path 639 to 665
Anemias
 Anemia – reduction of total circulating red cell mass below normal limits; reduces oxygen-carrying capacity of
blood, leading to tissue hypoxia; usually diagnosed based on reduction in hematocrit and hemoglobin
concentration of blood
 Morphologic characteristics providing etiologic clues include red cell size, degree of hemoglobinization (reflected
in color of RBCs), and shape
o In general, microcytic hypochromic anemias caused by disorders of hemoglobin synthesis (most often
iron deficiency), and macrocytic anemias often stem from abnormalities that impair maturation of
erythroid precursors in bone marrow
o Normochromic, normocytic anemias have diverse abnormalities
 Specific abnormalities of red cell shape seen through peripheral smear can give clue to cause
 Most useful red cell indices are MCV, MCH, MCHC, red cell distribution width (coefficient of
variation of red cell volume)
 Anemic patients appear pale and weakness, malaise, and easy fatigability are common complaints
o Lowered oxygen content of circulating blood leads to dyspnea on mild exertion
o Hypoxia can cause fatty change in liver, myocardium, and kidney – if fatty changes in myocardium are
sufficiently severe, cardiac failure can develop and compound tissue hypoxia caused by deficiency of O2
in blood; on occasion, myocardial hypoxia can manifest as angina, particularly when complicated by
preexisting coronary artery disease
o With acute blood loss and shock, oliguria and anuria can develop as result of renal hypoperfusion
o CNS hypoxia can cause headache, dimness of vision, and faintness
Anemias of Blood Loss
 Acute blood loss – effects mainly due to loss of intravascular volume, which can lead to cardiovascular collapse,
shock, and death; clinical features depend on rate of hemorrhage and whether bleeding is external or internal
o If patient survives, blood volume is rapidly restored by intravascular shift of water from interstitial fluid
compartment, resulting in hemodilution and lowering of hematocrit
o Reduction in oxygenation triggers increased secretion of erythropoietin from kidney, which stimulates
proliferation of committed erythroid progenitors (CFU-E) in marrow
 Takes 5 days for progeny of CFU-Es to mature and appear as newly released red cells
(reticulocytes) in peripheral blood
 Iron in hemoglobin recaptured if RBCs extravasate into tissues, whereas bleeding into gut or out
of body leads to iron loss and possible iron deficiency, which can hamper restoration of normal
RBC counts
o If bleeding is sufficiently massive to cause decrease in blood pressure, compensatory release of
adrenergic hormones mobilizes granulocytes from intravascular marginal pool and results in
leukocytosis
o RBCs start out normocytic normochromic, but as marrow production increases, there is an increase in
reticulocyte count (reticulocytosis) which reaches 10-15% after 7 days; reticulocytes are larger than
normal and have blue-red polychromatophilic cytoplasm
o Early recovery from blood loss accompanied by thrombocytosis, which results from increase in platelet
production
 Chronic blood loss – induces anemia only when rate of loss exceeds regenerative capacity of marrow or when
iron reserves depleted and iron deficiency anemia appears
Hemolytic Anemia
 Hemolytic anemias share
o Premature destruction of RBCs and shortened RBC life span below normal 120 days
o Elevated erythropoietin levels and compensatory increase in erythropoiesis
o Accumulation of hemoglobin degradation products released by RBC breakdown derived from
hemoglobin
o Physiologic destruction of senescent red cells takes place within mononuclear phagocytes, which are
abundant in spleen, liver, and bone marrow; triggered by age-dependent changes in RBC surface
proteins, which lead to recognition and phagocytosis
o

In great majority of hemolytic anemias, premature destruction of RBCs also occurs within phagocytes
(extravascular hemolysis) – if persistent, extravascular hemolysis leads to hyperplasia of phagocytes
manifested by varying degrees of splenomegaly
 Generally caused by alterations that render RBC less deformable
 Principal clinical features are anemia, splenomegaly, and jaundice
 Some hemoglobin inevitably escapes from phagocytes, which leads to variable decreases in
plasma haptoglobin (α2-globulin that binds free hemoglobin and prevents its excretion in urine)
 Because much of pathologic destruction of RBCs occur in spleen, patients often benefit from
splenectomy
o Intravascular hemolysis may be caused by mechanical injury (trauma caused by cardiac valves,
thrombotic narrowing of microcirculation, repetitive physical trauma [i.e. marathon running or bongo
drum beating]), complement fixation (occurs in situations where antibodies recognize and bind RBC
antigens), intracellular parasites (malaria), or exogenous toxic factors (clostridial sepsis, which results in
release of enzymes that digest RBC membrane)
 Manifested by anemia, hemoglobinemia, hemoglobinuria, hemosidernuria, and jaundice
 Large amounts of free hemoglobin released from lysed RBCs promptly bound by haptoglobin,
producing complex that is rapidly cleared by nononuclear phagocytes
 As serum haptoglobin depleted, free hemoglobin oxidizes to methemoglobin (brown in color)
 Renal proximal tubular cells reabsorb and catabolize much of filtered hemoglobin and
methemoglobin, but some passes out in urine, making it red-brown
 Iron released from hemoglobin can accumulate in tubular cells, giving rise to renal
hemosiderosis (iron overload disorder)
 Heme groups derived from hemoglobin-haptoglobin complexes catabolized to bilirubin in
mononuclear phagocytes, leading to jaundice
 No splenomegaly
o Uncomplicated hemolytic anemias – excess serum bilirubin unconjugated; level of hyperbilirubinemia
depends on functional capacity of liver and rate of hemolysis; excessive bilirubin excreted by liver into GI
tract leads to increased formation and fecal excretion of urobilin and often leads to formation of
gallstones derived from heme pigments
o Anemia and lowered tissue oxygen tension trigger production of erythropoietin, which stimulates
erythroid differentiation and leads to appearance of increased numbers of erythroid precursors
(normoblasts) in marrow
 Compensatory increases in erythropoiesis result in prominent reticulocytosis in peripheral blood
 Phagocytosis of RBCs lead to hemosiderosis most prominent in spleen, liver, and bone marrow
 If anemia is severe, extramedullary hematopoiesis can appear in liver, spleen, and lymph nodes
 With chronic hemolysis, elevated biliary excretion of bilirubin promotes formation of pigment
gallstones (cholelithiasis)
Hereditary spherocytosis (HS) – inherited disorder caused by intrinsic defects in RBC membrane skeleton that
render RBCs spheroid, less deformable, and vulnerable to splenic sequestration and destruction
o Highest in northern Europe
o Autosomal dominant inheritance pattern in 75% of cases (rest are more severe form of disease usually
caused by inheritance of 2 different defects [heterozygosity])
o Remarkable elasticity and durability in normal RBC attributable to physicochemical properties of
membrane skeleton, which lies closely apposed to internal surface of PM – chief protein component
(spectrin) consists of α and β polypeptide chains which form intertwined helical flexible heterodimers;
head regions of spectrin dimers self-associate to form tetramers, while tails associate with actin
oligomers – each actin oligomer can bind multiple spectrin tetramers, creating a 2D spectrin-actin
skeleton connected to cell membrane by 2 distinct interactions (first involves proteins ankyrin and band
4.2 and binds spectrin to transmembrane ion transporter (band 3), and second involves protein 4.1 and
binds tail of spectrin to another transmembrane protein [glycophorin A])
o HS caused by mutations that lead to insufficiency of membrane skeletal components, cutting RBC
lifespan to 10-20 days (instead of 120) – pathogenic mutations most commonly affect ankyrin, band 3,
spectrin, or band 4.2 (proteins involved in first tethering reaction) because complex important in

stabilizing lipid bilayer; most mutations cause shifts in reading frame or introduce premature stop
codons such that mutated allele fails to produce any protein; defective synthesis of affected protein
reduces assembly of skeleton as a whole and results in decrease in density of membrane skeleton
components
 Compound heterozygosity for 2 defective alleles understandably results in more severe
membrane skeleton deficiency – young HS RBCs normal in shape but deficiency of membrane
skeleton reduces stability of lipid bilayer, leading to loss of membrane fragments as RBCs age in
circulation
 Loss of membrane relative to cytoplasm forces cells to assume smallest possible diameter for
given volume (sphere)
o Beneficial effects of splenectomy prove spleen has cardinal role in premature demise of spherocytes
 Normal RBCs must undergo extreme deformation to leave cords of Billroth and enter sinusoids;
because of spheroidal shape of HS RBCs and reduced deformability, spherocytes trapped in
splenic cords, where they are eaten by phagocytes
 Splenic environment exacerbates tendency of HS RBCs to lose membrane along with K+ and H2O
– prolonged splenic exposure (erythrostasis), depletion of RBCs’ glucose, and diminished RBC pH
contribute to abnormalities
 After splenectomy, spherocytes persist, but they can’t become trapped in absent spleen
o Spherocytes are most specific morphologic finding – small, dark-staining (hyperchromic) RBCs lacking
central zone of pallor
 Spherocytosis distinctive but not pathognomonic, since other forms of membrane loss, such as
autoimmune hemolytic anemias, also cause formation of spherocytes
o Features also common to all hemolytic anemias – reticulocytosis, marrow erythroid hyperplasia,
hemosiderosis, mild jaundice
o Cholelithiasis (pigment stones) occur in 40-50% of affected adults
o Moderate splenic enlargement characteristic (happens in a few other hemolytic anemias) – results from
congestion of cords of Billroth and increased numbers of phagocytes needed to clear spherocytes
o Diagnosis based on family history, hematologic findings, and lab evidence
o 2/3 of patients have RBCs abnormally sensitive to osmotic lysis when incubated in hypotonic salt
solutions, which causes influx of water into spherocytes with little margin for expansion
o HS RBCs have increased MCHC due to dehydration caused by loss of K+ and H2O
o Severity varies greatly – small minority (mainly compound heterozygotes), HS presents at birth with
marked jaundice and requires exchange transfusions; 20-30% of patients have it so mild it is virtually
asymptomatic (RBC survival readily compensated for by increased erythropoiesis; in most,
compensatory changes outpaced, producing chronic hemolytic anemia of mild to moderate severity
o Generally stable clinical course sometimes punctuated by aplastic crises, usually triggered by acute
parovirus infection (infects and kills RBC progenitors, causing RBC production to cease until effective
immune response commences in 1-2 weeks)
 Because of reduced life span of HS RBCs, cessation of erythropoiesis even for short time leads to
sudden worsening of anemia, and transfusions may be necessary to support patient until
immune response clears infection
o Hemolytic crises produced by intercurrent events leading to increased splenic destruction of RBCs (e.g.,
infectious mononucleosis) – less significant than aplastic crises
o Gallstones can produce symptoms
o Splenectomy treats anemia and its complications, but brings risk of sepsis
Hemolytic disease due to RBC enzyme defects: Glucose-6-phosphate dehydrogenase (G6PD) deficiency
o Abnormalities in hexose monophosphate shunt or glutathione metabolism resulting from deficient or
impaired enzyme function reduce ability of RBCs to protect themselves against oxidative injuries and
lead to hemolysis
o G6PD reduces NADP to NADPH while oxidizing glucose-6-phosphate; NADPH then provides reducing
equivalents needed for conversion of oxidized glutathione to reduced glutathione, which protects
against oxidant injury by catalyzing breakdown of compounds such as H2O2
o G6PD deficiency – recessive X-linked trait; several hundred genetic variants known, but most harmless
o

G6PD- and G6PD Mediterranean cause most of clinically significant hemolytic anemias in this class
 G6PD- present in 10% of American blacks
 G6PD Mediterranean prevalent in Middle East
 High frequency of variants in each population from protective effect against malaria
o G6PD variants associated with hemolysis result in misfolding of protein, making it more susceptible to
proteolytic degradation
o G6PD B – most common normal variant, half-life longer than G6PD- (only moderately), but half-life of
G6PD Mediterranean is more markedly abnormal
o Because mature RBCs do not synthesize new proteins, G6PD- or G6PD Mediterranean enzyme activities
fall quickly to levels inadequate to protect against oxidant stress as RBCs age, so older RBCs much more
prone to hemolysis than younger ones
o Episodic hemolysis characteristic of G6PD deficiency caused by exposures that generate oxidant stress –
most common triggers are infections in which oxygen-derived free radicals produced by activated
leukocytes (viral hepatitis, pneumonia, and typhoid fever) – also can be initiated by drugs and certain
foods (oxidant drugs like antimalarials [primaquine and chloroquine], sulfonamides, nitrofurantoins);
some drugs cause hemolysis only in more severe Mediterranean variant; most frequently cited food is
fava bean, which generates oxidants when metabolized – favism is endemic in Mediterranean, Middle
East, and parts of Africa where consumption is prevalent
o Uncommonly, G6PD deficiency presents as neonatal jaundice or chronic low-grade hemolytic anemia in
absence of infection or known environmental triggers
o Oxidants cause both intravascular and extravascular hemolysis in G6PD-deficient patients – exposure of
RBCs to high levels of oxidants causes cross-linking of reactive sulfhydryl groups on globin chains, which
become denatured and form membrane-bound precipitates (Heinz bodies) which are dark inclusions in
RBCs stained with crystal violet
 Heinz bodies can damage membrane sufficiently to cause intravascular hemolysis
 Less severe membrane damage results in decreased RBC deformability
 As inclusion-bearing RBCs pass through splenic cords, macrophages pluck out Heinz bodies and,
as a result of membrane damage, some partially devoured cells retain abnormal shape,
appearing to have a bite taken out of them
 Other less severely damaged cells revert to spherocytic shape due to loss of membrane SA
 Both bite cells and spherocytes trapped in splenic cords and removed rapidly by phagocytes
o Acute intravascular hemolysis marked by anemia, hemoglobinemia, and hemoglobinuria, usually 2-3
days after exposure to oxidants – hemolysis tends to be greater in those with highly unstable G6PD
Mediterranean variant
 Since only older RBCs at risk for lysis, episode is self-limited since hemolysis ceases when only
younger G6PD-replete RBCs remain, even if administration of offending drug or food continues
 Recovery phase heralded by reticulocytosis
 Since hemolytic episodes occur intermittently, features related to chronic hemolysis
(splenomegaly, cholelithiasis) absent
Sickle cell disease – common hereditary hemoglobinopathy that occurs primary in those of African descent
o Caused by point mutation in 6th codon of β-globin that leads to replacement of glutamate residue with
valine residue – abnormal properties of resulting Hgb (HbS) responsible for disease
o 8-10% of African Americans are heterozygous for HbS (asymptomatic sickle cell trait)
o In patients with sickle cell disease, almost all Hgb in RBCs are HbS (α2βs2)
o Prevalence stems from protection of HbS against falciparum malaria
o HbS molecules undergo polymerization when deoxygenated – initially RBC cytosol converts from freely
flowing liquid to viscous gel as HbS aggregates form; with continued deoxygenation aggregated HbS
molecules assemble into long needle-like fibers in RBCs, producing distorted sickle shape
o Presence of HbS underlies chronic hemolysis, microvascular occlusions, and tissue damage
o Variables that affect rate and degree of sickling
 Interaction of HbS with other types of hemoglobin in cell – in heterozygotes, 40% of hemoglobin
is HbS and rest is HbA, which interferes with HbS polymerization, so RBCs in heterozygotes do
not sickle except under conditions of profound hypoxia

o
o
o
o
HbF inhibits polymerization of HbS even more than HbA, so infants do not become
symptomatic until 5-6 months of age when level of HbF normally falls
 Those with hereditary persistence of HbF have much less severe sickle cell disease
 HbC (lysine substituted for glutamate at 6th AA residue of β-globin) – HbSC cells (50%
HbS instead of 40% in HbAS cells) tend to lose salt and water and become dehydrated,
which increases intracellular concentration of HbS; increased tendency for HbS to
polymerize, so HbSC disease is milder than sickle cell disease (2-3% of American blacks
asymptomatic HbC heterozygotes and 1/1250 has HbSC disease)
 MCHC – higher HbS concentrations increase probability that aggregation and polymerization will
occur during given period of deoxygenation, so intracellular dehydration, which increases
MCHC, facilitates sickling; conditions that decrease MCHC reduce disease severity – occurs when
individual is homozygous for HbS but also has coexistent α-thalassemia, which reduces Hgb
synthesis and leads to milder disease
 Intracellular pH – decrease in pH reduces oxygen affinity of Hgb, thereby increasing fraction of
deoxygenated HbS at any given oxygen tension and augmenting tendency for sickling
 Transit time of RBCs through microvascular beds – transit times in most normal microvascular
beds too short for significant aggregation of deoxygenated HbS to occur, and as a result sickling
is confined to microvascular beds with slow transit times
 Transit times slow in normal spleen and bone marrow, which are prominentlyaffedcted
in sickle cell disease, and also in vascular beds that are inflamed (movement of blood
through inflamed tissues slowed because of adhesion of leukocytes and RBCs to
activated endothelial cells and transudation of fluid through leaky vessels) – inflamed
vascular beds prone to sickling and occlusion
 Sickle RBCs may express elevated levels of several adhesion molecules implicated in
binding to endothelial cells – sickle cells induce some degree of endothelial activation,
which may be related to adhesion of RBCs and granulocytes, vaso-occlusion-induced
hypoxia, and other insults
As HbS polymers grow, they herniate through membrane skeleton and project from cell ensheathed by
only lipid bilayer, causing influx of Ca2+, which induces cross-linking of membrane proteins and activates
ion channel that permits efflux of K+ and H2O, causing RBCs to become increasingly dehydrated, dense,
and rigid – eventually, most severely damaged cells converted to end-stage, nondeformable, irreversibly
sickled cells, which retain sickle shape even when fully oxygenated
 Severity of hemolysis correlates with percentage of irreversibly sickled cells, which are rapidly
sequestered and removed by mononuclear phagocytes (extravascular hemolysis)
 Sickled RBCs mechanically fragile, leading to some intravascular hemolysis as well
Microvascular occlusions dependent upon RBC membrane damage and other factors, such as
inflammation, that tend to slow or arrest movement of RBCs through microvascular beds (not by degree
of sickling or number of sickled cells)
 Leukocyte count correlates with frequency of pain crises and other measures of tissue damage
 Stagnation of RBCs within inflamed vascular beds results in extended exposure to low oxygen
tension, sickling, and vascular obstruction
Free Hgb released from lysed sickle RBCs can bind and inactivate NO (potent vasodilator and inhibitor of
platelet aggregation) – reduced NO increases vascular tone (narrowing vessels) and enhances platelet
aggregation, both of which contribute to RBC stasis, sickling, and (in some instances) thrombosis
Full-blown sickle cell anemia morph shows variable numbers of irreversibly sickled cells, reticulocytosis,
and target cells that result from red cell dehydration
 Howell-Jolly bodies (small nuclear remnants) present in some RBCs due to asplenia
 Bone marrow hyperplastic as a result of compensatory erythroid hyperplasia
 Expansion of marrow leads to bone resorption and secondary new bone formation, resulting in
prominent cheekbones and changes in skull that resemble crew-cut in X-rays
 Extramedulary hematopoiesis can appear
 Increased breakdown of Hgb can cause pigment gallstones and hyperbilirubinemia
o

In early childhood, spleen is enlarged up to 500 g by red pulp congestion caused by trapping of sickled
RBCs in cords and sinuses – with time, chronic erythrostasis leads to splenic infarction, fibrosis, and
progressive shrinkage, so that by adolescence or early adulthood only a small nubbin of fibrous splenic
tissue left (autosplenectomy)
o Infarctions caused by vascular occlusions can occur in bones, brain, kidney, liver, retina, and pulmonary
vessels (sometimes producing cor pulmonale)
o In adults, vascular stagnation in subcutaneous tissues leads to leg ulcers (rare in children)
o Causes moderately severe hemolytic anemia (Hct 18-30%) associated with reticulocytosis,
hyperbilirubinemia, and presence of irreversibly sickled cells
o Vaso-occlusive crises (pain crises) – episodes of hypoxic injury and infarction that cause severe pain in
affected region; most commonly involved sites are bones, lungs, liver, brain, spleen, and penis
 In children, painful bone crises extremely common and often difficult to distinguish from acute
osteomyelitis – frequently manifest as hand-foot syndrome or dactylitis of bones of hands or
feet or both
 Acute chest syndrome – particularly dangerous vaso-occlusive crisis involving lungs that
presents with fever, cough, chest pain, and pulmonary infiltrates
 Pulmonary inflammation (such as may be induced by simple infection) causes blood flow to
become sluggish and “spleen-like”, leading to sickling and vaso-occlusion, compromising
pulmonary function, creating potentially fatal cycle of worsening pulmonary and systemic
hypoxemia, sickling, and vaso-occlusion
 Occlusive crises most common cause of patient morbidity and mortality
o Sequestrian crises – occur in children with intact spleens; massive entrapment of sickle RBCs leads to
rapid splenic enlargement, hypovolemia, and sometimes shock; survival requires treatment with
exchange transfusions
o Aplastic crises – stem from infection of RBC progenitors by parovirus B19, which causes transient
cessation of erythropoiesis and sudden worsening of anemia
o Chronic hypoxia responsible for generalized impairment of growth and development, as well as organ
damage affecting spleen, heart, kidneys, and lungs
o Sickling provoked by hypertonicity in renal medulla causes damage that eventually leads to
hyposthenuria (inability to concentrate urine), which increases propensity for dehydration
o Increased susceptibility to infection with encapsulated organisms due to altered splenic function, which
is severly impaired in children by congestion and poor blood flow, and completely absent in adults
because of splenic infarction
 Defects in alternative complement pathway also impair opsonization of bacteria
 Pneumonia, influenza septicemia, and meningitis are common causes of death, particularly in
children – can be reduced by vaccination and prophylactic antibiotics
o Testing for HbS involves mixing blood sample with oxygen-consuming reagent such as metabisulfite,
which induces sickling of RBCs if HbS present
o Hemoglobin electrophoresis used to demonstrate presence of HbS and exclude other sickle syndromes,
such as HbSC disease
o Prenatal diagnosis possible by analysis of fetal DNA obtained by amniocentesis or chorionic biopsy
o 90% of patients survive to age 20, and about 50% survive beyond 50
 Mainstay of treatment is inhibitor of DNA synthesis (hydroxyurea) which causes increase in RBC
HbF levels, and anti-inflammatory effect from inhibition of WBC production – decreases crises
related to vascular occlusions in both children and adults
Thalassemia syndromes – group of disorders caused by inherited mutations that decrease synthesis of HbA
o α chains encoded by identical pair of α-globin genes on chromosome 16, while β chains encoded by
single β-globin gene on chromosome 11
o β-thalassemia caused by deficient synthesis of β chains, and α-thalassemia caused by deficient synthesis
of α chains
o Hematologic consequences of diminished synthesis of one globin chain stem from Hgb deficiency and
from relative excess of other globin chain, particularly in β-thalassemia
o

Thalassemia syndromes endemic in Mediterranean basin, Middle East, tropical Africa, Indian
subcontinent, and Asia – among most common inherited disorders; prevalence because of protection
against malaria by heterozygous carriers
o Defects in globin synthesis impair RBC production and contribute to pathogenesis
β-thalassemias caused by mutations that diminish synthesis of β-globin chains – causative mutations have 2
categories: β0 mutations associated with absent β-globin synthesis and β+ mutations characterized by reduced
but detectable β-globin synthesis
o More than 100 different causative mutations, mostly consisting of point mutations
 Splicing mutations – most common cause of β+-thalassemia; most mutations in introns with a
few in exons; some mutations destroy normal RNA splice junctions and completely prevent
production of normal β-globin mRNA, resulting in β0-thalassemia and others create ectopic
splice site in intron; because flanking normal splice sites remain, both normal and abnormal
splicing occurs and some normal β-globin mRNA made, resulting in β+-thalassemia
 Promoter region mutations – reduce transcription by 75-80%; some normal β-globin
synthesized, so mutations associated with β+-thalassemia
 Chain terminator mutations – most common cause of β0-thalassemia; 2 subtypes: most common
creates new stop codon in exon and other introduces small insertions or deletions that shift
mRNA reading frames – both block translation and prevent synthesis of any functional β-globin
o Deficit in HbA synthesis produces underhemoglobinized hypochromic, microcytic RBCs with subnormal
oxygen transport capacity; imbalance in α- and β-globin synthesis causes diminished survival of RBCs
and precursors
 Unpaired α chains precipitate within RBC precursors, forming insoluble inclusions that cause
membrane damage and other effects – many RBC precursors succumb to membrane damage
and undergo apoptosis (70-85% of RBC precursors in severe β-thalassemia suffer this), which
leads to ineffective erythropoiesis
 RBCs released from marrow bear inclusions and membrane damage and are prone to splenic
sequestration and extravascular hemolysis
o In severe β-thalassemia, erythropoietic drive in setting of severe uncompensated anemia leads to
massive erythroid hyperplasia in marrow and extensive extramedullary hematopoiesis
 Expanding mass of RBC precursors erodes bony cortex, impairs bone growth, and produces
skeletal abnormalities
 Extramedullary hematopoiesis involves liver, spleen, and lymph nodes and can in extreme cases
produce extraosseous masses in thorax, abdomen, and pelvis
 Metabolically active erythroid progenitors steal nutrients from other tissues that are already
oxygen-starved, causing severe cachexia (muscle atrophy) in untreated patients
o Ineffective erythropoiesis causes excessive absorption of dietary iron by suppressing circulating levels of
hepcidin (critical negative regulator of iron absorption); low levels of hepcidin and iron load of repeated
blood transfusions inevitably lead to severe iron overload unless preventative steps taken
 Secondary injury to parenchymal organs, particularly iron-laden liver, often follow and can
induce secondary hemochromatosis
o Clinical classification based on severity of anemia, which depends on genetic defect and heterozygosity
vs homozygosity
 Those with 2 β-thalassemia alleles (β+/ β+, β+/ β0, or β0/ β0) have severe, transfusion-dependent
anemia (β-thalassemia major)
 Heterozygotes with one β-thalassemia gene (β+/ β or β0/ β) usually have mild asymptomatic
microcytic anemia (β-thalassemia minor or β-thalassemia trait)
 β-thalassemia intermedia – those with milder variants of β+/ β+ or β+/ β0 thalassemia and
unusual forms of heterozygous β-thalassemia; some patients have 2 defective β-globin genes
and an α-thalassemia gene defect, which lessens imbalance in α- and β-chain synthesis
 Rare cases – individuals have single β-globin defect and 1-2 extra copies of normal α-globin gene
(stemming from gene duplication) that worsens chain imbalance; show role of unpaired α-globin
chains in pathology
o

β-thalassemia major – most common in Mediterranean countries, parts of Africa, and SE Asia; manifests
6-9 months after birth as hemoglobin synthesis switches from HbF to HbA
 RBCs may completely lack HbA (β0/ β0 genotype) or contain small amounts (β+/ β+ or β0/ β+)
 Major RBC hemoglobin is HbF and can sometimes have high levels of HbA2 (more often normal
or low levels of it)
 Blood smears show severe RBC abnormalities, including marked variation in size (anisocytosis)
and shape (poikilocytosis), microcytosis, and hypochromia
 Target cells (Hgb collects in center of cell), basophilic stippling, and fragmented RBCs common
 Inclusions of aggregated α chains efficiently removed by spleen and not easily seen
 Reticulocyte count elevated, but lower than expected for severity of anemia because of
ineffective erythropoiesis
 Variable numbers of poorly hemoglobinized nucleated red cell precursors (normoblasts) seen in
peripheral blood as result of stress erythropoiesis and abnormal release from sites of
extramedullary hematopoiesis
 In untransfused patient, there is striking expansion of hematopoietically active marrow
 In bones of face and skull, burgeoning marrow erodes existing cortical bone and induces new
bone formation, giving rise to crew-cut appearance on x-ray
 Phagocyte hyperplasia and extramedullary hematopoiesis contribute to enlargement of spleen
 Hemosiderosis and secondary hemochromatosis (2 manifestations of iron overload) occur in
almost all patients – deposited iron often damages heart, liver, and pancreas
 Clinical course is brief unless transfusions given – untreated children suffer from growth
retardation and die at an early age from effects of anemia; in those who survive long enough,
cheekbones and other bony prominences enlarged and distorted
 Hepatosplenomegaly due to extramedullary hematopoiesis
 Blood transfusions improve anemia and suppress complications related to excessive
erythropoiesis, but can result in cardiac disease due to progressive iron overload and secondary
hemochromatosis (important cause of death in heavily transfused patients, who must be
treated with iron chelators to prevent or reduce complications)
 With transfusions and iron chelation, survival into third decade is possible, but overall outlook
remains guarded – bone marrow transplantation only therapy offering cure
o β-thalassemia minor – much more common than β-thalassemia major and affects same ethnic groups
 Most patients heterozygous carriers of β+ or β0 allele
 Usually asymptomatic with mild anemia (if any)
 Peripheral blood smear typically shows some RBC abnormalities, including hypochromia,
microcytosis, basophilic stippling, and target cells
 Mild erythroid hyperplasia seen in bone marrow
 Hemoglobin electrophoresis reveals increase in HbA2 (α2δ2) to 4-8% of total hemoglobin (usually
around 2.5%) – reflection of elevated ratio of δ-chain to β-chain synthesis
 HbF levels generally normal or occasionally slightly increased
 Diagnosis important to differentiate from hypochromic microcytic anemia of iron deficiency and
for genetic counseling
 Iron deficiency can usually be excluded through measurement of serum iron, TIBC, and serum
ferritin
 Increase in HbA2 diagnostically useful, particularly in individuals who are at risk for both βthalassemia trait and iron deficiency (good to know for women of childbearing age)
α-thalassemias – caused by inherited deletions that result in reduced or absent synthesis of α-globin chains
o Normally, there are 4 α-globin genes and severity of α-thalassemia depends on how many α-globin
genes affected
o Anemia stems from lack of adequate hemoglobin and effects of excess unpaired non- α chains (β, γ, and
δ), which vary in type at different ages
 Newborns with α-thalassemia have excess unpaired γ-globin chains that form γ4 tetramers
(hemoglobin Barts), whereas older children and adults have excess β-globin chains that form β4

tetramers (HbH) – since these chains are more soluble and form more stable homotetramers
than α chains, hemolysis and ineffective erythropoiesis less severe than in β-thalassemias
o Variety of molecular lesions give rise to α-thalassemias, but gene deletion is most common cause of
reduced α-chain synthesis
 Clinical syndromes determined and classified by number of α-globin genes deleted
 More severe forms caused by more α-globin gene deletions (each normally contributes equally
so no one gene is more valuable than another)
o Silent carrier state – deletion of a single α-globin gene, which causes barely detectable reduction in αglobin chain synthesis – completely asymptomatic or slight microcytosis
o α-thalassemia trait – caused by deletion of any 2 α-globin genes (α/ α, -/- more common in Asian
populations and α/-, α/- more common in Africa) – clinically identical, but have different implications for
children of affected individuals (at risk of clinically significant α-thalassemia HbH disease or hydrops
fetalis if parent has -/- haplotype)
 Have microcytosis, minimal or no anemia, and no abnormal physical signs; HbA2 levels normal or
low
o Hemoglobin H disease – caused by deletion of 3 α-globin genes; most common in Asian populations
 Synthesis of α chains markedly reduced, and tetramers of β-globin (HbH) form
 HbH has extremely high affinity for oxygen and is not useful for oxygen delivery, leading to
tissue hypoxia disproportionate to level of Hgb
 HbH prone to oxidation, which causes it to precipitate out and form intracellular inclusions that
promote RBC sequestration and phagocytosis in spleen, resulting in moderately severe anemia
resembling β-thalassemia intermedia
o Hydrops fatalis – most severe form of α-thalassemia caused by deletion of all 4 α-globin genes
 In fetus, excess γ-globin chains form tetramers (hemoglobin Barts) that have such high affinity
for oxygen that they deliver little to tissues – survival due to expression of ζ chains (embryonic
globin that pairs with γ chains to form functional ζ2γ2 Hb tetramer
 Signs of fetal distress become evident by 3rd trimester of pregnancy – must be saved by
intrauterine transfusion or they would die from severe tissue anoxia
 Fetus shows severe pallor, generalized edema, and massive hepatosplenomegaly similar to that
seen in hemolytic disease of newborn
 Lifelong dependence on blood transfusions for survival with associated risk of iron overload
 Bone marrow transplant can be curative
Paroxysmal Nocturnal Hemoglobinuria (PNH) – disease that results from acquired mutations in
phosphatidylinositol glycan complementation group A gene (PIGA), an enzyme essential for synthesis of certain
cell surface proteins
o Incidence of 1 in 2-5 million in U.S.
o Only hemolytic anemia caused by acquired genetic defect
o Proteins anchored into lipid bilayer by hydrophobic region that spans cell membrane (transmembrane
proteins) or attached to cell membrane through covalent linkage to phospholipid
glycosylphosphatidylinositol (GPI)
 In PNH, GPI-linked proteins deficient because of somatic mutations that inactivate PIGA
o PIGA – X-linked and subject to lyonization (random inactivation of on X chromosome in females),
resulting in single acquired mutation in active PIGA gene of any given cell being sufficient to produce
deficiency state
o Because causative mutations occur in hematopoietic stem cell, all of its clonal progeny (RBCs, WBCs, and
platelets) are deficient in GPI-linked proteins – typically mutant clone coexists with progeny of normal
stem cells not PIGA deficient
o Most normal individuals harbor small numbers of bone marrow cells with PIGA mutations identical to
those that cause PNH – these cells may increase in number in rare instances where they have selective
advantage, such as with autoimmune reactions against GPI-linked antigens
o PNH blood cells deficient in 3 GPI-liked proteins that regulate complement activity: decay-accelerating
factor CD55, membrane inhibitor of reactive lysis CD59, and C8 binding protein


Most important of above is CD59, which is potent inhibitor of C3 convertase that prevents
spontaneous activation of alternative complement pathway
 RBCs, platelets, and granulocytes deficient in GPI-linked factors abnormally susceptible to lysis
or injury by complement
 RBCs – intravascular hemolysis caused by C5b-C9 membrane attack complex; hemolysis
paroxysmal and nocturnal in 25% of cases; chronic hemolysis without dramatic
hemoglobinuria more typical
 Tendency for RBCs to lyse at night explained by slight decrease in blood pH during sleep,
which increases activity of complement
o Anemia variable, but usually mild to moderate in severity; loss of heme iron in urine (hemosiderinuria)
eventually leads to iron deficiency, which can exacerbate anemia if untreated
o Thrombosis is leading cause of disease-related death – about 40% of patients suffer from venous
thrombosis, often involving hepatic, portal, or cerebral veins
 Dysfunction of platelets due to absence of certain GPI-linked proteins and contributes to
prothrombotic state, as does absorption of NO by free hemoglobin
o 5-10% of patients eventually develop acute myeloid leukemia or a myelodysplastic syndrome, possibly
because hematopoietic stem cells suffered some type of genetic damage
o Diagnosed by flow cytometry, which provides sensitive means for detecting RBCs deficient in GPI-linked
proteins such as CD59
o Infusion of monoclonal antibody inhibitor of C5a greatly reduces hemolysis but exposes patients to
increased risk of serious or fatal meningococcal infections (as is true of individuals with inherited
complement defects)
o Immunosuppressive drugs sometimes beneficial for those with evidence of marrow aplasia
o Only cure is bone marrow transplantation
Immunohemolytic anemia – hemolytic anemias caused by antibodies that bind to RBCs, leading to premature
destruction; term immunohemolytic anemia preferred because in some instances, immune reaction initiated by
ingested drug – classified based on characteristics of responsible antibody
o Diagnosis requires detection of antibodies and/or complement on RBCs from patient – done using direct
Coombs antiglobulin test in which patient’s RBCs mixed with sera containing antibodies specific for
human immunoglobulin or complement – if present, they cause agglutination
 Indirect Coombs antiglobulin test – patient’s serum tested for ability to agglutinate
commercially available RBCs bearing particular defined antigens; used to characterize antigen
target and temperature dependence of responsible antibody
 Quantitative immunological tests available to measure such antibodies directly
o Warm antibody type – most common form; about 50% of cases idiopathic (primary) and others are
related to predisposing condition (autoimmune disorders like lupus, lymphoid neoplasms) or exposure
to drugs; most causative antibodies are IgG class (less commonly IgA antibodies culpable)
 RBC hemolysis mostly extravascular
 IgG-coated RBCs bind to Fc receptors on phagocytes, which remove RBC membrane during
partial phagocytosis; loss of membrane converts RBCs into spherocytes, which are sequestered
and removed in spleen
 Moderate splenomegaly due to hyperplasia of splenic phagocytes usually seen
 Primary version of disease – antibodies directed against Rh blood group antigens
 Drug-induced version mechanisms
 Antigenic drugs – hemolysis usually follows large, intravenous doses of offending drug
and occurs 1-2 weeks after therapy initiated (penicillin or cephalosporins); drugs bind to
RBC membrane and are recognized by anti-drug antibodies
o Sometimes antibodies bind only to drug (penicillin-induced) and sometimes
antibodies recognize complex of drug and membrane protein (quinidineinduced)
o Antibodies sometimes fix complement and cause intravascular hemolysis, but
more often act as opsonins that promote extravascular hemolysis within
phagocytes

Tolerance-breaking drugs – antihypertensive agent α-methyldopa is prototype of these
drugs, which induce production of antibodies against RBC antigens, particularly Rh blood
group antigens; about 10% of patients taking α-methyldopa develop autoantibodies and
about 1% develop clinically significant hemolysis
 Treatment centers on removal of initiating factors (i.e., drugs); when not feasible,
immunosuppressive drugs and splenectomy help
o Cold agglutinin type – caused by IgM antibodies that bind RBCs avidly at low temperatures (0-4oC)
 Less common than warm antibody (15-30% of cases)
 Antibodies sometimes appear transiently following certain infections, such as Mycoplasma
pneumoniae, Epstein-Barr virus, cytomegalovirus, influenza virus, and HIV – with these, disorder
is self-limited and antibodies rarely induce clinically important hemolysis
 Chronic cold agglutinin anemia occurs in association with certain B-cell neoplasms or as
idiopathic condition
 Clinical symptoms result from binding of IgM to RBCs in vascular beds where temperature may
fall below 30oC, such as fingers, toes, and ears; IgM binding agglutinates RBCs and fixes
complement rapidly
 As blood warms and recirculates, IgM is released, usually before complement-mediated
hemolysis can occur, but transient interaction is sufficient to deposit sublytic quantities of C3b
(excellent opsonin), which leads to removal of affected RBCs by phagocytes in spleen, liver, and
bone marrow
 Hemolysis variable in severity
 Vascular obstruction caused by agglutinated RBCs results in pallor, cyanosis, and Raynaud
phenomenon (discoloration of fingers and toes due to cold or stress)
 Chronic version caused by IgM antibodies – difficult to treat
o Cold hemolysin type – autoantibodies responsible for paroxysmal cold hemoglobinuria; causes
substantial, sometimes fatal, intravascular hemolysis and hemoglobinuria
 IgG antibodies bind to P blood group antigen on RBC surface in cool, peripheral regions of body
 Complement-mediated lysis occurs when cells recirculate to warm central regions, since
complement cascade functions more efficiently at 37oC
 Most cases seen in children following viral infections – transient disorder and most recover
within a month
 Hemolytic anemia resulting from trauma to RBCs – most significantly seen in individuals with cardiac valve
prostheses and microangiopathic disorders (artificial mechanic cardiac valves more frequently implicated than
bioprosthetic porcine valves)
o Hemolysis stems from shear forces produced by turbulent blood flow and pressure gradients across
damaged valves
o Microangiopathic hemolytic anemia most commonly seen with disseminated intravascular coagulation,
but can occur in thrombotic thrombocytopenic purpura (TTP), hemolytic-uremic syndrome (HUS),
malignant hypertension, systemic lupus erythematosus, and disseminated cancer
o Common pathogenic feature is microvascular lesion that results in luminal narrowing, often due to
deposition of fibrin and platelets, producing shear stresses that mechanically injure passing RBCs
o Traumatic damage leads to appearance of RBC fragments (schistocytes), burr cells, helmet cells, and
triangle cells in blood smears
Anemias of Diminished Erythropoiesis
 Most common and important classes associated with RBC underproduction are those caused by nutritional
deficiencies, followed by those that arise secondary to renal failure and chronic inflammation
 Also less common disorders that lead to generalized bone marrow failure, such as aplastic anemia, primary
hematopoietic neoplasms, and infiltrative disorders that lead to marrow replacement (such as metastatic cancer
and disseminated granulomatous disease)
 Megaloblastic anemias – impairment of DNA synthesis that leads to distinctive morphologic changes, including
abnormally large erythroid precursors and red cells; vitamin B12 and folic acid are coenzymes required for
synthesis of thymidine (T of DNA bases), and deficiency or impairment in metabolism results in defective nuclear
maturation due to deranged or inadequate DNA synthesis, with attendant delay or block in cell division
o
o
o
o
o
o
o
o
o
All types characterized by presence of RBCs that are macrocytic and oval (macro-ovalocytes); because
they are large and contain ample hemoglobin, most macrocytes lack central pallor of normal RBCs and
could appear hyperchromic, but MCHC not elevated
Marked variation in size and shape of RBCs with low reticulocyte count
Nucleated RBC progenitors occasionally appear in circulating blood when anemia severe
Neutrophils larger than normal (macropolymorphonuclear) and hypersegmented (5 or more lobules
instead of normal 3-4)
Marrow usually markedly hypercellular as result of increased hematopoietic precursors, which often
completely replace fatty marrow
 Most primitive cells (promegaloblasts) large with deeply basophilic cytoplasm, prominent
nucleoli, and distinctive fine nuclear chromatin pattern
 As cells differentiate and begin to accumulate Hgb, nucleus retains finely distributed chromatin
and fails to develop clumped pyknotic chromatin of typical normoblasts
 While nuclear maturation delayed, cytoplasmic maturation and Hgb accumulation proceed at
normal pace, leading to nuclear-to-cytoplasmic asynchrony
 Because DNA synthesis impaired in all proliferating cells, granulocytic precursors display
dysmaturation in form of giant metamyelocytes and band forms
 Megakaryocytes can be abnormally large and have bizarre, multilobate nuclei
Marrow hyperplasia is response to increased levels of growth factors like erythropoietin
Derangement of DNA synthesis causes most precursors to undergo apoptosis in marrow and leads to
pancytopenia
Anemia exacerbated by mild degree of RBC hemolysis of uncertain etiology
Pernicious anemia – specific form caused by autoimmune gastritis and attendant failure of intrinsic
factor production, which leads to vitamin B12 deficiency
 Vitamin B12 – complex organometallic compound called cobalamin; usually solely from dietary
intake; microorganisms are ultimate origin of cobalamin in food chain (plants and vegetables
contain little cobalamin save that contributed by microbial contamination) so strictly vegetarian
or macrobiotic diets do not provide adequate amounts of cobalamin; diet that includes animal
products contains significantly larger amounts and normally results in accumulation of
intrahepatic stores of vitamin B12 sufficient to last several years
 Absorption of B12 requires intrinsic factor, which is secreted by parietal cells of fundic mucosa
 Vitamin B12 freed from binding proteins in food through action of pepsin in stomach and binds
to salivary proteins (cobalophilins or R-binders)
 In duodenum, bound B12 released by action of pancreatic proteases; B12 then associates with
intrinsic factor and complex transported to ileum, where it is endocytosed by ileal enterocytes
that express intrinsic factor receptors on surfaces
 In ileal cells, B12 associates with transcobalamin II and is secreted into plasma; transcobalamin II
delivers B12 to liver and other cells of body, including rapidly proliferating cells in bone marrow
and GI tract
 Alternative uptake mechanism not dependent on intrinsic factor or intact terminal ileum
(absorbs up to 1% of oral B12, making it feasible to treat pernicious anemia with high doses of
oral B12)
 2 reactions require B12
 Methylcobalamin serves as essential cofactor in conversion of homocystein to
methionine by methionine synthase – methylcobalamin yields methyl group recovered
from N5-methyl FH4 (principal form of folic acid in plasma), and N5-methyl FH4 is
converted to tetrahydrofolic acid (FH4), which is crucial for conversion of dUMP to dTMP
(immediate precursor of DNA)
o FH4 deficit may be further exacerbated by internal folate deficiency caused by
failure to synthesize metabolically active polyglutamylated forms, stemming
from requirement of B12 in synthesis of methionine, which contributes carbon
group needed in metabolic reactions that create folate polyglutamates
o
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Lack of folate is proximate cause of anemia in B12 deficiency since anemia
improves with administration of folic acid
 Isomerization of methylmalonyl coenzyme A to succinyl CoA – requires
adenosylcobalamin as prosthetic group on enzyme methyl-malonyl-CoA mutase
o Deficiency of B12 leads to increased plasma and urine levels of methylmalonic
acid – interruption of reaction and consequent buildup of methylmalonate and
propionate (precursor) could lead to formation and incorporation of abnormal
fatty acids into neuronal lipids
o This predisposes to myelin breakdown and thereby produces neurologic
complications of B12 deficiency
 Rare individuals with hereditary deficiencies of methylmalonyl-CoA
mutase, while having complications related to methylmalonyl academia,
do not suffer from neurologic abnormalities seen in B12 deficiency
Pernicious anemia – somewhat more prevalent in Scandinavian and other Caucasian
populations, but can occur in all racial groups; disease of older adults (median age at diagnosis is
60 and rarely occurs in people younger than 30)
Genetic predisposition strongly suspected, but no pattern discerned
Many patients have tendency to form antibodies against multiple self-antigens – believed to
result from autoimmune attack on gastric mucosa (chronic atrophic gastritis marked by loss of
parietal cells [prominent infiltrate of lymphocytes and plasma cells] and megaloblastic changes
in mucosal cells similar to those found in erythroid precursors)
3 types of autoantibodies present in many, but not all, patients
 Type I antibody – in 75% of patients, blocks binding of vitamin B12 to intrinsic factor;
found in plasma and gastric juice
 Type II antibodies – prevent binding of intrinsic factor-vitamin B12 complex to ileal
receptor, found in large proportion of patients with pernicious anemia
 Type III antibodies – found in 85-90% of patients; recognize α and β subunits of gastric
proton pump (normally localized to microvilli of canalicular system of gastric parietal
cell; not specific for pernicious anemia or other autoimmune diseases (found in as many
as 50% of elderly persons with idiopathic chronic gastritis not associated with pernicious
anemia)
Autoreactive T-cell response initiates gastric mucosal injury and triggers formation of
autoantibodies, which may exacerbate epithelial injury
When mass of intrinsic factor-secreting cells falls below threshold (and reserves of stored B12
depleted), anemia develops
Common association of pernicious anemia with other autoimmune disorders, particularly
autoimmune thyroiditis and adrenalitis, consistent with underlying immune basis
Tendency to develop multiple autoimmune disorders, including pernicious anemia, linked to
specific sequence variants of NALP1 (innate immune receptor on chromosome 17p13)
Other disorders associated with B12 deficiency impair absorption of B12
 Achlorhydria (low stomach acid production) and loss of pepsin secretion (occurs in some
elderly individuals), B12 not readily released from proteins in food
 Gastrectomy – intrinsic factor not available for uptake in ileum
 Loss of exocrine pancreatic function – B12 cannot be released from R-binder-B12
complexes
 Ileal resection or diffuse ileal disease can remove or damage site of intrinsic factor-B12
complex absorption
 Tapeworms compete with host for B12 and can induce deficiency state
 Pregnancy, hyperthyroidism, disseminated cancer, and chronic infection – increased
demand for B12 can produce relative deficiency, even with normal absorption
Stomach typically shows diffuse chronic gastritis
Most characteristic alteration is atrophy of fundic glands, affecting both chief cells and parietal
cells (virtually no parietal cells)

o
Glandular lining epithelium replaced by mucus-secreting goblet cells that resemble
those in large intestine (intestinalization)
 Some cells and their nuclei can double in size (megoblsatic change)
 Tongue may become shiny, glazed, and beefy (atrophic glossitis)
 Gastric atrophy and metaplastic changes due to autoimmunity, not B12 deficiency, so
administration of B12 corrects megaloblsatic changes in marrow and epithelial cells of
alimentary tract, but gastric atrophy and achloryhdria persist
 CNS lesions found in ¾ of cases of florid pernicious anemia but can also be seen in absence of
overt hematologic findings
 Principal alterations involve spinal cord, where demyelination of dorsal and lateral tracts
occurs, sometimes followed by loss of axons
 Gives rise to spastic paraparesis, sensory ataxia, and severe paresthesias in lower limbs
 Less frequently, degenerative changes occur in ganglia of posterior roots and in
peripheral nerves
 Pernicious anemia insidious in onset, so anemia often quite severe by time affected person
seeks medical attention – course progressive unless halted by therapy
 Diagnosis based on moderate to severe megaloblastic anemia, leukopenia with hypersegmented
granulocytes, low serum B12, and elevated levels of homocysteine and MMA in serum
 Diagnosis confirmed by striking increase in reticulocytes and improvement in hematocrit
levels 5 days after parenteral administration of B12
 Serum antibodies to intrinsic factor highly specific for pernicious anemia
 People with atrophic and metaplastic changes in gastric mucosa associated with pernicious
anemia at increased risk of developing gastric carcinoma
 Elevated homocysteine levels are risk factor for atherosclerosis and thrombosis (B12 deficiency
may increase incidence of vascular disease)
 With parenteral or high-dose oral B12, anemia can be cured and peripheral neurologic changes
reversed or halted in progression, but changes in gastric mucosa and risk of carcinoma stay
Anemia of folate deficiency – folic acid (pteroylmonoglutamic acid); deficiency results in megaloblastic
anemia; FH4 derivatives act as intermediates in transfer of 1-carbon units such as formyl and methyl
groups to various compounds, and FH4 serves as acceptor of 1-carbon fragments from compounds such
as serine and formiminoglutamic acid, which then donate acquired 1-carbon fragments in reactions
synthesizing various metabolites, so FH4 is biologic middle man in series of swaps involving 1-carbon
moieties – biological processes depending on transfers are purine synthesis, conversion of
homocysteine to methionine (requires B12), and deoxythymidylate monophosphate synthesis (in 1st 2
reactions, FH4 is regenerated from 1-carbon carrier derivatives and is available to accept another 1carbon moiety and reenter donor pool)
 In synthesis of dTMP, dihydrofolate produced that must be reduced by dihydrofolate reductase
for reentry into FH4 pool (dTMP required for DNA synthesis) – suppressed synthesis of DNA
(common to folic acid and B12 deficiency) is immediate cause of megaloblastosis
 Major causes of folic acid deficiency are decreased intake, increased requirements, and
impaired utilization
 Humans entirely dependent on dietary sources for folic acid requirement – richest sources are
green vegetables such as lettuce, spinach, asparagus, and broccoli; certain fruits (lemons,
bananas, melons) and animal sources (liver) have lesser amounts – folic acid in foods largely in
form of folylpolygutamates
 Polyglutamates sensitive to heat, so boiling, steaming, or frying foods for 5-10 minutes
destroys 95% of folate
 Intestinal conjugases split polyglutamates into monoglutamates that are readily absorbed in
proximal jejunum; during intestinal absorption, they are modified to 5-methyltetrahydrofolate
(normal transport form of folate)
 Dietary inadequacies most frequently encountered in chronic alcoholics, poor, and very elderly
 In alcoholics with cirrhosis, other mechanisms of folate deficiency such as trapping of
folate in liver, excessive urinary loss, and disordered folate metabolism implicated


Megaloblastic anemia often accompanied by general malnutrition and manifestations of
other avitaminosis, including cheilosis (cracking of lips and corner of mouth), glossitis
(inflammation of tongue), and dermatitis
 Malabsorption syndromes, such as sprue, can lead to inadequate absorption, as can diffuse
infiltrative disease of small intestine (lymphoma)
 Certain drugs such as phenytoin and oral contraceptives interfere with absorption
 Requirements of folate increased, and thus relative deficiency seen, with pregnancy, infancy,
hematologic derangements associated with hyperactive hematopoiesis (hemolytic anemias),
and disseminated cancer – demands of increased DNA synthesis render normal intake
inadequate
 Folic acid antagonists such methotrexate inhibit dihydrofolate reductase and lead to deficiency
of FH4; with inhibition of folate metabolism, all rapidly growing cells affected, particularly cells of
bone marrow and GI tract; many chemotherapeutic drugs used in treatment of cancer damage
DNA or inhibit DNA synthesis through other mechanisms, but can also cause megaloblastic
changes in rapidly dividing cells
 Diagnosis of folate deficiency (versus B12 deficiency) can be made only by demonstration of
decreased folate levels in serum or RBCs; serum homocysteine levels increased, but
methylmalonate concentrations normal; no neurologic changes
 Folate does not prevent (and may even exacerbate) neurologic deficits typical of B12 deficiency,
so absolutely necessary to exclude B12 deficiency in megaloblastic anemia before initiating
therapy with folate
Iron deficiency anemia – most common nutritional disorder in world; average Western diet contains sufficient
iron to balance fixed daily losses; about 80% of functional iron found in Hgb; myoglobin and iron-containing
enzymes such as catalase and cytochromes contain the rest; storage pool represented by hemosiderin and
ferritin (15-20% of total body iron); women have smaller stores of iron than men because of blood loss during
menstruation and can easily develop iron deficiency due to excessive losses or increased demands associated
with menstruation and pregnancy
o Iron in body recycled extensively between functional and storage pools
o Iron transported in plasma by iron-binding glycoprotein (transferrin) synthesized in liver; normally about
1/3 saturated with iron; major function of plasma transferrin is to deliver iron to cells, including
erythroid precursors, which require iron to synthesize Hgb; erythroid precursors possess high-affinity
receptors for transferrin, which mediate iron import through receptor-mediated endocytosis
o Free iron highly toxic, so it must be sequestered by binding iron in storage pool tightly to either ferritin
or hemosiderin (ferritin is ubiquitous protein-iron complex found at highest levels in liver, spleen, bone
marrow, and skeletal muscles)
 In liver, most ferritin stored in parenchymal cells; in other tissues, it is found mainly in
macrophages
 Hepatocyte iron derived from plasma transferrin; storage iron in macrophages derived from
breakdown of RBCs
 Intracellular ferritin located in cytosol and lysosomes, where partially degraded protein shells of
ferritin aggregate into hemosiderin granules; iron in hemosiderin chemically reactive; with
normal iron stores, only trace amounts of hemosiderin found in body, principally in
macrophages in bone marrow, spleen, and liver; in iron-overloaded cells, most iron stored in
hemosiderin
 Plasma ferritin derived largely from storage pool of body iron, so its levels correlate with body
iron stores; storage iron pool can be readily mobilized if iron requirements increase (i.e., after
blood loss)
o Iron balance maintained by regulating absorption of dietary iron in proximal duodenum; no regulated
pathway for iron excretion, which is mostly through shedding of mucosal and skin epithelial cells; as
body iron stores rise, absorption falls, and vice versa
o Luminal nonheme iron (mostly in Fe3+ or ferric state) and must first be reduced to Fe2+ (ferrous) iron by
ferrireductases such as b cytochromes and STEAP3
 Fe2+ transported across apical membrane by DMT1

o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
Absorption of nonheme iron inhibited by substances in diet that bind and stabilize Fe3+ and
enhanced by substances that stabilize Fe2+; often less than 5% dietary nonheme iron is absorbed
About 25% of heme iron derived from hemoglobin, myoglobin, and other animal proteins absorbed –
moved across apical membrane into cytoplasm through transporters; metabolized to release Fe2+, which
enters common pool with nonheme Fe2+
Iron that enters duodenal cells either transported to blood or stored as mucosal iron – distribution
influenced by body iron stores
Fe2+ destined for circulation transported from cytoplasm across basolateral enterocyte membrane by
ferriportin (process coupled to oxidation of Fe2+ to Fe3+, which is carried out by iron oxidases hephaestin
and ceruloplasmin); newly absorbed Fe3+ binds to transferrin, which delivers iron to RBC progenitors in
marrow
DMT1 mediates uptake of functional iron derived from endocytosed transferrin across lysosomal
membranes into cytosol of RBC precursors in bone marrow
Ferriportin plays role in release of storage iron from macrophages
Iron absorption regulated by hepcidin (small circulating peptide synthesized and released from liver in
response to increases in intrahepatic iron levels); hepcidin inhibits iron transfer from enterocyte to
plasma by binding to ferriportin and causing it to be endocytosed and degraded, causing hepcidin levels
to rise and iron becomes trapped within duodenal cells as mucosal ferritin and is lost as cells are
sloughed – thus high hepcidin levels (present when body has enough iron) inhibit iron absorption
When body stores of iron are low, hepcidin synthesis falls
By inhibiting ferriportin, hepcidin suppresses iron release from macrophages, which are important
source of iron used by erythroid precursors to make hemoglobin
Alterations in hepcidin have central role in diseases involving disturbances of iron metabolism; anemia
of chronic disease caused in part by inflammatory mediators that increase hepatic hepcidin production
 Rare form of microcytic anemia caused by mutations that disable TMPRSS6 (hepatic
transmembrane serine protease that suppresses hepcidin production when iron stores low);
affected patients have high hepcidin levels, resulting in reduced iron absorption and failure to
respond to iron therapy
 Primary and secondary hemochromatosis – hepcidin activity inappropriately low, causing
systemic iron overload
 Primary hematochromatosis associated with mutations in hepcidin or genes that
regulate hepcidin expression
 Secondary hemochromatosis can occur in diseases associated with ineffective
erythropoiesis, such as β-thalassemia major and myelodysplastic syndromes
 Ineffective erythropoiesis suppresses hepatic hepcidin production, even when iron stores high
Absorption of inorganic iron enhanced by ascorbic acid, citric acid, amino acids, and sugars in diet, and
inhibited by tannates (found in tea), cabonates, oxalates, and phosphates
Dietary iron inadequacy occurs in
 Infants at high risk due to very small amounts of iron in milk
 Impoverished who can have suboptimal diets
 Elderly who have restricted diets with little meat because of limited income or poor dentition
 Teenagers subsisting on junk food
Impaired absorption found in sprue, other causes of fat malabsorption (steatorrhea), and chronic
diarrhea
Gastrectomy impairs iron absorption by decreasing HCl and transit time through duodenum
Chronic blood loss is most common cause of iron deficiency in Western world; iron deficiency in adult
men and postmenopausal women in Western world must be attributed to GI blood loss until proven
otherwise
Iron deficiency produces hypochromic microcytic anemia
At outset of chronic blood loss or other states of negative iron balance, reserves in form of ferritin and
hemosiderin may be adequate to maintain normal Hgb and Hct levels as well as normal serum iron and
transferrin saturation
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Progressive depletion of reserves first lowers serum iron and transferrin saturation levels
without producing anemia – at this stage, increased erythroid activity in bone marrow
 Anemia appears when iron stores completely depleted and is accompanied by low serum iron,
ferritin, and transferrin saturation levels
o Mild to moderate increase in erythroid progenitors in bone marrow
o Diagnostic significant finding is disappearance of stainable iron from macrophages in blue marrow
(tested by Prussian blue stains)
o Peripheral smears have microcytic hypochromic RBCs with enlarged zone of pallor with only narrow
peripheral rim; poikilocytosis in form of small elongated red cells (pencil cells) also seen
o Dominating signs and symptoms frequently relate to underlying cause of anemia (disease, malnutrition,
pregnancy, and malabsorption)
 In severe and long-standing iron deficiency, depletion of iron-containing enzymes in cells
throughout body causes koilonychias (“spoon nails”), alopecia (hair loss), atrophic changes in
tongue and gastric mucosa, and intestinal malabsorption
 Depletion of iron from CNS may lead to appearance of pica, in which affected individuals
consume non-foodstuffs such as clay or food ingredients such as flour, and periodically move
their limbs during sleep
 Esophageal webs appear with microcytic hypochromic anemia and atrophic glossitis to complete
triad of major findings in rare Plummer-Vinson syndrome
o Diagnosis ultimately rests on lab studies – both Hgb and Hct low (usually to moderate degree) in
association with hypochromia, microcytosis, and modest poikilocytosis; serum iron and ferritin low and
TIBC (reflecting elevated transferrin levels) is high
 Low serum iron with increased IBC results in reduction of transferrin saturation
 Reduced iron stores inhibit hepcidin synthesis, and serum levels fall
o Uncomplicated iron deficiency – oral supplementation produces increase in reticulocytes in 5-7 days
followed by steady increase in blood counts and normalization of RBC indices
Anemia of chronic disease – impaired RBC production associated with chronic disease most common cause of
anemia in hospitalized patients in U.S.; associated with reduction in proliferation of erythroid progenitors and
impaired iron utilization; chronic illnesses associated can be grouped into chronic microbial infections
(osteomyelitis, bacterial endocarditis, lung abcess), chronic immune disorders (rheumatoid arthritis and regional
enteritis), neoplasms (carcinomas of lung and breast or Hodgkin lymphoma)
o Occurs in setting of persistent systemic inflammation and associated with low serum iron, reduced TIBC,
and abundant stored iron in tissue macrophages
o Certain inflammatory mediators (such as IL-6) stimulate increase in hepatic production of hepcidin,
causing erythroid precursors to be starved for iron in the midst of plenty; progenitors do not proliferate
adequately because erythropoietin levels inappropriately low for degree of anemia
o Anemia usually mild and dominant symptoms are those of underlying disease
o RBCs can be normocytic and normochromic or hypochromic and microcytic
o Presence of increased storage reduced TIBC readily rule out iron deficiency as cause of anemia
o Only successful treatment is treating underlying condition, but some patients, particularly those with
cancer, benefit from administration of erythropoietin
Aplastic anemia – syndrome of chronic primary hematopoietic failiure and attendant pancytopenia (anemia,
neutropenia, and thrombocytopenia) – can be autoimmune, inherited, or acquired
o Most cases of known etiology follow exposure to chemicals and drugs; certain drugs and agents
(including many cancer chemotherapy drugs and benzene) cause marrow suppression that is dose
related and reversible
o Can arise in unpredictable, idiosyncratic fashion following exposure to drugs that normally cause little or
no marrow suppression (chloramphenicol and gold salts cause this)
o Persistent marrow aplasia can appear after variety of viral infections, most commonly viral hepatitis of
non-A, non-B, non-C, non-G type (5-10% of cases)
o Whole body irradiation can destroy hematopoietic stem cells in dose-dependent fashion; people who
receive therapeutic irradiation or are exposed to radiation in nuclear accidents at risk for marrow aplasia
o

Fanconi anemia – rare autosomal recessive disorder caused by defects in multiprotein complex required
for DNA repair; marrow hypofunction becomes evident early in life and is often accompanied by
multiple congenital anomalies such as hypoplasia of kidney and spleen and bone anomalies most
commonly involving thumbs and radii
 Inherited defects in telomerase found in 5-10% of adult-onset aplastic anemia (required for
cellular immortality and limitless replication); results in premature hematopoietic stem cell
exhaustion and marrow aplasia
 More common are abnormally short telomeres found in marrow cells of as many as half of
patients
o 65% of cases are idiopathic (don’t know what caused it)
o 2 major etiologies: extrinsic immune-mediated suppression of marrow progenitors or intrinsic
abnormality of stem cells
o Few remaining marrow stem cells from aplastic anemia marrows have genes involved in apoptosis and
death pathways upregulated
o Immunosuppressive drugs (cyclosporine) produce responses in 60-70% of patients (possibly suppressing
or killing autoreactive T-cells)
o In some instances GPI-linked proteins may be targets (link with PNH)
o Occasional transformation of aplasias into myeloid neoplasms, typically myelodysplasia or acute myeloid
leukemia; association with abnormally short telomeres; karyotypic aberrations in many cases – possibly
caused by marrow insult or predisposition to DNA damage results in sufficient injury to limit
proliferative and differentiative capacity of stem cells and if damage is extensive enough, aplastic
anemia results
o Markedly hypocellular bone marrow largely devoid of hematopoietic cells; often only fat cells, fibrous
stroma, and scattered lymphocytes and plasma cells remain; marrow aspirates often yield little material
(dry tap), so aplasia best appreciated in marrow biopsies
o Nonspecific pathological changes related to granulocytopenia and thrombocytopenia – mucocutaneous
bacterial infections and abnormal bleeding
o If anemia necessitates multiple transfusions, systemic hemosiderosis can appear
o Can occur at any age and in either gender; onset usually insidious
o Anemia can cause progressive weakness, pallor, and dyspnea; thrombocytopenia heralded by petechiae
(red to purple spots that look like tons of tiny bruises) and ecchymoses (hematoma larger than 1 cm)
o Neutropenia manifests as frequent and persistent minor infections or sudden onset of chills, fever, and
prostration
o Splenomegaly absent and if present, diagnosis of aplastic anemia should be seriously questioned
o Reticulocytopenia is a rule
o RBCs usually slightly macrocytic and normochromic
o Bone marrow transplantation provides 5-year survival of over 75%
o Older patients and those without donors often respond well to immunosuppressive therapy
Pure red cell aplasia – primary marrow disorder where only erythroid progenitors suppressed; in severe cases,
RBC progenitors completely absent from marrow; may occur in association with neoplasms (thymoma and large
granular lymphocytic leukemia), drug exposures, autoimmune disorders, and parovirus infection
o Most cases have autoimmune basis
o When thymoma present, resection leads to hematologic improvement in about half of patients, possibly
because tumor is source of marrow suppressive cells
o In patients without thymoma, immunosuppressive therapy often beneficial
o Plasmapheresis may be helpful in patients with pathogenic autoantibodies, such as neutralizing
antibodies to erythropoietin that appear de novo or following administration of recombinant
erythropoietin
o Special form of RBC aplasia occurs in individuals infected with parvovirus B19, which preferentially
infects and destroys RBC progenitors; normal individuals clear parvovirus infections in 1-2 weeks, so
aplasia is transient and clinically unimportant
o In persons with moderate to severe hemolytic anemias, even brief cessation of erythropoiesis results in
rapid worsening of anemia, producing aplastic crisis
o
In the severely immunosuppressed (i.e., HIV patients), ineffective immune response sometimes permits
infection to persist, leading to chronic RBC aplasia and moderate to severe anemia
 Myelophthisic anemia – marrow failure in which space-occupying lesions replace normal marrow elements;
most common cause is metastatic cancer, most often breast, lung, or prostate; any infiltrative process (e.g.,
granulomatous disease) involving marrow can produce
o Feature of spent phase of myeloproliferative disorders
o All responsible diseases cause marrow distortion and fibrosis, which act to displace normal marrow
elements and disturb mechanisms that regulate egress of RBCs and granulocytes from marrow – causes
abnormal release of nucleated erythroid precursors and immature granulocytic forms
(leukoerythroblastosis) into peripheral smears and appearance of teardrop-shaped RBCs deformed
during tortuous escape from fibrotic marrow
 Chronic renal failure – almost invariably associated with anemia that tends to be roughly proportional to
severity of uremia; dominant cause is diminished synthesis of erythropoietin by damaged kidneys, which leads
to inadequate RBC production
o Extracorpuscular defect reduces RBC life span
o Iron deficiency due to platelet dysfunction and increased bleeding
o Administration of recombinant erythropoietin results in significant improvement of anemia – optimal
response may require concomitant iron replacement therapy
 Hepatocellular liver disease – whether toxic, infectious, or cirrhotic; associated with anemia attributed to
decreased marrow function
o Folate and iron deficiencies caused by poor nutrition and excessive bleeding can exacerbate anemia
o Erythroid progenitors preferentially affected
o Depression of WBC count and platelets less common but can occur
o Anemia often slightly macrocytic due to lipid abnormalities associated with liver failure, which cause
RBC membranes to acquire phospholipid and cholesterol as they circulate in peripheral blood
 Endocrine disorders – particularly hypothyroidism; may be associated with mild normochromic, normocytic
anemia
Polycythemia
 Abnormally high RBC count, usually with corresponding increase in Hgb level
o Relative RBC increase – when there is hemoconcentration due to decreased plasma volume
 Results from dehydration (prolonged vomiting or diarrhea, excessive use of diuretics, water
deprivation)
 Associated with obscure condition (stress polycythemia or Gaisböck syndrome); patients
hypertensive, obese, and anxious (stressed)
o Absolute RBC increase – when there is increase in total RBC mass
 Primary when it results from intrinsic abnormality of hematopoietic precursors
 Secondary when RBC progenitors responding to increased levels of erythropoietin
 Polycythemia vera – most common cause of primary polycythemia; myeloproliferative disorder associated with
mutations that lead to erythropoietin-independent growth of RBC progenitors
 Familial mutations in erythropoietin receptor induce erythropoietin-independent receptor activation
 Secondary polycythemias – caused by compensatory or pathologic increases in erythropoietin secretion
o Erythropoietin-secreting tumors can cause
o Inherited defects that lead to stabilization of HIF-1α (hypoxia-induced factor that stimulates
transcription of erythropoietin gene