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Tutorial Report

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

Melati Nurul Utami

1218011104

Ellya Rahmawati

1218011044

Putri Giani P

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Hani Zahiyyah

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Rizky Indria Lestari 1218011132

Ika Agustin Putri H

1218011076

Zygawindi N

1218011167

Program Study Medical Education


Medical Faculty
Lampung University
2014

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

Bandar Lampung, March 2014

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

1. Explain about Hemostasis and interferences of Hemostasis!


Hemostasis is the cessation of bleeding events as the body's reaction to the
injury. A balanced hemostasis mechanism occurs due to the interaction of four
factors:
1. Vascular factors.
2. Platelet factor.
3. Coagulation Factors.
4. Fibrinolysis factors.

The function of the hemostasis process are :


1. Prevent the escape of blood from blood vessels intact.
It depends on :
a. The blood vessel integrity.
b. Normal platelet function.
2. Stop bleeding from injured blood vessels. The process that occurs
after an injury is :
a. Vasoconstriction of blood vessels.
b. Platelet plug formation.
c. The process of blood clotting.

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.

2. What is diagnosis and differential diagnosis in cases ?


Diagnosis for Bimo is suspect Hemophilia. This can be seen from clinical
manifestations and laboratory result, aPTT is elongated. Clinical manifestations
that happens to Bimo are bleeding is difficult to stop, often get knee swelling,
and got bruise when he get minor trauma. And also have family history, His
uncle have similar condition. To get certain diagnosis, Bimo must have the
other laboratory test, including PTT.
Blood test - if a doctor suspects a child may have hemophilia a blood test can
determine whether the patient has hemophilia A or B, and how severe it is.
Blood tests can be performed from the time of birth onwards.

3. What is classification of Hemophilia ?

4. Explain how the pathogenesis and pathofisiology of Hemophilia ?


One of the most common hemophilia A mutations results from an inversion of
the intron 22 sequence,which is present in 40% of cases of severe hemophilia
A. The defect is an absence or low level of plasma factor VIII. Approximately
half of the patients havemissense or frameshift mutations or deletions in the
factor VIII gene. This mutation leads to a severe clinical form of haemophilia
A.

In many cases of hemophilia, there is no family history of the disease, and at


least 30 percent of the cases of hemophilia are a result of spontaneous (de
novo) mutations.

5. Clinical manifestations of Hemophilia ?


Signs and symptoms of hemophilia vary depending on how deficient you are in
clot- forming proteins called clotting factors. If levels of your deficient clotting
factor are very low, you may experience spontaneous bleeding. If levels of
your deficient clotting factor are slightly to moderately low, you may bleed
only after surgery or trauma.

Signs and symptoms of spontaneous bleeding may include:


1. Many large or deep bruises
2. Joint pain and swelling caused by internal bleeding
3. Unexplained and excessive bleeding or bruising
4. Blood in your urine or stool
5. Prolonged bleeding from cuts or injuries or after surgery or tooth
extraction
6. Nosebleeds without a known cause
7. Tightness in your joints
8. In infants, unexplained irritability
9. Unusual bleeding after immunizations

6. Explain how to diagnosis of Hemophilia ?


1. History: Symptoms (time), diet, medical history and family history of
hemophilia.
2.

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:
-

Blood edge: at the beginning of the normal (hemoglobin, leukocytes,


platelets)

Bleeding time normal

Clotting time extends

Normal platelet

Prothrombin Time (PT) and thromboplastin time (TT) Normal

Plasma thromboplastin time (PTT) or APTT elongated, or can not be


elongated in mild hemophilia

Diagnosis must: reduced levels of clotting factors


- In Hemophilia A will experience a lack of factor VIII
- Hemophilia B will experience a deficiency of factor IX.

7. Why the bleeding in cases difficult to stop?


The bleeding difficult to stop because there is interference with the hemostasis.
From the case we can know that the patient is suspect hemofilia. We know that
hemofilia is an X- linked recessive hemorrhagic disease due to mutations in the
F8 gene (hemophilia A or classic hemophilia) or F9 gene (hemophilia B).
Hemofilia commonly suffered in male but carrier in female.

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.

8. How to do therapy of Hemophilia?


It has to be comprehensive covering the provision of replacement factor VIII
for hemophilia A and F IX for hemophilia B, treatment and rehabilitation,
especially when there are joints, education and psychosocial support for
patients and their families.

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

1. Explain about Hemostasis and interference of Hemostasis!


Freezing Process Of Blood
1. Intrinsic pathway:
In this path all the materials necessary for blood clotting processes
contained within the blood stream. These materials typically circulate in an
inactive precursor form (inactive), and some of them are proensim and
cofactors.
2. Extrinsik Pathway:
In this pathway necessary ingredients derived from vascular tissue
injured/damaged (tissue factor/ tissue thromboplastin).

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

molecular weight kininogen, Ca2 + and platelet

phospholipids. These trails form factor Xa (active).

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.

Another important interaction between extrinsic and intrinsic trajectory is that


tissue factor complex with factor VIIa to activate factor IX also the intrinsic
trajectory. Actually, the formation of a complex between tissue factor and
factor VIIa is now seen as an important process involved in initiating blood
coagulation in vivo. Physiological meaning of the early stages of intrinsic

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

thrombocytopenia, thrombotic thrombocytopenia


b. Purpura disseminated intravascular
c. Koagulasi,
3) Abnormal platelet distribution,

15

4) Loss due to dilution, in the form of massive blood transfusion in


patients with bleeding store.
3. Coagulation disorders
Could be due to hereditary or acquired, which generally interfere with
coagulation factors.
a. Herediter: hemophilia A and hemophilia B
b. Acquired: vitamin K deficiency and liver disease
4. Impaired platelet function
Divided into two types, namely:
a. Acquired
1) due to anti-platelet drugs such as aspirin,
2) hiperglobulinemia,
3) Myeloproliferative and Myelodysplastic disorders, and
4) Uremia.
b. Herediter
1) Trombastenia,
2) Bernard Soulier Syndrom,
3) Storage Disease

2. What is diagnosis and differential diagnosis in cases ?


How is hemophilia diagnosed?
The majority of patients with hemophilia have a known family history of the
condition. However, about one-third of cases occur in the absence of a known
family history. Most of these cases without a family history arise due to a
spontaneous mutation in the affected gene. Other cases may be due to the
affected gene being passed through a long line of female carriers.
If there is no known family history of hemophilia, a series of blood tests can
identify which part or protein factor of the blood clotting mechanism is
defective if an individual has abnormal bleeding episodes.

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:

Above 5% - mild hemophilia

17

1% to 5% - moderate hemophilia

Less than 1% - severe 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:

Tingling sensation in the joint

Pain in the joint

Irritation in the joint

If left untreated, the patie nt may eventually expe rience:

More severe pain in the joint

Joint stiffness

The affected area becomes swollen, tender and hot

Joint bleeds most commonly affect the:

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:

Several large or deep bruises

Joint pain or swelling

Unexplained bleeding or bruising

Blood in feces (stools)

Blood in urine

Unexplained nosebleeds

Unexplained gum bleeding

Tightness in the joints

Intracranial he morrhage (bleeding inside the skull)


About 1 in every 30 patients with hemophilia will have intracranial
hemorrhage at least once during their lives. This should be treated as a medical
emergency. Spontaneous intracranial hemorrhage is rare and in many cases
bleeding inside the skull will be the result of a blow to the head.
Symptoms of intracranial he morrhage include:

A bad headache

Vomiting

Confusion

Fitting (Convulsion)

Loss of balance

Slurred speech, or other speaking difficulties

Stiff neck
19

Vision problems

Loss of coordination

Some of the facial muscles do not work (sometimes all of them)

Differential Diagnosis
Disease/Condition

Differentiating
Signs/Symptoms

Differentiating Tests

Diagnosis is based on various


tests, including von Willebrand
Family history is usually
factor (VWF) antigen, VWF
positive, including both females
activity (ristocetin cofactor or
and males, due to an
collagen-binding assay), factor
autosomal dominant pattern of
VIII assay, and VWF multimers.
inheritance.
Von Willebrand disease
Bleeding symptoms may be
(VWD)
similar to mild congenital
hemophilia, although patients
with von Willebrand disease
tend to have more mucosal
bleeding symptoms.

Most clinicians agree that VWF


levels <30 international
units/dL are consistent with a
diagnosis of von Willebrand
disease. [28] However,
repeated testing is often
needed to confirm the
diagnosis.
Platelet aggregation studies
are the diagnostic test of
choice to diagnose most
platelet disorders. [31]

Platelet dysfunction

Bleeding pattern is typically


mucocutaneous and not
musculoskeletal as in
hemophilia.

Specific platelet agonists (ADP,


epinephrine, collagen,
ristocetin, and arachidonic
acid) are used to assess
platelet aggregation by
measuring optical density.
Platelet electron microscopy
can also be used to evaluate
platelet ultrastructure.

Deficiency of other
Musculoskeletal bleeding is
coagulation factors
uncommon.
(e.g., factor V, VII, X XI,

Specific coagulation factor


assays are needed to establish

20

Disease/Condition
or fibrinogen)

Differentiating
Signs/Symptoms

Differentiating Tests

There have been cases of


diagnosis.
thrombosis reported in people
with fibrinogen or factor VII
deficiency.
Combined factor V and VIII
deficiency may be mistaken for
mild hemophilia A, but should
be suspected when the PT is
prolonged, and/or there is
parental consanguinity.
Bleeding is primarily mucosal in
origin.

Ehlers-Danlos
syndrome

Musculoskeletal bleeding is
uncommon.

Diagnosis based on clinical


findings, along with genetic
testing and/or tissue biopsy.

Skin hyperextensibility, joint


laxity present.
Bleeding is primarily mucosal in
origin.

Scurvy

Musculoskeletal bleeding is
uncommon.
History of a restricted diet,
sepsis, HIV, critical illness, or
pancreatitis may be present.

Diagnosis based on clinical


findings, along with a reduced
serum vitamin C level.

Bleeding is primarily mucosal in


origin.

Fabry disease

Musculoskeletal bleeding is
uncommon.
Typical skin lesions
(angiokeratomas), pain in the
extremities, renal and heart
disease, typical ocular signs.

Diagnosis based on clinical


findings, along with genetic
testing.

21

Disease/Condition

Differentiating
Signs/Symptoms

Inconsistent history of how


trauma occurred is typical.
Child abuse

Disseminated
intravascular
coagulation

Physical exam may show


injuries in various healing
stages or with an obvious
pattern, bruises, inflicted
burns, fractures.

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.

Unlike in acquired hemophilia,


platelet count is decreased.
Absence of factor VIII autoantibodies.

3. What is classification of Hemophilia ?


Hemophilia may be classified as severe, moderate or mild. This is based on the
levels of Factor VIII or Factor IX depending on the type of hemophilia. The
clinical presentation is also an indication of the severity of hemophilia.

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

Hemophilia C: This person has low levels of or is missing completely factor 11


(Also called FXI or factor XI deficiency) Hemophilia C is 10 times more rare
than type A. Factor XI deficiency is different because it can show up in both
males and females.
Von Willebrands Disease: A bleeding disease similar to Hemophilia that
affects both males and females equally. It is caused by a deficiency of a blood
clotting protein called Von Willebrand factor. Von Willebrand factor circulates
attached to factor VIII and is necessary to form a clot.
Von Willebrands Disease occurs in 1 - 2% of the population. It is a genetic
bleeding disorder that can be inherited from either parent, unlike hemophilia. It
affects males and females equally.
Von Willebrand Disease can be difficult to diagnose. A blood clotting test can
be performed to measure the amount and characteristics of von Willebrand
Factor. Because levels can vary, sometimes a blood clotting test may need to
be repeated. A person who might have von Willebrand Disease should be
referred to a hematologist who specializes in diagnosing and treating bleeding
disorders.

4. Explain how the pathogenesis and pathofisiology of Hemophilia ?


Hemophilia A results when mutations occur in the factor VIII gene located on
the long arm of the X-chromosome (X-q28). The disease occurs almost
exclusively in males. Figure 1241 shows the inheritance pattern of hemophilia
A and hemophilia B. All the sons of affected hemophilic males are normal,
whereas all the daughters are obligatory carriers of the factor VIII defect. Sons
of carriers have a 50 percent chance of being affected, whereas daughters of
carriers have a 50 percent chance of being carriers themselves.

24

Figure 1241.

Inheritance pattern of hemophilia A. All daughters of a hemophilic male are


carriers of hemophilia, whereas all sons are normal. Daughters of carriers have a
50% chance of being a carrier, whereas sons of carriers have a 50% chance of
having hemophilia. (X, normal; Xh , abnormal X chromosome with the
hemophilic gene; Xh Y, hemophilic male; XX, normal female; XXh , carrier
female; XY, normal male; Y, normal.)

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

One of the most common mutations, accounting for 40 to 50 percent of


patients, is a unique "combined gene inversion and crossing over" that disrupts
the factor VIII gene. The mechanism is homologous recombination between
the F8A sequence that lies within intron 22 and one of the homologous
extragenic sequences of the F8A gene 5' to the factor VIII gene. During
meiosis, crossing over of homologous sequences occurs between the F8A gene
lying within intron 22 and one of the extragenic homologous F8A sequences 5'
to intron 22. Thus, the transcription of the complete factor VIII sequence is
interrupted (Fig. 1243). Figure 1243 shows a common inversion and
crossing over but homologous recombinations can occur with either of the
extragenic genes. Occasionally, there are duplications of a2 or a3 genes 5' to the
intron 22 a1 gene such that there are four possible types of inversion. The
"inversioncrossing over" mutations result in severe hemophilia, and
approximately 50 percent of these patients are susceptible to developing
antibody inhibitors that neutralize factor VIII coagulant function.
Figure 1243.

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

is transcribed in a reverse manner from exon 1 through exon 22. Homologous


recombination with the extragenic a2 gene is also possible. In some individuals
there are two a2 or a3 extragenic sequences giving rise to four possible types of
the "inversioncrossing over" mechanism.
(From Antonarakis SE, Kazazian HH, Tuddenham EG: Mo lecular etiology of
factor VIII deficiency in hemophilia A. Hum Mutat 5:1, 1995, with permission.)
In many cases of hemophilia, there is no family history of the disease, and at
least 30 percent of the cases of hemophilia are a result of spontaneous (de
novo) mutations. Because the restriction fragment enzyme TaqI recognizes the
sequence TCGA, CpG mutations at this site can be directly detected by loss of
a TaqI cleavage site. Codons for the amino acid arginine (CGA) are frequently
affected by mutations at CG doublets. A CT transition often results in a stop
codon (Fig. 1244). A stop codon results in synthesis of a truncated factor VIII
molecule and usually is associated with severe hemophilia. However, as shown
in the figure, a GA transition results in a missense mutation, which often
leads to a dysfunctional factor VIII molecule that may be associated with mild,
moderate, or severe hemophilia. Some missense mutations result in the
production of normal or near-normal amounts of factor VIII antigen, while the
coagulant activity may be dramatically or only slightly reduced. Many other
single-base substitutions have been described, resulting in hemophilia of
varying degrees of severity.

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

5. Clinical manifestations of Hemophilia ?


Emergency signs and symptoms of hemophilia may include:
1. Sudden pain, swelling, and warmth of large joints, such as knees, elbows,
hips and shoulders, and of the muscles of your arms and legs
2. Bleeding from an injury, especially if you have a severe form of
hemophilia
3. Painful, lasting headache
4. Repeated vomiting
5. Extreme fatigue
6. Neck pain
7. Double vision
8. Babies with hemophilia
At first, because of limited mobility, a baby with hemophilia usually won't
have many problems related to hemophilia. But as your baby begins to move
around, falling and bumping into things, superficial bruises may occur. This
bleeding into soft tissue may become more frequent the more active your
child becomes.
The symptoms of hemophilia
What are the first signs of hemophilia in a young child?
What are the symptoms of hemophilia in an older child or adult?
Are symptoms less severe as children get older?
What causes the bleeding?
What are the symptoms of bleeding into the brain?
What other kinds of bleeding are serious?
What does a hemorrhage into a joint look like and feel like?
What are the first signs of hemophilia in a young child?
Babies have sharp teeth and bite their gums and tongue, often causing
bleeding. This and bruises from falls are usually the first signs of hemophilia.

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 joints (knees, elbows, ankles, shoulders, hips, wrists in


descending order of frequency)

Bleeding into soft tissues and muscles (the ileopsoas muscle around the
hip, calf, forearm, upper arm, Achilles tendon, buttocks)

Bleeding in the mouth from a cut, bitten tongue or loss of a tooth


(especially in children)

Blood in the urine (hematuria)

Surface bruising.

Are symptoms less severe as children get older?


Yes, in many children, symptoms become less severe as children move into
adolescence and young adulthood. The reason for this is not that their
hemophilia is any less serious. Factor VIII and IX levels remain constant
throughout life. However, hemophiliacs learn to avoid some of the situations
that lead to hemorrhages.
What causes the bleeding?
Bleeding is often caused by minor injury - a bump or a slight twist of a joint.
However, many hemorrhages, especially among severe hemophiliacs, happen
for no apparent reason. This is even truer in joints that have bled often in the
past. The more a joint has bled, the easier it bleeds again with no external
cause.
Even hemorrhages in the brain often have no apparent cause. Brain
hemorrhages are the leading cause of death from bleeding in hemophilia.

30

Therefore it is important to recognize the symptoms of a brain hemorrhage


very quickly.
What are the symptoms of bleeding into the brain?
Some of the following symptoms may occur in a person with bleeding in the
brain:
o

Persistent or increasing headache

Repeated vomiting

Sleepiness or a change in normal behaviour

Sudden weakness or clumsiness of an arm or leg

Stiffness of the neck or complaints of pain with neck movement

Complaints of seeing double

The development of crossed eyes

Poor balance when walking, a lack of coordination

Convulsions or seizures (fits).

What other kinds of bleeding are serious?


Any bleeding in a vital area is serious. Important examples are:
1. Bleeding in the neck, throat or tongue (this could block the airway)
2. Bleeding in the ileopsoas muscle across the front of the hip (this could
pinch important nerves to the leg)
3. Bleeding in the forearm or calf (this could pinch important nerves to the
hand or foot)
4. Bleeding in joints, especially knees, ankles and elbows (repeated bleeds
in joints can lead to loss of range of motion, muscle loss, and
destruction of the joints themselves).

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.

Explain how to diagnosis of Hemophilia ?


Accurate diagnosis is essential for the optimal management of hemophilia.
Testing for hemophilia should be performed at a highly experienced
specialized coagulation laboratory. Laboratories that do not frequently perform
these specialized tests may not be able to accurately establish a diagnosis.
Most people with hemophilia are diagnosed at an early age. However, those
with mild hemophilia may not be diagnosed until adulthood when they
experience a bleeding episode due to trauma or surgery.
Hemophilia is diagnosed with blood tests to determine if clotting factors are
missing or at low levels, and which ones are causing the problem. If you have a
family history of hemophilia, it is important that your doctors know the clotting
factor your relatives are missing. You will probably be missing the same one.
If you know you are a carrier of hemophilia, the testing for hemophilia in your
newborn usually occurs soon after birth. These tests can be run on blood
obtained from the umbilical cord or drawn from the newborn's vein. You may

32

be advised to delay some procedures, such as circumcision, until after you


learn whether your child has hemophilia.
Some families with a history of hemophilia may want to request prenatal
testing, or testing before birth. This testing can be done early in pregnancy,
allowing your family to make informed decisions and preparations. UCSF has
genetic counselors who are available to help you with prenatal testing, if
desired.
If you are pregnant and think you could be a carrier, or if you have a child
diagnosed with hemophilia and are expecting another child, it is important to
tell your obstetrician.
There are three ways to determine if you are a carrier:
Family tree If you have a son with hemophilia and have another son,
brother, father, uncle, cousin or grandfather with the disorder, then you are a
carrier. No additional tests are needed.
Clotting factor If the clotting factor level in your blood is below 50 percent
of normal, you are probably a carrier and have mild hemophilia. If the clotting
factor level is above 50 percent, you still may be a carrier, since other
conditions can elevate the factor level. Other tests may be necessary.
DNA test A DNA test can look for the mutation that caused hemophilia in
your son or another relative, and compare it to your DNA.
The majority of patients with hemophilia have a known family history of the
condition. However, about one-third of cases occur in the absence of a known
family history. Most of these cases without a family history arise due to a
spontaneous mutation in the affected gene. Other cases may be due to the
affected gene being passed through a long line of female carriers.
If there is no known family history of hemophilia, a series of blood tests can
identify which part or protein factor of the blood clotting mechanism is
defective if an individual has abnormal bleeding episodes.
33

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 after circumcision of the penis goes on for a long time.

Bleeding goes on for a long time after drawing blood and heel sticks
(pricking the infants heel to draw blood for newborn screening tests).

Bleeding in the head (scalp or brain) after a difficult delivery or after


using special devices or instruments to help deliver the baby (e.g.,
vacuum or forceps).

Unusual raised bruises or large numbers of bruises. If a child is not


diagnosed with hemophilia during the newborn period, the family might
notice unusual bruising once the child begins standing or crawling.

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

The CBC is normal in people with hemophilia. However, if a person with


hemophilia has unusually heavy bleeding or bleeds for a long time, the
hemoglobin and the red blood cell count can be low.
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 clo t. The results
of this test will show a longer clotting time among people with hemophilia A or
B.
Prothrombin Time (PT) Test
This test also measures the time it takes for blood to clot. It measures primarily
the clotting ability of factors I (1), II (2), V (5), VII (7), and X (10). If any of
these factors are too low, it takes longer than normal for the blood to clot. The
results of this test will be normal among most people with hemophilia A and B.
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.

7. Why the bleeding in cases difficult to stop?


Question has been answered at step 3

36

8. How to do therapy of Hemophilia?


Management of hemophiliacs have to be comprehensive covering the provision
of replacement factor VIII for hemophilia A and F IX for hemophilia B,
treatment and rehabilitation, especially when there are joints, education and
psychosocial support for patients and their families.
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.
For hemophilia A F VIII concentrate given at a dose of 0.5 x weight (kg) x
desired levels (%). F VIII given every 12 hours while the F IX given every 24
hours for hemophilia B.
F VIII or IX levels are desirable depending on the location where the bleeding
for bleeding joints, muscles, mouth and nasal mucosa levels of 30-50% is
required. Gastrointestinal bleeding, urinary tract, retroperitoneal area and the
central nervous system as well as trauma and surgery recommended levels of
60-100%.

Duration of administration depends on the severity of bleeding or type of


action. For dental, or epistaxis, given for 2-5 days, whereas extensive surgery
or laceration given 7-14 days. For rehabilitation as the hemarthrosis can be
given any longer.
Cryoprecipitate can also be given to hemophilia A in which a bag of
cryoprecipitate contains about 80 UF VIII. Likewise, the antifibrinolytic drugs
such as epsilon amino-caproic acid or tranexamic acid. Aspirin and nonsteroidal anti- inflammatory drugs should be avoided because it may interfere
with hemostasis.

37

F VIII or IX prophylaxis can be given to patients with severe hemophilia with


the goal of reducing the incidence of disability hemarthroses and joints. WHO
and WFH recommends primary prophylaxis starting at age 1-2 years and
continue throughout life. Prophylaxis is given based on the Malm protocol
was first developed in Sweden, namely the provision of F VIII 20-40 U/kg
every other day at least 3 days per week or F IX 20-40 U/kg twice per week.

For mild and moderate hemophiliacs, desmopressin (1 - deamino - 8 - arginine


vasopressin, DDAVP) an anolog vasopressin can be used to increase the
endogenous levels of F VIII in the circulation , but is not recommended for
severe hemophilia. Mechanism of action is still not clear, the alleged drug induced stimulation of vWF from the mistress (Weibel - Palade bodies) that
stabilize F VIII in plasma. DDAVP can be administered intravenously,
subcutaneously or intranasally
Hemophiliacs are encouraged to exercise regularly, wear appropriate protective
equipment for sports, avoid strenuous exercise or physical contact. Weight loss
should be maintained, especially if there are abnormalities in the joints due to
excess weight aggravate arthritis. Mouth and dental hygiene should also be
considered. Vaccination is given as a normal child, especially against hepatitis
A and B vaccine administered through subcutaneous, not intramuscular. The
school should be notified if a child is suffering from hemophilia that can help
patients when needed.
Efforts to determine the nature of haemophilia carrier status and genetic
counseling is integrated in the management of hemophilia. Genetic counseling
should be given to the patient and family. Counseling includes hemophilia
disease itself, treatment and prognosis, patterns of descent, the detection of the
nature and implications for the future of the patient and the nature of the
carrier. Detection of haemophilia in the fetus can be done especially when the
type of gene mutation already known. Samples can be obtained through
chorionic villus sampling or actions amnionsintesis.

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

viral (AAV) and

non-viral.

Intravenous

administration of gene vectors capable of expressing factor VIII or factor IX 3


cheaper but can trigger an immune response thereby blocking the next

39

administration if more than one injection is needed to achieve therapeutic


levels of factor VIII or factor IX.
In the ex vivo approach to gene therapy, cell implants modified by using the
tools that can be retroviral vectors and non-viral vectors. Unlike the in vivo
approach to gene therapy, recurrent implantation of cells ex vivo therapy does
not trigger an antibody response. However, the cost is relative ly more
expensive when compared with in vivo gene therapy approach.

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

Almost all neonates are vitamin K deficient, presumably as a result of deficient


vitamin K nutriture in the pregnant mother during the third trimester and
because of the lack of colonization of the colon by bacteria that produce
vitamin K in the neonate. However, this deficiency is further aggravated in
some patients by inadequate dietary intake of vitamin K. This disorder is more
prevalent in breast-fed babies, because human milk, in contrast to cow's milk,
contains only 15 g/L of vitamin K.
Neonates with hemorrhagic disease of the newborn have a prolonged
prothrombin time (PT) and partial thromboplastin time (PTT). However, it is
critical to determine whether the prolongation of these times is a manifestation
of the deficiency of the vitamin K-dependent proteins due to vitamin K
deficiency or to decreased synthetic capacity of the liver in newborns.
Elevation of the abnormal (des--carboxy) prothrombin (PIVKA-II) antigen
level is indicative of vitamin K deficiency, because this form of prothrombin
appears only when post-translational -carboxylation is impaired, but not when
protein synthesis is impaired. Administration of vitamin K (100 g) corrects
the deficiency state and usually does not need to be repeated in the otherwise
healthy infant.
Prophylactic vitamin K has been in use for in- hospital births for the past 40
years. Vitamin K (100 g to 1 mg) is administered intramuscularly to the
newborn immediately after birth. At these doses, vitamin K administration
carries little morbidity and can prevent hemorrhagic disease of the newborn.
Some of these vitamin K protocols are under revision and have been updated.
ACQUIRED VITAMIN K DEFICIENCY
Dietary Deficiency States and Antibiotics
The requirement for vitamin K is sufficiently low relative to the vitamin K
content of a normal diet, and clinically significant vitamin K deficiency does
not occur as a result of inadequate dietary intake. Although sensitive markers
of vitamin K deficiency, such as abnormal (des--carboxy) prothrombin

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

frozen plasma should be

administered. Because of the risk of transmission of viral infection, the use of


blood products must be weighed carefully. There is no role for available
concentrates of the vitamin K-dependent proteins because of the risk of
transmission of viral disease .
The approach to the treatment of vitamin K deficiency depends on the clinical
setting and the severity of bleeding. Except in the face of serious internal
bleeding, reversal of the vitamin K deficiency by the administration of vitamin
K is generally adequate. If the PT is significantly prolonged to indicate that a

46

bleeding complication may be induced by intramuscular injection, that route of


administration of vitamin K 1 should be avoided. Because the delivery of
vitamin K by the subcutaneous route is variable, intravenous vitamin K 1
(Aquamephyton, 10 to 15 mg) is the recommended approach because it ensures
rapid delivery. However, intravenous vitamin K 1 does require monitoring
because of early reports of severe allergic reactions with the intravenous route
of administration; care must be given to initiate rapid reversal of an untoward
reaction. With vitamin K, the PT should return toward the normal range within
12 hours and should have corrected within 24 to 48 hours. Serious bleeding
complications attributed to vitamin K deficiency, such as intracranial bleeding,
must be reversed immediately. Despite the rapid action of vitamin K,
administration of vitamin K should be preceded by the infusion of fresh frozen
plasma. This blood component contains all the vitamin K-dependent bloodclotting proteins. In sufficient quantities, fresh frozen plasma can correct or
nearly correct the PT and the bleeding tendency.
Patients with vitamin K deficiency without bleeding manifestations can be
treated with oral vitamin K or, as in patients with chronic vitamin K deficiency
resulting from malabsorption syndromes, with subcutaneous vitamin K.

2. Explain about Von Willebrand disease!


Von Willebrand disease (vWD) is the most common hereditary coagulation
abnormality described in humans, although it can also be acquired as a result of
other medical conditions. It arises from a qualitative or quantitative deficiency
of von Willebrand factor (vWF), a multimeric protein that is required for
platelet adhesion. It is known to affect humans and dogs (notably Doberman
Pinschers), and rarely swine, cattle, horses, and cats. There are three forms of
vWD: inherited, acquired, and pseudo or platelet type. There are three types of
hereditary vWD: vWD Type I, vWD Type II, and vWD III. Within the three
inherited types of vWD there are various subtypes. Platelet type vWD is also
an inherited condition.

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.

Signs and symptoms


The various types of vWD present with varying degrees of bleeding tendency,
usually in the form of easy bruising, nosebleeds and bleeding gums. Women
may experience heavy menstrual periods and blood loss during childbirth.
Severe internal or joint bleeding is uncommon (which mostly occurs in type 3
vWD).

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

VIII binding" assay. Detection of vWD is complicated by vWF being an acute


phase reactant with levels rising in infection, pregnancy and stress.

Other tests performed in any patient with bleeding problems are a complete
blood

count

(especially

platelet

counts),

APTT

(activated

partial

thromboplastin time), prothrombin time, thrombin time and fibrinogen level.


Testing for factor IX may also be performed if hemophilia B is suspected.
Other coagulation factor assays may be performed depending on the results of
a coagulation screen. Patients with von Willebrand disease will typically
display a normal prothrombin time and a variable prolongation of partial
thromboplastin time.

Laboratory findings in various platelet and coagulation disorders (V - T)


Condition

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

Liver failure, early

Prolonged

Unaffected

Unaffected

Unaffected

Liver failure, end-

Prolonged

Prolonged

Prolonged

Decreased

stage

49

Laboratory findings in various platelet and coagulation disorders (V - T)


Condition

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

diagnosis testing should be done at a facility with immediate on-site processing


in their own specialized coagulation laboratory.
Classification and types
Classification
The four hereditary types of vWD described are type 1, type 2, type 3, and
pseudo or platelet-type. Most cases are hereditary, but acquired forms of vWD
have been described. The International Society on Thrombosis and
Haemostasis's (ISTH) classification depends on the definition of qualitative
and quantitative defects.
Type 1
Type 1 vWD (60-80% of all vWD cases) is a quantitative defect which is
heterozygous for the defective gene. The production of von Willebrand factor
vWF is decreased. Decreased levels of vWF are detected at 10-45% of normal,
i.e. 10-45 IU.
Many patients are asymptomatic or may have mild symptoms and not have
clearly impaired clotting which might suggest a bleeding disorder. Often the
discovery of vWD occurs incidentally to other medical procedures requiring a
blood work-up. Most cases of Type 1 vWD are never diagnosed due to the
asymptomatic or mild presentation of Type I and most people usually end up
leading a normal life free of complications with many being unaware that they
have the disorder.
Trouble may however arise in some patients in the form of bleeding following
surgery (including dental procedures), noticeable easy bruising, or menorrhagia
(heavy menstrual periods). There are also a minority of cases of Type 1 which
may present with severe hemorrhagic symptoms.

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

are structurally abnormal, or subgroups of large or small multimers are absent.


Four subtypes exist: 2A, 2B, 2M and 2N.
Type 2A

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

Type 2M von willebrand disease is a qualitative defect of von Willebrand


factor characterized by its decreased ability to bind to glycoprotein1 (GP1)
receptor on the platelet membrane and normal capability at multimerization.
The vWF antigen levels are normal. The ristocetin cofactor activity is
decreased and high molecular weight large vWF multimers are present in the
circulation.
Type 2N (Normandy)

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

higher than in average capillaries, and the risk of bleeding is increased


concomitantly.
In more severe cases of type 1 vWD, genetic changes are common within the
vWF gene and are highly penetrant. In milder cases of type 1 vWD there may
be a complex spectrum of molecular pathology in addition to polymorphisms
of the vWF gene alone. The individual's ABO blood group can influence
presentation and pathology of vWD. Those individuals with blood group O
have a lower mean level than individuals with other blood groups. Unless ABO
groupspecific vWF:antigen reference ranges are used, normal group O
individuals can be diagnosed as type I vWD, and some individuals of blood
group AB with a genetic defect of vWF may have the diagnosis overlooked
because vWF levels are elevated due to blood group.

Genetics

von Willebrand disease types I and II are inherited in


an autosomal dominant pattern.

von Willebrand disease type III (and sometimes II)


is inherited in an autosomal recessive pattern.

55

The vWF gene is located on chromosome twelve (12p13.2). It has 52 exons


spanning 178kbp. Types 1 and 2 are inherited as autosomal dominant traits and
type 3 is inherited as autosomal recessive. Occasionally type 2 also inherits
recessively.
Epidemiology
The prevalence of vWD is about 1 in 100 individuals. However the majority of
these people do not have symptoms. The prevalence of clinically significant
cases is 1 per 10,000. Because most forms are rather mild, they are detected
more often in women, whose bleeding tendency shows during menstruation. It
may be more severe or apparent in people with blood type O.
Therapy
For patients with vWD type 1 and vWD type 2A, desmopressin (DDAVP) is
recommended for use in cases of minor trauma, or in preparation for dental or
minor surgical procedures. DDAVP stimulates the release of von Willebrand
factor (vWF) from the Weibel Palade bodies of endothelial cells, thereby
increasing the levels of vWF (as well as coagulant factor VIII) 3 to 5-fold.
DDAVP is available as a preparation for intranasal administration (Stimate)
and as a preparation for intravenous administration.
DDAVP is contraindicated in vWD type 2b because of the risk of aggravated
thrombocytopenia and thrombotic complications.
DDAVP is probably not effective in vWD type 2M and is rarely effective in
vWD type 2N. It is totally ineffective in vWD type 3.
For women with heavy menstrual bleeding, estrogen-containing oral
contraceptive medications are effective in reducing the frequency and duration
of the menstrual periods. Estrogen compounds available for use in t he
correction of menorrhagia are Ethinyl Estradiol and Levonorgestel (levona,
Nordette, Lutera, Trivora). Administration of Ethinyl Estradiol diminishes the
secretion of luteinizing hormone and follicle stimulating hormone from the
pituitary, leading to stabilization of the endometrial surface of the uterus.
56

Desmopressin (DDAVP) is a synthetic analog of the natural antidiuretic


hormone vasopressin. Overuse of Desmopressin (DDAVP) can lead to water
retention and dilutional hyponatremia with consequent convulsion.

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.

3. Explain how the pathofisiology of Hemophilia!


Hemophilia is an X-linked recessive hemorrhagic disease due to mutations in
the F8 gene (hemophilia A or classic hemophilia) or F9 gene (hemophilia B).
The disease affects 1 in 10,000 males worldwide, in all ethnic groups;

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

synovial thickening and synovitis in response to the intraarticular blood. After


a joint has been damaged, recurrent bleeding episodes result in the clinically
recognized "target joint," which then establishes a vicious cycle of bleeding,
resulting in progressive joint deformity that in critical cases requires surgery as
the only therapeutic option. Hematomas into the muscle of distal parts of the
limbs may lead to external compression of arteries, veins, or nerves that can
evolve to a compartment syndrome.
Bleeding into the oropharyngeal spaces, central nervous system (CNS), or the
retroperitoneum is

life threatening

and

requires

immediate therapy.

Retroperitoneal hemorrhages can accumulate large quantities of blood with


formation of masses with calcification and inflammatory tissue reaction
(pseudotumor syndrome) and also result in damage to the femoral nerve.
Pseudotumors can also form in bones, especially long bones of the lower limbs.
Hematuria is frequent among hemophilia patients, even in the absence of
genitourinary pathology. It is often self- limited and may not require specific
therapy

4. Explain about laboratory tests for Hemophilia!


Indications for Testing

Spontaneous or prolonged bleeding suggestive of coagulation disorder

Family history of hemophilia

Acute or recent onset bleeding accompanied by prolonged partial


thromboplastin time (PTT)

Initial Laboratory Testing for Coagulation Disorders

CBC with platelet count normal in hemophilia A, B

Prothrombin time (PT)/PTT


o

PT normal in hemophilia A, B

PTT prolonged in moderate and severe hemophilia

May not be prolonged in mild cases or in female carriers

59

Prolonged PTT that corrects in a mixing study suggests factor


deficiency

PTT that does not correct with mixing study suggests an


inhibitor

Incubated mixing studies are often necessary to identify FVIII


inhibitors

Thrombin clotting time and plasma concentration of fibrinogen normal


in hemophilia A and B

Laboratory Testing for Hemophilia

FVIII activity level decreased in hemophilia A

FIX activity level decreased in hemophilia B


o

Not reliable for carrier status detection in females

Measurement in neonate may need to be repeated when family history


of mild disease exists

von Willebrand factor (VWF) level normal


o

Because VWF is a carrier for FVIII, von Willebrand disease (VWD)


should be ruled out in patients with decreased FVIII levels

A rare subtype of VWD (type 2N) has isolated low FVIII activity with
normal VWF level and mimics hemophilia A

Specialized coagulation or genetic testing can be used to


distinguish these disorders

Genetic testing
o

Patient risk should be calculated by a clinical geneticist using


laboratory results and family history

Confirm the causative F8 or F9 mutation in affected individuals

Determine carrier status in at-risk females

Screening Tests
Screening tests are blood tests that show if the blood is clotting properly. Types
of screening tests:

60

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. The CBC is normal in people with hemophilia. However, if a
person with hemophilia has unusually heavy bleeding or bleeds for a long
time, the hemoglobin and the red blood cell count can be low.

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.

Prothrombin Time (PT) Test


This test also measures the time it takes for blood to clot. It measures primarily
the clotting ability of factors I (1), II (2), V (5), VII (7), and X (10). If any of
these factors are too low, it takes longer than normal for the blood to clot. The
results of this test will be normal among most people with hemophilia A and B.

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

Levels of Factor VIII (8)


or IX (9) in the blood

Normal (person who does not have


hemophilia)

50% to 100%

Mild hemophilia

Greater than 5% but less than 50%

Moderate hemophilia

1% to 5%

Severe hemophilia

Less than 1%

5. Explain about gene mutation in Hemophilia!


Mutation Hemophilia A
Hemophilia A is the most common X-linked coagulation disorder, with an
incidence of about one in 5,000 males. About half the families with severe
disease have a large genomic inversion of the factor VIII gene that separates
the first 22 exons from the final 4 exons. This inversion results from a hotspot
of recombination between a 9.5-kb region in intron 22 (int22h1) and either of
two extragenic, distal homologs, int22h2 and int22h3; int22h2 andint22h3 are
more than 99% identical to one another. Recombination produces an inversion
because the extragenic homologs are in the opposite orientation relative
toint22h1. 1,2
The inversions are detected by Southern blotting, which is slow and laborintensive. A rapid and inexpensive test is of particular clinical utility, because
carrier testing is often paid out-of-pocket due to insurance issues and
confidentiality; a low-cost test may facilitate more optimal use of genetic
services. This difficult 9.5-kb region previously has been refractory to
polymerase chain reaction (PCR) amplification, presumably due to the
presence of a 3.5-kb GC island, which includes a 1-kb segment with a GC
content of 79%. In addition, an optimal PCR-based assay requires more
genomic sequence flanking the homologs.

62

We have developed a single-tube PCR assay that combines overlapping PCR3


with long-distance PCR4 to achieve the genetic diagnosis of inversions causing
hemophilia A (Fig1). The inversion was detected by performing PCR directly
from genomic DNA with four primers that differentiate the wild-type,
inversion, and carrier. Two primers, P and Q, are located within the factor VIII
gene at positions 1212 bp and +1334 bp flanking int22h1. Two pr imers, A
and B, are located at 167 bp and +118 bp flanking int22h2 and
int22h3.Segments PQ (12 kb) and AB (10 kb) are produced in hemizygous
wild-type males. Males with hemophilia due to the inversion produce PB (11
kb) and AQ segments (11 kb) along with the 10-kb AB segment from the
nonrecombined extragenic homolog. Female carriers produce PQ, PB + AQ,
and AB segments. In all cases, an AB segment serves as an internal control
because at least one copy of int22h2 or int22h3remains intact. The three
segment sizes are readily separated on a 0.6% agarose gel. High yield and
reproducible amplification depended on three unusual modifications to
standard

long-distance

PCR

protocols:

(1)

high

concentrations

of

dimethylsulfoxide (DMSO) additive; (2) substantially increased amounts of


Taq andPwo DNA polymerases; and (3) 50% deaza-dGTP

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

denaturation at 94C, 30 cycles were performed at 94C for 12 seconds, 65C


for 30 seconds, and 68C for 12 minutes for 10 cycles, (the remaining 20
cycles were performed for 12 minutes with 20 more seconds for each
additional cycle). The primers used are as follows: P = GCC CTG CCTG TCC

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

6. Explain about treatment of Hemophilia!


Treatment Options For Patients Without Inhibitors
Effectiveness Of Plasma- Derived Products
Transfusion of blood, plasma-derived products, or even tissues with healthy
cells has been a treatment option for hemophilia patients since the 1970s
(Farrugia 2002). The coagulating factors in whole blood and in plasma
fractions were used as replacements for any absent or dysfunctional factors in
hemophiliacs (Ludlam et al. 2006). Plasma products currently available are
fresh frozen plasma, freeze-dried concentrates, and cryoprecipitate (slowly
thawed plasma precipitate) (Ingram 1976). Ingram (1976) notes that blood
transfusions were the earliest most successful treatment of hemophilia.
Transfusions with healthy blood not only provide the patient with missing
clotting factors necessary for coagulation, but also replenish blood volume
depleted during excessive bleeds. Although therapeutic benefits are p resent, reinjection with product is necessary as live tissue cells and cell products must be
replenished over time.
The success of the treatment lies in the methods used to cleanse blood and
plasma products from pathogens. After the adoption of moderate dry heating,

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.

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Recombinant Factor VIIa


Effectiveness
Activated recombinant factors VII (rFVIIa), or Novoseven, function by binding
directly or in conjunction with other clotting factors to platelets at the site of
trauma (Monroe et al. 1997). rFVIIa may activate thrombin and other factors
downstream of FVIII and FIX, thereby bypassing the immunoactivating step in
coagulation. In a trial (Shaffer and Phillips 1997) of 67 bleeds, an 85%
effectiveness rate has been reported with this drug in patients with inhibitors. A
larger-scale study conducted by Key et al. (1998) reports on a 92% success rate
in the 614 bleeding episodes that were treated with this method.
rFVIIa has been shown to effectively stop bleeding episodes with one dose as
opposed to treatment with FEIBA (Joshi et al. 2006). Re-bleeds after infusion
were associated with 50% of patients in the Key et al study. However, patients
using rFVIIa have been shown to be resistant to treatment when the product is
used over a prolonged period of time (Hayashi et al. 2004).
A Cochrane review on which treatment method arrests bleeding in people with
hemophilia A with inhibitors has shown that there have not been appropriate
trials to clarify the relative effectiveness of recombinant factor VIIa
concentrate compared to human plasma-derived concentrates (Hind 2004).

Immune Tolerance And Immunosuppressive Treatments


Effectiveness
Another method of combating inhibitors is to decrease the body's immune
response to coagulating factor treatment. One approach is to make the immune
system tolerant with either high-dose (Mannucci 2003) or low-dose (MauserBunschoten et al. 1995) factor treatments. Mauser-Bunschoten et al. (1995)
injected 25 U/kg of FVIII every other day for a period of 0.5 to 28 months into
patients found to produce inhibitors against the factor. Patients were said to be
immune tolerant when half the injected FVIII remained in the patients' blood
while retaining its coagulating activity and the inhibitor concentration
decreased below a set value. Twenty-one out of the 24 patients in this study

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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

cyclophosphamide are also a suitable combination for effective inhibitor


reduction (Holme et al. 2005).

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

can be synthesized in a wide variety of cells (High 2006). With continuous


supply of gene product, gene therapy could potentially cure hemophilia
(VandenDriessche et al. 2001). Gene therapy has been successful in greatly
improving, if not curing, hemophilia in dogs (Chuah et al. 2001) and in mice
with knock-out mutations for the coagulating factor genes.
There are generally two approaches to gene integration into cells. Genes can be
integrated into highly reproducing stem cells so that all the daughter cells
express the gene and its product. The other approach is to integrate genes into
long- living cells such as skeletal muscle, cardiac muscle, and central nervous
system cells that will be present in the body long enough to continuously
express the target genes (High 2006). Cell types that have been considered for
gene integration include fibroblasts, epithelial cells, endothelial cells, and bone
marrow cells. Target cells may be removed from a patient, harvested in a
culture medium, engineered to express a target gene, and then implanted back
into the body (Mannucci 2003).
The procedures by which genes are delivered into the target cells are divided
into two categories: viral- mediated and non-viral- mediated gene transfer.
Generally, viral vector- mediated transfers have been found to be more efficient
than non- viral- mediated transfers. Viral vectors are created by removing
genetic material from viruses and replacing that material with the genetic
material of the gene of interest. The machinery that incorporates genetic
material into the host genome is already present in the virus. Viral vector
transfers employed in coagulating factor integration have included the use of
lentiviral (such as HIV), retroviral, and adenoviral vectors (VandenDriessche et
al. 2001). The use of HIV- mediated transfer of FVIII has been successfully
demonstrated in knock-out mice such that therapeutic levels of the clotting
factor were produced (Kootstra et al. 2003). Adeno-associated viral vectors
were used to transmit FIX into humans with a 10% restoration of clotting
factor function in one hemophilia B patient (Ponder 2006).

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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.

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7. Explain about complication of Hemophilia!


INHIBITORS
In some patients with hemophilia, the immune system produces an antibody
that inhibits the action of replacement blood products and prevents clot
formation. This antibody is known as an inhibitor. The presence of an inhibitor
makes the treatment of bleeding episodes more difficult. An inhibitor destroys
the clotting factor before it has a chance to stop the bleeding. The reason
inhibitors develop is uncertain; however, they occur more frequently in people
with severe forms of hemophilia, particularly factor VIII deficiency, because of
their need for more frequent infusions. Inhibitors tend to develop within the
first one to three years of treatment, typically between the 50th and 100th
exposure days.

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

The tragedy of the HIV/AIDS crisis gave rise to heightened vigilance


surrounding the safety of the nations blood supply and blood products. HIV
transmission by factor concentrates in the United States has not occurred since
1986 due to viral inactivation methods used in manufacturing blood products.
While new screening methods and product processing procedures are now in
place, continued improvements and steadfast oversight are needed to ensure
that this tragedy is not repeated.

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.

Transmission of hepatitis A remains a risk for people with bleeding disorders


who use plasma-derived products. This is because hepatitis A virus can resist
the viral inactivation methods used to manufacture plasma products. Hepatitis
A is preventable. MASAC recommends all patients with bleed ing disorders
receive a hepatitis A and B vaccination. Currently, there is no vaccination for
hepatitis C.

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REFERENCE

1. Anthony S. Fauci, 2008. Harrisons Internal Medicine, 17th Edition, USA,


McGraw Hill
2. Hoffbrand, A.V. 2006. Essensial Haematology, 5th Ed. United Kingdom :
Blackwell Publishing Ltd.
3. Hoffman, R. 2009. Hematology : Basic Principles and Practices, 5 th Ed.
Philadelphia : Elsevier Inc.
4. Williams. Hematology, 7th Ed. McCraw-Hill Medical

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