Download MLAB 1315- Hematology Fall 2007 Keri Brophy

MLAB 1315- Hematology
Keri Brophy-Martinez
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Chapter 5: The
Erythrocyte
Erythrocyte Maturation
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Erythropoiesis
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Production and maturation of erythrocytes
Erythropoietin (EPO)
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The growth factor that stimulates RBC production
Released in response to decreased levels of oxygen in the body tissues
Hormone produced and released by the kidneys which acts on
committed RBC stem cells to stimulate red cell maturation and release
into the blood
With normal levels of EPO stimulation and normal red cell lifespan,
about 1% of the red cells in the blood are newly released red cells
called reticulocytes. Aged rbc’s are primarily removed by the spleen.
Deficient O2 delivery to the tissues causes the kidney to increase EPO
release to accelerate red cell production
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Normal RBC in adults
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Male
4.7 - 6.1 x 106/µl
Female
4.2 - 5.4 x 106/µl
Infants and children - normals vary by age
Maturation Sequence of Erythrocytes
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Stem cell - an unspecified
cell that gives rise to a
specific specialized cell, such
as a blood cell
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Multipotential and cannot be
identified morphologically
Can self-renew and
differentiate
CFU-GEMM: granulocyte,
Stem Cell
CFU-GEMM
BFU-E
erythrocyte, monocyte,
megakaryocyte
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BFU-E: burst forming unit
CFU-E: colony forming unit
CFU-E
EPO
Mature RBC
Maturation Sequence of Erythrocytes
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Rubriblast (Pronormoblast)
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Size = 14-20 µm
Cytoplasm
 Deeply blue (basophilic
 Scant amount, may have
a perinuclear halo
 No granules
Nucleus
Large and round
 Reddish-purple with fine
chromatin
 1-2 nucleoli (may be
bluish)
N:C ratio ( nuclear:
cytoplasmic)= 4:1
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Maturation Sequence of Erythrocytes
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Prorubricyte (basophilic
normoblast)
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Size = 10-16µm
Cytoplasm
 Deeply basophilic
indicating RNA activity
needed to produce
hemoglobin (no
hemoglobin is present at
this stage)
 No granules
Nucleus
 Round, large
 Chromatin more clumped
 No nucleoli
N:C ratio = 4:1
Maturation Sequence of Erythrocytes
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Rubricyte (polychromatic
normoblast)
Size = 10-12µm
 Cytoplasm
 Blue-gray to pink-gray
(pink indicates that
hemoglobin production
has begun)
 Slight increase in amount
 Nucleus
 Round and smaller
 Chromatin more clumped,
irregular
 No nucleoli
N:C ratio = 4:1
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Maturation Sequence of Erythrocytes
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Metarubricyte Nucleated RBC
(orthochromic normoblast)
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Size: 8-10 µm
Cytoplasm
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Nucleus
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Pinker indicating larger
amounts of hemoglobin
production
Increased amount
Tightly condensed
chromatin (pyknotic)
No nucleoli
Mitosis ends at this stage
(no more DNA synthesis)
Nucleus is extruded at
end of this stage
N:C ratio = 1:1
Maturation Sequence of Erythrocytes
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Reticulocyte (diffusely
basophilic or
polychromatophilic
erythrocyte
 Size: 8-10µm
 Cytoplasm
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Diffusely basophilic due to
residual RNA
Stain with new methylene
blue to see fine reticulum
strands
Hemoglobinization is not
complete
No nucleus present
Present in circulation for
1-2 days
Lab Methods
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New Methylene Blue is a supravital stain it is used to
stain reticulocytes. They cannot be identified as
reticulocytes from Wright’s stain.
Maturation Sequence of Erythrocytes
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Mature erythrocyte
 Size = 7-8µm
 Volume = 80-100 fL
 Cytoplasm
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Pink/red
Biconcave shape
Nucleus - none
Present in circulation for
about 120 days
Red Blood Cell Membrane
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Development
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Trilaminar, three-dimensional structure
Outermost layer: glycolipids, glycoproteins
Central layer: cholesterol, phospholipids
Inner layer: cytoskeleton
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spectrin
 Composed of alpha & beta chains
 Join to form a matrix which strengthens the membrane against sheer
force and controls biconcave shape
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ankrin
membrane proteins
Red Blood Cell Membrane
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Function
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Shape
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Provide deformability, elasticity
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Provides the optimum surface to volume ratio for respiratory
exchange AND is essential to deformability
Allows for passage through microvessels
Provides permeability
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Allows water and electrolytes to exchange
RBC controls volume and H2O content primarily through
control of sodium and potassium content
Red Blood Cell Metabolism
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Metabolism
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These pathways are essential for oxygen transport and
maintaining the physical characteristics of the RBC.
Embden-Meyerhof glycolytic pathway
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Generates 90% of energy needed by RBC’s
Glucose is metabolized and generates two molecules of ATP
(energy).
Hexose monophosphate shunt
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Metabolizes 5-10% of glucose.
NADPH is end product
Protects the RBC from oxidative injury.
Most common defect is deficiency of the enzyme glucose-6phosphate dehydrogenase (G-6PD).
If the pathway is deficient, intracellular oxidants can’t be
neutralized and globin denatures then precipitates. The
precipitates are referred to as Heinz bodies. (Must use supravital
stain to visualize them.)
Red Blood Cell Membrane
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Methemoglobin reductase pathway
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Maintains iron in the ferrous (Fe2) state.
In the absence of the enzyme (methemoglobin reductase),
methemoglobin accumulates and it cannot carry oxygen.
Leubering-Rapaport shunt
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Allows the RBC to regulate oxygen transport during
conditions of hypoxia or acid-base imbalance.
Permits the accumulation of 2,3-DPG which is essential for
maintaining normal oxygen tension, regulating hemoglobin
affinity
Checkpoint
Which erythrocyte metabolic pathway is
responsible for providing the majority of
cellular energy?
 For regulating oxygen affinity?
 For maintaining hemoglobin in a reduced
state?
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Checkpoint
Embden-Meyerhof :90-95%
 Rapoport-Leubering shunt: oxygen affinity
 Hexose-Monophosphate shunt/
Methemoglobin Reductase pathway: iron
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Red Blood Cell Metabolism: Summary
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Three Areas of RBC metabolism are crucial for RBC survival
and function.
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RBC membrane
Hemoglobin structure and function
RBC metabolic pathways= cellular energy
Erythrocyte Destruction
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Breakdown of the RBC
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Toward the end of 120 day life span of the RBC, it begins to break
down. This is about 1% of RBC’s per day.
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The membrane becomes less flexible.
The concentration of cellular hemoglobin increases.
Enzyme activity, especially glycolysis, diminishes
Aging RBC’s or senescent RBC’s are removed from the circulation
by the reticuloendothelial system (RES) which is a system of
fixed macrophages. These cells are located all over the body, but
those in the spleen are the most efficient at removing old RBC’s.
Extravascular hemolysis
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90% of RBC’s are destroyed extravascularly.
Occurs in splenn, liver and bone marrow
The RES cells lyse the RBC’s and digest them. Components of the RBC are
recycled.
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Iron is transported by transferrin to the bone marrow to be recycled into
hemoglobin.
Amino acids from globin are recycled into new globin chains.
The protoporphyrin ring from heme is broken and converted into biliverdin
(green).
Biliverdin is converted to unconjugated bilirubin and carried to the liver by
albumin, a plasma protein.
Bilirubin is conjugated in the liver and excreted into the intestine, where
intestinal flora convert it to urobilinogen.
Most urobilinogen is excreted in the stool, but some is picked up by the blood
and excreted in the urine.
Conjugated (indirect) and unconjugated (direct) bilirubin can be used to monitor
hemolysis.
Refer to pg.65
Intravascular hemolysis
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5-10% of RBC’s are destroyed intrasvascularly
RBC breakdown occurs within the blood vessels.
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The free hemoglobin α and β dimers that are released into the
bloodstream is picked up by a protein carrier called haptoglobin.
The haptoglobin-hemoglobin complex is large and cannot be excreted
in the urine. It is carried to the liver where the RES cells and the
breakdown process occurs as above.
If there is an increase in intravascular hemolysis, the haptoglobin is
used up and free hemoglobin is excreted in the urine
(hemoglobinuria).
Free hemoglobin may also be oxidized to methemoglobin which is then
broken down extravascularly or to methalbumin which is bound to
albumin
Refer to page 66