Laboratory Tests Bleeding Disorders

1

LABORATORY
APPROACH TO
BLEEDING DISORDERS
Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar
Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes

2

OVERVIEW
1.
2.
3.
4.

Normal coagulation homeostasis
Classification of bleeding disorders
Clinical evaluation of bleeding disorders
Laboratory evaluation of bleeding disorders
a. Screening tests
 SCREENING TESTS FOR PRIMARY HEMOSTASIS
1. Peripheral smear
2. Platelet count
3. Bleeding time
4. Platelet function analysis
 SCREENING TESTS FOR SECONDARY HEMOSTASIS
1. Clotting time
2. Prothrombin time
3. Activated partial thromboplastin time
4. Thrombin time
 Summary of screening tests
b. Specific tests
TESTS FOR PLATELET FUNCTIONS
1. tests for platelet adhesion
2. Test for platelet aggregation
3. Test for platelet release reaction
4. Test for clot retraction
5. Test for platelet procoagulant activity
6. Test for glycoproteins on platelet surface
7. Test for abnormalities in arachidonic acid metabolism
 TESTS FOR COAGULATION FACTORS
1. coagulation factor assays
2. Quantitative estimation of fibrinogen
3. Thromboplastin generation test
4. Mixing test based on PT or aPTT
5. FXIII qualitative assay
6. Paracoagulation test
 TESTS FOR FIBRINOLYSIS
1. Detection of fibrinogen and fibrin degradation products
2. Detection of cross linked fibrin D dimmers


3

* NORMAL COAGULATION HOMEOSTASIS
1. Normal coagulation involves interaction of vessel walls, platelets and coagulation factors
2. Problems at any one of these steps may lead to abnormal homeostasis

Vessel injury

Collagen exposure

Platelet adhesion

FXII activatn

Vasoconst
Platelet release reaction
riction
5-ht

Tissue thrombo
plastin

BLOOD COAGULATION

TXA2

Platelet aggregation
Fibrinogen + thrombin
Primary hemostatic plug
Fibrin
Stabilized hemostatic plug


4

* CLASSIFICATION OF BLEEDING DISORDERS


5

* CLINICAL EVALUATION OF BLEEDING DISORDERS
History from the patient can tell whether the disorder is –
1. Localized or generalized
2. hereditary or acquired
3. Platelet disorder or coagulation disorder
Localized
1. Localized bleeding

Generalized
1. recurrent episodes of bleeding in response
to trivial trauma
2. bleeding from more than one site
3. spontaneous bleeding
4. excess bleeding following minor surgeries
like tooth extraction, tonsillectomy,
circumcision

Hereditary
onset early in life
similar disorder in close relatives
past history of similar episodes
history of disorder only in males on
the maternal side for many
generations – X linked Hemophilia A or
B
5. history of consanguineous marriage,
both males and females only from the
current generation affected –
Autosomal recessive disorders like
afibrinogenemia, von willebrand
disease, Factor V or Factor X deficiency
6. Bleeding in both males and females,
bleedin in one parent, bleeding in all
generations – Autosomal dominant
disorders like von willebrand disease,
hereditary hemorrhagic telengectasia
1.
2.
3.
4.

Sex affected
Family history
Petechiae, bleeding gums,
epistaxis, menorrhagia
Deep hematoma in muscle
and joints
Delayed bleeding from same
site (12-24 hrs after)
Previous history of bleeding

Acquired
usually secondary to disorders of
1. liver
2. uremia
3. hematologic malignancies
4. carcinoma
5. sepsis

Platelet disorders/
Vascular disorders
Female
Negative
Common

Coagulation
disorders
Male
Positive
Rare

Rare

Common

Rare

Common

Not present

Present since childhood


6

* SCREENING TESTS FOR PRIMARY HEMOSTASIS

(i) Platelet count
1. MANUAL METHOD:
Blood + 1% Ammonium oxalate

counted on neubauer’s chamber
(small roughly spherical refractile particles)

Normals:
Platelet count

1,50,000 to 4,00,000 lakhs/mm3

2. AUTOMATED METHOD:
1. A cell counter analyses platelets based on its size.
2. Other platelet associated parameters like MPV, PDW and reticulated platelets are also
obtained
MPV (mean platelet volume)
In some platelet disorders, abnormally large platelets are released in circulation
MPV increased
1. myeloproliferative disorders
2. peripheral destruction of platelets

MPV normal
1. Thrombocytopenia due to impaired
platelet production like
thrombocytopenia

PDW (platelet distribution width)
Measure of degree of variation in platelet size
PDW increased
Myeloproliferative disorder

PDW normal
Secondary or reactive thrombocytosis

Reticulated platelets:
Concept analogous to reticulocytes.
Increased
Peripheral destruction

Normal
Aplastic anemia


7

(ii) Peripheral Smear
For details on peripheral smear examination, refer to separate notes on the same. Only
salient points related to platelet examination will be described here.
Examination of platelets on peripheral smear helps to rule out falsely low counts on
automated methods, like due to clumping.
Total count
RBC
WBC
Platelet

Associated abnormalities in other cell lines
Fragmented RBCs indicate DIC with thrombocytopenia
Abnormal cells with thrombocytosis/thrombocytopenia (leukemias)
Normal count
1 platelet per 500-1000 red cells
Giant platelets
Myeloproliferative neoplasms
Bernard soulier syndrome
ITP
Discrete isolated platelets
Glanzmann’s thrombasthenia
without clumping in finger prick
Uremia
smear


8

(iii) Bleeding time
Definition:
Time required for bleeding to cease following skin puncture/incision.
Methods:
1. Duke
a. lobe is punctured
b. method is not standardized
2. Ivy
a. On volar surface of forearm with lancet
b. Depth of 2 – 2.5 mm under standard venous pressure of 40 mm Hg
c. Blood oozing is blotted with filter paper
d. Time taken to stop bleeding is noted
3. Template
a. uses same site as Ivy
b. larger surgical blade is used for incision, depth 6-9 mm
Normals:
Ivy’s method

2 to 7 minutes

Causes of prolonged bleeding time:
See chart on page 4


9

(iv) Platelet function analysis
Principle:
Blood aspirated through
Capillary into machine
collagen
epinephrine/
ADP

Blood passes through
Aperture in
Machine which is coated with
Platelet agonists such as
Collagen/epinephrine/ADP

The PFA 100 ANALYSER

Platelet adhesion, activation
And aggregation closes the
Aperture overtime

This time known as closure time is indicator of
Platelet function

Abnormals:
Closure time is prolonged in
1. Platelet disorders
2. Von willebrand disease

not prolonged in vascular disorders


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* SCREENING TESTS FOR SECONDARY HEMOSTASIS

(i) Clotting time
Definition:
Time required for whole blood to clot in a glass test tube at 37˚ C
Normals:
Clotting time

5-15 mins

Abnormals:
1. Prolonged in defects of common/intrinsic pathway$
2. But is less sensitive, prolonged only when factor levels drop to <1% of normal, time is
not prolonged in mild / moderate deficiency


11

$ see next page for coagulation pathways details
Pathways of coagulation


12


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(ii) Prothrombin time (PT) and Activated partial thromboplastin time
(aPTT)
Precautions and prerequisites:
1. Venepuncture the anticubital vein, with minimal trauma to avoid tissue thromboplastin
release
2. Donot collect from indwelling catheters to avoid mixing with tissue fluids
3. Use plastic syringes with 20-21 G needles. Donot use glass syringes/bottles as glass
activates contact factors and initiates clotting through intrinsic pathway.
4. Prolonged tourniquet should be avoided as it causes increased fibrinolysis
5. Anticoagulant used in sodium citrate (3.2%) as it causes rapid chelation of calcium & FV
and FVIII remain relatively more stable in it, blood:anticoagulant ratio = 1:9
6. Allow blood to flow gently down the sides of tube/container
7. thoroughly mix blood and anticoagulant
8. transport the tightly sealed tube to lab without delay
9. if labile coagulation factors are to be measured, maintain tube at 4˚C
10. Coagulation studies should be carried out within 2 hours of collection
PROTHROMBIN TIME:
Method:
1. Prepare platelet poor plasma (PPP)

Allow blood in citrate tube to stand

centrifuge at 3000-4000 rpm for 15/20 min

Platelets, RBC and WBC will settle down

Use supernatant
(for platelet function studies, we need to use platelet rich plasma)
2. Take 25 µl PPP in a cuvette A. Take 25 µl tissue thromboplastin + 25 µl calcium chloride
in another cuvette B.
3. Place PPP cuvette in reader
4. Start optic reader
5. Put contents of cuvette B (total 50 µl) into cuvette A
6. Obtain readings of PT and INR
7. A control sample must be run along with patient’s


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Principle:
1. Tissue thromboplatin activates factor VII and initiates the extrinsic pathway of
coagulation
2. Thromboplastin also provides phospholipids for certain coagulation reactions
3. PT measures activity of Extrinsic
and
common pathways
FVII

F X, V, II, I

CONCEPT OF INR
1. The international normalized ratio (INR) was introduced in an attempt to standardize
the PT.
2. In its original manifestation, the PT was very variable because different thromboplastins
were non-standardized and derived from many varied sources. PTs performed on the
identical specimen by different laboratories were inconsistent.
3. The concept behind the INR is that differences between the thromboplastins are
accounted for by a calculation:INR = [PT (patient) ÷ PT (Control)]ISI
The INR has no units (it is a ratio) and is determined to one decimal place.
ISI, or international sensitivity index is a function of the thromboplastin reagent.

Normals:
PT
INR

11-16 Seconds
0.9 – 1.1

Abnormals:
PT is prolonged in
1. Vitamin K deficiency – (F II, VII, IX, and X are vit K dependent factors out of which F VII
and X are important in extrinsic and common pathways) Extrinsic and common
pathways are affected
2. Oral anticoagulant therapy – PT is standard for monitoring anticoagulant therapy
Recommended Therapeutic Range for Oral Anticoagulant Therapy.
INDICATION
INTERNATIONAL NORMALIZED RATIO (INR)
Treatment of venous thrombosis
Treatment of pulmonary embolism
Prevention of systemic embolism
Tissue heart valves
Acute myocardial infarction
Atrial fibrillation

2.0 - 3.0

Recurrent embolism
Mechanical heart valve
Antiphospholipid antibodies

2.5 - 3.5

3. DIC – exhaustion of coagulation factors
4. Inherited deficiency of coagulation factors

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ACTIVATED PARTIAL THROMBOPLASTIN TIME:
Method:
1. Prepare platelet poor plasma as above
2. Take 25 µl sample in cuvette, add 25 µl aPTT reagent (which contains activator#) to it
3. Incubate for 180 secs
4. Place cuvette in optic area
5. Add 25 µl calcium chloride when ready
6. A control should always be run along with the patient’s
#

Activator is not present in PT reagent, activators used can be kaolin, elite, ellagic acid, silica etc.

Normals:
aPTT

30-40 seconds

Principle:
1. The activator activates intrinsic pathway of coagulation.
2. Thus PT is altered in intrinsic and common pathway defects.
Abnormals:
aPTT is prolonged in:
1. Inherited deficiencies of factor VIII (Hemophilia A) and Factor IX (Hemophilia B)
2. Non specific inhibitor antibodies against F VIII e.g. Lupus inhibitor
a. Donot act directly but block interaction of FVIII with other clotting factors
3. DIC
4. Heparin
a. Inhibits factor XII, XI and X through antithrombin III
b. Heparin therapy is monitored through aPTT
5. Vit K deficiency
Affects Factor IX


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(iii) Thrombin Time (TT)
It is a test for plasma fibrinogen levels.
Method:
1. Platelet poor plasma + thrombin reagent
2. Time required for clotting is noted
Principle:
1. Plasma fibrinogen is cleaved by thrombin (activated factor II) to form fibrin clot
Normals:
TT

11.6 – 20.7 seconds

Abnormals:
TT is prolonged in
1. Afibrinogenemia (<100 mg/dl) or dysfibrinogenemia
2. Fibrinogen or fibrin degradation products (interfering substances)
3. presence of heparin in plasma
4. chronic liver disease


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*SUMMARY OF SCREENING TESTS
DEFECT IN

BT

Thrombocytopenia
Extrinsic pathway
Intrinsic pathway

N
N

N
N

Common pathway

N

PT

APTT

N

N

N

Platelet fuction/
vascular disorders/ vW
defects

PLATELET
COUNT
N

N

N

Multiple pathways (DIC)
Clot stabilization

N
N

N/
N

N

N

N

COMMON CAUSES
Von willebrand disease
Storage pool defects
Other platelet affecting
disorders
All causes of
thrombocytopenia
See causes of prolonged PT
See causes of prolonged
aPTT
Causes of either prolonged
PT or prolonged aPTT
DIC/Liver disease
FXIII deficiency


18

*TESTS FOR PLATELET FUNCTION

(i) Test for platelet adhesion (Glass bead column test)

Blood

Platelets counted before entering glass column

Glass column with beads, adhesion of platelets
Occurs here
Aggregation also occurs, hence is non specific

Platelets are counted after passing through
Glass column

Normals:
>25 % retention of platelets

Normal

Abnormals:
1. <25% retention is observed with von willebrand’s disease


19

(ii) Test for platelet aggregation
Method:
Prepare platelet rich plasma
Add aggregating agent and put in aggregometer

As platelets aggregate, more light passes through
Two waves are obtained on graph from the machine, primary wave and secondary wave
(biphasic) corresponding to degree of platelet aggregation against time


20

Aggregating agents used
ADP / Epinephrine

Curve type
Biphasic

Collagen / ristocetin / Arachidonic acid

Monophasic curve

Normals:
Bi phasic ADP curves

Biphasic epineph

Monophasic collagen

Monophasic ristocetin

monophasic AA

See control lines in the graphs


21

Abnormals:
1. GLANZMANN’S THROMBASTHENIA

1.
2.
3.
4.

In Glanzmann’s thrombasthenia, Platelet GPIIb/IIIa is defective.
This receptor is important for platelet aggregation.
All agents induce aggregation through this receptor except ristocetin.
Hence in Glanzmann’s thrombasthenia, aggregation is defective for all agents except
ristocetin.


22

2. BERNARD SOULIER SYNDROME AND VON WILLEBRAND DISEASE

1. In Bernard soulier syndrome there is deficiency of GpIb/IXa, whereas in von willebrand
disease, there is deficiency of von willebrand factor.
2. Aggregation is there in response to all agents except ristocetin

BS and VWD can be differentiated by addition of normal plasma. If the aggregation is seen
now, it means the disease is VWF because normal plasma is a source of VWF.


23

3. STORAGE POOL DEFECTS
1. In this disorder, there is defective granule release from platelets
2. Due to defective release reaction, secondary aggregation is defective
3. Hence there is no secondary wave in response to ADP/epinephrine/collagen and only
partial aggregation in response to ristocetin.

4. ASPIRIN LIKE DEFECT (DEFECT IN COX PATHWAY)
1. Absent aggregation to arachadonic acid.
2. Primary wave aggregation only with ADP.
3. Decreased or absent aggregation with collagen
Disorder

Aggregating agents
ADP/EPINEPHRINE/COLLAGEN
PRIMARY WAVE
SECONDARY WAVE
BS syndrome
N
N
vWD
N
N
Glanzmann’s
D
D
Storage pool$
N
D
$
Aspirin like
D
D
Aspirin like in
N
D
response to AA$
(AA used)
(AA used)
N normal D deficient AA arachidonic acid
$
Measure of platelet release reaction


RISTOCETIN
D
D
N
N
D
D

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(iii) Tests for platelet release reaction

Indirect Tests

Direct Tests

The secondary wave of aggregation in
Response to ADP/epinephrine/collagen
is a measure of release reaction

1. estimation of secretion of
dense core granules
2. estimation of secretion of
alpha granules

Dense core granules

Alpha granules

Contents of platelet granules
adenosine diphosphate (ADP)
adenosine triphosphate (ATP)
ionized calcium (which is necessary for several steps of the
coagulation cascade)
histamine
serotonin

insulin-like growth factor 1
platelet-derived growth factor
TGFβ
platelet factor 4 (which is a heparin-binding chemokine)
other clotting proteins (such as thrombospondin, fibronectin,
and von Willebrand factor)


25

Abnormals:
1. Storage pool deficiency
2. Aspirin like defect (COX deficiency)

(iv) Tests for clot retraction
1. Clot retraction is the "shrinking" of a blood clot over a number of days. In so doing, the
edges of the blood vessel wall at the point of injury are slowly brought together again to
repair the damage.
2. Clot retraction is dependent on release of multiple coagulation factors from platelets
trapped in the fibrin mesh of the clot. Thus, failure to retract can be a sign of
thrombocytopenia or Glanzmann’s thrombasthenia.

(v) Tests for platelet procoagulant activity
This test measures amount of residual prothrombin activity after whole blood is allowed to
clot completely.
Whole blood
In plain tube

allowed to clot

Citrated plasma
(same pt)

serum with residual
prothrombin

Perform PT
(PT1)

Perform PT
(PT2)
PT1
= PLATELET PROCOAGULANT ACTIVITY
PT2

Interpretation:
Presence of residual prothrombin (PCA >1) may be due to deficiency of coagulation factors
or of platelet phospholipids.

(vi) Tests for detection of glycoproteins on platelet surface (Flow
cytometry)
GpIb/IXa
Defective in Bernard soulier syndrome

GpIIb/IIIa
Defective in Glanzmann’s thrombasthenia


26

(vii) Tests for defects in Arachidonic acid metabolism (AA)
1. Arachidonic acid is necessary for TXA2 generation which activates platelets.
2. Normal aggregation in response to Arachidonic acid shows defects in arachidonic acid
metabolism pathway


27

*TESTS FOR COAGULATION FACTORS

(i) Coagulation factor assays
Principle:

Patient’s plasma

+

standard plasma deficient in
Factor to be tested in various
Dilutions

perform PT
and aPTT

Normal

Prolonged

Patients plasma
Contains that
Factor

patients plasma
doesn’t contain
that factor

Normals:
All factors

50-150 % or 50-150 U/dl


28

(ii) Quantitative estimation of fibrinogen
Normals:
Fibrinogen levels

200-400 mg/dl

Abnormals:
Decreased fibrinogen
Increased fibrinogen

Afibrinogenemia, dysfibrinogenemia, DIC
MI, trauma, neoplasia, inflammatory conditions

Methods:
1. Coagulable protein method (based on thrombin time)
thrombin
Fibrinogen

fibrin
Thrombin time

thrombin time

α

1
concentration of fibrinogen

It means there is a linear relationship between concentration of fibrinogen and
thrombin time.
By comparing thrombin time of the test sample with thrombin time of known
fibrinogen standard in the same system, the concentration of sample can be
obtained by using a standard graph.
2.
3.
4.
5.

Immunological method (RIA based)
Heat precipitation test
Chemical precipitation test
Weighing of clot


29

(iii) Thromboplastin generation test (TGT)
Method:
STAGE I
Sample
+
Phospholipid
+
Calcium

FXa-V complex
(aka prothrombinase) Generation of
Prothrombinase

Prothrombin

thrombin

Fibrinogen

Fibrin

estimation of clotting time
STAGE II
if clotting time is abnormal, PT and aPTT are done and substitutions studies are carried out
to determine deficient factors in plasma and serum.
Prepared Serum$
+
Sample
+
Phospholipids
+
calcium
Adsorbed plasma$$
+
Sample
+
Phospholipids
+
Calcium

estimation of clotting
time

estimation of clotting
time

$

prepared serum contains FV and VIII
adsorbed plasma – FIX and X
Both contain – FXI and XII
$$


30

Ad Plasma substitution defective
Serum substitution defective
Both substitution defective

F V / VIII abnormal
F IX/X abnormal
F XI/XII abnormal

PT tests – F V, X
aPTT tests – F VIII,IX, XI, XII
Result interpretation:
History
Bleeding
Bleeding
Bleeding
Bleeding
Bleeding
No bleeding

Coagulation screen
PT
aPTT
N
P
P
N
N
P
P
N
N
P
N
P

TGT
Plasma defect
Plasma defect
Serum defect
Serum defect
Both defect
Both defect


Interpretation
F VIII deficient
F V deficient
F IX deficient
F X deficient
FXI deficient
F XII deficient

31

(iv) Mixing test based on PT/aPTT
Done to differentiate clotting factor deficiency from prolonged PT/aPTT due to inhibitors
Method:
Prolonged PT or aPTT in patient suspected of bleeding disorder

Repeat PT/aPTT after adding normal plasma (patient plasma 50% + normal plasma 50%)

PT/aPTT normal

PT/aPTT still prolonged

Incubate at 37 deg and repeat

Due to immediate acting inhibitor

PT/aPTT prolonged

PT/aPTT still normal

Delayed acting
Inhibitor

coagulation factor deficiency
perform TGT


32

(v) FXIII Qualitative assay (Urea clot lysis test)

Done when all other tests for hemostasis are normal.
FXIII provides stability to clot formed.
Method:
Fibrin

Fibrin clot
+5M urea

dissolves Unstable clot

FXIII def

Doesnot dissolve

FXIII

Normal
FXIII

(vi) Paracoagulation tests (protamine sulphate test & ethanol gelation
test)
Tests done to detect ongoing intravascular coagulation.
Rationale:
In circumstances such as after a major surgical procedure or liver disease

We need to know whether active coagulation is going on in plasma

This would be indicated by presence of soluble (non polymerized) fibrin monomers in
plasma

Method:
Platelet poor plasma + protamine sulphate/
Ethanol

gel formed – fibrin
monomer +nt
(active coagulation +nt)

No gel formed – fibrin
Monomer absent
(active coagulation –nt)


33

*TESTS FOR FIBRINOLYSIS
These are latex agglutination tests for

Fibrinogen and Fibrinogen degradation
Products (FDPs)#

D-dimers#

#Actual clot degradation process
plasmin
Fibrinogen

fibrinogen degradation
products

Thrombin
Fibrin polymer
FXIII
Cross linked fibrin
Plasmin
D dimer

Application:
1. for detecting DIC – (d dimer more specific)
2. pulmonary embolism
3. DVT
4. severe pneumonia
5. MI


Laboratory Tests Bleeding Disorders

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