9 Total LeucocyteCount

LEARNING OBJECTIVES m the b.i;me mav~ The lymphocytes and monocytes

also develop frc>mthe stem cells in the bone marrow.
Afte r comple ting this p ractical you WILL b e able to: In adults, l:f.!!!p hocytes are produced pri wacil)', in
1. Explain the importance of performing total the lymphoid tissue Qymph nodes and SJ?leen) and
leucocyte count (TLC) in practical physiology. @ icondarily in tlhe bone marrow. Granulocyres develop
2. Explain the structure and functions of leucocytes. inthe followillg sequence: -
3. Perform TLC by manual method (using the
Pleuripotcnt stem cells
principle of hemocytometry).
4. List the precautions and sources of error of TLC.
t
Urtlpotent stem cells
5. State the composition and function of each t
constituent of Turk's Auid. Myeloblast
6. State the normal value of TLC. t
Promyelocyte
7. Define leucocytosis, leucocytopenia and leukemia.
8. List the common causes of leucocytosis and t
Myclocytc
leucocytopenia. eutrophil, Eosinophil, Basophil)
9. Name the types of leukemia.
You MAY also be able to:
1. Describe the steps of leucopoiesis.
•
Mctamyclot:yte
( cutrophil, Eosinophil, Basophil)
.,,

2. Describe the fate of leucocytes. 't

~ t
3. State the principle of automated method of TLC.
4. Explain the variation in leucocyte count in
eutrophiJ
•
Eosinophil Basophil

different conditions. The number of circulating leucocytes is very precisely

5. Briefly describe the types of leukemia.
INTRODUCTION
Leucocytes (white blood cells) are nucleated cells that a re
involved in the defence mechanism of the body. Unlike
red cells, white cells use the bloodstream primarily
for tr~rtation to ibeir place af function in the
bo y-t1ssues. Leucocytes are classified as granulocytes
and agranulocytes. Granulocytes are neutrophils)
eosinophils and basophils, and agranulocytes are
lymphocytes and monocytes.
W BC "'"~e... = <is-- ~oJ.)Jn.
controlled. Though there is no definite feedback control
system like tha1l for red cells, a number of chemical
substances released from the site of destruction of
'
leucocytes affect the development of the cells. The
substances that re ulate the development of leucocytes
are loosely called anulo oietins. These substances are
various types of mter et! ms an co~ stimulating
factors, and other c~'kines. I.Ls.., CS f ,,, CKL
Fun_
ctions
The granulocy,:es act as phagocyric scavengers. They
engulf and destroy invading microorganisms, and clea·r
the body of unwanted paniculate materials such as
dead or injured tissue cells. The neutrophils are said >

Formation to be the first line of defence against acute bacterial

Development of leucocyres is called leucopoiesis.
In the embryo, white blood cells develop in the
mesod~rm and migrate secondarily in the blood
vessels{b.fter b~ granulocytes develo p exclusively
invasion. They kill organisms by phagocytosis.
The monocytes are also phagocytes and are thought
to be the second line of defence against microbial
invasion. The monocytes enter the tissues where they
form tissue macrophages (reticuloendothelial system)
 Total Leucocyte Count 53

ha ocytose m the tissues. easy. Counting is done using a microscope under low-
Lym hoc es an lasma ce act as immunocytes power objective and with knowledge of the volume of
and maintain the 2ch;;'s immunity. Plasma cells are fluid examined and the dilution of the blood obtained.
not normally found in blood, but they are formed The number of white cells per mm3 of undiluted whole
from B lymphocytes under specific immunologic blood is calculated.
stimulation. Plasma cells produce antibodies that
in3ctivate antigens. Requirements
I. Apparatus
Life histor 1. Microscope
Radioactive labelling has shown that the entire 2. Hemocytometer (WBC diluting pipette and
maturation process, from myeloblast to neutrophil, counting chamber)
takes about three days. The leucocytes, especially the 3. Equipment for sterile finger prick
granuolcytes that circulate in blood, are marginatcd 4. Watchglass
on vessel walls (m argination) and sequestered in closed 5. C overslip_ _ _ _ _ __

I capillaries. Neutrophils have a half-life of about six

hours in the circulation. After their immigration into
the tissues, they never return to the bloodstream, and
survive in the tissues f~r a few days. The lifespan of
granulocytes is about four to eight days. Their entire
population turns over about two and a half times each
n.@3
Compositio11
1. 1 per cent glacial acetic acid.solution
jlmd. )
diluting fluid is also known as@ rk '.r

2. Gentian yjql ~ stain or aqueous methylene blue

(0.3 per cent w/v)
day . Th us, over 100 billion cells are produced by the 3. Distilled water
bone marrow each day. Lymphocytes survive for Fu11diot1 ef each constituent
about 80 to 100 days. Monocytes, after their activities Acetic acid causes destruction of red cells
J.
in circulation for few hours, enter different tissues of (hemolysis).
the body where they transform themselves into tissue Gentian violet stains the nuclei of leucocytes
, ' ·~ d stay in the ,;,sues for a long time (a Distilled water acts as a solvent

Procedure
Normal ~Q_unt Se~-ez tA?tth AGE- 1. Clean and dry the pipette, watchglass, coverslip and
Adults : 4,000-ll,000/ mm3 ofblood Neubauer's chamber thoroughly .
Newborns : 10,000- 25,000/mm3 of blood 2. Take enough WBC diluting fluid in a watchglass. l \: 2.0
Infants : 6,000-18,000/mm3 of blood 3. Prick the finger under aseptic conditions and wipe
3
Children; · 5 OP°=J~/mm of blood off the first drop of blood. Allow a good-sized blood
There is ~; sex differenc; ~een in RBC count. drop to form on the finger tip spontaneously (do
not squeeze).
METHODS OF COUNTING 4. Touch the blood drop with the tip of the pipette
and suck blood exactly up to the 0.5 mark.
White blood cells are counted by two methods: If blood is drawn above the 0.5 mark, bring the
non-autom ated (manual) and automated. The manual blood column to the 0.5 mark by tapping the tip
cell count is less accurate, but is still used widely in of the pipette on the palm or finger, or by using
developing countries because of its lower cost. nonabsorbent material. Do not use gauze or conon
J
for this adjustment, because the liquid portion of
Manual Method the sample inside the stem will be drawn into the
absorbent material, leaving a higher concentration
Principle of cells inside the stem.
Blood is diluted with an acid solution that removes red 5. Wipe the tip of the pipette and maintain the blood
cells by hemolysis and accg1t11~ of wbife level at the 0.5 mark by holding the pipeue in a
cells. The counting of the white cells then becomes horizontal position.
 "->. M~~ 10'111'U.O.. -;;: : N O , ~ C..ClllL& 1'n
---
l
54 -chapter 9
6. Dip the tip of the pipette into the diluting fluid
(in the watchglass) well below the surface of the
liquid.
7. Suck WBC diluting fluid exactly up to the
11 mark. While the bulb is being filled, you may tap
the pipette with a finger to knock the bead down
below the surface of the solution in the bulb. This
will help prevent the formation of bubbles.
8. While removing the pipette from the diluent,
maintain the level of the mixture at the
11 mark by closing the pipette tip with the index
finger.
9. Hold the pipette horizontally and close both ends
of the WBC pipette, then gently mix the contents
of the bulb. For mixing, shake the pipette at right
angles to its long axis for a few seconds. The glass
bead in the pipette should move from one side to
the other.
10. After mixing, place the pipette in a horizontal
position to prevent any loss of its contents until the
cell count is completed.
11. Discard the first two drops of fluid from the pipette
as the fluid in the stem does not contain cells.
12. Charge the Neubauer's chamber as described in
Chapter 6 and allow two minutes for the cells to
settle down.
13. Place the charged chamber on the stage of the
microscope and adjust it for observation under low
power.
14. Focus the Neubauer's chamber under the low-power
objective and check for uniform distribution of cells
in the WBC squares. If the cells are not uniformly
distributed, clean the Neubauer's chamber and
recharge it.
15. Count the total number of WBCs in the
four corner squares under the low-power objective.
To avoid counting the same cells again, count the
white cells present in the square and those present on
its left and lower lines. Ignore those on its right and
its upper lines. WBCs appear similar to clumped red
cell debris or stained particles. They are identified as
clear, nucleated arid refractile bodies.
16. Draw the WBC squares in your notebook and enter
the observation. Calculate the final result.
Dilution obtained
The volume of the bulb is 10 (11 - 1 = 10). The stem
of the pipette (from the tip of the pipette to mark
1) contains diluting fluid that does not take part in
.l."'9,· .... VO\WTI-C. ':"b D\O.l.Ol \V\ 1.-q.,
2-_1>,l.uHo"' ~"'~
x N~or ~-
CJJun~.D
the dilution. Dilution (mixing of blood with diluting
fluid) occurs only in the bulb. Thus 10 volumes of
diluted blood (in the bulb) contain 0.5 volumes of
blood and 9.5 volumes of diluting fluid, giving a
dilution of 0.5 in 10. Thus, ~ n obtained is
1 in 20, or 20 times. ~
Calculation
Area of 4 WBC squares = 4 x 1 = 4 mm2
Volume of 4 WBC squares= 4 x 1/ 10 = 4/10 mm3
Dilution factor = 1:20
Cells in 4/10 mm3 volume of diluted blood = n
Therefore, cells in 1 mm3 volume of diluted blood =
n X 10/ 4
Therefore, cells in 1 ~ e of undiluted blood =
nx 10/ 4 x 20 ~
Precautions
(Same as precautions taken for RBC count; for details
see Chapter 7.)
1. The pipette, coverslip and Neubauer's chamber
should be dry and thoroughly cleaned. ;I
2. The prick should be at least 3 mm deep. Do not
squeeze to get blood.
i
3. Wipe off the first drop of blood.
4. Draw blood exactly up to the 0.5 mark
5. Wipe the tip of the pipette.
6. Suck the WBC diluting fluid exactly up to the 11
mark.
7. Air bubbles should not enter while pipetting the
fluid.
8. Before charging the chamber, gently mix the
contents of the bulb of the pipette.
9. Discard the first two drops of fluid.
10. Do not overcharge or undercharge.
11. Do not allow air bubbles to enter the chamber.
12. Once the counting chamber is filled (charged),
complete the counting as early as possible before
the fluid begins to dry.
13. If the distribution of cells is not uniform, discard
and recharge.
14. Avoid counting the same cells twice.
Sources of error
Fortunately, error in the leucocyte count is not as
critical as error in the red cell counts. Even an error as
high as 20 per cent does not affect the result much. The
 10"'1'~''- , _ -· •~ n\J ·· \l--'J
C.\·Ytrn \ C.~'-q. 15ti~ ~
b~ ._J)'\ rn1c...ro C1"i 55
""':II 1{') e.c,W @) ~
possible sources of error may be 1) errors inherent in migration are called{cbemoattrac an The white
t he method or, technical errors. cells, once they reachtlie site of invasion, engulf
1,
and digest the foreign substances (pbagocytosis). All
A. Errors inherent in the method granulocytes, especially n~rophils, and mo~ytes
1. Envr of visual count: One potential error is kill the organisms by phagocytosis. Lymphocytes are
mistaking dirt or clumped red cell debris for the principal cells of the body's immune system.
leucocytes. This error decreases by making a 0
second count in the Neubauer chamber of the Clinical Significance · Pi"'-l.J~===-
opposite side.
2. Error dtte to distrib11tio11 ef cells: Distribution can
Total leucocyte count is a part of the routine
never be perfectly uniform even with thorough hematologic investigation used to assess the nature
and severity of an infection, extent of spread of the
mixing and charging. If there is clumping of
leucocytes, recharge the chamber. disease and the body's defence capability. In · some
diseases, alteration in leucocyte count alone may
B. Technical errors be diagnostic, but frequently the leucocyte count is
1. Improper volume measurement of blood and ordered with other investigations, especially with the
diluent differential leucocyte count, to aid in diagnosis. When
2. Use of defective pipettes the total leucocyte count increases above the normal,
3. Improper charging of the chamber the condition is called leuracytosis, and when the count
4. Improper counting decreases below norm.J, le_!:,coqtopenia.
5. Wrong calculation , \_~ ~
&~ v Conditions that Alter
Additional inform~ · "·"1
1>.1
moJ\"- Total Leucocyte Count
In case of a very high~ ucocytee co n, as seen · 0
__ ..,...._, ~err, \1'
leukemia, dilution may av to e increased to 100
times or more. The ~1!.1 ette can be used for this
- - --. ~\.<. : ~,~clioin
-I.Leucocytosis
Physiological
.J./~~p ~ ,.
· ~ ·--J v-!)eC- ~~~
~
p-!1rpose . .Blood is sucked up to mark 1 of the RBC 1. Newborns and infants: Count is as high as
pipette and then diluted 100 times. Calculation is 15,000-20,000/mrn3. cilseul~o
according to the dilution. ~
:.(p,\. ;:'(3)0 . 2. Physical exercise: During physical exercise, ~
--F--- ,7,t✓✓- circulation becomes more dynamic, which causes
Automated Method 0.od ~ ·('disruption of m argrnauon of the leucocytes along
~\\ irf\ClA'(. -
ot e um. ere ore, leucocytes
Automated cell counting is done either by impedance their margination pool into the
counting or by light scattering technology. The ;:.!::=~~ ~~ ~~.Ji.Siijeral circulation). This is
principle is the same as that described in Ch apter 7.
!;;,;s;;;;;;iii~=~o~s~ Leucocytosis does not
ormation of cells.
DISCUSSION 3. llowing food intake, the
Physiological Significance body's :::::---:..--..~ -b<- reases; this increases the
body's(1im1 era ecause of this, circulation
The total leucocyte count is performed to assess the improv~~ s causes cfisruption of margination of
subject's ability to defend his body against microbial leucocytes and results in leucocytosis.
invasions. The leucocytes are a P:!£ ofthe body's defen5e 4. Exposure to sun and increased enviro nmental
system that provides protection against infections. temperature.
The leucocytes constitute the mobile defences of the 5. Pregnancy: Leucocytosis occurs in spite of
body. T hey can(e,_ass thro ugh the vascular endothelium hemodilution. The exact cause of leucocytosis in
a§ enter the tissues 6 means otfoped~eucocytes pregnancy is not known, but it may possibly be due
4
migrate to the site of mJury 111 response to chemical to the action of hormones on leucopoiesis.
substances released by the microorganisms, or by the 6. Parturition: Leucocytosis occurs due to the
injured oJ:.-lllkc~d cells. T he process of migration is combined effects of tissue injury, hemo rrhage and
called ~ n d the substances that promote severe exertion.
 56 Chapter 9

7. Pain, nausea and vomiting 4. Aplastic anemia: H ypoplasia of bone marrow

8. Menstruation decreases all the cell counts.
9. Emotion and anxiety 5. Cytotoxic therapy: Treatment of malignant
II. Pathological diseases by cytotoxic drugs
1. Acute bacterial infection: Infection with gyogeni~ 6. Drugs (especially in sensitive individuals): I
Chloramphenicol
bacteria Qocalised or generalised) is the com.moue t
Sulpha drugs
.- j
cause of · leucocytosis. Examples of bacterial
infections are boils, abscess and pneumonia. Aspirin
But, there are a few bacterial infections in which 7. Hypersplenism i
l
leucocyte count decreases. One of the typical 8. Starvation and malnutrition
xamples of leucocytopenia in acute bacterial Leucocytopenia usually occurs due to neutropenia.
infection is typhoid fever. (' JL...oaib)
2. Chronic bacteri~ infection, for example, Leukemia
t~berc~l~sis. ( ~ ~ )
3. TISsue mmey Leukemia is a cancerous disease of the blood forming
Infarction tissues. Uncontrolled roliferation of one or more
Burns (NoarO-- of the various he";!.TAPPieric cells occurs, and t ese
Surgery progressively displace the ~ r-cellular elemeg ts.
4. H emorrhage
5. Neoplasia Definition
6. Stress states and hyperactivity Leukemia is a malignant neoplasia of hemopoietic
Convulsions cells in which there is abnormal proliferation of
Severe colic leucocytes and their precursors. It results in appearance
7. Inflammatory disorders of abnormal and immature cells with very high
Certain collagen diseases leucocytos1s tn t he penpheral bto od, and infiltration

8.
Rheumatic fever
Metabolic disorders such as diabetic ketoacidosis
of tissues by leukemic cells. There is increased
infiltration of bone marrow by the roliferatin
..
9. Corticosteroid therapy ( D~) cells. Th tota eucoc e count is usuall ve
except m the ~ leukemic or leukemic form o
LeuJ;oc_Y!_o_penia leukemia. U~ ly, the prolifer ·on involves the
I. Physiological leucocytic senes; occasionally, erythroid precursors
Ph siological decrease in leucoc e count is very o~ megakaryocytes may also be involved in the
xposure to severe cold ay , s~times d1sea e process.
c ease the tota WBC count. ~
II. P athological mo..~'\Nll~e<\) Causes
1. Infections The exact cause of leukemia is not known. Some of the
Typhoid fever (!n ~ i n ~ ) probable causes are:
Paratyphoid fever l. H eredity and genetic predisposition
Early phases of many viral infections such as 2. Environmental factors, especially exposure to
infectious hepatitis. t D \cf,) ga~ma radiation producing genetic mutation or
2. Overwhelmingsepsis: Inseveresepsis,consumption chromosomal aberration f '-' 1
of neutrophilsexceeds production. 3. V ~ gs l ~R ~\Jim ..
3. Replacement of hemopoietic tissue in the bone
marrow by neoplastic infiltrative cells:
4· So rt. _
~
i 1_ ~~
..11 c... "-=
Nl.(J.14.lLI.DY'-

A) Acute leukemia ~:J~'~'.< Classification

6) Lymphoma ----:/ u ~ •~ Leukemia is broadly classified into two main categories:
'l-) Multiple myeloma ~ ou)° ' myeloid (myelocytic) and lymphocytic leukemia.
D) Myelofibrosis These two types are again classified into acute and
 Total Leucocyte Count 57

d' >9 .
affected twice as frequently as women. Patients present
chronic forms on the basis of the clinical course and
the number of blast cells present. nonspecific symptoms. I-:Jmphadenopatf?y is the outstanding

~
Acute IY.ffil!.hoblastic leukemia (ALL)
ALL is primarily a disease of childr~ oung
adults. This constitutes 80 per cent of childhood acute
leukemias. It rarely occurs in adults and the elderly.
The most common mode of presentation is with
symptoms of anemia or hemorrhage, infective lesions
of the mouth and pharynx, fever, prostration, headache
and malaise. There is generalised lymphadenopathy,
splenomegaly and hepatomegaly. The typical blood
picture consists of anemia, thrombocytopenia and
moderate or marked increase in leucocytes, the majority
of which are blast cells ~ymphoblasts; 60-80 per cent).
pf?.ysical sign. Hepatosplenomegaly may be present. Mild
to severe increase in leucocyte count is seen. More than
90 per cent of leucocytes are lymphocytes.
OSPE
I. DiluLe the blood (from the given sample) for
total leucocyte count.
Steps:
1. Select the WBC pipette and ensure that it is
dry and clean.
2. Take adequate diluting fluid in the watchglass.
3. Mix blood thoroughly by gently shaking the

~
sample.
Acute myeloblastic leukemia AML 4. Suck blood exactly up to the 0.5 mark. ff blood
This primarily affects adults betw~ ges of is drawn above the mark, the extra blood is
15 and 40. years. It constitutes only 20 per cent of removed by tapping with a finger tip (not by
childhood leukemias. The presentation is like that of touching with absorbent material).
ALL, but lymphadenopathy and hepatosplenomegaly 5. Wipe the tip of the pipette.
is not common. Blood picture presents anemia, 6. Suck diluting fluid up to the 11 mark. While
thrombocytopenia and moderate to high leucocytosis. dra1wing the fluid, avoid entry of air bubbles.
j
More than 60 per cent of leucocytes in the peripheral 7. Gently mix the contents of the bulb and keep
blood are blast (myeloblast) cells. the pipette on the t~ble.

Chronic my~loid leukemia (CML) ~ II. Charge the Neubauer's chamber for total WBC
This form of leukemia accounts f o ~ per cent count.
of all cases of leukemia. It is primarily a disease of Steps,:
adults of 30- 60 years with peak incidence in the fourth 1. Clean the coverslip and Neubauer's chamber.
and filth decades of life. Onset is usually slow with 2. Plaice the coverslip on the platform of
nonspecific features like anemia, weight loss, weakness N€::ubauer's chamber.
and easy fatiguability. Splenomega!;· is the 011tstandi11gpqysical 3. Mix the contents of the bulb of the WBC
sign. Hepatomegaly may be present, but lymph node pipette.
enlargement is rare. Markedly elevated total leucocyte 4. Di:scard two drops of the fluid from the
count, usually more than one lakh cells per mrn3 of pipette.
blood, is seen. Neutrophils and metamyelocytes 5. Touch the tip of the pipene with the edge of
constitute most of the circulating cells. Blasts cells are the coverslip.
rarely present except in the blastic crises. 6. Slowly release fluid from the pipette (fluid
moves by capillary action) in such a way that
Chronic lymphoc~ leukemia (CLL) fluid spreads just beneath the coverslip and
CLL is the most indolent of all leukemias. It occurs does not spill into the guners and does not e
' typically in persons over 50 years of age. Men are contain air bubbles. Y.:. ~
,c"ff'Qr')k--. ~mp~-- •( c.i..:
l - --
~~emlO.. -I
e ~ L~F\~ ~fi(J.J.~- 9,Q"'/~ d,\\._J ( P>L.L)
·
\ , ..1\ / F)('.U,.\e -') :ttQ~/o (h\ \d ( f,ML)
4 hl\Je\01u--~
C..Y"\"troh,e, ➔ Sp\.ertafl\~~ Ct,)tnL')
\..J '"", ___ ~
58 Chapter 9

<
VIVA
1. Which diluting fluid-is used for determinatin g total leucocyte count and what is its composition? What is the
function of each component? f
2. Why are two drops of blood discarded from the pipette before charging the eubauer's chamber?
3. Why is Jbe dilution obtained 1 in 20, and not 1 in 22?
4. In which condition is the RBC pipette used for white cell counting?
5. What are the precautions for performing total leucocyte count? What are the possible sources of error in total
leucocyte count?
6. What are the functions of the white bead present in the WBC pipette?
7. What are the functions of leucocytes?
8. What is the physiological significance of performing a tq,tal leucocyte count?
9. What are the causes of physiologica l leucocytosis?
10. What is the mechanism of leucocytosis in physical exercise?
11. What are the pathological causes of leucocytosis and leucocytopenia?
12. What is leukemia? What are the types of leukemia?
13. What are the most frequently occurring leukemias in children and in adults?