Peripheral Blood Smear - Its Preparation, staining and morphology

1. P S -
S T A I N I N G
A N D
M O R P H O L O
G Y
S U D I PTA

2. What is a peripheral smear ?
• A peripheral blood smear or blood film is a thin layer of blood smeared on a microscope glass slide and then
stained in such a way to allow the various blood cells to be examined microscopically.

3. Peripheral Smear Preparation
• Wedge technique
• Coverslip technique
• Automated Slide Making and Staining

4. 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
• Grease free washed glass slides
• Blood capillary tube or micropipette 10 µL

5. 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 and date.
9. The slides should be rapidly air dried by
waving the slides or using an electrical fan.

6. The shape of blood film

7. 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.

9. 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
i. If the hematocrit is increased, the angle of the spreader slide should be decreased.
ii. If the hematocrit is decreased, the angle of the spreader slide should be increased.

10. 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.

11. Blood films

12. Aim of blood smear
• Blood films are usually examined to investigate hematological
disorders and, occasionally, to look for parasites within the blood
such as malaria, filaria.
• Examination of thin blood films is important in the investigation
and management of anemia, infections, and other conditions which
produce changes in the appearance of blood cells and differential
white cell count.
• A blood film report can provide rapidly and at low cost, useful
information about a patient’s condition.

13. STAINING OF PBS

14. Brief History
• The beginnings of modern-day blood staining can be traced back to Ehrlich,
who in 1877, was the first scientist to divide the aniline dyes into acidic and
basic categories.
• Three years later , the malarial parasite was discovered, and a rigorous search
for an improved blood stain ensued – In 1888, Chenzinsky discovered a stain
composed of cationic dye methylene blue and anionic eosin.
• Malachowski modified that stain with a markedly improved color range
and depth
• Regrettably, when he published his results in Berlin in 1891, he did not give the
necessary details as to how his method might be reproduced.
• 3 weeks later the young Russian protozoologist Romanowsky published similar
findings in an article so celebrated that all future stains of analogous
composition were named as Romanowsky-type stains
[Romanowsky noted that a neglected, moldy methylene blue solution was more effective
in producing the desired red plasmodial chromatin bodies than was a fresh solution]

15. Thionine

16. EOSIN-Y EOSIN-B

18. Romanowsky staining
It includes:
• May-Grunwald –Geimsa stain
• Wright’s stain
• Leishman’s stain
• Jenner’s stain
• Field’s stain.

19. • Wright’s Stain
• Components
• Stock solution:
• Wright’s stain 0.25gm to 0.5 gm
• Absolute acetone free methanol 100 ml
• Buffer (pH 6.8):
• KH2PO4 6.63 g
• Na2HPO4 2.56 g
• Distilled water 1,000 ml
• May–Grunwald Stain – 0.3 gm in 100 ml methanol
• Jenner – 0.5 gm in 100 ml methanol ( no over night incubation)
• Leishman’s Stain – 0.2 gm in 100 ml methanol
• Giemsa – 1 gm in 100 ml methanol

20. After drying
the smear
After drying the smear is
flooded with Wright stain
solution for 3 to 5 minutes,
Staining does not occur at
this stage
Buffer water of pH 6.8 is added
on the smear already
containing the stain solution.
(No washing)
Mixing of stain solution and
buffer water is done by blowing
in a figure of ‘8’ manner and kept
for 10 to 12 minutes for the
actual staining
Thoroughly washed under
slow running tap water.
Air drying and
examined under
light microscope
using a
temporary
mounting media
Wright staining followed in our Lab

21. Fixation is done
by immersing in
a jar of
methanol for 5–
10 min
Fixed smears are transferred to
a staining jar containing
May–Grunwald stain freshly
diluted with an equal volume
of buffered water and allowed
to stain for about 15 min.
Transferred
to a jar containing Giemsa’s
stain freshly diluted with 9
volumes of buffered water
without washing
After staining for 10–15 min,
slides are transferred to a jar
containing buffered water, pH
6.8
Rapid washing in 3 to 4
changes of water, and allow to
stand undisturbed
in water for a 2–5 minutes
Air drying and
examined under
light microscope
using a
temporary
mounting media
May–Grunwald–Giemsa Stain

22. Colour responses of blood cells to Romanowsky staining
• Cellular component
• Nuclei
• Nucleoli
• Chromatin
• Cytoplasm
• Erythroblast
• Erythrocyte
• Reticulocyte
Colour
Purple
Light blue
Dark blue
Dark pink
Grey–blue

23. Colour
Blue
Pink
Grey–blue
Pink
Pink/orange
Blue
Blue
Cytoplasm
• Lymphocyte
• Metamyelocyte
• Monocyte
• Myelocyte
• Neutrophil
• Promyelocyte
• Basophil

24. Granules
• Promyelocyte(primary granules) Red or purple
• Basophil
• Eosinophil
• Neutrophil
• Toxic granules
• Platelet
Purple black
Red–orange
Purple
Dark blue
Purple

25. Other inclusions
• Auer body
• Cabot ring
• Howell-Jolly body
• Döhle body
Purple
Purple
Purple
Light blue

26. 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

27. Factors giving rise to faulty staining
• Appearances
❖Too blue
Causes
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

28. Too pink Incorrect proportion of azure
B-eosin Y
• Impure dyes
• Buffer pH too low
• Excessive washing in buffer
solution

29. • Pale staining • Old staining solution
• Overused staining solution
• Incorrect preparation of
stock
• Impure dyes, especially
azure A and/or C
• High ambient temperature

30. •Neutrophil granules
not stained
•Neutrophil granules Dark
Blue/black (pseudo-toxic)
Insufficient azure B
Excess azure B
• Other stain anomalies Various
contaminating
dyes and metal salts

31. • Stain deposit
on film
Stain solution left in
uncovered jar
Stain solution not filtered
Inadequate fixation or
prolonged storage before
fixation
• Blood collected into heparin
as anticoagulant
• Blue background

32. Precautions
• PBS should be fixed as soon as possible after drying; background of
dried plasma may stain pale blue.
• Smear should not come in contact with water before fixation is
complete.
• Methanol must be stored in a bottle with a tightly fitting stopper
and not left exposed to the atmosphere, especially in humid
climates.

33. Examination of PBS

34. • Patient identification should be checked and confirmed and the microscope
slide matched with the corresponding CBC report.
• The film should be examined macroscopically to confirm adequate spreading
and to look for any unusual spreading or staining characteristics.

35. • PBS should be compared with the CBC report and a judgement
made as to whether the WBC, hemoglobin concentration (Hb), MCV
and platelet count are consistent with the smear.
(i) a poorly mixed or partly clotted or
hemolyzed specimen;
(ii) a specimen that is too small so that the
instrument has aspirated an inadequate volume;
(iii) the blood film and CBC being derived from
different blood specimens.

36. • On placing a pbs under the microscope, the first decision to be made is whether
or not it is suitable for further examination. Spreading, fixation and staining
must be satisfactory and there should be no artefactual changes produced by
excess EDTA or prolonged storage, e.g., spherocytosis, crenation or echinocytic
changes in red cells, degeneration of neutrophils and lobulation of some
lymphocyte nuclei
• RBCs are just touching but not overlapping.
• WBCs should be distributed regularly without undue concentration along the edges or
in the tail (can be seen in a very thin smear)

37. Evaluation of PBS• 1. RBC
•
•
•
•
•
Size
Shape
Color
Arrangement
Inclusions
• 2. WBC
• Total counts
• Differential counts
• Abnormal WBC
• 3. Platelets
• Counts
• Abnormality
•4. Parasites

38. Red cell Morphology
• Terms used to describe a normal red cell morphology
• normocytic, which means that the cells are of normal size
• normochromic, which means that the cells have the normal concentration
of hemoglobin (i.e., when central pallor occupies ≤ one‐third of the red cell
diameter portion)

39. • Micro and Macrocytic red cells

40. • Anisocytosis
• Anisocytosis is an increase in the variability of erythrocyte size beyond that which is
observed in a normal healthy subject.

41. • Dimorphic picture
• Indicates the presence of two distinct populations of red cells.
• Often applied when there is one population of hypochromic, microcytic cells and another
population of normochromic cells, either normocytic or macrocytic.

42. Chromasia
1. Hypochromia
➢ When central pallor is more than 1/3 of the red cell diameter
➢ Usually associated with microcytosis
(question – 1. Name 2 conditions where this association is not seen?
(thal trait , microspherocytosis, liver disease)
2. What are leptocytes ?)
2. Hyperchromia
➢ The term ‘hyperchromia’ is rarely used in describing smears.
➢ Deep staining of the red cells with a lack of central pallor
➢ May be seen in two circumstances: first, in the presence of macrocytes and second,
when cells are abnormally rounded

43. Leptocytes
• Thin red cells with large
unstained central area.
• Seen in
– Severe iron deficiency
anemia
– Thaleasaemia

44. 3. Anisochromasia
• Anisochromasia describes an increased variability in the degree of staining or haemoglobinisation of the red
cell
• In practice, it usually means that there is a spectrum of staining from hypochromic to normochromic.
• Indicates a changing situation, such as iron deficiency developing or responding to treatment or anaemia of
chronic disease developing or regressing.

45. 4. Polychromasia
• Polychromasia or polychromatophilia describes red cells that are pinkish-blue as a consequence of uptake
both of eosin, by hemoglobin, and of basic dyes, by residual nucleic acids.
• considerably larger than mature erythrocytes and, as a consequence of a reduced hemoglobin
concentration, are less dense
• have an irregular, multilobate surface

46. • Q. What is the normal percentage of polychromatophils in PBS?

47. • Variation of shape/Poikilocytosis
• Elliptocytes
• Spherocytes
• Target cells
• Spiculated cells
➢ Keratocytes
➢ Schistocytes
➢ Acanthocytes
➢ Echinocytes

48. Elliptocytes/ ovalocytes
• Both are oval in shape.
• A cell with a long axis
more than twice its short
axis should be designated
an elliptocyte
• while a cell with the long
axis less than twice its
short axis is designated
an ovalocyte

49. 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

50. Target cells
• Target cells have an area of increased staining, which appears in the
middle of the area of central pallor
• Formed as a consequence of a redundant membrane in relation to the
volume of the cytoplasm.
• They may also be thinner than normal cells.
• In vivo they are bell-shaped as demonstrated on scanning electron
microscopy.
• Flatten on spreading to form the characteristic target cell seen on light
microscopy

51. Q. What is the fancy name for Target cells ?

52. Echinocytes
• Also called crenated cells
• Erythrocytes that have lost their disc
shape and are covered with 10–30
short blunt spicules of fairly regular
form
• 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)

53. • Q – What is the usual mechanism of echinocyte formation in an
otherwise normal blood sample?

54. • Lysolecithin formation in plasma
• Depletion of ATP

55. Acanthocytes
• Acanthocytes are cells of
approximately spherical shape
bearing between 2 to 20 spicules
that are of unequal length and
distributed irregularly over the red
cell surface.
• Probably results from a
preferential expansion of the
outer leaflet of the lipid bilayer.
• Seen in Abnormal phospholipid metabolism
• Abetalipoproteinemia
• Inherited abnormalities of red cell membrane protein
• Splenectomy

56. Keratocytes
• Have pairs of spicules;
usually single pair.
• Sometimes termed as
Bite cell or helmet cell
• Seen in
– Mechanical damage
– Removal of Heinz body by
pitting action of spleen.

57. Schistocytes
• These are fragmaented
erythrocytes.
• According to ICSH these
are red cell fragments with
sharp angles and straight
borders, small crescents,
helmet cells, keratocytes
and microspherocytes
• Quantification is per 1000
erythrocytes, with more
than 1% schistocytes being
regarded as significant

58. 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.

59. 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

60. Teardropcells
• One side of cells is
tapered and other is
blunt.
• Usually found when
there is marrow failure
or fibrosis

62. Red blood cell inclusions
Name of Inclusion
• Howell-Jolly body
• Basophilic stippling
• Pappenheimer body
• Heinz body(supravital only)
• Crystals
• Cabot rings
• Nucleus
• Content
• DNA
• RNA
• Iron
• Denatured hemoglobin
• Hemoglobin-C
• Mitotic spindle
remnants
• DNA

63. 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
– Pyrimidine-5’-nucleotidase def.

64. 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
– Abnormal erythropoiesis • Howell-Jolly Bodies

65. 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

66. 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

67. Cabot Rings
• These are Ring shaped or 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,

68. Malarial stippling
• Fine granules of
plasmodium vivax
• On wright stain these
are fine , purplish red
• Red cells are larger than
normal

69. Rouleaux Formation
• Alignment of red cells one
upon another so that they
resemble stacks of coins.
• Occurs inParaproteinemia
( monoclonal
gammopathy)
• Elevated plasma
fibrinogen or globulin
level

70. Agglutination
• It is more irregular and
round clumping than
linear rouleaux
• Seen with cold
agglutinin
• Anti RBC antibody
• Autoimmune hemolytic
anemia
• Macroglobulinemia

71. • Discrepancies in Red cell parameters –
Falsely high RBC –
a) Numerous large platelets
b) Hyperlipidaemia
c) Cryoglobulinemia/
Cryofibrinogenemia
Falsely Low RBC –
a) Cold agglutinins (Rarely warm
autoantibodies)
b) EDTA‐dependent pan-
agglutination
c) In vitro red cell lysis due to
mishandling of Extreme
microcytosis or fragmentation
Falsely high MCV –
a) Storage of blood at room
temperature
b) Cold agglutinins and EDTA‐
dependent pan-agglutinins
c) very high WBC count
d) Hyperosmolar states (e.g.
hypernatraemia, )
e) Excess K2EDTA
Falsely Low MCV –
a) Increase in ambient
temperature
b) Hypo‐osmolar states (e.g.
hyponatraemia
c) Repeated mixing of sample
leading to increased
oxygenation
Falsely high RBC
a) Poorly mixed specimen
b) High WBC
c) Hyperlipidaemia,
[endogenous or due to
parenteral nutrition]
d) hypergammaglobuline
mia

72. WBC MORPHOLOGY

73. ▪ 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

74. • 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 verticallyinoppositetothe previousdirection.
• Procedure is repeated until 100 WBCS have been counted (zig zag motion)
Scanning technique for WBC differential count and
morphologic evaluation

75. Manual differential counts
• These counts are done in the same area as
WBC and platelet estimates with the red cells
barely touching.
• Count 100 WBCs under 40x/100x including
all cell lines from immature to mature.
✓Reporting results
• Approximate absolute number of cells/µl = %
of cell type in differential x white cell count

76. •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 we have actually counted 110 cells among which
100 cells are WBCs.
That means in 1 cell there will 100/110 WBC.
The corrected white count is (100/110) x 5000 = 4545.50

77. Leukocytes Normally Present in Blood
Neutrophils ( polymorphonuclear leucocytes)
Band form
Eosinophils
Basophils
Lymphocytes
Monocytes

78. 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
• small percent has four lobes
and occasionally five lobes.

79. 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)

80. • Left-shift: non-
segmented neutrophil >
5%
– Increased
bands Means acute
infection, usually
bacterial

81. Myeloblast (0%)
Promyelocyte (0%)
Myelocyte (<0.5%)
Metamyelocyte (<1%)
Band form (5 to 8%)
Bilobed neutrophils (30-35%)
Trilobed neutrophils (40-50%)
Tetra lobed neutrophils (15-20%)
Penta lobed (<3%)
Hexalobed (0%)
Left Shift
Right Shift

82. Granules
• Toxic granulation-
increase in staining
density and number of
granules
• Seen with Bacterial
infections and other
inflammation
• Administration of G-CSF
• Anaplastic anemia

83. • Hypogranular and
agranular neutophils
poorly stained
• seen in Myelodysplastic
syndrome

84. Alder–Reilly anomaly
• Granules are large,
– discrete,
– stain deep red
– may obscure the
nucleus
– Neutrophil function
is Normal

85. Chédiak-Higashi Syndrome
• Granules are
– also seen in other
leukocytes like
lymphocytes
– Giant
– Scanty azurophilic
– functional defect occur

86. 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

87. May–Hegglin anomaly
• inclusions occur in all
types of leucocytes
except lymphocytes.
• contain small basophilic
cytoplasmic granules

88. Vacuoles in neutrophils
• In Fresh blood smear
• vacuoles seen in
– severe sepsis
– as an artifact with
prolonged standing

89. 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

90. 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.

91. 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

92. 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

93. Exogenous neutrophil inclusions

94. EOSINOPHILS
• 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

95. • Eosinopenia- seen with prolonged steroid administration.
• Eosinophilia-
asthma
Allergic
conditions
• severe eosinophilia-
- parasitic infection
– reactive eosinophilia
– Eosinophilic leukaemia
– Idiopathic hypereosinophilic syndrome
– T-cell lymphoma, B-cell lymphoma
and acute lymphoblastic leukaemia.

96. BASOPHILS• 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

97. MONOCYTES
• 2-10% of total wbc count
• Size- largest circulating leucocyte, 15–18µm
in diameter
• Cytoplasm- grey blue, may be vacuolated
• Nucleus- large , curved , horse shoe
shape/kidney/ fetal shaped
• No segmentation occur
• Chromatin- fine evenly distributed
• Increase in chronic infections and
inflammatory conditions such as
– TuberculosisandCrohn’sdisease,
– Chronic myeloid leukaemias
– Acute leukaemias with a
monocytic component
– Infectious mononucleosis

98. 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

99. 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

100. Reactive lymphocytes
• Have slightly larger
nuclei with more
open chromatin
• Abundant cytoplasm
that may be
irregular.
• Seen in -infectious
mononucleosis
– viral infections

101. What are Downey cells
Type I: Small cells with minimum cytoplasm, indented
nucleus/irregular nuclear membrane, and condensed chromatin.
▪Type II: Larger cells with abundant cytoplasm; the lymphocyte
cytoplasm seems to hug the red cells. Type II is the most common
type of Downey cell.
▪Type III: Cells with large moderate basophilic cytoplasm and nucleus
with coarse chromatin. Nucleoli are apparent

102. WBC precursors

104. Leucoerythroblastic blood films
• A blood film is referred to as leucoerythroblastic if it contains NRBC
and granulocyte precursors.

105. • Discrepancies in WBC parameters –
Falsely high TLC–
a) Presence of NRBCs
b) Liver disease
c) Cold agglutinins
d) Numerous giant platelets or megakaryocyte
fragments
e) Platelet aggregates
f) Cryoglobulinemia/ Cryofibrinogenemia
g) Malarial parasites
h) Systemic mycoses
i) abnormal haemoglobins (in some counters)
Falsely Low RBC –
a) Cell lysis caused when blood is more than 3
days old
b) Storage at room temperature for 24 hours or
more
c) Storage at 4°C for more than 24 hours
(Horiba)
d) Leucocyte or leucocyte and platelet
aggregation due to an antibody or to
alteration of the cell membrane or to the
presence of neoplastic cells
e) In vitro red cell lysis due to mishandling of
Extreme microcytosis or fragmentation

106. Platelates
• Size -1-3µm
• Normal count - 280 ±130×109/µl
• Non nucleated cells derived from cytoplasmic
fragments of Megakaryocytes
• Has purple red granules.
• Liliac color

108. 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

109. Thrombocytosis
• Reactive thrombocytosis
✓Post infection
✓Inflammation
✓Juvenile rheumatoid arthritis
✓Collagen vascular disease
• Essential thrombocythemia

110. Platelet morphology: Giant platelets
• Platelates seems to be
size of rbcs.
• Seen in
– May –Hegglin anomly
– Bernard Soulier
syndrome
– Alport syndrome
– Storage pool syndrome

111. Platelet clumping

112. q. How to to manual platelet counting?

113. • 10 oil immersion fields counting
• Average
• Multiply by 15000.

114. • Discrepancies in Platelet parameters –
Falsely Low platelet count–
a) Partial clotting of specimen
b) Activation of platelets during venepuncture with
consequent aggregation
c) Activation of platelets during cardiopulmonary bypass
d) EDTA‐induced platelet aggregation (more common in viral
infections, particularly hepatitis A, CMV and inluenza A)
e) EDTA‐induced platelet degranulation and swelling
f) Lipiodol‐induced platelet clumping following
chemoembolisation
g) Platelet satellitism
h) Platelet phagocytosis by neutrophils and monocytes
i) Storage of blood at 4°C for more than 24 hours
j) Giant platelets falling above upper threshold for
platelet count
k) Heparin addition to blood sample in patient with
antibodies to heparin‐platelet factor 4
Falsely High platelet count –
a) Microcytic red cells or red cell fragments failing
below upper threshold for the platelet count
b) Presence of Microspherocytes (in hereditary
spherocytosis, burns)
c) Inadvertent heating of blood sample
d) White cell fragments counted as platelets
(fragments of leukaemic blast cells, hairy cells
or lymphoma cells)
e) Haemoglobin H disease
f) Hypertriglyceridaemia or hyperlipidaemia

115. 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

116. 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

117. 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

118. Plasmodium falciparum
Erythrocytes throughout
this series are not
enlarged or distorted.
• Early trophozoites
• Accole form
• Crescent (‘banana-
shaped’) gametocyte

119. double chromatin dot

120. 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

121. Ealy trophozoit in the form of thick ring with large chromatin
dot

122. Leishmaniasis (Leishman–Donovan
bodies)

123. African trypanosomiasis
(Trypanosoma brucei gambiense)
American trypanosomiasis
( T. cruzi);

124. microfilaria

125. Thank
you