MODERATOR - DR.

NEHA YADAV

SPEAKER - DR. DEEPAK VERMA

• The name “white blood cell” or Leukocyte is derived from the fact that after centrifugation of
a blood sample, these cells accumulate in the buffy coat, a thin, typically white layer of cells
between the sedimented red blood cells and the blood plasma.
• White blood cells are about 1% of all blood cells, they are continually at war as cells of the
immune system involved in defending the body against both infectious diseases and foreign
materials.
• WBC count can be used for diagnosis, treatment response analysis and follow up of various
pathological conditions. It can detect many hidden infections and undiagnosed medical
conditions.

• Granulocytes are Leukocytes characterized by the presence of differently staining granules in

their cytoplasm when viewed under light microscope. These granules are membrane-bound
enzymes which primarily act in the digestion of endocytosed particles. There are three types
of granulocytes: Neutrophils, basophils, and eosinophils, which are named according to their
staining properties.
• Agranulocytes are Leukocytes characterized by the apparent absence of granules in their
cytoplasm. Although the name implies a lack of granules but these cells do contain non-
specific azurophilic granules, which are lysosomes. The cells include lymphocytes and
monocytes.
• MANNUAL - PBS examination - for TLC and DLC estimation
Hemocytometer - for TLC estimation

• AUTOMATED - Coulter counters - for TLC and DLC estimation

Flowcytometers - for TLC and DLC estimation
• A properly prepared blood smear is
essential to accurate assessment of
cellular morphology.
• The wedge smear is the most convenient
and commonly used technique for
making PBS.
• Direct capillary blood or anticoagulated
blood is used.
• PBS should be smooth without
irregularities, holes, or streaks.
• When the slide is held up to light, the
featheredge of the smear should have a
“rainbow” appearance.
• The whole drop is picked up and spread.
• STAINING OF PBS FOR TLC and DLC ESTIMATION -

• Staining is commonly done by Leishman's stain.

It is a Romanowsky type polychromatic stain, composed of -
Methanol (acetone free - to avoid cell membrane damage) - fixative (fixes proteins by
dehydration and precipitation. carbo and proteins are not fixed but ppted along with protein)
Methylene blue stains basic materials (nucleus and the basophilic granules)-blue-grey
Eosin stains acidic materials (hemoglobin, eosin granules)-orange-red

• The air dried smear is placed horizontally on a staining rack and covered with the undiluted
stain. Take care not to overflow with excess stain.Preferably add just the enough number of
drops to cover the smear. To standardize, count the number of drops required to cover the
film (so that double the number of buffered-water can be added later).
• Then leave the slide for 2-3 minutes so that undiluted stain can act as fixative and also
partially stains the smear.
• Now add twice the volume of pH 6.8 buffered water to dilute the stain, taking caution that
the stain should not overflow (which will make the dilution inaccurate). Mix the water with
the stain underneath by gently blowing with a straw or using a plastic bulb pipette.
• Allow to stain for 10–12 minutes (time may require adjusting). In this method, better
ionization during the dilution by the aqueous buffer in this step is necessary to complete the
staining.
• Wash off the stain with clean tap water. Wipe the back of the slide clean and stand it in a
draining rack to air dry.
• The stained smear should grossly appear neither too pink nor too blue.

• Unfortunately, even in well-spread films, the distribution of the various cell types is not
totally random. Polymorphs and monocytes predominate at the margins and the tail;
lymphocytes predominate in the middle of the film.
• This separation probably depends on differences in stickiness, size and specific gravity of the
different types of cells.
 METHODS OF PBS EXAMINATION
• LONGITUDINAL METHOD - In this the
slide is examined along the length of slide
from head to tail end. This method nullify the
effect of NON RANDOM distribution of cells
(so this method is preferred method for DLC
estimation, but it is difficult to identify cell
morphology at head end).
• For DLC estimation Inspect the film from the
head to the tail and if fewer than 100 cells
are encountered in a single narrow strip,
examine one or more additional strips until at
least 100 cells have been counted.
• Each longitudinal strip represents the blood
drawn out from a small part of the original
drop of blood when it has spread out
between the slide and spreader.
 TRANSVERSE METHOD -
• In this method the leukocytes are counted (for TLC estimation) and identified
(for DLC estimation) in the area of ideal thickness (=MONOLAYERED - where RBCs
are just approximated, neither overlapped nor too far from each other).
• Counting is done across the whole width of slide avoiding the lateral edges.
 at least 8-10 feilds are examined under high dry
(40x) objective by transverse method and leukocytes are counted ,
average number is calculated and then estimated TLC is -
 slide is examined by longitudinal or transverse method for
identification and counting of different types of leukocytes, at least upto count of
100 leukocytes. DLC is expressed as % value of TLC.
Absolute number can be calculated by using TLC and % value of DLC.
(for ex. Absolute neutrophil count = Total leukocyte count x neutrophil percent)

DLC can be performed under high dry (40x) or oil immersion (100x) objective but
40x is not recommended since the magnification is too low to see adequately all
the features of the cells.

If abnormal cells are present in small numbers, they are more likely to be
detected when 200-500 cell counts are performed than with a 100 cell count.

Nucleated RBCs and abnormal immature white blood cells (blasts) if present,
should be mentioned separately as % count of TLC (= per 100 WBC).
• It is done by selective staining of particular cells and lysis of other interfering cells, and
then counting them in a defined counting volume in a counting chamber, under
microscope.
• These counting chambers are k/a HEMOCYTOMETERS, because these are originally
designed to count blood cells.
• However, the precision and accuracy are highly dependent on the number of counted
cells and, at a reasonable level of effort, are subject to fluctuations of up to 10%.
• Various chambers available are Neubauer, Bürker, Rosenthal, Thoma, Schilling and Türk.
• Among these neubauer counting chamber is widely used for counting of various blood
cells.
• some other uses of counting chambers are sperm counting, recording growth over time
in cell cultures, for preparation of yeast, Cell processing for downstream analysis where
accurate cell numbers are needed( ex. PCR, flow cytometry) and Measurement of cell
size (the real cell size can be inferred by scaling it to the width of a hemocytometer
square, which is known).
 GENERAL FEATURES OF THE NEUBAUER’S
CHAMBER
• Neubauer’s chamber is a thick glass plate (70x30x4mm). The counting region
consists of two square shaped ruled areas. There are depressions on either side
and in between the areas on which the squares are marked thus giving an “H”
shape.
• Each ruled area is 3mm x 3mm, divided into 9 large squares of 1mm x 1mm.
• The ruled area is 0.1 mm lower than the rest of the chamber. So that when a
cover slip is kept on the counting region, there is a gap of 0.1 mm between the
cover slip and the ruled area. So volume of each large sq is 0.1mm3.
• The four large sqaures placed at the corners are used for white blood cell count.
The total volume of these 4 large sq is 4x0.1mm3 = 0.4mm3.
• 1mm3 = 1 micro L = 10-6 L = 10-3 mL.
• 1ml = 1000 micro L
• SAMPLE COLLECTION - Blood may be collected either by finger prick method (capillary
blood) or venepuncture method (venous blood), of which venous blood is preferred.
• ANTICOAGULANT - Ethylene diamine tetra acetic acid (EDTA , esp. dipotassium salt) is the
preferred anticoagulant for cell counts as it gives the best preservation of cell
morphology (morphology is preserved even after 2-3 hours of blood collection).
Recommended concentration is 1.5 +/- 0.25 mg /ml of blood.
• WBC DILUTING FLUID(TURK'S FLUID) - It is prepared by distilled water, glacial acetic acid
(a weak acid to lyse RBCs) and 1 % gentian voilet (stain the nuclei to locate the WBCs
under microscope) , in a ratio of 97:2:1. A pinch of THYMOL may be added to inhibit
fungal growth. Gention voilet also has some fungus inhibiting property. Turk's fluid is not
isotonic and it is stable at room temperature (25°C ± 5°C).
• COVER SLIP - Special optically plain cover glasses of defined thickness (most commonly
0.4mm thick) are required. Other cover glasses must not be used.
• The well mixed whole blood sample is drawn
upto .5 mark and then immediately WBC
diluting fluid is drawn upto 11 mark while
rotating the pipet between the thumb and
forefinger to mix the specimen and diluent.
Hold the pipet upright to prevent air bubbles
in the bulb.
• The bulb has capacity of 10, so finally fluids
in bulb are .5 part blood sample and 9.5 part
dilutent, giving the desired ratio of 1:20.
• Mix the contents of the pipet for 3-5
minutes to ensure even distribution of cells,
and to ensure lysis of RBCs.
• Expel unmixed and relatively cell-free fluid
from the capillary portion of the pipet
(usually 4 drops).
• Clean the Neubauer chamber and the cover slip with 70% EtOH. Put the glass
cover slip on the Neubauer chamber central area. Use a flat surface to place
the chamber, like a table.
• After removing the rubber tube horizontally, Place the forefinger over the top
(short end) of the pipet, hold the pipet at a 450 angle, and touch the pipet tip
to the junction of the cover glass and the counting chamber.
• Allow the mixture to flow under the cover glass until the chamber is
completely charged. Similarly, fill the opposite chamber.
• If the mixture overflows into the moat or air bubbles occur, clean and dry the
chambers, remix the contents of the pipet, and refill both chambers again.
• Allow the cells to settle for about 3 minutes. Under low-power magnification
and reduced light, focus on the ruled area and observe for even distribution of
cells.
For TLC estimation Cells are counted in 4 large corner squares under 10x
magnification.
In each of the four squares, conduct the count as indicated by the "snake-like" lines
to avoid any missed area.

If Cells are touching the 4 perimeter sides of a corner square, only count cells on 2
sides, either the 2 outer sides or 2 inner sides, to avoid double counting.

A variation of more than 10 cells between any of the four areas counted or a
variation of more than 20 cells between sides of the hemacytometer indicate
uneven distribution and require the procedure to be repeated.
 - Suppose number of cells counted in all 4 large corner sq is N.
• Total volume of these 4 sq is 4 x 0.1mm3 = 0.4 mm3

0.4 mm3 volume has N cells

So 1mm3 volume has N / 0.4 = 10 N /4 CELLS
Dilution factor is 20, so cells in undiluted original sample is 20 x 10 N /4
=

In cases where the WBC count is very high, as in leukemia, the dilution should be made in the
RBC diluting pipet to make the dilution of 1:100.

In cases of severe leukopenia, the WBC diluting pipet should be filled upto 1 mark (instead of
0.5 mark) and then upto 11 mark to make the dilution of 1:10.
 Improper collection of blood specimens.
 Wet or dirty pipets, cover glasses and hemacytometers.
 Poor condition or inaccurate calibration of pipets. Pipets must be in good
condition and calibrated to have maximum error of ±1 percent.
 Poor pipetting technique.
 Failure to expel 2 or 3 drops in the pipet tips before charging the
hemacytometer.
 Overfilling the chamber of the hemacytometer, which causes high counts.
 Uneven distribution of cells in the counting chamber.
 Inaccuracy or carelessness in marking counts.
 Diluent which is cloudy or contains debris.
 Improper mixing of anticoagulated blood sample before use.
 Improper mixing of blood and dilutent in WBC pipette.
• When nucleated red blood cells (NRBCs) are present, they will be included in
the total WBC, which is really a ‘total nucleated cell count (TNCC).
• So TNCC = UnCorrected TLC = WBC + NRBC.
• NRBC should also be included in the differential count, as a percentage of the
TNCC and reported in absolute numbers in the same way as the different
types of leucocytes.
• If they are present in significant numbers (more than 5%), the TNCC should be
corrected to obtain the true total WBC.
• There is 2 methods for corrected TLC counting depending on the method used
for NRBC counting in PBS.
 (A) If NRBCs are counted per 100 WBC then -

Corrected TLC = TNCC x 100 / 100 + NRBC per 100 WBC

(B) If NRBCs are counted per 100 TNCC then -

Corrected TLC = TNCC - TNCC x NRBC per 100 TNCC / 100

• For example suppose TNCC = 8000 and NRBC per 100 WBC is 25, then as per 1st equation
Corrected TLC = 8000 x 100 / 100 + 25
= 6400

To use second eq we have to calculate NRBC per 100 TNCC. 25 NRBC per 100 WBC = 25 WBC per 125
TNCC = 20 WBC per 100 TNCC.
Now Corrected TLC = 8000 - 8000 x 20 / 100
= 6400.
• Except the newborns (1st 3-4 days) presence of nucleated RBCs are abnormal in the peripheral blood.
• These cells have a dark, dense nucleus in the center of a bluish (polychromatophilic) or red
(orthochromatic) cell periphery.
• They are mostly seen in cases of marked stimulation of the bone marrow.
 Some common causes are -
Severe Acute bleeding
Severe hemolytic anemia, (e.g. thalassemia)
Megaloblastic anemia
Congenital infections, (e.g. congenital syphilis, CMV, rubella)
Postsplenectomy or hyposplenic states: Spleen normally removes nucleated RBCs
Leukoerythroblastic reaction, seen with extramedullary hematopoiesis and bone
marrow replacement
Some Fungal and mycobacterial infections
High WBC count with left shift
Dyserythropoietic anemias
Hypoxia.
 Automated counters are based on flow cytometry principle that is by suspending cells in
a stream of fluid and passing them by an electronic detection apparatus.
 Detection of cells may be IMPEDANCE based or LASER (optic) based.
Impedance based counters are k/a COULTER COUNTERS. This method is widely used for
routein WBC counting.
Laser based counters are k/a FLOW CYTOMETERS. They use differences in light scatter or
optical properties, either alone or in combination. Another recent advancement in
hematology analyzers is incorporation of argon laser technology, allowing integration of
some flow cytometric data using specific fluorochrome stains. With use of advanced
flowcytometers with cytochemical fluoresence markers more than 18 subclasses of WBCs
can be identified and counted.

 Each instrument presents a pictorial representation of the hematological data registered

as either histogram or scatterogram. Histogram is a graphic representation of blood cells,
produced from thousands/millions of signals generated by the cells passing through
detector where they are differentiated by their size, and frequency of occurrence in the
population thus provides objective record of variation in cell sizes within the population
under study.
 COULTER COUNTERS

• A Coulter counter is an apparatus for VOLUME dependent COUNTING and

SIZING of particles suspended in electrolytes.
• It is used for cells, bacteria, and virus or virtually for any particle.
• A typical Coulter counter has one or more microchannels that separate two
chambers containing electrolyte solutions.
• The Coulter Principle relies on the fact that particles moving in an electric field
cause measurable disturbances in that field.
• It states that when a particle move through an orifice(Microchannel)
concurrent with an electric current, produce a change in impedance that is
proportional to the VOLUME of the particle due to displacement of electrolyte
caused by the particle.
 FLOWCYTOMETERS
• For TLC and DLC estimation principle of scattering of light by cells is used (optical scatter).
• Laser light is preferred because of its properties of intensity, stability, and monochromatism.
• The blood cell is subjected to fluid dynamics which leads them to flow in a single line. Each
cell will pass through a detection device called a flow cell.
• This flow cell have a laser device focused on it and as the cell passes through the laser light
path, it will scatter light in several directions.
• The unit has one detector that captures the forward scatter light (FSL) and a second detector
that captures the side scattered light (SSL) at 90° or orthogonal angle.
• The cell with its internal and external features determines the quantity and character of the
light scatter.
• FSL is used to determine cell density or volume which relates to the cell size.
• SSL is used to determine cell contents revealing information about the nuclear complexity
and cellular granularity.
• By using both of these each cell is determined by its size, volume, density, and other nuclear
and cytolasmic properties.They can Analyze up to 10,000 cells per second.
• COMBINED IMPEDANCE AND OPTICAL COUNTERS - Some of the newer hematology analyzers
have combined impedance and optical methods together within one instrument, thereby
allowing for optimal use and integration of the data generated by each method.

• THE CONCEPT OF “VCS” TECHNOLOGY - This was developed by Coulter instruments and
employs three principles of evaluating the leukocyte- Volume, Conductivity and Scatter.
Volume - measured by low voltage direct current
Conductivity - measured by high frequency radio waves
Scatter - measured by scattering of laser light by cells.

All automated cell counters are screening devices. Abnormalities must be verified by
examination of blood film by an expert observer.
• DATA OUTPUT FROM AUTOMATED COUNTERS ARE IN FORM OF -

1- NUMERICAL DATA
2- HISTOGRAMS (coulter counters)
3- SCATTER DIAGRAMS (laser based optic scatter flowcytometers)
4- Three dimensional cubical diagrams (VCS Technology)
• A histogram is a vertical bar chart that
shows the distribution of a set of data.
• It will make it easy to see where the
majority of values fall in a measurement
scale, and how much variation is present.
• One SD on either side of the mean
encompasses about 70% of the population;
two SD on either side of the mean
encompasses about 95% of the population.
Similarly, 99% of the population would be
within 3 SDs.
• Because coulter counters uses volume of
cells to count and differentiate them, their
histograms are k/a volume histograms.
The automated counter sets a lower discriminator
(LD) fluctuating between 30 and 60 fL and an upper
discriminator (UD) fixed at 300 fL. The number of
cells between the UD and the LD is the WBC count.
WBC histograms consist of two troughs (T1,T2) and
3 peaks - TRIMODAL CURVE.
T1 is between 78 and 114 fL and T2 is < 150 fL.
The peak between the LD and T1 represents small
cell population i.e lymphocytes. The volume of the
cells range from 35 to 90 fL.
The peak that lies between T1 and T2 represents
the mid cell population which includes the
eosinophils, basophils, monocytes, blasts and
promyelocytes. Volume of the cell ranges from 90
to 160 fL.
The peak after T2 represents large cell population
which is neutrophils. Volume ranging between 160
and 300 fL.
FLAGGING AND ABNORMAL WBC HISTOGRAM
• Whenever there is any parameter which is abnormal and does not fit
into the normal expected graph, the analysis system will flag those
areas.
• These situations demand a visual review of the patient’s blood smear
for confirmation.
ACUTE LYMPHOCYTIC LEUKEMIA
• The undiluted sample histogram
shows a curve which neither starts
nor ends at the baseline. This is due
to an extremely high WBC count.

 The diluted sample histogram shows

a predominantly lymphocytic curve
which begins approximately 20%
above the normal baseline. This is
due to the presence of microblasts
(lymphoblasts) and rare NRBC.
Both mixed cell and neutrophil data
are suppressed with the lack of a
neutrophil peak.
CHRONIC LYMPHOCYTIC LEUKEMIA
• The histogram shows a
predominant lymphocytic
population curve.
• There appears to be little or no
neutrophil peak. Both mixed cell
and neutrophil data were
suppressed.
• The lymphoctic curve starts at base
line, indicating the population of
mature lymphocytes.
ACUTE MYELOID LEUKEMIA

• The histogram shows a single curve

which peaks in the mixed cell area at
approximately 90 fL this is consistent
with the location of myeloblasts on
the trimodal WBC distribution.
 SEPTICEMIA

• The WBC histogram shows very little

valley between the mixed cells and
neutrophil populations.
• This is due to the presence of
immature neutrophils that lie on the
ascending neutrophilic slope.
• The trimodal histogram demonstrates
an increased neutrophil population
and a decreased lymphocyte
population.
 optic scatter flowcytometry

• Scattergram is a graph representing the distribution of two variables in a

sample population.
• One variable is plotted on the vertical axis (forward angle light scatter-FALS)
and the second is plotted on the horizontal axis (side scatter-SS).
• A scattergram demonstrates the degree or tendency with which the variables
occur in association with each other.
• The scores or values of each sample unit are usually represented by dots.
• The positions of dots that appear on the scattergram screen are determined
by the degree of side scatter (SS), degree of forward angle light scatter (FALS),
absorption of light by each cell, and dyes (if any used).
• Low forward and side scattering
angles are indicative of lymphocytes.
• As the angle of forward and side
scattering increases monocytes
appear on the cytogram followed by
neutrophils.
• Eosinophils have the highest forward
and side scattering angles.
• The basophil requires a special
window and detection technology.
• If a NRBC is present, it will most likely
be counted as a WBC.
The leukocyte count may alter beyond physiological limits in various pathological conditions,
giving valuable clues for clinical diagnosis.
Normal Reference range may vary between laboratories.
Patient results should always be interpreted using the units and reference ranges from the
laboratory that produced the results.
Below are reference ranges for leucocytes/WBCs -
• Total WBCs: 4,500–11,000/mm3 for adult (women and men) -
Neutrophils 50–70% (2000-7500/Cumm)
Lymphocytes 25–35% (1500-4000/Cumm)
Monocytes 4–6% (200-800/Cumm)
Eosinophils 1–4% (40-400/Cumm -Diurnal variation seen)
Basophils 0.5–2% (20-100/Cumm)

Leukocytosis is leukocyte count above the normal range in the blood.

Leukopenia is leukocyte count below the normal range in the blood.

The mechanism that causes leukocytosis/leukopenia can be of several forms:

Increased/decreased release of leukocytes from bone marrow storage pools,
Decreased/increased margination of leukocytes to vessel walls,
Decreased/increased extravasation of leukocytes from the vessels into tissues,
Increase/decrease in number of precursor cells in the marrow.
• At birth, the total leucocyte count is high; neutrophils predominate, reaching a
peak of 13.0 x 109/l within 8 h for neonates of >28 weeks’ gestation and 24 h
for those delivered at <28 weeks.
• The count then falls to 4.0 x 109/l over the next few weeks and then to a level
at which the count remains steady.
• The lymphocytes decrease during the first 3 days of life often to a low level of
2.0–2.5 x 109/l and then rise up to the 10th day; after this time, they are the
predominant cell (up to about 60%) until the 5th to 7th year, when they give
way to the neutrophils.
• From that age onwards, the levels are the same as for adults.
• There are also slight sex differences; the total leucocyte count and the
neutrophil count may be slightly higher in girls than in boys, and in women than
in men. After the menopause, the counts fall in women so that they tend to
become lower than in men of similar age.
SHIFT TO LEFT : Increased bands and other
precursors in peripheral blood indicates rapid
production of new neutrophils.
Usually caused by excessive demand of
defence system during acute processes like
acute infection.

SHIFT TO RIGHT : (Increased PMNs)

indicates increased mature neutrophils
caused by chronic processes like pernicious
anemia, hepatic disease, etc.
LEUKEMOID REACTION -

It is a hematological disorder that

simulates leukemia due to high WBC
counts (≥30,000–50,000) and
presence of some premature
leukocytes (band forms,
metamyelocytes, myelocytes, and
even promyelocytes).

Neutrophils often show toxic

granules along with Dohle bodies and
cytosolic vacuolation.

NAP score is increased.

The circulating white blood cells are

• Döhle bodies are light blue-gray, oval,
basophilic, leukocyte inclusions located
in the peripheral cytoplasm of
neutrophils. They measure 1-3 µm in
diameter. They are thought to be
remnants of the rough endoplasmic
reticulum.

• Toxic granulations are dark coarse

granules found in granulocytes,
particularly neutrophils. Along with
Döhle bodies and cytosolic vacuolation,
which are two other findings in the
cytoplasm of granulocytes, toxic
granulations are a peripheral blood film
finding suggestive of an inflammatory
 CHRONIC LYMPHOID LEUKEMIA
• Persistent lymphocytosis
• Absolute lymphocyte count usually
greater than 5000 /mm3
• Lymphocytes are small, mature
looking with high N/C ratio, round to
oval nuclei with clumped chromatin.
• Smudge cell is a characteristic
feature.
LYMPHOCYTIC LEUKEMOID REACTION
• Transient reactive lymphocytosis
• Absolute lymphocyte count usually
less than 5000 /mm3
• Reactive lymphocyes are large sized
with abundant cytoplasm and dark
blue edges.
THANK YOU