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Hema Diff count notes

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Peripheral Blood Smear
- Differential count and morphology
- Rough estimate if % of different
WBC
- Identify and count (100-to be
converted into %)
White Blood Cells
- Neutrophils:
• Polymorphonuclear nuetrophil
Ø AKA Segmenters
Ø 3-4 lobes separated
Ø With thin filamentous
tubes
Ø Granulated
Ø 2nd largest WBC in PBS
• Band/ stab
Ø Immature neutrophil
Ø Lacks filamentous
structure
Ø Nucleus is continuous
• Lymphocyte
Ø Smaller than neutrophil
Ø Large nucleus to
cytoplasm ratio
Ø “scanty
cytoplasm/nucleus”
Ø lighter color than
neutrophil
• Monocyte
Ø Largest in PBS
Ø Brain-like convolutions
Ø Consists of vacuoles if
ingests debris or
activated fxn
Ø Comes from
MEGAKARYOCYTE in the
Bone marrow
• Eosinophil
Ø Bilobed
Ø Orange or pinkish
granules
•
•
Basophil
Ø Most rare
Ø 0-0.5 or 1 range
Ø deep blue violet granules
nRbc
Ø error in Diff count
Ø lymphocytes are larger
Ø sam size as rbc
White
blood Cell
Segementer
Neutrophil
(50-70)
Band
Neutrophil
(0-5)
Lymphocyte
(18-42)
Monocyte
(2-11)
Eosinophil
(1-3)
Basophil
(0-2)
nRBC
SPA 3HMT 2019
1
Standard blood Smear
- similar base line
Staining procedure (FEMDB)
1. Fixative (6 dips)
2. Eosin (4 dips)
3. Methylene Blue (6 dips)
4. Distilled Water/Buffer (6 dips)
• both sides
• serve as drainage o excess blood
4. lateral platform
• pillars
• where thick cover slips are
placed
NOTE: Average count of 2 central platforms
Hemacytometer
- Insrtrument that counts Blood Cells
- Made up of glass
- Includes
• Neubauer’s Slide
• Thick cover slip
• Thoma Pipette (WBC/RBC)
Ø For dilution and mixing
diluted blood
Ø WBC: Bigger bulb than
RBC pipette and with
white bead
Ø RBC: smaller bulb than
WBC with red bead
• Improve Neubauer cytometer
Ø 2 sections (upper and
lower)
Counting chamber
Parts of nuebauer:
1. Central platform
• 0.1 depth
• ruled area/ grid lines
2. transvere groove/ canal
• divides central platform
3. moats
• shallow depression
Fuchs-rosental counting chamber
- total area of primary square is 16
mm sq
- secondary square is divided into 16
smaller squares
- depth of 0.2 mm
Speirs-levy counting chamber
- 0.2 mm depth
Primary square:
- 9mmsq
- whole square
Secondary Square:
- 9 squares
- 1mmsq
Tertiary squares(64-total):
- 4 corner secondary squares are
counted
- 16 (0.0625mmsq) per 4 corner
secondary square
SPA 3HMT 2019
2
-
4 ruled sections
primary square: 10mm sq
secondary square(10): 1 mm sq
MICROSCOPY PROCEDURE
- cedar wood oil
- LPO to focus
- OIO to diff count (count 100 to
differentiate)
Site for counting
- Monolayer: no overlapping cells
- Not in the feathery edge (may have
distorted morphology of cells that
can cause misinterpretation)
- Not in the Thick area ( WBC are
small and clustered and overlapped
with RBC precence of Rouleaux
(stacked RBC) may be seen)
Method of diff count
1. Four field meander
2. Two-field
3. Exaggerated battlement
4. Strip differential
- Most commonly used
Counting rule
-
Count all cells inside tertiary square
Count all overlapping inverted L
-
=
Formula:
𝒕𝒐𝒕𝒂𝒍 𝒄𝒆𝒍𝒍𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅
(𝒂𝒓𝒆𝒂)(𝒅𝒆𝒑𝒕𝒉 = 𝟎. 𝟏𝒎𝒎)(𝒅𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝒐𝒇 𝒃𝒍𝒐𝒐𝒅)
Area:
WBC = 4
RBC = 0.04
Dilution of blood
- Done with thoma pipettes
- Change the aspirator or barrel of
tuberculin syringe
- Aspirate until 0.5 unit or mark of
stem
- 0.5 blood + diluent
- bore is found at the end
Difference of WBC And RBC Thoma
Pipette
WBC
RBC
WBC count
Use
RBC count
Smaller (10
Bulb
Larger (100
units)
units)
White
Bead
Red
larger
Bore size
Smaller
11 units
Last
101 units
graduation
1:20
Dilution
1:200
Hypotonic:to
Diluting
Isotonic: to
remove RBC
fluid
maintain
RBC
morphology
≤ 12 WBC
Difference ≤ 20 RBC
in number
between
each 2°
square
Computation # of cells x
# cells x 50
short cut
10,000
UNIT
WBC / µL
RBC / µL
WBC/cumm
RBC/cumm
SPA 3HMT 2019
3
RBC diluting fluids:
1.
-
Dacie’s fluid or Formol citrate
best RBC diluting fluid
40% formaldehyde (10ml)
3% w/v trisodium citrate (990ml)
2.
-
Hayem’s
Not recommended
Allows yeast growth
Clump cells (from patients with liver
cirrhosis
No corrosive effect
Mercuric chloride,cp (0.50 g)
Sodium sulfate, cp crystalline (5g) or
anhydrous (2.65g)
Sodium chloride,cp (1g)
Distilled water (200mL)
-
3. Gower’s
- Prevent rouleaux
- Precipitate protein (hemoglobinemia
and hyperglobulinemia)
- Glacial acetic acid (16.65 g)
- Sodium sulfate, cp crystalline (6.25)
- Distilled water (100mL)
- OR
- Sodium chloride ( 0.85g)
- Sodium sulfate ( 12.50 g)
- Glycerine (33.30 g)
- Distilled water ( 200 ml)
4.
-
Toisson’s
High specific gravity
Stains WBC
Support fungal growth
Sodium Chloride (1g)
Sodium sulfate (8g)
Glycerine ( 30 g)
Methyl violet ( 0.025g)
Distilled water (200 ml)
5.
-
Bethell’s
Sodium sulfate , crystalline ( 5g)
Sodium chloride (1g)
Glycerine (20 g)
Sodium merthiolate 1:1000 soln
(2ml)
Distilled water (200ml)
6.
-
NSS
In cases of emergency
Excessive rouleaux
Agglutinated cells
Stable
Preservative
NaCl (0.85g)
Distilled water (100 mL)
7. 3.8 sodium citrate
- sodium citrate (3.80g)
- distilled wate (100 ml)
WBC diluting fluid
1. 1%-3% acetic acid with gentian
violet
- Glacial acetic acid (2g)
- Gentian violet (1ml)
- Distilled water (100ml)
2. 1% HCl
- HCl (1ml)
- Distilled water (100mL)
3.
-
Tuerk’s
Glacial acetic acid (2ml)
Methyl violet (1mL)
Distilled Water (100ml)
SPA 3HMT 2019
4
NORMAL VALUES:
SPA 3HMT 2019
5
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