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Blok Hematoimunology
Scenario 2
Created by:
Group 10
Ade Marantika
1218011002
Istighfariza Shaqina
1218011084
Alfianita Fadhilla
1218011010
M Nikhola Risol
1218011099
Christhopher P.P.P
1218011030
1218011104
Ellya Rahmawati
1218011044
Putri Giani P
1218011117
Hani Zahiyyah
1218011063
1218011076
Zygawindi N
1218011167
PREFACE
Assalammualaikum wr.wb
Alhamdulillah, Praise God we pray to God Almighty for His blessings and grace
so that we can prepare a report this tutorial discussion.
This report was prepared to fulfill the task subjects hematoimunology. To the
faculty involved in the course of this block, we say thank you for all the guidance
and the guidance that has been given so that the report can be completed.
We realize that there are many flaws in the writing of this report, both in terms of
content, language, and so on. Therefore, we apologize for the shortage. This is due
to the still limited knowledge, insight, and skills. In addition, criticism and
suggestions from readers are we expected, to the perfection of this report and
repair for all of us.
Hopefully this report can provide benefits and can add knowledge to us.
Wassalammualaikum wr.wb
Complier
CONTENTS
Preface ....................................................................................................................2
Contents...................................................................................................................3
Scenario ..................................................................................................................4
Step 1.......................................................................................................................5
Step 2.......................................................................................................................6
Step 3.......................................................................................................................7
Step 4......................................................................................................................12
Step 5......................................................................................................................41
Step 6......................................................................................................................42
Step 7......................................................................................................................43
References......................74
Scenario 2
Bleeding in Tooth Expulsion
Bimo, 9 years old, accompanied by his mother to put off his tooth. After
expulsion, the blood is not stopping. Then, Bimo is taken care in the hospital to
observe the bleeding. When he learned to walk, Bimo often get knee swelling if
he felt down, also he easily got bruise when he get minor trauma. His uncle also
have similar condition. In physical examination there`s no organomegaly found.
The laboratory findings result that aPTT 80 second (referral score 31-47 second)
and platelet count 200.000/L (referral score 150.000-400.000/L)
Step 1
Clarify Unfamiliar Term
1. What is aPTT ?
Activated Partial Thromboplastin Time (APTT) Test
This test measures how long it takes for blood to clot. It measures the clotting
ability of factors VIII (8), IX (9), XI (11), and XII (12). If any of these clotting
factors are too low, it takes longer than normal for the blood to clot. The results
of this test will show a longer clotting time among people with hemophilia A or
B.
Step 2
Define The Problem
1. Explain about Hemostasis and interferences of Hemostasis!
2. What is diagnosis and differential diagnosis in cases ?
3. What is classification of Hemophilia ?
4. Explain how the pathogenesis and pathofisiology of Hemophilia ?
5. Clinical manifestations of Hemophilia ?
6. Explain how to diagnosis of Hemophilia ?
7. Why the bleeding in cases difficult to stop?
8. How to do therapy of Hemophilia?
Step 3
Brainstorm Possible Hypothesis or Explanation
If there is an injury to the blood vessels, the blood vessels will undergo
vasoconstriction, so that blood flow is blocked, and spent too little blood, as
well as contact between the platelets with the vessel wall is quite long.
Contact the platelets in the blood vessels will lead to platelet adhesion to the
collagen network. This process requires the presence of platepresence of
platelet glycoprotein 1b, and von Willebrand factor from blood vessels.
Platelet adhesion will experience a release of ADP (Adenosine DiPhosphat)
and A2 tromboxan that will lead to agegrasi platelets, thus forming an
unstable platelet plug.
Platelets are experiencing will issue a stretcher age gresi Platelet factor 3
(PF3), which will stimulate the blood clotting process. The blood clotting
process will produce threads of fibrin threads.
Fibrin suture thread that happens will bind unstable platelet plug it to become
a stable platelet plug.
Physical examination:
Bleeding is difficult to stop, especially when the large blood vessels in the
joints or muscles after trauma or surgery. When there is bleeding in
muscles and joints (hemarthrosis), the patient will feel tingling and heat in
the place in question. Furthermore, there will be severe pain and swelling.
In general, bleeding in the joints resulting in joint unusable.
3. Laboratory examination:
-
Normal platelet
10
Blood coagulation is a host defense system that maintains the integrity of the
high-pressure closed circulatory system. After tissue injury, alterations in the
capillary bed and laceration of venules and arterioles lead to extravasation of
blood into soft tissues or external bleeding. To prevent excessive blood loss,
the hemostatic system, which includes platelets, endothelial cells, and plasma
coagulation proteins, is called into play. Immediately after tissue injury, a
platelet plug is formed through the processes of platelet adhesion and
aggregation. Blood coagulation may be considered a mechanism for rapid
stabilization of an otherwise unstable platelet plug with a fibrin clot. A series of
interdependent enzyme- mediated reactions translates the molecular signals that
initiate blood coagulation into a major biologic eventthe formation of the
fibrin clot.
If there is bleeding, especially acute joint area, then action RICE (rest, ice,
compression, elevation) immediately. Joint bleeding rested and immobilized.
An ice pack or a cold wet towel, and then performed the emphasis or splinting
and elevate bleeding area. Patients should be given a replacement factor within
2 hours after hemorrhage. or gene therapy can be done.
11
Step 4
Arrange Explanation into Tentative Solutions
Combined intinsik and extrinsic factors that will lead to changes in the X factor
X factor is active, and then together form the threads of fibrin.
Running intrinsic
Running intinsic involves factors XII, XI, IX, VIII and X in addition
prekalikrein,
high
The track begins with a "contact phase" with prekalikrein, high molecular
weight kininogen, factor XII and XI are exposed on the surface of negatively
charged activating. In vivo, the possibility of active proteins on the surface of
endothelial cells. If the components assembled on the surface of the contact
phase activator, factor XII is activated into factor XIIa during proteolysis by
kallikrein. Factor XIIa will attack prekalikrein to produce more kallikrein again
by causing reciprocal activation. Once formed, factor XIIa activates factor XI
into Xia, and also releases bradykinin (vasodilator) of high molecular weight
kininogen.
12
Xia factor in the presence of Ca2 + ions activate factor IX, a serine protease
enzyme into, namely factor IXa . The next deciding factor Arg - Ile bond in
factor X to produce 2- chain serine protease, namely factor Xa. This latter
reaction requires the assembly of components, called tenase complex, on the
surface of activated platelets, namely Ca2 + and factor IXa and factor X. We
must note that in all reactions involving zymogen containing Gla (factors II,
VII, IX and X), Gla residues in the amino terminal region of the molecule that
serves as a high-affinity binding sites for Ca2+. For tenase complex assembly,
platelets must first be activated to open the acidic phospholipids (anionic).
Phosphatidyl serine and inositol fosfatoidil are normally found on the side of
the state does not work. Factor VIII, a glycoprotein, is not a precursor
proteases, but which serves as a cofactor for factor IXa resepto and X on the
surface of platelets. Factor VIII is activated by thrombin with a very small
amount to form factor VIIIa, which is subsequently inactivated by thrombin in
the process of further breakdown.
Extrinsic Tracks
Running involves extrinsic tissue factor, factor VII, X, and Ca2+ and generate
factor Xa. Production begins at the site of factor Xa -tissue injuries with tissue
factor expression in endothelial cells. Tissue factor interacts with factor VII
and activate it ; factor VII is a glycoprotein containing Gla, circulate in the
blood and is synthesized in the liver. Tissue factor works as a cofactor for
factor VIIa to promote enzymatic activity to activate factor X. deciding factor
VII Arg - Ile bond in the same X factor that was cut by the trajectory of the
intrinsic tenase complex. Activation of factor X creates a significant
relationship between intrinsic and extrinsic path.
13
trajectory, which also involves the factor XII, prekalikrein and high molecular
weight kininogen. Actual trajectory can be more important than intrins ic
fibrinolysis than in coagulation, as kallikrein, factor XIIa and Xia can cut
plasminogen, and kallikrein can activate urokinase single-chain.
Inhibitors of tissue factor path ( TFPI : tissue factor fatway inhibitior ) is the
main physiological inhibitor that inhibits coagulation. This form of protein
inhibitors that circulate in the blood and bound lipoproteins. TFPI directly
inhibits factor Xa by binding to the enzyme near its active site. Then the factor
Xa - TFPI complex is complex hinders factor VIIa - tissue factor.
Last Trajectory
At the same last trajectory, factor Xa generated by the trajectory of DAK
intrinsic extrinsic, will activate prothrombin (II) to thrombin (IIa) which then
converts fibrinogen into fibrin.
Prothrombin activation occurs on the surface of activated platelets and require
assembly kompelks protrombinase consisting of anionic phospholipids of
platelets, Ca2+, factor Va, factor Xa and prothrombin.
14
Interference hemostasis
In his book, Capita Selecta Hematology, Hoffbrand AV et al mention that
hemostasis disorders (abnormal bleeding) can be caused by several things
below:
1. Vascular Abnormalities
Vascular disorders are a heterogeneous group o f state groups, which signed
by easy bruising and spontaneous bleeding from small blood vessels.
Abnormalities underlying blood vessels lie in itself or in the perivascular
connective tissue. In this circumstances, the test filters were normal
standard members. Normal bleeding period, another test was also normal
hemostasis. Vascular abnormalities, there are two types of hereditary
hemorrhagic telangiectasia is a hereditary, and connective tissue disorders.
Other types are derived vascular defects.
2. Thrombocytopenia
Thrombocytopenia was defined as a platelet count of less than
100.000/mm3. Usually characterized by spontaneous skin purpura, mucosal
bleeding, and prolonged bleeding after trauma. Some causes of
thrombocytopenia include:
1) Failure of platelet production. A's common cause of thrombocytopenia
is usually also part of a generalized suppression of bone marrow
failure megakarisit selective toxicity can be caused by drugs or viral
infection.
2) Increased destruction of platelets, It is divided into several types
namely:
a. Trombositopenia immune, including ITP, due to infection,
purpura
pascatranfusi,
because
drug- induced
immune
15
16
The platelet (a blood particle essential for the clotting process) count should be
measured as well as two indices of blood clotting, the prothrombin time (PT)
and activated partial thromboplastin time (aPTT). A normal platelet count,
normal PT, and a prolonged aPTT are characteristic of hemophilia A and
hemophilia B. Specific tests for the blood clotting factors can then be
performed to measure factor VII or factor IX levels and confirm the diagnosis.
Genetic testing to identify and characterize the specific mutations responsible
for hemophilia is also available in specialized laboratories.
Carrier of hemophilia
Since men with the genetic mutation will have hemophilia, a man who does not
have the condition cannot be a carrier of the disease. A woman who has a son
with known hemophilia is termed an obligate carrier, and no testing is needed
to establish that she is a carrier of hemophilia.
Women whose carrier status is unknown can be evaluated either by testing for
the clotting factors or by methods to characterize the mutation in the DNA. The
DNA screening methods are generally the most reliable.
Prenatal diagnosis is also possible with DNA-based tests performed on a
sample obtained through amniocentesis or chorionic villus sampling. Most
individuals are seen and tested by consultants who specialize in genetically
linked diseases.
What are the symptoms of he mophilia?
Hemophilia symptoms vary, depending on the degree of blood clotting factor
(coagulation factor) deficiency and they also depend on the nature of any
injury.
Three levels of hemophilia are recognized, according to the level of clotting
factor amounts in the blood. These are often expressed as percentages of
normal:
17
1% to 5% - moderate hemophilia
Mild hemophilia
People with inherited mild hemophilia may not have any symptoms until an
event occurs which wounds the skin or tissue, such as a dental procedure or
surgery, and results in prolonged bleeding. In societies where male
circumcision is carried out soon after birth, mild hemophilia will be detected
earlier. Joint bleeding is uncommon.
Moderate hemophilia
Those with inherited moderate hemophilia will be noticeable early on. The
child will bruise easily and may also experience internal bleeding symptoms,
especially around the joints, and after a blow or a fall. Bleeding that occurs
inside a joint is usually referred to as a joint bleed.
Symptoms of a joint bleed:
Joint stiffness
Ankles
Knees
Elbows
...and may less commonly affect the shoulders, hips or other joints.
Any surgical intervention, circumcision, dental procedure or injury will result
in prolonged bleeding in a person with hemophilia.
18
Severe hemophilia
Symptoms are similar to those found in moderate hemophilia, but occur more
frequently and are usually more severe.
A child with severe hemophilia will often bleed for no apparent reason, often
referred to as spontaneous bleeding. Most commonly, in early childhood from
about 18 months of age, the nose or mouth start to bleed or apparently
spontaneous bruises appear, particularly on the legs. Parents are sometimes
suspected of causing non-accidental injury (deliberate harm) to their children.
Symptoms of he mophilia type bleeding may include:
Blood in urine
Unexplained nosebleeds
A bad headache
Vomiting
Confusion
Fitting (Convulsion)
Loss of balance
Stiff neck
19
Vision problems
Loss of coordination
Differential Diagnosis
Disease/Condition
Differentiating
Signs/Symptoms
Differentiating Tests
Platelet dysfunction
Deficiency of other
Musculoskeletal bleeding is
coagulation factors
uncommon.
(e.g., factor V, VII, X XI,
20
Disease/Condition
or fibrinogen)
Differentiating
Signs/Symptoms
Differentiating Tests
Ehlers-Danlos
syndrome
Musculoskeletal bleeding is
uncommon.
Scurvy
Musculoskeletal bleeding is
uncommon.
History of a restricted diet,
sepsis, HIV, critical illness, or
pancreatitis may be present.
Fabry disease
Musculoskeletal bleeding is
uncommon.
Typical skin lesions
(angiokeratomas), pain in the
extremities, renal and heart
disease, typical ocular signs.
21
Disease/Condition
Differentiating
Signs/Symptoms
Disseminated
intravascular
coagulation
No differentiating
signs/symptoms to acquired
hemophilia.
Underlying causal condition
(e.g., acute promyelocytic
leukemia) is present.
Differentiating Tests
CBC may reveal anemia, which
may be chronic in neglected or
malnourished children.
Liver and pancreatic enzymes
may be elevated if abdominal
trauma.
Imaging may reveal abnormal
x-rays with evidence of
fractures, or abnormal brain or
abdominal imaging due to
bleeding.
22
Severe Hemophilia
Clotting factor levels less than 1% (< 0.01 IU/ml)
Spontaneous bleeding hemarthroses (joint bleeding) and muscle hematomas
Moderate Hemophilia
Clotting factor levels between 1% and 5% (0.01 IU/ml to 0.05 IU/ml)
Bleeding with mild trauma or minor surgery.
Mild Hemophilia
Clotting factor levels between 5% and 40% (0.05 IU/ml to 0.4 IU/ml)
Excessive bleeding with severe trauma or major surgery.
Hempophilia A or classic hemophilia: A person with this type of hemophilia
has low levels of or is completely missing factor 8 (Also called FVIII or factor
VIII deficiency) 80% of people with hemophilia have Type A Hemophilia.
Factor VIII deficiency usually manifests in males.
In about 30% of cases, there is no family history of this bleeding disorder and it
is just a spontaneous genetic mutation. About 1 in 5,000 males born in the
United States has hemophilia. All economic groups and races are affected
equally.
Hemophilia B: This person has low levels of or is completely missing factor 9
(Also called FIX or factor IX deficiency) 20% of people with hemophilia have
Type B Hemophilia. Factor IX deficiency usually manifests in males.
Hemophilia B was originally called "Christmas Disease" when it was first
diagnosed in 1952. About 30% of cases of Hemophilia B are caused by
spontaneous genetic mutation.
Hemophilia B is much less common than Hemophilia A. It occurs in about 1 in
25,000 male births, and affects about 3,300 individuals in the United States. All
races and economic groups are affected equally.
23
24
Figure 1241.
Hemophilia A can result from multiple alterations in the factor VIII gene.
These include gene rearrangements; missense mutations, in which a single base
substitution leads to an amino acid change in the molecule; nonsense
mutations, which result in a stop codon; abnormal splicing of the gene;
deletions of all or portions of the gene; and insertions of genetic elements. The
genetic defects leading to hemophilia have been reviewed.
25
Schematic of inversion and crossing over at intron 22. Inversion and crossingover of the a3 gene with its homologous sequence a1 nested within intron 22 are
shown. Middle panel: When crossing over of the a1 gene nested within intron 22
and the a3 gene extragenic to factor VIII occurs, a portion of the factor VIII gene
26
27
Figure 1244.
Examples of mutations and CG doublets. The red box denotes exon 26. A CT
transition results in a stop codon (TGA), whereas a GA transition results in
substitution of a glutamine for an arginine residue.
Large deletions in the factor VIII gene almost always are associated with
severe hemophilia. O n the other hand, a small deletion that does not change the
reading frame of the gene may result in milder disease. Patients with large
deletions who have no detectable factor VIII antigen are more susceptible to
the development of antifactor VIII antibodies, although antibodies clearly also
occur in patients without deletions.
28
29
Until the age of 2, bleeding into joints is uncommon. Most bleeds are surface
bruises. When babies are learning to walk, they fall frequently and suffer
many bumps and bruises.
Bleeding into the joints, soft tissues and muscles is seen more frequently after
the age of two.
What are the symptoms of hemophilia in an older child or adult?
Common symptoms of hemophilia are:
o
Bleeding into soft tissues and muscles (the ileopsoas muscle around the
hip, calf, forearm, upper arm, Achilles tendon, buttocks)
Surface bruising.
30
Repeated vomiting
What does a hemorrhage into a joint look like and feel like?
A hemorrhage into a joint, if untreated, goes on for days. This is what
happens.
The first sign is a feeling of tightness in the joint but no real pain. The joint
feels a little puffy to the touch.
31
As the hours pass, the joint becomes hot to the touch. Fully flexing or
extending the joint becomes painful. Weight bearing becomes difficult. By
this time, the joint is visibly swollen.
As the bleeding continues and the swelling increases, all movement in the
joint is lost. The joint becomes fixed in a slightly flexed position in an
attempt to relieve the interior pressure in the joint. The pain at this point can
be excruciating.
The bleeding slows after several days when the joint is so full of blood that
the pressure inside the joint cavity is equal to the pressure inside the broken
blood vessels. Slowly, the bleeding stops and the long process of absorbing
the blood in the joint cavity begins.
After several hemorrhages like this, the joint is permanently damaged.
6.
32
The platelet (a blood particle essential for the clotting process) count should be
measured as well as two indices of blood clotting, the prothrombin time (PT)
and activated partial thromboplastin time (aPTT). A normal platelet count,
normal PT, and a prolonged aPTT are characteristic of hemophilia A and
hemophilia B. Specific tests for the blood clotting factors can then be
performed to measure factor VII or factor IX levels and confirm the diagnosis.
Genetic testing to identify and characterize the specific mutations responsible
for hemophilia is also available in specialized laboratories.
Many people who have or have had family members with hemophilia will ask
that their baby boys get tested soon after birth. About one-third of babies who
are diagnosed with hemophilia have no other family members with the
disorder. A doctor might check for hemophilia if a newborn is showing certain
signs of hemophilia.
Diagnosis includes screening tests and clotting factor tests. Screening tests are
blood tests that show if the blood is clotting properly. Clotting facto r tests, also
called factor assays, are required to diagnose a bleeding disorder. This blood
test shows the type of hemophilia and the severity.
Families With a History of Hemophilia
Any family history of bleeding, such as following surgery or injury, or
unexplained deaths among brothers, sisters, or other male relatives such as
maternal uncles, grandfathers, or cousins should be discussed with a doctor to
see if hemophilia was a cause. A doctor often will get a thorough family
history to find out if a bleeding disorder exists in the family.
Many people who have or have had family members with hemophilia will ask
that their baby boys get tested soon after birth. In the best of cases, testing for
hemophilia is planned before the babys delivery so that a sa mple of blood can
be drawn from the umbilical cord (which connects the mother and baby before
birth) immediately after birth and tested to determine the level of the clotting
34
factors. Umbilical cord blood testing is better at finding low levels of factor
VIII (8) than it is at finding low levels of factor IX (9). This is because factor
IX (9) levels take more time to develop and are not at a normal level until a
baby is at least 6 months of age. Therefore, a mildly low level of factor IX (9)
at birth does not necessarily mean that the baby has hemophilia B. A repeat test
when the baby is older might be needed in some cases. Learn more about the
inheritance pattern for hemophilia.
Families With No Previous History of Hemophilia
About one-third of babies who are diagnosed with hemophilia have no other
family members with the disorder. A doctor might check for hemophilia in a
newborn if:
-
Bleeding goes on for a long time after drawing blood and heel sticks
(pricking the infants heel to draw blood for newborn screening tests).
Those with severe hemophilia can have serious bleeding prob lems right away.
Thus, they often are diagnosed during the first year of life. People with milder
forms of hemophilia might not be diagnosed until later in life.
Screening Tests
Screening tests are blood tests that show if the blood is clotting properly. Types
of screening tests:
Complete Blood Count (CBC)
This common test measures the amount of hemoglobin (the red pigment inside
red blood cells that carries oxygen), the size and number of red blood cells and
numbers of different types of white blood cells and platelets found in blood.
35
36
37
38
Gene therapy
Gene therapy is the use of genes as medicine. Gene therapy includes the
transfer of therapeutic genes or genes that have been fixed to specific cells of a
person to correct abnormalities in the person's genes. How to transfer the gene
can be done by using viruses that are no longer dangerous as gene transfer tools
(vector) and then injected to the patient's body and then do a repair genes in
target cells (in vivo gene transfer approach). Can also be done by using stem
cells (immature cells that would divide or develop into cells with different
functions) of the patients is by modifying the stem cells of the patient in the
laboratory and then genetically repaired on- implants- it back into the patient's
body (in vivo gene therapy approach)
Gene therapy in patients with hemophilia can be done in two types , namely the
approach of gene transfer and ex - vivo gene transfer approach to in- vivo . At
the approach of ex - vivo gene transfer to cultured cells isolated from patients
with hemophilia , genetically modified and then after the cells are able to
produce the necessary clotting factors put back into the patient's body. The
cells will then produce blood clotting factors in a sustainable manner .
The cells can be cultured skin fibroblasts, endothelial cells, keratinocyte,
hepatocytes, hematopoietic progenitor cells, and myoblasts. At the approach of
in- vivo gene transfer of genetic modification is done in- situ is in the patient's
body. Tools such as gene transfer vector is inserted into the patient's body
which will conduct the genetic modification on the desired network, which in
this case is a modification of the liver which is the producer of the blood
clotting factors in normal human primary.
Gene therapy in patients with hemophilia using gene vectors have been capable
of expressing factor VIII or factor IX. Retroviral vectors can be, lentiviral,
adenoviral,
adeno-associated
non-viral.
Intravenous
39
40
Step 5
Formulating Learning Objectives
1. Explain about Acquired Interference of Hemophilia!
2. Explain about Von Willebrand disease!
3. Explain how the pathofisiology of Hemophilia!
4. Explain about laboratory tests for Hemophilia!
5. Explain about gene mutation in Hemophilia!
6. Explain about treatment of Hemophilia!
7. Explain about complication of Hemophilia!
41
Step 6
Learning independence
--
42
Step 7
Sharing Result
1. Explain about Acquired Interference of Hemophilia!
Hemostatic disorders can conveniently be classified as either hereditary or
acquired . Alternatively, hemostatic disorders can be classified according to the
mechanism of the defect. Of the acquired disorders, the thrombocytopenias are
the most frequently encountered entities. Thrombocytopenias can result from
reduced production of platelets, excessive destruction caused by antibodies or
other consumptive processes, or pooling of platelets in the spleen, as in
hypersplenism.
43
Vitamin K Deficiency
FORMS AND DISTRIBUTION
The vitamin K family of chemical compounds has many members. Vitamin K 1
(2-methyl-3-phytyl-1,4-naphthoquinone) is the major form of the vitamin
found in plants. Animal tissue and bacteria produce menaquinones, a series of
vitamin K forms similar in structure to vitamin K 1 but with various lengths of
unsaturated polyprenyl groups at the 3 position.
NUTRITIONAL SOURCES
Vitamin K is an essential fat-soluble vitamin. The diet is the primary source of
vitamin K in humans. Leafy green vegetables in particular are a good source of
vitamin K, although vitamin K 1 is widely distributed in the normal human diet.
A contribution to adequate vitamin K intake in humans may be provided by the
vitamin K 2 synthesized by intestinal bacteria. The daily dietary requirement for
the vitamin has been estimated to be 100 to 200 g/day.
PHYSIOLOGY
Vitamin K is absorbed in the ileum. The presence of bile salts and normal fat
absorption are required for effective uptake. The storage pool of vitamin K is
modest. In the absence of a dietary source of the vitamin, this storage pool can
be exhausted within 1 week in an otherwise normal person. Such a deficiency
does not generally lead to clinical manifestations, because the vitamin K
synthesized by gut flora is available to provide suboptimal but adequate
synthesis of vitamin K-dependent proteins.
HEMORRHAGIC DISEASE OF THE NEWBORN
Hemorrhagic disease of the newborn caused by vitamin K deficiency deve lops
during the first week of life, usually between days 2 and 7 .Clinical
manifestations include bleeding in the skin or from mucosal surfaces,
circumcision, or venopuncture sites. Rarely, internal bleeding, including
retroperitoneal or intracranial hemorrhage, is the primary manifestation of
hemorrhagic disease of the newborn. These ominous complications are the
rationale for the use of vitamin K prophylaxis in neonates.
44
45
antigen, indicate that diet truly depleted of vitamin K can lead to mild vitamin
K deficiency, no evidence shows that an inadequate diet alone can have clinical
manifestations. Bacteria in the large intestine produce functional forms of
vitamin K. In the absence of dietary vitamin K, small amounts of vitamin K in
the large intestine are absorbed passively and prevent severe vitamin K
deficiency. In patients medicated with antibiotics that destroy the intestinal
flora, this vitamin K source is eliminated. A common setting of vitamin K
deficiency is the case of inadequate or minimal dietary intake treated
simultaneously with antibiotics . This form of vitamin K deficiency occurs
within 1 to 3 weeks after depletion of body stores of vitamin K.
Malabsorption syndromes are commonly associated with vitamin K deficiency.
Defects in the enterohepatic circulation due to biliary disease interfere with
absorption of fat-soluble vitamins in the ileum. Primary biliary cirrhosis,
cholestatic hepatitis, and other causes of cholestasis may lead to impaired
absorption of vitamin K. Intestinal malabsorption, as in sprue or regional
enteritis, also impairs vitamin K use. Older adults have evidence of mild
vitamin K deficiency, presumably because of intestinal malabsorption.
THERAPY FOR VITAMIN K DEFICIENCY
Vitamin K deficiency is treated by the administration of vitamin K 1 . The
preferred route of administration depends on the urgency for correcting the
bleeding tendency and on the risk of inducing local hematoma formation. For
severe or life-threatening bleeding,
fresh
46
47
vWD Type I is the most common type of the disorder and those that have it are
typically asymptomatic or may experience mild symptoms such as nosebleeds
although there may be severe symptoms in some cases. There are various
factors that affect the presentation and severity of symptoms of vWD such as
blood type. vWD is named after Erik Adolf von Willebrand, a Finnish
pediatrician who first described the disease in 1926.
Diagnosisa
When suspected, blood plasma of a patient needs to be investigated for
quantitative and qualitative deficiencies of vWF. This is achieved by
measuring the amount of vWF in a vWF antigen assay and the functionality of
vWF with a glycoprotein (GP)Ib binding assay, a collagen binding assay, or a
ristocetin cofactor activity (RiCof) or ristocetin induced platelet agglutination
(RIPA) assays. Factor VIII levels are also performed because factor VIII is
bound to vWF which protects the factor VIII from rapid breakdown within the
blood. Deficiency of vWF can therefore lead to a reduction in factor VIII
levels. Normal levels do not exclude all forms of vWD, particularly type 2
which may only be revealed by investigating platelet interaction with
subendothelium under flow (PAF), a highly specialized coagulation study not
routinely performed in most medical laboratories. A platelet aggregation assay
will show an abnormal response to ristocetin with normal responses to the
other agonists used. A platelet function assay (PFA) will give an abnormal
collagen/adrenaline closure time and in most cases (but not all) a normal
collagen/ADP time. Type 2N can only be diagnosed by performing a "factor
48
Other tests performed in any patient with bleeding problems are a complete
blood
count
(especially
platelet
counts),
APTT
(activated
partial
Prothrombin
Partial
Bleeding
Platelet
time
thromboplastin
time
count
Unaffected
Unaffected
time
Vitamin K
Prolonged
Normal or
deficiency or
mildly
warfarin
prolonged
Disseminated
Prolonged
Prolonged
Prolonged
Decreased
Unaffected
Prolonged or
Prolonged
Unaffected
intravascular
coagulation
Von Willebrand
disease
unaffected
Hemophilia
Unaffected
Prolonged
Unaffected
Unaffected
Aspirin
Unaffected
Unaffected
Prolonged
Unaffected
Thrombocytopenia
Unaffected
Unaffected
Prolonged
Decreased
Prolonged
Unaffected
Unaffected
Unaffected
Prolonged
Prolonged
Prolonged
Decreased
stage
49
Prothrombin
Partial
Bleeding
Platelet
time
thromboplastin
time
count
time
Uremia
Unaffected
Unaffected
Prolonged
Unaffected
Congenital
Prolonged
Prolonged
Prolonged
Unaffected
Factor V deficiency
Prolonged
Prolonged
Unaffected
Unaffected
Factor X deficiency
Prolonged
Prolonged
Unaffected
Unaffected
Unaffected
Unaffected
Prolonged
Unaffected
Unaffected
Unaffected
Prolonged
Decreased
afibrinogenemia
as seen in amyloid
purpura
Glanzmann's
thrombasthenia
Bernard-Soulier
syndrome
or
unaffected
Factor XII
Unaffected
Prolonged
Unaffected
Unaffected
Unaffected
Shortened
Unaffected
Unaffected
deficiency
C1INH deficiency
The testing for vWD can be influenced by laboratory procedures. There are
numerous variables in the testing procedure that may affect the validity of the
test results and may result in a missed or erroneous diagnos is. The chance of
procedural errors are typically greatest during the preanalytical phase (during
collecting storage and transportation of the specimen) especially when the
testing is contracted out to an outside facility and the specimen is frozen and
transported long distances.[2] Diagnostic errors are not uncommon, and there is
a varying rate of testing proficiency amongst laboratories with error rates
ranging from 7% to 22% in some studies to as high as 60% in cases of
misclassification of vWD sub-type. To increase the probability of a proper
50
Type 2
Type 2 vWD (20-30%) is a qualitative defect and the bleeding tendency can
vary between individuals. There are normal levels of vWF, but the multimers
51
The vWF is quantitatively normal but qualitatively defective. Its ability to bind
to the glycoprotein1 (GP1) receptor on the platelet membrane is diminished,
resulting in decreased platelet adhesiveness and aggregation and abnormally
low ristocetin cofactor activity. The ability of the defective von Willebrand
factors to coalesce and form large vWF multimers is also impaired, resulting in
decreased quantity of large vWF multimers. Only small multimer units are
detected in the circulation. Therefore von Willebrand disease type 2A is
characterized by qualitatively defective von Willebrand factor with decreased
ability to bind to platelet glycoprotein1(GP1) and decreased capability at
multimerization. Von Willebrand factor antigen assay is normal. Ristocetin cofactor activity is low and large vWF multimers are reduced or absent.
Type 2B
This is a "gain of function" defect. The ability of the qualitatively defective von
Willebrand factor to bind to glycoprotein1 (GP1) receptor on the platelet
membrane is abnormally enhanced, leading to its spontaneous binding to
platelets and subsequent rapid clearance of the bound platelets and of the large
vWF multimers. Thrombocytopenia may occur. Large vWF multimers are
reduced or absent from the circulation.
The ristocetin cofactor activity is low when the patient's platelet-poor plasma is
assayed against formalin- fixed, normal donor platelets. However, when the
assay is performed with the patient's own platelets (plate let-rich plasma), a
lower-than- normal amount of ristocetin causes aggregation to occur. This is
due to the large vWF multimers remaining bound to the patient's platelets.
Patients with this sub-type are unable to use desmopressin as a treatment for
bleeding, because it can lead to unwanted platelet aggregation and aggravation
of thrombocytopenia.
52
Type 2M
This is a deficiency of the binding of vWF to coagulation factor VIII. The vWF
antigen test is normal indicating normal quantity of vWF. the ristocetin
cofactor assay is normal. Assay for coagulation factor VIII revealed marked
quantitative decrease equivalent to levels seen in hemophilia A. This has led to
some vWD type 2N patients being misdiagnosed as having hemophilia A.
Type 3
Type 3 is the most severe form of von Willebrand disease (homozygous for the
defective gene) and is characterized by complete absence of production of
vWF. The von Willebrand factor is undetectable in the vWF antigen assay.
Since the von Willebrand factor protects coagulation Factor VIII from
proteolytic degradation, total absence of vWF leads to extremely low Factor
VIII level, equivalent to that seen in severe hemophilia A with its clinical
manifestations of life threatening external and internal hemorrhages. The
inheritance pattern of vWD type 3 is autosomal recessive while the inheritance
pattern of hemophilia A is x- linked recessive.
Platelet-type
(also known as pseudo- vWD or platelet-type vWD)
Platelet-type vWD is an autosomal dominant genetic defect of the platelets.
The von Willebrand factor is qualitatively normal and genetic testing of the
von Willebrand gene and vWF protein reveals no mutational alteration. The
defect lies in the qualtatively altered glycoprotein1 (GP1) receptor on the
platelet membrane which increases its affinity to bind to the von Willebrand
53
factor. Large platelet aggregates and high molecular weight vWF multimers are
removed from the circulation resulting in thrombocytopenia and diminished or
absent large vWF multimers. The ristocetin cofactor activity and loss of large
vWF multimers are similar to vWD type 2B.
Acquired von Willebrand disease
Acquired vWD can occur in patients with autoantibodies. In this case the
function of vWF is not inhibited but the vWF-antibody complex is rapidly
cleared from the circulation.
A form of vWD occurs in patients with aortic valve stenosis, leading to
gastrointestinal bleeding (Heyde's syndrome). This form of acquired vWD may
be more prevalent than is presently thought. In 2003 Vincentelli et al. noted
that patients with acquired vWD and aortic stenosis who underwent valve
replacement experienced a correction of their hemostatic abnormalities but that
the hemostatic abnormalities can recur after 6 months when the prosthetic
valve is a poor match with the patient. [7] Similarly, acquired vWD contributes
to the bleeding tendency in people with an implant of a Left Ventricular Assist
Device (LVAD), a pump that pumps blood from the left ventricle of the heart
into the aorta. Large multimers of vWF are destroyed by mechanical stress in
both conditions.
Thrombocythemia is another cause of acquired von Willebrand disease, due to
sequestration of von Willebrand factor via the adhesion of vast numbers of
platelets. Acquired vWD has also been described in the following disorders:
Wilms' tumour, hypothyroidism and mesenchymal dysplasias.
Pathophysiology
For the normal function of the coagulation factor, see von Willebrand factor.
vWF is mainly active in conditions of high blood flow and shear stress.
Deficiency of vWF therefore shows primarily in organs with extensive small
vessels, such as the skin, the gastrointestinal tract and the uterus. In
angiodysplasia, a form of telangiectasia of the colon, shear stress is much
54
Genetics
55
For patients with vWD scheduled for surgery and cases of vWD disease
complicated by clinically significant hemorrhage, human derived medium
purity Factor VIII concentrates, which also contain von Willebrand factors, are
available for prophylaxis and treatment. Humate P, Alphanate and Koate HP
are commercially available for prophylaxis and treatment of von Willebrand
disease. Monoclonally purified Factor VIII concentrates and reco mbinant
Factor VIII concentrates contain insignificant quantity of VWF and are
therefore not clinically useful.
Development of alloantibodies occur in 10-15% of patients receiving human
derived medium purity Factor VIII concentrates and the risk of allergic
reactions including anaphylaxis must be considered when administering these
preparations. Administration of the latters is also associated with increased risk
of venous thromboembolic complications.
Blood transfusions are given as needed to correct ane mia and hypotension
secondary to hypovolemia. Infusion of platelet concentrates is recommended
for correction of hemorrhage associated with platelet-type von Willebrand
disease. The antifibrinolytic agents Epsilon amino caproic acid and Tranexamic
acid are useful adjuncts in the management of vWD complicated by clinical
hemorrhage. The use Topical thrombin JMI and Topical Tisseel VH are
effective adjuncts for correction of hemorrhage from wounds.
57
hemophilia A represents 80% of all cases. Male subjects are clinically affected;
women, who carry a single mutated gene, are generally asymptomatic. Family
history of the disease is absent in 30% of cases and in these cases, 80% of the
mothers are carriers of the de novo mutated allele. More than 500 different
mutations have been identified in the F8 or F9 genes of patients with
hemophilia A or B, respectively. One of the most common hemophilia A
mutations results from an inversion of the intron 22 sequence, and it is present
in 40% of cases of severe hemophilia A. Advances in molecular diagnosis now
permit precise identification of mutations, allowing accurate diagnosis of
women carriers of the hemophilia gene in affected families.
Clinically, hemophilia A and hemophilia B are indistinguishable. The d isease
phenotype correlates with the residual activity of FVIII or FIX and can be
classified as severe (<1%), moderate (15%), or mild (630%). In the severe
and moderate forms, the disease is characterized by bleeding into the joints
(hemarthrosis), soft tissues, and muscles after minor trauma or even
spontaneously. Patients with mild disease experience infrequent bleeding that
is usually secondary to trauma. Among those with residual FVIII or FIX
activity >25% of normal, the disease is discovered only by bleeding after major
trauma or during routine presurgery laboratory tests. Typically, the global tests
of coagulation show only an isolated prolongation of the aPTT assay. Patients
with hemophilia have normal bleeding times and platelet counts. The diagnos is
is made after specific determination of FVIII or FIX clotting activity.
Early in life, bleeding may present after circumcision or rarely as intracranial
hemorrhages. The disease is more evident when children begin to walk or
crawl. In the severe form, the most common bleeding manifestations are the
recurrent hemarthroses, which can affect every joint but mainly affect knees,
elbows, ankles, shoulders, and hips. Acute hemarthroses are painful, and
clinical signs are local swelling and erythema. To avoid pain, the patient may
adopt a fixed position, which leads eventually to muscle contractures. Very
young children unable to communicate verbally show irritability and a lack of
movement of the affected joint. Chronic hemarthroses are debilitating, with
58
life threatening
and
requires
immediate therapy.
PT normal in hemophilia A, B
59
A rare subtype of VWD (type 2N) has isolated low FVIII activity with
normal VWF level and mimics hemophilia A
Genetic testing
o
Screening Tests
Screening tests are blood tests that show if the blood is clotting properly. Types
of screening tests:
60
Fibrinogen Test
This test also helps doctors assess a patients ability to form a blood clot. This
test is ordered either along with other blood clotting tests or when a patient has
an abnormal PT or APTT test result, or both. Fibrinogen is another name for
clotting factor I (1).
Clotting Factor Tests
Clotting factor tests, also called factor assays, are required to diagnose a
bleeding disorder. This blood test shows the type of hemophilia and the
severity. It is important to know the type and severity in order to create the best
treatment plan.
61
Severity
50% to 100%
Mild hemophilia
Moderate hemophilia
1% to 5%
Severe hemophilia
Less than 1%
62
long-distance
PCR
protocols:
(1)
high
concentrations
of
63
Schematic of the PCR assay. The four primers (P, Q, A, and B) are represented
by arrows and their positions are indicated. The upper box represents int22h1,
and the dashed lines indicate flanking sequences. The lower box represents
int22h2 andint22h3, and the wavy lines indicate the flanking sequences.
Deleterious inversions can occur by recombination betweenint22h1 and either
int22h2 or int22h3 (dotted lines). Amplified products in male patients with the
wild-type and the inverted factor VIII genes and a carrier female. PCR was
performed in 25 L with 250 ng of genomic DNA, a mixture containing 50
mmol/L Tris.HCl, pH 9.2, 2.25 mmol/L MgCl2, 7.5% DMSO, 16 mmol/L
(NH4)2SO4, 250 mmol/L each of dGTP and deaza-dGTP, 500 mmol/L of the
other dNTPs, and 3.3 U of Expand Long Template DNA polymerases
(Boehringer
Mannheim,
Mannheim,
Germany).
After 2
minutes of
64
ATT ACA CTG AT GAC ATT ATG CTG AC; Q = GGC CCT ACA ACC
ATT CTG CCT TTC ACT TTC AGT GCA ATA; A = CAC AAG GGG GAA
GAG TGT GAG GGT GTG GGA TAA GAA; B = CCC CAA ACT ATA
ACC AGC ACC TTG AAC TTA CCC TCT. Primer concentrations were 0.4,
0.4, 0.12, and 0.12 mol/L, respectively.
In conclusion, a PCR assay for factor VIII gene inversions has been developed
for patient screening, carrier testing, and prenatal diagnosis of severe
hemophilia A. The method is simple, rapid, reproducible, inexpensive,
nonisotopic, and amenable to automation. This approach may be helpful in
analyzing other inversions, deletions, and translocations in the genome
65
strong dry heating, and wet heating of blood products in the 1980s, the
occurrence of hemophilia patients contracting the deadliest of blood-borne
pathogens, Human Immunodeficiency Virus (HIV) and Hepatitis C, has not
been documented (Mannucci 2003). According to Ludlam et al. (2006),
detection methods such as nucleic-acid screening and incorporation of products
that reduce viral activity have made blood products safe from hepatitis B
(HBV), hepatitis C (HCV), HIV, and human T-cell lymphotropic virus
(HTLV). The current focus is on the use of reagents that are totally
independent of human plasma (Ludlam et al. 2006).
Recombinant Factors
Effectiveness
Recombinant clotting factors treatments deliver clotting factors that the
hemophilia patients are missing. For example, in the most common type of
hemophilia, hemophilia A, patients receive factor VIII replacements (rFVIII)
and in the second most common type of hemophilia, hemophilia B, patients
receive factor IX replacements (rFIX) (Meng et al. 2006). Recombinant factors
are derived from DNA and although albumin is used in synthetic steps, it is not
included in the final product. Newer recombinant factors have been produced
that use no human proteins in the synthetic or final stages of production. These
factors are thought to be safer in terms of viral transmission (Mannucci 2003).
However, recovery time for patients using rFIX has been shown to be slower
than those for plasma-derived factors (White et al. 1998).
Eighty percent of uncontrolled bleeds have been effectively eliminated with a
single dose of recombinant factors and subsequent use increases the success
rate to 90-95% (Mannucci 2003). Lusher et al. (1993) treated 95 moderate to
severe hemophiliac children with rFVIII for an average of 1.5 years with an
average of 34.9 infusions per individual. Injections of rFVIII were given in
response to excessive bleeds and prior to surgical/dental procedures. Lusher et
al. (1993) reported that all patients responded well to the treatment with minor
side effects experienced by 3 patients. Batorova and Martinowitz (2002)
suggest a different method of administering injections. They favor continuous
66
injection of clotting factor (at a rate that corresponds with its clearance from
the body), which prevents highs and lows in coagulating factor level and
promotes homeostasis, stopping large bleeds before they occur. This method
also reduces the overall amount of factor required for treatment. Clearance of
recombinant factors is noted to occur through the low-density lipoprotein
receptor and low-density lipoprotein receptor-related protein. Admission of
antagonists for these receptors may decrease recombinant factor clearance
(Saenko and Ananyeva 2006).
Activated Prothrombin Complex Concentrates
Efectiveness
Activated Prothrombin Complex Concentrates (aPCC) contain Factor Eight
Inhibitor Bypassing Activity (FEIBA) (Mannucci 2003) and have been used to
treat hemophilia patients with inhibitors for the past 30 years (Luu and
Ewenstein 2004). FEIBA works by activating the synthesis of thrombin by
stimulating prothrombinase (Turecek et al. 2004), thereby bypassing the
synthesis of factors IX and VIII (Sjamsoedin et al. 1981). FEIBA contains
precursor hormones to clotting factors FVII, FIX, FX, and prothrombin but
only contains trace amounts of the factors themselves. Therefore, no immune
response is seen with this product. Although FEIBA is synthesized from human
plasma, it undergoes a stringent, vapor- heated purification process and Turecek
et al (2004) suggest that there are no reported cases of viral transmission from
this product.
Negrier and coworkers (1997) reported on a study of FEIBA use in 60
hemophiliacs with inhibitors. The product was used in 433 bleeding events.
The study found that FEIBA was successful at stopping bleeds in 81% of
patients. Similarly, Sjamsoedin et al. (1981) report on a 64% success rate for
the 15 patients in their trial which lasted 15 months. Although these results are
promising, this rate is still lower than the effectiveness rate of multiple
recombinant factor infusions (Mannucci 2003). Further, Hayashi et al. (2004)
noticed that some patients experienced resistance to treatment over a prolonged
period of time.
67
68
reached immune tolerance through this method. High-dose factor treatment has
been successful in 70% of patients (Manno 2005).
Drugs that suppress the immune system are also effective at decreasing the
destructive response of the body towards circulating coagulating factor.
Rituximab is an anti-CD20 antibody that destroys existing B-cells which are
present in immune response (Wiestner et al. 2000). In a study (Stasi et al.
2004) of 10 patients with inhibitors to FVIII who received rituximab treatment,
8 patients were found to undergo remission. However, 3 patients later relapsed
and researchers noted that the most successful treatment option was a
combination of rituximab and other immunosuppressive agents.
Common immunosuppressive agents used to control inhibitors include
corticosteroids, cyclophosphamide, immunoglobulins (Ig) (Berezne et al.
2006), and prednisone (Yee et al. 2000). These drugs can be used alone or in
combination to reduce immune activity. Intravenous Ig in combination with
prednisone has been shown to raise platelet levels for a period of 2 to 6 weeks
with one infusion (Manno 2005). A combination of prednisone and
cyclophosphamide has been found to be an effective treatment option as well
(Yee et al. 2000). Shaffer and Phillips (1997) achieved remission of inhibitors
in the treatment of 9 patients with this method when the drugs were given daily
during
an
average
12
weeks
of
treatment.
Corticosteroids
and
Gene Therapy
Effectiveness
Treating hemophilia with gene therapy appears promising because the disease
is caused by a single gene defect and because only a small increase (5%) (Gan
et al. 2006) in gene product could essentially transform a severe form of
hemophilia into a mild one. Over activation of the gene up to 150% of its
action has also not caused any adverse effects (VandenDriessche et al. 2001).
Another advantage is that although clotting factors are made in the liver, they
69
70
Non-viral methods use naked DNA plasmids that not only include the gene of
interest but also include genes encoding for protein products that will
incorporate the target gene into the mammalian genome (Margaret et al. 2003).
Yant et al. (2000) successfully transferred genes for FIX into mice through the
non-viral method and achieved a greater than 5 month expression of clotting
factor. Cells that were genetically engineered to express clotting factor genes
can be administered to patients via grafting surgery or injections
(VandenDriessche et al. 2001).
From 2001 to 2003, 5 clinical trials involving human hemophilia subjects and
gene therapy were approved. In one of the trials, 13 patients with severe
hemophilia A were given intravenous retroviral vector with the FVIII gene.
However, the level of FVIII produced was only 1% of the desired level. In a
second trial, the FVIII gene was administered to 6 patients through the nonviral method. Although levels of clotting factor rose in 4 out of 6 patients,
levels of factor returned to pretreatment levels after a year of treatment. These
two studies show the possible limitations of gene therapy including gene
silencing and immunological response. Possible solutions to remedy these
problems include the following: RNA repair, which will assist in the packaging
of larger genes such as those for FVIII; genetically modified endothelial cells,
which may prove to be better target cell for gene transfer; and genetically
modified stem cells, which could potentially be stimulated to produce high
levels of clotting factors (Margaret et al. 2003). As of yet, there have been
limited human models that were able to produce continuous coagulating factor
expression. Further, human models were unable to produce the same degree of
coagulating factor production achieved in animal models (Mannucci 2003).
Though gene therapy is promising, it is currently not a viable option for mass
use among hemophilia patients.
71
JOINT DAMAGE
One of the major complications of hemophilia is joint damage or hemophilic
arthropathy that can occur when there is bleeding into joints. This is the most
common clinical complication of hemophilia. Bleeding into knees, elbows,
ankles, shoulders, and hips can lead to chronic swelling and later joint
deformity. Many people with severe hemophilia can suffer from painful,
debilitating joint bleeds and associated mobility issues that severely impede
their quality of life.
HIV/AIDS
In the late 1970s-and 80s people with hemophilia were treated with blood
products derived from thousands of donors. When the U.S. blood supply
became contaminated by HIV, the products used as treatment for thousands of
people with bleeding disorders also became contaminated. More than 50% of
the hemophilia population became infected with HIV prior to 1985. The
tremendous impact of HIV/AIDS on the hemophilia community is still with us.
So many families have lost loved ones. This devastation has placed an
emotional, health, ethical and financial burden on affected families and the
entire community as well.
72
HEPATITIS
Hepatitis viruses were also transmitted in blood products used by persons with
bleeding disorders. Todays blood products are much safer than those of the
past. As of 1997, there have been no reports of hepatitis C transmission
through clotting factor treated with newer processes.
There are six main hepatitis viruses, which cause problems ranging from mild
chronic infections to liver failure. Almost 95% of all hepatitis cases are
hepatitis A, B, or C. Some hepatitis viruses can be asymptomatic for many
years and may never become chronic. Others can progress to liver cancers, end
stage liver disease, and other life threatening conditions.
73
REFERENCE
74