CHAPTER 8

DIFFERENTIAL WHITE
CELL COUNT
 Objectives
 After completion of this chapter, the student will be
able to: -
 Define differential leukocyte count (DLC)
 Discuss the general features to be considered in the DLC
 Identify each type of white cells morphologically
 Perform a 100 cell differential count
 Define a relative white cell count and absolute white cell
count
 Perform WBC and platelet estimate on a stained peripheral
blood smear
 Objectives cont’d..
 Discuss the clinical implications of the
differential leukocyte count.
 Identify mature and immature leukocytes.
 Identify morphological abnormalities of
leukocytes.
 Identify reactive lymphocytes, including clinical
situations associated with their presence.
 Identify possible sources of error and their
remedies in DLC.
 Perform QC in DLC.
 Outline
 Introduction
 Clinicalsignificance
 Methods of counting
 Evaluating staining properties
 Reporting the Differential White Cell
Count
 Interpretation of Differential White cell
count
 QC in DLC
 8.1 Introduction
I. Differential white cell count:
 Is the enumeration of the relative proportions
(percentages) of the various types of white cells
as seen in stained blood films.
 Is used to determine the relative numbers of each
type of leukocyte.
 A smear is examined and each time a leukocyte is
seen, it is both tallied and classified on a special
counter.
 The counting and classifying continues until 100
cells have been observed.
 Diff count cont’d…
 The differential cell count also includes: -
An evaluation of RBC morphology,
Platelet morphology and numbers, and
General WBC morphology and estimation.
 With a manual differential count, inaccuracy or
deviation from the true count results both from: -
Maldistribution.
Misidentification of cells.
 Diff count cont’d..
Maldistribution of cells
 The different types of white cell are not
distributed evenly over a slide.
The tail of the film contains:-
 MoreNeutrophils.
 Fewer lymphocytes.

 N.B. Monocytes are fairly evenly distributed

along the length of the film.
 Diff count cont’d..
 Large cells are preferentially distributed at
the edges of the film.
 Maldistribution of cells is aggravated:-
If a film is too thin, or
If a spreader with a rough edge has been used.

II. Clinical significance of Diff count

 To correlate the distribution of different
leukocytes present in the circulation with
different disease conditions.
 Diff count cont’d…
III. Principle of Differential white cell count
 After taking blood sample, blood film is prepared, and after
staining with Romanowsky stains, 100, 200, 300, etc., cells
will be counted; then the percent distribution of each cell is
calculated.
IV. Type of specimen: - EDTA anticoagulated venous or
capillary blood
V. Equipment and Reagents
 Microscope
 Diff counter
 Oil immersion
8.2. Methods of Diff. WBC Counting
 Variousmethods of tracking over a slide
have been proposed to attempt to overcome
errors due to Maldistribution: -
The Longitudinal Strip Method.
The Battlement (Lateral strip,
crenellation) Method.
The Modified (Exaggerated)
Battlement Method.
 A. The Longitudinal Strip Method
 A method of tracking along the length of the film.
 The cells are counted using the 40x dry or 100x oil immersion
objectives in a strip running the whole length of the film until 100
cells are counted.
 If all the cells are counted in such a strip, the differential totals will
approximate closely to the true differential count.
 Longitudinal Method cont’d..
Advantage of the method:
It compensates for Maldistribution
between the body and the tail.
Disadvantage of the method:-
 It does not compensate for
Maldistribution between the centre and
the edge.
 i.e., It does not allow for include the
Neutrophils and Monocytes at the edges
of the film.
 Longitudinal Method cont’d..
 Difficultyin identifying contracted heavily
stained cells in the thicker parts of the film
(head part).
However, this problem is minimized in a
well made film and in practice brings little
difference to results.
 B. The Battlement Method
 Counting is performed by moving from side to
side across the width of the slide in the counting
area just behind the feather edge where the cells
are separated from one another and are free from
artifacts.
Advantage: -
 It compensates for Maldistribution between the
centre and the edge.
 Battlement Method Cont`d..
Disadvantage: -
 It does not compensates for Maldistribution
between the body and the tail.
Since the customary 100-cell differential
count will not cover a very large
proportion of the length of the blood film.
 C. The Modified Battlement Method
 In this method, two fields are counted close
to the edge parallel to the edge of the film,
then four fields at right angles, then two
fields parallel to the edge and so on.
 A modified battlement track is a
compromise between the two methods.
 Modified Cont`d..
 At least 100 cells should be counted.
 If there is white cell aggregation, the
Maldistribution of cells is so great that an
accurate differential count is impossible.
 Of the three methods: -
The lateral strip method appears to be
the method of choice because it averages
out almost all of the disadvantages of the
two other methods.
 Multiple manual registers or electronic
counters are used for the count.
 Diff count cont’d...
 Note: - All elements of the blood film must be
observed while performing the differential
count.
 If any abnormality is seen in the smear, it should
be confirmed and reported.
The following points should be checked:
Erythrocytes: - size, shape, distribution, and
degree of hemoglobinization.
Presence of inclusion bodies.
Presence of nucleated red cells, if so, the total
leukocyte count must be corrected.
 Diff count cont’d...
Leukocytes: -
 Mature,Immature and Atypical.
 Neutrophils: -
Average number of lobes.
Hyper segmented.
Hypo segmentation.
Hyper granulated ones? Vacuolation.
Toxic granulation, other inclusions.
 Estimation etc..
 Diff count cont’d...
 Platelets: -
Estimation (10-20/HPF)
Do they look normal?
Are there many giant or bizarre forms?
 Hemoparasites: -
 Malaria, borrelia, babesia, etc.
VI. Procedure
 Using the 10x dry objective, scan the smear for:
Proper staining.
 Diff count cont’d..
 WBC distribution.
 Presence of platelet clumps.
 Presence of light blue fibrin strands which may
indicate that the blood was not well mixed or
partially clotted.
 Any other unusual or irregular characteristics of
cells. Platelet Clump
 Using the 40x dry objective,
perform WBC estimate.
(see the estimation below)
 Performing a WBC Estimate
Under 40x High Dry
 Look for estimate area which has 200 RBCs that
are partially overlapping with many single red
cells.
 Determine a WBC estimate using the following
conversion information (hpf=high power field):
2-4 cells/hpf = 4.0 – 7.0x10 9/L
4-6 cells/hpf = 7.0 – 10.0x10 9/L
6-10 cells/hpf = 10.0 – 13.0x10 9/L
10-20 cells/hpf = 13.0 – 18.0x10 9/L
 Estimate Cont`d..
 Estimates: Remember!
Remember!
Estimates
Estimates
Are reported as decreased, are
are not
not
Increased, or adequate. reported!
reported!
Are used for QC purpose.
 Locate a thin area of the smear in which the
RBCs are evenly distributed, only slightly
overlap, and the central pallor or “whites-of-
their-eyes” is seen.
 Place a drop of oil on the slide and switch to the
oil immersion objective.
 Procedure cont’d…
 Change the objective to 100x oil immersion.
 Count 100 white cells including all cell lines
from immature to mature.
 Properly record and report results of differential
cell counts.
 Note: reference ranges and any abnormal
results.
Evaluating of Staining Properties
 Locate a cell that can be positively
identified, such as a poly- segmented
Neutrophils.
 Evaluating of Staining Cont`d..
 Note the staining characteristics of the
nucleus and cytoplasmic granules.
 Relate these features to any unidentified
cells that may be present.
 Myelocytes and metamyelocytes, if present,
are recorded separately from Neutrophils.
 Band (stab) cells are generally counted as
Neutrophils but it may be useful to record
them separately.
 Evaluating cont’d...
An increase may point to an
inflammatory process even in the
absence of an absolute Leucocytosis.
 Remember that cells don’t always appear
“picture” perfect.
 The actual staining color of cells may vary
from what is seen in textbooks or even in
images.
.
 Cell component of staining Colour
Cell component Staining color
Chromatin (including Howell–Jolly bodies) Purple
Promyelocyte granules and Auer rods Purplish-red
Cytoplasm of lymphocytes Blue
Cytoplasm of monocyte Blue grey

Cytoplasm rich in RNA (i.e. ‘basophilic cytoplasm’) Deep blue

Döhle bodies Blue-grey

Specific granules of neutrophil, granules of Light Purple or pink
lymphocytes, granulomere of platelets
Specific granules of basophiles Deep purple
Specific granules of eosinophils Orange

Red cells Pink

 Segmented Neutrophils
 Approximately 40-75%
in peripheral blood.
 2-5 lobes of nucleus.
 Joined by thin
filament.
 Faint pink cytoplasm
sprinkled with pink or
purple neutrophilic
granules.
 Small Lymphocyte
 Approximately
40% in peripheral
smear.
 Small round
nucleus, clumpy,
chunk chromatin
pattern.
 Notice the
monocyte on the
top right.
 Large Lymphocyte
 Large lymphocytes
are often confused
with Monocytes.
 Remember - the
lymphocyte nuclear
chromatin is
compact, with blocks
of chromatin.
 The nuclear shape
may be round, oval
or indented.
 Approx. 5% in peripheral smear. Monocyte
 Large, convoluted, brainy looking
nucleus with lacy (delicate)
chromatin.
 Pale, gray blue, ground glass
cytoplasm (numerous fine
azurophilic granules).
 Monocyte nucleus can sometimes
take many shapes from a band
shape, to an oblong shape, to a
convolutes brainy shape.
 What is important to remember is
that the chromatin is lace, open
weaved, and foamy looking.
 Monocytes vs. Lymphocytes
 Monocytes and lymphocytes are often confused.
 The chromatin pattern of the monocyte is light
purple and arranged in loose, lacy or linear
strands.
 The nucleus may be round, lobulated or folded.
 Basophils
 Approximately 0-2% in peripheral blood.
 Nucleus is bi-lobed and obscured by granules.
 Cytoplasm is pale, washed out and contains intense
large blue black granules.
 Granules are scattered throughout the cell and it is
hard to distinguish any distinct nuclear characteristics.
 Eosinophils
 Approximately 2-7% in peripheral blood.
 Bi-lobed or banded nucleus.
 Large, red-orange and distinct granules.
 To identify the Eosinophils rely on the granules. The
granules are very individualized looking.
 If the stain is right, then the cell will look red orange.
 If not, the cell will be

washed-out looking.
 What Does a Normal Adult
Blood Smear Look Like?
Segmented Neutrophils ~60%
Lymphocytes ~30%
Monocytes ~5%
Eosinophils, Basophils ~3-5%

 Remember, percentages are approximate

and may differ with ethnic groups and in
disease conditions.
What normal cell has not been discussed?
 What About Bands Neutrophils?
 This cell (also called a stab) is a precursor cell to
the segmented Neutrophils.
 The nucleus is C-shaped or horseshoe shaped
containing no filament and has no tight or pinched
constriction
 Cytoplasm is pink gray with granules
 Approximately 2-6% in
normal differential count.
If unsure of
identification, classify
as a more cell, the
segmented Neutrophils.
 What About Immature
Granulocytes?
 Metamyelocytes. Myelocytes.
 Promyelocyte. Myeloblast.
Metamyelocytes
 Also called a juvenile.
 Rarely seen normally in the peripheral blood.
 Nucleus characteristically indented or kidney-
shaped.
 The nuclear chromatin is clumped and condensed.
 Specific granules appear the same as in the band.
Band (top) and Metamyelocytes (bottom)
 Myelocytes
 Should never be seen in the peripheral
blood.
 The nucleus is round or oval and often
eccentrically located in the cell.
 The nuclear chromatin is beginning to
clump; no nucleoli are visible.
 A moderate amount of patchy blue
cytoplasm can be seen.
 The cell is characterized by the presence
of light pink or tan (neutrophilic) specific
granules.
Myelocytes
 Promyelocyte
 Should never be seen in the peripheral
blood.
 Large cell.
 The nucleus may be eccentric & slightly
indented with nucleoli still visible.
 The nuclear chromatin is loose and open.
 The cell is characterized by the presence
of few to many, prominent, dark red or
purple cytoplasmic granules.
Promyelocyte
 Myeloblast
 Should never be seen in the peripheral
blood.
 Large cell (size may vary).
 The nucleus is large and round, with
one or more visible nucleoli.
 The nuclear chromatin is loose and
open.
 The cytoplasm is scanty to moderate
and deeply basophilic.
Myeloblast
Observe the two cells and identify the difference

(2) Promyelocyte

(1) Myeloblast, which

shows the start of
azurophilic granulation
(arrow)
 Abnormal Inclusions
in Granulocytic cells
Neutrophils: -
Toxicgranulation.
Dohle bodies.
Vacuoles.

Toxic granulation
 Large, blue-black or deep purple granules
in the cytoplasm of Neutrophils.
 Toxic Granulation Cont`d..
 Represent primary or “azurophilic” (Bright blue)
granules.
 Seen associated with acute infections, drug
poisoning and burns.
 Dohle Bodies
 Small, round or oval light-blue staining areas in
the cytoplasm of Neutrophils.
 Represent aggregates of rough endoplasmic
reticulum.
 Seen in patients with infections, burns and after
exposure to cytotoxic agents.
 May also be seen in conjunction with toxic
granulation.
 Vacuoles
 Clear areas or “holes” in the cytoplasm of
Neutrophils.
 Seen when the neutrophilic cytoplasm begins to
degenerate or if the cell has been actively
phagocytic.
 Seen in patients with infections and can be noted
along with toxic granulation and Dohle bodies.
 N.B. Also can be seen as
an artifact associated with
old EDTA anticoagulated
blood.
 Toxic Granules

Dohle body Vacuoles

 Reactive Changes in Lymphocytes
 Response to a variety of viral and non-viral
stimuli produce diverse nuclear and/or
cytoplasmic changes in the lymphocytes.
 Associated with viral infections: -
E.g., Epstein-Barr virus (EBV),
cytomegalovirus (CMV), human
immunodeficiency virus (HIV).
 Also seen in response to drugs or non-viral
organisms such as toxoplasmosis.
 Reactive Lymphocytes cont`d..
Characteristics: -
 Irregular shape of cytoplasm and/or
nucleus.
 Abundant dark blue cytoplasm, overall
bigger cell.
 Cytoplasmic tags, sharp ridges; scalloped
by red cells.
 Increased number of reddish granules or
vacuoles in cytoplasm.
 Fine nuclear chromatin pattern; may see
nucleoli.
Reactive Lymphocytes
 8.3. Reporting the Differential
White Cell Count
 The differential leukocyte count expressed
as the percentage of each type of cell.
 It should be related to the total leukocyte
count.
 The results reported in absolute numbers
as the absolute value gives better clinical
indication.
 Absolute vs. Relative Count
Absolute count Relative count
 Derived by  Strictlythe percent
multiplying the counted in the
percentage of the differential WBC.
identified cell
The relative number
times the total
is then compared to
white cell count.
the reference range
for normal white cell
differentials.
 Absolute vs. Relative Cont`d…
Absolute cont`d.. Relative Cont`d..
 If 40% lymphocytes are  So, it is possible for
counted and the white an individual to
cell count is 5.0x109/L have a relative
then the absolute increase, an
lymphocyte count is absolute increase.
5.0x109/L x 0.4 or  A relative and
2.0x109/L
absolute values
 This number is then
increase in a
compared to the healthy particular cell.
reference range.
 Approximate Absolute and
Relative Reference Ranges
Values may vary according to geographic location
Healthy Adult
Cell Type Absolute Relative
Segmented
2.0-7.0 x 109/L 40.0-75.0%
Neutrophil

Lymphocyte 1.5-4.5 x 109/L 20-45%

Monocyte 0.2-0.8 x109/L 2-10%

Eosinophils 0.04-0.4 x 109/L 0-7%

Basophil 0.02-0.1 x 109/L 0-2%

 Quality control
 Well prepared and well stained blood film
which has 3 zones (Head, body, tail).
 WBC should contain blue nucleus along
with a lighter staining cytoplasm.
 RBC should have good quality of color
ranging from buff pink to orange.
 Platelet should be blue with granules and
no nucleus.
 Sources of error

 Use of unclean slide and improper

smear preparation and staining
technique.
 Counting cells in an area not
suitable for counting.
 Misidentification of white cells.
 Interpersonal skill.
8.4. Interpretation of Differential WBC
 For proper interpretation the relative count
is of little use by itself.
 For example, the fact that a sample may
have 60% polymorphs is of little use.
 A patient sample may have 60%
Neutrophils and a total leukocyte count of
8.0 x 109/L giving 4.8 x 109/L
Neutrophils, which is quite normal.
 Interpretation cont’d...
 But if the patient has 60% Neutrophils in a
total leukocyte count of 3.0 x 109/L, then the
patient’s Neutrophils count is1.8 x 109/L
Neutrophils.
In this case the patient has granulocytopenia.
1.Neutrophil
1.1 Neutrophilia/neutrophilic Leucocytosis:
 An increase in the number of circulating
Neutrophils above normal (>2.0-7.0 x 109/L).
 Interpretation cont’d...
 This is due to: -
Overwhelming infections.
Metabolic disorders: uremia, diabetic
acidosis.
Drugs and chemicals: lead, mercury,
potassium chlorate.
Physical and emotional stress.
Hematological disorders: myelogenous
leukemia.
Tissue destruction or necrosis: burns, surgical
operations.
 Interpretation cont’d...
1.2.Neutropenia:
 A reductionof the absolute Neutrophils
count below 2.0 x 109/L.
Myeloid hypoplasia.
Drugs (Cloroamphenicol,
phenlybutazone).
Ionizing radiation.
 Interpretation cont’d...
1.3. Hyper granular Neutrophils
(Neutrophils with toxic granules)
Neutrophils with coarse blue black or
purple granules.
Indicative of severe infection or other
toxic conditions.
1.4. Vacuolation
Seen in progressive muscular dystrophy.
 Interpretation cont’d..
1.5. Hyper segmentation:
Neutrophils with more than six lobes to their
nucleus (as many as ten or twelve may be
seen).
Indicative of megaloblastic erythropoiesis
(vitamin B12 and/or folic acid deficiency), iron
deficiency anemia and uremia.
1.6. Agranular Neutrophils:
Neutrophilsdevoid of granules.
Having a pale blue cytoplasm (features of
leukemia).
 Interpretation cont’d...
2. Eosinophils
2.1. Eosinophilia: -The count above 0.5 x 109/L.
Occurs during: -
 Allergic diseases: bronchial asthma,
seasonal rhinitis.
 Intestinal parasitic infections: e.g.
trichinosis, taeniasis.
 Skin disorders.
 Chronic myelogenous leukemia.

2.2. Eosinopenia: - An Eosinophils count

below 0.04 x 109/L.
 Interpretation cont’d...
 Occurs : - In acute stress due to secretion of
adrenal glucocorticoid and epinephrine.
In acute inflammatory states.

3. Basophiles
 Basophilia: - The count above 0.2 x 109/L in
rare condition.
Occurs : -

In allergic reactions.
 Chronic myelogenous leukemia.

 Polycythemia Vera.
 Interpretation cont’d...
4. Monocyte
4.1. Monocytosis: -The count is >1.0 x 109/l.
Occurs during: -
 Recovery from acute infections.
 Tuberculosis.
 Monocytic leukemia.

4.2.Monocytopenia: - The count is <0.2 x 109/l.

Occurs during:
 Treatment with prednisone
 Hairy cell leukemia
 Interpretation cont’d..
5.Lymphocytes
5.1. Lymphocytosis:- Absolute lymphocyte
count above 4.0 x 109/L in adults and above
8.0 x 109/L in children.
Seen during: -
 Infectious lymphocytosis associated with
coxackie virus.
 Other viral infections: Epstein-Barr virus,
cytomegalovirus.
 Interpretation cont’d..
 Acuteand chronic lymphocytic leukemia.
 Toxoplasmosis.

5.2. Lymphocytopenia: -
 A lymphocyte count below 1.0 x 109/l in
adults and below 3.0 x 109/l in children.
Seen in: -
 Immune deficiency disorders: HIV/AIDS.
 Drugs, radiation therapy.
 Interpretation cont’d...
Atypical lymphocytes:-
 Large cell; abundant pale blue cytoplasm
with peripheral Basophilia, may have
azurophilic granules.
They are primarily seen in: -
 Infectious mononucleosis which is an
acute, self-limiting infectious disease of the
reticuloendothelial tissues, especially the
lymphatic tissues
 Summary of Differential WBC Count
 Use properly prepared and stained blood
smears.
 Establish a good counting area.
 Properly identify white cells.
 Provide an estimate of total white cells and
platelets.
 Record cellular morphology (RBC, WBC,
and Platelet).
 Properly record and report results noting
reference ranges and abnormal results.
 Review Questions
1. Define differential leukocyte count.
2. What is the importance of reporting the
differential leukocyte counts in absolute
number?
3. What other elements of the blood film should be
evaluated while doing the differential leukocyte
count?
4. Describe three tracking methods of diff counting
with their advantage and disadvantage.
5. List at least five factors affecting the differential
white cell count and their remedies.