1. DLC BY PERIPHERAL SMEAR
COMPARISION WITH AUTOMATED
DIFFERENTIAL COUNT
Dr Nikhil Bansal
J.N.M.C.,Wardha

2. ACKNOWLEGMENT
 Skill based project is an art to enhance the creativity
of a student and to increase his/her interest in clinical
basis.
 As per student manner I firstly would like to thank
“Dr. S. Vagha Ma’am” for arranging such project. I
also thank “Dr Samarth Shukla Sir” for helping us
whenever required. My pay my heartful gratitude to
“Dr.Vijay Kumar Dembra Sir” for guiding us
throughout the project.
 Last but not the least I thank all my “group mates”
for co-operating with me and lending a helpful hand
when required.

3. INTRODUCTION
WBC or leukocyte is the colourless and
nucleated formed element of blood. Leukocyte
play very important role in defense mechanism of
body.
Leukocyte are classified into 2 types namely –
1. Granulocyte-with granules
2. Agranulocyte- without granules
The granulocyte are-Neutrophills
Eosinophills
Basophills
The agranulocyte are-Monocytes
Lymphocytes

4. MORPHOLOGY
Neutrophils-neutrophils have fine or small granules in
the cytoplasm granules appear violet in colour. The
nucleus is multilobed.nucleus has 4-5 lobes.
diameter – 10-12 microns
Eosinophils-eosinophils have larger granules in the
cytoplasm which stain bright red colour.the nucleus is
bilobed.
diameter-10 and 14 microns
Basophils-basophils also have coarse granules in the
cytoplasm.the granules stain purple blue with basic dyes
like methylene blue.
diameter-8 to 10 microns

5. Monocytes-monocytes are largest leucocytes with
diameter of 14 to 18 microns.the cytoplasm is clear
without granules.the nucleus is
kidney,round,oval,horseshoe shaped.
Lymphocytes-the lymphocyte also have clear cytoplasm
without granules.the nucleus is ovel shaped occupying
the whole of the cytoplasm.depending upon the size the
lymphocyte are divided into two group as:
large lymphocytes-the younger cell with a diameter of
10 to 12 microns.
Small lymphocytes-the older cell with a diameter of 7
to 10 microns
Depending upon the function,the lymphocytes are divided
into 2 types as:
T lymphocytes-concerned with cellular immunity
B lymphocytes-concerned with humoral immunity

7. NORMAL COUNT OF WBC
Total WBC count – 4,000 to 11,000/cu.mm of
blood.
Differential WBC count:
LEUCKOCYTE PERCENTAGE ABSOLUTE
VALUE
Neutrophils 50 to 70 % 3000-6000
Lymphocytes 20 to 30 % 1500-2700
Eosinophils 2 to 4 % 150-450
Monocytes 2 to 6 % 200-600
Basophils 0 to 1 % 0-100

8. CONDITION IN WHICH
ALTERATION IN DLC
Neutrophilia-increased in neutrophil
count called neutrophilic leukocytosis.this
occurs in the following condition:
1) Acute infections
2) Metabolic disorders
3) Injection of vaccines
4) After acute hemorrhage

9. Lymphocytosis - increasd in lymphocyte
count is called lymphocytosis and this
occurs in
1) Diptheria
2) Infectious hepatitis
3) Mumps
4) Malnutrition
5) Rickets
6) Syphilis

10. Eosinophilia-increasd in eosinophil count
is called eosinophilia and this occurs in
1) Allergic condition
2) Asthma
3) Blood parasitism
4) Scarlet fever

11. Monocytosis-increasd in monocytes
count is known as monocytosis and
occurs in
1) Tuberculosis
2) Syphilis
3) Malaria
4) Kala azar

12. Basophilia-increased in basophill count is
called basophilia and it occur in
1) Smallpox
2) Chicken pox
3) Polycythemia vera

13. AIM & OBJECTIVES
“ To determine the Differential leukocyte
count of the given sample in peripheral
smear and compare it with the
automated cell counter ’’
 To compare differential leukocyte count
in peripheral smear with automated
cell counter.
 To check the accuracy of automated cell
counter.

14. MATERIAL & METHOD
MATERIAL:-
 Cell counter
 Microscope
 Cotton
 Glass slide
 Leishmen’s stain
 Distilled water
 Needle
 Staining track

16. METHODS:
1) Slide method
-place a drop of blood in the centre of a clean glass
slide 1 to 2 cm from one end.
-place another slide with smooth edge at an angle
of 30-45 0 near the drop of blood.
-move the spreader backward so that it makes
contact with drop of blood.
-then move the spreader forward rapidly over the
slide.
-a thin peripheral blood film is thus prepared.
-Dry it and stain.

18.  Qualities of a good blood film-
1) It should not cover the entire surface of
slide.
2) It should have smooth and even appearance.
3) It should be free from waves and holes.
4) It should not have irregular tail.
Parts of a thin blood film-consists of 3 parts
1) Head-i.e. the portion of blood film near the
drop of blood.
2) Body-i.e. the main part of blood film.
3) Tail-i.e. the tapering end of the blood film.

19. 2) cover glass method-
-Take a no.1 (22 mm square)clean cover slide.
-Touch it on to the drop of blood.
-place it on another similar cover glass in cross wise direction
with slide containing drop of blood facing down.
-pull the cover glass quickly.
-Dry it and stain.
3) spine method-
This is an automated method
-place a drop of blood in the center of a glass slide.
-spin at a high speed in a special centrifuge cytospin.
-blood spreads uniformly.
-dry it and stain it.

20. PROCEDURE FOR STAINING
-pour leishman’s stain drop wise on the slide and wait
for 2 min.this allows fixation of the PBF in methyl
alcohol.
-add double the quantity of buffered water drop wise
over the slide.
-mix by rocking for 8 mints.
-wash it water for 1 to 2 mints.
-dry in air and examine under oil immersion lens of the
microscope.

21. COMPONENT OF LEISHMEN’S
STAIN
-preparation dissolve 0.2g of powdered
leishmens dye in 100 ml of acetone free
methyl alcohol in a conical flask.
-warm it to 50˚c for half an hour with
occasional shaking.
-cool it and filter it.

22. OBSERVATION
NAME A/S MRD
CELL COUNTER(%) PEREPHERAL SMEAR (%)
NO.
LYMP GRA MID P L E M B
H
1 SAINA 4/F 987808 21.0 74.7 4.3 75 22 02 01 00
2 KAVITA 22/F 907818 40.0 54.6 5.4 55 40 03 02 00
3 SUMAN 60/F 789149 21.6 74.3 4.1 76 20 02 02 00
4 SINDHUB 52/F 906676 46.4 47.4 06 48 47 03 02 00
AI
5 NOOTAN 6/F 906739 27.4 67.9 4.7 70 27 02 01 00

23. NAME A/S MRD
NO. CELL COUNTER(%) PEREPHERAL SMEAR (%)
LYMP GRA MID P L E M B
H
6 RAVI 7/M 908240 44.7 48.7 6.6 49 47 05 01 00
7 DEVASHIS 5/M 908241 50.0 42.4 7.6 47 50 02 01 00
8 SHALU 27/F 908285 31.6 63.4 5.0 65 32 02 01 00
9 MOTIRAN 72/M 908277 27.7 66.9 5.4 66 30 03 01 00
M
10 SUNITA 20/F 908266 31.0 61.5 7.5 65 32 02 01 00

24. RESULT
- As per the above observation we can see
that there isn’t much of a difference
between cell counter reading and the
reading by peripheral smear.
- Hence after the comparative study
between the two we can conclude that
cell counter reading are equally efficient
and automat zed reading scan be
preferred over the manual to save time.

25. DISCUSSION
Causes of alteration in DLC count-
1) Errors in calculation
2) Increased erythropoietin in a blood sample.
3) Improper admixture of anticoagulant in a blood sample.
4) If blood sample is clotted.
5) If sample collected in a plain blub.
6) Overstaining.
7) Suboptimal staining.
As per the causes mentioned above before going to
analysis or observe for differential leukocyte count one
must nn of all those things to avoid errors in differential
leukocyte count.
Accordingly some of the precautions to be taken by
examining the peripheral smear or analyzed by
automated cell counter