Peripheral blood smear examination

(slide preparation and reporting)

 Role of peripheral blood examination
• Evaluation of anemia
• Evaluation of thrombocytopenia/ thrombocytosis
• Identification of abnormal cells (blasts/abnormal
promyelocytes/atypical lymphoid)
• Infections like malaria, microfilaria
• Inclusions like basophilic stippling, Howell-Jolly
bodies, Cabot ring
 Peripheral Blood Smear
• Objective:
1. Peripheral Smear Preparation
2. Staining of Peripheral Blood Smear
3. Peripheral Smear Examination
 Peripheral Smear Preparation
• Wedge technique
• Coverslip technique
• Automated Slide Making and Staining
 WEDGE BLOOD SMEAR
• Specimen:
 Peripheral blood smear made from EDTA-
anticoagulated blood.
 Smears should be made within 1 hour of blood
collection from EDTA specimens stored at room
temperature to avoid distortion of cell
morphology
 Blood smears can also be made from finger prick
blood directly onto slide.
• Equipment
• Spreaders
• Clean slides
• Blood capillary tube or micropipette 10
µL
SMEAR PREPARATION
1.Place a drop of blood, about 2-3 mm in
diameter approximately 1 cm from one
end of slide.
2.Place the slide on a flat surface, and hold
the other end between your left thumb
and forefinger.
3.With your right hand, place the smooth
clean edge of a second (spreader) slide on
the specimen slide, just in front of the
blood drop.
4.Hold the spreader slide at a 30°- 45 angle,
and draw it back against the drop of blood
6.Allow the blood to spread almost to the
edges of the slide.
7.Push the spread forward with one light,
smooth moderate speed. A thin film of
blood in the shape of tongue.
8.Label one edge with patient name, lab id
The shape of blood film
 Characteristics of A Good Smear
1. Good smear is tongue shaped with a
smooth tail.
2. Does not cover the entire area of the slide.
3. Has both thick and thin areas with gradual
transition.
4. Does not contain any lines or holes.
 The thickness of the smear

Is determined by:
1. The angle of the spreader slide. (the
greater the angle, the thicker and shorter
the smear).
2. Size of the blood drop.
3. Speed of spreading
 The thickness of the spread
1. If the hematocrit is increased, the angle of
the spreader slide should be decreased.
2. If the hematocrit is decreased, the angle of
the spreader slide should be increased
Common causes of a poor blood smear

1. Drop of blood too large or too small.

2. Spreader slide pushed across the slide in a
jerky manner.
3. Failure to keep the entire edge of the
spreader slide against the slide while making
the smear.
4. Failure to keep the spreader slide at a 30°
angle with the slide
5. Failure to push the spreader slide completely across
the slide.

6. Irregular spread with ridges and long tail: Edge of

spreader dirty or chipped; dusty slide

7. Holes in film: Slide contaminated with fat or grease

and air bubbles.

8. Cellular degenerative changes: Delay in fixing,

inadequate fixing time or methanol contaminated with
water.
Blood films
Slide fixation and staining
 Romanowsky staining
It includes:
• May-Grunwald –Geimsa stain,
• Jenner’s stain,
• Wright’s stain,
• Leishman’s stain and,
• Field’s stain.
 Components of Giemsa stain
It mainly composed of
• Eosin Y and,
• Azure B- Methylene Blue
• Eosin-It is acidic component of stain and stain
basic component of cells like hemoglobin.

• Azure B – it is basic component and stains the

acidic component of cells like DNA and RNA
(nucleus of WBC)
 Materials
• Slide holder or rack
• Stain reagent
• Methanol : fix the cells on the slide

• Buffer:
• Used to maintain an adequate pH.
• 0.05M Na2PO4 (pH 6.4)
• Distill water kept in glass bottle for at least 24hours (pH
6.4-6.8)
 Methodology
• Put the smear into methanol jar and fix it for 1 -2 minute.
• Remove excess methanol from the smear
• Insert the smear into Wright’s stain jar and leave the
stain for 2 minutes.
• Insert smear into a buffer jar and allow to stand for 4-8
minutes
• Rinse thoroughly with a steam of distilled water
• Allow to air dry
• Note: time varies from manufacturers, thus ensure to
follow the exact time in the kit manual of each procedure
 Colour responses of blood cells to
Romanowsky staining
• Cellular component Colour
• Nuclei Chromatin Purple
• Nucleoli Light blue
• Cytoplasm
• Erythroblast Dark blue
• Erythrocyte Dark pink
• Reticulocyte Grey–blue
Cytoplasm colour
• Lymphocyte Blue
• Metamyelocyte Pink
• Monocyte Grey–blue
• Myelocyte Pink
• Neutrophil Pink/orange
• Promyelocyte Blue
• Basophil Blue
Granules
• Promyelocyte(primary granules) Red or purple
• Basophil Purple black
• Eosinophil Red–orange
• Neutrophil Purple
• Toxic granules Dark blue
• Platelet Purple
Other inclusions
• Auer body Purple
• Cabot ring Purple
• Howell-Jolly body Purple
• Döhle body Light blue
 Factors influence smear staining method

• Blood smear may be under or over stained based

on the following
• Concentration of the stain used
– Low concentration: pale coloured cells (under
staining)
– High concentration: dark stained smear (over stained)
• Time of exposure the stain and the buffer
– Too long: overstaining,
– Too short: understaining
Factors giving rise to faulty staining
• Appearances Causes
Too blue Eosin concentration too low Incorrect
preparation of stock
stock stain exposed to bright daylight
Batch of stain solution overused
• Impure dyes
• Staining time too short
• Staining solution too acid
• Smear too thick
• Inadequate time in buffer solution
– Too pink Incorrect proportion of azure
B-eosin Y
• Impure dyes
• Buffer pH too low
• Excessive washing in buffer
solution
• Pale staining Old staining solution
• Overused staining solution
• Incorrect preparation of
stock
• Impure dyes, especially
azure A and/or C
• High ambient temperature
• Neutrophil granules Insufficient azure B not
stained
• Neutrophil granules Dark Excess azure B
Blue/black (pseudo-toxic)

• Other stain anomalies Various

contaminating
dyes and metal salts
• Stain deposit Stain solution left in
on film uncovered jar
Stain solution not
filtered
• Blue background Inadequate fixation or
prolonged storage
before fixation
• Blood collected into heparin
as anticoagulant
 Peripheral blood smear examination
Macroscopic
•of the smear view : quality
•Any abnormal
present particles
•The Microscopic analysis
•begins
(10x), on lower power
•to assess quality
preparation of the
•assess whether
agglutination, red cell
excessive
rouleaux
platelet formation
aggregation or
is
present;
•Assess the
distributionnumber,
and staining
of the leucocytes; and
• find an area where the red cells are evenly
distributed and are not distorted.
• On high power(40x)-
• to obtain a WBC estimate
• All of the detailed analysis of the cellular
elements using high power or oil
immersion(100x).
Normal peripheral smear
Area too thin to examine the slide
Too thick area
 Evaluation of PBS
• 1. RBC
• Size
• Shape
• Color
• Arrangement
• Inclusions

• 2. WBC
• Total counts
• Differential counts
• Abnormal WBC
• 3. Platelets
• Counts
• Abnormality
•4. Parasites
 RBC
• In the blood from healthy person RBCs are
– Circular , Homogenous disc nearly of uniform
size(6–8 µm)
– deep pink cytoplasm with Central pallor <1/3rd
 Various changes in RBCs
•
Hypochromic
• Dcrease in Hemoglobin • hypochromia
content of RBC
• increase in central
pallor(>1/3rd)
• Decrease in MCH and

MCHC
• Seen in Iron Deficiency
anemia
• thalassaemia
Hyperchromia

• Red cells stain

deeply
• Have less central
pallor,
• Increase in MCH
• Seen in
Megaloblastic
anemia
• Hereditary
spherocytosis(MCH
is normal but MCHC
is increased)
• Anisochromia – presence of hypochromic cells and
normochromic cells in the same film. Also called
dimorphic anemia.
• Seen in
– Sideroblastic anemia
– Some weeks after iron therapy for iron deficiency anemia
– hypochromic anemia after transfusion with normal cells.
Dimorphic blood picture
Polychromatophillia
Blue grey tint of red cells
Due to Hb and
RNA(Residual) in young
cells.
• Larger than normal and
may lack central pallor.
• Implies Reticulocytosis
• Seen in
• Hemolysis
• Acute blood loss
 Variation In Size
• Anisocytosis- Variation in size of the red blood
cells
• Normal MCV is -80-100 fl
• Microcytes ( MCV <80 fl)
• Macrocytes (MCV >100fl)
• Anisocytosis is a feature of most anemias.
 Microcytes
• Size of RBC is
reduced(<80fl)
• Seen when hemoglobin
synthesis is defective
1. Iron deficiency anemia
2. Thalassemia
3. Anemia of chronic
disease
4. Sideroblastic anemia
Macrocytes
• When MCV of RBC is
Increased(>100fl)
• Macro- ovalocytes are
seen in Megaloblastic
anemia
• Myledysplastic
syndrome.
• Round Macrocytes seen
in Alcoholism, Liver
disease.
Shape
• Variation in shape is called Poikilocytosis.
• It is of following types-
• Elliptocytes
• Spherocytes
• Target cells
• Schistocytes
• Acanthocytes
• Keratocytes
• Echinocytes
Elliptocytes

• Elipitical in shapes
• Most abundant in
hereditary elliptocytosis
• Seen in –
1. Iron deficiency anemia
2. Myelofirosis with
myeloid metaplasia
3. Megaloblastic anemia
4. Sickle cell anemia
Spherocytes
• Nearly spherical
• Diameter is smaller than
normal
• Lack central pale area or
have a smaller , eccentric,
pale area
• Seen in
– hereditary spherocytosis
– Some cases of
autoimmune hemolytic
anemia
– direct physical or
chemical injury
Target cells
• Cells in which central round
stained area and peripheral
rim of cytoplasm
• Seen in Thalassaemia
• Chronic liver disease
• Hereditary hypo-
betalipoproteinemia
• Iron deficiency anemia
• Hemoglobinopathies (Hb C,
Hb H, Sickel cell anemia
• Postsplenectomy
Schistocytes
• These are fragmaented
erythrocytes.
• Smaller than normal red cells
and of varying shape
• Seen in
• Genetic disorder
– Thalassaemia
– congential dyserythropoietic
anemia.
• Acquired disorder of RBC
formation
– Megaloblastic
– Dyserythropoietic
• Mechanical stress MAHA
• Direct thermal injury
 Acanthocytes

Red cells with small no of

spicules of inconstant length,
thickness and shape ,
irregularly disposed over the
surface.
• Seen in Abnormal
phospholipid metabolism
• Abetalipoproteinemia
• Inherited abnormalities of
red cell membrane protein
• Splenectomy
 Echinocytes
• Also called crenated cells
• Numerous, short, regular
projection
• Commonly occur as an artifact
during preparation of film
• Hyperosmolarity
• discocyte–echinocyte
transformation
• Overnight stored blood at 20 C
before films are made.
• Premature infant after exchange
transfusion
• water contaminating the Wright’s
stain (or absolute methanol)
Keratocytes
• Have pairs of spicules
either one or two pairs.
• Sometimes termed as
Bite cell or helmet cell
• Seen in
– Mechanical damage
– Removal of Heinz body
by pitting action of
spleen.
Leptocytes
• Thin red cells with large
unstained central area.
• Seen in
– Severe iron deficiency
anemia
– Thaleasaemia
Stomatocytes
• Red cells with central
biconcave area appears slit like
in dried film.
• Wet film it appears as cup-
shaped.
• Seen in
– Artifact
– south-east Asian
ovalocytosis
– liver disease,
– alcoholism,
– myelodysplastic syndromes.
Sickle cell
• Cells are sickle (boat
shape) or crescent
shape
• Present in film of
patient with
homozygosity for Hb S.
• Usually absent in
neonates and rare in
patients with high Hb F
percentage
Tear drop cells
• One side of cells is tapered
and other is blunt.
• Seen in
– Myelofibrosis
– thalassemia
– Hemoglobin E heterozygous
+ homozygous
●HbH disease
●HbC trait
●Hb Lepore heterozygous +
homozygous
●HbO Arab disease
●HbD disease
●Iron defciency
●Hb Lepore trait
 Structure
• Basophilic stippling (Punctate basophilia)
• Howell – jolly Bodies
• Cabot Rings
• Malarial Stippling
• Rouleaux formation
 Red blood cell inclusions
• Name of Inclusion Content
• Howell-Jolly body DNA
• Basophilic stippling RNA
• Pappenheimer body Iron
• Heinz body(supravital only) Denatured hemoglobin
• Crystals Hemoglobin-C
• Cabot rings Mitotic spindle
remnants
• Nucleus DNA
 Basophilic Stippling
• Presence of irregular basophilic
granules with in Rbc which are
variable in size .
• Stain deep blue with Wright’s stain
• Fine stippling seen with
– Increased polychromatophilia
– Increased production of red cells.
• Coarse stippling
– Lead and heavy metal poisoning
– Disturbed erythropoiesis
• Megaloblastic anemia
• Thalassaemia
• infection
• liver disease
– Unstable Hb
 Howell-Jolly Bodies

• Smooth single large round

inclusions which are remnant of
nuclear chromatin.
• Seen in
• Single –
– Megaloblastic anemia
– Hemolytic anemia
– Postsplenectomy
• MULTIPLE –
– Megaloblastic anemia
• Howell-Jolly Bodies
– Abnormal erythropoiesis
 Pappenheimer Bodies
• These are small single or
multiple peripherally sited
angular basophilic (almost
black) erythrocyte inclusions.
• Smaller than Howell–Jolly
bodies.
• composed of haemosiderin.
• Their nature can be confirmed
by Perls’ stain.
• Seen in
– Sideroblastic erythropoiesis
– Hypospenism
– Myelodysplastic syndrome
– Hemolytic anemia
 Heinz bodies
• Seen on supravital stains
• Not seen on Romanowsky stain.
• Purple, blue, large, single or multiple
inclusions attached to the inner
surface of the red blood cell.
• Represent precipitated normal or
unstable hemoglobins.
• seen – Postsplenectomy
• Oxidative stress
– Glucose-6-phosphate
dehydrogenase deficiency,
– Glutathione synthetase deficiency
– Drugs
– Toxins
– Unstable hemoglobins
Cabot Rings
• These are Ring
shaped ,figure of eight or
loop shaped
• Red or Reddish purple
with Wright’s stain and
have no internal structure
• Observed rarely in
– Pernicious anemia,
– Lead poisoning,
Malrial stippling
• Fine granules of
plasmodium vivax
• On wright stain these
are fine , purplish red
• Red cells are larger than
normal
Rouleaux Formation

• Alignment of red cells one

upon another so that they
resemble
stacks of coins.
• Occurs in
• Paraproteinemia
( monoclonal gammopathy)
• Elevated plasma fibrinogen
or globulin level
Agglutination

• It is more irregular and

round clumping than
linear rouleaux
• Seen with cold
agglutinin
• Anti RBC antibody
• Autoimmune hemolytic
anemia
• Macroglobulinemia
WBC MORPHOLOGY
 Before evaluating leucocyte following must be
seen-
Film is well made
Distribution of cells is uniform
Staining is satisfactory
 While scanning estimate the total leucocyte count
 Differential count is done at oil immersion
Scanning technique for WBC differential count and
morphologic evaluation

• Ten microscopic fields are examined in a vertical

direction from bottom to top or top to bottom
• Slide is horizontally moved to the next field
• Ten microscopic fields are counted vertically.
• Procedure is repeated until 100 WBCS have been
counted (zig zag motion)
 Manual differential counts
• These counts are done in the same area as WBC
and platelet estimates with the red cells barely
touching.
• This takes place under × 100 (oil) using the
zigzag method.
• Count 100 WBCs including all cell lines from
immature to mature.
 Reporting results
• Absolute number of cells/µl = % of cell type in
differential x white cell count
• If 10 or more nucleated RBC's (NRBC) are
seen, correct the
White Count using this formula:
Corrected WBC Count =
WBC x 100/( NRBC +
100)
Example : If WBC = 5000 and 10 NRBCs
have been counted
Then 5,000× 100/110 = 4545.50
The corrected white count is 4545.50
Leukocytes Normally Present in Blood
• GRANULOCYES
Neutrophils ( polymorphonuclear leucocytes)
Eosinophils
Basophils
• Agranulocytes
Lymphocytes
Monocytes
POLYMORPHONUCLEAR
NEUTROPHILS
• 40 to 80 percent of total WBC
count(2.0–7.0 ×109/l )
• Diameter - 13 µm
• segmented nucleus and
pink/orange cytoplasm with
fine granulation(0.2-0.3µm)
stain tan to pink with Wright’s
• Lobes -2-5
• Neutrophils usually have
trilobed nucleus.
• small percent has four lobes
and occasionally five lobes.
Band forms
• neutrophils has either a
strand of nuclear material
thicker than a filament
connecting the lobes, or a U-
shaped nucleus of uniform
thickness.
• Up to 8% of circulating
neutrophils are unsegmented
or
partly segmented (‘band’
forms)
• Left-shift: non-
segmented neutrophil >
5%
– Increased
bands Means acute
infection, usually
bacterial
Granules
• Toxic granulation-
increase in staining
density and number of
granules
• Seen with Bacterial
infections and other
inflammation
• Administration of G-CSF
• Anaplastic anemia
• Hypogranular and
agranular neutophils
poorly stained
• seen in Myelodysplastic
syndrome
Alder–Reilly anomaly
• Granules are large,
– discrete,
– stain deep red
– may obscure the
nucleus
– Neutrophil function
is Normal
 Chédiak-Higashi Syndrome
• Granules are
– also seen in other
leukocytes like
lymphocytes
– Giant
– Scanty azurophilic
– functional defect occur
 Dohle Bodies
• Small, round or oval, pale
blue-grey structure
• Found at periphery of
neutrophil.
• Contains Ribosomes and
Endoplasmic reticulum
• Seen in – Bacterial infection
– inflammation
– administration of G-CSF
– during pregnancy
 May–Hegglin anomaly
• inclusions occur in all
types of leucocytes
except lymphocytes.
• contain small basophilic
cytoplasmic granules
 Vacuoles in neutrophils
• In Fresh blood smear
• vacuoles seen in
– severe sepsis
– as an artifact with
prolonged standing
 Nuclei
• Hypersegmentated
neutrophil
• def.-presence of
neutrophils with six or more
lobes or the presence of
more
than 3% of neutrophils with
at least five lobes.
• seen in Megaloblastic anemia
– uraemia
– iron deficiency.
– Drugs-cytotoxic treatment
with Methotrexate
– hydroxycarbamide
 Pelger–Huët Cells
• Pelger–Huët anomaly
• Benign inherited
condition.
• Neutrophil nuclei fail to
segment properly.
• Majority of circulating
neutrophils have only
two discrete equal-sized
lobes connected by a
thin chromatin bridge.
 Pseudo-Pelger cells
• Pseudo-Pelger cells or
the acquired Pelger–
Huët anomaly
• Acquired condition
• Morphologically similar
to Pelger–Huët anomaly
• seen in Myelodysplastic
syndromes,
• Acute myeloid
leukaemia with
dysplastic maturation,
• Occasionally in
chronic myelogenous
leukaemia
 Pyknotic Neutrophils (Apoptosis)
• Small numbers of dead
or dying cells may
normally be found in
the blood
• seen in infections
– invitro after standing for
12-18 hrs
– Nuclei-round dense,
featureless
– Cytoplasm-dark pink
 EOSINOPHILS
•
Eosinopenia- seen with prolonged steroid
administration.
• Eosinophilia- allergic conditions hay fever,
asthama
• severe eosinophilia- parasitic infection
– reactive eosinophilia
– Eosinophilic leukaemia
– Idiopathic hypereosinophilic syndrome
– T-cell lymphoma, B-cell lymphoma
and acute lymphoblastic leukaemia.
 BASOPHILS
•
 MONOCYTES
• 2-10% of total wbc count
• Size- largest circulating leucocyte,
15–18µm in diameter
• Cytoplasm- grey blue
• Nucleus- large , curved , horse
shoe shape
• No segmentation occur
• Chromatin- fine evenly
distributed
• Increase in chronic infections and
inflammatory conditions such as
– Tuberculosis and Crohn’s
disease,
– Chronic myeloid leukaemias
– Acute leukaemias with a
monocytic component
– Infectious mononucleosis
 LYMPHOCYTES
• 20-40% of total wbc count
• two types
1. Small lymphocyte(6-10µm)
2. Large lymphocyte(12-15µm)
• Nucleus-single, sharply
defined, stain dark blue on
Wright’s stain
• Cytoplasm- Pale blue
• Large lymphocytes less
densely stain nuclei &
abundant cytoplasm
• Few round purple(azure)
granules are present
 Turk cells
• Türk’ cell (immunoblasts)-
Transformed lymphocyte seen
in bacterial and viral infection
• Size 10-15 µm
• Nucleus- Round,
• Large nucleolus, and
abundant, deeply basophilic
cytoplasm
 Reactive lymphocytes
• Have slightly larger
nuclei with more
open chromatin
• Abundant cytoplasm
that may be
irregular.
• Seen in -infectious
mononucleosis
– viral infections
 Malignant lymphoid cells
• Commonest malignancy
is Chronic lymphocytic
leukemia- composed
almost exclusively of
small lymphocytes.
• Some times few larger
nucleolated cells
• Lymphocytes predominate in the blood films
of infants and young children.
 Platelates
•
 Thrombocytopenia
• Decreased production
– Aplastic anemia
– Acute leukemia
– Viral infections *Parvovirus *CMV
−Amegakaryocytic thrombocytopenia (AMT)
• Increased destruction
– Immune thrombocytopenia
– Idiopathic thrombocytopenic purpura (ITP)
– Neonatal alloimmune thrombocytopenia (NAITP)
– Disseminated intravascular coagulation (DIC)
– Hypersplenism
• Pseudothrombocytopenia- due to clumpping of pltelates in
EDTA bulb
 Thrombocytosis
• Reactive thrombocytosis
Post infection
Inflammation
Juvenile rheumatoid arthritis
Collagen vascular disease
• Essential thrombocythemia
 Platelet morphology: Giant platelets
• Platelates seems to be
size of rbcs.
• Seen in
– May –Hegglin anomly
– Bernard Soulier
syndrome
– Alport syndrome
– Storage pool syndrome
Peripheral Blood Cells
 changes on prolonged storage of sample
• EFFECT ON COUNT-
• Less marked in blood in ACD, CPD or Alsever’s solution than in
EDTA.
• At room temperature blood is stable up to 8 h.
• RBC
– Swell up the PCV and MCV increases
– Osmotic fragility increases
– Erythrocyte sedimentation rate decreases
– At 4ͦ C up to 24 h
– Reticulocyte count- Unchanged upto 24 h at 4 C
– Hemoglobin Unchanged upto 2-3 days
 Changes in Morphology
• At 3 h changes start occure
• By 12–18 h these become striking
• RBC- progressive crenation and sphering
• Netrophils- nuclei stain more homogeneously
– Nuclear lobes may become separated
– Cytoplasmic margin may appear ragged
– Vacuoles appear in the cytoplasm
– Lymphocytes and monocytes undergo similar
changes
Changes in netrophils
Changes in monocytes
changes in lymphocytes
 Apoptotic changes
• Cell shrinkage
• Cytoplasmic condensation
around the nuclear
membrane
• Indentations in the nucleus
• Followed by nuclear
fragmentation.
• Cell remnants form dense
basophilic masses (the
apoptotic bodies)
 Disadvantages of the Peripheral Blood
Smear
• Experience is required to make technically adequate
smears.
• Non-uniform distribution of white blood cells
• Larger leukocytes concentrated near edges and
lymphocytes scattered throughout.

• Non-uniform distribution of RBCs

– Small crowded red blood cells at the thick edge

– Large flat red blood cells without central pallor at the

feathered edge
 EXAMINATION OF BLOOD FILMS
FOR PARASITES
• thick film- when parasites are scanty
• thin film – identification of species
• STAINING OF FILM
– by Leishman’s stain at pH 7.2
 Plasmodium falciparum
Erythrocytes throughout
this series are not • Crescent (‘banana-
enlarged or distorted. shaped’) gametocyte
• Early trophozoites
• Accole form
double chromatin dot
Schizonts are commonly seen in P. vivax infection and appear as
large bodies containing 12 to 24 nuclei and a loose pigmented
body. This photograph shows an early schizont of P. vivax on
the left and mature schizonts
Ealy trophozoit in the form of thick ring with large chromatin
dot
Leishmaniasis (Leishman–Donovan bodies)
African trypanosomiasis
(Trypanosoma brucei gambiense)
American trypanosomiasis ( T.
cruzi);
microfilaria
Thank you