BMS2008
Hematology
Anemia
Erythrocyte Destruction
• Breakdown of the RBC
▫ Toward the end of 120 day life span of the RBC, it begins to
break down.
- The membrane becomes less flexible.
- The concentration of cellular hemoglobin increases.
- Enzyme activity, especially glycolysis, diminishes
Removal
▫ 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.
Erythrocyte Destruction
• Two Paths
• Extravascular
• Intravascular
• About 90 % of RBC destruction is extravascular.
• Extravascular destruction occurs in the
macrophages of the spleen, liver and bone
marrow.
• Extravascular destruction is the most efficient
method of cell removal.
• The remaining 10% occurs intravascularly, where
the RBCs lyse and release their hemoglobin into
the bloodstream.
Extravascular Destruction
• Hostile surrounding in the spleen, stress the RBC
• Glycolysis slows, ATP production ends
• Intracellular sodium increases, potassium decreases
allowing water to enter the cell
• Water enter the cell- RBCs loose flexibility
• RBC’s are now trapped in spleen
Extravascular Destruction
• The RES cells lyse the RBC’s and digest
them. Components of the RBC are
recycled.
▫ Amino acids from globin are recycled
into new globin chains
▫ Iron is transported by transferrin to
the bone marrow to be recycled into
hemoglobin or stored in the
macrophage
▫ The protoporphyrin ring from heme is
broken and converted into biliverdin
▫ 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 (direct) and unconjugated
(indirect) bilirubin can be used to
monitor hemolysis.
Intravascular Destruction
▫
▫
▫
▫
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
are and the breakdown process
occurs as in extravascular
destruction.
If there is an increase in
intravascular destruction, the
haptoglobin is used up and free
hemoglobin is excreted in the
urine (hemoglobinuria).
Hemopexin is another plasma
protein that assists in salvaging
iron.
Anemia
Anemia
●
●
●
Anemia is the inability of the blood to supply the
tissue with adequate oxygen for proper
metabolic function.
Clinically, anemia is defined as a decrease in
the normal concentration of hemoglobin or
erythrocytes.
Anemia is not a disease, but an expression of an
underlying disorder or disease.
Development of Anemia
●
Anemia occurs if:
●
●
Erythrocyte loss or destruction exceeds the
maximum capacity of bone marrow erythrocyte
production OR
Bone marrow erythrocyte production is impaired
or abnormal
Causes of anemia
● Acute blood loss (hemorrhage)
● Accelerated destruction of RBC’s (immune or non●
●
●
●
●
●
immune)
Nutritional deficiency (iron, folate or B12)
Bone marrow replacement (e.g. cancer)
Infection
Toxicity
Hematopoietic stem cell arrest or damage
Hereditary or acquired defect
Erythropoiesis: Production and maturation of
erythrocytes in the bone marrow
Rubriblast
Pronormoblast
Proerythroblast
Rubricyte
Reticulocyte
Polychromatic
normoblast
Diffusely basophilic
erythrocyte
Polychromatic
erythroblast
Polychromatophilic
erythrocyte
Prorubricyte
Metarubricyte
Mature RBC
Basophilic
normoblast
Orthochromatic
normoblast
Erythrocyte
Basophilic
erythroblast
Orthochromatic
erythroblast
Discocyte
11
Anemia Classifications
●
Functional
● Uses absolute and corrected reticulocyte count,
Reticulocyte production index (RPI, also called a
corrected reticulocyte count), and serum iron for
classification
● The normal reticulocyte count ranges between 0.5 %
to 2.5% in adults and 2% to 6% in infants
●
Types
▪ Survival Defects (Increased Destruction)
▪ Proliferation Defects (Decreased production)
▪ Maturation Defects
Anemia Classifications
●
Morphologic
● Uses erythrocyte indices (MCV) for
classification
●
Types
▪ Macrocytic (size: Larger) / Normochromic (color: Normal)
▪
Causes: Folate or B12 deficiency, liver disease,
alcoholism
▪ Normocytic / Normochromic
▪
Causes: bone marrow failure, hemolytic anemia, chronic
renal failure, leukemia, metastatic malignancy
▪ Microcytic / Hypochromic (paler than normal)
▪
▪
Most common anemia
Causes: iron deficiency, sideroblastic anemia, thalassemia,
chronic diseases
Reticulocyte production index
Megaloblastic anemia: A form of
macrocytic anemia, a blood
disorder that happens when your
bone marrow produces stem cells
that make abnormally large red
blood cells.
Myelodysplasia: A group of
cancers in which immature blood
cells in the bone marrow do not
mature or become healthy blood
cells.
Aplastic anemia: A condition
that occurs when your body stops
producing enough new blood
cells.
Thalassemia: An inherited blood
disorder that causes your body to
have less hemoglobin than
normal.
Sideroblastic anemia: A type of
anemia
that
results
from
abnormal utilization of iron during
erythropoiesis.
Diagnosis of anemia
●
●
●
Clinical history
Physical signs such as paleness, fatigue, weakness
and shortness of breath
Laboratory tests
●
●
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CBC (complete blood count)
Examination of the blood smear
Reticulocyte - measures effective erythropoiesis
Bone marrow examination
Iron studies - iron, total iron-binding capacity (TIBC), ferritin
Vitamin B12 and folate
Erythropoietin level
Laboratory Tests for
Measurement of Anemia
Hemoglobin
●
Reference values
▪
▪
●
Moderate anemia:
●
●
Male: 14-17.4 g/dl
Female: 12-16 g/dl
7-10 g/dl
Severe anemia:
●
<7 g/dl
Hematocrit
● Reference values
●
●
Male: 42-52%
Female: 36-46%
Parameters of the CBC (complete blood count)
●
●
●
Red Blood Count or RBC
Hemoglobin: grams per deciliter (g/dl)
Hematocrit : the volume percentage of red blood cells in blood
●
●
Note: the approximate relationship of the hemoglobin to the hematocrit is
1:3. This may vary with the cause of the anemia and the effect on the
RBC indices, especially the MCV.
RBC indices
●
MCV - (mean corpuscular volume) Average red blood cell size, mean
cell volume
●
●
●
●
Normal:80-100 fL (femtoliters: 10−15 litres)
Derived from RBC histogram OR calculate
Used to classify RBCs as normocytic, microcytic or macrocytic
Indicates the average volume of the red cells
Calculation:
Hct % x 10
RBC count (millions/mm3)
RBC Indices con’t
●
MCH (mean corpuscular or mean cell hemoglobin)
-Hemoglobin amount per red blood cell, mean cell hemoglobin weight
●
●
●
Normal: 28-34 pg
A measurement of the hemoglobin content in RBC’s
Calculation:
Hgb x 10
RBC
MCHC (mean corpuscular hemoglobin concentration)
- The amount of hemoglobin relative to the size of the cell (hemoglobin
concentration) per red blood cell, mean cell hemoglobin concentration
●
Normal: 32-36 %
●
Used to classify RBCs as normochromic, or hypochromic
●
A measure of the concentration of hemoglobin in the average RBC
Calculation:
Hgb x 100
Hct
Parameters of the CBC
(complete blood count)
● When the population of RBCs varies in size, the MCV is a less
reliable tool to describe the erythrocyte population.
● Therefore, the RDW or red cell distribution width is usedRDW -Red
Cell Distribution Width
● Calculated index used to identify anisocytosis, a condition when the red
blood cells are unequal in size
● Normal: 11.5-14.5%
Calculation: Standard deviation of MCV x100
Mean MCV
Reticulocyte
●
●
●
●
Adult reference range: 0.5 - 2.5%
Useful in determining the response to the anemia and the
potential of the bone marrow to manufacture RBC’s.
Expressed as a percentage of the RBC’s.
When anemia is present, it is helpful to correct the retic using
the patient’s hematocrit in order to assess appropriate bone
marrow response
A supravital stain called New Methylene Blue is used to stain
reticulocytes. On a Wright’s stained smear, reticulocytes appear
as bluish red cells. The term used for retics on Wright’s stain is
polychromasia.
Corrected retic% = reticulocyte % X
Patient hct
Normal hct* based on age and sex
[*Normal female hct = 42%]
[*Normal male hct = 45%]
Reticulocyte
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●
●
Prematurely released reticulocyte remain in the blood and
take from ½ to 1 ½ days longer to mature. This will cause
even the “corrected” reticulocyte to be elevated, so a
calculation must be performed to correct for this situation to
obtain the reticulocyte production index (RPI).
A maturation time table is used for this calculation.
Indicator of the adequacy of the bone marrow response in
anemia
●
●
RPI>2: good bone marrow response
RPI<2: inadequate response
RPI =
corrected reticulocyte
Maturation time in days
Adult Reference Ranges
Red Blood Cells
Male: 4.5-5.5 x 106 /µl
Female: 4.0-5.0 x 106 /µl
Hemoglobin
Male: 14-17.4 g/dl
Female: 12-16 g/dl
Hematocrit
Male: 42-52%
Female: 36-46%
MCV
80-100 fL
MCH
28-34 pg
MCHC
32-36 %
Reticulocyte
0.5-2.5%
RDW
11.5-14.5%
The “Normal” RBC
●
Biconcave disc
●
Area of central
pallor
●
Approx. size 7 µm
RBC Size Variations
● Alterations in the size of the RBC is called anisocytosis.
● Correlate with MCV and RDW
25
Normocytic
● MCV (mean corpuscular volume) 80-100 fL
Macrocytes
● 8 μm or larger in diameter
● MCV of greater than 100 fL
● Evaluate macrocytic cells for:
◦ shape (round versus oval)
◦ color (red versus blue)
◦ pallor (if present)
◦ Pallor is a condition in which a person's skin and
mucous membranes turn lighter than they usually are.
27
Macrocytes
● Macrocytes arrive in peripheral circulation by three
main ways:
◦ Impaired DNA synthesis leading to decreased number of
cellular divisions, resulting in a larger cell
⚫Vitamin
B12/Folate deficiency
◦ Accelerated erythropoiesis ending in a premature release of
reticulocytes
◦ Conditions in which membrane cholesterol and lecithin are
increased
⚫obstructive
liver disease
28
29
Microcytes
● Diameter less than 7 μm
● MCV less than 80 fL.
● Any defect impairing hemoglobin, heme,
or globin synthesis results in microcytic,
hypochromic RBCs.
● Decrease in hemoglobin synthesis
results in increased cellular division and,
consequently, small cells.
30
Microcyte
31
RBC Color Variations
● Correlates
with MCHC (mean
corpuscular hemoglobin
concentration)
● Reference range for MCHC= 32-36%
32
Normochromic
●
Normal hemoglobin
content
●
MCHC 32-36 %
Hypochromia
● Any RBC having area of central pallor
greater than 3 μm.
● Direct relationship between amount of
hemoglobin in red cell and appearance of
red cell when stained.
● Any problem with hemoglobin synthesis
results in some degree of hypochromia.
34
Hypochromia
●
●
MCHC <32
Most frequently seen in iron deficiency
anemia. See in thalassemias,
hemoglobinopathies, and sideroblastic
anemias. May also see hypochromia in
lead poisoning.
35
Hypochromia Grading
36
Polychromasia
● Occurs when immature RBCs are released into
peripheral blood stream.
● Blue-gray in color
● Larger than normal RBCs
● Basophilia is a result of residual RNA
fragments involved in hemoglobin synthesis.
37
Polychromasia
● Cells are actually reticulocytes.
● Common to find a few polychromatic cells
on a normal peripheral blood smear.
● Reticulocyte count should reflect the
degree of polychromasia present.
38
Polychromasia
● Causes of:
◦
◦
◦
◦
acute and chronic hemorrhage
hemolysis
regenerative red cell process
newborns
● Excellent indicator of therapeutic
effectiveness for correcting iron deficiency
anemia or vitamin therapy.
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Polychromasia Grading
40
Hyperchromasia
● Does
not exist!!!!!!
Although we can have cells with a MCHC
greater than 36%, we generally do not use the
term hyperchromasia as a descriptive term.
41
RBC Shape Variations
● Alterations in the shape of the RBC is called
poikilocytosis.
42
Target Cells (Codocytes)
● Occur due to an increased red
blood cell surface area.
● Appear as "targets" on
peripheral blood smear.
● Have a pale central area with
most of the hemoglobin
around the rim of the cell.
● Are always hypochromic.
43
Target Cells (Codocytes)
● Mechanism in formation is related to excess
membrane cholesterol and phospholipid, and to
decreased cellular hemoglobin.
● Osmotic fragility is decreased.
44
So what is Osmotic Fragility?
● It is a test to measure RBC resistance to
hemolysis
● The quicker the hemolysis occurs, the
greater the osmotic fragility
● What affects osmotic fragility?
◦ Surface to volume ratio
◦ Cell membrane permeability
Target Cells (Codocytes)
●
Seen in patients with:
◦
◦
◦
◦
◦
◦
Liver disease
Hemoglobin C Disease or Trait
Post-splenectomy
Iron Deficiency Anemia
Any Hemoglobin Abnormality
Can be artifactual
46
Spherocytes
● Have a low surface-to-volume
ratio.
● Smaller than normal red cell;
hemoglobin relatively
concentrated; and, have no
area of central pallor.
● Shape change is irreversible.
47
Spherocytes
● Several
mechanisms
for
formation, but all involve loss
of membrane; aging, antibody
coating or genetic defect
● Is the final stage for red cells
before they are sequestered in
the spleen.
48
Spherocytes
● Seen
◦
◦
◦
◦
in patients with:
Activated complement
Immune Hemolytic Anemia
Hereditary Spherocytosis
Post-Transfusion
49
Wait! What is Complement?
● Complement refers to a complex set of 14 distinct
serum proteins that are involved in three separate
pathways of activation.
● Major Functions
◦ Promote the inflammatory response by
opsonization which enhances susceptibility of
coated cells to phagocytosis.
◦ Alter biological membranes to cause direct cell
lysis.
Ovalocytes and Elliptocytes
● Ovalocytes may appear normochromic or hypochromic;
normocytic or microcytic.
● Hemoglobin concentrated at both ends
● Exact mechanism of formation unknown.
51
Ovalocytes and Elliptocytes
● Ovalocytes associated with:
◦ Myelodysplastic Syndromes
◦ Thalassemias
◦ Megaloblastic Processes
● Elliptocytes associated
with:
◦ Iron Deficiency Anemia
◦ Hereditary Elliptocytosis
◦ Idiopathic Myelofibrosis
52
Stomatocytes
● Red cell of normal size
● Slit-like central area of
pallor
● Exact mechanism of
formation unknown
● Usually artifactual
● Increased osmotic
fragility
53
Stomatocytes
●
Associated with following disorders:
◦ Hereditary Stomatocytosis
◦ Hemolytic, Acute Alcoholism
◦ Rh Null Phenotype
54
Sickle Cells (Drepanocytes)
● Sickle cells are known by a
variety of names. They can
be called drepanocytes,
cresent cells, oat cells or
boat cells.
● Have at least one pointed
end.
● Surface area of cell much
greater than normal cell.
55
Sickle Cells (Drepanocytes)
●
●
Low oxygen tension
causes hemoglobin to
polymerize, forming
tubules that line up in
bundles to deform cell.
Most sickle cells can
revert back to normal
shape when
oxygenated.
56
Sickle Cells (Drepanocytes)
● Associated with the following
disorders:
◦ Sickle Cell Anemia
◦ Hemoglobin C Disease
57
Acanthocytes
● Acanthocytes are also
●
●
●
●
●
known as thorn cells or
spur cells.
Normal or slightly smaller
size
Possess 3-12 thorny
projections of uneven
length along periphery of
cell membrane.
Projections are blunt
Acanthocytes lack an area
of central pallor.
Acanthocytes appear
saturated with hemoglobin,
but the MCHC is normal.
58
Acanthocytes
● Specific mechanism of
formation unknown.
● Contain increased
cholesterol-to-phospholipid
ratio.
● Surface area increased
● Susceptible to removal by
spleen
59
Acanthocytes
●
Possible pathologies include:
◦
◦
◦
◦
◦
Alcohol Intoxication
Pyruvate Kinase Deficiency
Congenital Abetalipoproteinemia
Vitamin E Deficiency
Post-Splenectomy
60
Fragmented Cells
● Includes:
◦ Burr Cells
◦ Helmet Cells
◦ Schistocytes
● Fragmentation is defined as
a loss of a piece of cell
membrane that may or may
not contain hemoglobin.
● The shapes of these cells
varies based on shear
forces and the presence of
fibrin strands in the
circulation.
61
Fragmented Cells
Two pathways that lead to fragmentation:
●
◦
◦
Alteration of normal fluid circulation (vasculitis,
malignant hypertension, heart valve replacement).
Intrinsic defects of red cell that make it less
deformable (spherocytes and antibody-covered red
cells).
62
Burr Cells (Echinocytes)
● Red cells with 10-30
evenly spaced
spicules over the
surface of the cell.
● Normocytic and
normochromic.
● In large numbers, are
an artifact of sample
contamination.
63
Burr Cells (Echinocytes)
● "True" burr cells occur in small numbers in uremia,
heart disease, stomach cancer, bleeding peptic
ulcers, and in patients with untreated
hypothyroidism.
● Seen in liver disease, renal disease, and burn
patients.
● May occur in any situation that causes change in
tonicity of intravascular fluid (dehydration).
64
Helmet Cells (Bite Cells)
● Helmet cells or bite cells
are
another
type
of
fragmented cell that has
distinctive
projections
surrounding an empty area
of the RBC membrane.
● Usually
have
two
projections surrounding an
empty area of red cell
membrane.
● Looks as if cell has had a
bite taken out of it.
● Caused by spleenic pitting
and impalement of the
RBC on fibrin strands
65
Helmet Cells (Bite Cells)
● In conditions where red cells
have large inclusion bodies
(such as Heinz bodies
● G6PD deficiency
● May be seen in patients with
pulmonary emboli, and
disseminated intravascular
coagulation (DIC)
66
Schistocytes
● Schistocytes are another type
●
●
●
●
●
of fragmented cell.
Extreme cell fragmentation
Cell is missing whole pieces
of membrane.
Schistocytes are split or cut
indicating there has been
some sort of trauma to the cell
membrane.
Schisotocytes can appear
triangular or comma shaped.
Causes bizarre shapes of red
cells.
67
Schistocytes
● Caused by loss of
membrane by mechanical
means
● See in patients with
microangiopathic
hemolytic anemia, DIC,
heart valve surgery, or
severe burns.
68
Teardrop Cells
● Appear as pear-shaped cells.
● Length of tail varies. May be
microcytic,
macrocytic.
● Exact
normocytic,
formation
unknown.
or
process
● Commonly seen in red cells
that contain large inclusion
bodies.
● There is a correlation with
cells that have inclusions and
the rate of teardrop formation.
● As these cells with inclusions
attempt to pass through
circulation, the parts of
cells holding the inclusion
not get through and the
gets pinched, leaving a
end.
the
the
can
cell
tail
69
Teardrop Cells
● Most commonly seen
in idiopathic
myelofibrosis,
thalassemia, and iron
deficiency anemia.
70
Agglutination
● Irregular clumps of RBCs
from antigen-antibody
reactions
● See in cold hemagglutinin
disease and paroxysmal
nocturnal
hemoglobinuria(PNH)
● Reported as “RBC
agglutination noted”
71
Agglutination
● Use of saline will not disperse
clumps;
however,
warming
specimen helps to break clumps
up.
● On a patient that demonstrates
cold agglutinins, the MCHC will
be falsely elevated. elevated.
● The best way to resolve
agglutination is to warm the
specimen to 370 C to disperse
the clumps.
72
Rouleaux
● Appears as a stack of coins
● Use of saline disperses
formation of stacks
● Correlates well with elevated
sedimentation rate.
73
Rouleaux
● Caused by increased or
abnormal plasma proteins
● Result of protein deposits
on the erythrocyte
membrane
● Seen in patients with
multiple myeloma,
Waldenstrom's
macroglobulinemia, and
chronic inflammatory
disease.
74
Howell-Jolly Bodies
● Howell-Jolly bodies are dark
●
●
●
●
purple or violet spherical
granules in the erythrocyte.
Are nuclear remnants
containing DNA.
Are 1-2um in size and appear
singly around periphery of red
cell membrane.
They appear singly around the
edge of the RBC membrane.
Develop during periods of
accelerated or abnormal
erythropoiesis.
75
Howell-Jolly Bodies
● Spleen usually removes them;
however, during times of
erythroid stress, spleen cannot
keep up with formation of
inclusions and increased
amounts are found in the
peripheral blood.
● Seen following splenectomy, in
thalassemia, hemolytic
anemias, and in megaloblastic
anemias.
76
Basophilic Stippling
● Cells
exhibiting
basophilic
stippling
have
bluish-black
granular inclusions throughout
the entire cell. The granules can
vary from fine to coarse in
appearance.
● Contain aggregated ribosomes
● Stippling may be the result of the
RBCs drying on the blood
smear.
● May be seen in lead poisoning,
defective or accelerated heme
synthesis and thalassemia.
77
Basophilic Stippling
● May be classified as three
forms:
◦ Diffuse or fine - looks like fine
blue dusting.
◦ Coarse - dots are larger and
more easily defined.
◦ Punctate - coalescing of
smaller forms. Very prominent
and easily defined.
Siderotic Granules and
Pappenheimer Bodies
● Pappenheimer
●
●
●
●
bodies
granules
are
that
clusters of
contain iron.
These inclusions are often
referred as siderotic granules
since they contain iron.
These inclusions are found at
the edge of the RBC.
Siderotic granules are small,
irregular, magenta inclusions
seen along the periphery of
the cell membrane.
Appear in clusters.
Prussian blue stain required
for confirmation
Cabot Rings
● Found in heavily
stippled cells
● Appear in figure-eight
configuration
● Causes of:
◦ Megaloblastic anemias
◦ Homozygous
thalassemias
◦ Post-splenectomy.
Sideroblasts/Siderocytes
●
●
●
Sideroblasts are nucleated
erythrocyte that has
stainable iron granules
Siderocytes are
◦ Non-nucleated
erythrocyte containing
iron granules
Must use Prussian blue
stain to identify
Siderocyte
Siderotic Granules and
Pappenheimer Bodies
● When Pappenheimer bodies
are observed in Wright’s
stain,
they
are
called
Pappenheimer bodies.
● When
these cells are
observed using an iron stain,
they are referred to siderotic
granules.
● Causes of:
◦ Sideroblastic anemias
◦ Any condition leading to
hemochromatosis.
◦ Hemoglobinopathies
◦ Post-splenectomy patients.
Heinz Bodies
● Formed
as
result
of
denaturation or precipitation
of hemoglobin.
● Heinz bodies are large
inclusions found at the
edge of the cell.
● They are rigid and severely
distort cell.
● Supravital stains used to
visualize
◦ I.E. Crystal violet,
◦ brillant cresyl blue
Heinz Bodies
●
Causes of:
◦ Alpha thalassemias
◦ Glucose-6phosphate
deficiency (G6PD)
◦ Any of unstable
hemoglobin
syndromes.
◦ Red cell injury from
chemicals.
85