Haem Morphology

The Bare Basics

Dr Saba Jamal
Dr Masood Raza
Dr Ziauddin University Hospital
Clinical Laboratories Version 1, April 2004
 “I am careful not to confuse
excellence with perfection”.

“Excellence is what I can reach for…

Perfection is God's business”.

Michael J. Fox
 CONTENTS
•EXAMINATION OF PERIPHERAL FILM

•WHITE BLOOD CELLS

•RED CELL MORPHLOGY

•PLATELET MORPHOLOGY

•DISEASE MORPHOLOGY

•MALARIA MORPHOLOGY

Click your desired destiny

Parts of a peripheral blood smear
 Head, body and tail

1-Head
2-Body
3-Tail

1-Head

2-Body

3-Tail
Cross-sectional method
of scanning slides
 Change Bad Habits

 Often students select areas where the red cells are

piled on top of one another

 Not only can red cell morphology not be examined in

such areas, but it is also impossible to study
peripheral white blood cell morphology
 Too Thick

Individual red cell morphology is difficult

to study in such areas, white cells stain
very deeply and are difficult to
identify.
 Too Thin

The red cells appear flattened with empty

spaces in between.
 Good Area

The correct area of the slide to be examined for

differential count is usually towards the body, in an
area where most of the red cells are almost, but not
quite touching.
THE CORRECT SELECTION OF THE
AREA
Too thin Too thick Good area

Click center for higher magnification

 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
WHITE BLOOD CELLS
Contents
•Identification of white blood cells
•Neutrophils
•Eosinophils
•Basophils
•How granulocytes are formed
•Lymphocytes
•Monocytes

Click your desired destiny

WHITE BLOOD CELLS
Contents (contd)
-Abnormal Cells in Peripheral Blood

-Reactive changes in Neutrophils

-Leukoerythroblastic blood picture

-Pelger-Huet Anomaly of Neutrophils

- Chediak Higashi syndrome

Click your desired destiny
 WHITE BLOOD CELLS
 Keys To The Lock of cell Identification
-Key I
-Key II
-Key III
 Tricks of the trade
 Exercise

Click your desired destiny

Identification of Cells
 WHITE BLOOD CELLS;
- Granulocytes . Neutrophils .
Eosinophils . Basophils
- Lymphocytes
- Monocytes
- Plasma cells
 Normal Smear

RBC’s are normal. They

have a zone of central
pallor, 1/3 the size of
RBC’s. Minimal variation
in size & shape. Few
plateletes & segmented
neutrophils seen.
Neutrophils
The segmented neutrophil is the
mature granulocyte, also termed
polymorphonuclear leucocyte.

The nucleus consists of 3 to 5

segments, connected by short,
thread-like filaments. The
nuclear material is clumped,
 Neutrophils
Morphologic Spectrum
 Neutrophils
Morphologic Spectrum
(contd)
 Neutrophils
Morphologic Spectrum (contd)
 Hypersegmented neutrophil
 Neutrophil with 5 or more
nuclear segments is known
as hypersegmented
neutrophil

 It is usually seen in
megaloblastic anaemia

 Other conditions
-Uremia , IDA,
cytotoxic therapy

Ref: Dacie and Lewis , Practical hematology ,ninth edition

Eosinophils
The eosinophilic leucocyte usually
has a bi-lobed nucleus with coarse
chromatin.

The cytoplasm is
filled with eosinophilic
granules of different
sizes.
 Eosinophils (contd)
Eosinophil Bilobed neutrophil

Eosinophils are usually and neutrophils may be

occasionnally bilobed
Eosinophils (contd)
Usually eosinophils
have bi-lobed nuclei.
with coarse
chromatin.

However, single lobed

eosinphils may be
seen
Basophils

•Usually unsegmented or
bilobed

•Dark purple cytoplasmic

granules

•Granules usually hide

the nuclear morphology
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
Metamyelocyt
Band
Mature cell
polymorph
e
Ref: CAP Color Atlas of Hematology
Eric F Glassy, M D
 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.
 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.
Lymphocytes Morphologic
Spectrum
Lymphocytes Morphologic
Spectrum (contd)
Lymphocytes Morphologic
Spectrum (contd)
Are nucleoli present only in the
blasts?
 Nucleoli ?
 Nucleoli represent the site of synthesis of
ribosomal RNA
 Nucleoli become prominent in any cell
during period of protein synthesis,
not necessarily in blasts.

Reactive lymphocyte
showing
 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
 Monocytes
Morphologic Spectrum (contd.)
 Abnormal Cells in Peripheral
Blood
 The cells discussed in the following section
are not normally seen in peripheral blood
BUT
Are normally formed & present
in the bone marrow & are discussed in the
sequence of maturation
They appear in the peripheral smear when
the patient is sick
Abnormal Cells in Peripheral
Blood
 Myeloblasts
 Promyelocytes
 Myelocytes
 Metamyelocytes
 Band cells
 Other cells
Lymphoblasts
Plasma cells
Click your desired destiny
 Myeloblasts
Size………..15-20 um
N/C ratio ; 7:1-5:1
Cell shape; round to oval
Nuclear shape; round to oval
Chromatin; fine reticular
Nucleoli………distinct 2-5
Cytoplasm; Scant basophilic

Key features N/C ratio

6

High N/C ratio 5

4

Fine reticular chromatin with nucleoli

3
2
N/ C

1
0
Nuc le us c y t o pla sm
 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
 Promyelocyte
Size……….12-24 um
N/C ratio; 5:1 to 3:1
Cell shape; round to oval
Nuclear shape; round to oval
Chromatin; finely reticular
Slightly clumped
Nucleoli ….distinct (1 to 3)
Cytoplasm; blue, numerous
azurophilic granules
Key features N/C ratio
Usually larger than blast, blue 4

cytoplasm with nucleoli & 3

numerous azurophilic granules 2

N/ C

Other features 1

Collection of Auer rods may be present

0
Nu c le us c y t o pla s m
 Myelocyte
Size………10-18 um
N/C ratio; 1:1
Cell shape; round to oval
Nuclear shape; oval slightly
indented flattened
Chromatin; variable amount
of clumping
Nucleoli……absent
Cytoplasm; bluish pink ,
contains both primary &
secondary granules N/C ratio
Key features
1. 5

Absence of nucleoli, chromatin 1

clumping & presence of both 0 .5

N/ C

azurophilic & lilac granules

0
Nuc le us c y t o pla sm
 Metamyelocyte
Size…………..10-18 um
N/C ratio; 1:1
Cell shape; round to oval
Nuclear shape; indented
kidney shaped
Chromatin; clumped
Nucleoli……None
Cytoplasm; pink, many specific
granules rare azurophilic granules
N/C ratio
Key features 1

0 .8

Absence of nucleoli, chromatin 0 .6

clumping & presence of both 0 .4 N/ C

azurophilic & lilac granules 0 .2

0
Nu c l e u s c y t o pla sm
 Band cell
Size…………..10-18 um
N/C ratio; 1:2
Cell shape; round to oval
Nuclear shape ; S, C, U, or lobulated

Chromatin; coarse clumped

Nucleoli……None
Cytoplasm; pink, plentiful
mainly specific granules
rare N/C ratio
Key azurophilc
features granules 2

Nucleus,coarse, clumpy, deeply 1.5

indented (more than half of the 1

N/C

theoretical round) nucleus. 0.5

0
Nucleus cytoplasm
Band Neutrophils
Granulopoiesis- All Stages
 Other Cells
 Lymphoblasts

 Plasma Cells
 Lymphoblasts

Lymphoblasts are seen in acute lymphoblastic leukemia,

should be differentiated from 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

Lymphoblasts (L3)/ Burkitt type

These are medium to large cells with intensly

basophilic, moderately abundant cytoplasm showing
prominent vacoulation .
Nucleus shows stipple chromatin & nucleoli
 Reactive lymphocytes

Key Features
Abundant cytoplasm,
fine nuclear
chromatin and
often nucleoli.
Causes
Viral infections.
Plasma Cells

The nucleus of plasma

cells is usually eccentric
and has a coarse
Chromatin structure.

The cytoplasm is
basophilic and a
perinuclear halo is often
seen.
Leishman X 40
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
Vacuolated Neutrophil
? Artifact ?sepsis

To avoid artifactual neutrophil vacuolation

, prepare the smear immediately
 Neutrophils with left shift
Key Features
Neutrophils with
premature granulocytes
eg myelocytes,
metamyelocytes or
band cells.
Associated conditions
Bacterial infection
Acute inflammation
Cytokines How toxic changes develop ?
Cell stimulated by cytokines
Cells Maturation
rapidly More cut short;
exit from spontaneous persistance
bone membrane of synthetic
marrow internalization machinery
in cell
Left Dohle
Enhanced Shift Bodies
lysosomal enzyme
production & Toxic vacuolization
packaging
resulting in large
granules
Toxic
Granulation
Reference; CAP Color Atlas of Hematology
Eric F Glassy, M D
Leukoerythroblastic blood picture
Key Features
Neutrophils with
premature
granulocytes
and normoblasts

Associated conditions
• Severe infection
•Bone marrow infiltration
•Myelofibrosis with
myeloid metaplasia
 Pelger-Huet Anomaly of
Neutrophils
Benign inherited
condition
Key Features
 Bilobed nuclei
 Chromatin coarsely clumped
 Granular content normal

D/D
Pseudo-Pelger Heut anomaly
 Pseudo-Pelger Heut anomaly

Key Features
 Bilobed nuclei
 Chromatin coarsely clumped
 Hypogranular cytoplasm

Associated conditions
Muelodysplastic syndrome
 PELGER-HUET PSEUDO- PELGER-HUET
ANOMALY ANOMALY

 Inherited disorder  Acquired condition

vs
 Benign  Associated with CML ,
Myelodysplastic
syndrome
 Irregular nuclear pattern
 Coarsely clumped bilobed with hypogranular
nucleus with normal cytoplasm
azurophilic granules

Ref: Dacie and Lewis Practical Hematology 9th Edition

 CHEDIAK-HIGASHI ANOMALY

Key Features
Scanty giant azurophilic
granules

Associated disease
Autosomal disorder of X 40 X 100
lysosomal granules

D/D
Should be differentiated from basophils
Keys to the lock of cell identification
 Question ?

 In identifying a cell, what details of the cell

does one notice?
Key I: Cell size

Small Medium Large

Note :- compare the cell with surrounding RBCs

 KEY II:
Characteristics of the nucleus;

- 1) Relative size, size of the nucleus

compared to the amount of cytoplasm is
expressed as the “nuclear to cytoplasmic
ratio”( N/C ratio)
KEY II:
Characteristics of nucleus;
 In which of the following cell N/C ratio is
high?
A B
 KEY II:
Characteristics of the nucleus;
 N/C ratio is high in A as compared to B,
i.e.volume of the nucleus is more as
compared to the cytoplasm in A:
A B

A B
KEY II:
Characteristics of the nucleus;
Multilobed

Bilobed

Mononuclear
KEY II:
Characteristics of the nucleus;
3)Chromatin Pattern;

Smooth & Granular Clumped with visible parachromatin

( visible lighter lines in chromatin
i.e.nucleus)

Parachromatin
 Normal & abnormal peripheral blood cells
usually show following pattern of chromatin
clumpiness
Fine CLUMPY
Monocytes Lymphocytes Neutrophils

Myeloblast Promyelocyte Myelocyte Metamyelocyte Band cell Neutrophil

 KEY III:
Characteristics of Cytoplasm
 Relative Amount; i.e. amount of cytoplasm
relative to the nucleus
 Color; staining properties
 Texture; smooth or foamy
 Granular or Non-Granular;
- color of the granules;
eosinophilic or basophilic etc
- size of the granules;
fine, coarse or clumped
KEY III:
Characteristics of Cytoplasm
 Relative Amount; i.e. amount of cytoplasm
relative to the nucleus
KEY III:
Characteristics of Cytoplasm
Color; staining properties
Pink Grey Light Blue Medium
blue

Deep Blue
KEY III:
Characteristics of Cytoplasm
 Texture;
smooth or foamy
KEY III:
Characteristics of Cytoplasm
Granular Non-Granular
 KEY-III:
Characteristics of Cytoplasm
 Granular or Non-Granular;- color of the granules;
eosinophilic or basophilic etc
Fine Coarse Coarse Fine
Coarse PLUMP - size of the granules;
fine, coarse or plump
Tricks of the trade

A step forward
 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
 Tips for Eosinophils
 Usually has bilobed nucleus. Not Abnormal
 Cytoplasm has reddish orange to pinkish orange
plump granules, usually very dense.

Morphological Spectrum of Eosinophils

Usually it is not difficult to recognize eosinophils,
when they have typical orange granules

But in some cases when the granules do not have

the typical orange color, it is a difficult to
distinguish b/w neutrophils & eosinophils

Eosinophil Neutrophil
In these cases, the color of the granules resemble the
pinkish orange color of the RBC’s whereas the
neutrophil granules resemble the purple color of the
nucleus but much lighter purple

Eosinophil Neutrophil

Compare the color of the cytoplasmic granules

with the nucleus & the RBC’s
 A Basophil? So Obvious
 Generally unsegmented or Bilobed nucleus
 Cytoplasm has dark purple plump granules, notice
that the granules are darker than the nucleus, almost
blackish purple & tend to hide the nucleus.
Basophil or Neutrophil Series?
Basophil !!! But WHY?
Sometimes the basophil granules tend to
degranulate

Notice that some very dark &

plump granules are still present.
 Identify Basophils in the
following:

A B C D E
 ANSWER
A B C D E

Blast Lymphocyte Blast Basophil Blast

Note : In basophils, the granules sometimes completely
overlap the nucleus & makes it look like lymphocytes etc.
However, it has a granular surface rather than smooth as

in the lymphocytes.
 Lymphocyte: A no brainer
 Lymphocytes occur in different sizes, the relative
amount of cytoplasm also varies.
 Generally , the larger the lymphocyte, the more
abundant the cytoplasm. The shade of blue also
varies.
Monocytes - a cell with multiple faces
 Nucleus, variable in shape; round, kidney shaped,
lobulated or folded.Chromatin is fine (not clumped).
 Cytoplasm;abundant & blue-gray, fine azurophilic
granules.Vacuoles may be present.

Note the variable shapes of

the nucleus, the shades of the cytoplasmic color, the
number & prominence of granules
EXERCISE
???????
Basophil
?????????
Lymphocyte
??????????
Promyelocyte
???????
Neutrophil
?????????
Neutrophil with Toxic
Granulation
?????
Monocyte
???????
Blast
?????????
Lymphocytes
????????
Monocyte
????????
Neutrophil
??????
Basophil
?????????
Eosinophil & Neutrophils
??????
Neutrophil with Vacuoles
????????
Monocyte
???????
Basophil
??????
Dohle Body in Neutrophil
????????
Eosinophil
????????
Lymphocyte & neutrophil
???????
NRBC
 RBC Morphology

 Erythropoiesis
 Abnormal RBC morphology
 Anaemias
 RBC Quiz
Click your desired destiny
ERYTHROPOIESIS
Proerythroblast

 The earliest erythroid

element.
 Round to oval nucleus.
 Sometimes cytoplasm
bulges to form a small ear
shaped process.
 Pale blue cytoplasm
 Lacy evenly distributed
cytoplasm.
 Usually prominent nucleoli.
 Basophilic erythroblast
•Size……….10-17 um
•N/C ratio; 6:1
•Cell shape; round to oval
•Nuclear shape; round to oval
•Chromatin; Open with
thickened strands
•Nucleoli ….Absent in late forms
•Cytoplasm; Deep blue

Key features N/C ratio

Open, beady chromatin pattern
6

Royal blue cytoplasm 4

3
N/ C

Faint or absent nuleoli 2

1
0
Nu c le us c y t o pla sm
Polychromatophilic erythroblast
•Size……….10-15 um
•N/C ratio; 4:1
•Cell shape; round or oval
;
•Nuclear shape; round to oval may
be eccentrically placed
•Chromatin; Clumped
•Nucleoli ….Absent
•Cytoplasm; Blue gray to pink
gray
Key features N/C ratio
Clumped chromatin with absent 4

nucleoli
3

Blue gray to pink gray cytoplasm

N/ C

0
Nuc le us c y t o pla s m
 Orthochromic erythroblast
•Size……….8-12 um
•N/C ratio; 1:2
•Cell shape; round to oval
•Nuclear shape; round to oval
•Chromatin; Pyknotic, may be
eccentrically placed or extruding
•Nucleoli ….Absent
•Cytoplasm; Pink orange
Key features
N/C ratio
Pyknotic, extruding nucleus 2

Cytoplasm pink orange with 1. 5

minimal basophilia 1
N/ C

0.5

0
Nuc le us c y t o pla s m
Normoblast
 Reticulocyte
•Size……… Slightly larger than
normal erythrocyte
•Cell shape; round to oval ;
•Cytoplasm; blue to green with
precipitated RNA occurring
as dark network
Key features
•At least two dark purple granules after supra-vital
staining
•Usually a network of dark fibrillar and granular
material visible.
•Often larger than surrounding mature red cells
 Mature red cell
 Size… Same size as the
nucleus of a small lymphocyte
that is 7.2 um

• Cell shape; round 7.2

m
• Cytoplasm; Pink

• Center pallor; 1/3 size of the

cell

Normal RBC ,suspended in fluid is biconcave.

Hemoglobin is comparatively less in the center, showing
central pallor on dried film
 Erythrocyte shapes

Shape depends on water content

Osmotic effects of solutes,
especially ions

(a): discocyte, (b) stomatocyte and (c) echinocyte

Erythroid series
Normochromic Normocytic

Key features
RBCs slightly
smaller than
small lymphocyte

Central pallor 1/3

the size of RBC
Normochromic Normocytic
 Abnormal RBC morphology
CONTENTS

Inadequqate haemoglobin formation

•Hypocromia
Abnormal erythropoiesis
•Anisocytosis
•Poikilocytosis
•Microcytosis
•Macrocytosis
Regenerating marrow
• Polychromasia
• Erythroblastaemia
Click your desired destiny
Abnormal RBC morphology
CONTENTS (contd)
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
Click your desired destiny
Abnormal RBC morphology
CONTENTS (contd)

Miscellaneous erythrocyte abnormalities

•Target cells
•Echinocytes
•Acanthocytes
•Stomatocytes
•Dimorphic blood picture
Normal RBCs & abnormal plasma
•Rouleaux formation
•Agglutination
Click your desired destiny
 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
Pathophysiology

Microcytosis is the body’s attempt to

conserve oxygen carrying capacity
within limitation of circulatory system

These cells circulate easily in capillaries

and still deliver as normal amount of
oxygen to the tissues as possible

Ref; Color Atlas of hematology

CAP Hematology and clinical microscopy
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
 Normocytic normochromic
vs
Microcytic /hypochromic

Normocytic normochromic Microcytic /hypochromic

 Macrocytosis

Round
Round vs Oval
 Round Macrocytes

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

x10 x 40
 Round Macrocytes
With normal With increased
reticulocyte count reticulocyte count

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

Causes
 Liver disease  Regenerating marrow
 Hypothyroidism  Hemolytic anaemia
 Alcoholism  Acute blood loss
 Chronic obstructive  Neonates
pulmonary disease
Which test is necessary if round macrocyte
are present on the peripheral film ?

Reticulocyte count should be done to categorize two

main types of round macrocytosis that is

round macrocytosis with normal reticulocyte count

&
round macrocytosis with increased reticulocyte count
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

Reticulocytes are immature RBCs which have

shed their nucleus but still retain ribosomes,
mitochondria, remnants of golgi bodies and
other cytoplasmic granules
What information is obtained from
reticulocyte count ?

Reticulocyte count is a
non-invasive test
that reflects the bone marrow activity
 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
Reticulocytes
 Erythroblastaemia

Key features
Piknotic 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)
Pathophysiology

Rule of thumb Physical damage to red cells

Causes
 Microangiopathic hemolytic
anaemia
-TTP
RBC -Hemolytic uremic syndrome
-DIC
 Malignant hypertension
 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
 Microspherocytes
Pathophysiology

Membrane loss of RBCs leads to spherocyte formation

 Intrinsic abnormalities Rule of thumb

Abnormality occurs due to Normal red cell membrane

deficiency of spectrin,
ankyrin or band 3

Ankyrin
Spectrin dimer

Uncoupling of Microvesicle formation Surface area deficiency

lipid bilayer& skeleton & membrane loss leading to spherocytosis
 Microspherocytes
Pathophysiology (contd)

 Immune hemolysis

Damaged
RBC reseals
and forms
microspherocyte
Antibodies
attached to red cell
Fc receptors on splenic Red cell membrane is
macrophages bind lost during pitting
to antibodies antigen antibody complex
Tear Drop Cells
 Tear drop Cells
Pathophysiology
1- RBC inclusion and pitting function of spleen

Red cell stretch

RBC containing to abnormal
inclusion passes length and result
through slit in in permanent
the basement damage to
membrane of membrane
splenic cord
 Tear drop Cells (contd)
Pathophysiology
2- Abnormal vasculature
in spleen and bone marrow

Normal RBC Red cell stretched to

attempts to squeeze abnormal length for a long
through abnormal period of time
vasculature of and results in permanent
spleen & bone damage to membrane
marrow
Red cell inclusions
Basophilic Stippling
Coarse basophilic stippling
Basophilic stippling
Pathophysiology
On electron microscopy
Reticulocytes contain ribosomes & mitochondria

Fine basophilic stippliiling

 Non-pathologic
 Usually seen in
reticulocytosis and
regenerating marrow

During the process of drying,

ribosomes and mitochondria clump together
and may result in fine basophilic stippling
 Coarse Basophiic stippling
Pathophysiology
Abnormal ribosomes in Aggregation of ribosomes
reticulocyte during process of drying

Causes
 Lead poisoning
In certain pathological conditions RNA
degradation is impaired and the
 Thalssaemia
ribosomes have greater tendency to  Refractory anaemia
form larger granules.  Megaloblastic anaemia
This is known as coarse basophilic  Sideroblastic anaemia
stippling  Congenital dyserythropoietic
anaemia
 Liver disease
 Infections
 Basophilic Stippling

Fine vs Coarse

•Seen in reticulocytes  Abnormal aggregates of

ribosomes & polyribosome in
•May be an artifact reticulocytes
of slow drying

•Not clinically significant  Clinically significant , suggest

impaired Hb synthesis
Howell Jolly Bodies
Key features
•Spherical blue-black
inclusions of red cells
•One to 2 µm in diameter
•These are nuclear
fragments of condensed
DNA removed by spleen
Associated conditions
 Severe hemolytic anemias
 Megaloblastic anaemia
 Splenectomy/splenic atrophy
 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
Miscellaneous RBC abnormalities
Target Cells
 Target Cells
Red cells with central round stained area and
peripheral rim of hemoglobinized cytoplasm
appear as target

Normal biconcave RBC Change of normal

biconcave shape of RBC
RBC with normal
central pallor Target cell

Increase in surface area to volume ratio of

erythrocytes either due to increase in red cell
surface area or due to decreased intracellular
hemoglobin
Causes of target cell formation
•Artifacts of air drying

•Haemoglobin C

•Decreased volume
 Iron deficiency anaemia
 Thalassaemia
 Haemoglobinopathies

•Increased surface membrane

 Liver disease
 LCAT deficiency
 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
 Burr cells
 Most common cause is an artifact;
-Improperly prepared smear
-Slow drying thick smear aged blood
-”Glass artifact” diffusion of basic
substances from glass slide causing an
elevation in pH
 Rare causes
uremia , chronic renal failure, heparin therapy
 Burr cells
Pathophiology
Red cell membrane is composed of lipid bilayer
with inner and outer leaflets

Inner leaflet
Outer leaflet } Lipid bilayer

In some conditions like increase pH,

decrease albumin, exposure to anionic
phenothiazine the area of outer leaflet of lipid
bilayer is increased as compared to inner layer

This results in evenly spaced projections

over the entire surface

Note: The process is entirely reversible

 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
 Spur Cells
Normal red cell membrane
Composed of lipid bilayer

Interaction between plasma lipids and red cell membrane lipids

Abnormal amount of lipid accumulates

on the outer half of lipid bilayer
resulting in spicule formation of
the membrane
 Spur Cells

Spur cells from a patient with severe liver disease

 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
 Dimorphic Blood Picture
Causes
 After blood transfusion
 Combined iron and
folate/B12 deficiency
 Sideroblastic anaemia

Key features
Two distinct population of red cells seen.
Normal RBCs suspended in
abnormal plasma
 Rouleaux Formation

Causes Rule of thumb

All the diseases associated with hypergamma
globulinaemia
 Multiple myeloma
 Chronic liver disease
 Chronic inflammatory
conditions
 Malignant lymphoma
 Rouleaux Formation
Pathophysiology
25 nm

Zeta potential
 RBCs have negative charge known as zeta potential around the surface
 This keeps RBCs 25 nm apart
In some diseases
plasma proteins are increased

Plasma proteins
reduce zeta potetial

RBCs nonspecifically
join in the form of stack of coins
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)
ANEMIAS
 Anaemia!

Anaemia is defined as haemoglobin level or the

hemocrite below the lower limits of normal
range with reference to age, sex and
geographic location

Anaemia is not a diagnosis, but sign of a

disease which should be investigated.
 Classification of Anaemia

Pathophysiological Morphlogical

• Acute blood loss •Microcytic/Hypochromic

•Impaired production •Macrocytic

•Increased destruction •Normocytic /Normochromic

 Hypochromic/ Microcytic Anaemias

Defect in haem synthesis Impaired globulin synthesis

eg.Iron Defeciency Anaemia eg. Thalassaemias

Sideroblastic Anaemia
Hypochromic/ Microcytic Anaemias
Pathophysiology
Unavailability of iron Protoporphyrin
after several steps
•Decreased iron stores
eg Iron deficiency anaemia
Fe Incorporation of
•Normal iron stores but iron with
decreased release of protoporphyrin
iron from macrophages
eg Anaemia of chronic disorder
Haem synthesis

Defect in haem synthesis

•
Iron not incorporated with
protoporphyrin Fe + protoporphyrin
eg lead poisoning,sideroblastic anaemia
IX
 Iron Deficiency Anaemia

Note; Hypochromia, anisocytosis, poikilocytosis &microcytosis

 Sideroblastic Anaemia
Bone marrow Peripheral blood smear
Ringed sideroblasts Siderocyte

Defective haem synthesis with Siderocytes are red blood

abundant iron in the mitochondria cells containing Pappenheimer
On prussion blue staining for iron, bodies (Small peripherally sited
basophilc black siderotic granules.)
erythroblasts appear as ringed
sideroblasts
 Macrocytic anaemias

With round macrocytes With oval macrocytes

Cause Cause
•Reticulocytosis •Megaloblastic anaemia
•Liver disease eg B12/folate deficiency
•Hypothroidism •Chemotherapeutic drugs
• MDS eg hyroxyurea
• Alcoholism
• Pregnancy
•Aplastic anaemia
Round macrocytosis
Anaemia Secondary to Liver
Disease
.

Note; moderate round macrocytosis & target cells

Megaloblastic Anaemia

Note; Oval Macrocytes & megaloblast

Normocytic/Normochromic anaemia
With polychromasia/increased reticulocyte count
 Acute blood loss

RBC RBC
 Hemolytic anaemia
Normocytic/Normochromic anaemia
With polychromasia
?acute blood loss ?haemolytic anaemia

Clinical correlation recommended

Hereditary Haemolytic anaemia

Beta- Thalassemia Major

Note; Microcytes,taeget cells,acanthocytes & normoblast

 Peripheral Blood Smear of
Thalassemia Major
Thalassemia Major

Note; Microcyted,target cells & several normoblasts

Aquired Haemolytic Anaemia
P.falciparum:
Autoimmune Hemolytic Anaemia (IgG)

Note; Polychromasia, spherocytosis, and a nucleated red cell.

Normocytic/Normochromic anaemia
Without polychromasia/
normal or decreased reticulocyte count

 Anaemia of chronic renal failure

 Anaemia of chronic disease

 Bone marrow infiltration

 Cytotoxic and other drugs

 Anaemia of chronic renal failure
Pathophysiology

Normal individual Chronic renal failure

Decreased Anaemia
Erythropoiesis erythropoiesis
Hypoxia Hypoxia

Erythropoitin Decreased
release from kidney erythropoitin
release from kidney
Anaemia of Renal Disease

Burr cells ,normocytic normochromic red cells

Anaemia of Chronic Disease

Note; red cells are of nomal morphology

Pancytopenia
RBC QUIZ
????????
Normochromic Normocytic
???????
Microcytic, Hypochromic
?????????
Oval Macrocytes
????????
Diagnosis
Thal Major
Arrow:?
1: ?
2: ?
 Arrow:
1: burr-cell
2: elliptocyte
????????
 Oval Macrocytosis:
Megaloblastic Anaemia
????????
Megaloblastic Anaemia

Oval macrocytes
??????
 Iron Deficiency + Normal:
Dimorphic Picture

Two populations - one microcytic-hypochromic,

the other normocytic-normochromic.
??????????
Rouleaux Formation
???????
Howell Jolly Bodies
?????????
Crenated RBC’s
????????
Basophilic Stippling? Schizont?
Coarse Basophilic Stippling
???????
Target cells
?????????
Stomatocytes
?????????
Microcytic Hypochromic
???????
Microspherocytes
???????
Polychromasia
???????
Sickle Cells & Target Cells
????????
Microspherocytes
?????????
Tear Drop cells
???????
 Megaloblastic Anemia,
macroovalocytosis, hypersegmentation,
thrombocytopenia
????????
Burr cells (Echinocytes)
????????
Cabot Ring
???????
Spherocytes
???????
Basophilic Stippling
??????
NRBC
Platelet Morphology
Contents

•Morphologic spectrum of platelets

•Formation of platelets
•Large Platelets

•Giant Platelet
•Platelet aggregates/clumps
 Platelets
•These are the smallest
particles in the blood.

•Lack nuclei.

•Basophilic cytoplasm
with azurophilic
granules.
Morphologic spectrum of platelets
 Formation of Platelets
 Platelets are formed by the
cytoplasmic fragmentation
of megakaryocyte
 Megakaryocyte is the
largest cell of bone marrow
 Mature magakaryocytes are
multilobulated and
granular.
 Megakaryocytes extrude
pseudopodia in the
sinusoids of bone marrow
& fragment into platelets of
various sizes
 Large Platelets
Key features
Irregular shape
Larger than normal size
Associated conditions
 Any condition associated with
increased platelet production.
 ITP
 Myeloproliferative disorders
What corrective action should be
taken if large platelets are seen?

Platelet count should be performed

manually because instrument counts
large platelets as RBC’s and
instrumental platelet count is usually
lower than true value.
 Giant Platelet

Key features
Extremely large platelets
May be larger than red cell
Associated conditions
 Hereditary disorder, Bernard
Soulier Syndrome
 Myeloproliferative disorders
 Thrombocytosis

Associated conditions
 Reactive eg, after bleeding, IDA
 Myeloproliferative disorders
Platelet aggregates/clumps

May result from inadequate mixing of blood

In vitro aggregation of platelets may cause
pseudo thrombocytopenia
Extreme Thrombocytosis &
clumps
Disease Morphology
Disease Morphology
Content
What is leukaemia ?
AML
ALL
CML
CLL
Hairy cell Leukemia
Multiple Myeloma
EXERCISE
 What is Leukaemia?
 A neoplastic proliferation of haematological cells

 Clonal in nature

 May involve mature or immature cells

 Types
Acute
Chronic
 Acute Leukaemias
 Unregulated proliferation of immature
hematopoetic cells.

 Rapid onset.

 Broadly divided into

Myeloid (AML)
Lymphoid (ALL)

 Subclassified by morphology,
immunophenotyping & cytogenetics.
General features of Acute
Leukaemia

 Symptoms usually of rapid onset

 Petechiae, bruising, epistaxis
 Fatigue and lethargy
 Infections
 Bone pain
 Hepatosplenomegaly
 Lymphadenopathy
Lab findings of Acute Leukaemia
 Normocytic, normochromic anaemia of variable
degree.

 Platelet count usually low.

 White count low, normal or high.

 Blasts may or may not be seen on blood smear.

 More than 20% blasts seen on bone marrow.

Myeloblasts Lymphoblasts
 Role of cytochemical stains in
Acute Leukaemia

Myeloperoxidase
 It is very important to classify acute leukaemia because
management of AML & ALL is quite different.
 Identification of Myeloblasts and lymphoblasts is not
enough on morphological basis.
 Cytochemical stains are used to demonstrate lineage
specific cellular components eg,
Myeloperoxidase for AML
Non-specific esterase for Monocytic lineage
 Myeloperoxidase stain

MPO is a cytochemical stain that stains

azurophilic granules containing peroxidase
enzyme and is used classification of leukaemia
 Immunophenotyping
 Cytochemical stains eg, meyloperoxidase/sudan back
cannot distinguish between ALL & subgroups of AML
like AML(M0),AML(M7)

 Immunophenotyping is helpful in these case.

 It is used to demonstrate lineage and maturation

specific cellular antigens / CD markers
Cytogenetics

 Used to demonstrate presence of clonal

abnormality

 May be disease specific

 May have prognostic significance

 Also used to monitor relapse, further

mutation etc
 Acute Myeloid Leukaemia
 Approximately 80% of adult and 20% of childhood
acute leukaemia

 May occur as de novo event or secondary to a

‘preleukaemic’ phase – myelodysplasia

 May be associated with

Radiation
Chemicals Chemotherapeutic agents
 Chromosomal abnormalities eg t(15;17) in AML M3
may be present
 FAB Classification of AML

 M0 Undifferentiated
 M1 Myeloid without differentiation
 M2 Myeloid with differentiation
 M3 Acute Promyelocytic Leukaemia
 M4 Myelomonocytic Leukaemia
 M5 Monoblastic Leukaemia
 M6 Acute Erythroleukaemia
 M7 Acute Megakaryoblastic leukaemia
 Laboratory Diagnosis
 More than 30% blasts in bone marrow.

 Cytochemical stains; myeloperoxidase,/SBB positive

for blasts in most of the sub groups

 Immunophenotyping; shows lineage specific markers

 Some specific cytogenetic abnormalities

eg t(15;17) in APML
 AML

Medium to large size blasts showing finely reticular

chromatin ,2-3 nucleoli & scant to moderate granular
cytoplasm
 AML-M0

AML- MO is type of AML in which <3% blasts

are positive for MPO/Sudan black
 Acute Myeloid Leukemia
(AML) FAB M1
 More than 3% blasts are positive for
myeloperoxidase (MPO) or Sudan black B

 Less than 10% nucleated bone marrow cells are

maturing granulocytes.

 Absent/rare Auer rods

 Commonly CD13 and/or CD33 are positive

 CD34 may be positive

AML M0 AML M1

Non-granular Fine azurophilic granules

are present
AML-M1
 AML-M1

Note; Medium to large size blasts showing finely

reticular chromatin ,2-3 nucleoli & moderate granular
cytoplasm . Mature cells are not seen.
AML-M1
AML-M1
AML-M1
 Myeloperoxidase stain

Blasts are positive for myeloperoxidase stain

Acute Myeloid Leukemia (AML)
FAB M2

 More than 3% blasts are MPO or Sudan black B

positive

 More than 10% mature myeloid cells

 Auer rods common

 Monocytic cells <20%

 AML-M2

AML- M2 is classified when >3% blasts in the

bone marrow are reactive for MPO/Sudan black
& > 10% nucleated cells are mature myeloid cells
 AML- M2

Note; moderate to large blasts with

fine chromatin and several mature
myeloid cells.
 AML-M2

Note; moderate to large blasts with moderate

cytoplasm and some mature cells.
AML-M2 with dysplasia
 Acute Myeloid Leukemia
(AML) FAB M3
(Acute Promyelocytic Leukaemia)
 Majority of cells are abnormal promyelocytes
showing heavy granulation in classic type.

 Characteristic cells containing bundles of Auer rods calle

“faggot cells” are usually present

 Immunophenotypic markers
HLA-DR negative
CD34 negative
CD13 and CD33 positive
 Cytogenetics; t(15;17)
 AML-M3, Faggot Cells

Smear shows promyelocytes containing abundant

azurophilic granules & faggot cells with multiple Auer
rods
 Acute Promyelocytic Leukemia

Showing blackish purple large azurophilic granules

 Faggot Cells

Cells showing multiple Auer rods arranged in

bundles are called Faggot cells
Usually seen in AML M3
 AML Acute Promyelocytic
FAB M3, Microgranular Variant
 Less abundant or finer granules.

 Marked irregular nuclear foldings with

monocytoid appearance.

 “Faggot cells” are present.

 HLA-DR negative
 AML-M3 variant

Smear shows abnormal micro-granular promyelocytes.

Nuclei are markedly lobulated.
 AML FAB M3: Acute Promyelocytic
Microgranular Variant

Note: Promyelocytes are hypogranular and moncytoid in appearance

AML-M3 Myeloperoxidase

Note; promyelocytes & blasts are reactive for

myloperoxidase stain
 Acute Myeloid Leukemia
(AML) FAB-M4

 More than 30% blasts in BM

 >20% are of marrow cells are of monocyte lineage

 > 5x109/l monocytic cells in peripheral blood

 Monocytic cells are reactive for non- specific esterase

Acute Myeloid Leukemia
(AML) FAB- M4
 AML FAB-M5: Acute
Monoblastic/Monocytic
More than 80% of nucleated marrow cells are of monocytic series
 M5a:
> 80% of monocytic cells are monoblasts
 M5b:
<80% of monocytic cells are monoblasts. Promonocytes &
monocytes are predominent

 Blasts in M5 are negative for myeloperoxidase & positive for

nonspecific esterase staining. However, promonocytes may have
scattered myeloperoxidase & sudan black granules
 AML-M5a

Smear shows monoblasts with abundant cytoplasm,

few nucleoli are also seen
 AML-M5b

Promonocytes have characteristic nuclear foldings

and delicate creases in nuclear membrane
AML FAB M6: Acute Erythroleukaemia
 The erythroid component comprises >50% of all
nucleated cells

 Remaining 30% of non-erythroid cells are myeloblasts

 Myeloblasts are positive for myeloperoxidase stain.

 Erythroid precusors are frequently PAS positive

 Dyserythropoiesis with nuclear multinucleation and

pyknosis

 Glycophorin A, CD71 (transferrin), and RC-1 positive

 AML FAB M6 Acute
Erythroleukemia

Erythroblasts with basophilia and vacuoles

AML FAB M7: Acute Megakaryoblastic

 At least 30% of nucleated cells are blasts

 More than 50 % are magakaryblasts are identified by

platelet peroxidase, immunophenotyping or electron
microscopy.

 Increased reticulin is common causing myelofibrosis.

 AML FAB M7

Note; abnormal megakaryocytes

with hypolobulation
Acute Lymphoblastic Leukaemia
 Predominates in children.

 Lymphadenopathy and moderate

hepatosplenomegaly common.

 Higher risk of
CNS involvement
Testicular involvement

 T-ALL associated with mediastinal mass

 FRANCH AMMERICAN BRITISH
FAB classification
 L1 – Homogenous, small blasts, with scanty
cytoplasm. Regular nuclear shape, indistinct nucleoli.

 L2 – Heterogenous, large blasts. Variable nucleus

and cytoplasm, nucleoli present

 L3 – Homogenous, large cells, basophilic with

cytoplasmic vacuolation.

ALL L3 has bad prognosis, otherwise ALL FAB

classification is no more important prognostically
 Lab findings
 More than 30% blasts in bone marrow.

 Blasts are negative for myeloperoxidase stain

 Immunological classification –
-B cell lineage
-T cell lineage

Cytogenetics – certain specific abnormalities affecting

prognosis
 ALL

Blasts of small size with scanty cytoplasm, fine

to coarse chromatin & inconspicuous nucleoli
 ALL-L1

Blasts of small size showing coarse chromatin &

inconspicuous nucleoli and scanty agranular cytoplasm
 ALL-L2

Blasts of heterogeneous size, coarse chromatin ,

nucleoli are present
ALL-L2
 L3

Chromatin is coarsely reticular, cytoplasm is deeply

basophilic and contains several vacuoles
 Chronic Leukaemias
 Much slower onset

 May be diagnosed incidentally

 Proliferation of mature cells

 Usually confined to adults

 May affect myeloid or lymphoid cell lines

Chronic Myeloid Leukaemias

Typical Ph chromosome
t(9;22)(q34;q11)
Has 3 phases
- Chronic Phase
- Accelerated Phase
- Transformation to
myeloid or lymphoid acute
leukaemia
 Chronic Myelogenous Leukemia
.

Leukocytosis, increased basophils , eosinophils and

granulocytes with all stages of maturation
CML-High Power

Showing all stages of maturation

 Chronic lymphoid leukaemias
 Disease of older age

 Gradual advancement

 Lymphadenopathy

 Hepatosplenomegaly

 Lymphocytosis
 CLL

Marked lymphocytosis with predominant mature

lymphocytes, round to slightly oval nucleoli
Nuclear chromatin is clumped
Lymphoma cells in peripheral blood

Lymphoma cells may be seen in peripheral

blood & cannot be differentiated from CLL
without immunophenotyping
 Hairy Cell Leukaemia

 Disease of middle age

 Splenomegaly
 Pancytopenia
 Bone Marrow/Splenic
infiltration with hairy
cells
 Typical morphology,
 Cytochemistry
Hairy Cell Leukemia

Note wispy cytoplasmic projections

 Multiple Myeloma

-Clonal proliferation of Ig
secreting B cells
-Monoclonal gammopathy
-Bone pain, lytic lesions
-Risk of renal failure
-Hypercalcaemia
-Primarily disease of
elderly
 Multiple Myeloma

Several plasma cells with eccentric nuclei &

occasional prominent nucleoli
 EXERCISE

DISEASE MORPHOLOGY
????????
ALL
????????
ALL-L2
??????
ALL-L3
????????
1.Blast
???????
Blasts
???????
AML-M3 Faggot Cells
?????
Plasma cells
MALARIA MORPHOLOGY

 Vector of disease
 Malaria diagnosis
 P.falciparum
 P.vivax
 QUIZ

Click your desired destiny

 Anopheles spp.
(mosquitos)
Of the insects that serve as vectors for parasitic
diseases, this genus is arguably the most important.

Of the approximately 400 species of Anopheles,

about two dozen serve as vectors
for malaria (Plasmodium spp) in humans.
Oocyst on Mosquito Gut
Plasmodium sp: The genus Anopheles includes
more than400 species of mosquitoes.
Many may act as vectors of human diseases such as
malaria, filariasis and some arbovirus.
.

Eggs present a pair of lateral floats and are

laid singly on the water surface,
while larvae lay in a horizontal position under the water
surface
Sporozoites of Plasmodium

Approximate length of each = 10 µm. This life cycle

stage is produced by the oocyst , migrates to the
mosquito's salivary glands, and is injected when the
mosquito feeds.
Malaria diagnosis:
whereas thin film gives more information on
parasite morphology and permits an easier
morphologic differentiation, Thick Smear
is more sensitive allowing a concentration of
plasmodia (10-15 folds) and it is the standard
reference diagnostic test.
P.falciparum
Trophozoites, thick smear, Giemsa stain
 Malaria Diagnosis

•Thick stain needs careful staining & experience in

examining slides;

•Quantification of baseline parasitemias necessary

for monitoring the response to therapy.

•N. of parasites counted x 100 = % Parasitic

Load 1000 RBC’s
 P.falciparum:
In thick films red blood cells are not visible
and leucocytes and parasites appear smaller
than in thin smears.

Trophozoites have a ring or comma shape,

with one or two dots of chromatin.
The pigment, when present, is compact.
P.falciparum:
Trophozoites in Giemsa-stained thick films have a
wide range of shapes.
P.falciparum: Trophozoites & Gamatocyte
P.Falciparum trophozoite in
peripheral blood
P. falciparum: sometimes trophozoites
appear at the edge of the red blood cell
(applique form).
Erythrocytes maintain regular shape and
size.
P. falciparum: late trophozoites and schizonts usually
are not observed in peripheral blood unless in severe
infections.

Cerebral malaria: late trophozoites with

a coarse granule of pigment in peripheral blood.
P.falciparum, thin smear, Giemsa stain.
P.falciparum: micro- and macrogametocytes
have an evident malaric pigment, scattered
through in the cytoplasm in the
microgametocyte.

Microgametocyte, Giemsa thick smear.

P.falciparum: micro- and macrogametocytes are
easily recognized by their crescentic, cigar- or
banana-like shape.

Microgametocytes have a diffuse chromatin,

while macrogametocytes have thickened
chromatin.

Microgametocyte, Giemsa thin smear.

P.falciparum
 P. falciparum: trophozoites are small rings
with single or double small chromatin dots,
and regular cytoplasm; multiple infection and
high parasitemia (>5%) are common.
Dots or cleft (Maurer's) can be observed on
the infected RBCs.

P.falciparum trophozoites, thin smear, Giemsa stain.

Ring stages of Plasmodium falciparum.

Note the multiple infections of some cells

P.Falciparum
 P.Falciparum
Pathogenesis
Severe P.falciparum infections are clinically
characterized by potentially fatal manifestations

Celebral malaria: parasitized RBCs in brain vessels (H&E stain).

P.falciparum: Rosetting of infected and
uninfected red blood cells and cytoadherence of
parasitized erythrocytes to the vascular
endothelium, play a crucial role in sequestration
of parasites and obstruction of brain vessels.

Induction of host cytokines and soluble

mediators such as oxygen radicals
and NO play an important role in the
pathogenesis of the infection.
 P.Falciparum
Pathogenesis (contd)
P.falciparum: The brain appears oedematous,
hyperaemic and with pigment deposition; the
capillaries, expecially of the white matter, appear
dilated and congested and obstructed by
parasitized RBCs.
P Falciparum H & E
 P.Falciparum
Pathogenesis (contd)

P.falciparum: Renal failure may result from

sequestration of RBCs and alteration of the
renal microcirculation.
Glomerular and interstitial vessels present
RBCs adhering to the endothelium.
 P.Falciparum
Pathogenesis (contd)

P.falciparum: Renal failure may also result from

releasing of compounds secondary
to intravascular haemolysis (not haemoglobin
itself) that can cause acute tubular necrosis
especially in the presence of dehydratation and
acidosis.
 P.vivax
Species identification is possible on the
basis of the appearance of parasites of
each of the four malaria species. Shape and
size of asexual parasites and of macro- and
microgametocytes, developmental stages
in peripheral blood, modifications of
infected erythrocytes, presence of dots or
clefts on the red blood cells are the main
differential characteristics.
P.vivax: Young trophozoites are small with single
(rarely double) chromatin, with a loop of thin
cytoplasm.

The red blood cell is slightly enlarged and a few

Shuffner's dots are present.

Parasitemia range from 0.5 to 2%, multiple

infection is rare.
P.vivax: The trophozoites increases in size
and the cytoplasm becomes ameboid with
rapid movements ("vivax").
The red blood cell enlarges and prominet
Shuffner's dots are present.

(Thin smear, Giemsa)

P.vivax: In more advanced stage of
development trophozoites occupy most
of the RBC, and have a large vacuole and fine
rods of pigment.
The nucleus increases in size.
P.vivax: Late trophozoites have a more dense
cytoplasm, and a large vacuole
P.vivax: In young schizonts the nucleus divides and
the vacuole disappears; the cytoplasm is dense.
P.vivax: In about 48hours schizogony is
completed.
Mature schizont may contain 12-24
merozoites.
In thick smears schizonts look smaller than in
thin smears and the Schuffner's dots are not
always visible.
P.vivax: Gametocytes are round or oval without
vacuole; most of the RBC is occupied by the
parasite.
Macrogametocytes have a compact chromatin mass
while microgametocytes have a more diffuse
nucleus stained pink.
 QUIZ

MALARIA MORPHOLOGY
????????
P.Vivax ( Amoeboid)
?????????
P.Vivax ( Schizont)
????????
P.Vivax
?????????
Plasmodium Vivax
???????
P.Falciparum; Applique form
??????
P.Falciparum; Double ring
???????
 P.Vivax
Female Gamete & Male Gamete
?????????
P.Falciparum; Multiple rings
???????
P.Falciparum; Mature schizont
???????
P.Falciparum; Gametocyte
?????????
P.Vivax( ring)
???????
P.Vivax ( gametocyte)
 References
• CAP Color Atlas of Heamatology
Eric F Glassy, M D

• Practical Hematology, Dacie and Lewis

ninth edition

• Williams Hematology, fifth edition

• Tumors of the Bone Marrow,

Richard Brunning &Robert McKenna,
Third series

• Multiple Hematology Web-sites

See you in the next version
with more improvements
Because

There is always room for

improvement