The document describes the process of performing a differential leukocyte count (DLC) from a blood smear. It involves preparing a blood smear, staining it with Leishman's stain, then examining it under a microscope. The key steps are identifying and counting 100 white blood cells to determine the percentage of each type present - neutrophils, lymphocytes, monocytes, eosinophils and basophils. An increased percentage of a particular type can indicate different diseases or infections. The DLC provides information about the immune status and helps diagnose conditions affecting white blood cell levels.

1. DIFFERENTIAL LEUCOCYTE COUNT

2. INTRODUCTION
 The differential WBC count is performed to
determine the relative number of each type of WBC
in the blood. In disease states a particular white
blood cell type shows increase in number in the
blood.

3. OBJECTIVES
 1. Describe the relevance and special importance ofpreparing and staining a
blood smear and doing the differential leukocyte count.
 2. Name the components of Leishman’s stain and explain the function of each.
 3. Prepare satisfactory blood films, fix and stain them, and describe the
features of a well-stained film.
 4. Identify different blood cells in a film, and indicate the identifying features of
each type of leukocyte.
 5. Differentiate between neutrophils, eosinophils, and basophils and between a
large lymphocyte and a monocyte.
 6. Carry out the differential count and express your results in their percentages
and absolute numbers.
 7. Describe the functions of each type of leukocyte
 8. List the conditions in which their numbers increase and decrease.

4.  AIM
 APPARATUS
 PRINCIPLE
 PROCEDURE
 OBSERVATION and CALCULATION
 RESULT
 COMMENT
 PRECAUTIONS
 DISCUSSION

5. A
PPARATUS
&
MATERIALS
 Microscope
 3 to 4 Glass slides
 sterile needle or Lancet
 Cotton swab, spirit
 Leishman’s stain
 Distilled water
 Staining rack
 Cedar wood oil

6. Leishman’s stain: It is probably one of the simplest and
most precise methods of staining blood for diagnostic
purposes.
Romanowsky stain, contains acidic and basic dye.
 i. Eosin. It is an acidic dye (negatively charged) and stains
basic (positive) particles—granules of eosinophils, and
RBCs a pink color.
 ii. Methylene-blue. It is a basic dye (positively charged)
and stains acidic (negatively charged) granules in the
cytoplasm, nuclei of leukocytes, especially the granules of
basophils, a blue-violet color.

7.  iii. Acetone-free and water-free absolute methyl alcohol.
The methyl alcohol is a fixative and must be free from
acetone and water. It serves two functions:
 a. It fixes the blood smear to the glass slide.
 The alcohol precipitates the plasma proteins, which then act
as a ‘glue’ which attaches (fixes) the blood cells to the slide so
that they are not washed away during staining.
 b. The alcohol preserves the morphology and chemical
status of the cells.

8. PRINCIPLE
 A blood film is stained with Leishman’s stain and
scanned under oil immersion, from one end to the
other. As each WBC is encountered, it is identified
until 100 leukocytes have been examined. The
percentage distribution of each type of WBC is then
calculated.
 Knowing the TLC and the differential count, it is
easy to determine the number of each type of cell
per mm3 of blood.

9. PROCEDURE
Prepare the blood smear
 Clean the slides, 2 to be covered with the
blood film and one to be used as spreader.
 Clean the finger with alcohol, allow it to dry
and then prick it with a disposable lancet to
obtain a drop of blood.
 Make a fine touch of one end of a slide with
the drop of blood (only a small amount is
required).
 Place the edge of the other slide on the
surface of the first slide just in front of the
drop of blood and at an angle of 45˚.

10.  Draw the spreader back until it makes contact with
the drop of blood.
 Push the spreader slowly and smoothly to the other
end of the slide.
 Allow the film to dry at room temperature i.e. the
blood smears should be air-dried

16. Smear Preparation

17. STAINING THE BLOOD SMEAR BY:-
 Put the dried slide on a staining rack.
 The blood smear should be stained as soon as
possible.
 Carefully drop Leishman's stain onto the blood film ( 8
to 10 drops )until the film is covered.
 Allow the stain to act for two minutes- fixation time.
 Add distilled water ( 20 drops) to the stain and mix by
blowing, this gives dilution of or 1:2
 The diluted stain should act for 10 minutes ( staining
time) -greenish scum
 Then wash it off with distilled water, continue washing
until the film has a pink color.
 Shake off excess water and allow it to dry at room temperature

18. EXAMINATION OF THE STAINED SMEAR
 For examining the blood smear a microscope with a
low-power objective (10 x) and an oil immersion
objective (100 x) is necessary.
 Place the slide (smear side up) ?? on the microscope
stage.
 Examine the blood smear using the low power (10 x)
objective. Choose an area where there are plenty of
WBCs. This area is usually located near the wedge
shaped end of blood smear.
 Place a drop of immersion oil on the selected site and
carefully change to the oil immersion objective (100x)
 Perform the differential cell count and, at the same
time examine the morphology of the WBCs.

19. Blood smear under microscope

20. Counting directions

21. COUNTING 100 CELLS
N N M N N N N E L N
N N N L N N Neutrophil = %
Eosinophil = %
Basophil= %
lymphocyte = %
Monocyte = %

22. CALCULATION
 Count each WBC seen and record on a differential cell
counter until 100 WBCs have been counted. For instance
if 25 of the 100 WBCs where lymphocytes, then the
percentage of lymphocytes is 25%.
 The normal range percentage of the different types of
WBCs is as follows:
 Neutrophils 50-70%
 Eosinophils 1-4%
 Basophils 0.4%
 Monocytes 2-8%
 Lymphocytes 20-40%

23.  Result
 comment

24. PRECAUTIONS
 1. The slides should be absolutely free from dust and
grease, because blood will not stick to areas where oils from
your fingers have been left.
 2. The edge of the spreader should be smooth and not
chipped, otherwise the slide would leave striations along or
across the smear. Leukocytes may also be caught in chipped
places and be carried towards the tail.
 3. When applying the slide to the blood drop from a finger-
prick, do not touch the skin with the slide, but only the
periphery (top) of the blood drop. This is to avoid taking up
epidermal squames or sweat.

25.  4. The blood should be spread immediately after taking it
on the slide. Any delay will cause clumping of cells due to
partial coagulation. This will give a ‘granular’ appearance to
the blood film, which is visible to the naked eye.
 5. The angle of the spreader should be 35° to 40°. The
more the angle of the spreader approaches the vertical, the
thinner the film, and the lesser the angle, the thicker the film.
 6. The pressure of the spreader on the slide should be
slight and even and the pushing should be fairly quick
while maintaining a uniform pressure throughout.

26.  7. The film should be dried by waving it in the
air immediately after spreading it. A delay can
cause not only clumping, but also crenation and
distortion of red cells in a damp atmosphere (if
water is allowed to slowly evaporate from the blood
plasma on the slide, crenation occurs due to
gradual increase in the concentration of salts).
 8. By turning the spreader over, you can use it to
make 4 blood films.

27.  11. Do not allow Leishman stain to become
syrupy (thick) or dry up on the slide as this is
likely to cause precipitation of the stain.
 12. When counting the cells, use the “battlement”
method. This is to avoid counting a cell twice.
 13. Avoid counting the leukocytes in the extreme
ends of the head or tail, and along the edges of
 blood film

28. DISCUSSION
 Magnification
 Why oil is placed while using this objective.
 Why cedar wood oil is preferred.
 Features of an ideal blood film
 1. The blood film should occupy the middle two-thirds (about 5 cm)
of the slide, with a clear margin of about 2 mm on either side.
 2. It should be tongue-shaped, i.e. broad at the head (starting point),
and taper towards the other end, but without any ‘tails’.
 3. It should be translucent, uniformly thick throughout, with no
vacant areas, striations (longitudinal or transverse), or
‘granular’ areas.
 4. It should be neither very thick nor very thin (this can be learned
only with practice). A thin film looks faintly pink against a white
surface, while

29.  a thick smear appears red. An ideal film appears ‘buff’
colored.
 5. The red cells, as seen under the microscope, should lie
separately from each other, just touching here and there,
but without any crowding, or rouleaux formation.

30.  Method used for identification and counting the
cells
 How to indicate the presence of the cell when
projected
 Relevance
 The differential count is done to find out if there is an
increase or decrease of a particular type of WBC. Knowing
the TLC, the absolute number of each type can be calculated.
This information is important in detecting infection or
inflammation, allergic and parasitic infections, and effects
of chemotherapy and radiation therapy.

31.  Why acetone free and water free
 Fixation time
 Staining time
 Absolute leucocyte count
 Any other cells seen in the smear
 Buffered water/ distilled water
 Tap water used?
 Approximate size of wbcs
 Dlc of child
 Physiological variations in count of leucocytes

32.  Absolute leukocyte count.
 The absolute values are more significant than the
DLC values alone. The reason is that the DLC may
show only a relative increase or decrease of a
particular type of cell with a corresponding change
in the other cell types.
 For example, a neutrophil count of 85% may suggest
neutrophilia, but if the TLC is, say, 8000/mm3, then the
absolute neutrophil count of 6800/mm3 (8000 × 85/100 =
6800) would be within the normal range.

33. TYPES OF WHITE BLOOD CELLS
33
• White blood cells:
• Are leukocytes
•There are five types of WBCs in two categories:
• Granulocytes
• Neutrophils
• Eosinophils
• Basophils
• Agranulocytes
• Lymphocytes
• Monocytes

35. NEUTROPHILS
35
•Size – 10-14 µm.
•Multilobed nucleus
•Light purple (neutral)
granules
•Other names is
Polymorphonuclear leukocyte
• Phagocytic in function
• Normal range = 50% - 70%
• Elevated in acute bacterial
infections

36. EOSINOPHILS
36
• Size – 10-15 µm
•Coarse red color granules
•Bi-lobed nucleus
• Defend against parasitic
worm infestations
• Normal range 1% - 6%
• Elevated in parasitic
worm infections and
allergic reactions

37. BASOPHILS
37
•Size – 10 – 15 µm
•Bilobed nucleus
•Deep blue granules
covering the whole nucleus
• Release histamine
• Release heparin
• Normal range- Less than
1%

38. MONOCYTES
38
• Largest of all blood cells
•Size – 12 – 20 µm
•Spherical, kidney-shaped,
oval or lobed nuclei
•No visible granules
• May leave bloodstream to
become macrophages
• Normal range - 2% - 10%
• Phagocytize bacteria, dead
cells, and other debris
•Increase count – in chronic
infections eg. Tuberculosis

39. LYMPHOCYTES
39
•Size – 7-9 or 10-15 µm
•Slightly larger than RBC
• Large spherical nucleus
surrounded by thin rim of
cytoplasm
•No visible granules
•Normal range -20% - 40%
•T cells and B cells
• Both important in
immunity
• B cells produce antibodies
•T cells involved in cell
mediated immunity
• Increased count is seen
in viral infections,
chronic infections

41. APPLICATIONS OF BLOOD SMEAR:
 to detect infection or inflammation,
 determine the effects of possible poisoning by
chemicals, drugs, chemotherapy, radiation, etc.
 DLC is also done to monitor blood diseases
like leukemia,
 or to detect allergic and parasitic infections.
 The determination of each type of WBC helps
in diagnosing the condition because a particular
type may show an increase or decrease.
 Can know the gender of the person

42.  Normal values. The normal values for
differential and absolute counts are given
below:
 Differential count (percent) Absolute
count (per mm3)
 Neutrophils 40–75 2000–7500
 Eosinophils 1–6 4–440
 Basophils 0–1 0–100
 Monocytes 2–10 500–800
 Lymphocytes (both) 20–45 1300–3500

43. WBC type high count may
indicate
low count may
indicate
Neutrophils bacterial infection,
burns, stress,
inflammation
radiation exposure,
drug toxicity, vitamin
B12 deficiency,
systemic lupus
erythematosus (SLE)
Eosinophils allergic reactions,
parasitic infections,
autoimmune diseases
drug toxicity, stress
Basophils allergic reactions,
leukemias, cancers,
hypothyroidism
pregnancy, ovulation,
stress,
hyperthyroidism
Lymphocytes viral infections, some
leukemias, Bacterial
infections.
prolonged illness,
radiation. AIDS,
immunosuppressio
n, treatment with
cortisol
Monocytes viral or fungal
infections,
tuberculosis, some
leukemias, other
chronic diseases
bone marrow
suppression,
treatment with
cortisol

44. Thank you