Blood & Lymphumanitoba.ca/faculties/medicine/units/pediatrics/... · Beta Thalassemia Thalassemia...

Notes compiled for Pediatrics

Blood & Lymph (Med I, Block 6, BL)

Contents

Class number Class Name Type Department Instructor

BL 008 Globin Gene Disorders T7 BC/HE Dr. B Triggs-Raine

BL 015 Pediatric Anaemia A PD/HE Dr. R A Yanofsky

BL 020 Coagulation A HE Dr. D Houston

BL 021 Platelet Production and Thrombocytopenia

L HE Dr. S Israels

BL 023 Platelet Function L HE Dr. S Israels

BL 046 Pediatric Oncology I L PD/HE Dr. R A Yanofsky

BL 047 Pediatric Oncology II T7 PD/HE Dr. R A Yanofsky

BL 052 Coagulation Testing T7 BC/HE Dr. D Houston / Dr. B Triggs-Raine

BL 053 Hemoglobinopathies L BC/HE Dr. D Houston

BL008 (T7) Genetic Testing: University of Manitoba Hemoglobinopathies Faculty of Medicine

Med II / BL008Instructor: Beth Spriggs

2008/2009

Objectives:

• Describe the globin gene loci and identify the common inherited abnormalities involving

them.

• Apply the principles of molecular genetics to the clinical diagnosis of sickle cell anemia

and thalassemias.

• Understand the approach to screening for defects in Hb

Required Reading:

Hoffbrand, Moss and Pettit (5th ed) pp. 72-93

Additional resources:

http://www.ctfphc.org/Full_Text/Ch20full.htm

http://www.emedicine.com/PED/topic2229.htm

Introduction:

Genetic disorders of the synthesis or function of hemoglobin (Hb), collectively known as

the hemoglobinopathies, comprise the largest group of single gene disorders in the world. The

world-wide incidence of severe cases of disease affecting normal Hb is approximately 300,000

per year. The high prevalence of these disorders is driven by the survival advantage that these

mutations confer against red cell parasitism by malaria.

Hemoglobinopathies can be divided into two general classes: 1) Disorders resulting from

reduced levels of Hb and referred to as thalassemias, and 2) Disorders resulting from altered

polypeptide structure of Hb and referred to as structural variants. Not only are they common,

but these disorders can have severe clinical manifestations. Some thalassemic patients suffer

from life-long transfusion-dependent anemia, while patients with sickle cell disease suffer

frequent pain and dysfunction of multiple organs.

Hemoglobinopathies are model disorders for genetic diagnostics. As the most abundant

protein in the red cell, abnormalities in hemoglobin produce morphological changes in the cells.

Hemoglobin protein is easily obtained in large amounts, and a crude lysate of red cells contains

relatively pure haemoglobin, facilitating its analysis at the protein level by techniques such as

electrophoresis. This is complemented by gene-based diagnostic approaches, which can

distinguish exact mutations and are invaluable in many instances for prenatal diagnosis.

Structure and Expression of the Globin Genes

Adult Hb, a tetramer of two α- and two β-globin chains, binds, carries and delivers

oxygen throughout the body. Understanding the complex organization and expression of the

globin gene family is important to understanding the pathogenesis and diagnosis of

hemoglobinopathies. The β-globin family is a cluster of five genes (ε- γG- γA- δ- β) on human

chromosome 11 and the α-globin gene family is a cluster of 4 genes (ζ2- ζ1- α2- α1) on human

chromosome 16. The order of the gene family members reflects their order of expression (see

below) with the first genes being expressed earliest in development.

The α globin family members, α2 and α1, are expressed early in development, and are

needed to form Hb both before and after birth. In contrast, the γ-globin genes account for most

beta-globin-like chains pre-birth, with the β-globin gene taking over production of most beta



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globin chains at about 3-6 months of age. As a result, severe α-globin gene mutations cause

problems before birth while mutations involving the β-globin gene do not become significant

until 3-6 months after birth. The major forms of normal Hb at different stages of development

are shown in the table below. Abnormal forms of Hb that are present when α-globin chains are

deficient include HbH (β4) and HbBarts (γ4).

Embryo Fetus Adult

ζ2ε2 α2γ2 α2β2 HbA (~97%)

ζ2γ2 α2δ2 HbA2 (2-3.5%)

α2ε2 α2γ2 HbF (<1%)

Thalassemias

Thalassemias result from reduced levels of the α- or β-globin chains. Clinically, the

classification of the disease is based on the severity of the condition, and genetically, it is based

on the number of mutant genes that are present.

Most mutations of α-globin genes are complete gene deletions and therefore do not result in

the synthesis of any α-globin chains. The mutant genes are represented as ‘-‘ in the following

table.

Gene

Status

Genetic

Classification

Clinical

Classification

Comments

αα/αα Normal Normal

-α/αα Silent carrier Normal DNA based detection

-α/-α α-thalassemia trait Thalassemia

minor↓MCV ↓MCH. Confirmed

using DNA based testing

--/αα α-thalassemia trait Thalassemia

minor↓MCV ↓MCH. Confirmed

using DNA based testing

--/-α α+ Thalassemia HbH Disease,

Thalassemia

intermedia or

major

Hb electrophoresis/ DNA

analysis

--/-- αo Thalassemia Hydrops fetalis Hb Barts present

A number of α-globin gene deletions have been described at the molecular level. The

most common single α-globin gene deletion, described as – α3.7 or as 3.7-kb rightward deletion,

is found in individuals of African, Mediterranean, Arabian, East Indian, and Southeast Asian

descent. Common two α-globin gene deletions include --SEA, --THAI, --FIL and these are

predominantly found in individuals originally from Southeast Asia. A small number of mutations

leading to α-thalassemia are not α-globin gene deletions but rather point mutations in the α-

globin gene that leads to reduced production of α-globin chains.

Beta-Thalassemia Classification



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The range of mutations that are present in the β-globin gene complicate the diagnosis of

this condition and prediction of the clinical consequences. In the table below, βo refers to a gene

that makes no β-globin chains and β+ refers to a gene that makes reduced amounts of β-globin

chain.

Gene

Status

Genetic

Classification

Clinical

Classification

Comments

β /β Normal Normal

β /βo Carrier Thalassemia minor Hematological/ Hb

electrophoresis/ DNA

analysis

β /β+ Carrier Thalassemia minor Hematological/ Hb

electrophoresis/DNA

analysis

βo/β+ Beta-Thalassemia Thalassemia

intermedia or major


analysis

β+/β+ Beta Thalassemia Thalassemia minor

to major


analysis

βo/βo Beta Thalassemia Thalassemia major Hb electrophoresis/ DNA

analysis

Hemoglobin E is a beta-chain variant with decreased protein synthesis, which therefore appears

clinically like β+ thalassemia. There have been more than 200 mutations in the β-globin gene

leading to beta-thalassemia, but in discrete populations only one or a few alleles comprise the

great majority of affected individuals.

Structural Abnormalities in Hb

There are many structural abnormalities in Hb, the best known being sickle cell disease.

This mutation in the β-globin gene, like several some other structural variants in Hb and like

thalassemia trait, confers resistance to malaria and thus has an elevated incidence in the areas

with a high frequency of malaria.

Approach to Screening

The frequency of sickle cell anemia is highest in Africa, and present at a lower frequency

in the Mediterranean region, in the Middle East and in India. Alpha and beta thalassemias are

found primarily in individuals of African, Mediterranean, Arabian, East Indian and Southeast

Asian descent.

1. RBC indices, including MCV (mean corpuscular volume), MCH (mean corpuscular

hemoglobin)

Carriers of alpha or beta thalassemia have MCV of less than 80 fL (normal is ~ 82 – 94

fL) and hypochromia.



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2. Hb electrophoresis or HPLC analysis of Hb

Many different Hb forms can be identified by Hb electrophoresis, which separates

hemoglobin molecules under non-denaturing conditions according to net charge. The

example on the left is taken from MD Images. Normal individuals will have 96-98% HbA,

less than 1% HbF and 2-3% HbA2. Carriers of β-thalassemia can be detected by increased

amounts of HbA2 and sometimes HbF. The commonest structural variants, sickle (S)

hemoglobin, hemoglobin C, and hemoglobin E introduce a change in net charge and are also

detected in this way. Individuals with HbH disease usually have HbH present, with reduced

amounts of HbA and HbA2. Individuals carrying the sickle cell trait will have both HbS and

HbA. Individuals affected with sickle cell anemia will have predominantly HbS. Gel

electrophoresis has been replaced by higher-resolution liquid chromatography techniques.

3. DNA-based approaches

Many common mutations leading to hemoglobinopathies can be identified directly using

DNA-based tests. Individuals identified by abnormal RBC indices and/or with abnormal Hb

patterns may be further investigated using DNA-based testing. Polymerase chain reaction-

based assays are fast and inexpensive, but presuppose knowledge of the mutations prevalent

in the population of which the patient is a member. Southern blotting techniques can be used

to detect gene deletions (of particular importance in the alpha thalassemias) and DNA

sequencing may be needed to identify rare mutations. For couples, whose offspring might be

at risk for a severe hemoglobinopathy, DNA-based testing is required to confirm and identify

the underlying mutations segregating within the family. If desired, prenatal diagnosis can be

used to identify at-risk fetuses.

______________________________________________________________________________

Case Study #1:

Jane and John, a couple of Afro-Caribbean descent, have had a son who was recently diagnosed

with sickle cell disease. Jane is now 6 weeks pregnant with their second child.



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1. What is the prognosis for their son?

2. What is the risk for sickle cell disease in future offspring of this couple?

3. Do you require molecular testing to confirm the diagnosis of sickle cell disease in the son?

Sickle cell disease can be diagnosed using fetal DNA extracted from either amniotic fluid or

chorionic villus. At the molecular level, sickle cell is caused by a single change in the β globin

chain. The glutamic acid at codon 6 is replaced with a valine in individuals with sickle cell

anemia. At the DNA level, this is a result of A to T substitution in the sixth codon (GAG to

GTG) of the β-globin gene (known as the HBB gene). Below is part of the genomic sequence

that includes the 5’ end of the HBB gene.

Note: The genomic sequence that is translated into mRNA is underlined.

1 ggctgggcat aaaagtcagg gcagagccat ctattgctta catttgcttc tgacacaact

61 gtgttcacta gcaacctcaa acagacacca tggtgcatct gactcctgag gagaagtctg

121 ccgttactgc cctgtggggc aaggtgaacg tggatgaagt tggtggtgag gccctgggca

181 ggttggtatc aaggttacaa gacaggttta aggagaccaa tagaaactgg gcatgtggag

241 acagagaaga ctcttgggtt tctgataggc actgactctc tctgcctatt ggtctatttt

301 cccaccctta ggctgctggt ggtctaccct tggacccaga ggttctttga gtcctttggg

361 gatctgtcca ctcctgatgc tgttatgggc aaccctaagg tgaaggctca tggcaagaaa

421 gtgctcggtg cctttagtga tggcctggct cacctggaca acctcaaggg cacctttgcc

481 acactgagtg agctgcactg tgacaagctg cacgtggatc ctgagaactt cagggtgagt

541 ctatgggacg cttgatgttt tctttcccct tcttttctat ggttaagttc atgtcatagg

601 aaggggataa gtaacagggt acagtttaga atgggaaaca gacgaatgat tgcatcagtg

Start of HBB protein



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4. If you were to use oligonucleotide primers shown below, HbS-F (forward) and HbS-R

(reverse), to PCR amplify the region of HBB containing the sickle cell mutation for analysis,

what would be the size of the PCR product you would generate? Remember that the reverse

primer is the reverse complementary sequence.

HbS-F 5’-AAAAGTCAGGGCAGAGCCATCTAT-3’

HbS-R 5’-GACGCTTGATGTTTTCTTTCCCCT-3’

5. How would using the restriction enzyme, DdeI, help you determine the genotype of the fetus?

DdeI C/TNAG where N is any nucleotide and “/” indicates where

GANT/C DNA strand is nicked.

What size of fragments would you expect if the individual is unaffected, or a carrier for HbS or

affected with sickle cell anemia?

6. The pictogram below is the actual results of the analysis described above for Jane, John and

their family. What is your interpretation with regards to the health of the fetus?

Lane M = Size standard

Lane 1 = John

Lane 2 = Jane

Lane 3 = Fetus

Lane 4 = Homozygous affected control

M 1 2 3 4 5 6

100 bp

200 bp

300 bp

400 bp

500 bp

307 bp

201 bp



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Lane 5 = Heterozygous control

Lane 6 = Homozygous normal control

____________________________________________________________________________

Case 2:

A geneticist sees a patient, Mary, who presents at 16 weeks gestation with the following

laboratory results.

MCV = 62.3 fL ; Hb = 86 g/L (Normal = 120-160 g/L)

Normal Hb electrophoresis pattern with HbA2 = 1.0%

She states that she is of Filipino descent and that she has been taking her prenatal vitamins which

contain iron daily.

1. What do you suspect this patient has? What test should be ordered to confirm your suspicions?

Molecular testing was performed. The results for Mary are as follows:

The α-globin gene cluster was analyzed by PCR specific for the --SEA/, --FIL/,

--MED/, -α3.7/ and -α4.2/ deletional forms of α-thalassemia. The results are consistent with

Mary being a compound heterozygote for the rightward single α-globin gene deletion and

the southeast Asian α-thalassemia-1 deletion (-α3.7/ --SEA).

2. What does Mary have? Is this possible?

.

Mary’s husband, Kris, is also of Filipino descent. He also has molecular testing done. His results

are as follows:

The α-globin gene cluster was analyzed by PCR specific for the --SEA/, --FIL/,

--MED/, -α3.7/ and -α4.2/ deletional forms of α-thalassemia. The results are consistent with

Kris being heterozygous for the Filipino α-thalassemia-1 deletion

( -- FIL/αα).

3. What does Kris have? What might you expect with respect to his RBC indices and

hemoglobin electrophoresis?

4. Comment on the risks to the fetus.

BL015 (A) Pediatric Anemias University of Manitoba

Faculty of Medicine Dr. Rochelle A. Yanofsky

2008-2009

4/2/2009 1

Objectives: 1. Understand the physiology of red blood cell production in childhood

including:

a. globin chain production; b. the physiologic nadir; and c. how the normal ranges for hemoglobin and MCV vary

throughout childhood. 2. Know how to approach anemia based on history, physical examination,

MCV and reticulocyte count. 3. Know the following about nutritional iron deficiency anemia:

a. the usual age at diagnosis; b. how to prevent and treat it; and c. the causes of failure to respond to iron.

Reference: Supplied supplemental notes. Optional reading (Reference text): (1) Nathan DG and Orkin SH (eds): Nathan and Oski’s Hematology of Infancy and Childhood, 6th edition. W.B. Saunders Co., Philadelphia, 2003. (2) Lanzkowsky P (ed): Manual of Pediatric Hematology and Oncology, 4th Edition. Elsevier Academic Press, 2005. (3) Health Canada Guidelines: Health Canada Website (http://www.hc-

sc.ga) under “Food and Nutrition” – Iron deficiency anemia, and vegetarian diets.

http://www.hc-sc.ga/

http://www.hc-sc.ga/



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ANEMIA IN CHILDHOOD

Introduction. There are numerous disorders that can lead to anemia in children (Table 2). The three most common causes of anemia in childhood are nutritional iron deficiency anemia, recent infection, and the minor (“trait”) forms of hemoglobinopathies and thalassemias. This assigned reading focuses on the physiology of red blood cell production in childhood, a general approach to the childhood anemias, and nutritional iron deficiency anemia (which is the most common cause of anemia in childhood). The reader is referred to the reference texts and to all of the other Med II lectures about anemia for a more detailed discussion of other anemias seen in childhood). Physiology of Red Cell Production and Normal Ranges for Hgb and MCV. The normal ranges for Hgb and MCV are different than the adult normal range and differ at different ages (Table 1). At birth, the normal Hgb for a term baby is 135 to 225 g/L, and the normal MCV is 95 to 121 fl, as compared to the normal range of 80 to 100 for MCV in adults. The more premature the baby, the lower the birth Hgb. There is normally little red cell production for the first two months of life in a term infant. During that time, the Hgb and MCV gradually decrease. The child grows, (i.e., the blood volume expands), and the senescent red cells gradually die off. The Hgb reaches a low point, called the physiologic nadir, at which point red cell production accelerates. A nadir Hgb as low as 90 g/L is reached at eight to 12 weeks of age in term infants; a nadir Hgb as low as 70 g/L (at four to eight weeks of age) is not uncommon in preterm infants who weighed 1,000 to 1,500 grams at birth. By the time the term infant is six months old, the normal MCV is lower than the adult normal range (at 70 to 86). The normal Hgb and MCV remain lower than the adult normal ranges until puberty (i.e., 12 years of age for girls and 18 years of age for boys). Table 1. Normal Ranges for Hemoglobin and MCV AGE HEMOGLOBIN (g/L) MCV (fl) 0-7 days *135-225 95-1217 days – 1 month *100-215 85-1261 month – 6 months *90-140 74-1156 months – 12 months *105-135 70-861 year – 5 years 115-135 75-875 years – 12 years 115-155 77-9512 years – 17 years 120-160 78-100Adult female 120-160 80-98Adult male 130-180 80-98*In children born at term. Adapted from Diagnostic laboratory services, Health Sciences Centre (Winnipeg, Manitoba) 2002-2003. Nutritional iron deficiency Anemia. Normal physiology. Most of the iron transfer from mother to child during gestation occurs during the third trimester of pregnancy. Iron stores are not used to a significant degree until after the physiologic nadir is reached and red cell production has accelerated. To become iron deficient, the body’s iron stores must be depleted and the amount of iron ingested inadequate to keep up with demands. An iron-deficient state precedes the development of



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iron deficiency anemia. Thus, nutritional iron deficiency anemia does not occur before six months of age in a term baby. It can occur, however, as early as age two to three months in a very premature infant. Prevention of Iron Deficiency. The use of iron-supplemented infant formulas is a reliable way of preventing nutritional iron deficiency in term infants. For preterm infants, especially those whose birthweight was less than 1,500 grams, additional (medicinal) iron supplementation should be provided, starting no later than two months of age. Iron is present in low concentrations in breast milk, but is very well absorbed and used. However, infants who continue to receive only breast milk after the first six months of life are also at increased risk for iron deficiency. Therefore, iron-containing foods should be introduced into the diet of breast-fed children by the age of six months. Nutritional iron deficiency anemia is preventable through appropriate feeding choices. The Health Canada guidelines recommend:

a. that exclusive breastfeeding be continued for the first six months of life; b. that iron-rich (or iron-fortified) foods be introduced at age six months; c. that iron-containing formulas be used and the introduction of cow’s milk be delayed until age 9 to 12

months; and d. where informed parents choose not to adhere to these recommendations, that the child be screened for

anemia at age 6 to 8 months and an iron supplement be provided, if necessary. Clinical Aspects of Nutritional Iron Deficiency Anemia. Nutritional iron deficiency anemia is the most common cause of anemia in childhood worldwide. Despite the promotion of breast feeding and an increased use of iron-fortified formulas and cereals, the prevalence of iron deficiency anemia in North American children aged one to three years is estimated to be 3% to 15%. Nutritional iron deficiency anemia is even more common in children from some ethnic minority groups (notably Asians and African Caribbean immigrants), as well as in children disadvantaged by poor socioeconomic circumstances. The prevalence of nutritional iron deficiency without anemia is even higher. Iron deficiency anemia has been associated with delayed mental and psychomotor development. These adverse consequences may not be fully reversible with treatment. Iron deficiency is also a risk factor for the development of strokes in infancy and childhood. Nutritional iron deficiency anemia usually occurs between the ages of six months and 24 months in children born at term. In preterm infants, iron deficiency can occur as early as two to three months of age in children who are breast fed or consume an infant formula that is not iron-supplemented. Cow’s milk is a poor source of iron. Furthermore, children may develop an allergy to cow’s milk protein, which causes occult blood loss in the stool, as well as hypoproteinemia. Cow’s milk is high in calories, therefore, children who drink a lot of milk are not hungry. In the term infant, the usual story is that the child’s formula was changed from an infant formula to whole cow’s milk before the age of one year and that he or she is consuming large amounts of cow’s milk, typically 40 to 60 ounces per day. (After the age of one, children should consume 24 ounces of milk or less per day). The child is often a picky eater and eats little if any meat. He or she is often chubby, pale, and edematous. The child may be very anemic, but is not very symptomatic for the degree of anemia, because the fall in Hgb has occurred gradually. Other children and adolescents at increased risk for the development of iron deficiency in the absence of an underlying disease are: vegetarians, menstruating or pregnant females and adolescents during their growth spurts.



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Diagnosis and treatment of nutritional iron deficiency anemia. The child with iron deficiency anemia typically has a microcytic anemia. The platelet count is often elevated in mild iron deficiency and may be slightly decreased in severe iron deficiency. Iron stores are depleted, as reflected by a low serum ferritin, a low serum iron (Fe), an increase in the total iron binding capacity (TIBC), a transferrin saturation of less than 12% to 16% and an elevated free erythrocyte protoporphrin level. However, tests used to assess iron stores can be misleading, as they may be affected by other coexisting conditions. For example, infection, inflammation and liver disease can cause a rise in the serum ferritin level. There is a diurnal variation in serum iron levels, and the serum iron levels can be affected by recent iron ingestion. The best test for iron deficiency is a one-month trial of therapeutic doses of oral iron (i.e., 3 mg to 6 mg of elemental iron per kilogram per day in divided doses to a maximum of 300 mg of elemental iron per day). In an otherwise healthy child, recovery from anemia is about two-thirds complete after one month. If the concentration of Hgb has increased significantly during the month, therapeutic doses of iron should be continued until the Hgb returns to normal and the iron stores have been repleted. It usually takes two to four months of iron at therapeutic doses to replenish the iron stores. The MCV should return to normal after about three to four months of iron therapy, by which point the old microcytic red cells should have died off. Failure to Respond to Iron Therapy. The most common cause of failure to respond to therapeutic doses of iron is poor compliance. If a child refuses to take medicine because of the unpleasant taste of ferrous sulfate (Fer-in-sol® syrup), other formulations of iron may be tried (e.g., cherry-flavored ferrous gluconate called Palafer® suspension). Nurses or pharmacists can assist with teaching the parent how to get the child to take the medication. Arrangements can even be made for a home-care nurse to come to the home and administer the medication to the child. It is almost never necessary to administer iron by the parenteral route. Other causes for failure to respond to iron therapy are: wrong diagnosis; combination of iron deficiency and some other disorder; inadequate dose of iron to meet needs; and extremely rarely, malabsorption. Other Causes of Anemia Due to Dietary Deficiencies. Vegetarian diets may result in severe protein-energy malnutrition and deficiencies of an iron, vitamin B12 and vitamin D. Babies of vegetarian mothers are also prone to dietary deficiency of vitamin B12, especially if breast-fed with milk of low vitamin B12 content. Goat’s milk is deficient in folic acid, iron and vitamin D. Infants whose diets consist primarily of goat’s milk may become deficient in any of these nutrients. A child who is folic acid deficient may present with a megaloblastic anemia. If in doubt about the adequacy of a diet or type of milk, the input of a dietician should be sought. If significant nutrient or vitamin deficiencies are noted in the diet, supplements can be introduced, or dietary changes made, before vitamin and/or nutrient deficiencies develop. Anemia of Acute Infection/Inflammation. The second most common cause of childhood anemia is a recent mild bacterial or viral infection, or an immunization with a live viral vaccine. The anemia is usually mild to moderate, and the MCV is either normal or low, with a blood smear that is otherwise normal. The anemia does not usually require treatment and resolves as the infection/inflammation resolve. This may take as long as three months. Transient erythroblastopenia of childhood (TEC) is a post-infectious cause of anemia that is unique to childhood. Diamond-Blackfan anemia is an inherited or sporadic anemia and is the main differential diagnostic consideration in children with TEC. Please refer to the reference texts for more information about these disorders.



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Thalassemias and Hemoglobinopathies. Pathophysiology. Thalassemias (e.g., the alpha- and beta- thalassemias) are characterized by a decrease in the rate of production of a normal globin chain while hemoglobinopathies (e.g., sickle cell trait and sickle cell disease) are characterized by the production of an abnormal globin chain. Alpha (α) globin chain disorders manifest themselves clinically at birth (or in utero), as alpha globin chain production reaches maximal levels by the second month of gestation. In contrast, beta (β) chain production is low at birth, but rises dramatically after birth, with large amounts of HgbA (α2β2) being produced by three months of age and the normal adult proportion of HgbA being achieved by six months of age. Beta chain disorders (e.g., sickle cell anemia or beta-thalassemia) do not cause anemia, abnormalities of red cell morphology, or clinical symptoms until after about two months of age, at which point a significant amount of beta chain is being made. However, beta chain disorders can be diagnosed at birth, or even in utero, using tests such as hemoglobin electrophoresis or more sophisticated studies. The diagnosis of sickle cell anemia (also called sickle cell disease) should be made as early in life as possible (ideally at birth), as serious problems such as overwhelming sepsis can occur by age two months. A detailed discussion of hemoglobinopathies and thalassemias is beyond the scope of this assigned reading. Please refer to the Med II lecture about globin chain disorders and to the reference texts for a more detailed discussion of these disorders. A General Approach to Childhood Anemias. A) Step I. The history and physical examination. The workup of anemia should begin with a complete history. The birth history should include information about prematurity and neonatal jaundice. The dietary history should document the amount and type of milk consumed per 24 hours, as well as the types of foods consumed (and avoided) and the frequency of consumption of those foods. In breast-fed infants, the dietary history should include a maternal dietary and medication history. A history of recent illnesses and infections also should be obtained, along with information about the general health of the child or adolescent. All medications taken should be documented.

A family history of hemolytic disorders should be sought. Questions asked should include a family history of anemia, jaundice, splenectomy and gallstones. The sex of the child should be noted (regarding X-linked disorders), along with his/her age. Information about the racial and ethnic background of the parents and their geographic descent should be obtained (regarding hemoglobinopathies, thalassemias and G6PD deficiency). Questions should also be asked to help determine the rapidity of onset and mechanism of the anemia. In the absence of a record of serial Hgb values, the rapidity of onset of the anemia can be assessed by evaluating how symptomatic the patient is from the anemia and by noting who realized the child was pale. Anemias due to decreased red cell production are usually of gradual onset. The child is not very symptomatic from the anemia for the degree of anemia. The parent has not usually noticed that the child is pale. Information should be obtained about blood loss (e.g., nosebleeds, menorrhagia, and overt or occult bleeding from the gastrointestinal tract), as well as about symptoms related to hemolysis (including jaundice, tea-colored urine, or urine that is described as dark red, brown or black if hemoglobinuria is present). Symptoms related to the cause of the anemia also should be sought. The physical examination should be complete, looking for findings related to the anemia as well as to the cause of the anemia. B) Step II. The initial laboratory workup.



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The initial laboratory workup should include a CBC and differential, platelet count, red cell indices, reticulocyte count (which reflects the marrow response to the anemia) and examination of the peripheral blood smear. These simple tests often provide clues as to the cause of the anemia. Is more than one cell line affected? What is the marrow response to the anemia? It is important to note that a “normal” reticulocyte count in the presence of anemia is a “low” reticulocyte count. Are there any clues on the blood smear regarding the cause of the anemia? The anemia should then be classified as microcytic, normocytic or macrocytic, using the correct normal ranges for age. It should be noted that the MCV reflects the average red cell size, and, thus, may not be as reliable a parameter in conditions in which there is marked variation in red cell size. The degree of variation in red cell size can be assessed by looking at the red cell distribution width (RDW) and the peripheral blood smear. In order to correctly interpret hemoglobin values and assess the response to therapy for anemia, the physician must be aware of other variables that affect the laboratory results. Hemoglobin (and hematocrit) values obtained by automated methods may vary by as much as 3% on repeated testing of the same blood sample. Furthermore, capillary blood samples obtained by skin prick have a higher hemoglobin (and hematocrit) concentration than do simultaneously collected venous samples. The capillary to venous hematocrit ratio is highest in the first five days of life. In healthy infants over five days of age, the hemoglobin (and hematocrit) performed on a capillary blood sample may only be 2.5% higher than the hemoglobin (and hematocrit) performed on a simultaneously collected venous sample. C) Step III. The systematic approach to childhood anemia. Having completed steps one and two, the physician should now consult Table 2, which is an approach to the differential diagnosis of childhood anemia based on MCV. The workup after this point will be guided by the information gained in steps one to three. Table 2: A Diagnostic Approach to Childhood Anemia (Based on MCV):* Microcytic anemias a) Iron deficiency b) Chronic disease c) Thalassemias and some hemoglobinopathies d) Lead poisoning e) Sideroblastic anemia f) Copper deficiency Normocytic anemias a) Reticulocyte count increased: hemorrhage or hemolysis b) Reticulocyte count low or “normal” and marrow normal: i) chronic disease ii) liver disease iii) renal disease iv) endocrine disorders (especially hypothyroidism) c) Reticulocyte count low or “normal” and marrow abnormal: i) aplastic anemia ii) red cell aplasia: including transient erythroblastopenia of

childhood, Diamond-Blackfan anemia, and aplastic crisis of hemolytic anemia

iii) marrow infiltration (malignancy, storage diseases, osteopetrosis)



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Macrocytic anemias a) Megaloblastic: due to deficiency of vitamin B12 or folate, including

aminoacidurias and inherited defects of metabolic pathways b) Non-megaloblastic:

i) marrow failure syndromes (including Fanconi’s anemia and Diamond-Blackfan anemia

ii) marked increase in reticulocyte count iii) hypothyroidism iv) Down’s syndrome v) liver disease vi) preleukemic (myelodysplastic) disorders * See reference text for more information regarding the disorders that cause anemia in childhood. SUMMARY. In order to properly diagnose and classify anemias in childhood, physicians must know what is normal, and utilize a systematic approach in the evaluation of these disorders. Primary-care physicians are in a key position to make significant inroads in the prevention and early detection of nutritional anemias of childhood, as well as in the prevention and early detection of inherited red cell disorders, such as sickle cell disorders and the thalassemias.



2008-2009

4/2/2009 8

SELF-STUDY QUESTIONS: 1. Compare the normal values for hemoglobin and MCV in the following patients:

(a) term newborn

(b) 11-month-old girl

(c) 11-year-old boy

(d) adult male

(e) adult female

2. What is the physiologic nadir and when does it occur? 3. Regarding nutritional iron deficiency anemia:

(a) at what age does it occur (in a child born at term, and a preterm child)?

(b) What information would you expect on history and what findings on physical

examination? (Be specific about the information in the dietary history).

(c) How would you make the diagnosis?

(d) What is the best test for iron deficiency? (be specific)

(e) What are the pitfalls of the tests used to assess iron stores?

(f) How would you treat the child and for how long (be specific)?

(g) List all of the possible causes of failure to respond to iron.

(h) What is the most common cause of failure to respond to iron?

-1-

BL020 (A) Coagulation University of Manitoba

Faculty of Medicine

Med II / BL020

Dr. D. Houston

2008/2009

Objectives:

After reading the assigned materials about secondary hemostasis and working through the case

questions, the student will be able to:

1. Explain, with the use of a diagram, the stages of the clotting cascade.

2. Describe how coagulation factors are activated.

3. Define what is measured by the screening tests of coagulation (PT, PTT, and thrombin

time) and show how they can be used to identify where in the cascade a defect lies.

4. Describe the principle of the mixing test, and how it is used to measure the levels of

specific coagulation factors and detect inhibitors.

5. Compare the typical clinical and laboratory presentations of: (i) Hemophilia, (ii) Factor

XII deficiency, (iii) Vitamin K deficiency, (iv) Liver disease, (v) Lupus anticoagulant.

Required reading:

Attached notes, and

Hoffbrand, Pettit and Moss, pages 290-302 (5th ed.)

NEW CONCEPTS IN COAGULATION

Our understanding of the fundamental

mechanisms of coagulation in vivo have been

revised substantially in the last few years.

Older terminology, particularly the distinction

of ‘intrinsic’ and ‘extrinsic’ pathways, though

still often used, does not fit well with the new

models and in my opinion should be

abandoned.

The current model is that physiological

coagulation always begins with the exposure

of blood to tissue factor. Tissue factor is a

transmembrane protein that is expressed

constitutively on cells in the adventitial

surface of blood vessels, in organ capsules, in

the dermis, and in the brain, but not under

normal circumstances by endothelial cells or

circulating blood cells. Tissue factor serves as

a co-factor to which circulating Factor VIIa

binds, enabling FVIIa to become catalytically

competent. Thus tissue factor provides a

‘hemostatic envelope’ that initiates

coagulation when the vasculature or vital

structures are breached. In the artificial

conditions of the prothrombin time assay,

where phospholipid and tissue factor are

present in high concentrations, Factor X is the

favoured substrate of the Factor VIIa/tissue

factor complex, but in vivo the major substrate

is Factor IX.

It is often argued that the coagulation cascade

is proof that God has it in for Med II students.

(This is wrong; it is professors that have it in

for Med II students. God has it in for the

professors, and the existence of medical

students is the proof.) A more sober reflection

suggests that the complexity of the cascade,

with multiple hard-to-memorize steps and

multiple components in each reaction

complex, acts as an extracellular signal

transduction pathway. Three purposes can be

imputed:

• it allows explosive amplification of a small

-2-

injury signal into a macroscopic clot, by

virtue of multiple positive-feedback loops

• it allows localization of reactions to sites of

injury, and rapid extinction of reactions in

areas with intact endothelium

• it permits multiple levels at which control

of this vital process can be regulated

The negatively charged phospholipid surface

required for assembly of the FIXa/FVIIIa/FX

complex and the FXa/FVa/prothrombin

complex is provided by activated platelets;

thus platelets serve a role in coagulation as

well as in primary hemostasis. Furthermore,

one of the principal agonists for platelet

activation is thrombin, so again there is cross-

talk between primary hemostasis and the

coagulation pathways.

Factor XII, prekallikrein, and high molecular

weight kininogen deficiencies, though

resulting in dramatic prolongation of the

partial thromboplastin time (PTT), are not

associated clinically with any bleeding

tendency. Factor XI deficiency is associated

with a mild and variable bleeding tendency,

much less severe than FVIII or FIX deficiency

(i.e. hemophilia A or B). It is now thought that

the physiologic activator of FXI is thrombin

on a platelet surface rather than FXII. FXI is

not required for initial clot formation, but it

serves in a feedback amplification loop to

enhance thrombin generation. High

concentrations of thrombin activate TAFI

(thrombin activatable fibrinolysis inhibitor).

Activated TAFI is a carboxypeptidase which

partially digests fibrin and renders it less

capable of supporting plasminogen activation,

thus making the clot more resistant to

fibrinolysis.

LABORATORY EVALUATION OF COAGULATION

Because the clinical history and examination

cannot give a precise diagnosis of hemostatic

defects, laboratory testing is essential to

confirm or rule out a suspected abnormality.

The routine first-level tests are the

prothrombin time (PT, usually reported as the

International Normalized Ratio or INR, which

corrects for differing reagent sensitivities

between laboratories) and activated partial

thromboplastin time (aPTT). A normal PT-

INR and aPTT rules out a major deficiency of

any of the clotting factors (except for FXIII).

Most laboratories can also measure a

fibrinogen level. The pattern of the findings

with these basic tests is informative (see table

in notes for BL019). Keep in mind that

complex situations with multiple coexisting

diagnoses can occur, and no algorithm can

encompass them all.

Perusal of that table from BL019 will lead the

more astute among you to realize that the PT-

INR is a far more useful test than the aPTT.

Indeed, an isolated abnormality of the aPTT

usually is a false positive, i.e. not associated

with a tendency to bleed: lupus anticoagulant,

heparin contamination of the sample, Factor

XII, HMWK or prekallikrein deficiency.

Pretty much all the aPTT adds is detection of

hemophilia, so an aPTT should be ordered

only when hemophilia is a diagnostic

consideration. An abnormal PT-INR, in

contrast, is almost always significant, and the

INR is more justifiably used as a screening

test for acquired hemostatic defects.

Prolongation of the PT or aPTT can in general

terms be due either to:

• a deficiency of one or more clotting factors,

or

• the presence of an inhibitory substance.

The next step in evaluating abnormal

screening tests is to perform a mixing test, in

-3-

Va

TissueFactor

VIIIaVII

VIIa

X

Xa

XI

XIa

IX

IXa

II

IIa

Fibrinogen

Fibrin

Fibrin

Fibrin

Fibrin

fibrin clot

XIIIa

TAFIa

XIII

TAFI

VIII

ActivatedPlatelet

Trauma

Cross

Cross

linked

linked

collagen& vWF

plateletaggregate

ADPTxA2

exposes:

pR

IMA

RY

HE

MO

STA

SIS

Primary and Secondary Hemostasis. Both primary and secondary hemostatic processesare triggered by exposure of materials in vessel wall, normally covered by the intactendothelium. Note the importance of cross-talk between these processes.

which the patient’s plasma is mixed 1:1 with

normal plasma, to distinguish between these

two possibilities. On the principle that 50% of

any clotting factor is enough to give a normal

clotting time, the mixed plasma should yield

a normal clotting time (i.e. it corrects) if the

patient’s problem is a deficiency of one or

more factors. On the other hand, dilution of an

inhibitory material by half will generally not

eliminate the inhibitory effect (remember that

concentration-effect curves generally have a

logarithmic X-axis), i.e. it doesn’t correct. If

the mixing test shows a deficiency, mixing

reactions using known factor-deficient

plasmas instead of normal plasma can be used

to identify the missing factor(s). For example,

if the patient has FVIII deficiency, his PTT

will correct on mixing with normal plasma

and with FIX-deficient plasma, but not with

FVIII-deficient plasma. If the mixing test

demonstrates an inhibitor, the substance

should be identified. Heparin can be

neutralized in the sample and the effect of

lupus anticoagulants reversed by the addition

of excess phospholipid.

Primary and Secondary Hemostasis

-4-

TissueFactor

‘Contact’Surface

VIIa

X

XI

XII

IX

K

II

IIa

Fa

IIa

IIa

Fibrinogen

Fibrin

Fibrin

precipitated fibrin

VIII

VIIIa

IXa

Va

Xa

XIa

XIIa

PK

PT / INR

aPTT

HMWK

= vitamin K dependent

= target of heparin/AT

Coagulation cascade in vitro. Coagulation proceeds as a stepwise series of catalytic reactions, which providesa mechanism for explosive amplification and also for multiple regulatory checkpoints. In each step, an inactiveprecursor is converted by proteolytic cleavage into an active form, designated by the suffix ‘a’. Each enzymeis a serine protease. Most of the enzymes in the cascade require the presence of a cofactor (tissue factor, FVaor FVIIIa, shown in ovals); in each case where a cofactor protein is involved, calcium and a negatively chargedphospholipid surface are also necessary for assembly of the reaction complex.The schema shown in this diagram is a representation of the reactions that occur in vitro in commonly usedclotting tests, and is worth committing to memory for that reason, but it is NOT a representation of our currentunderstanding of in vivo hemostasis. Clotting tests are performed with plasma that is anticoagulated with citrate,a chelator of calcium. Coagulation is initiated by the addition of tissue factor, phospholipid, and calcium(prothrombin time or PT) or by addition of a negatively charged ‘contact’ surface, phospholipid, and calcium(partial thromboplastin time or PTT), or by addition of thrombin (thrombin time). Thus, deficiencies of FVII, FX,FV, prothrombin, or fibrinogen will lead to a prolonged PT, whereas deficiency of FXII, HMWK, prekallikrein, FXI,FIX, FVIII, FX, FV, prothrombin, or fibrinogen will lead to a prolonged PTT. Only deficiency (or abnormality) offibrinogen, or presence of an inhibitor such as heparin or fibrin degradation products, will prolong the thrombintime. None of these tests detect deficiency of FXIII.

Coagulation in vitro: the PT-INR and aPTT

-5-

TissueFactor

VIIa

X

XI

IX

II

IIa

IIa

IIa

IIa

IIa

Fibrinogen

Fibrin

Fibrin

fibrin clot

XIIIaXIII

VIII

ActivatedPlatelet

Trauma

Cross

linked

Collagen

plateletaggregate

ADPTxA2

VIIIa

IXa

Va

Xa

Fibrin

IIa

XIa

Coagulation in vivo. Clotting is now thought to be initiated in almost all cases by the exposure of bloodto tissue factor; the phospholipid surface is provided by activated platelets. FVIIa/tissue factor preferentiallyactivates FX, but this path is quickly shut down by tissue factor pathway inhibitor (TFPI). Propagation ofcoagulation therefore requires ‘bypass’ through the intrinsic pathway: activation of FIX by FVIIa/tissuefactor, or activation of FXI by thrombin. Note that FXII, prekallikrein, and HMWK are not true coagulationfactors, since their deficiency does not result in a bleeding diathesis, although it does result in prolongationof the PTT in vitro. Note also the central role of thrombin and the positive feedback loops through itsactivation of FV, FVIII, FXI, and platelets (dashed lines). Factor XIII is the only coagulation enzyme that isnot a serine protease; instead, it is a transglutaminase that creates covalent cross-links between fibrinstrands.

XII

Coagulation in vivo

-6-

Protease complex

Enzyme(Factor IXa)

Activationpeptide

Coagulation complex assembly. Each step in the coagulation cascade involves the activation of an inactivezymogen by the removal of a small activation peptide, converting it into an active protease capable ofcatalyzing the next reaction in the cascade. The enzymes involved are serine proteases, and are closelyrelated to the digestive enzyme trypsin. Unlike trypsin, however, these enzymes cleave only very specificsubstrates. The specificity is conferred in two ways: by the specific conformation of the active site of theenzyme matching the corresponding peptide sequence it cleaves; and (in some cases) by the assembly ofa complex involving a protein cofactor. An example would be the assembly of the ‘tenase’ complex, consistingof FIXa (the enzyme), FX (the zymogen substrate), FVIIIa (the cofactor) and phospholipid, yielding the product,activated FXa. Thrombin requires no cofactor to activate FV, FVIII, or FXIII, but does require a cofactor(thrombomodulin) to activate Protein C.

Assembly of Coagulation Complexes

BLO21 Platelet Production and Thrombocytopenia University of Manitoba Faculty of Medicine

Med II / BLO21 Dr. Sara Israels 2008/2009

Objectives: After this lecture and reading the accompanying material and text the student, will be able to: 1. Describe the process of megakaryocyte maturation and platelet production. 2. Diagram the structure of a mature platelet showing dense granules, alpha granules,

Glycoprotein Ib, Glycoprotein IIb/IIIa and phospholipids. 3. List 3 mechanisms that cause thrombocytopenia and give examples of disease states

associated with each mechanism. 4. Identify three methods for treating ITP and the mechanism by which each method increases

the platelet count. 5. Identify appropriate indications for platelet transfusion.

Required reading: (1) Hoffbrand, Pettit and Moss, (Fifth Edition) pages 123-128, 264-265, 280-286, 288-289, 349. (2) George, J.N., Platelets, Lancet, 2000;355:1531-1539.

4/6/09

1

Platelet production and thrombocytopenia

Sara J. Israels M.D.

Dept. of Pediatrics & Child Health

University of Manitoba

Objectives

1. Describe the process of megakaryocyte maturation and platelet production.

2. Diagram the structure of a mature platelet showing dense granules, alpha granules, Glycoprotein Ib, Glycoprotein IIb/IIIa and phospholipids.

3. List 3 mechanisms that cause thrombocytopenia and give examples of disease states associated with each mechanism.

4. Identify three methods for treating ITP and the mechanism by which each method increases the platelet count.

5. Identify appropriate indications for platelet transfusion.

What do you need to form a blood clot?

1. A normal blood vessel

2. Platelets:

Normal number

Normal function

3. Coagulation factors:

I to XIII

Vessel wall

FactorsPlatelets

Maslak, P. ASH Image Bank 2005;2005:101324. Copyright ©2005 American Society of Hematology.

Megakaryocytes

Megakaryocyte ultrastructure

From stem cell to platelet

HOXA11CBFA2FOG1/GATA1FOG1/GATA2TPO

FOG1/GATA1FLI1(ETS1)TPO

commitmentdifferentiation

maturation Platelet production

Megakaryocyte maturation

2

(Italiano et al. JBC 1999)

Proplatelet formation

QuickTime™ and aVideo decompressor

are needed to see this picture.

proplatelet

Platelet ultrastructure

Platelet surface receptors

• Receptors involved in platelet adhesion to subendothelial matrix proteins– GPIb-IX-V: receptor for von Willebrand factor

– GPIaIIa and GPVI: receptors for collagen

Platelet surface receptors

• GPIIbIIIa (IIb3): receptor for fibrinogen, and other adhesive proteins. Must be activated to bind fibrinogen and von Willebrand factor. Required for platelet aggregation.

Vasculitis

Abuse

VWF studies Platelet function studies

1¡ hemostatic dysfunction

Normal

Single deficiency

Multiple deficiencies

Clotting factor studies

Correction

Inhibitor studies

No correction

mixing study with normal plasma

Abnormal

coagulation screen

PTT, PT

Normal

Acute, isolated

Acute, with other cytopenias

Chronic

Decreased

CBC with platelet count

History and physical exam

Standardized questionnaire or score

Diagnostic algorithm for bleeding

Primary hemostatic defects

Secondary hemostatic defects

Thrombocytopenia

Platelet count: thrombocytopenia

• CBC and blood film

– Platelet count

– MPV

– Blood film

Vessel wall

FactorsPlatelets

3

Plt ct. 45 X 109/L

Mechanisms of thrombocytopenia

Decreased production

Normal Aplastic Anemia

Bone marrow biopsies

•Isolated or in combination with other cytopenias

•Congenital or acquired

Decreased production•Isolated or in combination with other cytopenias

•Congenital or acquired

Increased destruction

Non-immune thrombocytopenia

•Microangiopathic hemolytic anemia

Increased destruction

Immune thrombocytopenias

4

Immune thrombocytopenic purpura

• Autoimmune disorder caused by destruction of antibody-sensitized platelets in the RE system

• Can be classified by:

– Age

– Duration: acute vs chronic

– Presence of an underlying disorder

• Infection

• Auto-immune disease


(Cines & Blanchette, NEJM 2002)


• Acute ITP– Common in young children

– Isolated severe thrombocytopenia

– Previously in good health

• 60% have a preceding infectious illness

– Sudden onset of bruising and petechiae

• 25% have significant epistaxis

(Kuhne et al. Lancet 2001)

Acute ITP

• Outcome is independent of management strategy

– 66% have recovered by 6 months

• Predictors of early remission:

– Abrupt onset

– Infectious prodrome

– Age < 10 years

– Wet purpura

– Platelet count < 5 x 109/L

Chronic ITP

• Lasting longer than 6 months

• 10-40% of cases of ITP depending on age and presenting features

• When to investigate:

– Atypical features

– Treatment failure

• To treat or not to treat?

– The risk of serious bleeding is low and not always predicted by the platelet count.

Treatment of ITP

1. Corticosteroids– Blocks antibody production

– Increase platelet production

2. High dose ImmunoglobulinImpairs clearance of autoantibody-coated platelets by the

reticulo-endothelial system (RE blockade)

3. Anti-D (RhoGAM)– Binds to D antigen on RBCs and produces RE blockade

4. Splenectomy– Removes primary site of platelet destruction

5

Treatment of ITP

5. Rituximab

o Anti-CD20 MoAb; targets B cells and inhibits antibody production

6. Romiplostim (Nplate)

o TPO mimetic; drives platelet production by megakaryocytes

Treatment of ITP

(Bromberg, NEJM 2006)

Immune thrombocytopenic purpura or is it?

• When does a low platelet count not mean ITP?

– Artifact: pseudo-thrombocytopenia

– Secondary thrombocytopenia

– Inherited thrombocytopenia:

• Bernard-Soulier syndrome

• MYH9 syndromes

• Velocardiofacial syndrome

• Gray platelet syndrome

• Wiskott-Aldrich syndrome

• Etc.

Platelet transfusion

• When is transfusion useful?

– Decreased production

• Congenital

• Acquired

– Increased destruction

• It depends on the cause

– Massive blood loss

– Platelet dysfunction

Platelet Transfusion Indications• Therapeutic transfusion

– Clinical bleeding

– Other hemostatic abnormality

– Recent surgical procedure

• Prophylactic transfusion

– Recommended thresholds ( X109/L):

• <10 in an asymptomatic patient

• 20 for a lumbar puncture

• 50 for surgical procedure

• 100 for ophthalmic or neurosurgery

Risks of platelet transfusion

• Transfusion reactions– Febrile reaction

– Allergic reaction

• Transfusion-transmitted infection– CMV

• Poor response– No increment in platelet count following

transfusion

6

Poor response to platelet transfusion

• Poor quality platelet concentrate

• Hypersplenism

• Non-immune destruction: sepsis

• Immune destruction (anti-platelet Abs)

– refractoriness ( anti-HLA Abs)

Summary

• Platelet production is a complex and active process.

• Thrombocytopenia is common in the tertiary care setting, usually secondary to other disease or therapy.

• The two most common mechanisms of thrombocytopenia are: decreased bone marrow production and increased peripheral destruction.

BLO23 Platelet Function and Dysfunction University of Manitoba and von Willebrand Disease Faculty of Medicine Med II / BL023

Dr. S. Israels 2008/2009

Objectives: After this lecture and reading the accompanying material and text, the student will be able to:

1. Construct a diagram that depicts the process of platelet adhesion, which includes subendothelial collagen, von Willebrand Factor and Glycoprotein Ib.

2. Construct a diagram that depicts the process of platelet aggregation including activation receptors, granule release, thromboxane synthesis, glycoprotein IIb/IIIa and fibrinogen.

3. Explain the role of von Willebrand Factor in hemostasis and why a deficiency leads to excess bleeding.

4. List useful clinical laboratory tests for measuring platelet function and von Willebrand disease. 5. Recommend 2 forms of therapy for hemorrhage in von Willebrand Disease and the

mechanism of the therapeutic effect. Required reading: (1) Hoffbrand, Pettit and Moss, (Fifth Edition) pages 265-270, 275-277, 286-289, 295-296.

(2) Lillicrap D. The basic science, diagnosis and clinical management of von Willebrand disease.

World Federation of Hemophilia 2004, monograph #35.

4/6/09

Platelet Function

Sara J. Israels M.D.

Dept. of Pediatrics & Child Health

University of Manitoba

Objectives

1. Understand the process of platelet adhesion.

2. Understand the process of platelet aggregation.

3. Understand the role of von Willebrand Factor in

hemostasis and why a deficiency leads to excess

bleeding.

4. Know the clinical laboratory tests for measuring platelet

function and von Willebrand disease.

5. Recommend 2 forms of therapy for bleeding in von

Willebrand Disease and the mechanism of the

therapeutic effect.

Platelet Functions

Haemostasis & ThrombosisAdhesionActivationSpreadingSecretionAggregationProcoagulant activityClot retraction

Regulation of vascular toneUptake of serotoninRelease of serotonin, thromboxane

InflammationAtherosclerosisAsthmaRenal diseaseChemotaxisPlt-leukocyte interactions

Host DefensePhagocytosis/internalizationof bacteriaRelease of microbicidal proteinsSuperoxide production

Tumor BiologyTumor growthTumor metastasisTumor killing

Vessel wall

FactorsPlateletsPlatelet Adhesion

Platelet Activation

Clinical Manifestations

• Easy bruising

• Epistaxis

• Gingival bleeding

• Menorrhagia

• Postpartum hemorrhage

• Gastrointestinal bleeding

• Bleeding post dental extractions

• Postoperative hemorrhage

Diagnostic tests

• Evaluation of 1° hemostasis

– Bleeding time

– Platelet function analyzer

• Quantitative assays

– Platelet count

– Flow cytometry for platelet antigens

• Functional assays

– Platelet aggregation

– Platelet adhesion

• Qualitative assays

– Light microscopy

– Electron microscopy

Measuring primary haemostasis

VS

Bleeding Time PFA-100

Closure Times

Dade Behring 1998

.

Turbidometric aggregometry

Flow cytometry

• Receptor numbers

• Granule contents

• Platelet activation

–Receptor activation

–Activation marker expression

–Platelet microparticles

Vasculitis

Abuse

VWF studies Platelet function studies

1¡ hemostatic dysfunction

Normal

Single deficiency

Multiple deficiencies

Clotting factor studies

Correction

Inhibitor studies

No correction

mixing study with normal plasma

Abnormal

coagulation screen

PTT, PT

Normal

Acute, isolated

Acute, with other cytopenias

Chronic

Decreased

CBC with platelet count

History and physical exam

Standardized questionnaire or score

Diagnostic algorithm for bleeding

Primary hemostatic defects

Secondary hemostatic defects

Thrombocytopenia

Disorders of Platelet Function

• Defects of platelet-agonist interaction

• Defects of platelet-vessel wall interaction

• Defects of platelet-platelet interaction

• Defects of platelet secretion:

– Storage pool deficiency

– Abnormalities of secretion pathways

• Defects of platelet-coagulant protein interaction

• Miscellaneous

Glanzmann Thrombasthenia

• Autosomal recessive inheritance

• Diagnosis

– Mucocutaneous bleeding

– Prolonged bleeding time/closure time

– Normal platelet count

– Impaired aggregation with all agonists

– Impaired clot retraction

– Defective expression of GPIIb/IIIa(aIIbb3�)

Dense granules

ADP

ATP

calcium

serotonin

pyrophosphate

a-granulesAlbumin

VWFFibrinogenFibronectin

ThrombospondinPF4

thromboglobulinFactor VIIIFactor V

MultimerinProtein S

PlasminogenPAI-1

a2-antitrypsina2 -antiplasmin

a2 -macroglobulinPDGFTGF-b

Lysosomes

Cathepsin D & E

Carboxypeptidase

b-glucuronidase

b-galactosidase

Acid phosphatase

Aryl sulfatase

Elastase

CD63

aIIbb3

GPIb

P-selectin

LAMP-2

HPS proteinP-selectin

aIIbb3

GPIb

CD9

GPV

CD36

PECAM-1

Granule membrane proteinsGranule contents

CD63

LAMP-1

LAMP-2

Thrombin

OCCS

-Storage Pool Deficiency

• Dense granule deficiency: deficient releasable ADP,

ATP, serotonin

• Diagnosis:

– Prolonged bleeding time

– Prolonged closure time(Collagen/Epi)

– Absent second wave of aggregation in response to

collagen and epinephrine

– Decreased no. of dense granules

– Elevated ATP/ADP ratio

Dense granule deficient

Dense granules

Acquired platelet function defects

• Drug effects

–Anti-platelet drugs

• Myeloproliferative and myelodysplastic disease

• Uremia

• Diabetes**

Aspirin Acetylsalicylic acid

(Israels & Israels 2002)

ASA

Therapeutic Platelet Inhibitors

Therapy

• Platelet transfusion

• Anti-fibrinolytic agents

• Desmopressin

• rFactor VIIa

Von Willebrand Disease

• 1926: “hereditary pseudohemophilia” described

by Erik von Willebrand in a family from the Aland

Islands, Finland

• 1950s: vWD is shown to be caused by a plasma

factor deficiency

• 1970s: the VWF protein is identified

• 1980s: laboratory tests for measuring vWF

protein and function are developed

Who was Erik von Willebrand?(1870-1949), a Finnish doctor who first described the

hereditary bleeding disorder. •Increases Factor VIII survival

•Supports platelet adhesion to vascular subendothelium

vWF Functions:

vWF Multimers Diagnostic tests for vWD

• Evaluation of 1°hemostasis– Bleeding time

– Platelet function analyzer

• Quantitative assays– vWF Antigen

– FVIIIC

• Functional assays– Ristocetin Cofactor

– Collagen binding

– Platelet aggregation with various conc. ristocetin

• Qualitative assays– Multimeric analysis

• Genotyping

vWD: Classification

• Type 1: partial deficiency (80% of vWD)

• Type 2: functional defects

– 2A: decreased function; absent HMW multimers

– 2B: increased affinity for platelet GPIb

– 2M: decreased function; abnormal multimers

– 2N: decreased affinity for FVIII

• Type 3: severe deficiency

vWD Treatment

• Desmopressin– Release of vWF from endothelial stores

– vWF increases 3-5 fold; lasts 8-10hrs

– Treatment of choice for Type 1

• Anti-fibrinolytic therapy– Stabilizes the clot

• vWF concentrate– Plasma-derived concentrates containing HMW multimers of

vWF

– For Types 2 and 3 who do not have N. vWF stores

Summary

• Much of what we have learned about platelet function is the result of studying naturally occurring platelet function disorders.

• Tests for these conditions are at best semi-quantitative and need to be interpreted in light of clinical data.

• Therapy can be effective, but is often not specific.

BL046 (L) Pediatric Oncology University of Manitoba


2008/2009 OBJECTIVES After attending this lecture, participating in discussion, reading the assigned materials, and reflecting upon them, the student will know: • How childhood cancers differ from adult cancers: biologic and

treatment aspects. ● The overall incidence of childhood cancer (as compared to the

incidence of cancer in adults. ● Current understanding of the etiology of childhood cancers. ● The types of cancer seen in childhood and the three most

common types of cancer seen ● The current overall long-term disease-free survival rate for

children with cancer and the reasons for the improved outcome. ● The pathophysiology, and the presenting signs and symptoms of

childhood A.L.L., as well as a general approach to diagnosis and treatment.

● How childhood Hodgkin Lymphoma and childhood Non-Hodgkin

Lymphomas differ from diseases with the same names in adulthood.

Reference Supplied supplemental notes.

NCI website (optional) Type in: PDQ Treatment summaries for physicians.

Hoffbrand AV, and Pettit JE. Essential Haematology, 5th Edition. (optional reading), pages 157-173 (acute leukemias) and pages 197-215 (lymphoma)

Version 27 November 2008 1



2008/2009

PEDIATRIC ONCOLOGY I INCIDENCE OF CANCER: Children (less than 15 years old) …………………………… 14.7 cases per 100,000 children/year Adults …………………………………………………………. 467.4 cases per 100,000 adults/year TYPES OF PEDIATRIC CANCER 1. Leukemia (ALL > ANLL > Chronic Leukemia) …………………………………………31.0% 2. CNS tumors ……………………………………………………………………………… 18.3% 3. Lymphomas (Hodgkin's, NHL)………………………………………………………… 13.8% 4. Neuroblastoma …………………………………………………………………………. 6.8% 5. Soft tissue sarcoma ……………………………………………………………………. 6.2% 6. Wilms' tumor ……………………………………………………………………………. 5.7% 7. Bone tumors ……….……….……….……….……….……….……….……….………. 4.7% 8. Retinoblastoma ……….……….……….……….……….……….……….……….… 2.5% 9. Germ Cell tumors …………………….………………………………………………… 2.4% 10. Liver tumors …………..…………..…………..…………..…………..………………… 1.3% 11. Other ………………….………………….………………….…………………………… 7.4% ETIOLOGY OF CHILDHOOD CANCER:

• Unknown in the majority of children. • Immunodeficiency disorders, some syndromes, and certain viral infections are associated

with an increased risk of developing certain types of cancers. • Radiation therapy and certain chemotherapy drugs increase the risk of developing a second malignancy.

LONG TERM SURVIVAL RATES Overall survival rate in 1962 was less than 10%; it was 77% in 2003. 1962 2003 A.L.L. …………………………………………………………………………. 4% 84% A.M.L. …………………………………………………………………………. 4% 50% Ewing's sarcoma ………………………………………………………………. 5% 65% Non-Hodgkin's lymphoma …………………..……………..……..………… 6% 84% Neuroblastoma ……………….……………….……………….…………….10% 69% Hodgkin's lymphoma ………. ………. ………. ………. ………. ……………50% 95% Retinoblastoma ……………………………………………………………….75% 94% Wilms' tumor ……………………………………………………………………50% 92% Rhabdomyosarcoma ……………….…….…….…….…….…….…….……. 30% 64% Osteosarcoma ………………………………………………………………… .20% 63%




2008/2009 CNS tumors ……………………………………………………………………35% 74% HOW DOES CHILDHOOD CANCER DIFFER FROM ADULT CANCERS? 1. Sarcomas (not carcinomas). 2. Undifferentiated histology frequent. 3. More rapid proliferation. 4. Propensity for early hematogenous spread. 5. More curable (Aim to cure). 6. Growth and developmental considerations. 7. Aggressive multimodal therapy. 8. Rx according to international protocols. 9. Rx at a 3o care centre. 10. Approach is multidisciplinary. (a) medical (b) psychosocial (c) holistic, involving the whole family 11. Long term follow-up essential. 12. Attempt to decrease side effects, late sequelae. 13. Role of prevention, early detection PURPOSES OF INTERNATIONAL GROUP STUDIES: 1. To find more effective therapy (Phase I, II, III trials). 2. To find ways to ↓ side effects (short term, long term). 3. To learn more about the etiology, natural history, and biology of malignant childhood diseases. 4. To identify factors (clinical and biological) which affect prognosis. 5. To stratify treatment according to relevant prognostic ("risk") factors. 6. To learn more about host biology, as it relates to drug metabolism, as well as treatment efficacy

and toxicity. TREATMENT OF PEDIATRIC CANCERS: A. General:

- rapid workup and institution of treatment - oncologic emergencies - first chance is best chance for cure

B. Role of Surgery: - biopsy (exceptions) - resect or debulk primary tumor (initially or following chemotherapy + XRT) - alleviate symptoms - (palliation)

C. Role of Chemotherapy: - to treat overt metastases - to treat presumed micrometastases - to shrink the primary tumor prior to local control measures (surgery and/or

radiotherapy) - (palliation)

D. Role of Radiation Therapy: - decreasing (why?) - used to treat the primary tumor, tumor bed, and overt metastases - limited or no role in non-hodgkin lymphoma, osteosarcoma




2008/2009

- (palliation) E. Role of BMT:

- evolving - rationale - current indications for BMT

F. Other - Biologic therapies: targeted therapies, differentiating agents, monoclonal antibodies, etc. Leukemias: The leukemias constitute the most common group of malignancies in childhood: 80% are A.L.L. (acute lymphoblastic leukemia), 15% are AML (acute myeloid leukemia, also called acute nonlymphoblastic leukemia) and the rest are chronic leukemias (either Chronic Myeloid Leukemia or Juvenile Myelomonocytic Leukemia). This is in contrast to leukemia in adults, where 14.1% are A.L.L., 34.4% are AML, 39% are CLL and 12.5% are CML. Of note is that children with Down syndrome are at increased risk for A.L.L. and A.M.L. Newborns with Down syndrome may also develop a transient myeloproliferative disorder (also called transient leukemia). Please see the assigned reading for the acute leukemias (BL043) for additional general information about A.L.L. and A.M.L. a) Childhood A.L.L.: Childhood A.L.L., which is the most common malignancy in childhood, will be covered in great detail in the lecture (BL046). The incidence of childhood A.L.L. is approximately 3 to 4 new cases per 100,000 children less than 15 years of age at diagnosis per year. A.L.L. can occur at any age. The peak incidence is between age two years and six years. Most children with A.L.L. who are one year of age or older at diagnosis have pre-B A.L.L. (Most of the others have pre-T cell A.L.L.). Mature B cell leukemias are treated as an advanced stage of Burkitt’s lymphoma, not as A.L.L. (see below under: Non-Hodgkin lymphomas). For most children and adolescents with A.L.L., treatment consists of systemic multi-agent chemotherapy (for a total of about 2.5 to 3.5 years) plus specific central nervous system preventive therapy. The intensity of treatment to be given is determined based on prognostic factors that are known to affect outcome. These include age and peripheral blood WBC at diagnosis, the presence of certain molecular or cytogenetic changes in the leukemia cells, rapidity of response to induction chemotherapy, and minimal residual disease measurements performed at the end of induction chemotherapy. Based on these prognostic factors, children are classified as having low risk, standard risk, high risk, very high risk, or infant A.L.L. About 80% of children with A.L.L. who are treated with current-state-of the art therapy are long terms survivors and are presumably cured. The “cure” rate is higher than this for children with low risk A.L.L. and much lower than this for children with very high risk A.L.L. or infant A.L.L. b) Childhood A.M.L.: For the most part, the clinical presentation, diagnosis and treatment of A.M.L. are very similar in children and adults. Of note is that children with Down syndrome who are diagnosed with A.M.L. prior four years of age generally have a more favorable outcome than children with A.M.L. who do not have Down syndrome. Furthermore, because of their more favorable outcome and also increased problems with acute toxicity from




2008/2009 therapy, children with Down syndrome who have A.M.L. and are diagnosed prior to age 4 years are treated with less intensive chemotherapy regimens (usually without stem cell transplantation) than children with A.M.L. who do not have Down syndrome. Transient myeloproliferative disorder of Down syndrome is beyond the scope of this lecture. Treatment of childhood A.M.L. includes intensive multi-agent chemotherapy and specific central nervous system preventive therapy. Biologic therapies may also be a component of the treatment regimen (e.g. ATRA for acute promyelocytic leukemia). In addition, some children and adolescents with A.M.L. will undergo allogeneic stem cell transplantation in first remission. Current long term survival rates for childhood A.M.L. range from about 50% to 80% (depending on the type of A.M.L.; the presence or absence of certain cytogenetic changes and molecular findings in the leukemia cells; the rapidity and completeness of the response to induction chemotherapy; whether the child has Down syndrome; and whether the treatment included allogeneic stem cell transplantation in first remission). NON-HODGKIN LYMPHOMA (NHL) Lymphomas comprise 15% of childhood malignancies, with Hodgkin lymphoma accounting for 8% and NHL accounting for 7% of the total. This is in contrast to lymphomas in adults, where non-hodgkin lymphomas comprise almost 90% of cases of lymphoma. The types of non-hodgkin lymphoma seen in children are also different than the types seen in adults. Most non-Hodgkin lymphomas seen in adults are indolent, whereas almost all cases of childhood non-hodgkin lymphoma are very aggressive, with undifferentiated histology and a propensity to early wide non-contiguous dissemination to the bone marrow and central nervous system. The primary tumor is often located in an extranodal site. Most cases of childhood NHL fall into one of into four main categories, based on histology, immuno-phenotype and the degree of cell differentiation.

a) Burkitt and Burkitt-like lymphoma/mature B-cell leukemia comprises 50% of cases of childhood NHL; it is a mature B-cell neoplasm, with a cell doubling time of about 24 hours. The most common site of origin in the non-endemic (i.e. non-African) form is in the abdomen - specifically, the lymphoid region of the distal ileum, appendix or cecum, or in retroperitoneal lymph nodes. Other possible primary sites include Waldeyer’s ring, the orbit, jaw, peripheral lymph nodes, skin, bone, brain, and the paraspinal region.

b) Diffuse large B-cell lymphoma (DLBL) comprises 10-20% of cases of childhood NHL; it is also a mature B-cell neoplasm. Of note is that the biologic features, clinical behavior and response of childhood DLBL to treatment differ from its adult counterpart with the same name. Pediatric DLBL can have a clinical presentation similar to that described above for Burkitt or Burkitt-like lymphoma. About 20% of pediatric DLBL originate in the mediastinum.

c) Lymphoblastic lymphoma, which comprises 20% of cases of childhood NHL and usually arises from a precursor T-cell. It most commonly presents with the rapid development of an anterior mediastinal mass and respiratory distress, often with an associated pleural effusion.

d) Anaplastic large cell lymphoma (ALCL) comprises about 10% of cases of childhood NHL and is usually of mature T-cell origin. ALCL can have a broad range of clinical presentations, including involvement of lymph nodes and a variety of extranodal sites (particularly skin and bone, and less often the gastrointestinal tract, lung, pleura, muscle, and other sites). It is often associated with fever and weight loss, and may have a prolonged waxing and waning course.




2008/2009


Other types of NHL are occasionally seen in childhood. Non-hodgkin lymphoma associated with immunodeficiency disorders usually have a mature B-cell phenotype and are more often DLBL than of Burkitt histology. Post-transplant lymphoproliferative disorders are also seen in children. A discussion of those disorders is beyond the scope of this lecture. Work-up and treatment: The diagnosis of NHL is made on tissue obtained by biopsy, or based on the findings in pleural fluid or bone marrow samples. Staging is done to determine the extent of involvement, particularly whether there has been dissemination to the bone marrow or CSF. Rapid staging is advised. Initiation of specific therapy is often a medical emergency, owing to the extremely rapid progression of NHL, frequently with the threat of complications due to the development of tumor lysis syndrome, airway obstruction or SVC syndrome (with mediastinal NHL’s) or spinal cord compression (by paraspinal NHL’s). Treatment begins with immediate assessment and stabilization of the child’s clinical and metabolic status. Once this has been achieved, multi-agent chemotherapy is initiated, including central nervous system prophylaxis. The details of the chemotherapy regimen depend on the type of NHL and the stage of disease. Children who present with a mediastinal mass are considered to have an advanced stage of disease. The only role for surgery is to biopsy the tumor. The only role for radiation therapy is as part of central nervous system prophylaxis in children with lymphoblastic lymphoma. The long term disease-free survival rate is approximately 80 to 90% overall for children with most types of non-hodgkin lymphoma. It is higher than this for children who present with localized disease. HODGKIN LYMPHOMA: The clinical aspects and treatment of Hodgkin Lymphoma are similar in children and adults, with the following exception: Because of the concern about the possible long term side effects of radiation therapy (including impaired growth of tissues in and near the radiation field, as well as the risk of a second malignant neoplasm), the dose of radiation therapy is kept as low as possible, if radiation therapy is given at all. Possible long term and/or late adverse effects of chemotherapy are also a concern. Approximately 90% of children and adolescents with Hodgkin disease are cured of their original cancer. Current areas of research include the identification of:

a. prognostic indicators (besides stage) that can help determine which children require more intensive, less intensive, or different treatment strategies for cure;

b. the types and frequency of late adverse effects arising from the treatment given; and c. effective treatment strategies for children with recurrent or refractory disease.

For additional general information about Hodgkin Lymphoma, please refert o BL035, and BL038.

BL047 (T6) Pediatric Oncology II University of Manitoba


2008/2009

1

Objectives: See objectives for BL046, Pediatric Oncology I TUTORIAL QUESTIONS: 1. A 9-year-old boy presents with a two month history of intermittent leg

pain. He is now refusing to walk. His automated CBC revealed the

following results:

Hgb 40 g/L

WBC 38.5x109L – 99% lymphocytes, 1% neutrophils

Platelet count 10x109/L

(a) What is the differential diagnosis and most likely diagnosis? (b) What would you do to make a definitive diagnosis (be specific)? (c) What other symptoms might be present on history? (d) What physical findings may be present? (e) What other lab abnormalities may be present on his bloodwork and imaging studies? (f) What are the most important prognostic factors for this disease? (g) How are treatment decisions made for children with this disease? (h) What are the principles of treatment? (i) What is the expected chance of cure?

BL047 (T6) Pediatric Oncology II University of Manitoba


2008/2009

2

2. A 14-year-old girl presents to the Emergency Room with a three week history of cough. She is in no respiratory distress. A chest x-ray is done and reveals the presence of a mediastinal mass (see x-ray). The emergency room physician pages you for advice. He wants to give the patient a dose of steroids now , if you agree.

(a) Provide a systematic approach to the differential diagnosis of a mediastinal mass in childhood. (b) What are the most likely possible diagnoses in this adolescent? (c) What emergency situations could arise related to the presence of the

mediastinal mass?

(d) How would you make a diagnosis? (e) What would you tell the emergency room physician about giving a dose

of steroids and why?

BL052 (T7) Coagulation Testing University of Manitoba

Faculty of Medicine

Med II / BL052

Barb Triggs-Raine

2008/2009

Note: replaces BL022 “Thrombin a molecular machine”

Objectives

1. Describe how quantities of proteins can be specifically measured by immunoassays

2. Describe how the function of proteins can be measured using biochemical assays, with

coagulation testing as an example

3. Describe the use of mixing tests to measure concentrations of functional clotting factors and

define deficiencies

4. Discuss how mutations or variants in protein expression can lead to significant disease, using

Factor V Leiden and the intron 22 inversion that commonly results in severe hemophilia A.

5. Compare the use of DNA-based (molecular) testing with protein-based biochemical testing for

diagnosis for hemostatic disorders

Readings: Hoffbrand, Moss and Pettit (5th ed.) pp. 270-277, 290-297, 306

METHODS OF TESTING COAGULATION

Laboratory testing plays a more central role in the

evaluation of hematological disorders than in

most other areas of medicine, and nowhere is this

more true than in the evaluation of hemostasis.

Testing may be performed to diagnose the reason

for bleeding in one patient, and the reason for

thrombosis in another patient, and to monitor

anticoagulant therapy in a third.

Some aspects of testing in coagulation rely on

functional biochemical analyses, often using

rather old-fashioned techniques based on timing

the visual detection of a fibrin clot. Even such old

approaches have been made more reproducible,

faster, and less expensive through the introduction

of automated analyzers. Other aspects are more

amenable to modern DNA-based analysis. Each

plays a useful role. The goal of this tutorial is to

use coagulation testing as an opportunity to

highlight the uses, advantages, and disadvantages

of these very different technologies in the

investigation of patients.

Hemophilia and Thrombophilia

Single-gene mutations can render patients prone

to bleed or prone to clot. An example of a

disorder that causes bleeding is hemophilia A. In

this disorder, a single mutation in the factor VIII

gene, which lies on the X chromosome, can lead

to a severe bleeding disorder, with spontaneous

events happening on the order of once a week.

Hemophilia is rare, affecting about one in 5000

boys. An example of a disorder that causes

clotting is Factor V Leiden. A single common

point mutation in factor V (a gene homologous to

factor VIII) renders the activated form of the

mutant factor V molecule insensitive to its

regulator, activated protein C. This mutation is

common, affecting about one in 20 people of

European background. The mutation confers an

increased risk of thrombosis, although the

spontaneous event rate is on the order of one

event per lifetime. Functional clotting-base assays

and gene-based assays are used in the evaluation

of both types of patients, and the results are often

complementary.

Clotting times

The most widely used technique for the

evaluation of coagulation is based on the

measurement of clotting time. In a clotting time

assay, an activator is added to patient plasma, and

the time until visible fibrin clot forms is recorded.

The plasma is generally anticoagulated with

citrate, which chelates extracellular calcium, but

which can be reversed by addition of extra

calcium. The commonly used clotting times are

the prothrombin time and partial thromboplastin

time. NOTE THAT THESE ASSAYS DO NOT

RECAPITULATE THE PHYSIOLOGY OF

HEMOSTASIS AS IT IS NOW UNDERSTOOD,

though they remain widely used and indeed

clinically very useful. Please refer to notes and

figures from BL019 and BL020.

In the prothrombin time, a source of negatively

charged phospholipid, purified tissue factor

protein, and calcium are added to citrated patient

plasma. Under the conditions used, a prothrombin

time (PT) reflects: (i) the activation of factor X by

the complex of tissue factor and factor VIIa,

yielding factor Xa; (ii) the activation of

prothrombin by the complex of factor Xa and its

cofactor factor Va, yielding thrombin; and finally

(iii) the clotting of fibrinogen by thrombin The

sequential series of reactions explains the lag time

to formation of clot. The prothrombin time is

sensitive to severe deficiencies of factor VII,

factor X, factor V, prothrombin, or fibrinogen.

The prothrombin time is generally reported

as the international normalized ratio, or INR.

In the activated partial thromboplastin time

(abbreviated aPTT or just PTT), a substance that

activates the ‘contact’ pathway is added, along

with negatively charged phospholipid and

calcium. The clotting time reflects the sequential

activation of factor XII and prekallikrein, factor

XI, factor IX, factor X, prothrombin, and

fibrinogen. Given the many steps in may come as

now surprise that the lag time is longer than for

the prothrombin time. Deficiencies of any of

those factors, or of the cofactors, factors V and

VIII, leads to prolongation of the aPTT.

In earlier times, clot was detected by tipping a

clear test tube back and forth and watching for

visually detectable clot formation. In most

automated instruments, what is monitored is the

turbidity of the solution: when fibrinogen in

solution clots, it turns cloudy, and this is readily

sensed by measuring light transmission through

the sample. Alternative approaches can be used;

one is to measure the activity of thrombin by

measuring its ability to cleave a small dye-

conjugated peptide, liberating the dye and

producing a readily-measured colour change.

A functional assay can be used to detect the factor

V Leiden mutation (and was indeed the basis for

the discovery of this mutation), based on

prolongation of the clotting time with addition of

activated protein C.

PCR-based assays

The factor V Leiden mutation is an example of a

founder effect: an ancestor in Asia Minor a little

over 20,000 years ago spread the mutation to all

of his/her offspring, ultimately encompassing

much of the population that subsequently

migrated into Europe. The single amino acid

substitution (506Arg→Glu) is the consequence of

a point mutation at the nucleotide level

(1691G→A). This is readily detected by PCR

techniques. This may be done in several ways, for

example:

• primers flanking the site of mutation may

be used to amplify the relevant segment,

and then the amplified segment may be

cleaved with a specific restriction

endonuclease, Mnl1, then the fragments

separated chromatographically; the

cleavage site for Mnl1 is lost in the

mutated sequence

• allele-specific PCR using primers that

recognize either the mutant or the normal

sequence may be used, under stringent

hybridizing conditions

• amplify the involved region with flanking

primers, then add a fluorescent probe

complementary to the normal sequence

spanning base 1691; then measure the

‘melting’ temperature, which will be

higher for the normal sequence (full base-

pair match with probe) than the mutant

sequence (1 base-pair mismatch with

probe sequence) [Light Cycler technique]

Southern methods

A large proportion of severe hemophilia A cases

arise because of a gene deletion (see figure 24.2

in Hoffbrand). This deletion can readily be

detected by Southern blotting. Genomic DNA is

digested with a restriction enzyme, subject to

electrophoresis, and transferred to filter paper. A

labelled DNA probe corresponding to the deleted

region of the factor VIII gene is than added;

absence of the expected band confirms the

deletion.

Sequencing

Because hemophilia A has in evolutionary times

conferred a major handicap to survival and

reproduction, it should be no surprise that most

cases occur in small kindreds or represent new

mutations (1/3 of cases). The factor VIII gene is

large (186,000 bases) and hundreds of different

mutations have been catalogued that will impair

translation of the message, expression of the gene

product, or function of the protein secreted. Thus

the only way to identify the mutation in many

patients is to sequence the entire gene. Despite

progressive advances in automated gene-

sequencing technology, this remains a laborious

and expensive proposition.

CASES

Case 1

A 25 year old woman develops pain and swelling in the right leg. Investigation with ultrasound reveals

the presence of a blood clot in the femoral vein. Prior to this she was well. Her mother and one of her

mother’s brothers have also suffered from venous thrombosis.

If the susceptibility to clotting in this woman and her family is due to Factor V 506Arg→Gln (Factor V

Leiden), describe:

a) how this mutation makes her more susceptible to clotting?

b) how you might devise a clotting-based assay to detect the defect?

c) how might you devise a molecular assay to detect the defect?

d) what are advantages to each approach?

Case 2

A 1 year old develops a hemarthrosis in his knee without any apparent trauma. His mother’s brother

died at the age of 5 from hemorrhage after a tonsillectomy.

a) how can you distinguish between hemophilia A (Factor VIII deficiency) and hemophilia B (Factor

IX deficiency)?

b) how do you measure the level of Factor VIII in his plasma?

c) assume the patient’s Factor VIII is <1% of normal. His mother is 16 weeks pregnant and wants to

know if the baby she is carrying also has hemophilia. How can this be approached?

BL053 (L) Hemoglobinopathies University of Manitoba

Faculty of Medicine

Med II / BL053

Dr. D. Houston

2008/2009

Objectives:

After attending this overview lecture, participating in discussion, reading the assigned materials,

and reflecting upon them, the student will be able to:

• List the common clinical features of sickle cell anemia, and which complications of the

disorder should be screened for

• Describe interventions used to manage sickle cell anemia, including transfusion,

hydroxyurea, analgesia, and supportive care

• Discuss the complications of chronic transfusion therapy, including iron overload, and

discuss chelation therapy

Reference: Hoffbrand, Pettit and Moss, pages 72-93 (5th ed.)

notes to follow

July-22-09

9:25 AM

BL053 notes Page 1

BL053 notes Page 2

BL053 notes Page 3

BL053 notes Page 4

BL053 notes Page 5

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