Dr Samson Ehe Teron SpPK

Clinical Pathology Laboratory

Prof Yohanes Hospital Kupang
 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
 Objective:
1. Peripheral Smear Preparation
2. Staining of Peripheral Blood Smear
3. Peripheral Smear Examination
 Wedge technique
 Coverslip technique
 Automated Slide Making and Staining
 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
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 and date.
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.
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
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
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.
It includes:
• May-Grunwald –Geimsa stain,
• Jenner’s stain,
• Wright’s stain,
• Leishman’s stain and,
• Field’s 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.

 AzureB – it is basic component and stains

the acidic component of cells like DNA and
RNA (nucleus of WBC)
 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)
 Distillwater kept in glass bottle for at
least 24hours (pH 6.4-6.8)
 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
 Cellular component Colour
 Nuclei Chromatin
Purple
 Nucleoli Light blue
 Cytoplasm
 Erythroblast Dark blue
 Erythrocyte Dark pink
 Reticulocyte Grey–blue
 Lymphocyte Blue
 Metamyelocyte Pink
 Monocyte Grey–blue
 Myelocyte Pink
 Neutrophil
Pink/orange
 Promyelocyte Blue
 Basophil Blue
 Promyelocyte(primary granules) Red or
purple
 Basophil Purple black
 Eosinophil Red–
orange
 Neutrophil Purple
 Toxic granules Dark blue
 Platelet Purple
 Auer body Purple
 Cabot ring Purple
 Howell-Jolly body Purple
 Döhle body Light blue
 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
 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
 Staindeposit 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
• Macroscopic view : quality of the smear
• Any abnormal particles present
• The Microscopic analysis
 begins on lower power (10x),

 to assess quality of the preparation

 assess whether red cell agglutination,

excessive rouleaux formation or platelet

aggregation is present;
 Assess the number, distribution 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).
 1. RBC
• Size
• Shape
• Color
• Arrangement
• Inclusions

 2. WBC
• Total counts
• Differential counts
• Abnormal WBC
 3. Platelets
• Counts
• Abnormality
4. Parasites
 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
1. COLOUR:
It is determined by hemoglobin
content of RBC.
1. Normochromic- Normal intensity of
staining.
2. Hypochromic-
3. Hyperchromic-
 Normal Hb conc. Male-150±20 g/l
 Female- 135±15 g/l
  hypochromia
 Dcrease in
Hemoglobin
content of RBC
 increase in central
pallor(>1/3rd)
Decrease in MCH
and MCHC
 Seen in Iron
Deficiency anemia
 thalassaemia
• 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.
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
 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.
 Sizeof RBC is
reduced(<80fl)
 Seen when
hemoglobin
synthesis is
defective
1. Iron deficiency
anemia
2. Thalassemia
3. Anemia of
chronic disease
4. Sideroblastic
 When MCV of RBC is
Increased(>100fl)
 Macro- ovalocytes are
seen in Megaloblastic
anemia
 Myledysplastic
syndrome.
 Round Macrocytes seen
in Alcoholism, Liver
disease.
 Variation in shape is called Poikilocytosis.
 It is of following types-
 Elliptocytes
 Spherocytes
 Target cells
 Schistocytes
 Acanthocytes
 Keratocytes
 Echinocytes
 Elipiticalin shapes
 Most abundant in
hereditary
elliptocytosis
 Seen in –
1. Iron deficiency
anemia
2. Myelofirosis with
myeloid metaplasia
3. Megaloblastic
anemia
4. Sickle cell anemia
 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
 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
 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
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
 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)
 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.
 Thin red cells with
large unstained
central area.
 Seen in
 Severe iron
deficiency anemia
 Thaleasaemia
 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.
 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
 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
 Basophilic stippling (Punctate basophilia)
 Howell – jolly Bodies
 Cabot Rings
 Malarial Stippling
 Rouleaux formation
 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
 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
 Smooth single large round
inclusions which are remnant
of nuclear chromatin.
 Seen in
 Single –
 Megaloblastic anemia
 Hemolytic anemia
 Postsplenectomy
 MULTIPLE –
 Megaloblastic anemia
 Abnormal erythropoiesis  Howell-Jolly 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
 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
 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,
 Fine granules of
plasmodium vivax
 On wright stain
these are fine ,
purplish red
 Red cells are
larger than normal
 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
 It is more irregular
and round
clumping than
linear rouleaux
 Seen with cold
agglutinin
 Anti RBC antibody
 Autoimmune
hemolytic anemia
 Macroglobulinemia
 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)
• 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
 GRANULOCYES
Neutrophils ( polymorphonuclear leucocytes)
Eosinophils
Basophils
 Agranulocytes
Lymphocytes
Monocytes
 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
 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
 Toxic granulation-
increase in
staining density
and number of
granules
 Seen with
Bacterial
infections and
other
inflammation
 Administration of
G-CSF
 Hypogranular and
agranular
neutophils poorly
stained
 seen in
Myelodysplastic
syndrome
 Granules are large,
 discrete,
 stain deep red
 may obscure the
nucleus
 Neutrophil
function is
Normal
 Granules are
 also seen in other
leukocytes like
lymphocytes
 Giant
 Scanty azurophilic
 functional defect
occur
 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
 inclusions occur in
all
types of
leucocytes except
lymphocytes.
 contain small
basophilic
cytoplasmic
granules
 InFresh blood
smear
 vacuoles seen in
 severe sepsis
 as an artifact with
prolonged standing
 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
anomaly
 Benign inherited
condition.
 Neutrophil nuclei
fail to segment
properly.
 Majority of
circulating
neutrophils have
only two discrete
equal-sized lobes
connected by a
 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
 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
 Normally 1-6%(
0.02–0.5 × 109/l)
 Size- 12–17 µm
 Nucleus- Bilobed
(spectacle shaped)
 Cytoplasm- Pale
blue
 Granules - Coarse
spherical
gold/orange
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.
 Rarest <1%
 Nucleus segments fold up
on each other resulting
compact irregular dense
nucleus(closed lotus
flower like)
 Granules-large, variable
size dark blue or purple
often obscure the nucleus
 Granules are rich in
histamine, serotonin and
heparin
 Increase in
myeloproliferative
disorder-CML
 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
 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
 Türk’ cell (immunoblasts)-
Transformed lymphocyte
seen in bacterial and viral
infection
 Size 10-15 µm
 Nucleus- Round,
 Large nucleolus, and
abundant, deeply basophilic
cytoplasm
 Have slightly
larger nuclei with
more
open chromatin
 Abundant
cytoplasm that
may be
irregular.
 Seen in -infectious
mononucleosis
 viral infections
 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.
 Size-1-3µm
 Normal count - 280 ±130×109/µl

 Non nucleated cells derived from cytoplasmic

fragments of Megakaryocytes
 Has purple red granules.
 Liliaccolor
 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
 Reactive thrombocytosis
 Post infection
 Inflammation
 Juvenile rheumatoid arthritis
 Collagen vascular disease
 Essential thrombocythemia
 Platelates seems
to be size of rbcs.
 Seen in
 May –Hegglin
anomly
 Bernard Soulier
syndrome
 Alport syndrome
 Storage pool
syndrome
 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
 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
 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)
• 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
 thick film- when parasites are scanty
 thin film – identification of species
 STAINING OF FILM
 by Leishman’s stain at pH 7.2
Erythrocytes
throughout this series  Crescent (‘banana-
are not enlarged or shaped’)
distorted. gametocyte
 Early trophozoites
 Accole form