Blood Smear Preparation

BLOOD SMEAR
PREPARATION
PREPARED BY: DANROE ARVEE BALAGAT, RMT

METHODS OF
SMEAR PREPARATION
WEDGE SMEAR
METHOD
WEDGE SMEAR
• Aka 2 slide method
• Smear slide
• Spreader

WEDGE SMEAR
• Procedure:
• Prepare clean glass slide
• Place a drop of blood

• Diameter: 2-3 mm
• 1 cm away from one end
• Just below the frosted end

WEDGE SMEAR
• Procedure:
• Spreader slide in front

of the drop
• Angle: 300- 400

WEDGE SMEAR
• Procedure:
• Spreader slide back to edge of drop
• Blood spreads across the end

WEDGE SMEAR
• Procedure:
• Push the spreader slide forward
• Continuous movement
• All the way to the end of the smear slide
• Maintain 300-400 angle

WEDGE SMEAR
• Procedure:
• Drying of slide
• Labelling of slide

EHRLICH METHOD
EHRLICH METHOD
• Aka
• 2 coverslip method
• Cover glass smear method

EHRLICH METHOD
• Procedure
• Drop of blood in a cover slip
• Place cover slip on a similar cover slip

• Crosswise direction
• Cover slip w/ blood should be facing down

EHRLICH METHOD
• Procedure
• Pull cover glass quickly
• Dry cover slip
• Labelling

ERLICH METHOD
• Advantage
• Even distribution of WBCs
• Disadvantage
• Difficult to prepare
• Too small for automated stainers
• Harder to label
• Easily broken

BEACON METHOD
BEACON METHOD
• Aka Coverslip- slide method
• Glass slide as smear slide
• Coverslip as spreader

SPUN SMEAR METHOD

SPUN SMEAR METHOD
• Aka Spinner’s Method
• Utilization of hemaspinner

SPUN SMEAR METHOD
• Procedure
• Insert slide into slide holder
• Place slide holder into the cradle

SPUN SMEAR METHOD
• Procedure
• Place one drop

• Each of the 2 holes in slide holder

SPUN SMEAR METHOD
• Procedure
• Close cover and spin

immediately
• Less than 3 seconds

SMEARS FOR
BLOOD PARASITES
• Preparation of
• Thick smear
• Thin smear

• Preparation:
THICK SMEAR
• Place a large drop of blood
• Spread blood into a circle

• Size of a dime
• 1.5 cm2

THICK SMEAR
• Preparation:
• Dry for at least 2 hours

THICK SMEAR
• Uses:
• Parasite detection
• Preliminary quantitation

THIN SMEAR
• Utilizes wedge smear technique
• Uses
• Morphology examination
• Species identification
• Quantitation

QUALITIES OF A GOOD PBS
• Gradual transition from thick to thin
• Should be at least 3 cm long

QUALITIES OF A GOOD PBS
• Smooth, no ridges, no bubbles, no holes & spaces
• Must have a feathery tail portion

TECHNICAL ERRORS ON BLOOD SMEARS
• Ridges/ Uneven distribution of blood
• Increased pressure on spreader slide
• Delay in making smear after drop is placed
• Movement in spreading is not continuous

• Holes in the smear
• Dirty slide
• Contamination with glove powder

• No feathered edge
• Spreader slide not pushed the entire

length of slide

• Streaks on the feathered edge
• Chipped / dirty spreader slide
• Pulling the spreader slide into the drop of

blood so that the blood is pushed instead of
pulled
• Delay in making smear

• Smear too thick and short
• Drop of blood is too big
• Angle of spreader slide is >400

• Smear too thin and long
• Drop of blood is too small
• Angle of spreader slide is < 300
• Spreader slide pushed too slowly

FIXATION
FIXATION
• Routine: Methanol
• Stop metabolic processes
• Maintains morphology of cells
• Mounts cells smear on the slide

STAINING
ROMANOWSKY STAINS
• Includes:
• Giemsa
• Wright’s
• Modified Wright-Giemsa
• Leishman
• Jenner
• May-Gruwald

ROMANOWSKY STAINS
• Contains:
• Methylene blue (Azure B)
• Eosin

ROMANOWSKY STAINS
• Optimum pH
• 6.8: Blood & bone marrow staining
• 7.2: Malarial parasites

PROPERLY STAINED SMEARS
• Nuclei of leukocytes: Purplish blue
• Neutrophilic granules: Reddish pink/ lilac
• Eosinophilic granules: Orange red

• Basophilic granules: Bluish black
• Platelets: Dark blue
• Cytoplasm of lymphocytes
• Light blue; Robin’s egg blue

• Cytoplasm of monocytes
• Bluish gray
• Bacteria
• Blue & granular

STAINING PROBLEMS
• Excessively blue stains
• Thick films
• Prolonged staining time
• Inadequate washing
• Too alkaline stains or diluents

STAINING PROBLEMS
• Excessively pink stains
• Insufficient staining
• Prolonged washing time
• Mounting coverslips before they are dry
• Too acid stains or diluents

SCANNING SMEARS
APPROPRIATE AREA
• Monolayer reading area
• Between feathery edge and thick edge

APPROPRIATE AREA

METHODS OF SCANNING
• Longitudinal method
• Cross-sectional method
• Battlement method
• Two field meander
• Four field meander

LONGITUDINAL METHOD
• WBCs counted in consecutive field from tail towards
head

BATTLEMENT METHOD
• Uses specific pattern of consecutive fields
• Count 3 fields along the edge
• Count 2 fields up
• Count 2 fields along the edge
• Count 2 fields down

CROSS-SECTIONAL METHOD
• Aka Crenellation Method
• WBCs counted in consecutive fields

• Blood film is moved from side to side

TWO FIELD MEANDER
• Two fields counted
• Thin portion
• Thick portion

FOUR FIELD MEANDER
• Four fields counted
• 2 Thin portion
• 2 Thick portion
• Convenient for very low WBC count

WBC
DIFFERENTIAL COUNT
WBC DIFFERENTIAL COUNT
• 100 WBCs counted & differentiated
• Neutrophil (Segmenters)
• Band cells
• Lymphocytes
• Monocytes
• Eosinophils
• Basophils
• Presence of immature cells

NEUTROPHIL

BAND CELL

LYMPHOCYTES

ATYPICAL LYMPHOCYTES
• Aka
• Reactive Lymphocytes
• Plasmacytoid Lymphocytes

MONOCYTES

EOSINOPHILS

BASOPHILS

METAMYELOCYTE
• Aka Juvenile Cell

• Deviations
• 50 cells
• WBC count <1x 109/L

• Deviations
• 200 cells
• Over 10% eosinophils
• Over 2% basophils
• Over 11% monocytes
• More lymphocytes than neutrophils

RELATIVE VALUES
• Neutrophil: 50-70%
• Band cell: 0-5%
• Lymphocyte: 18-42%
• Monocyte: 2-11%
• Eosinophil: 1-3%
• Basophil: 0-2%

ABSOLUTE VALUES
• Computed from relative values
Absolute value= Relative WBC x Total WBC Count
• Basis of absolute increase/ decrease of different

WBCs

ABSOLUTE VALUES
• Neutrophil: 2.3-8.1 x 109/L
• Band cell: 0-0.6 x 109/L
• Lymphocyte: 0.8-4.8 x 109/L
• Monocyte: 0.45-1.3 x 109/L
• Eosinophil: 0-0.4 x 109/L
• Basophil: 0-0.1 x 109/L

ABNORMAL ABSOLUTE COUNTS
• Neutrophils
• Neutrophilia
• >7-8 x 109/L
• Neutropenia
• < 1.75- 1.8 x 109/L
• Lymphocytes
• Lymphocytosis
• >5 x 109/L
• Lymphocytopenia
• <0.5 x 109/L
• Monocytes
• Monocytosis
• >1.5 x 109/L
• Monocytopenia
• < 0.4 x 109/L
• Eosinophils
• Eosinophilia
• >0.7 x 109/L
• Eosinopenia
• < 0.05 x 109/L
• Basophils
• Basophilia
• > 0.3 x 109/L
• Basopenia

RELATED
PROCEDURES
EOSINOPHIL COUNT
EOSINOPHIL COUNT
• Similar to manual leukocyte count w/ some
modifications

EOSINOPHIL COUNT
• Modifications
• Diluting fluid
• Volume of blood sample
• Hemocytometer
• Duration of cell settling

DILUTING FLUID
• Composition:
• Stains
• Phloxine
• Eosin
• Neutral red

DILUTING FLUID
• Composition:
• Propylene glycol
• Na2CO3
• Heparin

DILUTING FLUIDS
• Solutions:
• Pilot’s Solution
• Manner’s Solution
• Randolph’s Solution
• Hinkleman’s Solution
• Tannen’s Solution

VOLUME OF BLOOD SAMPLE
• Up to 1.0 mark
• Dilution of 1:10
• DF of 10

HEMOCYTOMETER
• 2-4 counting areas of Speirs-Levy
• 2 counting areas of Fuchs-Rosenthal

CELL SETTLING
• 15 to 20 minutes
• Settling of cells
• Lysis of cells
• Staining of eosinophils

SCHILLING COUNT
SCHILLING COUNT
• Granulocytes classified according to their granulations

SCHILLING COUNT
• Utilizes the Schilling hemogram
• Younger forms on the left

• Myeloblasts, Promyelocytes, Juvenile cells, Band
cells
• Mature forms on the right

• Segmenters, Eosinophils, Basophils, Lymphocytes,
Monocytes
SHIFT TO THE LEFT
• Increased immature forms
• 2 types
• Regenerative
• Degenerative

REGENERATIVE SHIFT
• Rapid production of WBCs
• Increased WBC count
• Early release of immature cells even before maturation
• Predominance of juvenile cells

DEGENERATIVE SHIFT
• Depression of leukopoietic centers
• Decreased WBC count
• Incomplete neutrophilic development
• Decreased lymphocytes, Eosinophil disappearance

ARNETH COUNT
ARNETH COUNT
• Neutrophils classified according to numbers of lobes
• Grouping of neutrophils
• Group I: 5%
• Group II: 35%
• Group III: 41%
• Group IV: 17%
• Group V: 2%
ARNETH COUNT
• Each form is counted & expressed in percentage of
the total neutrophil count
• Arneth index
• (%1-lobed + % 2-lobed cells) + ½ (% 3-lobed cells)

TRENDS
• Shift to the left
• Shift to the right

SHIFT TO THE LEFT
• Arneth index> 45%
• Increased 1- & 2-lobed cells
• Immature cells are in excess of the mature cells
• Pyogenic infections, hemorrhages, toxemias

SHIFT TO THE RIGHT
• If > 5% or more shows five lobes
• Pernicious anemia, sprue, anemia of pregnancy

WBC ESTIMATE
WBC ESTIMATE
• Should be part of differential count
• Validates WBC count

PROCEDURE
• Appropriate area for reading

PROCEDURE
• Using OIO or HPO
• Count number of WBCs in 10 fields
• Compute for average number of WBCs per field

• Divide total number by 10

PROCEDURE
• Compute for WBC estimate per mm3 / uL
• HPO: Use factor 2000
• OIO: Use factor 3000

SAMPLE COMPUTATION
• HPO: Average of 3/ field
3 x 2000= 6,000/ mm3
6.0 x 103/mm3
6.0 x 109/L
• OIO: Average of 3/field

3x 3000= 9000/ mm3
9.0 x 103/ L
9.0 x 109/L
ESTIMATION FACTOR
1. Perform automated WBC count for 30 samples
2. Prepare and stain peripheral blood smears for each

sample
3. Get average WBC count after scanning 10 fields

• Use either HPO/ OIO
ESTIMATION
4. For each specimen
FACTOR
• Divide automated count by average count
5. Compute for average ration of WBC count- WBCs/

HPO or OIO
• Add all numbers obtained in step 4
• Divide by 30

Blood Smear Preparation

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What are the different methods described for counting white blood cells?

What are the different methods described for counting white blood cells?

What are some of the modifications made to the eosinophil count compared to the manual leukocyte count?

What are some of the modifications made to the eosinophil count compared to the manual leukocyte count?

BLOOD SMEAR

PREPARED BY: DANROE ARVEE BALAGAT, RMT

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Prepare clean glass slide

• Place a drop of blood

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Spreader slide in front

• Angle: 300- 400

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Spreader slide back to edge of drop

• Blood spreads across the end

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Push the spreader slide forward

PREPARED BY: DANROE ARVEE BALAGAT, RMT

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Cover glass smear method

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Drop of blood in a cover slip

• Place cover slip on a similar cover slip

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Pull cover glass quickly

• Dry cover slip

PREPARED BY: DANROE ARVEE BALAGAT, RMT

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Glass slide as smear slide

PREPARED BY: DANROE ARVEE BALAGAT, RMT

SPUN SMEAR METHOD

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Insert slide into slide holder

• Place slide holder into the cradle

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Place one drop

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Close cover and spin

PREPARED BY: DANROE ARVEE BALAGAT, RMT

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Spread blood into a circle

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Dry for at least 2 hours

PREPARED BY: DANROE ARVEE BALAGAT, RMT

PREPARED BY: DANROE ARVEE BALAGAT, RMT

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Should be at least 3 cm long

• Must have a feathery tail portion

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Increased pressure on spreader slide

• Delay in making smear after drop is placed

• Movement in spreading is not continuous

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Contamination with glove powder

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Spreader slide not pushed the entire

PREPARED BY: DANROE ARVEE BALAGAT, RMT

• Chipped / dirty spreader slide