Laboratory Approach to coagulation disorders& Mixing studies

1. LABORATORY APPROACH TO
COAGULATION DISORDERS &
MIXING STUDIES
SUNILKUMAR P
HEMATLOGY& TRANSFUSION MEDICINE
ST.JOHN’S MEDICAL COLLEGE HOSPITAL , BANGALORE

2. Haemostasis : -
• Arrests bleeding from injured site.
• Maintain blood in fluid state in normal
vessels.

3. Trauma
Vessel constriction
+
Shed blood
Coagulation Platelet adhesion
Fibrin Platelet aggregation
Platelet
activation
Stable
haemostatic plug

4. CLOTTING FACTORS
• Fibrinogen
• Prothrombin
• Tissue factor
• Calcium
• Factor V
• Factor VII
• Factor VIII
• Factor IX
• Factor X
• Factor XI
• Factor XII
• Factor XIII
• Prekallikrein
• Hmwk
• platelets
• Factor I
• Factor II
• Factor III , Tissue thromboplastin
• Factor IV
• Proaccelerin, labile factor
• Proconvertin, stable factor
• Antihemophilic factor
• Christmas factor
• Stuart factor; stuart-prower
• Plasma thromboplastin antecedent
• Hageman factor
• Fibrin stabilizing factor
• Fletchet factor
• Fitzgerald factor

6. Evaluation of the patient
• History
• Physical Examination
• Laboratory Evaluation

7. History
–surgical challenges
–accidents & injuries
–dental extractions
–menstrual history

8. Physical Examination
• current hemorrhage
• intercurrent illnesses
–liver disease
• petechiae/ecchymoses

9. Type of Bleeding
• ecchymoses
• petechiae
• epistaxis
• deep soft tissue bleed
• hemarthroses
• GI bleeding

10. Laboratory Assessment
• Guided by history
• Screening tests:
• BT, CT, Platelet count,
– PT
– APTT
– thrombin time
– Fibrinogen

12. Specific Laboratory Tests
• Factor assays
• Euglobin Lysis test
• Urea solubility test
• D-Dimer
• Screening test for inhibitors
• Platelet function test

13. Blood Coagulation & Tests

14. FIRST LINE TESTS

15. PT APTT TT PLATELET
COUNT
CONDITION
N N N N Normal hemostasis
Disorders of PFT
Factor XIII deficiency disorder
VWD
↑ N N N VII deficiency
Early oral anticoagulant
Mild II ,V ,X deficiency
N ↑ N N VIII,IX,XI,XII ,prekallikerin,HMWK def
vWD disease
Circulating anticoagulants
↑ ↑ N N Vit K deficiency,oral anticoagulats,F V ,X,II
Deficiency
↑ ↑ ↑ N Liver disease,fibrinogen deficiency
Hyperfibrinolysis
N N N Low thrombocytopenia
↑ ↑ N Low Massive transfusion,Liver disease
↑ ↑ ↑ LOW DIC
ACUTE LIVER DISEASE

16. Prothrombin time. (PT)
Principle
The test measure the clot plasma in the presence of a
optimal conc. of tissue extract and indicates the overall
deficiency of the extrinsic clotting factors
Clinical Significance –
 PT reflects the overall efficiency of the extrinsic system.
 Most sensitive to changes in factor V, VII, X and
fibrinogen conc.
Normal range - 10 -14 seconds.
.

17. Activated partial Thromboplastin time
(APTT)
Principle
measures the clotting time of plasma after the
activation of contact factor but without added tissue
thromoplastin
Clinical significance
• Intrinsic system
• Deficiency of factor VIII, IX, XI, XII.
• Deficiency of common pathway(V,X,II,& I)
Normal range - 30 to 40 seconds.

18. Thrombin time
• Time taken by the citrated plasma to clot after
addition of thrombin in presence of calcium.
Fibrinogen Fibrin
• Increased value
– Decreased level of fibrinogen
– Qualitative abnormality of fibrinogen
– Presence of heparin / heparin like substance.
Normal range – 13 to 17 seconds
Thrombin

19. PT
TT
APTT
PT - 
APTT, TT, PLC - N
HMWK
XII
PK
XI
IX
VIII
VII
X
V
II
I
* Factor VII deficiency
• Anticoagulant therapy
• Vitamin Kdeficiency
• Liver disease

20. APTT - 
PT, TT, PLC - N
* Factor deficiency
* vWD
* Inhibitors
* Heparintherapy
PT
TT
APTT
HMWK
XII
PK
XI
IX
VII
X
V
II
I

21. PT
TT
APTT
PT, APTT - 
TT, PLC - N
HMWK
XII
PK
XI
IX
VIII
VII
X
V
II
I
* Common Pathway Factor deficiency
* Vitamin K deficiency
* Oral anticoagulant therapy
* Liver disease

22. PT
TT
APTT
PT, APTT, TT - 
PLC - N
HMWK
XII
PK
XI
IX
VIII
VII
X
V
II
I
* Hypo / dysfibrinogenemia
* Heparin
* Liver disease
* Systemic hyperfibrinolysis

23. PT
TT
* DIC
- Fibrin monomer
- Liver necrosis
APTT
APTT, PT,TT all 
PLC - low
HMWK
XII
PK
XI
IX
VIII
VII
X
V
II
I

24. PT
TT
APTT
PT, APTT- 
TT - N
PLC - 
HMWK
XII
PK
XI
IX
VIII
VII
X
V
II
I
Massive transfusion
with stored blood

25. Thrombocytopenia
Bone marrow biopsy to differentiate
 production
 destruction
PTAPTT
PT, APTT,TT-N
PLC - 
HMWK
XII
PK
XI
IX
VIII
VII
X
V
II
I TT

26. INHIBITORS-SCREENING TEST
• PRINCIPLE
• Inhibitors are Ab’s developed against factor VIII.
They are time dependent thus if factor VIII:C is added
to plasma containg an inhibitor and the mixture is
incubated, factor VIII:C will be progresively neutralized.
If the amount of factor VIII:C added and the duration of
incubation are standardized.
The strength of the inhibitor may be measured in
units according to how much of the added factor VIII:C
is destroyed.

27. REAGENTS
• APTT reagent
• Control plasma
• PROCEDURE
• Screening for coagulation inhibitors
• A. perform APTT of patient & control
• B. if prolonged do APTT with 1/2 patient + ½
control
• C. if there is no correction –suggest the presence
of inhibitors

28. PROCEDURE
PATIENT CONTROL MIXTURE
(0.5 ML PATIENT+0.5ML CONTROL)
PATIENT
PLASMA
1 ML
CONTROL
PLASMA
1 ML
MIXTURE 1ML
Incubate at 37c , perform the APTT at intervals of ½ hour, 1 hour and 2 hours
INTERPRETATION : The APTT s of incubated mixture gets prolonged with time, where as
fresh mixture remains the same in the presence of an inhibitor

29. FIBRINOGEN ASSAY
• PRINCIPLE :
• Fibriquik is based on a method described by
clauss. When thrombin is added to a sample
plasma, fibrinogen is converted enzymatically to
fibrin, fibrin in turn, undergoes polymerization to
form a fibrin network.
• factor XIII activated by thrombin, catalyzes
the formation of stabilizing crosslink to produce a
visible clot the time from addition of thrombin to
the formation of clot is inversely proportional to
fibrinogen level

30. Reagents
• Thrombin reagent
• Owner’s veronal buffer

31. Procedure
• Label a test tube for each sample to be tested
• Allow the reagents to R.T
• Prepare 1/10 dilution of patient plasma
( 0.1 ml of sample + 0.9 ml of owners veronal buffer).
TEST
Diluted patients plasma
(warm to 37c for at leat 2 min before testing)
0.2 ML
Fibrquik thrombin 0.2ML
Simultaneously begin timing for detection
Record time required for clot detection to the nearest 01 second
Reference Range : 146-389 mg/dl

32. UREA SOLUBILITY TEST
( To detect factor XIII deficiency)
• PRINCIPLE:
• Clots formed in the presence of factor XIII are
stable for at least 24 hrs in 5 mol. urea, where as
clots formed in the absence of factor XIII
dissolves rapidly
• REAGENTS :
• 5M urea solution
• Thrombin
• 0.025M cacl2

33. PROCEDURE
NEGATIVE CONTROL POSITIVE CONTROL TEST (PATIENT)
CONTROL PLASMA 0.2ML
EDTA PLASMA 0.2ML
PATIENT PLASMA 0.2ML
0.025 M Cacl2 0.2ML 0.2ML
THROMBIN 0.2ML
INCUBATE TUBES FOR 20 MIN
take 3ml of 5m urea in three other tubes, transfer the clots formed from above step to
these tubes , keep the tubes for 24 hrs at .R.T
INTERPRETATION :
NEGATIVE -- IF CLOT IS STILL PRESENT
POSITIVE -- IF CLOT IS DISAPPEAR

34. D-dimer
• PRINCIPLE :
• Fibrinosticon is an immunologic latex agglutination test
that utilizes latex prticles coated with a monoclonal
antibody specific for cross-linked D-diamer domain in
fibrin.
– These latex particles form macroscopic aggregates only in
the presence of soluble fibrin derivatives containing the D-
dimer domain.
– Because clot lysis by plasma results in a heterogeneous
population of fibrin degradation products with more than
one D-dimer domain per molecule, anti D-dimer antibody
coated to latex particles will cause agglutination of these
particles when the antigen is present

35. CORRECTION TEST USING PT & APTT
 Unexplained prolongation of PT ,APTT can be
corrected by simple correction test.
 Correction done by mixing patient plasma with
normal plasma.
 Failure to correction indicates presence of
inhibitor.
Specific factor deficiency can be identified.

36. Mixing studies in coagulation
• When PT and / or APTT are prolonged further
investigation done to identify specific
abnormality.
• Mixing studies are used to distinguish factor
deficiency from factor inhibitor (such as lupus
anticoagulant or specific factor inhibitor such
as Ab directed against factor VIII).

37. • PRINCIPLE
Mixing studies are used to determine whether
a prolonged PT or APTT is due to factor
deficiency or an inhibitor. Correction of the
abnormality by an additive reagent indicates
that the reagent contains the substance
deficient in the test sample

38. Correction – factor deficiency.
No correction- presence of inhibitors

39. Prolonged PT/APTT
Mixing studies
Corrected Not corrected
Factor deficiency Circulating
anticoagulants/
presence of inhibitors
Factor assays Special assays

40. REAGENTS
• Patient’s platelet poor plasma.
• Control platelet poor plasma.
• Aged serum/ plasma.
• Adsorbed plasma (with Al(OH)3 or bariumsulphate).
• Factor VIII/ IX deficient plasma.
Aged serum Adsorbed plasma
Factors present VII, IX, X, XI, XII I, V, VIII, XI, XII
Factor absent I, II, V, VIII II, VII, IX, X

41. • Adsorbed plasma
• Prepared using barium sulphate or
alluminium hydroxide.
• Removes factor VII,IX,X.
• Adsorbed plasma contains(I,V,VIII.XI,XII)

42. Method
• Perform PT/APTT on control and patients plasma.
• If prolonged, 50: 50 mixture of normal control
plasma or additive reagents and patient’s plasma is
used to perform mixing studies.
• Perform tests in duplicates to avoid time bias.
Correction studies using PT/APTT with normalplasma.
• A mixture of 50: 50 patient plasma and normal
plasma.
• Perform PT/ APTT in duplicates.
• Correction indicates factor deficiency

43. If APTT corrects by more than 50% of the difference
between clotting times of normal plasma and test
plasma good correction.
A poor correction ie, prolonged APTT on mixing
indicates presence of an inhibitor.
Eg:-
APTT test =60˝
Control = 35˝ 100%
• Correction with ½ patient +
½ control = 42˝ [50%]
60˝- 35˝ =25˝ [100%]
25/2 = 12.5˝ [50%] with out correction.

44. 60˝- 42˝ =18˝ [50%] after correction.
18˝ >12.5˝ Good correction.
• ½ patient + ½ control = 52˝ [50%]
60˝ - 52˝ = 8˝ [50%]
8˝ < 12.5˝ poor or no correction
2) Using aged serum.
½ patient serum + ½ aged serum.
Perform PT/APTT in duplicates.
Interpret the result.

45. interpretation
PT APTT Correction with
adsorbed plasma
PT APTT
Correction with aged
serum
PT APTT
Probable factor
deficiency
N N _ _ _ _ No factor
deficiency
N A _ C _ C XI/XII
N A _ NC _ C IX
N A _ C _ NC VIII
A N NC _ C _ VII
A A NC NC NC NC II
A A C C NC NC V
A A NC NC C C X

46. CORRECTION USING THROMBIN TIME
• PRINCIPLE
Test utilize certain physiochemical properties of
reagents to bind to inhibitor or abnormal
molecules and normalize the prolonged thrombin
time.
• REAGENTS
• patient’s and control platelet poor plasma.
• Protamine sulphate 1% and 10% in 9g/L NaCl.
• Toluidine blue 0.05g in 100mL of 9g/L NaCl.
• Bovine thrombin.

47. • PROCEDURE
• TT on 50:50 mixture of test and normal PPP.
• ½ Patient plasma + ½ protamine sulphate.
• ½ patient plasma + ½ toluidine blue.
• Interpret results
TT of test plasma corrected with
INTERPRETATION
NORMAL PLASMA PROTAMINE
SULPHATE
TOLUIDINE BLUE
C NC NC Fibrinogen
deficiency
VARIABLE C C Heparin
VARIABLE C NC High conc: of FDP

48. Reptilase time
• It is the modification of the TT in which the
purified enzyme reptilase is used to replace
thrombin. Reptilase isolated from the snake
Bothrop atrox. Thrombin splits small fibrino
peptide A and B from fibrinogen molecules
producing fibrin monomer to form a clot.
• REFERENCE RANGE
13 – 15 Sec

49. Interpretation
TT RT
Presence of heparin Prolonged N
Thrombin inhibitors Prolonged N
Decreased/absent
fibrinogen
Prolonged N
Warfarin Prolonged N
Dys fibrinogenemia Prolonged Prolonged
DIC Prolonged Prolonged
Liver disease Prolonged Prolonged

50. Limitations of mixing studies
• Be careful when thawing the pooled plasma
because prolonged incubation at 37°c will
selectively decrease factor V.
• The pooled normal plasma is stable for ~2hr at
room temperature.
• Some inhibitors are time or / and temperature
dependent.

51. REFERENCES
• Text book of Haematology William's,
chapter118,pg No-1883-1889.
• Text book of Hemostasis & Thrombosis,
columan, mardwill chapter 12.
• Text book of postgraduate hematology
Hoffbrand.
• Practical Haematology dacie and Lewis .

52. THANK YOU