2. TYPES OF CELL COUNTING
MANUAL
SEMI AUTOMATED
AUTOMATED
3. TYPES OF AUTOMATED
FULLY AUTOMATED MORPHOLOGY BASED
CoulterSTKS
SysmexSE series,XE2100
Bayer H Series Adiva
Abbot
Cella Vision DM 96
DiffMaster
SE-223 70
4. 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
5. 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
8. 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
9.
10. ELECTRICAL IMPEDANCE METHOD
The “Coulter Principle”
Direct current -
measurement of cell
volume
Radio frequency -
measurement of internal
cell structure.
11. 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
12. 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
13. 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.
15. 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)
18. WHOLE
WBC
• DC detection method
• Wbc count in 1µl of whole blood
LYM%
• WBC – Small cell ratio
• Ratio(%) lymphocyte to whole wbc
MXD%
• WBC- Middle cell ratio
• Ratio(%) of basophil,eosinophil&monocyte
to whole wbc
19. NEUT%
• WBC –Large cell ratio
• Ratio(%) of neutrophils to whole wbc
LYM#
• Absolute count of lymphocte in 1µl of
whole blood
MXD#
• Absolute count of middle cell in 1µl
of whole blood
20. NEUT#
• Absolute count of neutrophil in 1µl of whole blood
HGB
• Non cyanide Hb analysis method
• Volume(gm) of Hb in1dl of whole blood
RBC
• DC detection method
• Rbc count in 1µl of whole blood
21. HCT
• RBC pulse height detection method
• Ratio(%) of whole rbc volume in whole blood
MCV
• Mean rbc volume (fl) in whole blood
• HCT/ RBC
MCH
• Mean Hb volume(pg) per rbc
• HGB/ RBC
22. MCHC
• Mean Hb concentration (g/dl)
• HGB/HCT
RDW-CV
• Rbc distribution width-Coefficient of Variation
• Calculated from pts. defining68.26%of entire area
spreading from peak of rbc particle distribution curve
RDW-SD
• Rbc distribution width- Standard Deviation
• The distribution width(fl) at ht20% from bottom when
peak rbc particle distribution curve is taken 100%
23. PLT
• DC detection method
• Platelet count in1µl of blood
PDW
• Platelet distribution width
• Distribution width at ht 20%from bottom with peakof
platelet distribution curve taken 100%
MPV
• Mean volume of platelet (fl)
P-LCR
• Ratio of large platelet volume exceeding
12fl to the platelet volume
25. Overview of analysis
modes
Whole Blood Mode
o Analyse collected
sample in whole blood
status
Pre diluted mode
o Used in analyzing a
minute amount of
child’s blood e.g
collected from
earlobe
o Blood sample diluted
into 1:26
26.
27. 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.
29. 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
30. 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
31. 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
32. 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)
33. 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
34. 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
36. 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
37. 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.
38.
39. 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
40. 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
41. 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
42. 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 RNAfluoresce more strongly than the mature
retics found normally in PB.
o Size and Hb content of reticulocytes are measured by
light scatter
43.
44. 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
45. 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.
46. 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
47. 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.
48. 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
49.
50. 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
