from various sources

1. COAGULATION PROFILE
Dr.Mohamid Afroz Khan

2. HEMOSTASIS
Hemostasis
The termination of bleeding by mechanical
or chemical means or by the complex
coagulation process of the body, which
consists of vasoconstriction, platelet
aggregation, and thrombin and fibrin
synthesis.

3. HEMOSTASIS
Primary
Platelets
Secondary
Coagulation
Cascade

4. PRIMARY HEMOSTASIS
Endothelial
damage
Release of
Von
Willebrand
Factor (vWF)
Platelets
attach to vWF
via GP Ib/IX
Degranulation
attracts more
platelets
Platelet plug

5. SECONDARY HEMOSTASIS
• Platelet aggregation initiates secondary
hemostasis through the coagulation
cascade.
• Coagulation cascade is initiated by the
intrinsic or extrinsic pathway.
• The final cascade results in fibrin
deposition cross-linking platelets and clot
formation

7. Coagulation Cascade

8. HISTORICAL PERSPECTIVE
• ‘Coagulation cascade’ based upon the waterfall
hypothesis of Ratnoff & Davies and MacFarland.
• In 1977, Osterud and Rappaport recognized that
factor VIIa is able to activate factor IX to factor
IXa.
• Broze and colleagues factor VIIa–tissue factor
complex cannot directly activate factor X but has
to go through factor IX activation.
• Gailiani and Broze found that formed thrombin
can activate factor XI, resulting in amplification
of the coagulation system under stress.

9. APPROACH TO
COAGULATION DISORDERS

10. Clinical approach
1. Is the bleeding significant ?
2. Local Vs Systemic ?
3. Platelet Vs Coagulation disorder?
4. Inherited Vs Acquired ?

11. Findings Disorders of
Platelet
Disorders of
Coagulation
i) Petechiae
ii) Superficial
ecchymoses
iii) Deep dissecting
hematomas
iv) Hemarthosis
v) Bleeding from the
superficial cuts &
scratches.
vi) Positive family
history
vii) Bleeding from
mucous membrane
Characteristic
Characteristic,
usually small &
multiple
Rare
Rare
Persistent often
profuse
Rare
Prominent
Rare
Common, usually
large & solitary
Characteristic
Characteristic
Minimal
Common
May occur

12. History &
Clinical
Examination
Primary
Hemostasis
Secondary
Hemostasis

13. Disorder of secondary hemostasis
aPTT, PT,
Prolonged aPTT Normal PT
Factor XI
Factor XII, PK & HMWK
Prolonged aPTT & PT
Disorders of fibrinogen
Factor II
Factor V
Factor X
Combined def of Vit K
Dependent Factors
Normal aPTT Prolonged PT
Factor VII
Normal aPTT and PT
Factor XIII

14. Laboratory approach
• Large number of tests are essential to
diagnose spectrum of bleeding disorders.
• An investigative approach becomes cost
effective and patient friendly

15. Bleeding disorder
Defect / Deficiency
in plasma
coagulation
proteins
True protein
deficiency
An abnormal
protein
Missense,
Deletion &
translocation of
DNA
Inhibitor to active
site of protein
Immunoglobulins
Enhanced
clearance of
protein
Result of antigen
antibody complex
formation
Defect in platelet
number or function
Defect in adhesive
interaction
between platelet &
vessel wall

16. A short screening profile
2 To 7 minutes

17. Abnormalities
in Short
Screening
Tests
Extended
screening
tests
Specialized
Diagnostic
Tests.

18. ASSESSMENT OF COAGULATION
PROTEINS
(Assessment of secondary hemostasis)

19. General considerations
Sample collection
• Venous blood is used
• 21-gauge for adults
• 22- or 23-gauge needle for infants
• The blood should be mixed with sodium
citrate anticoagulant in the proportion 9
parts blood: 1 part anticoagulant.
• This should be 0.109M (3.2% trisodium
citrate dihydrate)

20. • Anticoagulant solution can be stored at 4°C for
up to three months.
• Use plastic or siliconized glass
• Test within 4 hours.
.
• Storage at -70°C or lower is preferable for
further testing

21. • 40 year old cascade hypothesis still has
merit in explaining mechanisms of clot
formation in screening tests.
• The coagulation proteins are classified as
members of intrinsic or extrinsic pathway.

23. Activated partial thromboplastin
time (aPTT)

24. • Principle –plasma is incubated with an
activator (which initiates intrinsic pathway
of coagulation by contact
activation).Phospholipids and calcium are
then added and clotting time is measured
• Reagents-kaolin 5 gm/l
-phospholipid
-calcium chloride 0.025 mol/l

25. • Method –
1.Mix equal volumes of phospholipid reagent and
calcium chloride solution in a glass test tube
and keep in a waterbath at 370 C
2.Deliver 0.1 ml of plasma in another test tube
and add 0.1 ml of kaolin solution.Incubate at
370 C in the waterbath for 10 minutes.
3.After exactly 10 minutes,add 0.2 ml of
phospholipid-calcium chloride mixture,start the
stopwatch,and note the clotting time.
Normal range- 30- 40 seconds

26. Causes of prolongation of APTT
1.Haemophilia A and B.
2.Deficiencies of other coagulation factors in
intrinsic and common pathways.
3.Presence of coagulation inhibitors
4.Heparin therapy
5.DIC
6.Liver disease

27. Test Methodology

28. Normal Values and Critical Limits
• 24 - 37 sec
• Statistically, the aPTT is slightly lengthened in
young individuals and slightly shortened in
older populations.
• Premature infants have prolonged aPTT values
which return to normal by 6 months of age.
Interferences
• Lipemia and hyperbilirubinemia interfere with
the detection of clot formation by photo-optical
methods.

29. Prothrombin Time and INR

30. Principle- tissue thromboplastin and
calcium are added to plasma and clotting
time is determined.The test determines
the overall efficiency of extrinsic and
common pathways.
Reagents-
1.Thromboplastin reagent
2.Calcium chloride 0.025 mol/litre

31. • Method-
1.Deliver 0.1 ml of plasma in a glass test
tube kept in water bath at 370 C.
2.Add 0.1 ml of thromboplastin reagent and
mix.
3.After 1 minute, add 0.1 ml of calcium
chloride solution.Immediately start the
stopwatch and record the time required
for clot formation.

32. Test Methodology

33. • The result is reported in seconds
(prothrombin time), or as a ratio
compared to the laboratory mean normal
control (prothrombin ratio, PTR).
• Normal Value: 11-16 seconds

34. Understanding the INR
• Need for standardization of PT results.
• The International Normalized Ratio (INR)
introduced by WHO.
• Calibration system was developed to relate any
PT ratio to a WHO standard.

35. • International Reference Preparation (IRP).
• ISI correlates the sensitivity of commercial
thromboplastin preparations to the IRP.
• By definition, the ISI of the first IRP was 1.0
• An additional term, the INR, was introduced to
compare a given prothrombin ratio measurement
to the IRP.
• Thus, the INR represents the prothrombin time
which would have been obtained if the IRP had
been used as a reagent in the test.

36. Calculating the INR
• ISI is supplied by manufacturer.
• If the ISI is known, the INR is easily calculated
by the following formula:

37. • INR should be maintained in the
therapeutic range for the particular
indication (INR of 2-3 for prophylaxis and
treatment of deep venous
thrombosis;INR of 2.5-3.5 for mechanical
heart valves).
• Therapeutic range provides adequate
anticoagulation for prevention of
thrombosis and also checks excess
dosage,which will cause bleeding.

38. Thrombin Time (TT)
• To perform this assay, purified exogenous
thrombin is added to plasma to determine the
time for clot formation.
• Direct measure of fibrinogen function.
• Used to asses if there is defect in fibrinogen
function.
• Prolonged in hypofibrinogenic states and in
dysfibrinogenemia.
• The normal range is 15 – 17 seconds.

39. Fibrinogen Assay (Clauss
Technique)
• Principle-diluted plasma is clotted with a
strong thrombin solution;the plasma must
be diluted to give a low level of any
inhibitors (e.g.FDPs and heparin).A
strong thrombin solution must be used so
that the clotting time over a wide range is
independent of the thrombin
concentration.

40. • Make dilutions of the calibration plasma in
veronal buffer to give a range of fibrinogen
concentrations (i.e. 1 in 5; 1 in 10; 1 in 20 and 1
in 40)
• 0.2 ml of each dilution is warmed to 370C & 0.1
ml of thrombin solution added & clotting time
measured
• A calibration curve prepared by plotting clotting
time in seconds against the fibrinogen conc in
g/l
• The normal range is approximately 1.8–3.6 g/l

41. Mixing studies
• Used to distinguish between factor
deficiencies factor inhibitors.
• If APTT is prolonged, patient’s plasma is
mixed with an equal volume of normal
plasma
• Plasma samples found to have abnormal
screening tests (i.e. PT/APTT) may be
further investigated.

42. • Abnormal screening tests are repeated on
equal volume mixtures (termed 50:50 below) of
additive and test plasma.
• The following agents can be used for mixing
tests:
1. Normal plasma
2. Aged plasma (deficient in factors V and VIII)
3. Adsorbed plasma(Adsorbed plasma is
deficient in factors II, VII, IX, and X - vitamin K-
dependent factors).
4. FVIII-deficient plasma
5. FIX-deficient plasma

44. Individual Factor Assays
• One staged assays based on PT and APTT
• Comparing the ability of dilutions of a standard
or reference plasma and test plasma to correct
the PT or APTT of a plasma known to be totally
deficient in the clotting factor being measured.

45. • Functional FVII activity is measured by a PT-
based, FVII deficient plasma clotting assay
• Use of r-human thromboplastin will yield
results that are more likely to reflect in vivo
FVII levels
• Therapeutic options include FFP,
prothrombin complex concentrates, and
recombinant FVIIa
• For major surgery, plasma FVII levels of at
least 20% are sufficient

46. One-Stage Assay of Factor VII
(based on PT)
• Principle-The assay of factor VII is based on the
PT. Assay compares the ability of dilutions of the
patient’s plasma and of a standard plasma to
correct the PT of a substrate plasma
• It is easily adapted to assay of prothrombin, factor
V or factor X
Reagents
• PPP from the patient
• Standard/reference plasma

47. • Factor VII-deficient (substrate)plasma-
Commercial or from a patient with known
severe deficiency
• Barbitone buffered saline
• Thromboplastin
Method
• Prepare 1 in 5, 1 in 10; 1 in 20 and 1 in 40
dilutions of the standard and test plasma in
buffered saline
• 0.1 ml of each dilution +0.1 ml of deficient
(substrate) plasma

48. • Mix and allow to warm to 370 C
• Add 0.1 ml of dilute thromboplastin and start
the stopwatch
• Record the clotting time
Calculation of Results
• Plot the clotting times of the test and standard
against concentration of factor VII on graph
paper
The normal range is 50–150 iu/d

50. One stage assay of Factor VIII
(based on APTT)
• Principle- based on APTT according to
the bioassay principle.
• Reagents-PPP. From the patient
Standard/reference plasma, factor VIII
deficient plasma (substrate),barbitone
buffered saline,reagent for APTT,plastic
tubes,ice bath.
Method-place the APTT reagent and CaCl2
37 degree celsius and patient’s,standard
and substrate plasma in the icebath until
used

51. • Make 1 in 10 dilutions of the test and standard
plasma in buffered saline in plastic tubes in the
ice bath
• Using 0.2 ml volumes, make doubling dilutions in
buffered saline to obtain 1 in 20 and 1 in 40
dilutions.
• Place 0.1 ml of three dilutions in glass tubes.
• Add to each dilutions 0.1 ml of freshly
reconstituted or thawed substrate plasma and
warm up at 37 degree celsius.
• Perform APTTs according to the laboratory
protocol.
• The dilutions should be tested at 2-min intervals
on the master watch.

52. • Normal range- 45- 158 iu/dl
Reduced factor VIII concentration is found in-
1.Haemophilia A
2.VWD,types I and III and some cases of type
II
3.Congenital combined deficiency of factors
VIII and V.
4.DIC

53. • In this example, the
FVIII concentration in
the test sample is 7 %
of that in the standard.
• If the standard has a
concentration of 85
IU/dl, the test sample
has a concentration of
85 IU/dl × 7% = 6 IU/dl.

54. Additional Special tests
• FDP Assays-FDPs are fragments produced by
proteolytic digestion of fibrinogen or fibrin by
plasmin.
• For determination of FDPs,blood is collected in
a tube containing thrombin (to remove all
fibrinogen by converting it into a clot) and
soybean trypsin inhibitor (to inhibit plasmin
and thus prevent in vitro breakdown of fibrin).
• A suspension of latex particles linked to anti-
fibrinogen antibodies is mixed with dilutions of
patients’s serum on a glass slide.If FDPs are
present ,agglutination of latex particles occurs.

55. • Increased levels occurs in
DIC,DVT,severe pneumonia and recent
MI.

56. • D-dimer assay- D-dimer is derived from the
breakdown of fibrin by plasmin and D-dimer
test is used to evaluate fibrin by plasmin and
D-dimer test is used to evaluate fibrin
degradation.
• Blood sample can be either plasma or serum.
• Latex or polystyrene microparticles coated
with monoclonal antibody to D-dimer are
mixed with patient’s sample and observed for
microparticle agglutination.

57. • D-dimers are raised in –
DIC,intravascularthrombosis
(MI,stroke,venous thrombosis,pulmonary
embolism) and during postoperative
period or following trauma.

58. • Reptilase time (assess the rate of
fibrinogen → fibrin conversion in the
presence of heparin.)

60. AUTOMATED COAGULOMETER

61. Caveron Alpha analyzer
• It is a fully automatic ,coagulation
measuring instrument used to perform all
plasma coagulation tests for routine and
for research for in vitro diagnostic use.
• It can analyze samples using
clotting,chromogenic and turbidimetric
methods.
• The Caveron Alpha consists of an
analyzer, a personal computer and an
optional printer.

62. • It operates on the photometric measurement
principle.
• This measurement method finds the
coagulation time by an optical detection of the
change of turbidity caused by the formation of
fibrin fibres.
• For chromogenic methods the change of
absorbance is detected after adding a
chromogenic substrate after plasma and
reagent incubation and for immunological
methods also the change of absorbance is
detected during the reaction of antigen and
antibody complex formation.A calibration
converts the results into concentration units.

63. THANK YOU