Peripheral blood Smear Preparation

Blood Smear Examination: Powerpoint prepared by Dr. Raihan Mannan

1. BLOOD SMEAR
EXAMINATION
Making Blood smear
Raihan Mannan
JR-1, Deptt. of Physiology
JNMCH, AMU, Aligarh

2. A WELL MADE AND WELL
STAINED SMEAR CAN PROVIDE:
Estimates of cell count
Proportions of the different types of WBC
Morphology
Objective:
1. Specimen Collection
2. Peripheral Smear Preparation
3. Staining of Peripheral Blood Smear
4. Peripheral Smear Examination

3. PREPARATION OF BLOOD SMEAR
There are three types of blood smears:
1.The wedge smear.
2.The cover glass smear.
3.The spun smear.
The are two additional types of blood smear used
for specific purposes
1.Buffy coat smear for WBCs < 1.0×10
9
/L
2.Thick blood smears for blood parasites .

4. WEDGE BLOOD SMEAR
Specimen : venipuncture, EDTA blood within 2
to 3 hours & collected to the mark on tube or by
pricking finger.
May change in RBCs morphology such as
Spiculated (crenated) cells if :
1.Excessive amount of anticoagulant to specimen
2.Old blood - long standing.
3.Warm environment (room temperature) may
hasten changes.

5. PROCEDUREPROCEDURE EE
Prick the finger or placing a drop of blood from mixed
sample on a clean glass slide.
Place the Spreader slide on the surface of slide just
infront of the drop of blood at an angle of 45 degree.
Draw the spreader backward so as to touch the drop
and hold there tilll the blood runs along the full width
of spreader
Spreader is then moved slowly and smoothly to the
other end of slide maintaining the angle of 45°
Allow the blood film to air-dry completely before
staining. (Do not blow to dry. The moisture from
your breath will cause RBC artifact.)

9. CHARACTERISTICS OF A GOOD
SMEAR
Smear should cover 2/3 of the entire slide
Smear is tongue shaped with no tails at the
end.
Neither too thin nor too thick (uniform)
Smear is smooth without irregularities,
holes or striations or air-gap
When held up in light: feathery edge should
show rainbow appearance

10. tail body head

11. MORPHOLOGIC CHANGES DUE TO
AREA OF SMEAR
Thin area- Spherocytes which are really
"spheroidocytes" or flattened red cells.
True spherocytes will be found in other
(Good) areas of smear.
Thick area - Rouleaux, which is normal
in such areas. Confirm by examining thin
areas. If true rouleaux, two-three RBC's
will stick together in a "stack of coins"
fashion..

12. COMMON CAUSES OF A POOR
BLOOD SMEAR
1.Drop of blood too large or too small.
2.Spreader slide pushed across the slide in a jerky
manner.
3.Failure to keep the entire edge of the spreader slide
against the slide while making the smear.
4.Failure to keep the spreader slide at a 30° angle with
the slide.
5.Failure to push the spreader slide completely across
the slide.
6.Irregular spread with ridges and long tail: Edge of
spreader dirty or chipped; dusty slide
7.Holes in film: Slide contaminated with fat or grease
8.Cellular degenerative changes: delay in fixing,
inadequate fixing time or methanol contaminated
with water.

13. PERIPHERAL BLOOD SMEAR
Examples of unacceptable smears

14. PERIPHERAL BLOOD SMEAR
Examples of unacceptable smears

15. SLIDE FIXATION & SLIDE FIXATION &
STAININGSTAINING
LEISHMAN'S STAINLEISHMAN'S STAIN

16. ROMANOWSKY PRINCIPLE ROMANOWSKY PRINCIPLE
Leishman's stain : a polychromatic stain
Acetone free methyl alcohol : as a fixative
fixes cells to slide (precipitation of protein)
Preseves the cells
Methylene blue: basic dye and stains cytoplasm,
nuclei of WBCs and granules of basophils
=== blue color
Eosin: acidic dye and stains RBCs and granules
of eosinophils
=== orange-red color
pH value of phosphate buffer is very important

17. FIXING AND STAINING PROCEDURE FIXING AND STAINING PROCEDURE
Fixing the blood smear:
Place the smear across two parallel glass rods.
Pour 8-10 drops of leishman’s stain. Leave it for about
2 min (fixation time)
Staining the smear:
After 2 min add an equal amount of buffer solution
and mix the stain by blowing air intermittently with
the help of dropper.
Leave the mixture on the slide for 10-15 min.
Wash the slide by running water, hold the slide
slanted and water is allowed to flow on thumb until
film gets a pinkish tinge.
Wipe clean the back of slide
Stand slide upright and let dry in air.

18. QUALITY OF STAINED SMEARQUALITY OF STAINED SMEAR
Smear is single-cell thick, no overlapping of cells
and uniformly distributed
Atleast 1 WBC per high power field (100x)
RBCs are stained light pink
Overstained smear:RBCs look bluish & WBCs
look purple
Understained smear: RBCs look very pale &
WBCs almost colourless

19. CAUSES & CORRECTION
Too Acid Stain:
1.insufficient staining time
2.prolonged buffering or washing
3.old stain
Correction:
1)lengthen staining time
2)check stain and buffer pH
3)shorten buffering or wash time

20. Too Alkaline Stain:
1.thick blood smear
2.prolonged staining
3.insufficient washing
4.alkaline pH of stain components
 Correction :
1)check pH
2)shorten stain time
3)prolong buffering time

21. TOO ACIDIC SUITABLE TOO BASICTOO ACIDIC SUITABLE TOO BASIC

22. PRECAUTIONS:
Slides should be clean & grease free.
Spreader should have a smooth clean edge.
Do not heat dry the smear.
Stored blood should not be used for making
smear.
Assess the quality of smear both grossly &
microscopically.

23. PERFORMING A PERFORMING A
MANUAL MANUAL
DIFFERENTIAL AND DIFFERENTIAL AND
ASSESSING RBC ASSESSING RBC
MORPHOLOGY MORPHOLOGY

24. Observing Observing
direction:direction:
Observe one field and record the number of WBC
according to the different type then turn to another field
in the snake-liked direction
*avoid repeat or miss some cells

25. MANUAL DIFFERENTIAL COUNTSMANUAL DIFFERENTIAL COUNTS
These counts are done in the same area as
WBC and platelet estimates with the red
cells barely touching.
This takes place under × 100 (oil) using
the zigzag method.
Count 100 WBCs including all cell lines
from immature to mature.
Reporting results
Absolute number of cells/µl = % of cell type
in differential x white cell count

26. N M N N N L N L N
N: NEUTROPHIL
L: LYMPHOCYTE
M: MONOCYTE
E: EOSINOPHIL
B: BASOPHIL

27. MORPHOLOGY MORPHOLOGY
OF WBCOF WBC
IN PERIPHERAL IN PERIPHERAL
BLOODBLOOD
Normal Normal

28. NORMAL PERIPHERAL BLOOD NORMAL PERIPHERAL BLOOD
SMEARSMEAR

30. NEUTROPHIL

31. NEUTROPHIL

33. EOSINOPHIL

34. EOSINOPHIL

36. BASOPHIL

37. BASOPHIL

38. LYMPHOCYTE

39. LYMPHOCYTE
Ly

41. MONOCYTE

42. MONOCYTE

45. ABNORMAL CHANGES
OF WBC MORPHOLOGY

46. ARNETH COUNT:

Left to shift or regenerative shiftLeft to shift or regenerative shift::
If N1+N2+N3 exceeds 80%If N1+N2+N3 exceeds 80%
Indicates hyperactive bone marrowIndicates hyperactive bone marrow
Causes:Causes:
1. Acute pyogenic infections.
2. Tuberculosis: Though there is lymphocytosis, a shift
to the left may be due to removal of older neutrophils
from the blood.
3. Hemorrhage.
4. Low-dosage irradiation is said to stimulate bone
marrow while heavy doses cause a shift to the right.

47. Right to shift or degenerative shift:Right to shift or degenerative shift:
If N4+N5+N6 exceeds 20%If N4+N5+N6 exceeds 20%
Indicates hypoactive bone marrowIndicates hypoactive bone marrow
Causes:Causes:
1. Bone marrow depression (hypoplasia and aplasia) due to
any factor.
2. Drugs, toxins, chemical poisons.
3. Megaloblastic anemia.
4. Septicemia.
5. Uremia.

48. LEFT SHIFT
N1 N2 N3 N4 N5&6

49. TOXIC GRANULATION

50. AUER BODIES(AUER ROD)

51. HYPERSEGMENTATION

52. Anisocytosis of neutrophil

53. vacuolization

54. Degeneration of nucleus

55. Dohle body