By Dr.

Richa Sharma
TYPES OF CELL COUNTING
MANUAL

SEMI AUTOMATED

AUTOMATED
TYPES OF AUTOMATED
FULLY AUTOMATED MORPHOLOGY BASED
 CoulterSTKS  Cella Vision DM 96
 SysmexSE series,XE2100  DiffMaster
 Bayer H Series Adiva  SE-223 70
 Abbot
DISADVANTAGES OF MANUAL
CELL COUNTING:
 Cell identification errors in manual counting:
mostly associated with distinguishing lymphocytes from
monocytes, bands from segmented forms and abnormal
cells (variant lymphocytes from blasts)

 Slide cell distribution error:

increased cell concentration along edges also bigger cells
found there i.e. monocytes, eosinophils, and neutrophils

 Statistical sampling error

ADVANTAGES OF AUTOMATED:
 Objective (no inter-observer variability)

 No slide distribution error

 Eliminate statistical variations associated with

manual count based on high number of cells counted

 Many parameters not available from a manual count,

e.g. MCV, RDW…

 More efficient and cost effective than manual method:

– Some cell counters can process 120-150 samples per hour
PRINCIPLES
OF AUTOMATED CELL
COUNTERS
Impedance (conductivity) system (Coulter)

Optical system (Light Scattering)

Flow cytometry

Selective lysis

Special Stains
ELECTRICAL IMPEDANCE
METHOD
The “Coulter Principle”
 Cell counting and sizing is based on the detection
and measurement of changes in electrical impedance
(resistance) produced by a particle as it passes through
a small aperture

 Particles such as blood cells are nonconductive but

are suspended in an electrically conductive diluent

 As a dilute suspension of cells is drawn through

the aperture, the passage of each individual cell
momentarily increases the impedance (resistance)
of the electrical path between two electrodes that
are located on each side of the aperture
ELECTRICAL IMPEDANCE METHOD
The “Coulter Principle”
Direct current - Radio frequency -
measurement of cell measurement of internal
volume cell structure.
OPTICAL METHOD
 Laser light is used

 A diluted blood specimen passes in a steady stream through

which a beam of laser light is focused

 As each cell passes through the sensing zone of the flow,scatters

the focused light

 Scattered light is detected by a photodetector and converted to

an electric impulse

 The number of impulses generated is directly proportional to

the number of cells passing through the sensing zone ina specific
period of time
 The application of light scatter means that as a single
cell passes across a laser light beam, the light will be
reflected and scattered.

 The patterns of scatter are measured at various angles.

 Scattered light provides information about cell

structure, shape, and reflectivity.

 These characteristics can be used to differentiate the

various types of blood cells and to produce scatter
plots with a three or five-part differential
Flow Cytometry
PRINCIPLE:A beam of light ( laser light) of a
single wavelength is directed onto a
hydrodynamically-focused stream of fluid. A
number of detectors are aimed at the point where the
stream passes through the light beam: one in line with
the light beam (Forward Scatter ) and several
perpendicular to it (Side Scatter ) and one or more
fluorescent detectors. Each suspended particle from
0.2 to 150 µm passing through the beam scatters the
ray, and fluorescent chemicals found in the particle or
attached to the particle may be excited into emitting
light at a longer wavelength than the light source.
Optical System
 This combination of scattered and fluorescent
light is picked up by the detectors, and, by
analysing fluctuations in brightness at each
detector (one for each fluorescent emission
peak),it is then possible to derive various types of
information about the physical and chemical
structure of each individual particle.
 Forward light scatter - cell size & volume
 Side light scatter- inner complexity of the particle
(i.e., shape of the nucleus, the amount and type of
cytoplasmic granules or the membrane roughness)
 Side fluorescence - RNA/DNA
information(nucleus)
Specific measurements
Analysis Parameter
• Whole WBC Count • MCV
• LYM% • MCH
• MXD% • MCHC
• NEUT% • RDW-CV
• LYM# • RDW-SD
• MXD# • PLT
• NEUT# • PDW
• HGB • MPV
• RBC Count • P-LCR
• HCT • Some extended
parameters
 • DC detection method
WHOLE • Wbc count in 1µl of whole blood
WBC

• WBC – Small cell ratio

• Ratio(%) lymphocyte to whole wbc
LYM%

• WBC- Middle cell ratio

• Ratio(%) of basophil,eosinophil&monocyte
MXD% to whole wbc
 • WBC –Large cell ratio
NEUT% • Ratio(%) of neutrophils to whole wbc

• Absolute count of lymphocte in 1µl of

LYM# whole blood

• Absolute count of middle cell in 1µl

MXD# of whole blood
 • Absolute count of neutrophil in 1µl of whole blood
NEUT#

• Non cyanide Hb analysis method

• Volume(gm) of Hb in1dl of whole blood
HGB

• DC detection method
• Rbc count in 1µl of whole blood
RBC
 • RBC pulse height detection method
• Ratio(%) of whole rbc volume in whole blood
HCT

• Mean rbc volume (fl) in whole blood

• HCT/ RBC
MCV

• Mean Hb volume(pg) per rbc

• HGB/ RBC
MCH
 • Mean Hb concentration (g/dl)
• HGB/HCT
MCHC

• Rbc distribution width-Coefficient of Variation

• Calculated from pts. defining68.26%of entire area
RDW-CV spreading from peak of rbc particle distribution curve

• Rbc distribution width- Standard Deviation

• The distribution width(fl) at ht20% from bottom when
RDW-SD peak rbc particle distribution curve is taken 100%
 • DC detection method
• Platelet count in1µl of blood
PLT

• Platelet distribution width

• Distribution width at ht 20%from bottom with peakof
PDW platelet distribution curve taken 100%

• Mean volume of platelet (fl)

MPV

• Ratio of large platelet volume exceeding

P-LCR 12fl to the platelet volume
REAGENT
 DILUENT

Cell Pack(approx. 30 ml)

 WBC/HGB LYSE REAGENT

StromatolyserWH(approx. 1 ml)
Overview of analysis
modes
 Whole Blood Mode  Pre diluted mode
o Analyse collected o Used in analyzing a
sample in whole blood minute amount of
status child’s blood e.g
collected from
earlobe
o Blood sample diluted
into 1:26
 Fully automated instruments perform TLC by
impedance or light scattering or both.
 Counts performed on diluted WBC in which red cell’s
are either lysed or rendered transparent.

FLAGS
 Although threshold set to exclude platelet from
being counted, giant platelet might be included.
 NRBC are usually included, hence. Count approx
more to TNCC than to WBC.
 FACTIOUS COUNTS

LOW COUNT
 Leucocyte agglutination

HIGH COUNT
 Failure to lyse RBC
 Microclots
 Platelet clumping
 Abnormal Hb
DLC
 Automated cell counters have a differential capacity of
doing three-part, five-part or seven-part differential
count
 Categorization based on different volume of various
cells& cytoplasmic shrinkage
Three-part differential
Can be produced by single channel by both impedance or light
scatter instruments.
It designates cell as:
a. Granulocyte/Large cell
b. Lymphocyte/Small cell
c. Monocyte/Middle cell
Five or Seven part differential
• Produced by two or more channels.
• Analysis dependent on volume and other physical
characteristic of cell or binding of certain dyes to
granules or activity of cellular enzyme.
• Technology used are Light scattering, Absorbance and
Impedence with low or high frequency
electromagnetic or radiofrquency current.
• Cell classification:-
a) neutrophil b)eosinophil c)basophil
d)monocyte e)lymphocyte
extended classification:- *large immature cells
*atypical lymphocytes
New White Cell Parameter
 Many instruments can flag presence of atypicalor
variant lymphocyte by alteration in size,impedence
and light scattering
eg: identify eosinophils by ability of their granules to
polarize light
detect a left shift or presence of blasts by reduced
light scattering of nuclei of more immature
granulocytes.
 The ADVIA uses TWO separate channels to
determine the white cell count:
1) Peroxidase channel
2) Basophil/lobularity channel
PEROXIDASE CHANNEL
 Specimen is diluted with peroxidase reagents, red cells
and platelets are lysed
 Dark precipitate forms in cells containing peroxidase:
– Neutrophils, eosinophils, monocytes
Lymphocytes, basophils, large unstained cells contain no
peroxidase and therefore do not stain
 A tungsten-based optical system is used to measure:
– Individual impulses are counted to determine WBC
– Stain intensity (absorbance) for each cell
– Cell size (by forward light scatter)
 Cells are plotted on a scattergram: scatter (size) on yaxis
and peroxidase activity (staining intensity) on the
x-axis
 Each cell is classified based on this information
 In this channel, basophils are classified with
lymphocytes
 Lymphocytes: Small, unstained cells
 Larger atypical lymphocytes, plasma cells, and some
blasts: “large unstained cells”
 Eosinophils: Strongest peroxidase activity and appear
smaller than neutrophils.
 Neutrophils are large and have moderate peroxidase
activity
BASOPHIL/LOBULARITY
CHANNEL
• Surfacant and phthalic acid are added to lyse the
RBCs and strip away the cytoplasmic membrane from
all leucocytes except the basophils
• Laser light signal from a low-angle forward scatter
plot identifies basophils, which, due to their retained
cytoplasm, are larger than other cells
• A high-angle detector is sensitive to the shape and
structure (lobularity and density) of remaining nuclei
Flags
DLC ordered

Automated Differential Performed

flags no flags
slide made automated DLC
reported

Review with Review with

Microscope Cellavision
FLAGS GENERATED BY COMBINATIONS
OF THE CHANNELS
 In addition to numerical data and various flags generated
by the peroxidase and the basophil channels
individually, other flags are generated by comparison of the
channels:
– Discrepancy of the two WBC counts: Can be due to nonlysis
of RBC in peroxidase channel
– Flag for immature granulocytes: Presence of more
peroxidase-positive cells than expected from the number
of PMN in basophil channel
– NRBC flag: Excess of cells in PMN in comparison with
sum of eosinophils and neutrophils in peroxidase channel
HAEMOGLOBIN
Measure Hb by modification of HiCN method with
Sodium lauryl sulphate(non-toxic)
Modification include :
a) alteration in conc. of reagent
b) temp & pH of reaction

A non ionic detergent used for rapid cell lysis & to

reduce turbidity by cell membrane and plasma lipid.
 Counted on either aperture impedance or light scattering
technology
 Renders red cell indices derived from RBC count
 ERRORS
 Inaccuracy of coincidence
a. Two cells passing from aperture being counted as one or
pulse being genrated during electronic dead time
b. Red cell agglutination
c. Recirculation of already counted cell
d. Counting of bubbles or lipid drops or micro organisums
 Faulty maintainance
 Use of diluent other than the one recommended by
manufacturer
hct & MCV
 Passage of cell through apperture of an impedence
counter or through beam of light of light scattering
instrument generates electrical pulse of ht proportional
to cell volume
 No. of pulses generated= RBC count
 Average pulse ht = MCV
 MCV× RBC Count= HCT
OR
Summation of pulse ht= HCT
MCV= HCT/RBC Count
 It’s seen that apperture impedance system measures
apparent volume which is greater than true volume of
RBC and is influenced by
shape factor
< 1.1- young and flexible RBC
1.1-1.2 – fixed RBC
1.5 – Spheres
Reticulocyte Count
o An automated retic count can be performed using the fact
that various fluoro-chromes combine with the RNA of the
reticulocytes. Fluorescent cells can then be enumerated
using a flowcytometer.
o An automated retic counter also permits the assessment of
retic maturity since the more immature reticulocytes have
more RNAfluoresce more strongly than the mature
retics found normally in PB.
o Size and Hb content of reticulocytes are measured by
light scatter
 Same principal of electrical or electro optical detection
 Upper Threshold needed to separate platelet from RBC
 Lower Threshold needed to separate platelet from
debris and noise
 3 technique for setting threshold:-
a. Platelet can be counted between two fixed threshold
2 and 20 fl
b. Platelet b/w fixed threshold can be counted with
subsequent fitting of curve, so that, platelet falling
outside threshold hold can be conted in computed
count
c. Threshold may vary automatically depending on
characteristic of individual blood sample to make
allowance for microcytic or fragmented RBC
NEW METHOD- BY FLOW CYTOMETRY
 Platelet in a blood sample labelled flourscently with
specific monoclonal or combination of antibody
and by measuring RBC : Platelet ratio the platelet
count can be measured.
 Antibody suitable for labeling of platelet Antigen are
CD41,CD42 and CD61.
ERRORS
Factiously low count
 giant platelet identified as RBC
 EDTA induced platelet clumping
 Satelitism

Factiously high count

 Markedly microcytic or fragmented RBC
 Cell fragments in leukemia
 Bacteria or fungi
Mean platelet volume
 Measured by same technology as for MCV.
 Dependent on technique of measurement and on
length &condition of storage before testing blood.
 Other parameters are:
 PDW
 Plateletcrit= MPV x platelet count.
graphics
 Display of automated cell counters include coloured or
black and white graphics

 Histogram of RBC, WBC & Platelet size

 Histogram of Red cell Hb concentration

 Scatter plots of size versus Hb conc

TRENDS
 Current trends include attempts to incorporate as
many analysis parameters as possible into one
instrument platform, in order to minimize the need to
run a single sample on multiple instruments (e.g CD4
counts, smear preparation)

 Such instruments are being incorporated into highly

automated combined chemistry/hematology
laboratories, where samples are automatically sorted,
aliquoted, and brought to the appropriate technique by a
robotic track system
Tha
nk
you