Practical PATHOPHYSIOLOGY
UNIT 3
INVESTIGATION OF HEMOSTASIS DISORDERS
LEARNING OBJECTIVES
At the end of this chapter, students are expected to:
1. Ask for and interpret the main laboratory investigations of primary hemostasis.
2. Ask for and interpret the main laboratory investigations of secondary hemostasis
(coagulation)
3. Ask for and interpret the main laboratory investigations of fibrinolysis.
4. Ask for and analyze the anticoagulant therapy monitoring.
I. HEMOSTASIS - PHYSIOLOGY
Normal hemostasis comprises mechanisms
operative immediately following an injury and
those acting over a longer period to maintain
hemostasis. The
immediate mechanism
consists principally of two components:
vasoconstriction due to active contraction of
the smooth muscle of the vessel wall and
platelet plug formation within the so-called
primary hemostasis. The maintenance
mechanism consists of the fibrin clot formation
produced by the coagulation system within the
secondary hemostasis classically occurring via
two pathways (Fig. 1).
Figure 1. Coagulation (secondary hemostasis):
intrinsic and extrinsic pathways.
Broken lines signify inhibitory activity.
a) PRIMARY hemostasis
Definition: hemostasis leads to hemostatic
plug and requires the activity of vascular and
platelet factors
Phases:
1. Vasoconstriction. An immediate and
transient response (less than 1 minute
duration), which reduces the blood loss from
the damaged zone.
2. Platelet plug formation. Subendothelial
structures (collagen, fibronectin), which are
exposed, due to the damage of the vascular
endothelium
initiates
the
adhesion,
aggregation, and secretion of platelets
(release of granule content)
platelet plug
formation
b) SECONDARY hemostasis (coagulation)
Definition: formation of fibrin clot; requires
the activity of plasma coagulation factors
Phases:
1. THROMBIN formation – through the
activation of 2 pathways:
Intrinsic pathway (contact phase)
became active when the blood is in
contact
with
the
subendothelial
structures (collagen)
Extrinsic
pathway
(tissue
factor
dependent phase) became active when
the blood is in contact with tissue
products (tissue thromboplastin - factor
III).
Activation of the intrinsic or extrinsic pathway
activates the common pathway.
2. FIBRIN formation
take place under the
action of thrombin and consist in generation of
fibrin polymers from fibrinogen with fibrin clot
formation.
Practical PATHOPHYSIOLOGY
II. LABORATORY INVESTIGATION OF
PRIMARY HEMOSTASIS
1. Platelet Count
Is the first step in evaluating disorders of primary
hemostasis. Platelets are the smallest cells in
the peripheral blood. Traditional counting
methods using a microscope and counting
chamber have been replaced by automated
counting on most standard haematology
analysers.
Indications:
Confirm
a
low
platelet
count
(thrombocytopenia),
which
can
be
associated with bleeding
Confirm an elevated platelet count
(thrombocytosis),
which
can
cause
increased clotting
Identify the possible cause of abnormal
bleeding, such as epistaxis, hematoma,
gingival
bleeding,
hematuria,
and
menorrhagia
Provide screening as part of a complete
blood count in a general physical
examination, especially upon admission to a
health care facility or before surgery.
Normal values: 150,000 - 450,000 cells/mm3
2. Bleeding Time (BT)
Is a coarse test that measures the time it takes
for a standardized incision to stop bleeding. It is
basically a screening test for disorders of
overall platelet function and/or vascular
defects.
There are two technical variants:
Duke's BT (which involves stabbing an
earlobe)
Ivy BT (which involves 2-3 small stabs with
a lancet on the forearm with serially blotevery 30 sec. with filter paper the blood
flowing from the wound till bleeding stops).
Normal values: 2-8 min (Ivy technique)
Pathological changes:
Prolonged in:
Thrombocytopenia
Bernard-Soulier syndrome
von Willebrand’s disease
Platelet function defect (acquired, e.g.
aspirin)
Glanzmann’s thrombasthenia
Hereditary telangiectasia
Liver disease
! BT is usually normal in coagulation
disorders.
3.
Platelet
special
tests
(adhesion,
aggregation and release tests)
Are rarely performed in routine lab practice, but
they are useful in von Willebrand' s disease
(vW), abnormalities of fibrinogen binding
(thrombasthenia) or of arachidonic acid
metabolism (i.e., cyclooxygenase inhibition
secondary to aspirin).
3. Peripheral Blood Smear
Examining a stained peripheral blood smear
under the microscope allows the examination of
red cells, white cells, and platelets.
Because platelet counts almost universally are
done by automated counter, it is important to
examine the blood smear to confirm the
presence of thrombocytopenia. Occasionally,
platelets clump in the automated counter,
causing an inappropriately low platelet count
(pseudothrombocytopenia);
but
adequate
numbers of platelets can be clearly discerned on
the smear. Unusual platelet morphology and
size are seen in inherited platelet disorders.
Also, evaluation of other blood cell morphology
may suggest an underlying disorder, e.g.,
fragmented
red
cells
in
thrombotic
thrombocytopenic purpura (TTP).
4. Bone marrow evaluation
Is a key investigation in haematology. It is used
for diagnostic purposes in the follow-up of
abnormal peripheral blood findings or it can be
used as an important staging procedure in
defining the extent of disease, e.g. lymphomas.
It is a helpful investigative procedure in
unexplained anaemia, splenomegaly or selected
cases of pyrexia of unknown origin.
Considering coagulation it is used primarily to
assess platelet production as it is critical for
determining
the
mechanism
of
the
thrombocytopenia:
an increased number of megakaryocytes
suggests that thrombocytopenia is the result
of
a
increased
peripheral
destruction/consumption of platelets
a decreased number of megakaryocytes
is strongly suggestive for failure of bone
marrow production as main cause of
thrombocytpenia.
Practical PATHOPHYSIOLOGY
III. LABORATORY INVESTIGATION OF
SECONDARY HEMOSTASIS
(COAGULATION)
1. Activated Partial Thromboplastin Time
(APTT or PTTK)
APTT evaluates the function of the intrinsic
pathway (factors XII, XI, IX, and VIII, specifically
the intrinsic thromboplastin system. APTT is the
time required for a fibrin clot to form after tissue
thromboplastin or phospholipid reagents similar
to thromboplastin and calcium are added to the
specimen. The APTT has additional activators,
such as kaolin (PTTK, K stands for kaolin),
celite, or elegiac acid, that more rapidly activate
factor XII, making this test fast and reproducible.
APTT is commonnly used to monitor the
anticoagulant therapy with standard heparin.
Indications:
Detect congenital deficiencies in clotting
factors, as seen in diseases such as
hemophilia A (factor VIII) and hemophilia B
(factor IX)
Evaluate response to anticoagulant therapy
with heparin or coumarin derivatives
Identify individuals who may be prone to
bleeding during surgical, obstetric, dental, or
invasive diagnostic procedures
Identify the possible cause of abnormal
bleeding, such as epistaxis, hematoma,
gingival
bleeding,
hematuria,
and
menorrhagia
Monitor the haemostatic effects of conditions
such as liver disease, protein deficiency, and
fat malabsorption.
Normal values: 22 - 35 sec.
Pathological changes:
Increased values: deficiency of factors VIII, IX,
X, XI or XII, HMW-kininogen, prekallikrein,
fibrinogen, factor V or II, inhibitors to above
factors, DIC, heparin and oral anticoagulants.
Decreased
values
are
found
with
hypercoagulable states.
2. Prothrombin Time (PT, formerly Quick
time)
The test measures the the coagulation time from
the moment of adding thrombloplastin and
calcium to a citrated blood specimen.
Prothrombin is a vitamin K–dependent protein
produced by the liver. PT reflects the function
of the extrinsic pathway including the common
pathway.
PT is commonly used to monitor oral therapy
with
warfarin
or
coumarin
type
anticoagulants.
Indications:
To differentiate between deficiencies of
clotting factors II, V, VII, and X, which
prolong the PT; and congenital coagulation
disorders, such as hemophilia A (factor VIII)
and hemophilia B (factor IX), which do not
alter the PT
To assess the response to anticoagulant
therapy with coumarin derivatives and
determine dosage required to achieve
therapeutic results
To identify the possible cause of abnormal
bleeding, such as epistaxis, hematoma,
gingival
bleeding,
hematuria,
and
menorrhagia
To identify individuals who may be prone to
bleeding during surgical, obstetric, dental, or
invasive diagnostic procedures
To monitor the effects of pathological
conditions such as liver disease, protein
deficiency, and fat malabsorption on
hemostasis
To screen for prothrombin deficiency
To screen for vitamin K deficiency.
Normal values: 11 - 14 sec.
Pathological changes:
PT is prolonged if plasma levels of factors VII,
X, II, V and I (fibrinogen) are < 40% of normal.
In order to evaluate factor deficiency, usually a
comparison is made between APTT and PT:
normal APTT with a prolonged PT can
occur only with factor VII deficiency
prolonged APTT with a normal PT could
indicate a deficiency in factors XII, XI, IX,
and VIII as well as VIII:C (von Willebrand
factor).
Increased in:
Afibrinogenemia, dysfibrinogenemia, or
hypofibrinogenemia
Biliary obstruction
Disseminated intravascular coagulation
Hereditary deficiencies of factors II, V, VII,
and X
Intravascular coagulation
Liver disease
Practical PATHOPHYSIOLOGY
Poor fat absorption (tropical sprue, celiac
disease, chronic diarrhea)
Presence of circulating anticoagulants (eg.,
in lupus)
Vitamin K deficiency
3. International Normalized Ratio (INR)
Nowadays a more precise method was adopted
in virtually all hospital laboratories and clinics for
assessing the intensity of anticoagulation with
oral drugs, especially warfarin: the International
Normalized Ratio.
INR compares the ratio of the patients’s PT to
the mean PT for a group of normal individuals.
The ratio is adjusted for the sensitivity of
laboratory’s thromboplastin determined by the
International Sensitivity Index (ISI). Thus the
formula for INR calculation is:
INR = (Patient PT/ MRI PT) ISI
Where:
PT = prothrombin time in seconds
MRI = geometric mean of reference interval
ISI = international sensitivity index supplied by
reagent manufacturer
Use of INR permits the physicians (i) to obtain
the appropriate level of anticoagulation
independent of laboratory reagents (because
commercial thromboplastins have different
potencies and markedly affect the resulting PT)
and (ii) to follow published recommendations for
intensity of anticoagulation.
Normal values: Satisfactory control of
anticoagulation requires an INR of 2.0 to 4.0.
2.0–3.0 for patients with pulmonary
embolism, deep vein thrombosis, valvular
heart diseas
2.5–3.5 for patients with prosthetic heart
valve or recurrent systemic embolism.
4. Thrombin Time (TT)
The test measures the final stage (common
pathway) of coagulation (i.e., the ability of
thrombin to convert fibrinogen to fibrin) by
measuring the coagulation time of citrated or
oxalated plasma after adding a known amount
of thrombin.
The
test do not differentiate disseminated
intravascular
coagulation (DIC, secondary
fibrinolysis) from primary fibrinolysis.
Normal values: 14 - 21 sec.
Pathological changes:
Increased values are find with: deficiency of
fibrinogen, inhibitors of thrombin or fibrinogen
(plasmin and the presence of fibrin degradation
products-FDP), DIC, heparin therapy.
Observation: In order to differentiate prolonged TT caused
by fibrinogen defects from those caused by the presence of
heparin one can use the protamine-thrombin clotting
time (protamine neutralizes the anticoagulant effect of
heparin and FDP). Prolonged TT and prolonged protamine
thrombin time are suggestive of fibrinogen defect.
Prolonged TT and normal protamine thrombin time are
suggestive of heparin.
5. Fibrinogen
Fibrinogen is a fibrillar protein produced by the
liver which loses two small peptides prior to
polymerization and becomes insoluble fibrin
during the clotting process. Also, it is a sensitive
acute phase protein whose concentration raises
several folds during inflammation, tissue
necrosis, vascular collagen diseases.
Indications:
To assist in the diagnosis of suspected
disseminated intravascular coagulation
(DIC), as indicated by decreased fibrinogen
levels
To diagnose congenital or acquired
dysfibrinogenemias
To monitor hemostasis in disorders
associated with low fibrinogen levels or
elevated levels that can predispose
patients to excessive thrombosis.
Normal values: 200 – 400 mg/dL
Pathological changes:
Increased in:
Acute myocardial infarction
Cancer
Eclampsia
Hodgkin’s disease
Inflammation
Multiple myeloma
Nephrotic syndrome
Pregnancy
Tissue necrosis
Administration of estrogens, oral
contraceptives and in pregnancy.
Practical PATHOPHYSIOLOGY
Decreased in:
DIC
Dysfibrinogenemia
Liver disease (severe)
Primary fibrinolysis
Observation: Evidence has shown that plasma levels of
fibrinogen above the reference range constitute an
independent risk factor for both coronary artery and
cerebrovascular diseases.
6. Specific factor assays can further define
abnormalities of the coagulation cascade once
they have been localized by means of screening
tests.
Indications:
Identify the presence of inherited bleeding
disorders
Identify the presence of qualitative or
quantitative factor deficiency
Specific factor assays may also be useful
in patients with normal screening tests but
compelling family or clinical evidence
suggesting a mild factor deficiency.
IV. LABORATORY INVESTIGATION OF
FIBRINOLYSIS
1. Fibrin Degradation Products (FDP, FSplitP)
FDP = degradation products of fibrinogen and
fibrin, with high molecular weight.
The test do not differentiate fibrinogen
fragments from fibrin fragments and it is not
possible to differentiate primary from secondary
fibrinolysis (DIC) on the basis of FDP alone.
Normal values: < 10 mg/L
Pathological changes:
Increased values are seen with: primary or
secondary fibrinolysis (all the causes of DIC),
during thrombolytic or defibrination therapy,
circulating plasminogen, thrombosis, pulmonary
embolism, myocardial infarction.
3.4 D-dimers
D-dimers = degradation products of crosslinked fibrin, with low molecular weight.
Indications:
To exclude, diagnose, and monitor diseases
and conditions that cause hypercoagulability,
such as deep vein thrombosis (DVT) and
pulmonary embolism
To diagnose DIC (along with other tests)
Normal values: 20- 400 g/L
Pathological values
Increased values:
A positive D-dimer indicates the presence
of an abnormally high level of cross-linked
fibrin degradation products.
May be due to DIC, recent surgery, or
trauma, infection, liver or kidney disease,
pregnancy, heart disease and some
cancers.
4. Laboratory approach in coagulation
disorders (Table 1)
Table 1. Routine Screening Tests of
Coagulation
Test
Interpretation
Platelet count
Platelet number
Peripheral blood smear Cellular
morphology
and number
Bleeding time (BT)
Platelet
&
vessels
function
Prothrombin time (PT)
Extrinsic pathway
Activated
partial Intrinsic pathway
thromboplastin
time
(APTT)
Thrombin time (TT)
Fibrinogen conversion
to fibrin
After performing the routine tests, one may
consider the following situations in which the
deficiency together with possible causes are
presented:
PT and APTT normal
Deficiency: VII
Causes: early liver disease, vitamin K
deficiency, warfarin or dicoumarol.
PT and APTT
Deficiency: X, II, V, I (fibrinogen)
Causes: single or multiple deficiency, e.g.
DIC, liver failure, vitamin K deficiency.
PT normal and APTT
Deficiency: XII, XI, IX, VIII
Causes: hemophilia A or B, von Willebrand
disease, single or multiple deficiency,
heparin, non-specific inhibitors of these
factors (e.g. lupus anticoagulants).
PT normal and APTT normal
Deficiency: XIII
Causes: normal patient, platelet abnormality,
single deficiency of factor XIII, LMW heparin.
Practical PATHOPHYSIOLOGY
DIC: Platelet count , fibrinogen , TT ,
FDP .
Vit. K deficiency: PT , assay II, VII, IX,
X ( ); give vit K and repeat after 48 h. If
PT will not correct to normal with vit. K it
means liver disease. If PT returns to
normal it means vit. K deficiency.
BT, APTT, PT are all normal, in the
presence of bleeding, one or more of the
following must be true: surgical problem
(suture deficiency), patient is hypothermic
(APTT, PT run in vitro at 37 degrees),
laboratory test error.
5. Anticoagulant therapy monitoring
5.1 Heparin (Standard, Unfractionated
Heparin, UFH)
Indications
Cornerstone for the prophylaxis, treatment of
acute venous thromboembolism and its
extension:
Prophylaxis and treatment of pulmonary
embolism
Prevention of post-operative deep
venous thrombosis and pulmonary
embolism in patients undergoing major
abdomino-thoracic surgery or who for
other reasons are at risk of developing
thromboembolic disease
Prevention of clotting in arterial and cardiac
surgery.
Diagnosis and treatment of acute and
chronic consumptive coagulopathies (DIC).
As an anticoagulant in blood transfusions,
extracorporeal circulation, dialysis procedures
and in blood samples for laboratory purposes.
Mechanism of action
Increases
the
inhibitory
effect
of
antithrombin on the serine proteases
thrombin, IXa, Xa, XIa, and XIIa with the
greatest effect upon thrombin.
Laboratory monitoring:
a. APTT
Assay 4-6 hours after bolus dosage and
every 24 hours thereafter
Target: prolonged APTT ratio to 1.5 to 2.5.
b. Platelet count
Daily, in order to detect heparin induced
thrombocytopenia (HIT)
If count drops 30-50%, consider HIT,
withdraw
heparin,
start
alternative
anticoagulant, order confirmatory test for
HIT. HIT is a severe and potentially fatal
form of thrombocytopenia which typically
occurs 2 to 5 days after heparin exposure.
When the clinical suspicion is high, heparin
should be replaced with one of the direct
thrombin inhibitors Lepirudin or Argatroban,
until the clinical situation is elucidated
c. Avoid concurrent use of aspirin and other
NSAIDs. Aspirin irreversibly inhibits platelet
function. Current evidence indicates that aspirin
can increase the risk of bleeding in patients
anticoagulated with heparin.
Overdose of heparin
Stop heparin and monitor APTT. Heparin
half-life is approximately 30 minutes. If
bleeding is severe, consider heparin
antagonist: protamine sulfate.
5.2 Low Molecular Weight Heparin (LMWH)
(Enoxaparin, Tinzaparin, Fondaparinux)
Indications
Prevention or treatment of thromboembolic
disease
Mechanism of action
Inhibits f. Xa
LMWH clearance is predictable and
requires little monitoring in uncomplicated
thrombosis
5.3. Vitamin K antagonists (VKA) - warfarin,
acenocoumarol
Indications
Treatment of arterial and venous thrombosis
to prevent clot propagation
Prevention of thromboembolic disease in
thrombophilia, atrial fibrillation, mechanical
heart valves, and high-risk surgery.
Mechanism of action
Prevents the vitamin K dependent activation
of factors II, VII, IX, and X, thus slowing
thrombin production.
Requires 2-7 days to reach therapeutic
levels.
To
achieve
immediate
anticoagulation, one should begin with
heparin.
Laboratory monitoring: the INR
Target INRs:
Myocardial infarction, most therapy and
prophylaxis of embolism: INR 2.0-3.0
Mechanical heart valves: INR 2.5-3.5.
5.4. Direct Thrombin Inhibitors (DTIs)
Indications
Practical PATHOPHYSIOLOGY
Substitute for heparin when HIT is
suspected or confirmed.
Laboratory monitoring: APTT is used to
prevent bleeding or thrombosis
Target APTT: 1.5-3.0 x mean of reference
interval.
5.5. Novel Oral Anticoagulants (NOAC) or
Direct Oral Anticoagulants (DOAC) are
direct inhibitors of factor II (dabigatran)
and X (rivaroxaban, apixaban), respectively.
Indications
Substitute for indirect oral anticoagulants
(warfarin and acenocoumarol).
Laboratory monitoring: not available.
Practical PATHOPHYSIOLOGY
CHECKPOINT
*1. Which test is used to follow up patients
undergoing treatment with vitamin K
antagonists?
A. INR
B. APTT
C. BT
D. TT
E. Platelets count
*2. What is the utility of Koller test?
A. To identify bleeding risk prior to surgical
interventions
B.
For
differential
diagnosis
between
hepatopathy and vitamin K deficiency
C. For DIC diagnosis
D. For differential diagnosis between primary
and secondary fibrinolysis
E. None of the above
*3. What kind of platelet dysfunction is von
Willebrand disease?
A. Adhesion defect
B. Aggregation defect
C. Secretion defect
D. Adhesion and aggregation defect
E. Thrombocytopenic disorder
*4. Which is the screening test for the
evaluation of both vascular and platelet
function?
A. BT
B. PT
C. APTT
D. TT
E. APTT and PT
5. A 2-year old boy is brought to the hospital for
a painful, swollen ankle after a minimal trauma.
In the past 48 h he refused walking and playing
due to the pain. Lab investigation shows:
Thrombocyte count: 384.000 /mm3
BT: 5 min
PT: 13 sec
APTT: 80 sec
Which is the most probable diagnosis?
Which investigation(s) you would further
recommend?
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6. A 70 year-old patient is transferred from the
emergency unit to the ICU because of fever
(40°C), epistaxis i hematuria. The pacient was
hospitalized for a posttraumatic hip fracture and
has a bladder catheter for one week.
Examination shows purpura and petechiae on
the thorax and superior limbs. Prolonged
belleding occurs at the site of the venopunction.
Lab investigation shows:
Thrombocyte count: 84.000 /mm3
BT: 13 min
PT: 17 sec
APTT: 48 sec
Which is the most probable diagnosis?
Which investigation(s) you would further
recommend?
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