powerpoint

1. Hematology automation

2. • Concept of counting blood cells automatically
began in the mid-1930s with a report describing
a photoelectric method for counting cells
passing through a capillary tube using darkfield
optics.
• In the late 1940s and early 1950s, two
instruments were introduced that proved to be
accurate as well as practical.
– The first was modification of the original capillary
method using darkfield optics – the darkfield optical
scan or light scatter.
– The second instrument was based on a completely
new nonoptical principle of cell counting: electrical
gating or electrical impedance.

3. • In order to count and size the particles
accurately, means of discriminating
between particles sizes:
– THRESHOLD
• Voltage limit with which a pulse is comparedVoltage limit with which a pulse is compared
• Only pulses that exceed the threshold are sized
and counted in which both lower and upper
threshold can be set
– Increasing the lower threshold eliminates unwanted
small pulses such as those caused by debris.
– An upper threshold eliminates large pulses.
• by manipulating the lower and upper thresholds, it
is also possible to produce a “window”
– Specific particle size range
• Have adjustable thresholds
– Capable of being calibrated to eliminate spurious counts
and produce clinically accurate counts

4. • CLASSIFICATION OF ELECTRONIC
CELL COUNTERS
• SINGLE PARAMETER PARTICLE
COUNTING INSTRUMENTS
– Automated cell counters not capable of
counting erythrocytes, leukocytes and
platelets simultaneously
• MULTIPARAMETER INSTRUMENTS
– Instrument capable of simultaneous RBC and
WBC counts, as well as determinations of Hb
concentration and MCV
• Using these data, the instrument calculates the
Hct, MCH and MCHC

5. • 2 CLASSIC CELL COUNTING PRINCIPLES:
• ELECTRICAL IMPEDANCE or LOW VOLTAGE
DIRECT CURRENT RESISTANCE
– Based on the fact that cells are relatively poor
conductors of electricity
– If two electrodes conducting an electrical current
through an electrolyte solution such as saline are
separated so that the only connection is through a tiny
aperture, any interference (such as blood cell) will
change the conductance.
– As cells are pulled through the aperture, the changes
in voltage that occur as the cells increase resistance
to the current are sensed by the instrument
– Good illustration of Ohm’s law: voltage = current x
resistance
• the magnitude of the voltage pulses produced by the cells is
directly related to their size

6. • OPTICAL DETECTION
– Utilizes both laser and non-laser lights
– Suspension of cells was pumped through a
narrow capillary tube in the path of darkfield
lighting
– Light pulses reflected by the cells were
collected with a series of mirrors and lenses
into the photomultiplier tube
– This information was then converted to cells
per microliter

7. • CELL COUNT
• The aspirated whole blood sample is divided into twotwo
aliquotsaliquots, and each is mixed with isotonic diluent.
• One dilution is delivered to the RBC aperture for
information about RBCs and platelets. The platelet count
is determined from a segment of the RBC histogram
within the range of 2 to 20 fL. The pulses obtained in this
range are classified as platelets for the raw count. Red
cells 36-360 fL.
• In some analyzers the WBC count is directly measured
by electric impedance after the red cells have been
destroyed by a lysing agent. The lysing agent also
shrinks the leukocyte cell membrane and cytoplasm;
therefore, the WBC count represents the measure of the
cell volume not native cell size. The diluent is saponin
solution.
• While the other dilution is delivered to the WBC bath,
where a lytic reagent is added to break down the RBC
stroma and release Hb. HemoglobinHemoglobin concentration is
measured by the amount of light transmittance at a
wavelength of 525nm.525nm. This information is sent to the
analyzer.

8. • ELECTRICAL IMPEDANCE
• INSTRUMENT COMPONENTS:
• Aperture tube
– Hollow glass tube with an open upper end that is
shaped to permit an airtight seal between the
aperture tube and the stopcock assembly.
– The lower end is closed except for a small aperture or
orifice. The size of the aperture is indicated on the
tube; several sizes are available (e.g. 200, 100, 70
and 50 um).
– It is suspended from the stopcock assembly and
contains an internal electrode as well as a tube that
may be used to deliver electrolyte solution into the
aperture tube.
• Stopcock assembly
– Connects the aperture tube to manometer

9. • Aperture current electrodes
– Function is to generate current across the aperture
and current polarity alternates with each count
– There are two platinum electrodes:
• Internal electrode within the aperture tube
• External electrode suspended by a wire next to the aperture
tube
• Manometer
– Specialized glass U-tube containing mercury. One
end is connected to the aperture tube through the
stopcock assembly, and the other end is open.
– Electrodes are placed in the manometer that is
activated by the passage of mercury.
• Vacuum system
– Consist of a simple rotary pump connected to a trap,
which in turn is connected to the stopcock assembly.

10. • Oscilloscope
– Displays visual representation of voltage pulses
caused by cells as they pass through the critical
volume.
– This depiction is a visual guide to the size and
number of particles being counted.
• Threshold dials
– Located on the front of the instrument. Both upper
and lower thresholds may be manipulated. These
dials are marked in units of 0 to 100.
• Aperture Current Settings
– A rotary switch on the front of the instrument controls
the amount of current passing between the internal
and external electrodes.
– Increases in aperture current will increase the voltage
pulse amplitude caused by individual cells.
• Digital Readout
– A five-digit numerical display records cells as they are
counted.

11. – An audible click is emitted as each thousand
is counted. These clicks create the cadence
or rhythm of the count; by listening to this
cadence, the operator can detect
malfunctions without having to watch
instrument constantly.
• Debris monitor
– Consist of modified microscope lens system
and back light focused on the aperture. The
image of the aperture is transmitted to a small
viewing screen so the operator can detect any
debris interfering with the aperture during a
count.

16. PRINCIPLE OF OPERATION
• Coulter counters manufactured by Coulter Electronics
operate on the principle of electrical impedance.
• Cells are suspended in an electrolyte solution. The
electrolyte is a good conductor of electrical current,
where as cells are relatively poor conductors.
• Electrical current is applied to the cell suspension
between two electrodes.
– One electrode (external) is in the cell suspension, and
the second electrode (internal) is within the tube
made of inert material (glass).
• The only connection between the two electrodes is a
small aperture (orifice) in the side of the tube.
• A measured volume of diluted cells are pulled through
the aperture. Differences in electrical resistance between
the two electrodes occur as the cells pass through the
aperture, causing changes in voltages (voltage pulses)
that are amplified and counted.

17. • Oscilloscope screens display the pulses
generated by the cells as they interrupt the
current.
• The size of the voltage pulse is directly
proportional to the size (volume) of the cell, thus
allowing discrimination and counting of specific-
sized cells through the use of threshold circuits.
• Pulses are collected and sorted according to
their amplitude by pulse height analyzers.
• The data are plotted on a frequency distribution
graph, or size distribution histogram, with the
relative number on the y axis and size (channel
number equivalent to specific size) on the x axis.

18. FACTORS AFFECTING SIZE OR
VOLUME MEASUREMENTS:
• Aperture size
– The red cell/platelet aperture smaller than the WBC
aperture to increase platelet counting sensitivity
• Protein buildup
– Protein buildup on the aperture decreases the size of
the orifice, thereby decreasing the flow of cells and
increasing the electrical resistance as the cells are
pulled through  results in lower cell counts and with
falsely elevated cell volumes.
– Older instruments require frequent cleaning and
newer instruments have incorporated “burn curcuits”
or other internal cleaning systems to prevent or slow
down protein buildup.
– Carryover of cells from one sample to the next is also
minimized by these internal cleaning systems

19. • Coincident passage
– Coincident passage of more than one cell at a time
through the orifice causes artificially large pulses 
falsely increased cell volumes and false decreased
cell counts.
– This count reduction, or coincident passage loss, is
statistically predictable (can be corrected
mathematically) because of its direct relationship of
cell concentration and the size or effective volume of
the aperture
– Correction is made and completed by the analyser
computer before final printout of cell counts from the
instrument.
• Orientation of the cell in the center of the
aperture and deformability of RBC
– Altered by decreased haemoglobin content

20. • Recirculation of cells
– Recirculation of cells back into the sensing zone
creates erroneous pulses and falsely elevated cell
counts
– A back-wash or sweep-flow mechanism has been
added to prevent recirculation of cell back into the
sensing zone, and anomalously shaped pulses are
edited out electronically.

21. OPTICAL DETECTION
• Cells pass through a flow cell on which a beam of light is
focused. As the cells interrupt the beam and collect
scattered rays at specific angles as individual cells pass
through the sensing zone.
• Analysis and conversion into digital form provides cell
counts and size information.
• Patterns of Scatter:
– Forward light scatter 0º : cell volume
– Forward low-angle scatter 2-3º : size or volume
– Forward high angle 5º-15º : refractive index of cellular
components
– Orthogonal light scatter 90º : reflection and refraction of internal
components’ complexity or granularity

22. • FLOW CYTOMETRY
• Instruments using light scatter methodology that
are capable of making multiple measurements of
individual cells processed in a flowing fluid are
termed “flow cytometers”.
• Many new terms used to describe the
characteristics of the flow cytometers:
– Laser
• Is an acronym for light amplification by stimulated emission
of radiation
• Differs from a beam of ordinary light in that it is emitted as a
single wavelength (monochromatic light)
• It is also coherent – it travels in phase (wave peaks and
valleys are together) from its source and enables detection of
the effects of interference.
• It has a little spread (low divergence)
• The fourth characteristic is its brightness (high power per
solid angle)

23. – Light scatter
• Summation of three independent processes:
– Diffraction – bending around corners and generally
dominating at small angles relative to the incident light
– Refraction – bending because of change in speed and
refractive index generally dominating at intermediate
angles
– Reflection – light rays turned back by the surface or
boundary of an obstruction and dominates at larger
angles.
– Flow cells
• Made of quartz instead of glass because quartz is
transparent and does not bend light that passes
through it.
• Allows UV light to pass where counting is done
and cell characteristics are measured.

24. – Sheath fluid
• Fluid that fills a flow cell and surrounds the sample
stream as it passes through the flow cell.
• It prevents flow cells from being coated by
reagents, cell stroma or other substance that may
bend rays of light; also facilitates laminar flow and
hydrodynamic focusing.

25. – Laminar flow
• Physics term that describes the flow properties of a
fluid moving relatively constant through a long
channel or pipe
• Particles within this fluid follow paths of
streamlines
• When laminar flow condition exists, all particle flow
in parallel lines as they travel through the flow cell
• Under these conditions, the second fluid (sample)
does not mix with the surrounding fluid (sheath)
• Dependent on flow velocity, channel diameter, fluid
density and the fluid viscosity coefficient plug into a
formula to give a value called the “Reynolds
number”.
– Flow is more laminar as the Reynolds number gets
further below the critical value, whereas flow becomes
turbulent when the critical value is exceeded.

26. – Hydrodynamic focusing
• Produced by symmetrically decreasing the
cross sectional area of the fluidic channel in
the flow cell and reducing the area in which
the fluid is flowing results to a faster-flowing
central fluid with narrowing of the central
sample stream.
• Done in order to narrow the sample stream
thus separating and aligning cells into a
single for passage through the sensing
zone

27. – Photodetectors
• Scatter detectors; used commonly are photodiodes
and photomultiplier tubes
• Photodiodes
– Light detectors that are not very sensitive but are
sufficient to detect forward scatter, which has a relatively
strong light level.
• Photomultiplier tubes
– Sensitive to weak light levels and, as the name implies,
multiply weak signals into stronger, useful signals.
– Blocker bar or darkfield stop
• Barriers that prevents direct (unscattered light)
from reaching the light scatter detectors

28. • Simultaneous measurement of multiple physical
characteristics of a single cell as the cell flows in
suspension through a measuring device
• Cells are stained in suspension
• Usually using laser light because of its intensity,
stability and monochromism
• In laser flow cytometers:
– Light scatter is used to measure intrinsic size and
granularity of the cell
– Fluorescence can be used to express extrinsic
features
– Fluorescent dyes used must react with the cellular
component of interest
• Examples: acridine orange, thioflavin T, pyronin Y, FITC and
phycoerythrin (PE)

30. PRINCIPLE OF OPERATION
• A suspension of stained cells is pressurized using gas and
transported through plastic tubing to a quartz flow chamber.
• In the flow chamber, the specimen is injected through a
needle into a stream called the sheath. The sheath and
specimen both exit the flow chamber through a 75-µm orifice.
• The laminar flow design confines the cells to the very center
of the saline sheath with the cells moving in single file.
• As the cells pass through the sensing zone and interrupt the
beam, light is scattered in all directions. Light scatter results
from the interaction between the process of absorption,
diffraction, refraction and reflection.
• The detection and conversion of scattered rays into electrical
signals is accomplished by photodetectors (photodiodes and
PMTs) at specific angles.
• Lenses fitted with blocker bars to prevent nonscattered light
from entering the detector are used to collect the scattered
light.

31. • A series of filters and mirrors separate the varying
wavelengths and present them to the photodetectors.
• Photodiodes convert light photons to electronic signals
proportional in magnitude to the amount of light
collected.
• PMTs are used to collect the weaker signals produced at
a 90 degree angle and multiply the photoelectrons into a
stronger, useful signals. Analog-to-digital converters
change the electronic pulses to digital signals for
computer analysis.
• Interaction between the laser beam and the cells
provides the information on the amount of light scattered
by each cell hit by the laser beam and the intensity of
fluorescence emitted
• Extrinsic properties make use of dyes and stains
• Intrinsic properties such as forward and right angle
scatter does not require dyes or stains for detection

32. – Side light scatter: light scattered at a 90◦
angle from the particle defines internalinternal
complexity and granularitycomplexity and granularity of the particle.
• Neutrophils and eosinophils produce a great deal
of side scatter due to their cytoplasmic granules.
– Forward scatter: light that continues in the
forward direction relates the particle size.
• Large cells such as monocytes and neutrophils
produce more forward scatter than nRBCs, and
normal lymphocytes.

34. INTERFERENCES CAUSING ERROR
Fragmented red cells or
very small red cells
These may cause red cell counts to be decreased and
may flag the platelet count as the red cells become closer
in size to the platelets and cause an abnormal platelet
histogram.
Platelet clumps or
satellitosis
These cause falsely decreased platelet counts.
Platelet clumps can be seen on the right side of the
platelet histogram. Decreased platelet counts are
confirmed by reviewing the peripheral smear.
Always scan the edge of the smear when checking
low platelet counts.
Giant platelets Giant platelets may be counted as RBC and result in a
spuriously low platelet and high red cell count.
Nucleated red cells The NRBC is counted in the WBC bath as leukocytes
and will falsely increase the WBC count and decreases
the RBC count.

35. Agglutination Low red cell counts and high MCVs can be caused by a
decreased number of large red cells or red cell
agglutinates. If agglutinated red cells are present, the
automated hematocrits and MCHCs are also incorrect.
Cold agglutinins cause agglutination of the red cells as
the blood cools. Cold agglutinins can be present in a
number of disease states, including infectious
mononucleosis and mycoplasma pneumonia infections. If
red cell agglutinates are seen on the peripheral smear,
warm the sample in a 37 degrees C heating block and mix
and test the sample while it is warm. Strong cold
agglutinins may not disperse and need to be redrawn in a
pre-warmed tube and kept at body temperature.
Increased WBC counts
(>50,000/cumm)
This may produce a proportional elevation in Hb
values because of increased cellular turbidity in
the WBC counting chamber.
High patient glucose
(>400mg/dl)
It results in intracellular hyperosmolality in RBCs
and may cause high MCV and hematocrit with a
low MCHC.
High plasma lipid
levels
These results in lipemic plasma may produce
turbidity in the WBC aperture and falsely
increase Hb, MCH and MCHC.

36. HISTOGRAMS
• Depicts the volume distribution of cells
counted, can be used for the evaluation of
one cell population or subgroups within a
population since size thresholds separate
cell populations on these histograms.
• Analysis provides valuable information of:
– anomalous cell distributions
– problems with the sample or instrument

37. • Patterns produced can be used as a QC-
tool in the identification of sample or
instrument problems.
• Graphic representations of cell
frequencies (y-axis) versus size (on x-
axis)
• In a homogenous population, curve
assumes a symmetrical bell shape or
Gaussian curve
• A wide or more flattened curve is seen
when the SD from the mean is increased

38. • ERYTHROCYTE HISTOGRAMSERYTHROCYTE HISTOGRAMS
• Reflects the native size of the erythrocytes or
any other particles in the erythrocyte range
• Counted in the RBC dilution with sizes ranging
from 36-360 fL.
• Red cell histograms are derived by plotting the
size of each red cell on x axis and the relative
number on the y axis. They are used to
determine the average size, distribution of size,
and to detect subpopulations.
• Normal RBC histogram:
– Single peaks should be normally between 70-110 fL;
peak should coincide with the MCV

40. • Abnormal RBC histogram:
– When MCV of the curve falls outside of the
normal range (80-100 fL)
– When the RDW is greater than 14.5% (two-
cell populations)
• “Toe”:
– Extended distribution between 100-200 fL;
represents RBC duplicates, triplicates,
agglutinated RBCs, aperture artifacts,
occasional WBC

41. • Red cell distribution width
– The red cell distribution width (RDW) is the coefficient
of variation of the curve
– Ratio of the width of the distribution curve (histogram)
to the mean red cell volume
– Index of red cell size variation
– Reveal the presence of distinct population of red cells
of different cell volume
– Normal RDW value: 11.6-14.6%
– The higher the RDW, the greater the variation in cell
size
– Variation may be caused either by abnormally large
or small cells, a mixture of the two, or cell fragments

42. • Red cell distribution histograms. In these
histograms, RBC volume (x-axis) is plotted vs.
the cell count (number of events counted (y-
axis). The mean corpuscular volume (MCV) is
the median value of the histogram distribution.
Microcytic red cells fall to the left portion of the
curve, while macrocytic red cells fall to the right.

43. ABNORMAL RED CELL
HISTOGRAMS
• Coincidence:
– The small tail to the right of the curve
represents coincidence, multiple cells passing
through the aperture at the same time. The
correction for coincidence takes place in the
cell count calculation and is not a concern.

45. • Microcytic red cells:
– The black curves on the red cell and platelet
histograms indicate expected or normal cell
distributions. The red curves demonstrate the
effect of very microcytic red cells on the
histograms. Since microcytic red cells are
present in the population it causes a left shift
of the curve to the red cell histogram. This
may also affect the platelet curve.

47. • Macrocytic red cells:
– An erythrocyte population that is larger than
the size of the normal is represented by a
curve that is more to the right of the normal
erythrocyte size distribution.

49. • Giant platelets:
– Platelets that are 6 microns in diameter or
larger, are considered giant platelets. The red
curves demonstrate the effect of giant
platelets on the red cell histogram. They
cause the right hand tail of the platelet
histogram to remain elevated and may be
seen at the left of the red cell histogram.

51. • Dimorphic red cell population
– This is a representation of a bimodal
distribution. A bimodal peak illustrating a
dimorphic RBC population (camel humps) can
be seen in such situations as cold agglutinin
disease, after transfusion of red blood cells
into a person with abnormally sized RBCs,
treated iron deficiency anemia, as well as
other conditions.

53. RBC Histogram as a QualityControl:
ABN / INDICATOR
PROBABLE CAUSE COMMENT
Left of curve does not
touch baseline
Schistocytes and extremely
small red cells
Review smear CBC and
Platelet histogram
Bimodal peak Transfused cells, therapeutic
response
Review Smear
Right portion of curve
extended
Red cell autoagglutination Review CBC & Smear
Left shift of curve Microcytes Review smear & CBC
Right shift of curve Macrocytes Review smear & CBC

54. RED CELL
CYTOGRAM/SCATTERGRAM
• RBC light scatter analysis produces two-dimensional
cytogram, plotting volume versus haemoglobin
concentration.
• Computer analysis of this cytogram enables enumeration
plus independent measurements of RBC volume and
direct measurement of individual red cell haemoglobin
concentration.
• An MCHC value is calculated in the traditional manner
(from Hb and Hct).
• The direct measurement of Hb concentration, termed the
CHCM (cellular haemoglobin concentration mean), is
unaffected by lipemia and icterus.
• Histograms of both RBC volume and the Hb distribution
are generated. The dispersion of both curve is reported
as RDW and HDW, respectively.

56. • RBC cytogram pattern from a normal
individual with a 9-part grid showing the
volume markers (a and b) and
haemoglobin concentration markers (x
and y). Most RBC plots in the central area.

57. LEUKOCYTE HISTOGRAM
• Does not display the native cell size rather
the size of the cell after lysis
– The lytic reagent causes a cytochemical
reaction that causes the cytoplasm to collapse
around the nucleus.
– Therefore, histogram of WBC differential
reflects the sorting of the cell according to
their relative size (size of the nucleus).
• Count and plot of cells on the WBC
aperture bath larger than 35 fL.

58. • Normal WBC histogram has three
distribution peaks:
– First peak – 45-90 fL, small mononuclear cell
population
– Second peak – 90-160 fL – minor population
of large mononuclear cells
– Third peak – 160-450 fL – normal mature
types of granulocytes

60. • The presence of increased numbers of immature
granulocytes, eosinophils, or variant lymphocytes is not
recognized, because there are only 3 categories in which
to place the differentiated leukocytes.
• Blasts forms generally are categorized with
lymphocytes as well as between lymphocytes and
monocytes, which may increase the interference at the
lymphocyte-monocyte interface.
• Increased numbers of eosinophils may blend in with
granulocytes or fall between monocytes and
granulocytes, thereby increasing the interference at that
interface.
• Increased numbers of granulocytes (bands,
metamyelocytes, myelocytes) generally fall the
granulocytes and may interfere at the monocyte-
granulocyte interface.
• Other interferences include lysis resistant RBCs (e.g.
sickle cells) and clumped platelets which increase
total lymphocyte count.

61. ABNORMAL WBC HISTOGRAM:
• Region code flags are used to signal
abnormalities in the distribution in a
printed warning or audible alarm.
• Each flag will appear simultaneously next
to the percentage and the absolute values
of the cell type(s) in question.

62. • R1 Flag
– It denotes interference in the valley (separation
between cell populations) to the left of the lymphocyte
subpopulation at approximately 35 fL.
– The interference can be caused by clumped or giant
platelets, nucleated RBCs, nonlysed red cells,
malarial parasites, fibrin strands, cryoglobulin or fat
globules attributable to total parenteral nutrition.
• R2 Flag:
– Flag indicates excessive overlap of cell populations at
the lymphocyte-mononuclear boundary
(approximately 90 fL).
– Abnormal cell types are that could be present are
variant lymphocytes, abnormal lymphocytes (plasma
or hairy cells), blasts, eosinophilia, monocytosis and
basophilia,.

63. • R3 Flag:
– This indicates an overlap of cells at the mononuclear-
granulocyte boundary (approximately 160 fL)
– Many of these are false-positive flags, but may
indicate neutrophilia, neutrophilic left shift,
eosinophilia, or a sample processed less than 30
minutes after collection.
• R4 Flag:
– Flag indicates truncation of the distribution at the
upper leukocyte threshold (450 fL) and is most often
triggered when granulocyte numbers increased.
• RM:
– This indicates interference of more than one region.
• Backlighting
– Indicates that the histogram curve does not start on
the baseline below 35 fL (the lower leukocyte
threshold).

65. Abn / Indicator
Probable Cause Comment
Tail extending
downward at extreme
left, or lymph peak
not starting at
baseline
NRBC, Platelet clumping, unlysed
RBC, cryoproteins, parasites
Review smear and
correct WBC for
NRBC
Peak to the left of
lymph peak or
widening of lymph
peak towards left
NRBC Review smear &
correct WBC for
NRBC
Widening of
lymph peak to
right
Atypical lymphs, blasts, plasma cells,
hairy cells, eosinophilia, basophilia
Review smear
Wider mono peak Monocytosis, plasma cells,
eosinophilia, basophilia, blasts
Review smear
WBC histogram
(lymph peak) does
not start at baseline
Giant platelets, NRBC, Platelet
clumping
Review smear, correct
WBC for NRBC
WBC HISTOGRAM AS A QUALITY CONTROL TOOL

66. Elevation of left portion of granulocyte Left Shift Review smear
Elevation of right portion of granulocyte
peak
Neutrophilia Review smear

67. DIFFERENTIAL SCATTERGRAM
• Automated differential counters which are
available now generally use flow cytometry
incorporated into a full blood counter rather than
being standard alone differential counters
• Automated counters provide a three-part or five-
to seven-part differential count.
• 3-part differential usually count
– Granulocytes or large cells
– Lymphocytes or small cells
– Monocytes(mononuclear cells) or (middle cells)

68. • 5-part classify cells to
– Neutrophils
– Eosinophils
– Basophils
– Lymphocytes
– Monocytes
• A sixth category designated “large unstained
cells” include cells larger than normal and lack
the peroxidase activity this include
– Atypical lymphocytes
– Various other abnormal cells.

69. • Other counters identifies 7 categories
including
– Large immature cells(composed of blasts and
immature granulocytes)
– Atypical lymphocytes (including blast cells).
• Analysis may be dependent on:
– Volume of the cell
– Other physical characteristics of the cells
• Sometimes the activity of cellular enzymes such as
peroxidase.
• Basophils are not classified in the peroxidase
because they appear in the same area as
lymphocytes, therefore require separate analysis in
a separate channel

72. • PEROXIDASE CHANNELPEROXIDASE CHANNEL
• The peroxidase cytochemistry has a shortened reaction
time and more sophisticated analysis.
• The computer establishes floating thresholds that fit
around the population clusters and identify cell types.
• Total leukocyte, neutrophil, lymphocyte, monocyte, and
eosinophil counts are determined on this channel.
– The tungsten lamp is used in counting the leukocyte and for
differentiating the granulocyte populations using an alkalinealkaline
peroxidase stainperoxidase stain to detect cell volume and light absorption.
– On the basis of these properties and the count from the basophil
channel, the WBCs are differentiated into the five traditional
categories.
• In addition, large unstained cells (LUCs) and cells with high
peroxidase activity (HPX) are identified.
• The LUCs correspond to variant lymphocytes, blasts, or any large
cell devoid of peroxidase activity
• Peroxidase channel display shows clusters of unstained
lymphocytes, unstained debris (platelets and red cell stroma),
slightly stained monocytes, moderately stained neutrophils and
intensely stained eosinophils.

74. • BASOPHIL-LOBULARITY CHANNELBASOPHIL-LOBULARITY CHANNEL
• The WBCs are selectively stripped of their
cytoplasm using an acid with a buffer that
enhances the natural resistance of the
basophils. Only the basophils are resistant to the
lysing agent.
• Using the RBC laser optics with two-angle light
scatter to analyse the effluent, leukocyte
subpopulations can be identified on the basis of
scatter information from the nuclei.
• In the basophil/lobularity cytogram, the
basophils are much larger than the nuclei of
other leukocytes and can be detected as those
cells above a set (volume) scatter threshold.

75. • The two-angle laser scatter detection method
separates the nuclei of the remainder leukocytes
according to their degree of lobulation.
– Segmented neutrophils, which have a large amount of
high-angle scatter, fall to the right on the X axis, and
mononuclear cells fall to the left.
– When the neutrophils are less mature (band and
metamyelocyte forms), their scatter signature is
different, and the cells move to the left on the X axis.
– Blasts give somewhat lower amount of high-angle
forward scatter based on their chromatin texture and
are separated from the mononuclear cells at the
extreme left of the X axis.
• The instrument keeps track of the ratio of highly
lobulated nuclei to nonlobulated nuclei and
reports of a lobularity index (LI). Decreased
lobularity results in a decreased LI (left shift).

76. • Discrepancies:
– Occur in the presence of nucleated or lysis-resistant
RBCs. NRBC areas from the lobularity and
peroxidase channels are compared and a flag set
when a comparison of the number of “blasts” detected
by nuclear shape and LUC number in the peroxidase
channel suggests that blasts are likely.
– Additional flags are set when analysis tolerance limits
are exceeded for “left shift” and atypical lymphocytes.
– Platelets are completely lysed in the basophil
channel, so the WBC count from this channel is
unaffected by platelet clumps or giant platelets, which
would alter the WBC count from the peroxidase
channel.

78. PLATELET HISTOGRAM
• Platelet counting and sizing in both the electrical
impedance and optical systems reflect the native cell
size. In the electrical impedance method, counting and
sizing take place in the RBC aperture. In the electrical
impedance system, the analyzer’s computer classifies
particles that are greater than 2 fL or less than 20 fL as
platelets.
• The raw data is sorted and histograms are then
smoothed (smooth curve) and tested against
mathematical criteria that eliminate nonplatelet particles
and finally fitted to a lognormal distribution curve. This
distribution curve has a range of 0 to 70 fL (fitted curve).
The final platelet count is derived from the integrated
area under this “best fit” log-normal curve.

79. The MPV (mean platelet volume) and the PDW (platelet distribution
width) are obtained from the platelet histogram as well.

80. ABNORMAL PLATELET HISTOGRAM
Small red cells:
o.
Since microcytic red cells only affect the right end of the platelet curve, the
black and red lines are superimposed for most of the curve

81. Giant platelets:
oThese are platelets that approach or exceed the size of the red cells.
They cause the right hand tail of the histogram to remain elevated and may
be seen at the left of the red cell histogram.

82. PLATELET HISTOGRAM AS A QUALITY CONTROL:
ABN / INDICATOR PROBABLE CAUSE COMMENT
Peak or spike at left end of
histogram (2-8 fl)
Cytoplasmic fragments Review smear
Spike towards right end of
histogram
Schistocytes, microcytes,
giant platelets
Review smear +
CBC ( MCV &
 RDW)
( MPV & 
PDW)
Bimodal peak Cytoplasmic fragments Review smear