Hemolysis

Hemolysis Increased cell destruction Rate of destruction exceeds the capacity

of the bone marrow to produce red blood cells (RBC)

Normal RBC survival time is 110-120 days

Approximately 1% of RBC are removed each day and replaced by the marrow to maintain the RBC count

During hemolysis RBC survival is shortened and increased marrow activity results in a heightened reticulocyte percentage

Hemolysis can be divided into two Intravascular hemolysis Extravascular hemolysis

Hemolysis

Extravascular hemolysis The degradation of Hb results in the

biliary excretion of heme pigments and increased fecal urobilinogen

Gallstones composed of calcium bilirubinate may be formed in children as young as 4 years of age

Hemolysis

Intravascular hemolysis Hb binds to haptoglobin and hemopexin both of

which are reduced Oxidized heme binds to albumin to form

methemalbumin which is increased When the capacity of these binding molecules is

exceeded, free Hb appears in the plasma (evidence of intravascular hemolysis)

When the tubular reabsorbtive capacity of kidneys for Hb is exceeded free Hb appears in the urine

Hemolysis

Hemolytic anemia Hemolysis

Increased cell destruction A feature of hemolytic anemia is a reduction

in the normal red cell survival of 120 days The premature destruction of RBC may result

from corpuscular abnormalities such as Hb defects, abnormalities of RBC enzymes or

defects of RBC membrane Other defects may result from

extracorpuscular abnormalities and may be due to immune or non-immune mechanisms

The approach to the diagnosis of hemolytic anemia should include Consideration of the clinical features

suggesting hemolytic disease Demonstration of the presence of

hemolytic process by laboratory means Establishment of the presice cause of

the hemolytic anemia by special hematologic investigations

Hemolytic anemia

The following clinical features suggest hemolysis Age: anemia and jaundice in an Rh(+) infant born

to a Rh(-) mother or a group A or B infant born to a group O mother

History of anemia, jaundice or gallstones in family Persistent/ recurrent anemia associated with

reticulocytosis Anemia unresponsive to hematinics Intermittent/persistent indirect hyperbilirubinemia

Hemolytic anemia-Clinical features(1)

Splenomegaly Hemoglobinuria Presence of multiple gallstones Chronic leg ulcers Development of anemia or

hemoglobinuria after exposure to certain drugs

Dark urine

Hemolytic anemia-Clinical features(2)

Reduced cell survival and evidence of accelerated Hb catabolism

Evidence of increased erythropoiesis

Hemolytic anemia-laboratory findings

Accelerated Hb catabolism Extravascular

Raised unconjugated bilirubin Raised fecal and urinary urobilinogen

Intravascular Hemoglobinuria Low/absent plasma haptoglobin Raised plasma methemalbumin


Increased erythropoiesis (response to a reduction in Hb) Reticulocytosis Increased MCV Increased normoblasts in peripheral blood Spesific morphological abnormalities

Sickled cells, target cells, spherocytes Erhytroid hyperplasia of bone marrow Expansion of marrow space

Prominence of frontal bones, broad cheek bones, widened intratrabecular spaces, hair-on-end appearance of skull radiographs


normal macrocytes

Hypochromic, microcytes

Target cells

schistocytes

Tests used to establish a spesific cause of hemolytic anemia (1) Membrane defects (Hereditary spherocytosis,

elliptocytosis, stomatosis, acantocytosis) Blood smear Increased RBC osmotic fragility

(spherocytes lyse in higher concentrations of saline than normal RBC)

Autohemolysis at 24 and 48 hours Enzyme defects (G6PD and pyruvate kinase)

Heinz body preparation Autohemolysis test Screening tests for enzyme deficiencies

Hemoglobin defects (sickle cell disease, thalassemias) Blood smear, sickle cell, target cell Sickling test Hemoglobin electrophoresis HbF determination

Tests used to establish a spesific cause of hemolytic anemia (2)

Immune hemolytic anemia Isoimmune

Mismatched blood transfusion Hemolytic disease of the newborn

Autoimmune Action of Ig Idiopathic, secondary to number of

conditions Coombs’ test (+)


Non-immune hemolytic anemia Infections, drugs, underlying

hematologic disease- microangiopathic HA, hypersplenism

Coombs’ test (-)


Congenital hemolytic anemias Membrane defects

Hereditary spherocytosis(HS) Enzyme defects

G6PD deficiency Hemoglobin defects

- thalassemia (quantitative hemoglobinopathies)

HbS (qualitative hemoglobinopathies) Hemolytic disease of the newborn (isoimmune)

ıntracorpuscular

Hereditary spherocytosis Familial hemolytic disorder Marked heterogenicity of clinical features

Asymptomatic condition Fulminant hemolytic anemia

The morphologic hallmark of HS Microspherocyte Caused by loss of membrane surface area Abnormal osmotic fragility

HS usually is transmitted as an autosomal dominant trait

An autosomal recessive mode of inheritance also occurs 20-25% of all HS cases

HS is encountered worldwide

Hereditary spherocytosis

An intrinsic genetic defect causes defects in membrane proteins

The major complications Aplastic or megaloblastic crisis Hemolytic crisis Cholecystitis and cholelithiasis Severe neonatal hemolysis

Hereditary spherocytosis

HS erythrocytes are caused by membrane protein defects resulting in cytoskeleton instability

Four abnormalities in red cell membrane proteins have been identified Spectrin deficiency alone (most common) Combined spectrin and ankyrin deficiency Band 3 deficiency(10-20% of patients) Protein 4.2 defects (common in Japan)

Hereditary spherocytosis- Pathophysiology

Spectrin deficiency Loss of erythrocyte surface

Spherical RBC Culled rapidly from the circulation by the spleen

Splenomegaly Hemolysis primarily confined to the spleen

Extravascular hemolysis Biochemical spectrin deficiency and the degree

of spectrin deficiency are reported to correlate with the extent of spherocytosis, the degree of abnormality on osmotic fragility test results and the severity of hemolysis


Ankyrin defects Ankyrin is the principal binding site for spectrin

on RBC membrane A proportional decrease in spectrin content

occurs although spectrin synthesis is normal 75-80% of patients with autosomal dominant HS

have combined spectrin and ankyrin deficiency Deletion of chromosome 8 are shown to have a

decrease in RBC ankyrin content


Anemia

Jaundice

Splenomegaly

Hereditary spherocytosis- Clinical findings

Clinical features of HS

Anemia or hyperbilirubinemia may be of such magnitude as to require exchange transfusion in the neonatal period

Anemia is mild to moderate Sometimes severe/not present

In patients with mild HS cholelithiasis may be the first sign of underlying disease

Moderate HS (most common, 60-75%) Mild HS (20-30%) Severe HS (5%, requires RBC transfusions)

Hereditary spherocytosis- Clinical findings(2)

Minimal or no anemia Reticulocytosis Increased MCHC Spherocytes on the peripheral blood smear

Howell-Jolly bodies may be seen Hyperbilirubinemia Abnormal osmotic fragility test

hemolysis of HS cells may be complete at a solute concentration that causes little or no lysis of normal cells

LDH increased Increased unconjugated bilirubin Looking for abnormalities in spectrin, ankyrin,

band 3 (not routine)

Hereditary spherocytosis- Laboratory findings

Neonates Phototherapy/exchange transfusion

Aplastic crisis RBC transfusion

Folic acid supplementation to prevent megaloblastic crisis

Splenectomy (after 6 years of age) Increased Hb level Decreased reticulocyte count Appereance of Howell-Jolly bodies and target cells Thrombocytosis

Hereditary spherocytosis- Treatment

Glucose 6 phosphate dehydrogenase deficiency (G6PD) X-linked disorder

Homozygous women are found in populations in which the frequency of G6PD is high

Heterozygous carrier women can develop hemolytic attacks

Polymorphic with more than 300 reported variants

The highest prevalance rates are found in tropical Africa, the Middle East, some areas of Mediterranean (severe forms)

G6PD enzyme catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate while reducing the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) to nicotinamide adenine dinucleotide phosphate (NADPH)

Glucose 6 phosphate dehydrogenase deficiency- Pathophysiology

NADPH Protects the cells against oxidative stress Required cofactor in many biosynthetic

reactions Maintains glutathion in its reduced form

Glutathion acts a scavanger for dangerous oxidative metabolites in the cell

Converts harmful hydrogen peroxide to water with the help of glutathion peroxidase

Glucose 6 phosphate dehydrogenase deficiency- Pathophysiology

The most common clinical feature is no symptoms

Symptomatic patients Neonatal jaundice

Appears by age 1-4 days Often requires exchange transfusion

Acute hemolytic anemia Results from stress factors such as oxidative drugs

or chemicals, infection or ingestion of fava beans Jaundice and splenomegaly may be present

during crisis

Glucose 6 phosphate dehydrogenase deficiency- Clinical findings

Anemia Reticulocytosis Activity of G6PD is low (after hemolysis) Indirect hyperbilirubinemia Serum haptoglobin levels will be decreased Formation of bodies which consist of

denaturated hemoglobin Heinz body

Glucose 6 phosphate dehydrogenase deficiency- Laboratory findings

Avoid oxidant drugs Antimalarial drugs, nitrofurantoin,

nalidixic acid, ciprofloxacin,methylene blue, chloramphenicol, phenazopyridine, vit K analogs, sulfonamides, acetanilid, doxorubicine, isobutyl nitratre, naphtalene, phenylhydrazine, pyridoxin

Exchange transfusion RBC transfusion

Glucose 6 phosphate dehydrogenase deficiency- Treatment

Thalassemia (Cooley’s anemia, Mediterranean Anemia) Genetically determined defect in Hb synthesis An inability to manufacture sufficient quantities of

globin chains In the adult there are 3 Hb types normally present

Hb A 22 (95% of total) Hb A2 22 (3% of total) HbF 22 (2% of total)

During fetal life the majority of Hb During embryonic life at least 2 different Hbs are

produced Gowers 2 22 chains Gowers 1 4 chains The manufacture of each of these chains is controlled by

spesific genes

In thalassemia there is a genetic failure in the production of globin chains

Failure of production of and chains is the most common thalassemia

a failure of beta chain production thalassemia

a failure of alpha chain production

Thalassemia

The genes controlling beta chain production are located on chromosome 11

thalassemia major If both genes fail

thalassemia minor If only one gene fails

Beta Thalassemia

Most common of thalassemias Beta chain production is less than normal Alpha chain production continues at a near

normal rate Decreased level of HbA Excess alpha chains stimulates the

increased production of delta chains Increased amount of HbA2

Rate of gamma chain production is greater Increased amount of HbF

Beta Thalassemia minor (Heterozygous) (B+)

Beta Thalassemia minor These patients are not severely anemic These patients can be provided

appropriate genetic counselling Hb, Hct are decreased RBC count is not as low as the Hb and Hct

Bone marrow produce the cells but cannot fill them with Hb

RBCs are microcytic and hypochromic Normal RDW

Beta Thalassemia minor MCV is slightly decreased MCH is decreased MCHC is normal WBC count is normal Reticulocyte count is relatively increased Bone marrow is either normal or undergoes

slight erythroid hyperplasia Serum iron,ferritin is normal Bilirubin slightly increase due to

intramedullary hemolysis

Hb studies HbA decreased HbA2 increased HbF slightly increased to normal

Beta Thalassemia minor

Complete failure of beta chain production Raised levels of HbA2 and HbF

HbF has a very high affinity for oxygen (poor oxygen deliverer)

Only functional Hb is HbA2 The patient is hypoxic

İncreased erythropoietin production Stimulates the marrow to maximum

Typical facial appereance Splenomegaly

Extramedullary hemopoiesis

Beta Thalassemia major(homozygous)(B0)

Beta Thalassemia major Patients develop a life threatening anemia by

one or two months (mostly often 6 months) Severe anemia (Hb:2-3 mg/dl)

Hct and RBC count are also decreased MCV, MCH, MCHC are all decreased RDW is increased Hypochromic microcytic RBC

Anisocytosis, poikilocytosis, target cells Reticulocytosis WBC is increased at the beginning

Beta Thalassemia major Bone marrow undergoes erythroid

hyperplasia Serum Fe increased/normal Ferritin increased/normal Hb electrphoresis

HbA decreased HbA2 variable HbF increased

The patients must be supported with blood transfusions which result in iron overload

Unless iron is removed with appropriate chelation therapy these patients die of hemosiderosis

Splenectomy When the yearly transfusion requirement of

packed red cells exceeds 200-250 ml/kg Bone marrow transplantation

Beta Thalassemia major

Alpha thalassemia Four genes coding for alpha chain

production These genes are located on

chromosome 6 There are at least five forms of

alpha thalassemia depending on the number and location of abnormal genes

All genes are abnormal There is no alpha chain production

No HbF production and death in utero At autupsy the cord blood shows severe

anemia There is no HbA and HbF on

electrophoresis most of the Hb is HbBart’s which consists of

4 gamma chains

Hydrops fetalis- Homozygous alpha thalassemia

Hemoglobin H disease Three genes are abnormal and one gene is

coding for alpha chains Limited production of HbF in utero and HbA

after birth The excess gamma chains form HbBart’s and

the excess beta chains form HbH Unstable hemoglobins precipitate in the cell Premature destruction in the marrow and spleen

with splenomegaly Infant is anemic at birth RBC and hct count are also decreased

MCV, MCHC, MCH decreased RDW is increased Microcytosis, hypochromia Reticulocyte count is slightly increased Bone marrow undergoes erythroid

hyperplasia Serum iron,ferritin increased Hb electrophoresis

Hb Bart’s increased at birth Hb Bart’s 2-10% later HbH 5-40% HbA and A2 decreased

Hemoglobin H disease

Heterozygous alpha thalassemia (minor) Depends on whether or not the two deleted genes

are on the same chromosome In alpha th O, both genes are absent from the same

chromosome In alpha th +, one gene is missing from each

chromosome In both forms

Minor changes Mild anemia MCV and MCHC are borderline low

Hb electrophoresis is normal with increased levels of HbBart’s if the cord blood is electrophoresed

Alpha thalassemia silent Only one of the four genes is

abnormal There is a near normal production

of alpha chains with very few if any clinical or laboratory changes

Beta thalassemia variants Delta/beta th

Similar to beta th Symptoms are milder HbA decreased HbF 5-15% HbA2 normal

HbLepore No normal beta or delta chain HbF 80-90% HbA absent HbA2 absent HbLepore 10%

(Homozygous) clinical and lab.findings are identical to Beta th

HbLepore HbA decreased HbA2 decreased HbLepore 10%

Beta thalassemia variants

(Heterozygous) clinical and lab.findings are identical to Beta th minor

Sickle cell anemia Qualitative hemoglobinopathy Valine is substituted instead of glutamine in

the sixth position on the globin molecule Sickle cell anemia is caused by

homozygosity for the sickle cell gene and is the most common form of sickle cell disease

The charge at this site is altered and allows for polymerization of Hb under conditions of hypoxia

Polymerization of sickle Hb distorts erythrocyte morphology causing a marked reduction in RBC

life span increases blood viscosity predisposes to episodes of

vasoocclusion

Sickle cell anemia

Children are normal at birth Onset of symptoms is unusual before 3-4

months of age High levels of HbF inhibits sickling

Moderately severe hemolytic anemia is often present by age 1 year

Pallor,fatigue, jaundice Predispozition to the development of

gallstones

Sickle cell anemia-clinical findings

Intense congestion of the spleen with sickled cells may cause splenomegaly in early childhood and results in functional asplenia as early as age 3

months Great risk for infection with encapsulated bacteria

Acute splenic sequesteration Sudden enlargement of spleen with pooling of red

cells Acute exacerbation of anemia, shock, death

Aplastic crisis Caused by infection with human parvovirus


Vasoocclusive crisis Hand-foot syndrome Abdominal pain Musculoskletal pain Stroke Acute chest syndrome

Fever, pleuritic chest pain, acute pulmonary infiltrates


Decreased Hb (7-10 g/dl) with normal MCV

Reticulocytosis Characteristic sickle cells Hb electrophoresis

HbS (SCA with 0 th; HbF and S) (sickle +th;HbS with lesser HbA)

Most infants with sickle hemoglobinopathies born in USA are now identified by neonatal screening

Sickle cell anemia-laboratory findings

Patient and family education Prevention of complications and

optimization of health All children should be immunized with

the conjugate pneumococcal vaccine At the age of 2 months all children

should begin penicillin prophylaxis At least at 5 years of age

Sickle cell anemia-treatment

Treatment of painful vaso-occlusive episodes Adequate hydration Correction of acidosis if present Administration of analgesics Maintenance of normal oxygen saturation Treatment of associated infection

RBC transfusion In acute exacerbation

Exchange transfusion Hydroxyurea Bone marrow transplantation

Sickle cell anemia-treatment

Sickle cell trait Individuals who are heterozygous

for the sickle gene Hb electrophoresis

HbA 60% HbS 40% Normal levels of A2 and F

No anemia, no hemolysis

Exposure to environmental hypoxia may precipitate splenic infarction or sequestration

Sudden death during exercise? Hematuria Bacteriuria Intraocular bleeding Genetic counselling is important

Sickle cell trait

HbE Hemoglobinopathy (not a thalassemia) Production of abnormal globin chains Beta chain variant in which lysine is

substituted for glutamic acid in position 26

Mild anemia Reticulocyte count slightly increased Serum iron, ferritin increased/normal

The catabolism of 1 gr Hb yields 35 mg of bilirubin

Red blood cell of the newborn has a shortened life span= 70-90 days Significant bilirubin load

Albumin binding of unconjugated bilirubin may be important in the prevention of toxicity (kernicterus)

In the hypoglycemic infant, glucuronide production may be limited and thus conjugation is impaired

The presence of β-glucuronidase in the bowel lumen during fetal life enables bilirubin to be reabsorbed and transported across the placenta for excretion by the maternal liver

Overproduction of bilirubin-Hemolytic disease of the newborn Blood group incompatibilities such as Rh,

ABO or minor blood groups exist between a mother and her fetus

Rh(-) mother can become sensitized to the Rh Antigen Improperly matched blood transfusion Occurance of fetal-maternal blood transfusion

During pregnancy, delivery, abortion, amniocentesis

Hemolytic disease of the newborn Rh antigen Maternal antibody production IgG crosses placenta into the fetal

circulation Reacts with the Rh Ag on fetal erythrocytes These antibody coated cells are recognized

as abnormal and are destroyed by the spleen

Production of bilirubin

Hemolytic disease of the newborn Mild hemolysis Severe anemia, erythroblastosis fetalis Cardiac decompensation, massive

anasarca, circulatory collapse Hydrops fetalis (abnormal fluid in two or

more fetal compartments) The use of anti-D gammaglobulin

(rhoGam) including antenatal administration at 26-28 weeks’ gestation

ABO incompatibility ABO incompatibility is limited to

mothers of blood group O and affects infants of blood group A or B

All group O individuals have naturally occurring anti-A and anti-B antibodies, previous sensitization is not necessary

Clinical disease is milder

spherocytosis eliptocytosis

poikilocytosis

stomatocytosis

acanthocytosis Fragmentation

hemolysis

Sickle cell anemia; target cells and sickled cells

Normal RBC

Target cells Heinz body anemia

Thalassemia; severe hypochromia

Anisopoikilocytosis, target cells

normal macrocytes

Hypochromic, microcytes

Target cells

schistocytes

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Hemolysis