RBC and WBC morphology

Parts of a peripheral blood smear

 Head, body and tail

1-Head

2-Body

3-Tail
Examples of unacceptable smears

A: Blood film with jagged tail made from a spreader with chipped end.
B: Film which is too thick
C: Film which is too long, too wide, uneven thickness and made on a
greasy slide.
D: A well-made blood film.
THE CORRECT SELECTION OF TH
AREA
Too thin Too thick Good area
 Good Habits
Always scan the feather edge

The "feather edge”or “tail”of the slide, should

be scanned at low power to assess
•white cells
•any large abnormal cells or
• platelet clumps
Principle
 White Blood Cells.
1. Check for even distribution and estimate the
number present (also, look for any gross
abnormalities present on the smear).
2. Perform the differential count.
Principle

 Red Blood Cells, Examine for :

1. Size and shape.
2. Relative hemoglobin content.
3. Polychromatophilia.
4. Inclusions.
5. Rouleaux formation or agglutination

 Platelets.
1. Estimate number present.
2. Examine for morphologic abnormalities.
Procedures

 Observations Under ×10

1. Check to see if there are good counting areas
available free of ragged edges and cell clumps.
2. Check the WBC distribution over the smear.
3. Check that the slide is properly stained.
4. Check for the presence of large platelets, platelet
clumps, and fibrin strands.
Observations Under× 40x : WBC Estimates

 Using the × 40 high dry with no oil.

 Choose a portion of the peripheral smear where there
is only slight overlapping of the RBCs.
Observations Under × 100: Platelet
Estimates
1. Use the oil immersion lens estimate the number of platelets
per field.
2. Look at 5-6 fields and take an average.
3. Multiply the average by 20,000.
4. Note any macroplatelets.
 Platelets per oil immersion field (OIF)
1) <8 platelets/OIF = decreased
2) 8 to 20 platelets/OIF = adequate
3) >20 platelets/OIF = increased
RBC Morphology
Normochromic Normocytic
Abnormal RBC morphology

Inadequqate haemoglobin formation

•Hypochromia
Abnormal erythropoiesis
•Anisocytosis
•Poikilocytosis
•Microcytosis
•Macrocytosis
Regenerating marrow
• Polychromasia
• Erythroblastaemia
Abnormal RBC morphology
Abnormal haemoglobin formation
•Sickle cells
Damage to red cells
•Fragmented red cells
•Bite cells
•Spherocytes
•Tear drop cells
Red cell inclusions
•Basophilic stippling
•Howell Jolly bodies
•Pappenheimer Bodies
Abnormal RBC morphology
Miscellaneous erythrocyte abnormalities
•Target cells
•Echinocytes
•Acanthocytes
•Stomatocytes
•Dimorphic blood picture
Normal RBCs & abnormal plasma
•Rouleaux formation
•Agglutination
 Inadequate haemoglobin formation
Hypochromia

Associated diseases
 Iron deficiency

 Thalassaemia

 Anaemia of chronic disease

 Sideroblastic anaemia

 Lead poisoning

Central pallor more than 1/3 of the red cell

 Abnormal
erythropoiesis
 Anisocytosis

Variation in size

MCV may
be normal

Associated with
increased RDW
 Poikilocytosis

Variation in shape
Non specific feature of
Abnormal erythropoiesis
Associated diseases
Iron deficiency anaemia
Thalasaemia
Megaloblastic anaemia
Myelofibrosis
 Microcytes

Compare with small lymphocyte which is slightly larger than a normal RBC
Microcytosis, hypochromia, &
poikilocytosis
 Elliptocytes

Key features
•Elongated red cells
•Ends are rounded
•Central pallor is present
Associated conditions
 Hereditary elliptocytosis
 Iron deficiency anaemia
 Megaloblastic anaemia
 Round Macrocytes

Key features:
•Larger than normal ,
round shaped
more than 100 fl
•Central pallor
is present
Round Macrocytes
 Round Macrocytes
With normal With increased
reticulocyte count reticulocyte count

Pathophysiology
Due to abnormal lipid composition Reticulocytes are larger than normal
of erythrocyte membrane RBCs

Causes
 Liver disease • Regenerating marrow
 Hypothyroidism • Hemolytic anaemia
 Alcoholism • Acute blood loss
 Chronic obstructive • Neonates
pulmonary disease
Oval Macrocytosis
Oval Macrocytosis
Pathophysiology
Defective DNA synthesis

Slow nuclear maturation

Nuclear cytoplasmic asynchrony

Formation of abnormally large cells

Oval macrocytosis
 Oval Macrocytes

Causes
 Megaloblastic anaemia
- Vitamin B12 deficiecy
- Folate deficiency
 Myelodysplastic anaemia
 Treatment with hydroxyurea
Regenerating marrow
 Polychromasia
Reticulocytosis Marked erythropoiesis
Key features Supra-vital stain

Larger than normal RBCs

Show bluish grey shades Bone marrow

Central pallor not present

Associated conditions
• Any condition associated
with reticulocytosis
• These are in fact
reticulocytes & can be confirmed with supra- vital
stains.
 Reticulocytes

Reticulocytes are immature RBCs which have shed

their nucleus but still retain ribosomes, mitochondria,
remnants of golgi bodies and other cytoplasmic
granules
 Reticulocyte count
Increased Decreased
 Regenerating marrow
• Aplastic anaemia
 Acute blood loss
• Drug suppression
 Haemolytic anaemia
• Megaloblastic anaemia
 Deficiency anaemias
receiving therapy
• Myelodysplastic
 Hypersplenism syndrome

 New born • Pure red cell aplasia

 Erythroblastaemia
Peripheral Blood Smear of
Thalassemia Major
Key features
Pyknotic eccentric nucleus
Cytoplasm pink to grey

Associated conditions
• Any condition associated
with severe haemolysis
• Exramedulary haematopoiesis
• Myelopthesic diseases.
 Erythroblastaemia

Normoblasts in haemolytic disease of new born

 Abnormal Haemoglobin
formation
 Sickle Cell
Key features
• Red cells with pointed
ends
• Crescent shape
• No central pallor
• Very dense hemoglobin

Associated disease
Hemoglobin SS
Hemoglobin SC
Hemoglobin SD
S- beta thalassaemia
Damaged red cells
 RBC Fragmentation

Synonym:
Schistocytes

Key features
•Two to three sharp
angles of spines
•Central pallor is not present
 Fragmented RBCs (schistocytes)
Causes
 Microangiopathic hemolytic
anaemia -
TTP
-Hemolytic uremic syndrome
-DIC
 Malignant hypertension
RBC
 Artificial heart valve
Schistocytes vs Microcyte

Key 1-Schistocytes with pointed ends & loss of central pallor

2-Microcytes showing central pallor
 Bite cells

Synonym: Bite cell with reticulocytosis

Keratocytes
Key features
•Pair of spicules
Central pallor is not present
•Helmet like cells are also included

Pathophysiology
Removal of Heinz body pitting action of spleen
Associated conditions
G6PD deficiency,unstable hemoglobin, Drug insult,
thalassaemias
 Microspherocytes

Key features
Smaller than normal RBCs
• No central pallor
• Very dense hemoglobin

Associated conditions
• Artifactual
• Hereditary spherocytosis
• Immune hemolytic anaemia
• Thermal injury
• Microangiopathic hemolytic anaemia
 Microspherocytes

Compare them with Microspherocytes in

small lymphocyte Microangiopathic hemolytic anaemia
 Basophilic stippling

 Hemolytic anemias
 Pyrimidine-5’nucleotidase deficiency

 Iron deficiency
 Thalassemias
 Lead poisoning

Diffuse fine or coarse blue dots in the red

cell representing usually RNA residue
 Howell Jolly Bodies

Howell jolly bodies seen in post splenectomy patient

 Pappenheimer Bodies
Key features
Small peripherally sited
basophilic inclusions
Their nature can be
confirmed by perl stain
Associated conditions
• Any condition associated
with iron over load
• Sideroblastic anaemia
• Thalassaemia
Target Cells
Causes of target cell formation

•Artifacts of air drying

•Haemoglobin C

•Decreased volume
 Iron deficiency anaemia
 Thalassaemia
 Haemoglobinopathies

•Increased surface membrane

 Liver disease
 Post splenectomy
 Echinocytes

Synonyms:
-Burr cells
-Crenated cells

•Spiculated red cells with 10-30 short evenly spaced

projections over the entire suface
•These retain the central pallor
 Acanthocytes
Synonym:
Spur Cells
Key features
•Red cell with
small number of
Spicules with irregular
thickness & shape.
•Central pallor is not
present
Causes
Abnormal lipid metabolism
Liver disease Compare
Splenectomy 1-burr cells with 2-spur cell
 Stomatocytes
Key features
•Red cell in which
Central pallor area
is slit like or mouth
like in dried Smear.

Causes
Artifact
Decreased pH
Liver disease
Alcholism
 Rouleaux Formation

Causes
All the diseases associated with hypergamma globulinaemia

• Multiple myeloma
• Chronic liver disease
• Chronic inflammatory
conditions
• Malignant lymphoma
Agglutination
Agglutination
Zeta potential

25 nm

IgM antibodies are formed against red cell antigens

These antibodies are large enough

to overcome the zeta potential gap

RBCs are joined together in the form of

clumps ( grape like clusters)
RBC QUIZ
????????
Normochromic Normocytic
???????
????????
Diagnosis
Thal Major
????????
Oval Macrocytosis:
Megaloblastic Anaemia
??????????
Rouleaux Formation
???????
Howell Jolly Bodies
????????
?????????
Microcytic Hypochromic
???????
Polychromasia
???????
Sickle Cells & Target Cells
????????
Microspherocytes
?????????
Tear Drop cells
???????
Megaloblastic Anemia

macroovalocytosis, hypersegmentation,
thrombocytopenia
????????
Burr cells
(Echinocytes)
???????
Spherocytes
??????
NRBC
White Blood Cells Morphology
Five Types of Leukocytes (WBCs)
Granular Leukocytes

Eosinophil

Neutrophil

Basophil
Neutrophil
 60-70% of all WBC’s
 Anatomy
 10-12 µm diameter
 2-6 nuclear lobes
 Fine, pale inconspicuous granules
 Physiology
 Respond first to bacteria damage
by chemotaxis
 Phagocytosis
 After engulfing pathogen releases
several chemicals
 lysozyme
 strong oxidants
 defensins
 Clues to recognize
Neutrophils
• Multiple lobes of the nucleus
 bilobation is not seen in normal neutrophils
•Cytoplasm has purple/basophilic
granules i.e.same as the color of the
nucleus.
BUT
lighter in shade i.e. light purple
Neutrophil granules can be fine or coarse
Eosinophil
 2-4% of all WBC’s
 Anatomy
 10-12 µm diameter
 2 connected nuclear lobes
 red/orange large, uniform
granules, do not obscure the
nucleus
 Physiology
 exit capillaries, enter tissue
fluid
 combat parasites
 histamine
 phagocytize antigen-antibody
complexes
Basophil  0.5-1% of all WBC’s
 Anatomy
 8-10 µm diameter
 bilobed or irregular nucleus
 round, blue-black granules may
obscure the nucleus
 Physiology
 exit capillaries to enter tissue
fluid
 mature into mast cells
 release heparin, histamine,
serotonin – stimulate
inflammation
 Hypersensitivity (allergic)
reactions
Agranular Leukocytes

Lymphocyte

Monocyte
Lymphocytes

 20-25% of all WBC’s

 Anatomy
 7-15µm
 nucleus large and dark
stained, round or
indented
 cytoplasm forms a pale
blue rim around the
nucleus
Monocytes
 3-8% of all WBC’s
 Anatomy
 14-19 µm
 indented or kidney-shaped
nucleus (not round)
 cytoplasm foamy
 Physiology
 slower to arrive but survive
longer
 enlarge, differentiate into fixed
and wandering macrophages
 remove microbes, cellular debris,
following injury
Platelet satellitism

 Platelet satellitism describes the

phenomenon of adherence of
platelets to white cells.
 It is an in vitro phenomenon of
no clinical significance. However
it is important that it is detected
since the platelet count will be
factitiously low.
How granulocytes are formed
?
 Myeloblast is the earliest
recognizable cell of the
granulocytic series
 It is normally present in the
bone marrow
 Large cell
 Fine reticular chromatin
 Nucleoli are present
 Fine azurophilic granules may
be present
How granulocytes are formed ?
Myeloblast Promyelocyte

Appearance of primary
azurophilc granules
 Promyelocyte Myelocyte

Primary azurophilic granules Specific (secondary)granules

Fine reticular chromatin appear, with primary azurophilc
granules. Note; sunrise effect
Slightly clumpy chromatin
Neutrophilic granules
Primary Secondary
Synonyms  Non specific/  Specific granules
Azurophilic granules
Size  Large  Small sand-like
Color  Blue-Black  Pink-red to Pink violet
Visibility  Appear in  Appear in myelocyte
promyelocytes,
however their synthesis
cease at myelocyte
stage
Consistency  Contains  Contains alkaline
myeloperoxidase phosphatase
Ref: Text book of Heamatology, Mckenzie, second edition
Myelocyte Metamyelocyte

Slight indentation of nucleus

 Metamyelocyte Band cell

Nuclear indentation Nuclear indentation

< half > half
Band cell Segmented neutrophil
 Maturation of
polymorphs
Myelocyte

Metamyelocyte

Band cell

Mature polymorph

Ref: CAP Color Atlas of Hematology

Eric F Glassy, M D
 Maturation of
polymorphs

Myelocyte
Metamyelocyte
Band
Mature cell
polymorph

Ref: CAP Color Atlas of Hematology

Eric F Glassy, M D
Granulopoiesis- All Stages
 Auer rods ?
•Auer rods are collection of
azurophilic granules containing
peroxidase lysosomal enzymes
organized in the form of rods

•Seen in 2-3% of myeloblasts

•Diagnotic feature of AML

Ref: Williams Hematology Fifth edition

Myeloblasts
Myeloblasts
 Morphological features
Myeloblasts Lymphoblasts

Size  Medium to large  Small to medium

Cytoplasm  Scanty to moderate  Scanty non-granular
finely granular
Auer rods  Present in 60-70%  Not present
Chromatin  Fine reticular  Fine to coarse
Nucleoli  Two to 3  Indistinct

Ref: Atlas of tumor pathlogy AFIP

 Small Lymphocytes
Normal lymphocytes are slightly
larger than erythrocytes, and
constitute more than 95% of the
peripheral blood lymphocyte pool.

They have condensed nuclear

chromatin with a thin rim of
cytoplasm. Nucleoli are rarely
seen.
 Reactive lymphocytes

Key Features
Abundant cytoplasm,
fine nuclear
chromatin and
often nucleoli.
Causes
Viral infections.
 Large Granular Lymphocytes
(LGL)

 The LGL has

cytoplasm,which is often
lighter stained than that of
small lymphocytes
 Azurophilic granules are
seen and the nucleus is
more variable in shape and
often eccentric. Some LGL
are natural killers, while
others are T-cells.
 Monocytes
o Monocytes are larger than
normal lymphocytes.
o Nucleus is kidney shaped
or lobulated, with fine to
coarse chromatin.
o Cytoplasm is slightly
basophilic or gray-blue with
occasional granules or
vacuoles.
 Monocytes
Morphologic Spectrum
 REACTIVE CHANGES IN
NEUTROPHIL MORPHOLOGY
Toxic Granulation in
Neutrophils
Key Features
Increased number of
coarse azurophlic
granules
Associated conditions
Bacterial infections
Other inflammatory
conditions
Dohle Body in Neutrophils

Key Features
Small round, or oval
pale blue – grey structure

Consist of ribosome or
endoplasmic reticulum
Associated conditions
Bacterial infection
May-Heglin anomaly
 Vacuolated Neutrophils

Associated conditions
Artifact of
prolonged standing

Severe sepsis
EXERCISE
???????
Basophil
?????????
Lymphocyte
??????????
Promyelocyte
???????
Neutrophil
?????????
Neutrophil with Toxic
Granulation
?????
Monocyte
???????
Blast
?????????
Lymphocytes
????????
Monocyte
????????
Neutrophil
?????????
Eosinophil & Neutrophils
??????
Neutrophil with Vacuoles
????????
Monocyte
???????
Basophil
??????
Dohle Body in Neutrophil
????????
Eosinophil
????????
Lymphocyte & neutrophil
???????
NRBC
Lymphocytosis