Hemoglobinopathies

Hemoglobin structure Hemoglobin structure

globin globin

globin globin

Hgb A tetramer

Globin chain synthesisGlobin chain synthesis cluster - chromosome 16

cluster - chromosome 11

Gower 1

Portland Embryonic

Gower II

F Fetal <1%

A2 1.5-3.5%Adult

A >95%

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HAEMOGLOBINOPATHIES

Qualitative

(structural)

Combined (Quantitative &

Qualitative)

Quantitative

ThalassemiaThalassemia

Heterogenous group of disorders due to an Heterogenous group of disorders due to an imbalance of imbalance of and and globin chain synthesis globin chain synthesis• thalssemia: thalssemia: -globin chain production decreased-globin chain production decreased• thalassemia: thalassemia: globin chain production decreased globin chain production decreased

The globin chains that are produced are normalThe globin chains that are produced are normal

Quantitative deficiency:Quantitative deficiency:• o thalassemia: No o thalassemia: No -globin chain is made-globin chain is made• + thalassemia: decreased + thalassemia: decreased -globin chain is made-globin chain is made

With 4 With 4 genes and 2 genes and 2 genes there is wide genes there is wide phenotypic variation phenotypic variation

Types of ThalassemiaTypes of Thalassemia thalthal: : excess of excess of globins globins, , leading toleading to formation of formation of globin tetramers globin tetramers

((44) that accumulate in the erythroblast ) that accumulate in the erythroblast , , leading to ineffective leading to ineffective erythropoiesiserythropoiesis.. Two types of mutations, the β0 in which no β globin Two types of mutations, the β0 in which no β globin chains are produced and β+, in which some β chains are produced but at chains are produced and β+, in which some β chains are produced but at a reduced rate. a reduced rate.

thalthal : : excess of excess of globins globins, , leadingleading to the formation of to the formation of globin globin tetramers (tetramers (44) called hemoglobin H. ) called hemoglobin H. Results inResults in hemolysis, generally hemolysis, generally shortening the life span of the red cell. Hemoglobin H-Constant Spring shortening the life span of the red cell. Hemoglobin H-Constant Spring disease is a more severe form of this hemolytic disorder. disease is a more severe form of this hemolytic disorder. MostMost severe severe formform is is thalassemia major, in which fetus produces no thalassemia major, in which fetus produces no globins, globins, which is generally incompatible with life.which is generally incompatible with life.

Demographics: ThalassemiaDemographics: Thalassemia

• Found most Found most frequently in the frequently in the Mediterranean, Africa, Mediterranean, Africa, Western and Western and Southeast Asia, India Southeast Asia, India and Burmaand Burma

• Distribution parallels Distribution parallels that of Plasmodium that of Plasmodium falciparumfalciparum

Genetics of Thalassemia

Alpha ThalassemiaAlpha Thalassemia Inadequate production of alpha chainsInadequate production of alpha chains Hemoglobin analysis normal; can be detected by Hemoglobin analysis normal; can be detected by globin gene globin gene

analysisanalysis Absence of 1-2 alpha chains Absence of 1-2 alpha chains

• CommonCommon• AsymptomaticAsymptomatic• Does not require therapyDoes not require therapy

Absence of 3 alpha chainsAbsence of 3 alpha chains• Microcytic anemia (Hgb 7-10)Microcytic anemia (Hgb 7-10)• SplenomegalySplenomegaly

Absence of 4 alpha chainsAbsence of 4 alpha chains• Hydrops fetalis (non-viable)Hydrops fetalis (non-viable)

chains Hgb (g/dl) MCV (fl) RDW

/ Normal Normal Normal

/- 12-14 75-85 Normal

-/- or - -/ 11-13 70-75

- -/- 7-10 50-60

- -/- - - - -

Laboratory Findings in Alpha Laboratory Findings in Alpha ThalassemiaThalassemia

Minor (Trait)Minor (Trait) / / ++ or or / / ° ° 10-13 10-13

IntermediaIntermedia ++//++ 7-10 7-10

MajorMajor ++//° or ° or °/°/° ° < 7 < 7

ClinicalSyndrome Genotype Hemoglobin (g/dl)

Beta ThalassemiaBeta ThalassemiaInadequate production of chains

Minor (Trait) Minor (Trait) / / ++ or or / / ° 90-94 3.5-8 1-10° 90-94 3.5-8 1-10

IntermediaIntermedia ++//++ 5-60 2-8 5-60 2-8 20-80%20-80%

MajorMajor ++//°° 2-10 2-10 1-6 1-6 >85>85

°/°/° ° 0 0 1-6 1-6 >94 >94

ClinicalSyndrome Genotype A A2 F

Beta Thalassemia - Hgb analysisBeta Thalassemia - Hgb analysis

Hemoglobin analysis: Increased levels of Hgb A2 and Hgb F

Signs and symptoms

Thalassemia carriers (trait): Usually no signs or symptoms are apparent,

except for a mild anemia. Carriers are usually initially detected

through screening, or when performing routine CBC (complete blood count). Later it can be confirmed using hemoglobin electrophoresis.

Signs and symptoms- Cont

Thalassemia major:

Signs such as paleness and growth retardation, are readily detectable since the first year of life. Those are mainly due to severe anemia. Later bone deformities and hepato-splenomegaly develops.

Laboratory diagnosisLaboratory diagnosis

Thalassemia minor:Thalassemia minor: -Blood smear shows hypochromia and -Blood smear shows hypochromia and

microcytosis (similar to Iron Deficiency Anemia).microcytosis (similar to Iron Deficiency Anemia). -Blood indices: MCV< 75 fl, Hb usually> 10, -Blood indices: MCV< 75 fl, Hb usually> 10,

Hematocrit> 30%, RDW < 14%.Hematocrit> 30%, RDW < 14%. -Hemoglobin A2 often elevated > 3%, sometimes -Hemoglobin A2 often elevated > 3%, sometimes

reaching 7-8%. reaching 7-8%.

Laboratory diagnosis- ContLaboratory diagnosis- Cont Thalassemia major:Thalassemia major: -Blood smear shows profound microcytic -Blood smear shows profound microcytic

anemia, with extreme hypochromia, anemia, with extreme hypochromia, tear drop, target cells and nucleated tear drop, target cells and nucleated RBCs.RBCs.

-Hemoglobin may be very low at 3-4 -Hemoglobin may be very low at 3-4 g/dl.g/dl.

Primary Laboratory InvestigationPrimary Laboratory InvestigationThalassemiaThalassemia

• Severe cases present withSevere cases present with• MicrocytosisMicrocytosis• HypochromiaHypochromia• PoikilocytosisPoikilocytosis• RBC counts higher than expected for the level RBC counts higher than expected for the level

of anemiaof anemia

Thalassemia majorThalassemia major

Prenatal diagnosisPrenatal diagnosis

Early prenatal diagnosis can be done using Early prenatal diagnosis can be done using first fetal blood sampling, and later chorion first fetal blood sampling, and later chorion villus biopsy and direct analysis of the villus biopsy and direct analysis of the globin genes.globin genes.

The error rate in experienced centers is now The error rate in experienced centers is now well under 1%.well under 1%.

Beta thalassemia major Beta thalassemia major treatmenttreatment

TransfusionTransfusion Iron chelationIron chelation stem cell transplantstem cell transplant

Management and treatmentThalassemia minor (trait) :

No need for any treatment, since the carriers are usually symptomless.

Thalassemia major:

The severe life-threatening anemia, requires regular life long blood transfusion, to compensate for damaged red blood cells.

Management and treatment- Cont

Thalassemia Major:

The continuous blood transfusion will eventually lead to iron overload, which must be treated with chelation therapy to avoid organ failure.

Source: Cooley’s Anemia Foundation

Management and treatment- Cont Thalassemia Major -Continued:

Other novel treatments like bone-marrow transplantation are very costly.

New treatments includes the use of oral chelators, to replace the chelation treatment using Desferal delivered by infusion under the skin through a battery-operated pump.

Gene therapy is also an option still researched

Prevention effortsPre marital screening to make sure that the couple are not both carriers.Provision of counseling and health education for the thalassemics, their families and the public .Provision of prenatal testing for thalassemia.Reduction of marriages between relatives.

Approach to Beta ThalassemiaApproach to Beta Thalassemia

Screening/counselingScreening/counseling

RBC transfusion therapyRBC transfusion therapy

Agents to increase hemoglobin F Agents to increase hemoglobin F (Hydroxyurea)(Hydroxyurea)

Bone marrow transplantation Bone marrow transplantation

Hemoglobinopathy-antenatal Hemoglobinopathy-antenatal diagnosisdiagnosis

Test partners of heterozygous or Test partners of heterozygous or affected individualsaffected individuals

Antenatal diagnosis from DNA Antenatal diagnosis from DNA obtained by chorionic villus obtained by chorionic villus sampling, or by amniocentesissampling, or by amniocentesis

Clinical PresentationsClinical Presentationsof Abnormal Hemoglobinsof Abnormal Hemoglobins

Sickling disorder Sickling disorder Thalassemia or microcytic anemiaThalassemia or microcytic anemia CyanosisCyanosis ErythrocytosisErythrocytosis Hemolytic anemiaHemolytic anemia Asymptomatic (screening or family study)Asymptomatic (screening or family study)

Structural Haemoglobin VariantsStructural Haemoglobin Variants

Mostly: single AA substitution.Mostly: single AA substitution. Over 300 variants.Over 300 variants. Many:Many:

Harmless, Accidently discovered Harmless, Accidently discovered (screening).(screening).

May alter Hb stability clinical May alter Hb stability clinical disorders.disorders.

The Sickling DisordersThe Sickling DisordersDefinitionDefinitionA group of disorders characterised by sickling of A group of disorders characterised by sickling of

RBC on deoxygenation (unstable Hb).RBC on deoxygenation (unstable Hb).Include:Include: Heterozygous state of Hb S (sickle cell Heterozygous state of Hb S (sickle cell traittrait, ,

genotype genotype A SA S).). Homozygous state of Hb S (sickle disease, Homozygous state of Hb S (sickle disease,

genotype S S).genotype S S). Compound heterozygous state of Hb S with Hb Compound heterozygous state of Hb S with Hb

SC, SD, SE and other structural variants.SC, SD, SE and other structural variants. Inheritance of sickle cell together with Inheritance of sickle cell together with

thalassemia.thalassemia.

Pathogenesis Pathogenesis

Substitution of valine for Substitution of valine for Glutamic acid at position 6 in the Glutamic acid at position 6 in the B chain (BB chain (B66 Glut valine) = Hb S. Glut valine) = Hb S.

Gene defect:Gene defect:Replacement of adenine by thymine Replacement of adenine by thymine

in the DNA code (GAG GTG).in the DNA code (GAG GTG).

Patho-physiology of sikle cell disease

Clinical picture Clinical picture In infancy:In infancy: Higher levels of Hb F in neonates protective 8 – Higher levels of Hb F in neonates protective 8 –

20 weeks.20 weeks. Chronic hemolytic anaemia + Jaundice.Chronic hemolytic anaemia + Jaundice. Growth & developmental retardation (skeletal Growth & developmental retardation (skeletal

deformities): Frontal bossing, short stature, deformities): Frontal bossing, short stature, inequalities between upper & lower segments, inequalities between upper & lower segments, deformities ………deformities ………

Anaemia & jaundice (ch. Haemolysis).Anaemia & jaundice (ch. Haemolysis). Dactylitis.Dactylitis. Splenomegaly resolves by infarcts (auto Splenomegaly resolves by infarcts (auto

spelenectomy).spelenectomy). Splenic sequestration.Splenic sequestration. Fulminant pnuemococcal septicemia.Fulminant pnuemococcal septicemia. Chronic leg ulcers.Chronic leg ulcers.

Paediatric emergencies

1 High fever ? Septicaemia.

2 Acute splenic sequestration.

3 Aplastic crisis.

4 Stroke.

5 Acute chest syndrome.

6 Hand-foot syndrome.

Pneumococcal septicaemia

Prophylactic penicillin works but pneumococcal vaccine does not work well when it is most needed.

Penicillin is given as monthly IM injections to ensure compliance.

Given 4 months to 4 years when last injection is given with 23V vaccine.

Protocol will need modifying with rapid emergence of penicillin resistance and conjugate vaccine.

Definition Sudden increase in spleen size, fall in Hb > 2g/dl, and reticulocytosis. The spleen size decreases spontaneously or post transfusion. Accounted for 24% deaths in first 10 years of study.

Acute Splenic Sequestration

Aplastic crisis

Human parvovirus B19 infection accounts for virtually all clinically defined episodes.

High secondary infection rate so that siblings of affected cases should be closely monitored.

Immunity appears to be life long with no recurrent episodes.

Most cases can be safely treated by outpatient transfusion.

PathologyPathology

• Mostly ischaemic in childhood Mostly ischaemic in childhood associated with blockage of major associated with blockage of major cerebral vessels.cerebral vessels.

• Mostly haemorrhagic in adults often Mostly haemorrhagic in adults often associated with berry aneurysms at associated with berry aneurysms at base of brain.base of brain.

Stroke

Prevention of first or recurrent strokesPrevention of first or recurrent strokes

• Chronic transfusion programmesChronic transfusion programmes• HydroxyureaHydroxyurea• Bone marrow transplantationBone marrow transplantation

Stroke

Chronic transfusion programmes Chronic transfusion programmes (problems)(problems)

• Regular supply of bloodRegular supply of blood• Transfusion-acquired infectionsTransfusion-acquired infections• Red cell alloimmunizationRed cell alloimmunization• Transfusion reactionsTransfusion reactions• Iron accumulation necessitating chelationIron accumulation necessitating chelation• Venous accessVenous access

Stroke

Bone marrow transplantationBone marrow transplantation• Cost and expertise involvedCost and expertise involved• Availability of HLA matched marrow (16%)Availability of HLA matched marrow (16%)• Mortality of 5-10% within 3 monthsMortality of 5-10% within 3 months• Long term effects of marrow ablationLong term effects of marrow ablation• Graft versus host diseaseGraft versus host disease• Long term effect on growth and fertilityLong term effect on growth and fertility

Stroke

Acute chest syndrome

• complex pathology resulting from infection, infarction, fat embolism, acute pulmonary sequestration.

• major cause of death at all ages after 2 years• monitor closely with

pulse oximetry • exchange transfusion

may be life-saving

Acute chest syndrome

Radiological change in 3d with acute pulmonary sequestration following gallbladder surgery

Hand-foot Syndrome

Hand-foot Syndrome

Note avascular necrosis of bone marrow of metacarpal (left) compared with normal marrow in proximal phalanx (right)

Painful CrisisPainful Crisis Hospitilization.Hospitilization. Resustation: IV fluid, Oxygen, Antibiotics.Resustation: IV fluid, Oxygen, Antibiotics. Analgesia:Analgesia:

• Given on regular basis (not on need basis).Given on regular basis (not on need basis).• Early in starting crisis.Early in starting crisis.• Prefered smaller, frequent than larger over Prefered smaller, frequent than larger over

longer periods (swings of euphoria& pain).longer periods (swings of euphoria& pain).• Inadequate doses= inadequate control.Inadequate doses= inadequate control. HaemolysisHaemolysis• Daily Hb, Retics TRANSFUSIONDaily Hb, Retics TRANSFUSION AplasiaAplasia

Laboratory DiagnosisLaboratory Diagnosissickle Cell Diseasesickle Cell Disease

1- Haemogram:1- Haemogram: Variable anaemia (Hb 6 – 8 gm/dl).Variable anaemia (Hb 6 – 8 gm/dl). TLC & Platalet: N, .TLC & Platalet: N, . retics (10 – 20%).retics (10 – 20%). Smear:Smear:

• Sickled erythrocytes, normoblasts, fragmented Sickled erythrocytes, normoblasts, fragmented RBCs, target cells (SC), basophilic stippling, RBCs, target cells (SC), basophilic stippling, Howell-Jolly & pappenheimer bodies Howell-Jolly & pappenheimer bodies (hyposplensim).(hyposplensim).

2- ESR:2- ESR:Low as sickle cells fail to form rouleaux.Low as sickle cells fail to form rouleaux.

3- Screening Tests3- Screening Tests a) Sickling test:a) Sickling test:

• In vitro deoxygenation of Hb to induce In vitro deoxygenation of Hb to induce sickling.sickling.

• So. Metabisultite + cell suspension and So. Metabisultite + cell suspension and immediately covered cause sickling.immediately covered cause sickling.

b) Solubility test:b) Solubility test:• Solubility testing-Dithionite tube testSolubility testing-Dithionite tube test Modification of sickling test:Modification of sickling test:

Lysing RBCs release Hb then add Lysing RBCs release Hb then add metabisulfite to Hb solution + DW form metabisulfite to Hb solution + DW form Dark pptate indicates Hb S.Dark pptate indicates Hb S.

Screening for Sickle Cell TraitScreening for Sickle Cell Trait and and DiseaseDisease

RBC lysate with RBC lysate with concentrated phosphate concentrated phosphate buffer and sodium buffer and sodium hydrosulfitehydrosulfite

Incubate 10-20 minIncubate 10-20 min

4- Hb Electrophoresis4- Hb Electrophoresis On alk pH or with agar strach gel.On alk pH or with agar strach gel. The electrophoretic pattern of normal & abnormal The electrophoretic pattern of normal & abnormal

Hb depends on the amount of –ve charges that Hb depends on the amount of –ve charges that they carry.they carry.

The net charge varies due to the gain of –ve The net charge varies due to the gain of –ve charges in AA substitution.charges in AA substitution.

Hb A > Hb S (B Hb A > Hb S (B Glu Glu val val) > Hb C (B) > Hb C (B Glu Glu lys lys))-ve mobility.-ve mobility.So, Hb S migerates slower than Hb A.So, Hb S migerates slower than Hb A.

Several ab Hb may overlap.Several ab Hb may overlap.e.g. Hb S & D, Hb C with E.e.g. Hb S & D, Hb C with E.

Hb SC & Hb SO Arab: the clinical data may be the Hb SC & Hb SO Arab: the clinical data may be the only clue.only clue.

5- HPLC & PCR Amplication5- HPLC & PCR Amplication

Hemoglobin electrophoresis:Identification of abnormal hemoglobins

HPLC:Sickle cell anemia (Hgb SS)

6- Prenatal diagnosis6- Prenatal diagnosis When parents are both sickle trait to determine fetal When parents are both sickle trait to determine fetal

homozygosity.homozygosity. prenatal measuring of the Bprenatal measuring of the Bss / B / Baa synthetic ratio in the synthetic ratio in the

fetal blood.fetal blood. May be made from:May be made from:

• 11stst trimester chronic villus sampling (10 wk gestation). trimester chronic villus sampling (10 wk gestation).• 22ndnd trimester fetal RBCs from umbilical cord or trimester fetal RBCs from umbilical cord or

trophoblast.trophoblast.• 33rdrd trimester DNA fetal fibroblast (amniotic fluid). trimester DNA fetal fibroblast (amniotic fluid).

Restriction endonuclease:Restriction endonuclease:To detect sickle mutation in the fetal B globin gene.To detect sickle mutation in the fetal B globin gene.

Olignucleotide probes specific for globin point Olignucleotide probes specific for globin point mutation.mutation.

PCR amplification followed by restriction enzyme PCR amplification followed by restriction enzyme digestion of amplified DNA.digestion of amplified DNA.

ARMS (Amplification Refractory Mutation System) ARMS (Amplification Refractory Mutation System) PCR.PCR.

Sickle Cell Trait (Hb A – S)Sickle Cell Trait (Hb A – S) The HETEROZYGOUS from Hb A – s.The HETEROZYGOUS from Hb A – s. Sickling does not occur under normal conditions.Sickling does not occur under normal conditions. CLINICAL FEATURES:CLINICAL FEATURES: No clinical disability unless extreme anoxia.No clinical disability unless extreme anoxia. May suffer vaso-occlusive crisis (hypoxia, May suffer vaso-occlusive crisis (hypoxia,

anasthesia, pregnancy, or flying in unpressurized anasthesia, pregnancy, or flying in unpressurized aricrafts).aricrafts).

Renal papillary necrosis cause haematuria, Renal papillary necrosis cause haematuria, hypothenuria (dil urine).hypothenuria (dil urine).

Racial background.Racial background.Sickling test if +ve during anasthesiaSickling test if +ve during anasthesia

adequate O2

Avoid postop. dehyd.

Management of Sickle Cell DiseaseManagement of Sickle Cell Disease

Maintenance TherapyMaintenance Therapy

1.1. Prenatal diagnosis programs.Prenatal diagnosis programs.

2.2. Avoid: Infection, Hypoxia, Dehyration, Avoid: Infection, Hypoxia, Dehyration, Exhaustion, Psychological stress.Exhaustion, Psychological stress.

3.3. Prophylactic:Prophylactic:• Oral penicillin infection & crisis.Oral penicillin infection & crisis.• Vaccination: pneumococcal, Vaccination: pneumococcal,

meningococcal & H. influenza.meningococcal & H. influenza.• Malaria (if endomic).Malaria (if endomic).

For Patients:For Patients:

1.1. Regular blood transfusion is not required.Regular blood transfusion is not required.2.2. Adaptation to anaemia is successful (Hb S has Adaptation to anaemia is successful (Hb S has

low Olow O22 affinity). affinity).3.3. Early & prompt treatment of infection.Early & prompt treatment of infection.4.4. Folic acid supplements.Folic acid supplements.5.5. Major surgical emergencies or recurrent crisis: Major surgical emergencies or recurrent crisis:

HYPERTREANSFUSION OR EXCHANGE. HYPERTREANSFUSION OR EXCHANGE.6.6. Pregnancy:Pregnancy:

• Good antenatal, folic acid.Good antenatal, folic acid.• If Hb or crisis regular transfusion.If Hb or crisis regular transfusion.

7.7. Oral contraceptive ? Veno-occlusive crisis Oral contraceptive ? Veno-occlusive crisis (contraindicated).(contraindicated).

8- Occular (Retinopathy): LASER 8- Occular (Retinopathy): LASER viterous Hage.viterous Hage.

9- Haematuria Warning sign 9- Haematuria Warning sign 10- CRF: as other causes, renal 10- CRF: as other causes, renal

transplant.transplant.11- Hip pain (aspectic necrosis): Hip 11- Hip pain (aspectic necrosis): Hip

replacement.replacement.12- Splenomegaly with hypersplenism, 12- Splenomegaly with hypersplenism,

sequestration crisis: splenectomy.sequestration crisis: splenectomy.

PriapismPriapism Stilbosterol 5 mg daily.Stilbosterol 5 mg daily. Conservative (1Conservative (1stst 24hs.: Hydration, Analgesia, 24hs.: Hydration, Analgesia,

Exchange transfusion).Exchange transfusion). Surgery: Cavernosus-Spongiosum shunt.Surgery: Cavernosus-Spongiosum shunt. Anticoagulants: little value.Anticoagulants: little value.

Leg UlcerLeg Ulcer Unsatisfactory, bed rest, debridment (often Unsatisfactory, bed rest, debridment (often

relapse).relapse). Skin graft limited success.Skin graft limited success. Transfusion dose not speed healing.Transfusion dose not speed healing.

New TherapiesNew TherapiesI- Antisikling Agents:I- Antisikling Agents:

Agents that increase Hb F.Agents that increase Hb F.a) Hydroxyurea:a) Hydroxyurea:

Painful crisis by:Painful crisis by:Hb F, cell volume, alter cell hydration, Hb F, cell volume, alter cell hydration,

WBCs.WBCs.Only in adults (child leukomogenic).Only in adults (child leukomogenic).

b) rhu EPO:b) rhu EPO:Additive effect with hydroxyurea.Additive effect with hydroxyurea.

c) 5-Azacytidine:c) 5-Azacytidine: Prevents methylation of the Y glob gene Hb F.Prevents methylation of the Y glob gene Hb F. Risk of 2ry malignancy.Risk of 2ry malignancy.

d) Short chain fatty acids:d) Short chain fatty acids:Butyrate Hb F.Butyrate Hb F.

II- Bone Marrow Transplantant:II- Bone Marrow Transplantant:Controversial.Controversial.

III- Gene Therapy:III- Gene Therapy: Potentially curative.Potentially curative. Gene transfer attempted with Gene transfer attempted with

variable results.variable results. Expression exogenous gene too low Expression exogenous gene too low

to be of benefit.to be of benefit.