CHAPTER 3.5 - DIFFERENTIAL COUNT Flashcards
Quantitative and qualitative study of the types of leukocytes
DIFFERENTIAL COUNT
Represents the percentage distribution of the different WBCs
DIFFERENTIAL COUNT
DIFFERENTIAL COUNT Includes:
o Quantitative & qualitative study of thrombocytes
oAge and morphologic abnormalities or RBCs
DIFFERENTIAL COUNT
4 General steps:
- Preparation of blood smear
- Staining of the smear
- Counting of the cells
- Reporting
Blood smears must be made within
2-3 hours
Advantages of EDTA blood smear
1. Can create
2. May be prepared at
3. Avoids
multiple slides
later time
platelet clumping
SOURCES OF SPECIMEN:
EDTA BLOOD
ANTICOAGULANT FREE BLOOD (CAPILLARY BLOOD)
Best for evaluating blood morphology
ANTICOAGULANT FREE BLOOD (CAPILLARY BLOOD)
ANTICOAGULANT FREE BLOOD (CAPILLARY BLOOD)
Advantages:
• Made at the patient’s side
• Artifacts are minimized
ANTICOAGULANT FREE BLOOD (CAPILLARY BLOOD)
• Disadvantages:
• Platelet clumping
• Few films can be made
METHODS OF BLOOD SMEAR PREPARATION
BM preparations; NOT for routine
Coverslip Method
PBS
Coverslip Method
ADV: superior leukocyte distribution
Coverslip Method
Coverslip - rotated 45° (16sided)
Coverslip Method
Never touch the coverslips with fingers
Coverslip Method
Erlich’s
Coverslip Method
Beacom’s
Glass Slide-Coverslip Method
Wedge Slide/Push Smear
Two-Glass Slide Method
Uses 2 slides
Two-Glass Slide Method
Film slide
Two-Glass Slide Method
Two-Glass Slide Method Pusher Spreader slide
30-45 degree
Two-Glass Slide Method Considerations:
1. Drop of blood (?) in diameter
2. (?) of spreader (smooth & swift)
3. (?) of patient
2-3mm
Speed
Gct
Two-Glass Slide Method SPREADER SLIDE:
Must be (?) than the stationary slide
Spreading edge:
With
narrower
clean, smooth, polished & thin
beveled edge
Two-Glass Slide Method DISADVANTAGES:
Tendency of (?) at the slide edges & feathered ends (ADV: easier to find abnormal cells)
(?) of nucleated cells
Greater (?) to cells
larger cells to settle
Poor distribution
trauma
prepares dual smears simultaneously ata constant angle and speed.
Miniprep/Hemaprep
glass slides are placed on a platform inside the instrument (0.2 ml WB)
Hemaspinner
Instrument spins causing the slide to be covered with a monolayer of cells
Hemaspinner
Uses beam of sensor light
Hemaspinner
Advantages of Hemaspinner
o Even distribution
o Consistency of preparation
o Large examination area
o Fewer broken cells
Disadvantages of Hemaspinner
o Longer preparation
o Smears can not be done outside the lab
o No specific location for abnormal cells
o Centrifugal force may cause distortions
finding reactive immature or abnormal cells that are present in small numbers
Buffy Coat Smear
Megaloblastic nucleated RBC or hypersegmented neutrophils
Buffy Coat Smear
WhenWBCcountis<1× 109/L
Buffy Coat Smear
Finding abnormal plasma cells
Buffy Coat Smear
Easier to locate bacteria & parasites
Buffy Coat Smear
For blood parasites
Thick Blood Smear
blood parasites
o Malaria
o Filaria
o Trypanosomes
o Spirochetes
CHARACTERISTICS OF A PROPERLY PREPARED WEDGE SMEAR
- Length: must occupy at least (?) of the slide
- Should be free of (?)
- Should have (?) appearance
- Should be (?) than the stationary slide
- Must have (?) from thick to thin
- (?) to allow proper fixation
- Should terminate in a (?)
- Should contain at least (?) in which 50% of the RBCs do no overlap each other; the remainder ma overlap in doublets or triplets and the central pallor should be clear
1/2 to 2/3
waves, holes, and ridges
smooth and even
narrower
gradual transition
Thin enough
feathery tail
10 LPF
EFFECTS OF THICK SMEAR:
o Excess plasma causes nucleated cells to shrink
o RBCs form rouleaux
EFFECTS OF THIN SMEAR:
Effects:
o Increased number of smudge el
o RBCs become artificially spheroid w/ distorted shape
o More nucleated cell on the edges
ARTIFACTS OF SMEAR PREPARATION
(Common in EDTA)
- Monocyte vacuolation
- Reactive lymphocytes
- RBC spiculation
- Dole bodies may disappear on standing
- Basophilic granules
- Drying artifacts
– vacuolated cytoplasm with convoluted nuclei & “swiss cheese” cvtoplasm
Reactive lymphocytes
Causes of Drying artifacts
Humid environment
Severe anemia
Water contamination of fixative
Types of Drying artifacts
- Red cell artifacts
- Hairy appearance of lymphocytes
- Shrinkage of normal leukocytes
METHODS OF DRYING BS
- Air Drying
- Use of low flame
- Use of oven
- Immersion in Methyl alcohol or absolute alcohol for 1 - 2 minutes
STAINING OF BLOOD SMEARS
Composition:
Tetramethylthionine; Trimethylthionine
Methylene blue
(+) charged
Methylene blue
Basic dye: DNA, RNA
Methylene blue
(-) charged
Eosin B/Y
Acidic dye: hemoglobin, eosinophil granules
Eosin B/Y
ROMANOWSKY STAINS
Composition
Buffer (pH: 6.4 – 6.8): Ionization
Fixative: Methanol
Wash water/Distilled water (NOT tap water)
ROMANOWSKY STAINS
Types
- Wright
- Giemsa
- Modified Wright-Giemsa
- Leishman
- May-Grunwald
- Jenner
- MacNeal
Wright’s Stain: PREPARATION
1g Wright’s powder
500 ml Acetone-free Methanol (Mallinckrodt methanol)
1 wk at 37°C then RT/Ref T
Wright’s Stain:
1 gm Giemsa powder/500 stain shortens the incubation
Wright’s Stain
Wright-Giemsa Stain: PREPARATION
9g Wright’s powder
1g Giemsa powder
2,910 ml Acetone-free Methanol (Mallinckrodt methanol)
Age for 30 days; shake 1/day
Incubate
Wright-Giemsa Stain
Sorenson’s PO buffer: PREPARATION
9.5g 1L
Anhydrous monobasic K2PO4 Dist. Water
9.5g 1L
Anhydrous dilbasic Na2PO4 Dist. Water
Mix according to pH desired
Sorenson’s PO buffer
METHODS OF STAINING
- Rack Method
- Dip Method
- Automated Methods
Macroscopic
pink to reddish-brown
Erythrocytes:
salmon pink
Platelets:
purple- blue to lilac cytoplasm with red-purple granules
PMN:
Nuclei =
Cytoplasm =
Granules =
purple-blue
pinkish tan
pink to lilac
Lymphocytes:
light blue cytoplasm
Monocytes:
faint blue-gray cytoplasm with tiny red-purple granules
Eosinophils:
with red-orange granules (pH meter)
Basophils:
with dark purple granules
PROCEDURE
1. Check slide identification
2. Perform patient specimen orientation 3. LPO-scan to review blood film
4. LPO-scan to review blood film
5. Oil Immersion Examination
OTHER CRITERIA:
50 Cells: WBC count is
100 cells:
< 1 x 109/L
o > 10% Eosinophils
o > 2% Basophils
o > 11% Monocytes
o Lymphocytes > Neutrophils