Meet the Red Cell
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K. Krishnan MD. FRCP,
FACP
The red cell
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Durability of red cell is
remarkable
No
nucleus
to
direct
regenerative processes
No mitochondria available
for
efficient
oxidative
metabolism
No
ribosomes
for
regeneration of lost or
damaged protein
No de novo synthesis of
lipids
RED CELL SURVIVAL
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Survives constant mechanical stress like hydrostatic pressure
and turbulence and shear stress
Survives biochemical stress of osmotic and redox fluxes as it
travels through the renal collecting system, sluggish vascular
system of the spleen, muscle and bone
Survives the ambient oxygen pressures occurring in the lungs
ALL CONSPIRE TO DAMAGE RED CELLS BUT IT SURVIVES
FOR 120 DAYS!!!
RED CELL SURVIVAL TOOLS
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SIMPLE but EXQUISITE
Adaptive membrane structures
Pathways of intermediary energy metabolism and redox
regulation and
Ability to maintain Hb in the soluble and nonoxidized form
The membrane and enzymes of the red cell are crafted to
protect the cell from external ravages of the circulation and
the internal assaults of the massive amount of iron rich and
oxidizing protein represented by the hemoglobin molecules
Basics of Erythropoiesis
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Erythropoiesis is the process of producing red cells.
Regulated by a series of steps beginning with the
pluripotent hematopoeitic stem cell
Erythroid cells come from a common
erythroid/megakaryocyte progenitor
Needs transcription factors, GATA-1 and FOG-1
(friend of GATA-1)
Basics of Erythropoiesis
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After lineage commitment is achieved, growth
factors and hormones regulate development
Erythropoeitin induces the committed progenitor to
expand in number.
Epo is regulated by Oxygen availability
Physiological regulation of RBC production
by tissue oxygen tension
Erythroid
marrow
Iron
Epo
Kidney tissue
pO2
folate
Red cell mass
Hb
concentration
B12
Erythropoeitin
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Glycoprotein
Released by specialised cells- the peritubular
capillary lining cells in the kidney
Small amount of Epo from hepatocytes
Oxygen tension in the kidney is the stimulus for Epo
production
Epo binds to specific receptors on the surface of
marrow erythroid precursors
Concept of the Erythron
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Dynamic organ made up a pool of rapidly
proliferating marrow erythroid precursors and a
large mass of circulating red blood cells
The size of the red cell mass reflects a balance
between production and destruction
Elements of Erythropoiesis
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Eythropoeitin production
Iron availability
Proliferative capacity of the bone marrow
Effective maturation of the red cell precursors
What are these?
What stains were used?
Reticulocyte Count
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An accurate reticulocyte count is
key to the initial classification of
anemia
Represent new, young, just
released red cells
Signature- supravital dye that
identifies the ribosomal RNA
Blue or black punctate spots
The residual RNA is metabolised
over time
Measure of red cell production
Reticulocytes

Reticulocytosis
 Acute
blood loss
 Splenic sequestration
 Hemolysis
 Immune
 Non-immune
 Infection
 Membrane

Reticulocytopenia
 Early
iron deficiency
 Primary bone marrow
failure
 Secondary bone
marrow failure
Use of reticulocyte count
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Two corrections necessary
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Adjusts reticulocyte count based on the reduced number of circulating red cells (with anemia the
reticulocyte percentage is increased but not the absolute number). The reticulocyte percentage is
multiplied by the ratio of the patient’s hemoglobin/hematocrit for the age and gender. This provides a
reticulocyte count corrected for the anemia
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For example, if the reticulocyte count is 8 and hemoglobin is 8, then the corrected reticulocyte count is
8/16 x 8=4
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A further correction of the corrected reticulocyte count (reticulocyte production index) is necessary for
an index of marrow production to account for the premature release of reticulocytes from the marrow
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Examine smear and see if there are polychromatophilic, macrocytes-these are prematurely
released reticulocytes called SHIFT RETICULOCYTES. If no polychromatic red cells are seen second
correction is not required
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These reticulocytes live in the peripheral blood for a longer time than normal reticulocytes and
hence provide a falsely high estimate of daily red cell production
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If polychromasia is present, the reticulocyte count corrected for anemia should be further divided
by a factor of 2.
Functional classification of anemias
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Marrow production defect
 Hypoproliferative
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Red cell maturation defect
 Ineffective
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erythropoeisis
Decreased red cell survival
 Blood
loss/hemolysis
Physiological classification of anemia
Anemia
CBC
reticulocyte
count
Index <2.5
Index >2.5
Red cell morphology
Hemolysis/hemorrhage
Normocytic normochromic
Hypoproliferative
Micro/macrocytic
Maturation disorder
Blood loss
Intravascular hemolysis
Metabolic defect
Membrane problems
Immune destruction
Frragmentation
Hypoproliferative anemias
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Serum iron, TIBC, renal and thyroid function, bone
marrow biopsy, serum ferritin
Microcytic, hypochromic anemia
Iron deficiency anemia
Microcytic hypochromic red cells
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Iron deficiency
Thalassemias
Lead poisoning
Sideroblastic anemias
Anemia of chronic
diseases
Thalassemic syndromes
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Hypochromic microcytic
red cells
“Chip munk” facies
Hemolytic anemia
Hepatosplenomegaly
Leg ulcers
Gallstones
High output heart failure
Endocrine dysfunction
Infections
Beta-thalassemic syndromes
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Microcytes
Bizarre poikilocytes
Tear drop cells
Target cells, nucleated red
cells
Extraordinarily folded red
cells called LEPTOCYTES
containing alpha-globin
inclusion bodies
What is the hematological defect?
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Failure of synthesis of the globin chains either alpha or beta
chains
Low supply of globin chains and not enough to form
hemoglobin tetramers
Leads to microcytosis and hypochromia
Unbalanced accumulation of the normal chain
Toxic inclusions and intramedullary hemolysis
Eythropoeitin surge but ineffective hematopoeisis
Builds up erythroid masses that does not produce hemoglobin
Alpha thalassemia syndromes
Alpha-thalassemia syndromes
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Hemoglobin H disease
Hemoglobin H inclusions
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Alpha-thalassemia
intermedia
Hemolysis and
splenomegaly
Supravital staining
Multiple small inclusions
due to excess betaglobin
Sickle-beta thalassemia
Hemoglobin C disease
Blood smear in a 43 year old man with history of a motor vehicle
accident 12 years ago.
What is this?
What may have
happened?
Stomatocytosis
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Slit-shaped central pallor
Usually alcoholic liver disease
and other liver diseases
 No hemolysis
Hereditary forms due to red cell
overhydration
 Na and water gain
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Hemolysis +
Target red cells
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Increased membrane surface
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obstructive liver disease due to
excess lipoprotein, cholesterol
and post-splenectomy states
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no hemolysis, cells are flexible
Volume loss
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Decreased Hb: iron deficiency,
thalassemia
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Poorly soluble hemoglobin: Hb
S, Hb C; interact with
membrane and cause water
loss
Acanthocytosis
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Irregularly spiculated red cells
with net gain in lipids and an
asymmetry between the 2 lipid
layers
Causes:
 Severe liver disease
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Abetalipoproteinemia
 Mcleod’s syndrome
Cholesterol loading causes spur
cell anemia and severe hemolysis;
no hemolysis if not cholesterol
loaded
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Hemolytic and non-hemolytic
What is the abnormality? What is the mechanism?
Spherocytosis
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Microspherocytosis:
deficiency of red cell
surface
Immune hemolytic anemias
Hereditary spherocytosis
Heinz-body hemolytic
anemia
Clostridial sepsis, Severe
burns
Hypophosphatemia
Spherocytosis
Mechanisms of Spherocytosis
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Loss of membrane lipids leading to a reduction in surface area
due to deficiencies of red cell-hereditary spherocytosis
Removal of membrane material form antibody coated red cells
by macrophages- Immune hemolytic anemia
Removal of membrane associated Heinz bodies with the
adjacent membrane lipids by the spleen- Heinz body hemolytic
anemia
Hereditary spherocytosis
Rabbit spleen showing how RBC need to elliptically deform in order to pass through the very narrow slits in the wall of
the splenic cords of Billroth and enter the sinusoids from which they can return to the circulation. A microspherocyte,
deprived of its excess surface area, cannot ellipitically deform and is thus trapped in the cords.
Osmotic fragility test
Lower panel: Hereditary spherocytosis-lysis occurs in mildy hypotonic solutions
Red cell membrane proteins
A model depicting the major proteins of the erythrocyte membrane is shown: α and β
spectrin, ankyrin, band 3, 4.1 (protein 4.1), 4.2 (protein 4.2), actin, and GP
(glycophorin).
Elliptocytosis
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Hereditary
elliptocytosis
Acquired elliptocytosis
Myelofibrosis
 Thalassemic syndromes
 Iron deficiency
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What is this?
Causes?
Cold agglutinin diseases
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Red cell autoantibodies
not cryoglobulins
Causes
 Monoclonal
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Idiopathic/chronic
B cell disorders
Polyclonal
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Benign
Postinfectious-mycoplasma,
EBV, HIV
Rouleaux
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Paraproteinemias
Rouleaux and Agglutination
Tear drop red cells
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Bone marrow infiltration
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Fibrosis
Tumors
Granulomas
What are these?
What stains were used?
Basophilic stippling
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Hemolytic anemias
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Pyrimidine-5’nucleotidase
deficiency
Iron deficiency
Thalassemias
Lead poisoning
Diffuse fine or coarse blue dots in the red cell
representing usually RNA residue
Mechanisms of basophilic stippling
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Many small bluish dots in portion of erythrocytes;
from staining of clustered polyribosomes in young
circulating red cells
Failure to digest/clear residual RNA due to
 Acquired
and congenital hemolytic anemias
 Lead poisoning (lead inhibits pyrimidine 5’ nucleotidase
which normally digests residual RNA)
What do you call these cells?
How was it stained?
Heinz bodies in red cells
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This is a positive Heinz Body preparation, with multiple red cells
containing Heinz Bodies, visible only with a supravital stain (methyl
crystal violet)
Heinz Bodies are large, blue-purple intracytoplasmic inclusions, mostly
attached to the inner cell membrane.
Heinz bodies consist of either precipitated normal or unstable
hemoglobin.
Represent oxidative injury to the red cell
These inclusions are found in cases of hemolysis due to unstable
hemoglobins, oxidant drugs (such as primaquine or dapsone),
hemolytic anemia associated with severe liver disease and G-6PD
deficiency and other enzymopathies.
Heinz-body hemolytic anemias
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Failure of mechanisms that
prevent autooxidation
(NADH/NADPH, catalase,
glutathione, peroxidase)
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Oxidative hemolysis
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Bite cells, Heinz bodies
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G6 PD deficiency
states
Nitrites, paraquat,
dapsone, hydrogen
peroxide
Unstable Hbs
Post-splenectomy
Bite cells or blister cells in G6PD oxidant hemolysis
Oxidant hemolysis and G6PD deficiency
Embden Meyerhof Glycolytic Pathway
Howell-Jolly bodies
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Usually one or at most
a few purplish
inclusions in the red
cell visible on routine
peripheral smear
exam
Mechanisms of Howell Jolly bodies
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The bodies represent aggregates of denatured
hemoglobin
Associated with states of splenic hypofunction or
splenectomy
What is this?
Hb C disease
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Intracellular and extracellular crystals
Hemoglobin C disease
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Hemoglobin C-2 normal alpha chains and 2 variant beta chains in which lysine has
replaced glutamic acid at position 6.
Unstable hemoglobin
Precipitates in red blood cells to form crystals. These intracellular crystals lead to a
decrease in red blood cell deformability and an increase in the viscosity of the
blood. The spleen effectively removes these crystal-containing cells.
The amino acid change in the hemoglobin C molecule impairs malaria growth and
development. It reduces parasitemia and confers protection against malaria.
Heterozygotes for hemoglobin C have a survival advantage in endemic areas. The
risk of malaria is lower still in persons who are homozygous for hemoglobin C.
In terms of geographic distribution, the hemoglobin C allele is found at the highest
frequencies in West Africa, where it has been associated with protection against
malaria. Also in African Americans and those of Sicilian ancestry
Macrocytic anemia
Macrocytosis
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Without megalobastosis
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Reticulocytosis
Liver disease
Aplastic anemia
MDS
Hypoxemia, smokers
With megalobastosis
Spurious increases: Cold agglutinin disease,
marked hyperglycemia, older individuals
Macrocytosis/megalobastosis
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B12 and folate moprhology is
the same
Smear: High MCV, macroovalcoytes, nuclear
hypersegmentation,
thrombocytopenia,
leucoerythroblastosis
Marrow: hypercellular,
orthochromatic megalobasts,
giant metamyelocyte
Cabot’ s rings
Hypersegmented Neutrophil
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Megalobastic anemia
Myelodysplastic
syndromes
Megalobast
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Orthochromic
megaloblast
Nuclear-cytoplasmic
asynchony
Macrocytosis and megaloblastosis
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Nuclear maturation defect
 B12,
folate, drug damage or myelodysplasia
 DNA metabolism
CBC in MAHA
Schistocytes
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Microangiopathic
process
Microangiopathic hemolytic anemia (MAHA)
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Damaged
microvasculature
Atrioventricular
malformations
Cardiac abnormalities
PNH
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This series of containers holds urine
of a patient with paroxysmal
nocturnal hemoglobinuria, showing
the episodic nature of the dark
urine (hemoglobinuria) during
intravascular hemolysis, usually
occurring at night. Early morning
urine is cola-colored. This may occur
at different times of the day and
vary from patient to patient. (This image
has been from the American Society of Hematology Slide
Bank, 3rd edition)
PNH
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Acquired chronic hemolytic anemia
Triad
 Intravascular
hemolysis
 Pancytopenia
 Venous thrombosis
The Ham Test
The Ham test (acidified
serum lysis) establishes
the diagnosis of
paroxysmal nocturnal
hemoglobinuria (PNH),
demonstrating a
characteristic
abnormality of PNH
red blood cells by
acidified fresh normal
serum. Here is a PNH
patient's (Pt) red blood
cells lysed by normal
serum at room
temperature (RT) and
at 37°C compared with
normal red cells (no
hemolysis) (control [C]).
Heated serum at 56°C
inactivates complement
and prevents hemolysis
in PNH cells.
(Taken from Image
bank American Society
of Hematology Slide
Bank, 3rd edition.
PIG-A mutation
Shortage of glycolipid molecule, GPI, due to a mutation in an X-linked gene called PIG-A
Somatic mutations and hence the patient’s marrow is a mosaic of PNH and normal cells
Type III PNH cells lacking CD 59 by flow cytometry