Hematology: Peripheral Blood Smear

Method
Coversli
p

Advantage
Bone Marrow preparation

Disadvantage
Difficult to master and to label
To thin which can break easily
Slip has to mount to the side

Good for WBC

even cell distribution

Wedge

Easy to label
Immature WBC (abnormal cells) can be
easily observed at the edges of the
smear

Glass slide & Coverslip

-

Beacons Method

Remember: Wedge Method/Double

slide/Slide spreader

used to transfer blood:

capillary tube
applicator sticks
apply blood: 1 cm from the edge of
the slide
drop of blood: 2-3 mm diameter
1.2 mm diameter of
capillary tube
spread immediately
angle: 30-45 (best angle: 30)
ideal spreading: smooth
ideal EDTA concentration: 1.25-1.75
nm/ml
K2EDTA best used to prevent crenation
terminate smear: half-inch at the end
observe all cells at the thin area
routine pH: 6.8

Types of Wedge Method:

a. Push-type
b. Pull-type
Criteria for good smear:

- 2/3 of the slide

tongue shape
occupy the whole width
feather edge
lateral edges are visible
film is smooth
no holes or spaces
transition from thick (near the heel)thin-feathered
rainbow appearance near the feather
edge under light

Larger cells/Artifacts go to the edge of the

smear which can contribute to relative
lymphocytosis
Pushing can introduce trauma to cells
(Basket cells, Smudge cells)

Lymphocytosis
- increased lymphocytes viral
- normal: 10-47%
a. Absolute/True Lymphocytosis
b. Relative/False Lymphocytosis
- false increase
- lymphocytes is about 50%
- large cells are pushed at the edge of
the smear
Different Poikilocytes:
Smudge cells
-

Nuclear remnants/fragments of
lymphocyte
looks like a finger

Basket cells
-

Cytoplasmic remnants/fragments of
granulocyte
looks like a net

Echinocyte
- no pathologic relevance
- diagnosis: equidistant, regular-sized
pointed spicules

Cells mistaken as Echinocytes:

o Burr Cells
- has pathologic relevance
- diagnosis: irregularly spaced,
irregular-sized blunt spicules
- caused by: increase blood urea
nitrogen
due to Kidney
problem/Renal
insufficiency
o
-

Acanthocyte
has pathologic relevance
diagnosis: irregularly spaced,
irregular-sized pointed spicules

JOsephine Aluyen

Stains:
o
o
o
o

Leishman
May Grunwald
Jenner-Giemsa
Wright-Giemsa
- crispier granules
- most used
Romanowsky
- modified Wright-Giemsa
- polychrome stain
Methylene Blue
- stains acidic (nucleic acid)
property of the cell
Eosin Y
- acidic
- stains basic (cytoplasm)
property of the cell
stains the protein part,
hemoglobin, eosinophilic
granules
- pink stain

Staining Process
1. Fixation
10% volumes/volume Methanol
- prevent water contamination
- 40% can attract humidity in the air
2. Addition of buffer solution
pH 6.4-6.8 aged distilled water
pH 6.4 0.05 M NaPO4
pH 7.2 observe Malarian
pigments/stiplings
3. Staining
Methylene Blue
Eosin Y
4. Washing remove excess stain
5. Drying
- airdry
- tissue paper - can contaminate

Microscopic:
Neutrophil
granules

Eosinophil
granules

pinkish-tan; neutral color &

pH
pick up staining
characteristics of both stain
striking bright red-orange
attracts more Eosin Y

Basophil
granules

blue-black
graying-blue = improper
stain

Monocyte

has fine azuredast

blurry appearance in
cytoplasm
red-blue-grayish with
ground glass appearance
blue
has scanty cytoplasm

Lymphocyte
Platelets
RBC

lilac; pinkish-tan; purple

red (Wrights Giemsa)
fade central pallor
sharp central pallor =
hypochromic

Too Blue
Thick smear
Alkaline stain
Inadequate washing

Too Red
Thin smear
Acid stain
Excessive washing

Heparinized blood:
heparin + Wright stain
Hyperproteinemic
patients
-

protein stains blue

Ideal Smear
Macroscopic:

ideal: purple
blue: slides stained after 1 week or
longer

Examination: Differential Count

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Macroscopic
- bluer overall than normal
due to hyperproteinemia
Roleux Formation
(Pseudoagglutination)
- holes all over the film (lipids)
check hemoglobin
- grainy appearance
agglutinated/clump RBC
(destroyed)
caused by antibodies against
RBC antigen
accumulation of nucleated cells
false elevated: leukocytes
- blue specks out at the feathered edge
increase WBC & platelet

100x OIO
Morphology is evaluated
WBC is generally performed
100 WBC is counted & classified to
obtain percentages of types
Segmenters can be distinguished
from Bands
Inclusions are seen
RBC: Howell-Jolly
WBC: Dohle bodies
Reactive or Abnormal cells are seen
RBC Morphology
o

Microscopic
-

must be examined at the heel

10x objectives
overall film quality, color, distribution
feather edge should be checked quickly
for WBC distribution
presence of Rouleaux formation
scanned quickly for any large abnormal
cells
presence of fibrin strands
specimen must be rejected
snowplow effect
presence of more than four times
the number of cells per field at the
edges / feather compared with the
monolayer area of the film

Size
discoid with central pallor
Anicocytosis
- reference: Lymphocytes
Shape
Poikilocytosis
- depends on severity of abnormal
shape
- can be reported using grading chart
- Skistocyte
- Target cell/Codocyte
- Sickle Cell
Grading Chart
Norma
l
Slight
+1
+2

40x High-dry or 50x OIO

efficient for validating/verifying
instrument values when a total
microscopic assessment of the film is not
needed

+3
+4

WBC estimate
select an area in which the RBCs are
separated from one another with
minimal overlapping
average WBC per field x2000
(x40 magnification)
average WBC per field x3000
(x50 magnification)

5%
5-10%
1025%
2550%
5075%
>75%

(+4) Nucleated RBC

- only in bone marrow
- anemic patients
excreted immediately
due to hypoxia
- orthochromatophilic stage
- mistaken from lymphocytes
scanty cytoplasm
false elevated
count

Hemoglobin concentration
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Central Pallor: 1/3 of total diameter

Hypochromic has sharp edge
Hyperchromic
Normochromic

Grading Chart of central pallor

+1
+2
+3
+4

2/3

Thin
ring

Distribution
not overlapping because of the zeta
potential that repels the RBC from
each other

antibodies against RBC antigen

Auto Anti-I
Mycoplasma pneumoniae
Auto Anti-i
Epstein-Barr: infectious
mononucleosis
Auto Anti-P
Paroxysmal cold hemoglobinuria
viral infection
Incompatible blood
HDN

Grading Chart
+1
+2
+3

o
-

Polychromasia
representing Reticulocyte
blue-red color
stains to see inclusions
New Methylene Blue
Brilliant Cresil Blue (BSB)

Grading chart
Sligh
t
+1
+2
+3
+4

1%
3%
5%
10%
>11
%

Inclusion
no inclusions
not graded, reported as negative
and positive
Sickle Cell
Popenheimer bodies Sideroblastic
Howell-jolly
Basophilic stiplings

Burr cells
Ovalocyte
Target cell
Stomatocyte
Bizzare RBC
Poikilocytes
Dacrocytes
(tear drop
cell)
Acanthocytes
Schistocytes
Spherocytes
Polychromato
philia

3-4/hpf
510/hpf
>10/hp
f

+1
1-5/hpf

+2
610/hpf

+3
>10/hpf

310/hpf

1120/hpf

>20/hpf

Platelet
o
o

average platelet per field x20000

average platelet per field xRBC count/200
RBC

WBC

Distribution
o Roleaux formation
- high protein (except Albumin)
- high ESR reading
+1
+2
+3
o
-

34/hp
5-10
>10

Agglutinated RBC
discoid shape is disrupted
grainy appearance of the smear

50x or 100x magnification

increased Neutrophil bacterial
increased Lymphocyte viral
increased Eosinophil allery/parasitic

shift to the left

immature WBC

shift to the right

Hypersegmented
Neutrophil
1 neutro w/ 6
lobes
5 neutro w/ 5
lobes
100 neutro
(lobe count)
JOsephine Aluyen

Diseases:

Poikilocytosis
Myelogenous Leukmia
- increased Basket cells
- especially in patients undergoing
chemotherapy
all cells (especially leukemic
cells) are fragile

Chronic Lymphoblastic Leukemia

(CLL)
- increased Smudge cells

Pyruvate-Kinase Deficiency
- presence of Burr Cells

Acanthocytosis
Abetalipoproteinemia
- presence of Acanthocytes
- absence of B-Lipoprotein
important for lipid
transportation

Normocytic
Leukemia
Myelofibrosis
Diamond Blackfan
Fanconi Anemia
other bone marrow problem
Macrocytic, Hyperchromic
Megaloblastic Anemia
- hypersegmented neutrophil
H. Spherocytosis
Microcytic, Hypochromic
Anemia of chronic disease
Thalassemia
- Thalassemia Minor male with
Fe deficiency
Iron deficiency
Sideroblastic (dimorphism)
Reticulocytosis
Hemolytic Anemia

McLeod Phenotype
- low Kell Antigen
affects RBC shape
causes Neuroacanthocytosis

Hyperproteinemia
Multiple Myeloma
- plasma defect
Macroglobulinemia (Waldens
Strongs)
with Agglutination (Antibody against
RBC Antigen)
Auto-immune Hemolytic Anemia
Hemolytic Transfusion Reaction
Hemolytic Disease of the Fetus &
the Newborn
Cold Heme Agglutinin Disease

JOsephine Aluyen

Problems/Troubleshooting
Bullet-shaped smear
- not ideal
- has a narrow area for observation
- caused when blood was spread before
the drop was fully spread along the
spreader slide
High accumulation of leukocytes
along the side & tail edges of the
film
Thick smear
overlapping of RBC causing falsely
elevated count

Jagged edge or Letter-A formation

-

Holes are present

-

Polycythemia patients or Newborn

- parameter: hematocrit
- many RBC
- viscous blood
decrease angle of spreading = 25
Anemic patients
- low hematocrit
- less viscous
increase angle of spreading
Uneven pressure of spreading

smear is not tongue-shape

Hesitation to smear
presence of parallel lines

remedy:
o smooth spreading
o frosted slide may be used
caused by:
o not smooth/dirty spreading
o chipped off/rough edge
must be avoided

remedy: clean the slide

causes:
o dirt
o oil droplets
o hyperlipidemia/lipemic or chyle
formation
determine if present: when EDTA
was spun, plasma turns cloudy
CBC parameter checked:
hemogloblin (lipids target
hemoglobin)
false elevation: hemoglobin
count

Lymphocyte is >40%
-

create a new smear and compare diff.

count

Fast spreading with Increased angle

Thick smear
overlap of cells
false (+): cell count
also caused by large amount of
blood
Short smear
also caused by small amount of
blood
Slow spreading with Decreased angle
- accentuates poor leukocyte
distribution by pushing larger cells
(monocytes, granulocytes) to the
very end & sides
affect accuracy of differential count
Thin smear
also caused by small amount of
blood
decrease cell count
increase Smudge cells
stretched cells = elongated RBC
false (+):
ovalocyte =
Megaloblastic
Anemia
JOsephine Aluyen

Artifacts
Thick areas
EDTA-Anticoagulant samples

increased plasma causes leukocyte

shrinkage
remains 3-D with
less cytoplasm

Monocytes demonstrate immediate

alteration
Platelet clumping
due to platelet autoagglutinins
o Satellitism
- tendency for platelets to adhere to
neutrophils in EDTA
- false (+): Thrombocytopenia
Vacuolization
Relative lymphocytes develop
vacuolated (Swiss cheese) cytoplasm
& convoluted/clover leaf nuclei similar
to pathologic bast cells
Toxic neutrophils become
vacuolated (autophagocytosis)

Delay in making blood film

Necrobiotic (dead leukocytes)

mistaken as nucleated RBC
Clot formation
- cause trapping of WBC & platelets
decrease count
Crenation of RBC or Echinocyte or Sea
Urchin Cell
- abnormal shape
- causes of crenation:
o delay spreading of smear
o high EDTA concentration
o K3 EDTA
o delay drying of smear was used
allows time for the cell to be in
contact with the surface of the
slide
changes in blood pH which
affects the morphology

Thin areas
-

Slow air drying

drying or moisture of RBC artifact

hairy lymphocyte cytoplasm
shrinkage of leukocytes

Severe anemia, in which excessive plasma

cause poor drying
regardless of technique
Preparing blood film in a humid
environment
An inadequate fixation period
Water contamination of the fixative or
staining solution
Excess buffer in the stain solution

o
o
o
o

Water artifacts
-

due to humidity when specimen was

blown and not air-dry
long been nuisance
affect morphology
bubbles, moth-eaten, holes,
refractive/shiny blotches in RBC
heavily demarcated central
pallor

glass effect enhances leukocyte

spreading

JOsephine Aluyen