HAEMOLYTIC HAEMOLYTIC ANAEMIAANAEMIA

Dr. Mustafijur Rahman (Rizvi)Dr. Mustafijur Rahman (Rizvi)RegistrarRegistrar

Medicine unit IIIMedicine unit III

What is hemolytic What is hemolytic anemia?anemia?•Hemolytic anemia is a Hemolytic anemia is a

disorder in which the red disorder in which the red blood cells are destroyed blood cells are destroyed prematurely.prematurely.

•RBCs are destroyed faster RBCs are destroyed faster than the bone marrow can than the bone marrow can produce them. produce them.

Features of HaemolysisFeatures of Haemolysis

•Red cell destruction overloads Red cell destruction overloads pathways for Hb breakdown , pathways for Hb breakdown , causing a modest rise in un-causing a modest rise in un-conjugated bilirubin in the blood conjugated bilirubin in the blood and mild jaundice.and mild jaundice.

•There is an increase in urinary There is an increase in urinary urobilinogen.urobilinogen.

Features of Features of Haemolysis(cont)Haemolysis(cont)

•LDHLDH

•Nucleated red cell precursors Nucleated red cell precursors appear in the blood as well as appear in the blood as well as reticulocytosis.reticulocytosis.

•Leuco-erythroblastic blood film.Leuco-erythroblastic blood film.

Features of Features of Haemolysis(cont)Haemolysis(cont)

•Activation of the bone marrow Activation of the bone marrow can result in a neutrophilia and can result in a neutrophilia and immature granulocytes immature granulocytes appearing in the blood to cause a appearing in the blood to cause a leuco-erythroblastic blood film. leuco-erythroblastic blood film.

Features of Features of Haemolysis(cont)Haemolysis(cont)

•The appearances of the red cells The appearances of the red cells may give an indication of the may give an indication of the likely cause of the haemolysislikely cause of the haemolysis

•Spherocytes are small, dark red Spherocytes are small, dark red cells which suggest autoimmune cells which suggest autoimmune haemolysis or hereditary haemolysis or hereditary spherocytosis. spherocytosis.

Features of Features of Haemolysis(cont)Haemolysis(cont)

•Sickle cells suggest Sickle cells suggest haemoglobinopathy.haemoglobinopathy.

•Red cell fragments indicate Red cell fragments indicate microangiopathic haemolysis. microangiopathic haemolysis.

Features of Features of Haemolysis(cont)Haemolysis(cont)

• The compensatory erythroid The compensatory erythroid hyperplasia may give rise to folate hyperplasia may give rise to folate deficiency, when the blood findings deficiency, when the blood findings will be complicated by the presence will be complicated by the presence of megaloblastosis. Measurement of of megaloblastosis. Measurement of red cell folate is unreliable in the red cell folate is unreliable in the presence of haemolysis and serum presence of haemolysis and serum folate will be elevated. folate will be elevated.

Intravascular haemolysisIntravascular haemolysis

• Free haemoglobin is released into Free haemoglobin is released into the plasma. the plasma.

• Decreased amount of haptoglobin.Decreased amount of haptoglobin.

• Methaemoglobin is produced.Methaemoglobin is produced.

• Methaemoglobin plus Methaemoglobin plus albumin>>>methaemalbumin.albumin>>>methaemalbumin.

Intravascular haemolysis(cont)Intravascular haemolysis(cont)

• Methaemoglobin is degraded and Methaemoglobin is degraded and any free haem is bound to a second any free haem is bound to a second binding protein termed haemopexin.binding protein termed haemopexin.

• Free haemoglobin may appear in the Free haemoglobin may appear in the urine. When fulminant, this gives rise urine. When fulminant, this gives rise to black urine, as in severe to black urine, as in severe falciparumfalciparum malaria infection. malaria infection.

• Haemosiderinuria, which is always Haemosiderinuria, which is always indicative of intravascular indicative of intravascular haemolysis. haemolysis.

Extravascular haemolysisExtravascular haemolysis

• Physiological red cell destruction in Physiological red cell destruction in the liver or spleen.the liver or spleen.

• No free haemoglobin in the plasma. No free haemoglobin in the plasma.

• Increased bilirubin, reticulocytosis.Increased bilirubin, reticulocytosis.

Compensated haemolytic Compensated haemolytic diseasedisease• Shortening of the red cell life span does Shortening of the red cell life span does

not necessarily result in anaemia as not necessarily result in anaemia as compensatory bone marrow compensatory bone marrow hyperplasia may increase red cell hyperplasia may increase red cell production six to eight fold and production six to eight fold and maintain a normal haemoglobin level. maintain a normal haemoglobin level. So anaemia is not invariable in So anaemia is not invariable in haemolytic disorders. But they show haemolytic disorders. But they show reticulocytosis or erythroid hyperplasia reticulocytosis or erythroid hyperplasia of bone marrow. of bone marrow.

MorphologyMorphology

•Most haemolytic anaemias Most haemolytic anaemias are either normocytic and are either normocytic and

normochromic or macrocytic normochromic or macrocytic and normochromic.and normochromic.

Types of Hemolytic Types of Hemolytic AnemiaAnemia

• Intrinsic(intra-corpuscular)Intrinsic(intra-corpuscular) - the - the destruction of the red blood cells due to a destruction of the red blood cells due to a defect within the red blood cells defect within the red blood cells themselves. These are mainly congenital.themselves. These are mainly congenital.

• Extrinsic(extra-corpuscular)Extrinsic(extra-corpuscular) - red blood - red blood cells are produced healthy but are later cells are produced healthy but are later destroyed by becoming trapped in the destroyed by becoming trapped in the spleen, destroyed by infection, or spleen, destroyed by infection, or destroyed from drugs that can affect red destroyed from drugs that can affect red blood cells.These disorders are acquired . blood cells.These disorders are acquired . These are immune or non-immune These are immune or non-immune mechanism.mechanism.

Aetiological classification of Aetiological classification of haemolytic anaemiahaemolytic anaemia

(A)(A)Due to intra-corpuscular Due to intra-corpuscular mechanisms:mechanisms:

1. Congenital1. Congenital

2. Acquired2. Acquired

Classification(cont.)Classification(cont.)

1. Congenital1. Congenital >Membrane defects >Membrane defects (Hereditary (Hereditary

spherocytosis, Hereditary spherocytosis, Hereditary elliptocytosis).elliptocytosis).

>Haemoglobin defects >Haemoglobin defects (Haemoglobinopathies, Thalassemia)(Haemoglobinopathies, Thalassemia)

>Enzyme defects >Enzyme defects (G6PD deficiency)(G6PD deficiency)

2. Acquired2. Acquired

(Paroxysmal nocturnal (Paroxysmal nocturnal haemoglobinuria)haemoglobinuria)

Classification(cont.)Classification(cont.)

(B) Due to extra-corpuscular (B) Due to extra-corpuscular mechanism:mechanism:

1.1. CongenitalCongenital (None) (None)

2. Acquired2. Acquired

Classification(cont.)Classification(cont.)

1.1. CongenitalCongenital (None) (None)

2. Acquired2. Acquired >Immune mechanisms >Immune mechanisms (Auto-immune acquired (Auto-immune acquired

haemolytic anaemia/warm and haemolytic anaemia/warm and cold antibody, Haemolytic disease cold antibody, Haemolytic disease of the newborn, Incompatibe of the newborn, Incompatibe blood transfusion,blood transfusion,

Drug induced haemolytic Drug induced haemolytic anaemia)anaemia)

>Non-immune mechanisms >Non-immune mechanisms (Cardiac haemolytic anaemia,(Cardiac haemolytic anaemia,

micro-angiopathic haemolytic micro-angiopathic haemolytic anaemia,anaemia,

March haemoglobinuria)March haemoglobinuria)

>Miscellaneous >Miscellaneous (Haemolytic anaemia due to direct (Haemolytic anaemia due to direct

action of drugs and chemicals, action of drugs and chemicals,

infection, burn, lead poisoning)infection, burn, lead poisoning)

Basic principle of diagnosis Basic principle of diagnosis of haemolytic anaemiaof haemolytic anaemia

• Following three questions Following three questions must be answered-must be answered-

1.1.Is the anaemia Is the anaemia haemolytic ?haemolytic ?

>Evidence of increased >Evidence of increased haemoglobin breakdown haemoglobin breakdown ( jaundice, hyper-( jaundice, hyper-bilirubnaemia,reticulocytosis,bilirubnaemia,reticulocytosis,

plasma haptoglobin, LDH).plasma haptoglobin, LDH). > Bone marrow regeneration.> Bone marrow regeneration.

2. If so, is the red cell 2. If so, is the red cell destruction intra-vascular or destruction intra-vascular or extra-vascular?extra-vascular?

> > Presence or absence of free Presence or absence of free haemoglobin haemoglobin

and haemoglobin breakdown products and haemoglobin breakdown products

in the plasma and urine.in the plasma and urine.

(Haemoglobinaemia , Haemoglobinuria, (Haemoglobinaemia , Haemoglobinuria,

Methaemalbuminaemia, Methaemalbuminaemia, Haemosiderinuria)Haemosiderinuria)

3. What is the aetiology?3. What is the aetiology?

By the clinical features By the clinical features and the results of the and the results of the special investigations. special investigations.

Auto-immune haemolytic Auto-immune haemolytic anaemiaanaemia

•Due to red cell autoantibodies(IgG Due to red cell autoantibodies(IgG or IgM, or more rarely IgE or IgA )or IgM, or more rarely IgE or IgA )

• If an antibody avidly fixes If an antibody avidly fixes complement, it will cause complement, it will cause intravascular haemolysis, but if intravascular haemolysis, but if complement activation is weak, complement activation is weak, the haemolysis will be the haemolysis will be extravascular. extravascular.

Types of auto-antibodyTypes of auto-antibody

•Warm antibodiesWarm antibodies bind best at 37 bind best at 37 °C and account for 80% of cases. °C and account for 80% of cases. The majority are IgG .The majority are IgG .

•Cold antibodiesCold antibodies bind best at 4 °C bind best at 4 °C but can bind up to 37 °C in some but can bind up to 37 °C in some cases. They are usually IgM and cases. They are usually IgM and bind complement. They account bind complement. They account for the other 20% of cases. for the other 20% of cases.

Warm autoimmune HAWarm autoimmune HA

Approximately 1/100 000 population Approximately 1/100 000 population per annum; it occurs at all ages but per annum; it occurs at all ages but is more common in middle age and is more common in middle age and in females. in females.

Causes of W AIHACauses of W AIHA• Idiopathic:Idiopathic: 50% cases. 50% cases.

• Lymphoid neoplasms:Lymphoid neoplasms: lymphoma, lymphoma, chronic lymphocytic leukaemia, chronic lymphocytic leukaemia, myeloma. myeloma.

• Solid tumours:Solid tumours: lung, colon, kidney, lung, colon, kidney, ovary, thymoma. ovary, thymoma.

• Connective tissue disease:Connective tissue disease: SLE, SLE, rheumatoid arthritis. rheumatoid arthritis.

• Drugs:Drugs: methyldopa, mefenamic acid, methyldopa, mefenamic acid, penicillin, quinidine. penicillin, quinidine.

• Miscellaneous:Miscellaneous: ulcerative colitis, HIV. ulcerative colitis, HIV.

W AHIA (Cont)W AHIA (Cont)

•Investigations Investigations

1.1.Evidence of haemolysis Evidence of haemolysis

2.2.Spherocytes on the blood film. Spherocytes on the blood film.

3.3.The diagnosis is confirmed by the The diagnosis is confirmed by the direct Coombs or antiglobulin test.direct Coombs or antiglobulin test.

W AIHA (cont)W AIHA (cont)

• A positive test requires about 200 A positive test requires about 200 antibody molecules to attach to each antibody molecules to attach to each red cell; with a very avid complement-red cell; with a very avid complement-fixing antibody, haemolysis may occur fixing antibody, haemolysis may occur at lower levels of antibody-binding. So at lower levels of antibody-binding. So the direct Coombs test can be the direct Coombs test can be negative in the presence of brisk negative in the presence of brisk haemolysis. haemolysis.

• The standard Coombs reagent will The standard Coombs reagent will miss IgA or IgE antibodies. miss IgA or IgE antibodies.

ManagementManagement

• Treatment of underlying cause. Treatment of underlying cause. • Removal of offending drugs.Removal of offending drugs.• Prednisolone 1 mg/kg orally. A response is seen Prednisolone 1 mg/kg orally. A response is seen

in 70-80% of cases but may take up to 3 in 70-80% of cases but may take up to 3 weeks.weeks.

• Once the haemoglobin has normalised and the Once the haemoglobin has normalised and the reticulocytosis resolved, the corticosteroid dose reticulocytosis resolved, the corticosteroid dose can be reduced slowly over about 10 weeks.can be reduced slowly over about 10 weeks.

• Transfusion support may be required for life-Transfusion support may be required for life-threatening problems, such as the threatening problems, such as the development of heart failure or rapid unabated development of heart failure or rapid unabated falls in Hb.falls in Hb.

Management(cont)Management(cont)

• If the haemolysis fails to respond to If the haemolysis fails to respond to corticosteroids or can only be stabilised by corticosteroids or can only be stabilised by large doses, then splenectomy should be large doses, then splenectomy should be considered. Good response in 50-60% of considered. Good response in 50-60% of cases. cases.

• If splenectomy is not appropriate, If splenectomy is not appropriate, azathioprine or cyclophosphamide may be azathioprine or cyclophosphamide may be considered. This is least suitable for young considered. This is least suitable for young patients, in whom long-term patients, in whom long-term immunosuppression carries a risk of immunosuppression carries a risk of secondary neoplasms. The anti-CD20 (B secondary neoplasms. The anti-CD20 (B cell) monoclonal antibody, rituximab, has cell) monoclonal antibody, rituximab, has shown some success in difficult cases.shown some success in difficult cases.

Cold auto-immune HACold auto-immune HA• Usually IgM, which bind to the red cells at 4 °C. It Usually IgM, which bind to the red cells at 4 °C. It

may cause intravascular haemolysis if may cause intravascular haemolysis if complement fixation occurs. complement fixation occurs.

• This can be chronic when the antibody is This can be chronic when the antibody is monoclonal, or acute or transient when the monoclonal, or acute or transient when the antibody is polyclonal.antibody is polyclonal.

• Causes: Causes: 1. Low grade B cell lymphoma1. Low grade B cell lymphoma 2. Mycoplasma pneumoniae infection2. Mycoplasma pneumoniae infection 3. IMN 3. IMN 4. 4. Paroxysmal cold haemoglobinuria is a Paroxysmal cold haemoglobinuria is a

very very rare cause seen in children in association rare cause seen in children in association

with with congenital syphilis. congenital syphilis.

C AHIA(cont)C AHIA(cont)• Chronic cold agglutinin diseaseChronic cold agglutinin disease This affects elderly patients and may This affects elderly patients and may

be associated with an underlying be associated with an underlying low-grade B cell lymphoma. low-grade B cell lymphoma.

It causes a low-grade intravascular It causes a low-grade intravascular haemolysis with cold, painful and haemolysis with cold, painful and often blue fingers, toes, ears or nose often blue fingers, toes, ears or nose (so-called acrocyanosis). (so-called acrocyanosis).

The blood film shows red cell The blood film shows red cell agglutination and the MCV may be agglutination and the MCV may be spuriously raised because the spuriously raised because the automated analysers count automated analysers count aggregates as single cells. aggregates as single cells.

Management(C AHIA) Management(C AHIA)

• Treatment is directed at any underlying Treatment is directed at any underlying lymphoma. lymphoma.

• If the disease is idiopathic, then patients must If the disease is idiopathic, then patients must keep extremities warm, especially in winter.keep extremities warm, especially in winter.

• Some patients respond to corticosteroid Some patients respond to corticosteroid therapy and blood transfusion may be therapy and blood transfusion may be considered, but the cross-match sample must considered, but the cross-match sample must be placed in a transport flask at a be placed in a transport flask at a temperature of 37 °C and blood administered temperature of 37 °C and blood administered via a blood-warmer. via a blood-warmer.

Alloimmune haemolytic Alloimmune haemolytic anaemiaanaemia• Alloimmune haemolytic anaemia is Alloimmune haemolytic anaemia is

due to an antibody against non-self red due to an antibody against non-self red cells. cells.

It has two main causes: It has two main causes:

• An unmatched transfusion of red cells An unmatched transfusion of red cells (a haemolytic transfusion reaction) (a haemolytic transfusion reaction)

• Maternal sensitisation to paternal Maternal sensitisation to paternal antigens on fetal cells (haemolytic antigens on fetal cells (haemolytic disease of the newborn). disease of the newborn).

Non-immune haemolytic Non-immune haemolytic anaemiaanaemia

• Physical trauma : Physical trauma : characterised by the presence characterised by the presence of red cell fragments on the blood film and markers of red cell fragments on the blood film and markers of intravascular haemolysis:of intravascular haemolysis:

A. Mechanical heart valvesA. Mechanical heart valves. . B. March haemoglobinuriaB. March haemoglobinuria. . C. Thermal injuryC. Thermal injury. .

D. Microangiopathic haemolytic anaemiaD. Microangiopathic haemolytic anaemia. .

Fibrin deposition in capillaries can cause severe red cellFibrin deposition in capillaries can cause severe red cell disruption. It may occur in a wide variety of conditions: disruption. It may occur in a wide variety of conditions:

1.1. disseminated carcinomatosis, disseminated carcinomatosis, 2.2. malignant or pregnancy-induced hypertension, malignant or pregnancy-induced hypertension, 3.3. haemolytic uraemic syndrome, haemolytic uraemic syndrome, 4.4. thrombotic thrombocytopenic purpura thrombotic thrombocytopenic purpura 5.5. disseminated intravascular coagulationdisseminated intravascular coagulation

NIHA(cont)NIHA(cont)

• Infection:Infection:

1.1. Plasmodium falciparumPlasmodium falciparum (blackwater (blackwater fever). fever).

2.2. Clostridium perfringensClostridium perfringens septicaemia septicaemia usually in the context of ascending usually in the context of ascending cholangitis, may cause severe intravascular cholangitis, may cause severe intravascular haemolysis with marked spherocytosis due haemolysis with marked spherocytosis due to bacterial production of a lecithinase to bacterial production of a lecithinase which destroys the red cell membrane.which destroys the red cell membrane.

NIHA(cont)NIHA(cont)

• Chemicals or drugs Chemicals or drugs Dapsone and sulfasalazine cause Dapsone and sulfasalazine cause

haemolysis by oxidative denaturation haemolysis by oxidative denaturation of haemoglobin. Denatured of haemoglobin. Denatured haemoglobin forms Heinz bodies in haemoglobin forms Heinz bodies in the red cells, visible on supravital the red cells, visible on supravital staining with brilliant cresyl blue. staining with brilliant cresyl blue.

• Arsenic gas, copper, chlorates, Arsenic gas, copper, chlorates, nitrites and nitrobenzene derivatives nitrites and nitrobenzene derivatives may all cause haemolysis.may all cause haemolysis.

Paroxysmal nocturnal Paroxysmal nocturnal haemoglobinuria (PNH)haemoglobinuria (PNH)

• This rare acquired non-malignant clonal This rare acquired non-malignant clonal expansion of haematopoietic stem cells expansion of haematopoietic stem cells deficient in GPI-anchor protein results deficient in GPI-anchor protein results in intravascular haemolysis and in intravascular haemolysis and anaemia because of increased anaemia because of increased sensitivity of red cells to lysis by sensitivity of red cells to lysis by complement.complement.

• Episodes of intravascular haemolysis Episodes of intravascular haemolysis result in haemoglobinuria, most result in haemoglobinuria, most noticeable in early morning urine which noticeable in early morning urine which has a characteristic red-brown colour. has a characteristic red-brown colour.

PNH (cont)PNH (cont)

• The disease is associated with an The disease is associated with an increased risk of venous thrombosis in increased risk of venous thrombosis in unusual sites such as the liver or unusual sites such as the liver or abdomen. abdomen.

• PNH is also associated with hypoplastic PNH is also associated with hypoplastic bone marrow failure, aplastic anaemia bone marrow failure, aplastic anaemia and myelodysplastic syndrome.and myelodysplastic syndrome.

• Management is supportive with Management is supportive with transfusion and treatment of thrombosis. transfusion and treatment of thrombosis. Recently the anti-complement C5 Recently the anti-complement C5 monoclonal antibody ecluzimab was monoclonal antibody ecluzimab was shown to be effective in reducing shown to be effective in reducing haemolysis. haemolysis.

Red cell membrane Red cell membrane defectsdefects• The red cell diameter is 8 μm but the The red cell diameter is 8 μm but the

narrowest capillaries 2 μm ( In the narrowest capillaries 2 μm ( In the spleen).spleen).

• When there is deficiency of one or more When there is deficiency of one or more proteins in the cytoskeleton, cells lose proteins in the cytoskeleton, cells lose their elasticity. their elasticity.

• Increase in mean cell haemoglobin Increase in mean cell haemoglobin concentration (MCHC), abnormal cell concentration (MCHC), abnormal cell shape and reduced red cell survival due shape and reduced red cell survival due to extravascular haemolysis. to extravascular haemolysis.

Hereditary spherocytosisHereditary spherocytosis

•Usually autosomal dominant Usually autosomal dominant inheritence.inheritence.

•25% of cases is new mutations. 25% of cases is new mutations.

•The incidence is approximately The incidence is approximately 1:5000 in developed countries.1:5000 in developed countries.

•The most common abnormalities The most common abnormalities are deficiencies of beta spectrin or are deficiencies of beta spectrin or ankyrin. ankyrin.

Normal RBC structureNormal RBC structure

Hereditary Hereditary spherocytosis(cont)spherocytosis(cont)

• Most cases are asymptomatic Most cases are asymptomatic compensated chronic haemolytic state.compensated chronic haemolytic state.

• Pigment gallstones are present in up to Pigment gallstones are present in up to 50% of patients and may cause 50% of patients and may cause symptomatic cholecystitis. symptomatic cholecystitis.

• Occasional cases are associated with Occasional cases are associated with more severe haemolysis; these may be more severe haemolysis; these may be due to coincidental polymorphisms in due to coincidental polymorphisms in alpha spectrin or co-inheritance of a alpha spectrin or co-inheritance of a second defect involving a different second defect involving a different protein. protein.

Hereditary Hereditary spherocytosis(cont)spherocytosis(cont)

The clinical course may be complicated by crises: The clinical course may be complicated by crises:

• A haemolytic crisisA haemolytic crisis ----this is rare, and usually ----this is rare, and usually associated with infection. associated with infection.

• A megaloblastic crisisA megaloblastic crisis ----this may occur as a ----this may occur as a first presentation of the disease in pregnancy. first presentation of the disease in pregnancy.

• An aplastic crisisAn aplastic crisis ----occurs in association with ----occurs in association with erythrovirus infection.Patients present with erythrovirus infection.Patients present with severe anaemia and a low reticulocyte count. severe anaemia and a low reticulocyte count.

Hereditary Hereditary spherocytosis(cont)spherocytosis(cont)

Investigations :Investigations :

•Screening of the familly Screening of the familly members. members.

•Flow cytometric tests--detecting Flow cytometric tests--detecting binding of eosin-5-maleimide to binding of eosin-5-maleimide to red cells, are recommended in red cells, are recommended in borderline cases.borderline cases.

Hereditary Hereditary spherocytosis(cont)spherocytosis(cont)

Management :Management :

1. 1. Folic acid prophylaxis, 5 mg Folic acid prophylaxis, 5 mg once weekly, should be given for once weekly, should be given for life.life.

2. Splenectomy (indications 2. Splenectomy (indications include moderate to severe include moderate to severe haemolysis with complications haemolysis with complications (anaemia and gallstones).(anaemia and gallstones).

Red cell enzymopathiesRed cell enzymopathies

• Anaerobic glycolysis via the Embden-Anaerobic glycolysis via the Embden-Meyerhof pathway generates ATP, and Meyerhof pathway generates ATP, and the hexose monophosphate shunt the hexose monophosphate shunt produces NADPH and glutathione to produces NADPH and glutathione to protect against oxidative stress. protect against oxidative stress.

Red cell Red cell enzymopathies(cont)enzymopathies(cont)

• Defects in the hexose Defects in the hexose monophosphate shunt pathway monophosphate shunt pathway result in periodic haemolysis induced result in periodic haemolysis induced by oxidative stress, whilst those in by oxidative stress, whilst those in the Embden-Meyerhof pathway result the Embden-Meyerhof pathway result in shortened red cell survival and in shortened red cell survival and chronic haemolysischronic haemolysis

Glucose-6-phosphate Glucose-6-phosphate dehydrogenase (G6PD) dehydrogenase (G6PD)

deficiencydeficiency

•This enzyme is pivotal in the hexose This enzyme is pivotal in the hexose monophosphate shunt pathway.monophosphate shunt pathway.

•Most common human enzymopathy, Most common human enzymopathy, affecting 10% of the world's affecting 10% of the world's population.The enzyme is encoded by population.The enzyme is encoded by a gene on the X chromosome. a gene on the X chromosome.

What is G6PDWhat is G6PD??

• It is an X-linked recessive inheritance. (males It is an X-linked recessive inheritance. (males usually affected and females are carriers)usually affected and females are carriers)

• Risk factors: being black, being male, or Risk factors: being black, being male, or having a family history of G6PD deficiency.having a family history of G6PD deficiency.

• G6PD enzyme functions in the Pentose-G6PD enzyme functions in the Pentose-Monophosphate shunt and in the process, Monophosphate shunt and in the process, catalyzes the reduction of NADP+ to NADPH catalyzes the reduction of NADP+ to NADPH required in triggering a cascade of events that required in triggering a cascade of events that can detoxify the harmful oxidant Hcan detoxify the harmful oxidant H22OO22..

Glycolytic PathwayGlycolytic Pathway

Glucose

Glucose-6-phosphate

ATP

ADP

Fructose-6-phosphate

hexokinase

isomerase

Fructose-1,6-bisphosphate

ATPADP

Glyceraldehyde-3-phosphate

Dihydroxy-acetone Phosphate

PFKinase

G-3-P Dehydrogenase

NAD+ NADH

1,3-Bisphosphoglycerate

Pyruvate

NAD+

Fe3+ + O2O2 + Fe2+

MetHb reductase

(2 Net ATP)

Drugs H2O2

•Know this diagram

PMP Generation of NADPHPMP Generation of NADPH

GlucoseGlucose-6-phosphate

6-Phosphogluconate

Ribose-5-phosphate

Fructose-6-phosphate

Glyceraldehyde-3-phosphate+

Glyceraldehyde-3-phosphate

Pen

tose

Shu

nt

ATPADP

G6PDehydrogenase

NADPH

NADP+

GSSG

GSH

GSH reductase

NADPH

NADP+

H2O

H2O2 O2

Catalase

•Know this diagram

How Drugs Affect G6PD Deficient How Drugs Affect G6PD Deficient Individuals?Individuals?

GlucoseGlucose-6-phosphate

6-Phosphogluconate

Ribose-5-phosphate

Fructose-6-phosphate

Glyceraldehyde-3-phosphate+

Pen

tose

Shu

nt

G6PDehydrogenase

NADPH

NADP+

GSSG

GSH

GSH reductase

NADPH

NADP+

H2O

↑H2O2O2

Catalase

NAD+

NADH

Fe2+ (oxyHb)

Fe3+

(metHb)

Drugs

Glyceraldehyde-3-phosphate

2 OH

Hemolysis

SuperoxideDesmutase(SOD)

(Fe2+)GSH Peroxidase

NADPH

(O2 )

Clinical featuresClinical features

• Acute drug-induced haemolysis to Acute drug-induced haemolysis to (e.g.) (e.g.) – Analgesics:Analgesics: aspirin, phenacetin aspirin, phenacetin – Antimalarials:Antimalarials: primaquine, quinine, primaquine, quinine,

chloroquine, pyrimethamine chloroquine, pyrimethamine – Antibiotics:Antibiotics: sulphonamides, sulphonamides,

nitrofurantoin, ciprofloxacin nitrofurantoin, ciprofloxacin – Miscellaneous:Miscellaneous: quinidine, probenecid, quinidine, probenecid,

vitamin K, dapsonevitamin K, dapsone

Clinical features (cont)Clinical features (cont)

•Chronic compensated haemolysis Chronic compensated haemolysis

• Infection or acute illness Infection or acute illness

•Neonatal jaundice: may be a Neonatal jaundice: may be a feature of the B- enzyme feature of the B- enzyme

•Favism, i.e. acute haemolysis after Favism, i.e. acute haemolysis after ingestion of the broad bean ingestion of the broad bean Vicia Vicia fabafaba

Laboratory featuresLaboratory features

• Non-spherocytic intravascular haemolysis Non-spherocytic intravascular haemolysis during an attack during an attack

The blood film will show:The blood film will show: • Bite cellsBite cells (red cells with a 'bite' of membrane missing) (red cells with a 'bite' of membrane missing)

• Blister cellsBlister cells (red cells with surface blistering of the (red cells with surface blistering of the membrane) membrane)

• Irregularly shaped small cellsIrregularly shaped small cells • Polychromasia reflecting the Polychromasia reflecting the

reticulocytosisreticulocytosis • Denatured haemoglobin visible as Heinz Denatured haemoglobin visible as Heinz

bodies within the red cell cytoplasm, if bodies within the red cell cytoplasm, if stained with a supravital stain such as stained with a supravital stain such as methyl violet methyl violet

Treatments may includeTreatments may include• Stopping use of offending drug.Stopping use of offending drug.

• For more severe cases, treat with: For more severe cases, treat with:

• corticosteroids (e.g. prednisone)corticosteroids (e.g. prednisone)

• intravenous immunoglobulin infusions intravenous immunoglobulin infusions

• immunosuppressive (e.g. azathioprine (Imuran) and immunosuppressive (e.g. azathioprine (Imuran) and cyclophosphamide (Cytoxan) cyclophosphamide (Cytoxan)

• Vitamin and mineral supplements (e.g. folic acid).Vitamin and mineral supplements (e.g. folic acid).

• Change in diet.Change in diet.

• If Hb levels If Hb levels ↓↓, treatment may include blood transfusion or , treatment may include blood transfusion or splenectomy (surgical removal of the spleen).splenectomy (surgical removal of the spleen).

• If physical damage to RBC, then treat w/ blood transfusions If physical damage to RBC, then treat w/ blood transfusions or simple iron supplements. or simple iron supplements.

• Iron - Taken during pregnancy and when iron levels are Iron - Taken during pregnancy and when iron levels are low.low.

• Erythropoietin (Procrit) - To increase RBC production in Erythropoietin (Procrit) - To increase RBC production in people w/ kidney problems.people w/ kidney problems.

ManagementManagement

• To stop any precipitant drugs To stop any precipitant drugs and treat any underlying and treat any underlying infection. infection.

• Transfusion support.Transfusion support.

HaemoglobinopathiesHaemoglobinopathies(abnormal HB)(abnormal HB)

•………….are characterized by the .are characterized by the production of structurally production of structurally defective haemoglobin due defective haemoglobin due to abnormalities in the to abnormalities in the formation of the globin formation of the globin moiety of the molecule. moiety of the molecule.

Normal human Normal human haemoglobinhaemoglobin

• Adult- Adult- Hb-A ----- Hb-A ----- alpha2/beta2alpha2/beta2

Hb A2Hb A2 ----- ----- alpha2/delta2alpha2/delta2

• Fetal-Fetal- Hb-F ------- Hb-F -------alpha2/gama2alpha2/gama2

Hb-Barts---Hb-Barts---gama4gama4

• Embryonic-Embryonic- Hb gower 1, Hb gower 2, Hb gower 1, Hb gower 2, Hb PortlandHb Portland

• Majority of the abnormal Hb differ from Majority of the abnormal Hb differ from the corresponding normal haemoglobin by the corresponding normal haemoglobin by the substitution of a single amino acid in the substitution of a single amino acid in one of their pairs of polypeptide chains.one of their pairs of polypeptide chains.

•Common abnormal haemoglobin:Common abnormal haemoglobin:

Hb-S------alpha2/beta2 (6 glu---> val).Hb-S------alpha2/beta2 (6 glu---> val).

Hb-C------alpha2/beta2 (6 glu---> lys).Hb-C------alpha2/beta2 (6 glu---> lys).

Hb-E-----alpha2/beta2 (26 glu--->lys).Hb-E-----alpha2/beta2 (26 glu--->lys).

Hb-D Hb-D punjabpunjab--alpha2/beta2(6 glu-> val).--alpha2/beta2(6 glu-> val).

Lab testLab test

•Hb electrophoresisHb electrophoresis is the most is the most useful method for the useful method for the demonstration of abnormal demonstration of abnormal haemoglobin.haemoglobin.

The thalassaemiasThe thalassaemias(Inadequate HB)(Inadequate HB)

The thalassaemias are a The thalassaemias are a heterogenous group of disorders heterogenous group of disorders with a genetically determined with a genetically determined reduction in the rate of reduction in the rate of synthesis of one or more types of synthesis of one or more types of normal haemoglobin polypeptide normal haemoglobin polypeptide chain.chain.

ClassificationsClassifications(A) (A) Alpha thalassaemia-Alpha thalassaemia--- Levels of Hb-A , -- Levels of Hb-A , Hb-F and Hb-A2 are equally depressed Hb-F and Hb-A2 are equally depressed since they all have alpha chains.since they all have alpha chains. Causes microcytic and hypochromic Causes microcytic and hypochromic anaemia.anaemia.(B)(B)Beta thalassaemia-Beta thalassaemia--- decrease -- decrease

production production of beta chain leads to a reduction in of beta chain leads to a reduction in

the amount of Hb-A in red cell. Causes the amount of Hb-A in red cell. Causes microcytic hypochromic anaemia. But microcytic hypochromic anaemia. But Total Hb is maintained in part by the Total Hb is maintained in part by the product of gama and delta chain. Thus product of gama and delta chain. Thus increase Hb-F or Hb-A2 is usually found.increase Hb-F or Hb-A2 is usually found.

Beta thalassaemiaBeta thalassaemia

•Beta 0 thalassaemiaBeta 0 thalassaemia --Complete --Complete absence of beta chain.absence of beta chain.

•Beta + thalassaemiaBeta + thalassaemia --- Incomplete --- Incomplete absence of beta chain.absence of beta chain.

•ClinicallyClinically --- --- 1.1. Beta thalassaemia Beta thalassaemia

major major (homozygous form (homozygous form

of disease).of disease).

2.Beta thalassemia minor.2.Beta thalassemia minor.

Beta thalassaemia majorBeta thalassaemia major

• Onset of anaemia is insidious.Onset of anaemia is insidious.

• Initial manifestation being pallor which is Initial manifestation being pallor which is usually obvious within the first year of life usually obvious within the first year of life and in severe cases within few weeks of and in severe cases within few weeks of birth. birth.

• Growth retardation is common.Growth retardation is common.

• Anorexia, nausea ,loss of fat and Anorexia, nausea ,loss of fat and recurrent fever occur.recurrent fever occur.

Beta thalassaemia major(cont)Beta thalassaemia major(cont)

•Splenomegally is obvious at the Splenomegally is obvious at the age of 3 years.age of 3 years.

•Moderate to marked Moderate to marked hepatomegally is also present.hepatomegally is also present.

•Clinical jaundice is uncommon Clinical jaundice is uncommon but is sometimes present.but is sometimes present.

Laboratory InvestigationLaboratory InvestigationThalassemiaThalassemia

• Variable hemogram results proportional Variable hemogram results proportional to the severity of the thalassemiato the severity of the thalassemia

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

level of anemialevel of anemia

Laboratory InvestigationLaboratory InvestigationThalassemiaThalassemia (cont)(cont)

• Findings in severe cases can mimic those Findings in severe cases can mimic those seen in other microcytic/hypochromic seen in other microcytic/hypochromic anemiasanemias

• Results of the reticulocyte count are variableResults of the reticulocyte count are variable• NRBCs may be present (contrast with iron NRBCs may be present (contrast with iron

deficiency anemia)deficiency anemia)• Hemoglobin electrophoresisHemoglobin electrophoresis

• Major test for identifying thalassemia and Major test for identifying thalassemia and hemoglobinopathyhemoglobinopathy

Treatment of beta-Treatment of beta-thalassaemia majorthalassaemia major

• Erythropoietic failure: Erythropoietic failure: Allogeneic bone Allogeneic bone marrow transplantation from human marrow transplantation from human leucocyte antigen (HLA)-compatible leucocyte antigen (HLA)-compatible sibling Transfusion to maintain Hb > 100 sibling Transfusion to maintain Hb > 100 g/L  Folic acid 5 mg dailyg/L  Folic acid 5 mg daily

• Iron overload: Iron overload: Iron therapy forbiddenIron therapy forbidden

   Iron chelation therapyIron chelation therapy

• Splenomegaly:Splenomegaly: causing mechanical causing mechanical problems, excessive transfusion needs problems, excessive transfusion needs Splenectomy.Splenectomy.

Course and TreatmentCourse and TreatmentThalassemiaThalassemia

• Untreated Untreated thalassemia thalassemia

• Major: Death in first or second decade Major: Death in first or second decade of life of life

• Intermedia: Usually normal life spanIntermedia: Usually normal life span

• Minor/Minima: Normal life spanMinor/Minima: Normal life span

Course and TreatmentCourse and TreatmentThalassemiaThalassemia

• Time of presentationTime of presentation• Related to degree of severityRelated to degree of severity• Usually in first few years of lifeUsually in first few years of life• Untreated severe Untreated severe thalassemia thalassemia

• --/--: Prenatal or perinatal death --/--: Prenatal or perinatal death

• --/---/- & --/ & --/cscs: Normal life span with chronic : Normal life span with chronic hemolytic anemiahemolytic anemia

X-ray skull of congenital X-ray skull of congenital haemolytic anaemiahaemolytic anaemia• Broadening of the diploic space with Broadening of the diploic space with

seperation of the tables and thickening seperation of the tables and thickening of the vault of the skull , especially of of the vault of the skull , especially of the frontal and parietal bones.the frontal and parietal bones.

• The medulla is less dense, giving a The medulla is less dense, giving a ground glass appearance and the ground glass appearance and the tables, especially outer, are thickened.tables, especially outer, are thickened.

• Bony trabeculae may develop at right Bony trabeculae may develop at right angles to the tables giving rise to ‘hair-angles to the tables giving rise to ‘hair-on-end’ or ‘brush’ appearance which is on-end’ or ‘brush’ appearance which is common in thalassemia major. common in thalassemia major.

Sickle Sickle haemoglobinopathyhaemoglobinopathy• …………are hereditary disorders in which the red are hereditary disorders in which the red

cells contain Hb-S.cells contain Hb-S.• The sickling of the red blood cells in the The sickling of the red blood cells in the

circulating blood has two major pathological circulating blood has two major pathological effects-effects-

1. The distorted and rigid cells1. The distorted and rigid cells block small vessels , impairing flowblock small vessels , impairing flow and causing ischaemia and infarction.and causing ischaemia and infarction. 2. Repeated sickle/unsickle cycles lead 2. Repeated sickle/unsickle cycles lead

to to loss of fragments of the cells and the loss of fragments of the cells and the

cellscells become spherocytic and fragile.become spherocytic and fragile.

What is Sickle Cell?What is Sickle Cell?

• People who have People who have Sickle Cell have sickle Sickle Cell have sickle shaped red blood shaped red blood cells, which causes cells, which causes complications complications because the blood because the blood cells are not able to cells are not able to reach certain parts of reach certain parts of the bodythe body..

Red blood cells Going through Red blood cells Going through VesselsVessels

• Abdominal and Abdominal and bone/joint painbone/joint pain

• BreathlessnessBreathlessness• Delayed growth and Delayed growth and

pubertypuberty• Fatigue and feverFatigue and fever• Jaundice (yellowed skin)Jaundice (yellowed skin)• Paleness Paleness • Rapid heart rateRapid heart rate• Greater risk for infectionGreater risk for infection• Adolescents and adults Adolescents and adults

can develop ulcers on can develop ulcers on their legstheir legs

• Chest painChest pain• Excessive thirstExcessive thirst• Poor eyesight, blindness Poor eyesight, blindness

– when blood can’t get – when blood can’t get to the back of eyes, to the back of eyes, they don’t have a they don’t have a constant nourishment, constant nourishment, causing people to not be causing people to not be able to see able to see

* About 30% of Jamaican patients with Sickle Cell develop ulcers in comparison to 1% of Americans

Diseases and Conditions people with Diseases and Conditions people with Sickle Cell are likely to develop:Sickle Cell are likely to develop:

•Acute chest syndrome

•Aplastic crisis

•Dactylitis – swelling of the hands and feet

•Painful crises: really painful episodes when blood cells are blocked from going to certain parts of the body – pain can occur anywhere, but it is usually in the chest, arms, and legs

•Enlarged spleen – sickle cells pool in the spleen, and in some cases there is no spleen in the body.

•Stroke

•Hematuria

An x-ray of a hand swollen from dactylitis

Who would you think has Who would you think has Sickle Cell?Sickle Cell?

• Indian girlIndian girl

•Hispanic boy

• Black boy• White girl

Who can get Sickle Cell?Who can get Sickle Cell?

• ANYONEANYONE can get Sickle Cell Disease. can get Sickle Cell Disease.

• It is most common in the following ancestries:It is most common in the following ancestries:o AfricanAfricano Central or South AmericanCentral or South Americano CubanCubano Indian Indian o Saudi ArabianSaudi Arabiano Mediterranean Mediterranean o Hispanics Hispanics

– People can live a relatively normal life with People can live a relatively normal life with Sickle Cell if they have the proper treatment.Sickle Cell if they have the proper treatment.

– The average life expectancy for males is The average life expectancy for males is 42.42.

– The average life expectancy for females is The average life expectancy for females is 4848. .

– In 2003 the oldest patient to have Sickle cell In 2003 the oldest patient to have Sickle cell was an was an 85 85 year old Jamaican woman.year old Jamaican woman.

Genetics of Sickle CellGenetics of Sickle Cell

• Sickle cell is an autosomal recessive disease.

Therefore, the child can only getSickle cell if both

parents are carriers, not if only one is and

the other is normal. Theyhave a 25%

chance of getting it if both are

Carriers

How is sickle cell disease How is sickle cell disease diagnosed?diagnosed?

– Many states now use hemoglobinopathy Many states now use hemoglobinopathy testing (looking at the blood for testing (looking at the blood for abnormalities of testing) as part of the abnormalities of testing) as part of the newborn screening blood tests between newborn screening blood tests between 2 and 7 days of age.2 and 7 days of age.

– Careful examination of family history.Careful examination of family history.– Physical examination – some symptoms Physical examination – some symptoms

are visible, such as jaundice. are visible, such as jaundice. – A hemoglobin electrophoresis can A hemoglobin electrophoresis can

determine if a person is a carrier.determine if a person is a carrier.

Main Treatment MethodsMain Treatment MethodsThere is no known cure for sickle cell anemia.There is no known cure for sickle cell anemia.

The four main treatment options are:The four main treatment options are:• Blood TransfusionsBlood Transfusions• Drug TreatmentDrug Treatment• Blood and Marrow Stem Cell TransplantationBlood and Marrow Stem Cell Transplantation• Gene TherapyGene Therapy

These main treatment options for the painful These main treatment options for the painful crisis involves heavy reliance on painkilling crisis involves heavy reliance on painkilling drugs and oral and intravenous fluids whose drugs and oral and intravenous fluids whose main functions are to reduce pain and main functions are to reduce pain and prevent complications. prevent complications.

Role of G6PDRole of G6PD

• Responsible for maintaining adequate Responsible for maintaining adequate levels of NADPH inside cell. levels of NADPH inside cell.

• The oxidation of NADPH back to NADP+ The oxidation of NADPH back to NADP+ is coupled with the reduction of oxidized is coupled with the reduction of oxidized glutathione (GSSG) to glutathione (GSH).glutathione (GSSG) to glutathione (GSH).

• Thus, NADPH keeps glutathione, a tri-Thus, NADPH keeps glutathione, a tri-peptide, in its reduced formpeptide, in its reduced form..

Role of G6PD ContRole of G6PD Cont’’d...d...

• Reduced glutathione Reduced glutathione (GSH) acts as a scavenger(GSH) acts as a scavenger for dangerous oxidative metabolites in the cell.for dangerous oxidative metabolites in the cell.

• GSH converts harmful hydrogen peroxide to GSH converts harmful hydrogen peroxide to waterwater catalyzed by the enzyme, glutathione catalyzed by the enzyme, glutathione peroxidase (catalase enzyme also detoxifies peroxidase (catalase enzyme also detoxifies HH22OO22).).

• If HIf H22OO22 cannot be detoxified by GSH or cannot be detoxified by GSH or catalase, hydroxyl radical formed from Hcatalase, hydroxyl radical formed from H22OO22 can be scavenged by Vit C/E.can be scavenged by Vit C/E.

G6PD DeficiencyG6PD Deficiency

• Red cells deficient in G6PD are unable to Red cells deficient in G6PD are unable to neutralize hydrogen peroxide - Hneutralize hydrogen peroxide - H22OO22 converts to hydroxyl radicals and this can converts to hydroxyl radicals and this can lead to oxidative damage/toxic injury.lead to oxidative damage/toxic injury.

• Impaired response to oxidizing drugsImpaired response to oxidizing drugs can can also induce hemolytic anemia (Individuals also induce hemolytic anemia (Individuals with G6PD deficiency are particularly with G6PD deficiency are particularly susceptible)susceptible)

Drugs that affect itDrugs that affect it

• Drugs that can precipitate this reaction Drugs that can precipitate this reaction include:include: anti-malarial agents anti-malarial agents sulfonamides (antibiotic) sulfonamides (antibiotic) aspirin aspirin non-steroidal anti-inflammatory drugs non-steroidal anti-inflammatory drugs (NSAIDs) (NSAIDs) nitrofurantoin nitrofurantoin quinidine quinidine quinine quinine othersothers

• Also: Also: exposure to certain chemicals such as those in exposure to certain chemicals such as those in mothballs and flava beans.mothballs and flava beans.

What are the What are the symptoms?symptoms?

• The most common symptoms include:The most common symptoms include:

– abnormal paleness or lack of color of the abnormal paleness or lack of color of the skinskin

– jaundice, or yellowing of the skin, eyes, and jaundice, or yellowing of the skin, eyes, and mouthmouth

– dark color to urinedark color to urine– feverfever– weaknessweakness– dizzinessdizziness– confusionconfusion– intolerance to physical activityintolerance to physical activity

Signs of anemia Signs of anemia include:include:

• pale skin and fingernailspale skin and fingernails

• rapid pulserapid pulse

• heart murmurheart murmur

• Enlarged spleen and liverEnlarged spleen and liver

Required TestsRequired Tests

• Blood tests are taken to measure levels of:Blood tests are taken to measure levels of:– red cells, assess size and shape of red cellsred cells, assess size and shape of red cells– measure the Hb levelmeasure the Hb level– determine the number of reticulocytes.determine the number of reticulocytes.

• Other blood tests may include:Other blood tests may include:

Coombs' testCoombs' test (direct and indirect) — checks for (direct and indirect) — checks for hemolytic anemia caused by an abnormal immune hemolytic anemia caused by an abnormal immune reaction. reaction.

Heinz body presentationHeinz body presentation — looks for a deficiency — looks for a deficiency in in amount of G6PD enzyme, which results in amount of G6PD enzyme, which results in hemolysis if hemolysis if certain medications or foods are certain medications or foods are ingested.ingested.

ReferencesReferences• Ezra E. W. Cohen, M.D., Section of Hematology/Oncology, Department of Medicine, The University Ezra E. W. Cohen, M.D., Section of Hematology/Oncology, Department of Medicine, The University

of Chicago, Chicago, IL. 11 Jan 2004 <of Chicago, Chicago, IL. 11 Jan 2004 <http://www.nlm.nih.gov/medlineplus/ency/article/000571.htm > >

• Updated by: Corey Culter, M.D. M.P.H., F.R.C.P.C., Department of Medical Oncology, Dana Farber Updated by: Corey Culter, M.D. M.P.H., F.R.C.P.C., Department of Medical Oncology, Dana Farber Cancer INsitiute; Instructor of Medicine, Harvard University, Boston, MA. Review provided by Cancer INsitiute; Instructor of Medicine, Harvard University, Boston, MA. Review provided by VeriMed Healthcare Network. Source: MEDLINEplus Medical Information. 11 Jan 2004 VeriMed Healthcare Network. Source: MEDLINEplus Medical Information. 11 Jan 2004 http://www.hlm.nil.gov/medlineplus/ency/article/000528.htm

• Ramez A. EthnasiosRamez A. Ethnasios. An Introduction to G6PD Deficiency. 7 Jan 2004 <. An Introduction to G6PD Deficiency. 7 Jan 2004 <http://www.rialto.com/g6pd/physiolo.htm> >

• Peggy Gulley, MD.Peggy Gulley, MD. Hemolytic Anemia Lecture. 9 Jan 2004 Hemolytic Anemia Lecture. 9 Jan 2004 <<http//:pathology.uthscsa.edu/MSII/Hemo.html>http//:pathology.uthscsa.edu/MSII/Hemo.html>

• Rebecca Elstrom, M.D., Division of Hematology-Oncology, University of Pennsylvania Medical Rebecca Elstrom, M.D., Division of Hematology-Oncology, University of Pennsylvania Medical Center, Philadelphia, PA. Review provided by VeriMed Healthcare Network. . 11 Jan 2004 <Center, Philadelphia, PA. Review provided by VeriMed Healthcare Network. . 11 Jan 2004 <http://www.umm.edu/blood/anehemol.htm> >

• Patrick Yorba, MD, Staff Physician, Department of Emergency Medicine, University of Virginia Patrick Yorba, MD, Staff Physician, Department of Emergency Medicine, University of Virginia Health Sciences Center. 11 Jan 2004 Health Sciences Center. 11 Jan 2004 http://www.emedicinehealth.com/articles/4893-6.asp?pd=1/11/2004%2010:25:15%20PM

• Faculty of Harvard Medical School © 1996-2003 Aetna InteliHealth Inc. 11 Jan 2004 <Faculty of Harvard Medical School © 1996-2003 Aetna InteliHealth Inc. 11 Jan 2004 <http://www.intelihealth.com/IH/ihtIH/WSIHW000/9339/21246.html> >

• Images taken from Images taken from http://health.allrefer.com/health/hemolytic-anemia-pictures-images.html> . 11 > . 11 Jan 2004 Jan 2004