;pe:oe01~

11 uifter~ntial Leucocyte Count ,

LEARNING OBJECTIVES

After completing this practical you WILL be able to:

1. Describe the importance of performing DLC in
the same field, which are approximately 7.5 µm in
diameter. The various characteristics of the nucleus
and the cytoplasm of the cells are considered for
identifying leucocytes. The features of the nucleus that
i
practical physiology. are considered include the shape and size compared
2. Select a good spreader slide. t o the rest of the cell, and the pattern of chromatins
3. Prepare a good smear for [Link] DLC. in the nucleus. T he features of t he cytoplasm th at are
4. Identify the leucocytes. considered include the presence or absence of granules,
5. Perform a differential count within the stipulated the nature of granules and their st aining characteristics
time. and colour, and the relative am ount of cytoplasm.
6. Describe the structure and functions of various
leucocytes. STRUCTURE AND FUNCTIONS OF
7. List the common causes of increase or decrease in LEUCOCYTES
different leucocytes.
You MAY also be able to: Neutrophils are the commonest leucocytes. They
1. Explain the functions of different leucocytes. constitute 50- 70 per cent of the total leucocytes.
2. State the physiological basis of alteration in cell Typically, there are two types: segmented and band
counts in different clinical conditions. neutrophils.

Segmented Neutrophils
INTRODUCTION

The types and numbers of each type of leucocyte . meter of neutrophils (Fig. 10.4) varies from 10
counted are traditionally reported as percentages. , 4 ~ d the nucleus forms a relatively small part
etermination of percentage distribution of ~
of t e cell. The nucleus can assume various shapes,
leucocytes in peripheral blood is known as differential but its usual configuration is lobular (a series of lobes
leucocyte count (DLC). Increase or decrease in connected by narrow st nds of chromatin filaments).
individual cell lines are reported separately and It usually contains -5 lo e , but sometimes may
give more meaningful info rmation to the physician contain more o nucleo are visible in the nucleus.
regarding the status of the leucocytes in the blood. The cytoplasm has a amt pink colour and contains a
Five types of leucocytes are encounter ed in normal lar ge number of fine p~ _gr~ les. About two-thirds
peripheral blood . They are divided into ·granulocytes of the granules are specific?"n ~ tro ilic ules, while
and agranulocytes. N eutrophils, eosinophils and the remaining one~third are az ilic granules.
basophils are granulocytes, and ly mphocytes and
monocytes are agranulocytes. In 15-30 per cent of Functions
monocytes, [Link] granules are seen. H owever, since N eutrophils are actively phagocyti&· The granules have
this is not a constant feature, mon ocytes are classified , . many ~ s and appear to be lysosomal
under agranulocytes. :o: in character. Phagocytosis occurs through a series
The size of the cells, whether small, medium or.C · of events, which include opsonisation, chemotaxis,
large, cannot be directly measured in che smear in the:! : ingestion and degranulation. During phagocytosis, a
micr?scopic field but c'.111 easily be compared with:p ; numb~r of chemicals are _released, such as hy~en
the size of the surrounding normal red blood cells in peroxide, and hypif1onte and hy ~ l raaicals,
 Different ial Leucocyte Count 71

all of which Stru~ture

form the They are about the @IJJe me as neutrophilD
infections. T he nucleus occupies a relative! reater orti n of
the cell. T he n eus is often irre lar in sha The
Band Neutrophils cytoplasm is colourless and contains coarse granules.
The nucleus and cytoplasm are very often totally
T hese neutrophils are younger neutrophils. The obscured by granules which are large, deeply stained
relative band neutrophil count in adults is l-5 per cent and blue-black or dee brown in colour.
(of the total neutrophils). G'Anct.? , ~ .
Functions fil\ c. &. AnT1-~
~tructure T he exact Jfunctih" basoph ~ not known aso~hil
Band neutrophils resemble segmented neutrophils granules contain rin, histah11ne and a sl,~ ::re,~.ng
except for the shape of their nucleus. The nucleus substance (SRS). The cells are(ppagoc C After their
may be rod- or band-shaped. There may be slight activities in the blood, they ent~e issue.
indentations, but without definite lobes. The number
of granules in band neutrophils is more than in
Monocytes
segmented neutrophils.
Monocyte.s constitute 2-8 per cent of the total
Functions leucocytes.
T he functions are the same as in segmen,,...._~
except that the cells are more active
Monce e:s i . 10.8 are the largest leucocytes,
Eosinophils 1 - m in diameter. The nucleus is large and
occupies 50 per cent of the cell. T he nucleus is
Structure frequently kidney-shaped or horseshoe-shaped, but
Eosinophils (Fig. 10.5) are slightly larger than it may be oval, lobular, notched or polymorphic.
neutrophils. Their diameter is 10- 14 µm . It never undergoes segmentation. N uclear chromatins
T he nucleus usually h lobes sometimes 3 . The are sharply segregated and distributed in a linear
lobes are plumper. · are visible. The arrangement of delicate strands which gives the nucleus
cytoplasm is packed with brick-red coarse granules. a stringy appearance. T he cytoplasm is abundant and is
T he cytoplasm is pale blue, but is usually obscured ground-glass or muddy blue in appearance. Sometimes,
by granules. T he granules are hard, firm bodies that it contains extremely fine pink granules. The granules
are not easily damaged. Eosinophilic granules are also are [Link].
highly refractile, a feature that is often valuable in -~,--..,..-,...; f-"{e,,
--=-:---.--r---- = , - ~
distinguishing them from neucrophilic granules. Functions .I""> mru:.w ,c. APU
. Monocytes are c ive a oc . ey remain
Functions ~ -R-nti-all.~,c.., ~Cl9Vict&n\J~9'9~&ln circulation for a few hours and then migrate into
Eosinophils are involved in defendi~the
- body from the tissues and transform clu;msehres iero tissue
allergic reactions. Eosiqm,hil ~ ml contain a ~coph?,!~S. They are the second line of defence
n~ber of che~als like 'MBP (maJOr asic proteins), ohhe body. T hey protect the body against bacteria,
pr'Brein X, ECP="'A (~ophil chemotactic factor of viruses, fungi and parasites. They also participate in
anaphylaxis) and lys~mes that neutralise allergens immunity . They act as the antigen-presenting cells,
and are also larvicidal and parasiticidal. tJ't1<;"7'- and also secrete a number of cytokines that mediate
various immunologic responses.
Basophils
Lymphocyt~s
Basophils are the~arest leucocyte.t)onst ituting 0 ~
cent of the total eucocyte population in peripheral Lymphocytes constitute 20-40 per cent of leucocytes
blood. in adults. But the count is slightly higher in infants
72 Chapter 11

and children, who have more lympho~ytes and less Normal Count
neutrophils.
For a differential leucoctye count, a mm1mum of
Structure 100 cells should be counted, though ideally, 200 or
Structurally, lymphocytes are of two types- small more cells should be counted to find out the more
and large. The majority (80 per cent) of the circulating accurate percentage of each type of leucocyte. The
lymphocytes are small. Only 20 percentoflymphocytes percentage distribution of cells in a normal leucocyte
are large. cQunt in adults is: ~.

Small lymphocytes eutrophil 50-70 per cent

The size of small lymphocytes (Fig. 10.7) is the same "'Eosinophil 1-4 per cent
as or slightly bigger than the red cells. Their diameter
"Basophil : 0-1 per cent
ranges from 7 to 10 µm. The cells are mainly occupied
by the nucleus. The nucleus is round or slightly Lymphocyte : 20-40 per cent
notched. Almost the entire nucleus stains deep purple. "M'onocyte : 2-8 per cent
The cytoplasm may not be present, but there may be a Infants and children have fewer neutrophils (40-60
thin rim of cytoplasm to one side at the periphery. per cent) and more lymphocytes (25-55 per cent).
Large lymphocytes
Large lymphocytes (Fig. 10.6) are the same size as or METHODS OF COUNTING
slightly larger than neutrophils. Their diameter varies
Principle
from 10 to 16 µm, but may be as much as 20 mm.
A blood film stained with the Leishman stain is
A large lymphocyte is confused with monocytes.
examined undef"'an oil-immersion objective and the
The nucleus is large and occupies about 90 per cent of
different types of white blood cells are identified.
the cell. The nucleus contains homogenous chromatin
The percentage distribution of these cells is then
with some clumping at the nuclear periphery.
determined.
This gives the nucleus of lymphocytes a compact ..
appearance in contrast to the spongy appearance of Re uirements
the nucleus of monocytes. The cytoplasm is clear and 1. Glass slides
nary blue in colour, in contrast to the hazy or turbid 2. Leishman stain
cytoplasm of monocytes. The cytoplasm contains 3. Microscope
no granules, but on very rare occasions, a few fine 4. Distilled water
azurophil granules may be seen. 5. Pasteur pipene
C)'I\ I.
Functions < ftM 1: ·
Lymphocytes mediate the llI!fJlunologic res12qnses ofthe
Procedure
1. Prepare blood smears and stain the smears with
body. Functionally, lymphocytes are divided broadly Leishman stain as described in Chapter 10.
into two categories: T lymphocytes and B lymphocytes. 2. First,examinethesmearunderalow-power~tive
The majority (80per cent) of the circulating lymphocytes for tneral scanning and ~sessing the quality of the
are T lymphocytes. T lymphocytes mediate cellular sme , and for stuaying the distribution panern of
immunity, which is concerned with viral and fungal the cells.
infections; .p~asitic infestation, cancer cells, transplant 3. Place a drop of cedar wood oil on the smear at the
rejection and rejection of tumour cells. B lymphocytes feather edge. Bring the oil-immersion objective into
mediate hurnoral immunity by producing antibodies position and make the lower end of the objective
~oncemed with protecting the body against bacterial touch the drop of oil. Using the fine adjustment
and other infections. B lymphocytes do not directly screw, adjust the objective and focus the cells.
synthesise antibodies, which are produced by plasma cells. 4. For counting cells, start from one end of the smear
B cells, on specific immunologic stimulation, transform and move the slide in a zigzag manner (Fig. 11.1).
themselves into plasma cells. Therefore, plasma cells are Count individual white cells that you come across
not normally found in the blood. and enter your observation in a table (as described
 Differential Leucocyte Count 73

below) in your rough notebook. Continue cour,[Link]

F
until 100 cells are counted.
5. Calculate the percentage of each leucocyte and
report your observation.

I Precautions

~
All the precautions described in the previous chapter are
Fig. 11 .1. Zigzag way of counting leucocytes for d1fferent1al applicable to this practical. In addition, the following
count Arrows indicate the direction of counting
precautions should be observed:

Observation and Result

Different leucocytes \ire placed in groups of 5, five lines indicating five cells (four vertical lines and one oblique
line placed over the four lines which indicates the fifth cell). In this way, 100 cells are counted. An example of
such type of counting is shown below.
Neutrophils I-H1 U-f-t 1-H-t 1-H-t u+t Uf1 u+t 1-H-t l-+t1 1-H-t I-H1 = 55 cells
Eosinophils w-+t u+t Uf1 = 15 cells
Basophils - nil
Lymphocyte 1-H-t w-+t 1-H-t u+t l-+t1 = 25 cells
Monocytes 1-H-t - 5 cells
T oral = 100 cells
Neutrophils : 55 per cent
Eosinophils : 15 per cent
Basophils : 0 per cent
Lymphocytes : 25 per cent
Monocytes : 5 per cent

Alternatively, a table of 100 small squares can be drawn and each cell counted can be entered in the small
squares of the table, till 100 cells are filled. On the right side of the table, against each row, the number of
different leucocytes present in that row can be entered in separate columns and finally, the percentage can be
taken as shown below.
- N E B L M
N 1N N L L- N N N E M 6 1 0 2 1
-
N L L N L E E N N N 5 2 0 3 0
N N N L E N E M N N 6 2 0 1 1
N -L L E N N N L N N 6 1 0 3 0
- -
li.i N N L L L N E N E 5 2 0 3 0
-
N N N L L N N N N M 7 0 0 2 1
N L L N E E N L E N 4 3 0 3 0
N N N L L N E M N N 6 1 0 2- 1
N L L E 1E N M L N N 4 2 0 3 1
-
1N N N L L L N E N N 6 1 0 3 0
-
55 15 0 25 5

Neutrophils 55 per cent

Eosinophils 15 per cent
Basophils 0 per cent
Lymphocytes : 25 per cent
Monocytes : 5 per cent
Inference: There is moderate eosinophilia of 15 per cent. Other leucocytes are in the normal range.
74 Chapter 11

1. The quality of the smear should be assessed before 4. Food intake

starting the count. The smear should be discarded 5. Emotional stress
if it is not good and a fresh smear should be 6. Exposure to cold
prepared.
B. Pathological
2. Count cells throughout the length of the smear,
1. Acute pyogenic infections, for example,
except the extreme ends of head and tail.
tonsillitis, pneumonia
3. Counting should be done in a zigzag pattern to
2. Noninfective inflammations, for example,
prevent double counting of a cell.
rheumatic fever
4. A minimum of 100 cells should be counted.
3. Noninflammatory conditions, for example,
5. The slide should be preserved for future rechecking
myocardial infarction, pulmonary
of the result. embolism ·
4. Acute hemorrhage
DISCUSSION
5. Muscle trauma, for example, following
Differential count is a frequently o rdered investigation surgery
in clinical medicine. Usually it is done along with 6. Leukemia, for example, chronic myeloid
other hematologic tests, like total leucocyte count, to leukemia
assess the ability of the subject to defend the body from 7. Toxic conditions, for example, ureIIlla,
microbial invasion, and also to assist in diagnosing the hepatic coma
disease. 8. Coticosteroid therapy

Clinical Significance ~ ~ eutropenia

Differemialcount isvitalforthediagnosisofanumber A. Physiological

blood-related diseases involving leucocytes or red c 1 Physiological neutropenia is very rare.
Its primary use is to identify changes in the distributi Sometimes, it occurs after chronic exposure to
of white cells, which may be related to a particu a severe cold.
disease like a specific infection (for example, typhoi B. Pathological
or a malignant condition (like leukemia). C linical 1. Starvation and debility
terms like increase in specific white cells (neutrophilia, 2. Typhoid and paratyphoid fever
eosinophilia, lymphocytosis and m onocytosis) or their 3. Aplastic anemia (bone marrow failure)
decrease (neutropenia, eosinopenia, lymphocytopenia 4. P arasitic infections, like malaria, kala azar
and monocytopenia) are based on the result of the· 5. Viral infections, like measles, influenza, viral
differential count. In addition, a study of the blood hepatitis
smear reveals morphological abnormalities of blood 6. H ype·rsplenism
cells, the presence of abnormal cells, and the presence 7. Drug-induced neutropenia
of blood parasites. Differential count also helps in
III. Eosinophilia
the approximate estimation of other cells (RBCs and
1. Allergic conditions ( '.' ~ ~~k.""
platelets). Morphological studies of red cells enable
a physician to recognise the hemoglo bin status and Bronchial asthma
types of anemia, and also helps to check reports on Urticaria
blood indices. Food allergy
Hay fever
Conditions that alter different cell counts 2. Parasitic infestations l-·{)~[Link])
H ookworm
I. N cutrophilia Filariasis
A. Physiological H ydatid disease
l. Exercise 3. Skin diseases (_ ·: ~ ~'"' g\iJ('. ~~
2. Pregnancy Psorias'is
3. Panuricion Pemphigus
 Differential Leucocyte Cou nt 75

4. Collagen diseases 2. Infectious mononucleosis

Periarteritis nodosa 3. ]Lymphocytic leukemia
5. H odgkin 's disease 4. Lymphomas·
6. ~ ddisoo 's disease 5. Viral infections
7. Certain leukemias
VIII. Lymphocytopenia
IV. Eosinopenia 1. lmmunosuppressive therapy
► 1. ACTH therapy 2. ACTH therapy
2. Cush,ing's dg ase 3. H odgkin's disease
3. £cute pyogenic infections l futt .\r,~ct\cyf')) 4. Bone marrow failure
4. Aplastic anemia_ IX. Moniocytosis
V. Basophilia 1. Protozoan diseases
Malaria
1. Chronic myeloid leukemia
Kala azar
2. Polycythemia
2. Hodgkin's disease
VI. Basophilopenia 3. Monocytic or myelomonocytic leukemia
It occurs rarely, as seen in severe septicemia or 4. ACTH therapy
aplastic anemia
X. Monocytopenia (rare)
VII. Lymphocytosis 1. Bone marrow failure
2. Aplastic anemia
1. Chronic infections 3. Siepticemia
T uberculosis
Pertussis (whooping cough) OSPE
Syphilis The OSPE discussed in the previous chapter are also
Brucellosis applicable to this chapter.

VIVA - - - - - - - - - - - - - -
All the questions asked in the p revious chapter can be asked here too. Additional questions are:
1. What are the funct~ons of neutroph1ls, eosinophils and basophils?
2. What are the functions of lymphocytes and monocytes?
3. What is the normal percentage distribution of different leucocytes in peripheral blood?
4. What is the clinical significance of performing DLC?
5. What are the conditions that increase or decrease the different types of leucocytes?
6. H ow do you calculate the absolute count of each type of leucocyte by using the value of TLC and DLC?
Ans. Absolute count of a cell is equal to the number of that cell counted in DLC divided by 100 x TIC
For example, if TLC = 7,000/ mm' of blood and DLC is,
eucrophils 55 per cent
Eosinophils : 15 per cent
Basophils : 0 per cent
Lymphocytes : 25 per cent
Monocy tes S per cent
Ab olute count of:
eutrophils (55/100) x 7,000 = 3850/mm3 ofblood
Eosinophils (15/100) x 7,000 - 1050/ mm 1 of blood
Lymphocytes (25/100) x 7,000 = 1750/ mm3 of blood
Monocytes (5/100) x 7,000 - 350/mm3 of blood