Hem atol ogy 1 WH GB |1
Peripheral Blood Smear
Midterms
Mr. Normel Adarve
o Coverslip method
o Coverslip and slide method
o Wedge method
o Automated
• Mini prep
• Centrifugal/Spinner
COVERSLIP METHOD
Advantages:
o Ideal for WBC observation
▪ For WBC differential
count
o Even WBC distribution
Disadvantages:
o Not ideal for RBC
observation
o Difficult to master
o Difficult to handle
o Difficult to label
o Easily breaks
WEDGE METHOD
• aka: Double-slide technique
o Uses 2 glass slides
• aka: Slide spreader technique
Note: Whenever you observe blood cells,
always locate the thin area of the smear.
Advantages:
o Easy to make
o Abnormal/large cells are
easily found at the feathered-
edge
Note: If there is an elevated WBC count,
check the feathered-edge for presence of
plus cells/immature cells.
❖ Leukemia
o Presence of immature cells
at the feathery edge
▪ immature granulocytes
▪ immature monocytes
▪ immature lymphocytes
❖ Leukemoid reaction
o Absence of immature cells at
the feathery-edge
Disadvantages:
o Normal WBCs except
lymphocyte are pushed to
the feathered-edges.
▪ Only the lymphocytes
will remain on the thin
area.
• False ↑
lymphocyte
differential
count
❖ False ↑ lymphocyte diff. count
o aka: relative lymphocytosis
o may reflect
pseudoneutropenia
▪ False ↓ neutrophil
differential count
• <37% diff.
count
▪ Neutrophils are
pushed to the
feathered-edges
Note: If the lymphocyte differential count
reaches >50%, suspect false (↑) increase.
What to do?
1. Do another wedge smear
2. Do another manual differential count
3. Compare both differential counts
Traumatic cells
Note: Pushing of blood cells to the edges
might introduce trauma to the cells.
1. Basket cells
➢ Cytoplasmic fragments of
granulocytes
Note: Too much basket cells on PBS will
result to false ↑ of platelet count
➢ Due to chemotherapy
 Hem atol ogy 1 WH GB |2

o Fast spreading
➢ Thicker smear
o Slow spreading
➢ Thinner smear
• Angle
❖ 30° – 40° angle of spreading
o Increase angle
➢ Anemia
• Hct% < Ref.
value
• Non-viscous
blood
•
o Decrease angle
2. Smudge cells ➢ Polycythemia
➢ Disintegrated nucleus of • Hct% > Ref.
lymphocytes value
Note: Too much smudge cells on PBS • Viscous blood
represent chronic lymphoblastic ➢ 20° - 25° angle
leukemia

• Drop of blood (diameter)

❖ 2 mm – 3 mm
• Speed
❖ Smooth spreading
 Hem atol ogy 1 WH GB |3

Characteristics of a well-made smear

1. Occupies at least 1/2 - 2/3 of slide
2. Occupies the entire width of slide
3. Has a tongue-shaped appearance
4. Must have a fine feathered-edge
5. No holes or spaces in the smear
6. Must have three portions:
a. Thick portion
b. Thin portion
c. Feathered-edge portion

Source: Atlas of Pediatric Peripheral Blood Smears (Hays & Jamieson)

Feathered-edge thin thick

SEMI-AUTOMATED/ AUTOMATED
Water artifacts/water contamination
Advantages:
1. Blowing the smear with mouth
o Even cell distribution
o Low number of traumatic Note: Air dry the smear to avoid
cells contamination
Note: After smearing, dry the slide right 2. Very high fixative (methanol)
away. concentration
When asked: Note: 10% Methanol is the ideal
fixative
What poikilocyte will you see under the
microscope if you delay the drying of blood PBS STAINING
smear? Echinocytes
1. Fixation
➢ aka: Crenated RBCs o Fixative
➢ aka: Sea urchin cells ▪ 10% methanol
➢ aka: Burr cells o Fix and preserve the cells
2. Buffer Solution
When asked:
o Maintain the pH of stain
What disease is associated with presence ▪ pH=6.8
of Burr cells? Renal insufficiency ❖ Very alkaline stain
➢ “Too blue” appearance
➢ Pyruvate kinase deficiency ❖ Very acidic stain
➢ Uremia ➢ “Too red” appearance
o Leads to anemia 3. Staining
o Kidney problem
▪ ↑ Blood BUN a. Eosin Y
b. Methylene blue
 Hem atol ogy 1 WH GB |4

Too blue Too red ➢ Small central pallor

❖ Hypochromic RBC
➢ Enlarged central pallor
Too thick smear Too thin smear
➢ Loss of hemoglobin
▪ (ATIS) Anemia
Too alkaline stain Too acidic stain a) Anemia of chronic disease
b) Thalassemia
Inadequate washing Excessive washing c) Iron deficiency anemia
d) Sideroblastic anemia
Use of heparinized Note: Hyperchromic RBCs don’t represent
blood high level of hemoglobin
Hyperproteinemia ❖ Poikilocytes with central pallor
disappearance
1. Hereditary spherocytosis
2. Sickle cell
Thick smear Thin smear ▪ Hgb SS
Fast spreading Slow spreading ▪ Hgb SC
▪ Hgb SD
High angle of Low angle of
spreading spreading Things to check in RBCs
A.) Size
Too large amount of Too small amount of
blood used (>3mm) blood used (<2mm) Variation
1. Slight anisocytosis
2. Moderate anisocytosis
3. Severe anisocytosis
➢ Possible anemia
❖ Normal RBC
➢ Biconcave disc shape
➢ No inclusions
➢ Central pallor ▪ Microcytic
o <3 μm diameter ➢ RBC < lymphocyte
o 1/3 of RBC size ➢ (ATIS) Anemia
▪ Normocytic
Shape ➢ RBC = lymphocyte
▪ Macrocytic
❖ Poikilocytosis
➢ RBC > lymphocyte
o Variation in RBC shape
➢ Megaloblastic anemia
Size ➢ Acquired aplastic anemia
➢ Newborns
❖ Anisocytosis
o Variation in RBC size Note: Lymphocytes are the reference cells
for checking RBC size
Central pallor
Note: High number of lymphocytes,
❖ Hyperchromic RBC reticulocytes, and presence of nucleated
➢ Disappearance of central pallor RBCs are normally found in newborns.
o Megaloblastic anemia
 Hem atol ogy 1 WH GB |5

Note: Nucleated RBCs are sometimes

Causes of megaloblastic anemia
1. Vitamin B12 deficiency
▪ Pernicious anemia
▪ Diphyllobothrium latum
(Tapeworm)
2. Follate deficiency
▪ Chronic alcoholism
Note: Whenever you report a microcytic-
hypochromic RBCs peripheral blood smear
report, check for the mean cell volume
(MCV). There should be no contradiction
between the two.
B.) Shape
Grading Scale for RBC Morphology
(poikilocytosis)
Normal 5%
mistaken by machines as lymphocytes
When asked:
What specific WBC may falsely elevate
(False ↑) when you see a lot of nRBCs.
Answer: Lymphocytes
▪ Relative lymphocytosis
Note: If you see >5 nRBCs, correct the
WBC count given by the machine.
CORRECTED WBC COUNT FORMULA:
𝑊𝐵𝐶 𝑐𝑡. 𝑥 100
;
𝑛𝑅𝐵𝐶 𝑜𝑏𝑠𝑒𝑟𝑣𝑒𝑑+100
C.) RBC distribution
D.) Hgb concentration
Diseases with normocytic-normochromic
blood picture but with low Hgb and Hct

Slight 5% - 10% 1. Bone marrow-associated anemia

▪ Myelophthisic anemia
1+ 10% - 25%
2. Kidney-associated anemia
2+ 25% - 50%
When asked:
3+ 50% - 75%
What is the expected blood picture of
4+ >75%
patients suffering with leukemia?
Note: 4+ (severe poikilocytosis) is common
on patients with β-Thalassemia major Normocytic-normochromic
E.) Presence of inclusions

Grading scale for central pallor size

Slight 1%
1+ 3% 1/2 RBC
diameter
2+ 5% 2/3 RBC
diameter
3+ 10% 3/4 RBC
diameter
4+ >11% Only the thin rim
of Hgb is left
 Hem atol ogy 1 WH GB |6

RETICULOCYTES ❖ Supravital stains

➢ To see inclusions of retics
1. New Methylene Blue (NMB)
2. Brilliant Cresyl Blue (BCB)
Inclusions that can’t be visualized by
Wright’s stain:
o Reticulocytes
o Hgb H
o Heinz bodies

Normal retic count in adults: (per 1000 RBCs)

0.5 – 1.5% (Adarve)
0.5 – 2.5% (Rodak)
Normal retic count in babies: (per 1000 RBCs)
2 – 6% (Adarve)
Factors that increase reticulocyte count:
1. Hypoxia
▪ Difficulty of breathing
▪ Triggers EPO secretion in
kidneys
➢ Bone marrow will
release reticulocytes
prematurely.
Note: Normal reticulocytes are released
from the marrow on the 3rd day of
maturation.
Severe hypoxia indirectly triggers immature
reticulocytes to be released on the 2 nd day
of maturation.
❖ Shift cells
➢ Immature reticulocytes
➢ Due to severe hypoxia
Causes of hypoxia
2. Emphysema
3. Lung problems
4. Heart problems