Hemolytic anemias are a group of disorders characterized by the premature destruction of red blood cells, either extravascularly by macrophages or intravascularly through complement activation or mechanical destruction. This results in increased amounts of hemoglobin being released into the circulation. Physical exam may reveal pallor, jaundice, splenomegaly, dark or red-brown urine, fever, or disease-specific symptoms. Laboratory findings include increased reticulocytes, low MCV, elevated bilirubin, low haptoglobin, plasma hemoglobin, or urinary hemoglobin. Hemolytic anemias can be classified based on abnormalities of the red blood cell interior, membrane, or extrinsic factors.

1. Hemolytic AnemiasHemolytic Anemias
Marcelo dos SantosMarcelo dos Santos
B.Med.Lab.ScB.Med.Lab.Sc..

2. Hemolytic AnemiasHemolytic Anemias
A group of disorders characterized byA group of disorders characterized by prematurepremature
destruction of red blood cells:destruction of red blood cells:
 extravascular (macrophages)extravascular (macrophages)
 intravascular (complement activation;intravascular (complement activation;
mechanical destruction)mechanical destruction)
In both cases, increased amounts of hemoglobinIn both cases, increased amounts of hemoglobin
are released into the circulationare released into the circulation

3. Hemolytic AnemiasHemolytic Anemias
 Physical examination:Physical examination:
- pallorpallor
- jaundicejaundice
- splenomegalysplenomegaly
- dark or red-brown urinedark or red-brown urine
- feverfever
- disease-specific symptomsdisease-specific symptoms

4. Hemolytic AnemiasHemolytic Anemias
 Laboratory findings:Laboratory findings:
- reticulocytesreticulocytes
- MCVMCV
- bilirubinbilirubin
- haptoglobinhaptoglobin
- plasma hemoglobinplasma hemoglobin
- urinary hemoglobin N orurinary hemoglobin N or

5. Hemolytic AnemiasHemolytic Anemias

6. Hemolytic AnemiasHemolytic Anemias

7. Hemolytic AnemiasHemolytic Anemias
 ClassificationClassification
1. Abnormalities of RBC interior:1. Abnormalities of RBC interior:
-enzimopathies-enzimopathies
-hemoglobinopathies-hemoglobinopathies
2. Abnormalities of RBC membrane:2. Abnormalities of RBC membrane:
-inherited cytoskeleton abnormalities-inherited cytoskeleton abnormalities
-paroxysmal nocturnal hemoglobinuria-paroxysmal nocturnal hemoglobinuria
-severe liver disease (spur-cell anemia)-severe liver disease (spur-cell anemia)

8. Hemolytic AnemiasHemolytic Anemias
3. Extrinsic factors:3. Extrinsic factors:
-splenomegaly (hypersplenism)-splenomegaly (hypersplenism)
-antibody-mediated (autoimmune)-antibody-mediated (autoimmune)
-mechanical (microangiopathic changes,-mechanical (microangiopathic changes,
prosthetic valves)prosthetic valves)
-toxic (infectious-malaria, heavy metals--toxic (infectious-malaria, heavy metals-
copper, venoms-cobra bite)copper, venoms-cobra bite)

9. RBC interior abnormalities:RBC interior abnormalities:
HemoglobinopathiesHemoglobinopathies

10. HemoglobinopathiesHemoglobinopathies
Caused byCaused by qualitativequalitative oror quantitativequantitative defects indefects in
the globin chains.the globin chains.
 Quantitative defects:Quantitative defects: point mutations orpoint mutations or
deletions causing change in RNA processingdeletions causing change in RNA processing
or translation and leading toor translation and leading to reduced amountreduced amount
of chainsof chains
 Qualitative defects: point mutations causingQualitative defects: point mutations causing
changes in the compositionchanges in the composition of the protein ofof the protein of
the chainsthe chains

11. ThalassemiaThalassemia

12. ThalassemiaThalassemia –– aa quantitativequantitative defectdefect
Decreased or absent synthesis of one of theDecreased or absent synthesis of one of the
chains of the hemoglobin tetramer:chains of the hemoglobin tetramer:
 InIn αα-thalassemia,-thalassemia, αα-chains are missing;-chains are missing;
inin ββ-thalassemia,-thalassemia, ββ- chains are missing- chains are missing
 Continued normal synthesis of unaffectedContinued normal synthesis of unaffected
chain leads to accumulation of unstablechain leads to accumulation of unstable
aggregates of excess chainsaggregates of excess chains
 Aggregates precipitate on the membraneAggregates precipitate on the membrane
and cause damage to RBC, leading to anemiaand cause damage to RBC, leading to anemia

13. β
β
αα αα
Normal hemoglobin production:
chain balance

14. Thalassemia: chain
imbalance

15. ThalassemiaThalassemia
 the world’s most common geneticthe world’s most common genetic
diseasedisease
 autosomal recessive inheritanceautosomal recessive inheritance
 millions of carriers and hundreds ofmillions of carriers and hundreds of
thousands of patients worldwidethousands of patients worldwide

16. World incidence ofWorld incidence of αα--
thalassemiathalassemia

17. World incidence ofWorld incidence of ββ-thalassemia-thalassemia

18. World Incidence of MalariaWorld Incidence of Malaria

19. ThalassemiaThalassemia

20. αα-Thalassemia-Thalassemia
 4 is the normal number of4 is the normal number of αα-globin-globin
genesgenes
 If one or two genes are missing :If one or two genes are missing :
αα-thalassemia trait, causing mild-thalassemia trait, causing mild
microcytosis with or without anemiamicrocytosis with or without anemia
 If three genes are missing:If three genes are missing:
αα-thalassemia (HbH disease)-thalassemia (HbH disease)

21. αα-Thalassemia-Thalassemia
GENOTYPE CBC data DesignationGENOTYPE CBC data Designation
4 genes4 genes MCV ~85MCV ~85 Normal stateNormal state
3 genes3 genes MCV 78-84MCV 78-84 Silent carrierSilent carrier
((αα--//αααα))
MCV 70-72MCV 70-72 αα-Thalassemia-Thalassemia
2 genes2 genes traittrait
((αα--/α/α-- oror αααα//----) Mild anemia) Mild anemia

22. αα-Thalassemia-Thalassemia
GENOTYPE CBC data DesignationGENOTYPE CBC data Designation
MCV 55-60MCV 55-60
1 gene1 gene Hb H diseaseHb H disease
((αα-/--) Hb 7-10-/--) Hb 7-10
0 genes0 genes Hydrops fetalisHydrops fetalis
(--/--)(--/--) (incompatible with(incompatible with life)life)

23. α−/α − α−/α −
α-thalassemia trait
100%
αα/− −α−/α −
Silent carrier-50%
Hemoglobin H desease-50%
αα/− − αα/− −
Normal state-25%
Silent carrier-50%
Hydrops Fetalis-25%

24. αα-Thalassemia-Thalassemia
 Israeli ethnic groups at risk: Yemenite, Iraqi,Israeli ethnic groups at risk: Yemenite, Iraqi,
Kurdish and Ashkenazi Jews; ArabsKurdish and Ashkenazi Jews; Arabs
 αα-thalassemia trait is differentiated from-thalassemia trait is differentiated from
ββ-thalassemia trait by Hb electrophoresis:-thalassemia trait by Hb electrophoresis: normalnormal
levels of Hb A2 and Flevels of Hb A2 and F
 Definitive diagnosis at DNA level: gene mappingDefinitive diagnosis at DNA level: gene mapping
and/or sequence analysisand/or sequence analysis
 Hb H disease is much less severe thanHb H disease is much less severe than ββ--
thalassemia major, with Hb around 7-8, and nothalassemia major, with Hb around 7-8, and no
need of transfusionsneed of transfusions

25. Severe hypochromia
HbH inclusion bodies

26. ββ-thalassemia-thalassemia
MinorMinor (trait) –(trait) – heterozygousheterozygous forfor ββ-globin gene-globin gene
mutationmutation
MajorMajor (disease) –(disease) – homozygoushomozygous forfor ββ-globin-globin
gene mutation:gene mutation:
 ββ++
-thalassemia: residual synthesis of-thalassemia: residual synthesis of
ββ-chains-chains
 ββoo
-thalassemia:-thalassemia: nono residual synthesisresidual synthesis
ofof ββ-chains-chains

27. ββ-thalassemia-thalassemia
Thalassemia minorThalassemia minor::
 Low MCV with mild anemiaLow MCV with mild anemia
 Normal RDWNormal RDW
 High levels of Hb F and Hb AHigh levels of Hb F and Hb A22
((lowlow in iron deficiency!)in iron deficiency!)
 On peripheral smear: no anisocytosis withOn peripheral smear: no anisocytosis with
abundance of target formsabundance of target forms

29. ββ-thalassemia-thalassemia
Thalassemia major:Thalassemia major:
 Very low hemoglobin and MCVVery low hemoglobin and MCV
 High RDWHigh RDW
 Mild reticulocytosisMild reticulocytosis
 Variable amounts of Hb FVariable amounts of Hb F
 Peripheral smear: prominent anisocytosis,Peripheral smear: prominent anisocytosis,
poikilocytosis, hypochromia, basophilic stippling,poikilocytosis, hypochromia, basophilic stippling,
numerous normoblastsnumerous normoblasts

31. ΤΤhalassemiahalassemia
Pathophysiology:Pathophysiology:
 Severe anemia due to absent or reduced globinSevere anemia due to absent or reduced globin
chains productionchains production
 Compensation by the body in an attempt toCompensation by the body in an attempt to
make more RBCs: expansion of bone marrowmake more RBCs: expansion of bone marrow
withwith ineffective erythropoiesisineffective erythropoiesis
 Production of blood in the spleen:Production of blood in the spleen:
extramedullary erythropoiesisextramedullary erythropoiesis

32. ΤΤhalassemiahalassemia
Clinical featuresClinical features::
 Untreated thalassemia major: severe anemia inUntreated thalassemia major: severe anemia in
first year of life with transfusion dependencyfirst year of life with transfusion dependency
 Increased erythropoiesis: hyperactive boneIncreased erythropoiesis: hyperactive bone
marrow, bone deformities, enlarged liver andmarrow, bone deformities, enlarged liver and
spleenspleen
 Early death: cardiac failure, iron overload fromEarly death: cardiac failure, iron overload from
hyperabsorption and multiple transfusionshyperabsorption and multiple transfusions

34. ΤΤhalassemiahalassemia
Therapy:Therapy:
 Blood transfusion (to keep hemoglobin above 8 g%)Blood transfusion (to keep hemoglobin above 8 g%)
 Aggressive treatmentAggressive treatment
of infectionsof infections
 Iron chelationIron chelation
Bone marrow transplantation (curative)Bone marrow transplantation (curative)

35. ΤΤhalassemiahalassemia
Prenatal diagnosis:Prenatal diagnosis:
 Lifelong cost of care per patient: over aLifelong cost of care per patient: over a
quarter of a million dollars, pain and sufferingquarter of a million dollars, pain and suffering
for the patientfor the patient
 In Israel: about 1 prenatal test a week forIn Israel: about 1 prenatal test a week for
thalassemiathalassemia
 In Gaza: Over 90 families with thalassemia,In Gaza: Over 90 families with thalassemia,
which use 25% of all the blood transfused inwhich use 25% of all the blood transfused in
Gaza (very few prenatal diagnoses areGaza (very few prenatal diagnoses are
performed)performed)

36. Thalassemia is a preventableThalassemia is a preventable
disease!disease!
In Cyprus and in Greece, extensive screeningIn Cyprus and in Greece, extensive screening
and public health education have led to aand public health education have led to a
nearly complete disappearance ofnearly complete disappearance of
thalassemic birthsthalassemic births

37. ΤΤhalassemiahalassemia
Prenatal diagnosis:Prenatal diagnosis:
 Involves identification of specific mutations in eachInvolves identification of specific mutations in each
family and therefore requires prior knowledge of afamily and therefore requires prior knowledge of a
specific family genotypespecific family genotype
 Uses PCR on amniotic fluid, CVS or embryo biopsyUses PCR on amniotic fluid, CVS or embryo biopsy
 Can be done in first trimester, results available in a fewCan be done in first trimester, results available in a few
days (or preconception)days (or preconception)
Errors: “maternal contamination”, technical problems,Errors: “maternal contamination”, technical problems,
mistaken paternitymistaken paternity

38. ThalassemiaThalassemia
Preimplantation genetic diagnosis:Preimplantation genetic diagnosis:
 Conventional IVF is performedConventional IVF is performed
 Embryo biopsy (2 cells) is performed atEmbryo biopsy (2 cells) is performed at
early stage of developmentearly stage of development
 DNA diagnosis is performed by PCRDNA diagnosis is performed by PCR
 Healthy embryo is selected forHealthy embryo is selected for
implantation: avoids need for terminationimplantation: avoids need for termination
of pregnancy in case of affected fetusof pregnancy in case of affected fetus

39. ThalassemiaThalassemia
For families who already have an affectedFor families who already have an affected
child, a baby who is HLA-compatible withchild, a baby who is HLA-compatible with
that child can be selected and become athat child can be selected and become a
donor for bone marrow transplantationdonor for bone marrow transplantation
This technique is in
use in Hadassah

40. Sickle-cell AnemiaSickle-cell Anemia

41. Sickle cell anemia-Sickle cell anemia-
aa qualitativequalitative defectdefect

42. Sickle cell anemiaSickle cell anemia
Molecular basisMolecular basis:: point mutationpoint mutation in thein the
ββ-globin gene-globin gene, leading to, leading to 1 amino acid1 amino acid
substitutionsubstitution in thein the ββ-globin chain-globin chain
GGAAG GG GTTGG
Glutamic AcidGlutamic Acid ValineValine

43. Polymerization of Hb under low oxygen tension

44. Sickle cell anemiaSickle cell anemia
Millions of carriers and hundreds ofMillions of carriers and hundreds of
thousands of homozygotes all over the world.thousands of homozygotes all over the world.
In Israel, patients (homozygotes) are mostlyIn Israel, patients (homozygotes) are mostly
Arabs, but carriers also seen among Jews:Arabs, but carriers also seen among Jews:
Yemenites, Bukharians, etc.Yemenites, Bukharians, etc.

46. Sickle cell anemiaSickle cell anemia
 Carrier (trait): normal blood count andCarrier (trait): normal blood count and
peripheral smear; no symptomsperipheral smear; no symptoms
 Homozygous (disease):Homozygous (disease): ““sick as hell anemiasick as hell anemia””
 chronic anemia (Hb 6-7)chronic anemia (Hb 6-7)
 susceptibility to infectionssusceptibility to infections
 growth retardationgrowth retardation
 vaso-occlusive phenomenavaso-occlusive phenomena

47. Sickle cell anemiaSickle cell anemia
RBC membrane changesRBC membrane changes::
 under low-oxygen conditions thereunder low-oxygen conditions there’’s as a
cation flux leading to characteristic changecation flux leading to characteristic change
in cell shapein cell shape
 sickle shape is reversible for a few cyclessickle shape is reversible for a few cycles
 hemolysis and compensatory reticulocytosishemolysis and compensatory reticulocytosis
(>20%) are prominent during the period of(>20%) are prominent during the period of
““sicklingsickling””

48. Sickle cell anemiaSickle cell anemia

49. Sickle cell anemiaSickle cell anemia
““SicklingSickling”” episodes:episodes:
 InfectionInfection
 DehydrationDehydration
 HypoxiaHypoxia
 Renal circulation (mild)Renal circulation (mild)

50. Sickle cell anemiaSickle cell anemia
The most important cause of morbidity inThe most important cause of morbidity in
patients with sickle cell anemia ispatients with sickle cell anemia is
vaso-occlusion:vaso-occlusion: reticulocytes and sickledreticulocytes and sickled
cells adhere to endothelium of small andcells adhere to endothelium of small and
large vessels, leading to tissue infarctionlarge vessels, leading to tissue infarction
and organ damageand organ damage

51. Sickle cell anemiaSickle cell anemia
The most common organs involved are:The most common organs involved are:
 Skin (ulcers)Skin (ulcers)
 Bones (painful crises, infections)Bones (painful crises, infections)
 Respiratory system (acute chest syndrome)Respiratory system (acute chest syndrome)
 Brain (infarction)Brain (infarction)
 Eyes (retinopathy)Eyes (retinopathy)

55. Sickle cell anemiaSickle cell anemia
Diagnosis:Diagnosis:
 Peripheral smearPeripheral smear
 Reticulocyte countReticulocyte count
 High MCV (secondary folic acid deficiencyHigh MCV (secondary folic acid deficiency
is common)is common)
 Hb electrophoresisHb electrophoresis

56. Sickle cell anemiaSickle cell anemia
Therapy during crisisTherapy during crisis::
 HydrationHydration
 AnalgesiaAnalgesia
 SteroidsSteroids
 AntibioticsAntibiotics
 Ventilatory supportVentilatory support
Do NOT give transfusions hyperviscosity!Do NOT give transfusions hyperviscosity!
as needed

57. Sickle cell anemiaSickle cell anemia
Exchange transfusionExchange transfusion: replacement of patient: replacement of patient’’ss
blood with equal amount of normal blood (toblood with equal amount of normal blood (to
reachreach Hb S level of <30-50%Hb S level of <30-50%))
 Acute chest crisisAcute chest crisis
 Stroke (life-long therapy)Stroke (life-long therapy)
 Multi-organ failureMulti-organ failure
 PriapismPriapism

58. Sickle cell anemiaSickle cell anemia
Chronic therapy:Chronic therapy:
 Prevention and early therapy of infectionsPrevention and early therapy of infections
 Local therapy for skin ulcersLocal therapy for skin ulcers
 Gallbladder surgeryGallbladder surgery
 Analgesia as neededAnalgesia as needed
 Hydroxyurea (increases levels of Hb F)Hydroxyurea (increases levels of Hb F)
 Bone marrow transplantationBone marrow transplantation