Practical Hematology Lab

- LAB 2 -

RBC’s Morphology
RBCS Abnormal Morphology

Peripheral Blood Morphology

RECORDING RBC MORPHOLOGY
1. Scan area using ×100 (oil immersion).
2. Observe 10 fields.
3. Red cells are observed for size, shape, hemoglobin content, and
the presence or absence of inclusions.
4. Abnormal morphology: Red cell morphology is assessed
according to See the following sample grading system. Note
that red cell morphology must be scanned in a good counting
area.
Two questions should be asked
1. Is the morphology seen in every field?
2. Is the morphology pathologic and not artificially induced?
Abnormal Erythrocyte Morphology
Is found in pathological states that may be
abnormalities in
I. Red cell distribution.
II. Size (anisocytosis).
III. Hemoglobin content – Color Variation .
IV. Shape (poikilocytosis).
V. The presence of inclusion bodies in erythrocyte.
I. Erythrocyte Distribution Abnormalities

Rouleaux formation
Stacking of RBCs due to increased plasma
proteins coating RBCs (resembling a stack of
coins)
Found in
• Hyperfibrinogenaemia
• Hyperglobulinaemia
Agglutination
Antibody-mediated Irregular clumping ,
temperature dependent

Found in
• Cold agglutinins
• Warm autoimmune hemolysis
Rouloux Formation Agglutination
II. Variation In Erythrocyte Size (Anisocytosis)
Anisocytosis
Variations in size (Microcyte and
Macrocyte)

Normocytic RBC’s
Normal size of RBC (8 μm) with
a range of 7 to 9 μm.
The nucleus of a small
lymphocyte (± 8 µm) is a useful
guide to the size of a red blood
cell).
 Microcytic
RBC cell smaller than the normal RBC ( <7 μm), and is
associated with a decrease in hemoglobin synthesis
Found in
• Iron deficiency anemia.
• Thalassaemia.
• Sideroblastic anemia.
• Lead poisoning.
• Anemia of chronic disease.
Macrocyte
RBC larger than the normal (<9 μm) and is the result of a
defect in nuclear maturation or stimulated erythropoiesis.
May be round or oval in shape, the diagnostic significance
being different.

Found in
• Folate and B12 deficiencies (oval)
• Ethanol (round)
• Liver disease (round)
• Reticulocytosis (round)
Example : Film Study
Most erythrocytes presented
in the picture are microcytes
(compare with the small
lymphocyte). The degree of
hemoglobinization is
sufficient. Normal platelets
and single ovalocytes are
present.
1. Microcyte
2. Normocyte
III. Variation In Erythrocyte Color
• A normal erythrocyte has a pinkish-red color with a
slightly lighter-colored center (central pallor) when
stained with a blood stain, such as Wright.

• The color of the erythrocyte is representative of

hemoglobin concentration in the cell.

• Under normal conditions, when the color, central pallor,

and hemoglobin are proportional, the erythrocyte is
referred to as Normochromic.
 Hypochromia
• Increased central pallor and decreased hemoglobin
concentration, the central pallor occupies more
than the normal third of the red cell diameter.
Found in
• Iron deficiency
• Thalassaemia
• any of the conditions leading
to Microcytosis
 Polychromasia
• Red cells stain shades of blue-gray as a consequence of
uptake of both eosin (by hemoglobin) and basic dyes (by
residual ribosomal RNA). Often slightly larger than normal
red cells and round in shape - round macrocytosis.

Found in
Any situation with reticulocytosis
– for example bleeding,
hemolysis or response to
heamatinic factor replacement.
What Abnormal Results Mean
This test is used to diagnose the cause of anemia.
The following are the types of anemia and their
causes:
• Normocytic/ normochromic (NC/NC) anemia is caused by
sudden blood loss, prosthetic heart valves, sepsis, tumor,
long-term disease or aplastic anemia.
• Microcytic/ hypochromic anemia is caused by iron
deficiency, lead poisoning, or thalassemia.
• Microcytic/ normochromic anemia results from a deficiency
of the hormone erythropoietin from kidney failure.
• Macrocytic /normochromic anemia results from
chemotherapy, folate deficiency, or vitamin B-12 deficiency.
 IV. Shape Abnormalities of Erythrocytes
• Poikilocytosis is the general term for mature
erythrocytes that have a shape other than the round,
biconcave disk.
• Poikilocytes can be seen in many shapes.(e.g.
Acanthocyte, Spherocytosis,…)
Shape Abnormalities of Erythrocytes
Terminology Description Condition
Target Cells Central Hemoglobin; target Liver Disease; Thalassaemia,
shaped Abnormal Hb; Iron Deficiency
Echinocyte Short specula's, equally- Uremia, Hypokalemia, Artifact
spaced
Acanthocyte Speculated, Irregular Liver disease (Alcohol), Post-
spleenoctomy.
Spherocyte Spherical, no central pallor HS, immune Hemolytic anemia
Shistocyte Fragmented RBC, Helmet MAHA, burns
cells
Ovalocyte Oval / Elliptical shaped Hereditary elliptocytosis,
Megaloblastic anemia.
Sickle Cell Bipolar speculated shape “ Hb S-containing
banana” shaped hemoglobinopathy
Teardrop cell Single elongated extremity Myelophthistic changes
Bite cells Irregular gap in membrane G6PD deficiency
Target cell
Red cell with a “target” or bull’s-eye appearance. The cell
appears with a central bull’s eye that is surrounded by a
clear ring and then an outer red ring.
Found in
• Obstructive liver disease
• Severe iron deficiency
• Thalassaemia
• Post splenectomy
• Lipid disorders
• Haemoglobinopathies (S and C)
Spherocytosis
Red cells are more spherical. Lack the central area of
pallor on a stained blood film.
Found in
• Hereditary spherocytosis
• Immune haemolytic anemia
• Zieve's syndrome
• Microangiopathic haemolytic
Stomatocyte
Red cells with a central linear slit or stoma. Seen as mouth-
shaped form in peripheral smear.

Found in
• Alcohol excess
• Alcoholic liver disease
• Hereditary stomatocytosis
Ovalocyte
An elongated oval cell. They are a result of a
membrane defect.
Found in
• Thalassaemia major.
• Hereditary ovalocytosis.
• Sickle cell anemia
Elliptocyte
The red cells are oval or elliptical in shape. Long
axis is twice the short axis.

Found in
• Hereditary elliptocytosis
• Megaloblastic anemia
• Iron deficiency
• Thalassaemia
• Myelofibrosis
 Schistocyte
Red cell fragments that are
irregular in shape and size. They
are usually half the size of the
normal RBC; therefore, they
have a deeper red color.

Found in
• DIC
• Micro angiopathic haemolytic
anemia
• Mechanical haemolytic anemia
Blister cell: pre keratocyte
Have accentric hallow area. Resemble a women's handbag
and may be called pocket-book cell.

Found in
Microangiopathic hemolytic anemia
Keratocytes (horn cell)
Part of the cell fuses back leaving two or three horn-like
projections. The keratocyte is a fragile cell and remains in
circulation for only a few hours.
Found in
• Uraemia
• Severe burns
• EDTA artifact
• Liver disease
Also called helmet cells
Degmacyte "bite cell"
• An abnormally shaped red blood cell with one or more
semicircular portions removed from the cell margin.
• These "bites" result from the removal of denatured
hemoglobin by macrophages in the spleen.
Found In
• G-6-PD deficiency, in which
uncontrolled oxidative stress
causes hemoglobin to
denature and form Heinz
bodies, is a common disorder
that leads to the formation of
bite cells.
Sickle Cells
Sickle shaped red cells.

Found in
Hb-S disease and trait
Echinocyte “Burr” (crenation ) cell:
Red cell with 30 or more, short blunt projections which
are regularly distributed on their surface
Found in
Usually artifactual— the result of slow drying under
humid conditions.
Sometimes are non - artifactual, indicating uremia or
pyruvate kinase deficiency.
• Hemolytic anemia
• Uremia.
• Megaloblastic anemia
Cells retain the central pallor.
Echinocytes (Burr Cells)
 Acanthocytosis (Spur Cells):
Red blood cells with irregularly spaced
projections, these projections very in width but
usually contain a rounded end

Found in
• Liver disease
• Post splenectomy
• Anorexia nervosa and starvation
Acanthocytes (Spur Cells)
 Dacryocytes (Teardrop)
Resembles a tear and usually smaller than the
normal RBC.
Found in
• Bone marrow fibrosis
• Megaloblastic anemia
• Iron deficiency
• Thalassaemia
Envelope Form Cell
Found in
• Thalassaemia
• Sickle cell anemia
 V. Erythrocyte Inclusions with Wright’s Stain
Inclusion Composition Appearance Condition

Basophilic stippling Precipitated Evenly dispersed - Lead poisoning

ribosomes fine or coarse Thalassaemia , -
granules other anemia.
Howell-Jolly bodies DNA in origin Dense, round blue Post – Splenectomy
Nuclear Fragment granule

Pappenheimer Iron-containing Small blue granules Anemia's

bodies granules in clusters

Heinz bodies Denatured Round blue G6PD

Hemoglobin precipitates
Cabot Rings Remnants of Reddish-blue thread Severe anemia,
Nuclear membrane like rings Lead poisoning.

Organism Small blue inclusion Malaria

Babesiosis
Howell-Jolly Bodies
Small round cytoplasmic red cell inclusion
with same staining characteristics as nuclei

Found in
• Post splenectomy
• Megaloblastic anemia
Siderotic Granules (Pappenheimer Bodies)
These are iron containing granules in red blood cells that
are seen because the iron is aggregated with
mitochondria and ribosomes. They appear as faint violet
or magenta specks, often in small clusters, due to staining
of the associated protein.
They are associated with severe anemias and
thalassemias. Pappenheimer bodies can be increased in
hemolytic anemia, infections and post-splenectomy.
Basophilic stippling
Considerable numbers of small basophilic
inclusions in red cells.

Found in
• Thalassaemia
• Megaloblastic anemia
• Hemolytic anemia
• Liver disease
• Heavy metal poisoning.
 Heinz Bodies
Represent denatured hemoglobin (methemoglobin - Fe+++)
within a cell.
With a supravital stain like crystal violet, Heinz bodies
appear as round blue precipitates.
Presence of Heinz bodies indicates red cell injury and is
usually associated with G6PD-deficiency.
Heinz Body Preparation. RBC are incubated supravitally in new
Methylene blue to identify precipitates of oxidatively denatured
hemoglobin.
 Cabot Rings
Reddish-blue threadlike rings in RBCs of severe anemia's.
These are remnants of the nuclear membrane or remnants
of microtubules and appear as a ring or figure 8 pattern.

Very rare finding in patients with

• Megaloblastic anemia.
• severe anemia's.
• lead poisoning.
• Dyserythropoiesis.

A - Cabot ring
B - Howell-Jolly body
Parasites of Red Cell
Two organisms are have a tendency to invade the RBCs.
1. All 4 species of the malaria parasite will invade RBCs.
We will see the Plasmodium of different species in
RBCs.
2. Theileria microti (Bebesia microti)
Malaria
 RBCs Abnormal morphology
Depiction of red blood
cell morphologies that
may appear on a
peripheral smear,
showing:
(A)basophilic stippling,
(B)Howell-Jolly bodies,
(C)Cabot's ring bodies
(D)Heinz's bodies.
 RED BLOOD CELL MORPHOLOGY
 A normal red blood cell should be approximately
the same size as a normal lymphocyte nucleus or 2
normal sized red blood cells should fit side by side
across a normal sized poly (not a hypersegmented
poly).

NO. of Field/ Oil imm. Grade Degree of abnormality

1-6 per oil imm. field 1+
7-10 per OIF 2+
11-20 per OIF 3+
> 20 per OIF 4+
 REPORTING RESULTS
 Where possible use macrocytic and microcytic,
rather than simply anisocytosis alone, when
describing red cell morphology.
 Use specific cell morphology when possible, rather
than simply reporting poikilocytosis.
 When red cells are normocytic, normochromic,
report out as NORMAL. When abnormal
morphology has been noted, DO NOT indicate
normal on the report form.
 EXAMPLE: 7-10 microcytic RBC's/OIF is reported
out as: 2+ microcytosis or Moderate microcytosis.
Determine A Quantitative Scale

1
Grading Inclusions

2
Blood Film In Some Cases
Normal Peripheral Smear
 Autoimmune Hemolytic Anemia

Spheroc
ytes
 Hereditary Spherocytosis

Spherocytes
Hereditary pyropoikilocytosis
Microangiopathic Hemolytic Anemia

Schistocytes
 Sickle Cell Anemia

Hb
SS
 Idiopathic myelofibrosis

Dacryocytes
 Iron Deficiency Anemia

Severe Hypochromia
 Treated Iron Deficiency Anemia

Mixed Population:
 Alpha Thalassaemia (a-/--)

Microcytic Hypochromia
Morphologic Changes in Liver Disease

Target Cells Spur Cells

 Hepatorenal Syndrome

Burr + Spur Cells