BLOOD SMEAR
PREPARATION
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
METHODS OF
SMEAR PREPARATION
PREPARED BY: DANROE ARVEE BALAGAT, RMT
WEDGE SMEAR
METHOD
WEDGE SMEAR
• Aka 2 slide method
• Smear slide
• Spreader
PREPARED BY: DANROE ARVEE BALAGAT, RMT
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
PREPARED BY: DANROE ARVEE BALAGAT, RMT
WEDGE SMEAR
• Procedure:
• Spreader slide in front
of the drop
• Angle: 300- 400
PREPARED BY: DANROE ARVEE BALAGAT, RMT
WEDGE SMEAR
• Procedure:
• Spreader slide back to edge of drop
• Blood spreads across the end
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
WEDGE SMEAR
• Procedure:
• Push the spreader slide forward
• Continuous movement
• All the way to the end of the smear slide
• Maintain 300-400 angle
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
WEDGE SMEAR
• Procedure:
• Drying of slide
• Labelling of slide
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
EHRLICH METHOD
EHRLICH METHOD
• Aka
• 2 coverslip method
• Cover glass smear method
PREPARED BY: DANROE ARVEE BALAGAT, RMT
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
PREPARED BY: DANROE ARVEE BALAGAT, RMT
EHRLICH METHOD
• Procedure
• Pull cover glass quickly
• Dry cover slip
• Labelling
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
ERLICH METHOD
• Advantage
• Even distribution of WBCs
• Disadvantage
• Difficult to prepare
• Too small for automated stainers
• Harder to label
• Easily broken
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
BEACON METHOD
BEACON METHOD
• Aka Coverslip- slide method
• Glass slide as smear slide
• Coverslip as spreader
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
SPUN SMEAR METHOD
SPUN SMEAR METHOD
• Aka Spinner’s Method
• Utilization of hemaspinner
PREPARED BY: DANROE ARVEE BALAGAT, RMT
SPUN SMEAR METHOD
• Procedure
• Insert slide into slide holder
• Place slide holder into the cradle
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
SPUN SMEAR METHOD
• Procedure
• Place one drop
• Each of the 2 holes in slide holder
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
SPUN SMEAR METHOD
• Procedure
• Close cover and spin
immediately
• Less than 3 seconds
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
SMEARS FOR
BLOOD PARASITES
• Preparation of
• Thick smear
• Thin smear
PREPARED BY: DANROE ARVEE BALAGAT, RMT
• Preparation:
THICK SMEAR
• Place a large drop of blood
• Spread blood into a circle
• Size of a dime
• 1.5 cm2
PREPARED BY: DANROE ARVEE BALAGAT, RMT
THICK SMEAR
• Preparation:
• Dry for at least 2 hours
PREPARED BY: DANROE ARVEE BALAGAT, RMT
THICK SMEAR
• Uses:
• Parasite detection
• Preliminary quantitation
PREPARED BY: DANROE ARVEE BALAGAT, RMT
THIN SMEAR
• Utilizes wedge smear technique
• Uses
• Morphology examination
• Species identification
• Quantitation
PREPARED BY: DANROE ARVEE BALAGAT, RMT
QUALITIES OF A GOOD PBS
• Gradual transition from thick to thin
• Should be at least 3 cm long
PREPARED BY: DANROE ARVEE BALAGAT, RMT
QUALITIES OF A GOOD PBS
• Smooth, no ridges, no bubbles, no holes & spaces
• Must have a feathery tail portion
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
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
PREPARED BY: DANROE ARVEE BALAGAT, RMT
TECHNICAL ERRORS ON BLOOD SMEARS
• Holes in the smear
• Dirty slide
• Contamination with glove powder
PREPARED BY: DANROE ARVEE BALAGAT, RMT
TECHNICAL ERRORS ON BLOOD SMEARS
• No feathered edge
• Spreader slide not pushed the entire
length of slide
PREPARED BY: DANROE ARVEE BALAGAT, RMT
TECHNICAL ERRORS ON BLOOD SMEARS
• 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
PREPARED BY: DANROE ARVEE BALAGAT, RMT
TECHNICAL ERRORS ON BLOOD SMEARS
• Smear too thick and short
• Drop of blood is too big
• Angle of spreader slide is >400
PREPARED BY: DANROE ARVEE BALAGAT, RMT
TECHNICAL ERRORS ON BLOOD SMEARS
• Smear too thin and long
• Drop of blood is too small
• Angle of spreader slide is < 300
• Spreader slide pushed too slowly
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
FIXATION
FIXATION
• Routine: Methanol
• Stop metabolic processes
• Maintains morphology of cells
• Mounts cells smear on the slide
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
STAINING
ROMANOWSKY STAINS
• Includes:
• Giemsa
• Wright’s
• Modified Wright-Giemsa
• Leishman
• Jenner
• May-Gruwald
PREPARED BY: DANROE ARVEE BALAGAT, RMT
ROMANOWSKY STAINS
• Contains:
• Methylene blue (Azure B)
• Eosin
PREPARED BY: DANROE ARVEE BALAGAT, RMT
ROMANOWSKY STAINS
• Optimum pH
• 6.8: Blood & bone marrow staining
• 7.2: Malarial parasites
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PROPERLY STAINED SMEARS
• Nuclei of leukocytes: Purplish blue
• Neutrophilic granules: Reddish pink/ lilac
• Eosinophilic granules: Orange red
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PROPERLY STAINED SMEARS
• Basophilic granules: Bluish black
• Platelets: Dark blue
• Cytoplasm of lymphocytes
• Light blue; Robin’s egg blue
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PROPERLY STAINED SMEARS
• Cytoplasm of monocytes
• Bluish gray
• Bacteria
• Blue & granular
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
STAINING PROBLEMS
• Excessively blue stains
• Thick films
• Prolonged staining time
• Inadequate washing
• Too alkaline stains or diluents
PREPARED BY: DANROE ARVEE BALAGAT, RMT
STAINING PROBLEMS
• Excessively pink stains
• Insufficient staining
• Prolonged washing time
• Mounting coverslips before they are dry
• Too acid stains or diluents
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
SCANNING SMEARS
APPROPRIATE AREA
• Monolayer reading area
• Between feathery edge and thick edge
PREPARED BY: DANROE ARVEE BALAGAT, RMT
APPROPRIATE AREA
PREPARED BY: DANROE ARVEE BALAGAT, RMT
METHODS OF SCANNING
• Longitudinal method
• Cross-sectional method
• Battlement method
• Two field meander
• Four field meander
PREPARED BY: DANROE ARVEE BALAGAT, RMT
LONGITUDINAL METHOD
• WBCs counted in consecutive field from tail towards
head
PREPARED BY: DANROE ARVEE BALAGAT, RMT
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
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
CROSS-SECTIONAL METHOD
• Aka Crenellation Method
• WBCs counted in consecutive fields
• Blood film is moved from side to side
PREPARED BY: DANROE ARVEE BALAGAT, RMT
TWO FIELD MEANDER
• Two fields counted
• Thin portion
• Thick portion
PREPARED BY: DANROE ARVEE BALAGAT, RMT
FOUR FIELD MEANDER
• Four fields counted
• 2 Thin portion
• 2 Thick portion
• Convenient for very low WBC count
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
WBC
DIFFERENTIAL COUNT
WBC DIFFERENTIAL COUNT
• 100 WBCs counted & differentiated
• Neutrophil (Segmenters)
• Band cells
• Lymphocytes
• Monocytes
• Eosinophils
• Basophils
• Presence of immature cells
PREPARED BY: DANROE ARVEE BALAGAT, RMT
NEUTROPHIL
PREPARED BY: DANROE ARVEE BALAGAT, RMT
BAND CELL
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
LYMPHOCYTES
PREPARED BY: DANROE ARVEE BALAGAT, RMT
ATYPICAL LYMPHOCYTES
• Aka
• Reactive Lymphocytes
• Plasmacytoid Lymphocytes
PREPARED BY: DANROE ARVEE BALAGAT, RMT
MONOCYTES
PREPARED BY: DANROE ARVEE BALAGAT, RMT
EOSINOPHILS
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
BASOPHILS
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
METAMYELOCYTE
• Aka Juvenile Cell
PREPARED BY: DANROE ARVEE BALAGAT, RMT
WBC DIFFERENTIAL COUNT
• Deviations
• 50 cells
• WBC count <1x 109/L
PREPARED BY: DANROE ARVEE BALAGAT, RMT
WBC DIFFERENTIAL COUNT
• Deviations
• 200 cells
• Over 10% eosinophils
• Over 2% basophils
• Over 11% monocytes
• More lymphocytes than neutrophils
PREPARED BY: DANROE ARVEE BALAGAT, RMT
RELATIVE VALUES
• Neutrophil: 50-70%
• Band cell: 0-5%
• Lymphocyte: 18-42%
• Monocyte: 2-11%
• Eosinophil: 1-3%
• Basophil: 0-2%
PREPARED BY: DANROE ARVEE BALAGAT, RMT
ABSOLUTE VALUES
• Computed from relative values
Absolute value= Relative WBC x Total WBC Count
• Basis of absolute increase/ decrease of different
WBCs
PREPARED BY: DANROE ARVEE BALAGAT, RMT
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
PREPARED BY: DANROE ARVEE BALAGAT, RMT
ABNORMAL ABSOLUTE COUNTS
• Neutrophils
• Neutrophilia
• >7-8 x 109/L
• Neutropenia
• < 1.75- 1.8 x 109/L
PREPARED BY: DANROE ARVEE BALAGAT, RMT
ABNORMAL ABSOLUTE COUNTS
• Lymphocytes
• Lymphocytosis
• >5 x 109/L
• Lymphocytopenia
• <0.5 x 109/L
PREPARED BY: DANROE ARVEE BALAGAT, RMT
ABNORMAL ABSOLUTE COUNTS
• Monocytes
• Monocytosis
• >1.5 x 109/L
• Monocytopenia
• < 0.4 x 109/L
PREPARED BY: DANROE ARVEE BALAGAT, RMT
ABNORMAL ABSOLUTE COUNTS
• Eosinophils
• Eosinophilia
• >0.7 x 109/L
• Eosinopenia
• < 0.05 x 109/L
PREPARED BY: DANROE ARVEE BALAGAT, RMT
ABNORMAL ABSOLUTE COUNTS
• Basophils
• Basophilia
• > 0.3 x 109/L
• Basopenia
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
RELATED
PROCEDURES
PREPARED BY: DANROE ARVEE BALAGAT, RMT
EOSINOPHIL COUNT
EOSINOPHIL COUNT
• Similar to manual leukocyte count w/ some
modifications
PREPARED BY: DANROE ARVEE BALAGAT, RMT
EOSINOPHIL COUNT
• Modifications
• Diluting fluid
• Volume of blood sample
• Hemocytometer
• Duration of cell settling
PREPARED BY: DANROE ARVEE BALAGAT, RMT
DILUTING FLUID
• Composition:
• Stains
• Phloxine
• Eosin
• Neutral red
PREPARED BY: DANROE ARVEE BALAGAT, RMT
DILUTING FLUID
• Composition:
• Propylene glycol
• Na2CO3
• Heparin
PREPARED BY: DANROE ARVEE BALAGAT, RMT
DILUTING FLUIDS
• Solutions:
• Pilot’s Solution
• Manner’s Solution
• Randolph’s Solution
• Hinkleman’s Solution
• Tannen’s Solution
PREPARED BY: DANROE ARVEE BALAGAT, RMT
VOLUME OF BLOOD SAMPLE
• Up to 1.0 mark
• Dilution of 1:10
• DF of 10
PREPARED BY: DANROE ARVEE BALAGAT, RMT
HEMOCYTOMETER
• 2-4 counting areas of Speirs-Levy
• 2 counting areas of Fuchs-Rosenthal
PREPARED BY: DANROE ARVEE BALAGAT, RMT
CELL SETTLING
• 15 to 20 minutes
• Settling of cells
• Lysis of cells
• Staining of eosinophils
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
SCHILLING COUNT
SCHILLING COUNT
• Granulocytes classified according to their granulations
PREPARED BY: DANROE ARVEE BALAGAT, RMT
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
PREPARED BY: DANROE ARVEE BALAGAT, RMT
SHIFT TO THE LEFT
• Increased immature forms
• 2 types
• Regenerative
• Degenerative
PREPARED BY: DANROE ARVEE BALAGAT, RMT
REGENERATIVE SHIFT
• Rapid production of WBCs
• Increased WBC count
• Early release of immature cells even before maturation
• Predominance of juvenile cells
PREPARED BY: DANROE ARVEE BALAGAT, RMT
DEGENERATIVE SHIFT
• Depression of leukopoietic centers
• Decreased WBC count
• Incomplete neutrophilic development
• Decreased lymphocytes, Eosinophil disappearance
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
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%
PREPARED BY: DANROE ARVEE BALAGAT, RMT
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)
PREPARED BY: DANROE ARVEE BALAGAT, RMT
TRENDS
• Shift to the left
• Shift to the right
PREPARED BY: DANROE ARVEE BALAGAT, RMT
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
PREPARED BY: DANROE ARVEE BALAGAT, RMT
SHIFT TO THE RIGHT
• If > 5% or more shows five lobes
• Pernicious anemia, sprue, anemia of pregnancy
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
WBC ESTIMATE
WBC ESTIMATE
• Should be part of differential count
• Validates WBC count
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PROCEDURE
• Appropriate area for reading
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PREPARED BY: DANROE ARVEE BALAGAT, RMT
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
PREPARED BY: DANROE ARVEE BALAGAT, RMT
PROCEDURE
• Compute for WBC estimate per mm3 / uL
• HPO: Use factor 2000
• OIO: Use factor 3000
PREPARED BY: DANROE ARVEE BALAGAT, RMT
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
PREPARED BY: DANROE ARVEE BALAGAT, RMT
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
PREPARED BY: DANROE ARVEE BALAGAT, RMT
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
PREPARED BY: DANROE ARVEE BALAGAT, RMT
