Download Hemoglobular Anemia

Hematology Pathophysiology: Hemoglobinopathies (Sarnaik + Pathoma)
NORMAL HEMOGLOBIN:

Structure:
Tetrameric protein with 4 peptide chains
o HbA= 2 alpha + 2 beta chains
o Molecule held together by interactions between the chains
Each Hb chain has a heme moiety (carries O2)
o Heme bound to globin chains by covalent bonds
o Iron in Fe2+ form (changes to Fe3+ form results in inability to carry O2)
Alpha1 beta2 interface is an important region for the unique O2 carrying capacity of Hb
o Abnormalities in this region will alter O2 affinity
Areas where chains are in contact with eachother OR the heme molecules are functionally important
o Have been highly conserved throughout evolution
Summary:
o Primary structure (sequence of amino acids)
o Secondary structure (folding into helix)
o Tertiary structure (binding of heme into heme pocket of helix)
o Quaternary structure (binding of four globin chains into a tetramer)

Function:
Function is oxygen transport and delivery to tissues
Relationship between O2 saturation and O2 tension is a sigmoid (S-shaped) curve
o Allows tissues to extract ~25% of O2 brought to it

O2 tension in venous system is 40mm (PO2 in venous system=40)

O2 saturation after unloading to tissues is 75%
o If the relationship were not this way, the Hb would only be able to unload O2 if the PO2 were to fall
very low
Important Values:
o P50: oxygen tension (PO2) at which Hb is 50% saturated (measure of the AFFINITY of Hb for O2)

Normal value is 26torr (under physiologic conditions)

Lower P50= higher affinity (LEFT-SHIFT)

Higher P50= lower affinity (RIGHT SHIFT)
 Lower pH
 Higher pCO2 (Bohr effect)
 Higher temperature
 Higher 2,3-BPDG levels

Genetics:
Synthesis of Hb control by genes that are switched on and off at certain stages of human life
o Results in different globin chain synthesis at different times
Alpha chain genes located on cs 16 (4 genes total)
o Alpha synthesized at high levels throughout prenatal and post natal periods
Beta chain and beta-like chain genes (delta, gamma) located on cs 11 (2 genes each)
o Beta chains begin to be synthesized ~9th week of gestation and continue to rise after birth

HbA detected around this time
o Gamma chains synthesized at high level in prenatal period and decline after birth

HbF declines but persists until ~9 months of age (switch to beta is complete)
 Therefore, B abnormalities do not present at birth

Small amount of HbF present in adults (<1%)
 Detected by exposing RBCs to acid (HbF resistant to acid)
o Delta chains made at low level both before birth and after

HbA2 normally present at 1-3% of total Hb

Diagnostically elevated in beta thalassemias*

Types of Hb:
HbA: alpha2beta2 (96%)
HbA2: alpha2delta2 (3%)
HbF: alpha2gamma2 (1%)
ABNORMAL HEMOGLOBIN SYNTHESIS:

Production of STRUCTURALLY Abnormal Globin Chains: HbS, HbC etc.
Recall that these are INTRINSIC RBC defects that result in hemolytic anemia (normocytic anemia)

Production of Structurally Normal but DECREASED Amounts of Globin Chains: thalassemias
Recall that these result in DECREASED Hb and MICROCYTIC anemia

Failure to Switch Globin Chain Synthesis: hereditary persistence of fetal Hb
THALASSEMIAS:

Definition: a genetic decrease in globin chain synthesis

Genetics: caused by mutations in globin gene clusters
Various defects
Deletional (usually alpha thalassemias) and non-deletional mutations (usually beta thalassemias)

Alpha Thalassemias:
Basics: most commonly DELETIONAL defects, although other mutations have been described
Types:
o Single Gene Deletion: silent carrier state
o Two Gene Deletion: mild microcytic, hypochromic anemia (similar to iron deficiency)
o Three Gene Deletion (HbH Disease): moderate microcytic, hypochromic anemia

Also hepatosplenomegaly due to extramedullary hematopoiesis

HbH results from tetramers of beta globin chains that form since they cannot combine with
alpha chain

Hb Barts can be detected in first few weeks of life with 3 gene deletions as well
o Four Gene Deletion (Hydrops Fetalis): incompatible with life, resulting in intrauterine death or hydrops
fetalis

HbBarts due to tetramers of gamma chains
Diagnosis:
o CBC: microcytosis and hypochromia with anemia (2 and 3 gene mutations)
o Demonstration of decreased alpha chains: globin chain synthesis measure in reticulcytes

Expensive and difficult test to perform
o Abnormal Hb Tetramers: demonstrated by cresyl blue stain and Hb electrophoresis (migrate faster
than HbA)

Hb Barts: gamma chain tetramers (prenatal or infancy)

HbH: beta chain tetramers (in adults)
o Routine Hb Electrophoresis: important to note that while this is useful for detecting tetramers in 3 and
4 gene deletions, it is not useful for 1 and 2 gene deletions

Why? All Hb types are decreased proportionately
o Restriction Mapping: reserved for prenatal diagnosis
Blood Smear:
o Microcytic, hypochromic RBCs
o Target cells
o Heinz bodies (beta chain tetramers- HbH)

Beta Thalassemias:
Types:
o Beta Thalassemia Trait/Thalassemia Minor (B/B0): one normal beta gene (not a disease)

HbA slightly decreased (90%)

HbA2 diagnostically elevated (>4%)

HbF may be slightly elevated (2-5%)
o Beta Thalassemia Intermedia (B+/B+ or B+/B0): miler disease because beta globin synthesis is NOT
completely suppressed
o Thalassemia Major (B0/B0): completely suppressed beta globin synthesis

HbA VERY low (<20%)

HbA2 diagnostically elevated (>4%)

HbF increased (70-80%; age-related)
Pathophysiology:
o Decreased beta chain synthesis leads to excess of alpha chains

Some of these used up by beta-like chains to form HbF and HbA
o Free alpha chains form tetramers (very insoluble)

Accumulate/precipitate in RBC and cause problems resulting in cell death
-
-
-
 Accumulation in cytoplasm  decreased RBC deformability
 Accumulation in membrane  increased K+ flux
 Accumulation in nucleus  G1 cell cycle arrest

Overall result of above changes:
 RBC lifespan very short (extravascular hemolysis due to removal by spleen)
 RBCs may be destroyed in bone marrow  ineffective erythropoeisis
o Lack of beta chains results in microcytic, hypochromic anemia

Attempts to increase red cell mass (expansion of marrow cavity, extramedullary
hematopoiesis)

Increased iron absorption
Clinical Features of Beta Thalassemia Major (B0/B0):
o Severe anemia at 4-6 months of age (switch to adult Hb and beta chains occurs)

Hemolytic anemia (removal of alpha tetramers by spleen) + microcytic, hypochromic anemia
(decreased Hb production)
o CBC:

Microcytic, hypochromic anemia

Evidence of compensation for hemolytic anemia (polychromasia and nucleated RBCs)
 Extravascular hemolysis due to removal of damaged RBCs by spleen (damage
caused by alpha chain tetramers)
 Increased RDW and reticulocytes
o Evidence of extramedullay hematopoiesis and marrow expansion:

Hepatosplenomegaly

Typical facies (bossing, chipmunk cheeks)

Thinning of bony cortex on X-ray

Osteoporosis (due to hypoparathyroidism)

Hair on end appearance of skull bones
Management of Beta Thalassemia Major:
o Chronic Transfusions: required to avoid growth failure and other consequences of severe anemia

Results in development of secondary hemochromatosis (iron overload)

Chelation therapy required due to iron overload:
 Desferoxamine (daily infusions via battery operated pumps)
 Desferasirox (oral qd; hepatic and renal issues)
 Desferiprone (oral qd; agranulocytosis and arthralgias)

Iron overload causes organ dysfunction:
 Liver  enzyme elevation
 Pancreas  diabetes
 Pituitary gland  growth failure and delayed puberty
 Heart  arrhythmias and cardiomyopathy

Increased risk of infection with Yersinia due to iron toxicity and chelation therapy
o Splenectomy: performed if there is an increased transfusion requirement from hypersplenism (ie.
trapping transfused RBCs in the spleen)

Increased risk of infections due to pneumococcus, hemophilus and meningococcus
(encapsulated organisms)
o Folic Acid Supplementation: due to increased requirements
o Bone Marrow Transplant: when chronic transfusions and chelation are not possible
Note that there is NO treatment for beta thalassemia minor:
o DO NOT TREAT WITH IRON  danger of iron overload
STRUCTURAL HEMOGLOBINOPATHIES:

Basics:
Cause: result from point mutation in alpha or beta globin gene, which produces an amino acid insertion in the
polypeptide chain of the Hb molecule  functional abnormality of Hb
Possible Results of Mutation:
o No physical abnormality and no clinical problem
o Increased tendency to aggregate (HbS, HbC)
o Instability of Hb molecule (hemolytic anemia)
o Altered O2 affinity (increased or decreased)
o Decreased O2 carrying capacity (HbM)

Sickling Disorders:
Types:
o SS (Sickle Cell Anemia): homozygous for mutant beta chain HbS

No normal beta alleles

No HbA
o SC: double heterozygosity with HbS and HbC (mutant beta chains)

No normal beta alleles

No HbA
o Sickle-B Thalassemia: double heterozygosity for HbS and B-thalassemia

One meta gene directs synthesis of HbS

One B gene is completely suppressed (SB0-thal) OR partially suppressed (SB+-thal)
 SB0-thal may be as severe as SS

HBA2 is elevated
o SO Arab or SD: double heterozygosity for HbS and HbO/D

Potentially as severe as SS
o SE: heterozygosity for HbS and HbE

HbE causes hypochromic thalassemic phenotype and decreases severity
o HbS with Hereditary Persistence of Fetal Hb: failure to switch from gamma to beta chain synthesis

Patients have high HbF and very mild clinical symptoms due to its protective effects
o Sickle Cell Trait: benign carrier state of HbS (10% incidence in Blacks)

Vast majority of people have no symptoms

Incurs resistance to malaria

Can cause hematuria and loss of urine concentration capacity

May also cause intravascular sickling with strenuous exercise at high altitudes OR flying at
high altitudes in an unpressurized aircraft
Inheritance:
o All sickling disorders are autosomal CODOMINANT

Since being heterozygous carries discernible clinical findings (although minor)

If both parents carry one abnormal gene, then each pregnancy has a 1 in 4 risk

If one parent has the disease and the other one abnormal gene, then each pregnancy has a 1
in 2 risk
Pathophysiology of Sickling:
o HbS: point mutation of AG leads to replacement of a glutamic acid with a valine

HbS is extremely insoluble when deoxygenated
 Therefore, low O2 conditions, acidosis and dehydration result in sickling

Polymers form in the RBC that causes the shape change to sickled form

Shape change results in:
 Increased rigidity
 Loss of deformability
 Increased adhesiveness to endothelial cells
 RBC membrane damage

All changes adversely affect flow properties of red cells through microvasculature  vasoocclusion
 Results in stasis, low pO2 in tissues, high acidity (accumulation of metabolites),
increased viscosity and FURTHER vaso-occlusion
 PMNs, platelets and the coagulation cascade also play a role in this process

Hemolytic anemia results (normocytic) due to membrane damage, which decreases RBC
lifespan to 15 days instead of 120 days
Clinical Features:
o In general, sickled cells lead to:

Vaso-occlusion (pain, ACS, stroke, priapism)

Chronic organ damage (spleen, liver, kidney, lung)

Shortened RBC survival (chronic anemia, aplastic crisis, sepsis, CHF)
o Hemolytic Anemia (Extravascular):

Pallor

Jaundice

Increased fatigue

Gallstones
-
-

Poor growth

Aplastic crisis following viral infections (can result in life-threatening decrease in Hb)
o Vascular Obstruction:

Cause: intravascular sickling

Result: episodic musculoskeletal pain (can be frequent and severe)
 Although termed “pain crises”, they are usually uncomplicated and NOT lifethreatening

Typical Areas of Obstruction:
 Bone pain/infarction: very common at all ages
o Dactylitis: swelling of the hands and feet due to bone infarction (early
childhood symptom)
o Osteonecrosis: of spine or femoral heads (often seen in adults; cause of
chronic pain)
 Spleen:
o Splenic Sequestration Crisis: sudden pooling of blood in the spleen with
hypovolemic shock (life threatening and recurrent syndrome in kids)
o Loss of Splenic Function (Autosplenectomy): due to vaso-occlusion and
fibrosis

Lifelong risk of severe bacterial infection (encapsulated)

Howell Jolly bodies on blood smear

Increased risk of Salmonella osteomyelitis
 Strokes: uncommon but highly recurrent (requires chronic transfusions to prevent)
 Acute Chest Syndrome: pulmonary infarction and/or pneumonia are common
causes (indistinguishable from one another)
 Kidneys:
o Loss of urine concentration capacity due to sickling in vessels around loop
of Henle (large volumes of dilute urine may deter patient from taking in
fluids to prevent dehydration)
o Hematuria (medullary infarcts)
o Glomerular nephropathy
o Disease is EXTREMELY variable in severity:

Factors affecting disease severity include:
 Presence of genetic markers (ie. beta gene haplotype)
 Co-inheritance of alpha-thalassemia (beneficial)
 Amount and distribution of HbF (higher levels are beneficial and prevent sickling)
Diagnosis:
o Clinical history and findings
o Demonstrate the presence of HbS

Solubility test (saponin lyses RBCs and releases Hb into solution of Na-diathionite, which
deoxygenates Hb; solution will become turbid if HbS present)

Hb electrophoresis (presence of HbS and absence of HbA)
 HbS moves slower than HbA

Modern technique is HPLC
o Demonstrate the presence of hemolytic anemia

Low Hb

Normocytic, normochromic RBCs

High reticulocyte counts

High bilirubin

Low haptoglobin
o Morphologic sickling on blood smears

Sickling is NOT seen with sickle cell trait
Management:
o Symptomatic and supportive care of pain episodes (analgesics, local heat packs, adequate hydration,
acid-base balance, avoid hypoxia and exposure to cold)
o Treatment of febrile episodes EARLY and AGGRESSIVELY with Abx (can die of fever if mismanaged)
o Blood transfusions in some scenarios:

Prevention of strokes in children

Treatment of pneumonia if respiratory distress is present


Severe anemia (Hb <5; usually with aplastic crisis or splenic sequestration)

Before general anesthesia

In problem pregnancies
o Early diagnosis by screening of newborns (allows for prevention of high childhood mortality)

Anticipatory guidance for care-givers

Routine prophylactic penicillin

Pneumococcal vaccine
o Psychosocial support

Pain attacks can be barrier to independent living and self-determination
o Non-directive genetic counseling for couples at risk

Offer prenatal diagnosis by CVS

Choice of therapeutic abortions for involved pregnancies
o Newer therapies:

Hyroxyurea to stimulate HbF production (protective against sickling)

Bone marrow or cord blood stem cell transplant
 Used in patients with bad outcome (ie. stroke in children)
o Future directions:

Anti-sickling agents (ie. membrane active drugs)

Gene therapy
Rarer Structural Hemoglobinopathies:
Unstable Hemoglobins:
o Cause: usually occur near the HEME POCKET, resulting in Hb that is unstable (results in tendency for
heme to separate from globin chain with small amounts of oxidative stress)

Denature Hb precipitates in the red cell and forms Heinz bodies (cells sequester to the spleen
and extravascular hemolysis and hemolytic anemia result
o Diagnosis:

Demonstration of hemolytic anemia

Detection of Heinz bodies by supravital staining

Heart precipitation test (exposure to 50 degree temperature produces a precipitate)

Hb electrophoresis not always helpful since Hb rapidly denatures
o Management:

Avoid oxidant drugs

Transfusions as needed

Splenectomy if anemia is severe
Hemoglobins with High O2 Affinity:
o Hb Bethesda (Prototype): amino acid substitution near the alpha1-beta2 interface, resulting in tight
binding of O2

Release of O2 to tissue is SLOW, resulting in inefficient tissue oxygenation

End result is increased Hb production due to high EPO levels
o Diagnosis:

High red cell mass

High arterial O2 saturation (>90%)
 A markedly LEFT shifted O2 dissociation curve

Presence of familial erythrocytosis (polycythemia)

Exclusion of other causes of polycythemia (Polycythemia Vera, cyanotic heart disease)
Hemoglobins with Low O2 Affinity:
o Hb Kansas (Prototype): amino acid substitution near the alpha1-beta2 interface, resulting in low O2
affinity

Hb picks up O2 poorly from the lungs resulting in high deoxyHb levels (cyanosis)

Clinically detectable cyanosis require >5gms/dl of deoxygenated Hb (found in patients with
chronic cyanosis)
o Diagnosis:

Right shifted O2 dissociation curve

Normal Hb level and RBC mass
o Management:

No specific management necessary or effective

Cyanosis is relatively well tolerated if strenuous activities are avoided
-
-
Hemoglobin M:
o Hb M Boston (Prototype): amino acid substitution close to the heme pocket, near the site of the Fe
molecule

Mutant Hb loses ability to keep Fe in ferrous state (constantly in ferric state- metHb)

MetHb is unable to carry O2 resulting in chronic cyanosis
o Diagnosis:

History of cyanosis since birth, with normal O2 saturation

Brown discoloration of freshly drawn blood (does not change with aeration)

Spectrophotometry confirms presence of metHb

Electrophoresis demonstrates abnormal Hb
o Management:

None required

Amount of HbM is not sufficient to cause physiological derangements
Hemoglobins C/D/E:
o Basics: structural variants synthesized at a lower rate than normal beta chains and comprise less than
half the total Hb in heterozygotes
o AC: heterozygous for HbC

Mild target cells

No anemia
o CC: homozygous for HbC (glutamic acid  lysine at position 6 on beta chain)

Mild hemolytic anemia (extravascular hemolysis)

Significant RBC changes (target cells, HbC crystals, microspherocytes)
o AE: heterozygous for HbE (HbE common in Far East)

Mild thalassemic phenotype

Mild micrcytosis and hypochromasia
o EE: homozygous for HbE

Moderate thalassemic phenotype

Significant hypochromasia, microcytosis and mild anemia
o E-B0 Thalassemia: heterozygous for both traits

Results in transfusion dependent thalassemic phenotype (major issue)*