1

Tutorial Report

Blok ―Hematoimunology‖

Scenario 2

Created by:

Group 10

Ade Marantika 1218011002

Alfianita Fadhilla 1218011010

Christhopher P.P.P 1218011030

Ellya Rahmawati 1218011044

Hani Zahiyyah 1218011063

Ika Agustin Putri H 1218011076

Istighfariza Shaqina 1218011084

M Nikhola Risol 1218011099

Melati Nurul Utami 1218011104

Putri Giani P 1218011117

Rizky Indria Lestari 1218011132

Zygawindi N 1218011167

Program Study Medical Education

Medical Faculty

Lampung University

2014

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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.

Wassalammu’alaikum wr.wb

Bandar Lampung, March 2014

Complier

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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

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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/μL (referral score 150.000-400.000/μL)

5

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.

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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?

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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.

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Platelets are experiencing will issue a stretcher agegresi 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 ?

9

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

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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.

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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 event—the 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.

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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.

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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

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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.

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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 of 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,

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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.

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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 hemophilia?

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

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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 patient may eventually experience:

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.

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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 hemophilia 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 hemorrhage (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 hemorrhage include:

A bad headache

Vomiting

Confusion

Fitting (Convulsion)

Loss of balance

Slurred speech, or other speaking difficulties

Stiff neck

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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

Von Willebrand disease

(VWD)

Family history is usually

positive, including both females

and males, due to an

autosomal dominant pattern of

inheritance.

Bleeding symptoms may be

similar to mild congenital

hemophilia, although patients

with von Willebrand disease

tend to have more mucosal

bleeding symptoms.

Diagnosis is based on various

tests, including von Willebrand

factor (VWF) antigen, VWF

activity (ristocetin cofactor or

collagen-binding assay), factor

VIII assay, and VWF multimers.

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 dysfunction

Bleeding pattern is typically

mucocutaneous and not

musculoskeletal as in

hemophilia.

Platelet aggregation studies

are the diagnostic test of

choice to diagnose most

platelet disorders. [31]

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

coagulation factors

(e.g., factor V, VII, X XI,

Musculoskeletal bleeding is

uncommon. Specific coagulation factor

assays are needed to establish

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Disease/Condition Differentiating

Signs/Symptoms Differentiating Tests

or fibrinogen) There have been cases of

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.

diagnosis.

Ehlers-Danlos

syndrome

Bleeding is primarily mucosal in

origin.

Musculoskeletal bleeding is

uncommon.

Skin hyperextensibility, joint

laxity present.

Diagnosis based on clinical

findings, along with genetic

testing and/or tissue biopsy.

Scurvy

Bleeding is primarily mucosal in

origin.

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.

Fabry disease

Bleeding is primarily mucosal in

origin.

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.

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Disease/Condition Differentiating

Signs/Symptoms Differentiating Tests

Child abuse

Inconsistent history of how

trauma occurred is typical.

Physical exam may show

injuries in various healing

stages or with an obvious

pattern, bruises, inflicted

burns, fractures.

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.

Disseminated

intravascular

coagulation

No differentiating

signs/symptoms to acquired

hemophilia.

Underlying causal condition

(e.g., acute promyelocytic

leukemia) is present.

Unlike in acquired hemophilia,

platelet count is decreased.

Absence of factor VIII auto-

antibodies.

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.

23

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.

24

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 124–1 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.

25

Figure 124–1.

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; XhY, 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.

26

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. 124–3). Figure 124–3 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

"inversion–crossing 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 124–3.

Schematic of inversion and crossing over at intron 22. Inversion and crossing-

over 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

27

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 "inversion–crossing over" mechanism.

(From Antonarakis SE, Kazazian HH, Tuddenham EG: Molecular 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. 124–4). 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.

28

Figure 124–4.

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. On 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.

29

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.

30

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)

o Bleeding into soft tissues and muscles (the ileopsoas muscle around the

hip, calf, forearm, upper arm, Achilles tendon, buttocks)

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

(especially in children)

o Blood in the urine (hematuria)

o 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.

31

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

o Repeated vomiting

o Sleepiness or a change in normal behaviour

o Sudden weakness or clumsiness of an arm or leg

o Stiffness of the neck or complaints of pain with neck movement

o Complaints of seeing double

o The development of crossed eyes

o Poor balance when walking, a lack of coordination

o 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.

32

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

33

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.

34

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 baby’s delivery so that a sample 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

35

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 infant’s 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.

36

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 patient’s 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

37

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 non-

steroidal anti- inflammatory drugs should be avoided because it may interfere

with hemostasis.

38

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 Malmö 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.

39

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

40

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.

41

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!

42

Step 6

Learning independence

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43

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.

44

Vitamin K Deficiency

FORMS AND DISTRIBUTION

The vitamin K family of chemical compounds has many members. Vitamin K1

(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 K1 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 K1 is widely distributed in the normal human diet.

A contribution to adequate vitamin K intake in humans may be provided by the

vitamin K2 synthesized by intestinal bacteria. The daily dietary requirement for

the vitamin has been estimated to be 100 to 200 µg/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.

45

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 µg/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-γ-carboxy) prothrombin (PIVKA-II) antigen

level is indicative of vitamin K deficiency, because this form of prothrombin

appears only when post-translational γ-carboxylation is impaired, but not when

protein synthesis is impaired. Administration of vitamin K (100 µg) 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 µg 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-γ-carboxy) prothrombin

46

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 K1. 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

47

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

administration of vitamin K1 should be avoided. Because the delivery of

vitamin K by the subcutaneous route is variable, intravenous vitamin K1

(Aquamephyton, 10 to 15 mg) is the recommended approach because it ensures

rapid delivery. However, intravenous vitamin K1 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 blood-

clotting 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.

48

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

49

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

time

Partial

thromboplastin

time

Bleeding

time

Platelet

count

Vitamin K

deficiency or

warfarin

Prolonged Normal or

mildly

prolonged

Unaffected Unaffected

Disseminated

intravascular

coagulation

Prolonged Prolonged Prolonged Decreased

Von Willebrand

disease

Unaffected Prolonged or

unaffected

Prolonged 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-

stage

Prolonged Prolonged Prolonged Decreased

50

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

Condition Prothrombin

time

Partial

thromboplastin

time

Bleeding

time

Platelet

count

Uremia Unaffected Unaffected Prolonged Unaffected

Congenital

afibrinogenemia

Prolonged Prolonged Prolonged Unaffected

Factor V deficiency Prolonged Prolonged Unaffected Unaffected

Factor X deficiency

as seen in amyloid

purpura

Prolonged Prolonged Unaffected Unaffected

Glanzmann's

thrombasthenia

Unaffected Unaffected Prolonged Unaffected

Bernard-Soulier

syndrome

Unaffected Unaffected Prolonged Decreased

or

unaffected

Factor XII

deficiency

Unaffected Prolonged Unaffected Unaffected

C1INH deficiency Unaffected Shortened Unaffected Unaffected

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 diagnosis. 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

51

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

52

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 co-

factor 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.

53

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

54

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

55

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

group–specific 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.

56

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 the

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.

57

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 anemia 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;

58

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 (1–5%), or mild (6–30%). 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

59

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

o PTT – prolonged in moderate and severe hemophilia

May not be prolonged in mild cases or in female carriers

60

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

o 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

o 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

o Confirm the causative F8 or F9 mutation in affected individuals

o 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:

61

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 patient’s 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.

62

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 labor-

intensive. 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.

63

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

64

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

for 30 seconds, and 68°C 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

65

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 μmol/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, re-

injection 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,

66

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

67

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.

68

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 (Mauser-

Bunschoten 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

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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 non-

viral 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.

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

surrounding the safety of the nation’s 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. Today’s 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. Harrison’s 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, 5th Ed.

Philadelphia : Elsevier Inc.

4. Williams. Hematology, 7th Ed. McCraw-Hill Medical