CHAPTER 3.5 - DIFFERENTIAL COUNT Flashcards by Arriane Cyrel (MTI)

Quantitative and qualitative study of the types of leukocytes

DIFFERENTIAL COUNT

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Represents the percentage distribution of the different WBCs

DIFFERENTIAL COUNT

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DIFFERENTIAL COUNT Includes:

o Quantitative & qualitative study of thrombocytes
oAge and morphologic abnormalities or RBCs

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DIFFERENTIAL COUNT

4 General steps:

Preparation of blood smear
Staining of the smear
Counting of the cells
Reporting

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Blood smears must be made within

2-3 hours

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Advantages of EDTA blood smear
1. Can create
2. May be prepared at
3. Avoids

multiple slides

later time

platelet clumping

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SOURCES OF SPECIMEN:

EDTA BLOOD

ANTICOAGULANT FREE BLOOD (CAPILLARY BLOOD)

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Best for evaluating blood morphology

ANTICOAGULANT FREE BLOOD (CAPILLARY BLOOD)

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ANTICOAGULANT FREE BLOOD (CAPILLARY BLOOD)

Advantages:

• Made at the patient’s side
• Artifacts are minimized

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ANTICOAGULANT FREE BLOOD (CAPILLARY BLOOD)

• Disadvantages:

• Platelet clumping
• Few films can be made

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METHODS OF BLOOD SMEAR PREPARATION

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BM preparations; NOT for routine

Coverslip Method

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PBS

Coverslip Method

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ADV: superior leukocyte distribution

Coverslip Method

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Coverslip - rotated 45° (16sided)

Coverslip Method

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Never touch the coverslips with fingers

Coverslip Method

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Erlich’s

Coverslip Method

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Beacom’s

Glass Slide-Coverslip Method

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Wedge Slide/Push Smear

Two-Glass Slide Method

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Uses 2 slides

Two-Glass Slide Method

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Film slide

Two-Glass Slide Method

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Two-Glass Slide Method Pusher Spreader slide

30-45 degree

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Two-Glass Slide Method Considerations:
1. Drop of blood (?) in diameter
2. (?) of spreader (smooth & swift)
3. (?) of patient

2-3mm

Speed

Gct

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Two-Glass Slide Method SPREADER SLIDE:
 Must be (?) than the stationary slide
 Spreading edge:
 With

narrower

clean, smooth, polished & thin

beveled edge

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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 1. Length: must occupy at least (?) of the slide 2. Should be free of (?) 3. Should have (?) appearance 4. Should be (?) than the stationary slide 5. Must have (?) from thick to thin 6. (?) to allow proper fixation 7. Should terminate in a (?) 8. 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)

1. Monocyte vacuolation 2. Reactive lymphocytes 3. RBC spiculation 4. Dole bodies may disappear on standing 5. Basophilic granules 6. 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

1. Red cell artifacts 2. Hairy appearance of lymphocytes 3. Shrinkage of normal leukocytes

METHODS OF DRYING BS

1. Air Drying 2. Use of low flame 3. Use of oven 4. 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

1. Wright 2. Giemsa 3. Modified Wright-Giemsa 4. Leishman 5. May-Grunwald 6. Jenner 7. 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

1. Rack Method 2. Dip Method 3. 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

CHAPTER 3.5 - DIFFERENTIAL COUNT Flashcards

(72 cards)