrombin time Reptilase time

Heparin Prolonged Normal

contamination
Dysfibrinogenemia Prolonged More Prolonged

Presence of FDP More Prolonged Prolonged

 MIXING
TESTS
 Plasma samples found to have abnormal screening tests,
i.e. PT/APTT, can be further investigated to determine the
cause of the abnormality.

 To differentiate a factor deficiency from the presence of

a
circulating inhibitor.

 Information on the nature of the defectcan

usually be obtained by mixing experiments.
 The agents which can be used r
fo mixing tests are as
follows
 Normal plasma

 Aged plasma

 Adsorbed plasma

 Factor VIII deficient

plasma

 Factor IX deficient plasma

INTERPRETATION OF RESULTS
FROM MIXED
Defect in TESTS
APTT Aged Adsorbed Normal
Tested plasma plasma plasma
Plasma
VIII Abnormal No Correction Correction
correction

IX Abnormal Correction No Correction

correction

XI/XII Abnormal Correction Correction Correction

Inhibitor Abnormal No No No
correction correction correction
Defect APTT Aged Adsorbed Normal
in plasma plasma
plasma plasma
II Abnormal Correction No Correction
Correction
V Abnormal No Correction Correction
Correction
VII Normal Correction No Correction
Correction

X Abnormal Correction No Correction

Correction
 FIBRINOGEN (MODIFIED S
CLAU ASSA S
Principle Y)
 Dilutions of a standard normal plasma with
known fibrinogen content are prepared in glyoxaline
buffer.
 The clotting time is measured after the
addition of thrombin.
 A graph of clotting times against the
fibrinogen concentration is constructed.
Reagent
s
 Reference plasma with known fibrinogen concentration

 Thrombin >30u/ml (concentration may vary according

source)
 Imidazole buffer (glyoxaline) pH 7.35

 Normal range : 1.5 – 3.5

g/L
TESTS TO EVALUATE
CIRCULATING
INHIBITORS
CLOTTING FACTOR INHIBITOR
SCREEN BASED ON
PT/APTT
Principle
 Coagulation inhibitors affecting the PT/APTT may be
immediate acting or time dependent.
 Test plasma containing an immediate-acting inhibitor will,
when mixed with normal plasma, show little or no
correction of the clotting time.
 Plasma with time-dependent inhibitors, on the other hand,
requires a period of incubation with normal plasma
before inhibitors can be detected
 LUPUS ANTICOAGULANT AND
ANTIPHOSPHOLIPID ANTIBODIES

 The criteria for presence of lupus anticoagulants are as

follows:

1. Prolongation of a phospholipid-dependent coagulation test;

2. Evidence of an inhibitor demonstrated by mixing studies;

3. Phospholipid dependency of the inhibitor.

 PROCEDURE : 3
STEP
 1ststep- Clotting assay like APTT screening tests
like Dilute Russell's Viper Venom Time (DRVVT)
Low-phospholipid APTT

 2nd step- Mixing studies

This distinguishes a factor deficiency from presence
of
inhibitor.

 3rd step - Accomplished by either reducing or adding

an excess of phospholipid to the test system
This is done using Platelet neutralizing procedure.
 The confirmatory test cut off is the value corresponding to
the mean of the individual percentage corrections as
follows:

 % correction = [(screen CT – Confirm CT)/Screen CT] x

100

 The result is confirmation of LA if the % correction is above

the cutoff value obtained.
LAB INVESTIGATIONS OF
FIBRINOLYTIC SYSTEM
DETECTION OF FIBRIN
DEGRADATION
PRODUCTS USING A
LATEX AGGLUTINATION
METHOD
Principle
 A suspension of latex particles is sensitized with specific
antibodies to purified FDP.
 The suspension is mixed on a glass slide with a dilution of
the serum to be tested.
 Aggregation indicates presence of FDPs.
 Hence, by testing different dilutions of the
unknown sample , a semi quantitative assay can be
performed.
Conditions with the range
between 10 – 40μ g /ml are:
 Acute venous thromboembolism
 Acute MI
 Severe pneumonia

Very high levels are seen in:

 DIC
 Thrombolytic therapy with
streptokinase
 D-DIMER
Principle TEST
 D-dimer is a marker specific for plasmin degradation of
fibrin.
 Its an FDP generated from FXIIIa crosslinked fibrin.
 Latex beads are coated with a monoclonal antibody
directed specifically against fibrin D dimer in human
plasma or serum.
Reagents
 Latex suspension
 Dilution buffer
 Positive & negative control
 METHOD

 Undiluted plasma is mixed with one drop of latex

suspension on a glass slide. Gently rock the slide for the
length of time mentioned in the kit.

 If agglutination is observe then dilute the plasma until

agglutination can no longer be seen.
 INTERPRETATIO
 N with undiluted plasma
Agglutination - D-dimer >
200mg/L

 The D-dimer value can be quantified by multiplying

reciprocal of the highest dilution showing a positive
result by 200 to give a value in mg/L.

NORMAL RANGE : <200 mg/L.

 EUGLOBIN LYSIS
TEST
Principle
 When plasma is diluted & acidified, the precipitate
(euglobin) that forms contains plasminogen activator,
plasminogen & fibrinogen.
 Plasminogen inhibitors are left out in the solution.
 The precipitate is redissolved, fibrinogen is
clotted with thrombin & the time for clot lysis is
measured.

Reagents
 Acetic acid – 0.01%
 Bovine thrombin – 10 U/ml
 Fresh PPP from patient & control
 Glyoxaline buffer
 NORMAL RANGE : 90 – 240
Min
 Abnormal values
Increased Decreased
Disseminated malignancies
Pregnanc Cirrhosis.
y Obese
Post- op
MI
 AUTOMATION IN
COAGULATION
STUDIES
 Introduced in early 20th century.

 Initial instruments were based on detection of

clot formation:

Coaguloviscometer
Nephelometer
Photoelectric
technique
 The 2 principles of clot based detection currently
used
are electromechanical and optical density.

Electromechanical instruments
 BBL Fibrosystem
 American Bioproducts STArt 4 Clot Detection Instrument

Optical Density Instruments

 Organon Teknika Coag-A-Mate XM
 Chromogenic / Clot detection instruments
 ADVANTAGE
S
 Faster and more accurate
 In case of an abnormal result can cross check
 Can avoid the problem of inter-observer
variation

DISADVANTAGES

 Availability of reagents
 High cost of reagents
 Strict Quality Control
 Availability of well trained technicians
QUALITY
CONTROL
 REFERENCE
1. McKenzie S B. Clinical laboratory
Shirlyn
Hematology; 2nd edition,2004.

2. Wintrobe’s hematology,10th edition,2nd volume

3. Text book of Hematology- Tejinder Singh

4. Robbins and Cotrans;Pathologic basis

of diseases, 8th edition

5. Dacie and Lewis Practical Hematology 11th edition.