Universidad Juárez del Estado de Durango

Facultad de Ciencias Químicas

Químico Farmacéutico Biólogo

Thrombosis Risk Testing

REALIZADO POR: LIZETH S ÁNCHEZ CASTRO
Etiology of Thrombosis

Thrombosis is a multifaceted disorder resulting from abnormali-

ties in blood flow, such as stasis, and abnormalities
in the coagulation system, platelet function,
leukocyte activation molecules, and the blood vessel wall.
Thrombosis is the inappropriate formation of platelet
or fibrin clots that obstruct blood vessels. These obstructions
cause ischemia (loss of blood supply) and necrosis (tissue
death). Thrombophilia
Physical,
Physical, chemical,
chemical, or
or biologic
biologic events
events such
such as
as chronic
chronic or
or acute
acute inflammation
inflammation

inappropriate
inappropriate and
and uncontrolled
uncontrolled platelet
platelet activation
activation

Uncontrolled
Uncontrolled triggering
triggering of
of the
the plasma
plasma coagulation
coagulation system
system

Inadequate
Inadequate control
control of
of coagulation-impaired
coagulation-impaired fibrinolysis
fibrinolysis
Prevalence of Thrombosis

At least 30% of the world’s people are expected to die of a thrombotic

condition, and 25% of initial thrombotic events are fatal.
Venous Arterial
thrombosis thrombosis

U.S. population is Cardiovascular disease causes 500,000

1 in 1000. premature deaths annually in the U.S.

Fragments of 500,000 cerebrovascular accidents

thrombi, called (strokes) result in 100,000 stroke-
emboli. related deaths.

About 80% of myocardial infarctions

and 85% of strokes are caused by
thrombi that block coronary arteries.
THROMBOSIS RISK FACTORS

J
Thrombosis Risk Factors
Associated with Systemic Diseases
Together, transient and chronic antiphospholipid (APL) antibodies
such as LA, ACL, and anti–β2-GPI may be detected in 1% to 2%
of the unselected population.
Chronic APL antibodies confer a risk of venous or arterial
thrombosis—a condition called the antiphospholipid syndrome
(APS).
Chronic APL antibodies often accompany autoimmune connective
tissue disorders, such as lupus erythematosus. Some appear in
patients without any apparent underlying disease.
Congenital Thrombosis Risk
Factors

Congenital thrombophilia is suspected when a thrombotic event occurs

in young adults; occurs in unusual sites such as the mesenteric, renal,
or axillary veins; is recurrent; or occurs in a patient who has a family
history of the disorder.

Because thrombosis is multifactorial, however, even patients with

congenital thrombophilia are most likely to experience thrombotic
events because of a combination of constitutional and acquired
conditions.
The prothrombin G20210A gene mutation is the second most
common inherited thrombophilic tendency in patients with a
personal and family history of deep vein thrombosis. All together,
protein C, protein S, and antithrombin deficiencies are found in
0.2% to 1.0% of the world population.
APC resistance is found in 3% to 8% of whites. Resistance
extends to Arabs and Hispanics, but the mutation is absent from
African and East Asian population.
LABORATORY EVALUATION OF
THROMBOPHILIA

When thrombophilia is suspected, it is important to assess all

known risk factors, because it is the combination of positive
results that determine the patient’s cumulative risk of
thrombosis.
Antiphospholipid Antibodies

APL antibodies arise as immunoglobulin M (IgM), IgG, or IgA

isotypes. Because they may bind a variety of protein-
phospholipid complexes, they are called nonspecific inhibitors.
Clinical Consequences of Antiphospholipid
Antibodies

Most APL antibodies arise in response to a bacterial, viral, fungal,

or parasitic infection or to treatment with numerous drugs and
disappear within 12 weeks. These are mostly transient
alloimmune APL antibodies and have no clinical consequences.
Performing the Clot-Based Lupus Anticoagulant
Mixing Study.
Anticardiolipin antibodys Immunoassay
Anti–β2-Glycoprotein I Immunoassay

IgM and IgG anti–β2-GPI

immunoassays are performed as a
part of the profile that includes
ACL assays.An anti–β2-GPI result
of greater than 20 GPL or MPL
units correlates with thrombosis
more closely than the presence of
ACL antibodies.
Antiphosphatidylserine
Immunoassay

For cases in which an APL antibody is suspected but the routine

LA, ACL, and β2-GPI assay results are negative.
the clinician may wish to order the antiphosphatidylserine immu-
noassay to detect APL antibodies specific for phosphatidyl-
serine.
A result greater than or equal to 16 IgG or 22 IgM
antiphosphatidylserine units is considered positive.
Activated Protein C Resistance and Factor V Leiden
Mutation
A mutation in the factor V gene
substitutes glutamine for arginine at
position 506 of the factor V molecule
(FV R506Q). The arginine is a
cleavage site for APC, so the
substitution slows or prevents
hydrolysis of the factor V molecule.
Antithrombin

Antithrombin is a serine protease inhibitor (serpin) that

neutralizes factors IIa (thrombin), IXa, Xa, XIa, and XIIa.
Antithrombin activity is enhanced by unfractionated heparin, low-
molecular-weight heparin, and synthetic pentasaccharide.
Antithrombin Deficiency

Acquired antithrombin deficiency occurs in liver disease, in

nephrotic syndrome, with prolonged heparin therapy, with
asparaginase therapy, with the use of oral contraceptives, and in
DIC, where antithrombin is rapidly consumed.
Antithrombin Reference
Ranges

Adult plasma antithrombin activity ranges from 78% to 126%.

Antithrombin antigen levels range from 22 to 39 mg/dL (68% to
128%) by latex microparticle immunoassay
Adult levels are reached by 3 months of age,
The levels remain steady throughout adult life except during
periods of physiologic challenge, such as pregnancy.
Antithrombin Activity Assay
The chromogenic substrate test for plasma antithrombin activity
detects quantitative and qualitative antithrombin deficiencies and
detects mutations affecting the proteolytic site, but not the
heparin binding site.
Heparin Resistance and the Antithrombin Assay
Antithrombin may be decreased during prolonged or intense
heparin therapy and may be totally consumed if the patient has a
congenital antithrombin deficiency. In this instance, heparin may
be administered in therapeutic or higher dosages, but it neither
exerts an anticoagulant effect nor is detected by the PTT.
Protein C Control Pathway

Thrombin is an important
coagulation factor because it
cleaves fibrinogen and activates
platelets and factors V, VIII, XI,
and XIII.

The thrombin-thrombomodulin
complex activates plasma protein
C, and APC binds free plasma
protein S.
Protein C and Protein S
Reference Ranges

Heterozygous deficiency of protein C or protein S leads to a 1.6-

fold to 11.5-fold increased risk of recurrent deep vein thrombosis
and pulmonary embolism. Protein S deficiency also has been
implicated in transient ischemic attacks and strokes, particularly in
the young.
The reference interval for both protein C and protein S activity and
antigen levels is 65% to 140%, and levels ordinarily remain
between 30% and 65% for heterozygotes.
Protein C and protein S assays therefore cannot be used to
identify a congenital deficiency when they are employed soon
after thrombosis or soon after the cessation of warfarin therapy,
during pregnancy, or in the presence of DIC, liver disease, renal
disease, or vitamin K deficiency.
Homozygous protein C or protein S deficiency results in neonatal
purpura fulminans, a condition that is rapidly fatal
Protein C Assays
Chromogenic assays.

The assay detects

abnormalities that affect the
molecule’s proteolytic
properties (active serine
protease site), but misses
those that affect protein C’s
phospholipid binding site or
protein S binding site.
a clot-based protein C assay
but the clinical condition continues to indicate possible protein C
deficiency, a clot-based protein C assay may detect abnormalities
at these additional sites on the molecule.
 Protein S Assays
A clot-based assay is performed by mixing the patient’s plasma
with protein S–depleted normal plasma to ensure normal levels of
all other factors

When there is clinical suspicion of

primary protein S deficiency based
on low activity level.
These assays detect most
quantitative congenital deficiencies
and aid in the diagnosis of
qualitative type II .
ARTERIAL THROMBOSIS
PREDICTOR
Arterial thrombotic disease in the form of peripheral vascular
disease, myocardial infarction (heart attack), and cerebrovascular
disease (stroke) arises from atherosclerosis.
The traditional predictors of arterial thrombosis risk are:
Elevated total cholesterol and low-density lipoprotein cholesterol
(LDL- C)
A high ratio of total cholesterol to high-density lipoprotein
cholesterol (TC : HDL-C) secondary to deficient HDL-C.
High-Sensitivity C-Reactive
Protein Measurement
The high-sensitivity CRP assay is an established clinical tool to
evaluate subtle chronic systemic inflammation and predict
cardiovascular or cerebrovascular disease.

Mild chronic elevations of CRP may predict atherosclerosis 6 or

more years before myocardial infarction or stroke.
High-Sensitivity C-Reactive Protein Reference Range and
Relative Risk
The high-sensitivity CRP mean for people who have no coro-
nary artery disease, as proven by angiography, is 0.87 mg/L. In
contrast, the mean for patients with three atherosclerotic arter-
ies is 1.43 mg/L.
Plasma Homocysteine
Homocysteine is a naturally occurring sulfur-containing amino-
acid formed in the metabolism of dietary methionine.
Homocysteine circulates in the form of various disulfides,
together called total homocysteine. The concentration of homo-
cysteine in plasma depends primarily on adequate protein intake
and levels of vitamin B6, vitamin B12, and folate.
Its concentration is regulated by three enzymes: cystathionine β-
synthase, which converts homocysteine to cystathionine in the presence
of vitamin B6; 5,10-methylenetetrahydrofolate reductase (MTHFR),
required for the remethylation of homocysteine to methionine in the folic
acid cycle; and methionine synthase, which requires vitamin B12
Clinical Significance of
Homocysteinemia

with relative risk ratios of 1.7 for coronary artery disease, 2.5 for
cerebrovascular disease, and 6.8 for peripheral artery disease.
Mild elevations of homocysteine are an independent risk factor
for occlusive arterial disease.
Homocysteine Test Principles

High-performance liquid chromatography has been a familiar

approach to homocysteine analysis.
An enzyme immunoassay is available from several dis-
tributors. In the Abbott AxSYM assay (Abbott Laboratories, Abbott
Park, Ill.)
 Fibrinogen Activity
Elevated fibrinogen makes blood more
viscous, which favors coagulation,
platelet activation, and formation of
atherothrombotic lesions.
Lipoprotein (a)

Lipoprotein (a) is an LDL with noteworthy thrombosis risk

prediction characteristics.
Lipoprotein (a) may contribute to thrombosis by its antifi-
brinolytic property. The molecule competes with plasminogen for
binding sites on newly formed fibrin polymer, which decreases
the plasmin activity available for clot degradation
DISSEMINATED INTRAVASCULAR
COAGULATION
In DIC, fibrin microthrombi partially occlude small vessels and
lead to consumption of platelets, coagulation factors, coagulation
control proteins, and fibrinolytic enzymes.

Fibrin degradation products (FDPs, traditionally called fibrin split

products, FSPs) become elevated and interfere with normal fibrin
formation. Many toxic and inflammatory processes are set loose.
Causes
Any disorder that contributes hemostatic molecules or promotes their endogenous secretion
may cause DIC.
Chronic DIC may be associated with vascular tumors, tissue
necrosis, liver disease, renal disease, chronic inflammation, use
of prosthetic devices, and adenocarcinoma. The malignancies
most associated with DIC are pancreatic, prostatic, ovarian, and
lung cancers; multiple myeloma; and myeloproliferative diseases.
Pathophysiology
Symptoms

The symptoms signaling DIC frequently are masked by the

symptoms of the underlying disorder
Thrombosis in the microvasculature of major organs may
produce symptoms of organ failure, such as renal function
impairment, adult respiratory distress syndrome, and central
nervous system manifestations.
Skin, bone, and bone marrow necrosis may be seen.
Laboratory Diagnosis
Specialized Laboratory Tests That May Aid
in Diagnosis
Treatment

Chronic DIC
Surgery, antiinflammatory agents, antibiotic

Acute DIC
therapies that replace missing platelets and coagulation
therapies that slow the clotting process
factors.

Heparin may be used for its antithrombotic properties to stop the uncontrolled activation of
the coagulation cascade.
HEPARIN-INDUCED
THROMBOCYTOPENIA
Heparin-induced thrombocytopenia (HIT), also called heparin-
induced thrombocytopenia with thrombosis, is an adverse effect
of treatment with unfractionated heparin
Cause and Clinical Significance
Between 1% and 5% of patients receiving unfractionated heparin
for more than 5 days develop an IgG antibody to heparin–platelet
factor 4 immune complexes.

In 30% to 50% of cases, the immune complexes that are formed

bind platelet Fc receptors, which leads to platelet activation,
thrombocytopenia, and formation of microvascular thrombi.
Patients may develop pulmonary emboli,
limb gangrene requiring amputation, stroke,
and myocardial infarction.
Platelet Count

Patients receiving heparin must have platelet counts performed

every other day.
Patients receiving heparin develop an immediate, benign, and
limited thrombocytopenia, sometimes called HIT type I.
This benign form of thrombocytopenia usually develops in 1 to 3
days, whereas immune-mediated HIT, sometimes called HIT type
II, develops after 5 days.
Treatment
When heparin-induced antibodies are detected, the
administration of heparin must be discontinued immediately.

Synthetic penta-saccharide, which mimics heparin’s antithrombin-

binding sequence, appears safe to use in HIT.