WEDGE BLOOD SMEAR

 Specimen : EDTA blood within 2 to 3

hours & collected to the mark on tube.
 Not's : May change RBCs morphology
such as Spiculated (crenated) cells if :
1. Excessive amount of anticoagulant to
specimen
2. Old blood - long standing.
3. Warm environment (room temperature)
may hasten changes.
 Procedure
• placing a drop of blood from mixed
sample on a clean glass slide.
• Spreader slide using another clean glass
slide at 30-40 degree angle.
• Control thickness of the smear by
changing the angle of spreader slide
• Allow the blood film to air-dry
completely before staining. (Do not blow
to dry. The moisture from your breath
will cause RBC artifact).
high HCT

small angle

low HCT

large angle
CHARACTERISTICS OF A GOOD SMEAR
1. Thick at one end, thinning out to a smooth
rounded feather edge.
2. Should occupy 2/3 of the total slide area.
3. Should not touch any edge of the slide.
4. Should be margin free, except for point of
application.
tail body head
MORPHOLOGIC CHANGES DUE TO AREA OF
SMEAR

• Thin area: Spherocytes which are really

"spheroidocytes" or flattened red cells.
True spherocytes will be found in other
(Good) areas of smear.

• Thick area: Rouleaux, which is normal in

such areas. Confirm by examining thin
areas. If true rouleaux, two-three RBC's
will stick together in a "stack of coins"
fashion.
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
8. Cellular degenerative changes: delay in fixing, inadequate
fixing time or methanol contaminated with water.
Biologic causes of a poor smear

1. Cold agglutinin: RBCs will clump

together. Warm the blood at 37° C for 5
minutes, and then remake the smear.
2. Lipemia: holes will appear in the smear.
There is nothing you can do to correct
this.
3. Rouleaux: RBC’s will form into stacks
resembling coins. There is nothing you
can do to correct this
Romanowsky staining
• Leishman's stain : a polychromatic stain
• Methanol : fixes cells to slide
• methylene blue stains RNA,DNA
 blue-grey color
• Eosin stains hemoglobin, eosin granules
 orange-red color
• pH value of phosphate buffer is very important
 Procedure
• Thin smear are air dried.
• Flood the smear with stain.
• Stain for 1-5 min. Experience will indicate
the optimum time.
• Add an equal amount of buffer solution and
mix the stain by blowing an eddy in the
fluid.
• Leave the mixture on the slide for 10-15
min.
• Wash off by running water directly to the
centre of the slide to prevent a residue of
precipitated stain.
• Stand slide on end, and let dry in air.
Too acidic Suitable Too basic
CAUSES & CORRECTION

 Too Acid Stain:

1. insufficient staining time
2. prolonged buffering or washing
3. old stain
 Correction:
1) lengthen staining time
2) check stain and buffer pH
3) shorten buffering or wash time
 Too Alkaline Stain:
1. thick blood smear
2. prolonged staining
3. insufficient washing
4. alkaline pH of stain components

 Correction :
1. check pH
2. shorten stain time
3. prolong buffering time
 Principle
 White Blood Cells
1. Check for even distribution and estimate
the number present (also, look for any
gross abnormalities present on the
smear).
2. Perform the differential count.
3. Examine for morphologic abnormalities.
 Red Blood Cells, Examine for :
1. Size and shape.
2. Relative hemoglobin content.
3. Polychromatophilia.
4. Inclusions.
5. Rouleaux formation or agglutination
 Platelets.
1. Estimate number present.
2. Examine for morphologic abnormalities.
 Observations Under 10X

1. Check to see if there are good counting

areas available free of ragged edges and cell
clumps.
2. Check the WBC distribution over the smear.
3. Check that the slide is properly stained.
4. Check for the presence of large platelets,
platelet clumps, and fibrin strands.
 Observing direction:

Observe one field and record the number of WBC according to the
different type then turn to another field in the snake-liked direction

*avoid repeat or miss some cells

WBC estimation Under 40X
• Using the × 40 high dry with no oil.
• Choose a portion of the peripheral smear
where there is only slight overlapping of the
RBCs.
• Count 10 fields, take the total number of
white cells and divide by 10.
• To do a WBC estimate by taking the average
number of white cells and multiplying by
2000.
Platelet estimation Under 100X
1. Use the oil immersion lens estimate the number of
platelets per field.
2. Look at 5-6 fields and take an average.
3. Multiply the average by 20,000.
4. Note any macroplatelets.

 Platelets per oil immersion field (OIF)

1) <8 platelets/OIF = decreased
2) 8 to 20 platelets/OIF = adequate
3) >20 platelets/OIF = increased
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.
Determine a quantitative scale

1
Reference values vary depending on age

Total WBC x 103 /µL 14 yr

Cell Type 4.5-11.0

Neutrophils % 50-65
Lymphocyte % 30-40
Monocyte % 0-10
Eosinophil % 0-4
Basophil % 0-1
Normal blood smear
Stab neurophile

• Diameter:12-16
• Cytoplasm : pink
• Granules: primary
secondary
• Nucleus: dark purple blue
• dense chromatin
Segmented neurophile

• Diameter: 12-16
• Cytoplasm : pink
• Granules: primary
secondary
• Nucleus: dark purple blue
• dense chromatin
• 2-5 lobes
SEGMENTED NEUTROPHIL
Basophils
• Diameter: 14-16
• Cytoplasm : full of granules
• Granules: large refractile,
orange-red
• Nucleus: blue
• dense chromatin
• 2 lobes like a pair of glass
EOSINOPHIL
Eosinophils
• Diameter: 14-16
• Cytoplasm : pink
• Granules: dark blue –black
obscure nucleus
• Nucleus: blue
BASOPHIL
Lymphocyte

• Diameter: small 7-9

large 12-16
• Cytoplasm: medium blue
• Granules: small agranular
large a few
• Nucleus: dark blue \round
dense chromatin
LYMPHOCYTE
Monocytes

• Diameter: 14-20
• Cytoplasm : grey blue
• Granules: dust-like lilac
color granules
• Nucleus: blue
• large irregularly shaped and
folded
MONOCYTE
LEFT-SHIFT AND RIGHT-SHIFT OF
NEUTROPHIL:

• Left-shift: non-segmented neutrophil >

5%
(Increased bands Means acute infection,
usually bacterial).

• Right-shift: hypersegmented neutrophil

>3% (Increased
hypersegmented neutrophile )
• Leukocytosis, a WBC above 10,000, is usually due to an
increase in one of the five types of white blood cells

 Neutrophilic leukocytosis neutrophilia

 Lymphocytic leukocytosis lymphocytosis
 Eosinophilic leukocytosis eosinophilia
 Monocytic leukocytosis monocytosis
 Basophilic leukocytosis basophilia