Download MBBS Practicals Physiology Differential Leukocyte Count

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Principle

Apparatus

Procedure

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Precaution


PRINCIPLE:

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BLOOD SMEAR IS PREPARED, STAINED WITH

LEISHMAN'S STAIN AND CELLS ARE IDENTIFIEDUNDER
OIL IMMERSION LENS.

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4-5 GLASS SLIDES
LANCET/ SPRIT/COTTON
LEISHMAN'S STAIN

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MICROSCOPE
CEDAR WOOD OIL
DISTILLED WATER
STAINING TRAY

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preparation of blood smear

staining of blood smear

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Examination of smear under oil immersion(100x) lens


selection of spreader

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which has smooth edge, without coarse or uneven edge should

be avoided.

wash out the grease or oil, if it present in the glass slides as

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well as at the edge of the spreader slide.

we have to use oil and grease free slides.

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tongue shaped ( head ,body, tail)

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should cover 2/3rd of the slide

Should not be thick

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(single cell thickness)

should not have marks or

blank spaces in the smear.

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Leishman's stain:

Belongs to Romanowsky group of stain.
Contains acidic and basic dye.

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Composition:

Methylene blue- basic dye, positively charged and stains negatively charged

[acidic] particles ( stains nucleus of WBCs, the cytoplasm and basophilic

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granules)

Eosin ?acidic dye, negatively charged and stains positively charged (basic)

particles (stains eosinophilic granules / RBCs)

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Acetone free methyl alcohol-(fixative fix the smear to the slide)


WRIGHT STAIN

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FIELD STAIN


MAKE SURE THE SLIDE IS DRY
POUR THE LEISHMAN'S STAIN DROP BY DROP

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TILL IT COVERS ENTIRE SMEAR (8-10 DROPS)

NOTE THE TIME ALLOW FOR 1-2 MINs

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(its known as FIXATION TIME)
ADD DOUBLE THE AMOUNT DROPS OF
DISTILLED WATER

WAIT FOR 6-8 MINS (FORMATION OF CATIONS

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AND ANIONS OF BASIC AND ACIDIC DYE
REPECTIVELY) its knows as STAINING TIME.

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TO DETERMINE THE DLC- (GRANULOCYTES

/AGRANULOCYTES)

TO STUDY THE MORPHOLOGY OF RBCS

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DETECT THE PRESENCE OF PARASITES LIKE MALARIA,

FILARIA

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SEX DETERMINATION CAN BE DONE BY IDENTIFICATION

OF BAR BODY

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FOCUS UNDER HIGH POWER (100X)

PUT ONE DROP OF CEDAR WOOD OIL

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1 0 - 1 4

CYTOPLASM PINK IN COLOUR

NUCLEUS HAVING 2 - 5 LOBES

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HAVING FINE PINK OR PURPLE GRANULES

PURPULE/PINK IN COLOUR

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1 0 - 1 4 MICRON/ NUCLEUS BILOBED PURPULE

COARSE ORANGE TO RED IN COLOUR

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BLUE COLOUR

GRANULES/ CYTOPLASM PINK COLOUR

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18-22/NOTCHED OR OVAL OR HORSE SHOE SHAPE

8 - 1 0 /NUCLEUS 2 - 3 LOBES/NOTPROPERLY

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NUCLEUS

VISIBLE BECAUSE OF GRANULES/

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NONE OR MODERATE COARSE AZURE GRANULES

COARSE BLUISH BLACK GRANULES OVERLYING
THE NUCLEUS

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CYTOPLASM SKY BLUE


LARGE LYMPHOCYTE

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SMALL LYMPHOCYTE

1 0 - 1 4

7 - 1 0

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SMALL ROUND NUCLEUS

NUCLEUS LARGE ROUND FILLS THE WHOLE CELL

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CYTOPLASM SKY BLUE

SKY BLUE THIN RIM

GRANEULS NONE OR FEW

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SKY BLUE/NONE OR MODERATEGRANEULES


CELLS

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SIZE

NUCLEUS

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CYTOPLASM

GRANULES

NEUTROPHIL

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1 0 - 1 4

2 - 5 LOBES

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PINK

FINE PINK OR PURPLE

PURPULE/PIN

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GRANULES

K

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EOSINOPHIL

1 0 - 1 4 MICRON

BILOBED PURPULE

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PINK

COARSE RED IN

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BLUE COLOUR

COLOUR

BASOPHIL

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8 - 1 0

2 - 3 LOBES/NOT

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PALE PINK

COARSE BLUISH BLACK

PROPERLY VISIBLE

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GRANULES OVERLYING

BECAUSE OF

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THE NUCLEUS

GRANULES

LARGE LYMPHOCYTE

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1 0 - 1 4

SMALL ROUND

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SKY BLUE

NONE OR FEW

NUCLEUS

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SMALL LYMPHOCYTE

7 - 1 0

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LARGE ROUND FILLS

SKY BLUE THIN RIM

SKY BLUE/NONE OR

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THE WHOLE CELL

MODERATE
GRANEULES

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MONOCYTE

1 8 - 2 2

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NOTCHED OR OVAL

SKY BLUE

NONE OR MODERATE

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OR HORSE SHOE

COARSE AZURE

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SHAPE

GRANULES

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NEUTROPHIL

50-70%

LYPHOCYTE

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20-40%

MONOCYTE

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2-8%

EOSINOPHIL

1-4%

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BASOPHIL

0-1%

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DRAW 100 SQUARES

IDENTIFY THE VARIOUS CELLS ENTER FIRST LETTER

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N-NEUTROHIL

E-EOSINOPHIL
B-BASOPHIL
L- LYMPHOCYTE

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M-MONOCYTE


NEUTROPHIL=

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? %

EOSINOPHIL =

? %

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LYMPHOCYTE=

? %

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MONOCYTE =

? %

BASOPHILS =

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? %

ABSOLUTE NEUTROPHIL =WBC count x neutrophil %= - - c u m m

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100


DETERMINATION OF THE PERCENTAGE DISTRIBUTION OF DIFFERENT

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TYPES OF NEUTROPHILS ON THE BASIS OF NUMBER LOBES IN THEIR
NUCLEUS

Normal count

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N1 - 2-10%

N2 - 20-30%

N3 - 40-50%

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N4 ? 10-15%

N5 ? 2-5 %

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SHIFT TO LEFT ?MORE YOUNGER CELLS (REGENARATIVE

SHIFT)

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SHIFT TO RIGHT - MORE OLDER CELLS (DEGENARATIVE
SHIFT)

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Textbook of Practical Physiology by

A.K.Jain

C.L. Ghai &

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G.K.Pal

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