Hematology Automation

TERMINOLOGIES
 Flags – abnormal distribution
 Threshold – voltage limit with which a pulse is compared
 Electrical gating – electrical impedance
 Window – range set by threshold
 Multiparameter instrument – instrument capable of counting
RBC, WBC, platelets and determining the Hb, MCV.
 Single parameter instrument – Not capable of counting WBC,
RBC, and platelets simultaneously
 Noise – contaminants
 Coefficient of variation – Allowable error
 Coincidence error – Error produced when two or more cells
enter the aperture at the same time
Coulter Principle

 Also known as Electrical impedance or

Aperture Impedance
 Presented by Wallace Coulter in 1956
 “High speed automatic blood cell counter and
cell size analyzer”
Principle

 Cell counting and sizing are based on the

detection & measurement of changes in
electrical impedance(resistance) produced by
a particle as it passes through a small aperture.
 The passing of cells increase the
impedance(resistance) of electrical path
between 2 submerged electrodes located on
each side of the aperture, because they are
poor conductors of electricity.
 The number of pulses generated during a specific
period is proportional to the number of particles
or cells.
 The amplitude(magnitude) of the electrical pulse
produced indicates the cell’s volume.
 The output histogram is a display of of the
distribution of cell volume & frequency,
 Each pulse on the x-axis represents size in
femtoliters(fL);the y-axis represents the relative
number of cells.
Common Paramaters

 RBC COUNT PDW

 WBC COUNT RDW
 Hemoglobin Leukocyte diff’l
 Hematocrit
 Platelet Count
 MCV
 MCH
 MCHC
BASIC CELL COUNTING PRINCIPLES

1. ELECTRICAL IMPEDANCE
2. OPTICAL SCATTER/LASER LIGHT SCATTER
ELECTRICAL IMPEDANCE
 a.k.a. ELECTRICAL RESISTANCE or Low Voltage
Direct Current (DC) resistance or “Coulter
Principle”. The first automated machine
 developed by Coulter in the 1950s.
 MOST COMMON methodology used.
 Radiofrequency (RF) a.k.a. ALTERNATING
CURRENT (AC) resistance is a modification
sometimes used in conjunction w/ DC electri-
cal impedancce.
 Cell counting & sizing is based on the detection &
measurement of the CHANGES in the Electrical
impedance produced by a particle as it passes
through a small aperture.
 BLOOD CELLS= poor conductors of electricity;
DILUENT= good conductors
 The number of pulses generated during a specific
period is PROPORTIONAL to the no. of particles
or cells.
 The amplitude (magnitude) of the electrical
impulse produced indicates the cell’s volume.
RADIOFREQUENCY
 a.ka. RF/ HIGH VOLTAGE electromagnetic current w/c
measure CONDUCTIVITY.
 may be used in conjunction w/ DC impedance.
 Total vol. of cell is PROPORTIONAL to the change in
DC.
 Cell interior density is PROPORTIONAL to Pulse size/
change in RF signal.
 2 dimensional distribution cytogram/ scatterplot= plots
conductivity(RF) & impedance (DC) of the cells.
 Scatterplot- displays the cluster of cells, the no. of dots
represents the conc. of a specific cell type(WBC)
 This is a newer application where high-
voltage electromagnetic current is used to
detect cell size,based on the cellular density.
 The radiofrequency pulse is directly
proportional to the nuclear size & density of a
cell.
 RF or conductivity is related to the nuclear-
cytoplasmic rtio, nuclear density, and
cytoplasmic granulation.
OPTICAL SCATTER/DETECTION

 uses LASER and /or NON LASER LIGHT.

 “FLOW CYTOMETERS”
 A diluted blood specimen passes in a steady
stream through a quartz flow cell past a
focused light source.
 LIGHT SOURCES:
 TUNGSTEN HALOGEN
 HELIUM NEON LASER
LASER

Light A mplification by Stimulated Emission of

Radiation

Most Common light source used in flow

cytometers bec. Of the properties of:
 INTENSITY
 STABILITY
 MONOCHROMATISM
Fundamentals of Laser Technology

 In 1917, Albert Einstein speculated that

under certain conditions, atoms or molecules
could absorb light or other radiation and then
be stimulated to shed this gained energy. In
the 1950s, physicists theorized how this
borrowed energy could be multiplied and
emitted in high quantities. A decade later,
new lasers were developed & used in medical
& industrial applications.
Fundamentals…

 The electromagnetic spectrum

PRINCIPLE

 In the optical or hydrodynamic focusing method

of cell counting and cell sizing,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 cell, it scatters the focused light.
Scattered light is detected by a photodetector &
converted to electrical impulse.The number
PRINCIPLE

 Of pulses generated is directly proportional

to the number of cells passing through the
sensing zone in a specific period.
 The application of light scatter means that a
single cell passes across a laser light beam,
the light will be reflected & scattered.
 The patterns of scatter are measured at
various angles(forward scatter 180 degrees, &
right angle 90 degrees).
 Scattered light provides information about
cell structure, shape, and reflectivity. These
characteristics can be used to differentiate
the various types of WBCs and to produce
scatter plots with a five-part differential.
 As each cell passes through the sensing zone
of the flow cell, it scatters the focused light.
 Scattered light is detected by a
PHOTODETECTOR & converted to
EELECTRICAL PULSES.
 The no. of pulses generated is DIRECTLY
PROPORTIONAL to the no. of cells passing
through the sensing zone in a specific period.
3 INDEPENDENT PROCESSES

 DIFFRACTION- bending of light around corners

using small angles.
 REFRACTION- bending of light because of the
change in speed using intermediate angle.
 REFLECTION -light rays are turned back by
obstruction using large angles.
*** The pattern of scatter are measured at various
angles.
***Angles of light scattered measured by different
flow cytometers.
ANGLES OF LIGHT SCATTER

 FORWARD-ANGLE LIGHT SCATTER( 0

DEGREES) - cell volume/ cell size.
 FORWARD-LOW-ANGLE LIGHT SCATTER(2-3
DEGREES)- can relate to size or volume.
 FORWARD-HIGH-ANGLE(5-15 DEGREES)=
allows for description of the refractive index of
cellular components.
 ORTHOGONAL LIGHT SCATTER(90 DEGREES) -
a.k.a SIDE SCATTER w/c correlates w/ degree of
internal complexity.
 Structures such as nuclei & cytoplasmic
granules are determined by the intensity of
light scattering at 90 degrees (Right angle
scatter)
BLOOD CELL HISTOGRAM

 Provide size distribution of the different cell

populations.
 VOL= cu.mm/ fL plotted against the relative
frequency for platelets,WBCs& RBCs.
 X-axis= Cell size
 Y-axis= no. of cells
RBC HISTOGRAMS

 vol./ sizes= 36fL - 360fL as RBCs but can

measure as small as 24fL
 LARGER RBC(NRBC)= curve will shift toward
the right.
 SMALLER RBC(microcytes)= curve will shift
toward the left.
 BIMODAL= there are 2 population of RBC.
 H.A., w/ schistocytes or Anemia w/ diff. size cell
populations.
WBC HISTOGRAMS

 Vol. size= LARGER than 45fL.

 3 NORMAL PEAKS
 FIRST PEAK (45-90fL)= small mononuclear
population of cells -lymphocytes
 SECOND PEAK(90-160fL)= minor population of
large mononuclear cells.(monocytes, blasts)
 THIRD PEAK (160-450 fL)=normal mature types
of granulocytes.
ABNORMAL WBC HISTOGRAM
PATTERNS
1. REGION CODE FLAGS
A. R1= left of the lympho peak (approx. 35fL)
B. R2= excessive overlap of cells populations of Lympho &
Mono (approx. 90fL)
C. R3= excessive overlap of cells populations of Mono&
Granulo(approx. 160fL)
D. R4= upper end of WBC threshold ( approx. 450 fL)
E. RM= “MULTIPLE REGION OVERLAP”
2. OTHER FLAGS
A. H= HIGHER THAN THE NORMAL LIMIT.
B. L= LOWER THAN THE NORMAL LIMIT.
PLATELET HISTOGRAM

 vol. size= 2-20 fL

 count is performed in takes place in RBC
bath.
 Threshold is re set.
 PDW is inversely proportional to platelet
count.
 AUTOMATED SMEARER
SYSMEX SP-1000i

o Completely automates manual procedure

o Removes the danger of handling open
vial blood specimens
o Reliable reporting of patient results to
clinicians because of more precision &
accuracy
o Eliminates unnecessary costs
o Ensures maximum efficiency and
minimum turnaround times
TECHNICAL FEATURES

 Fully Automated Blood Smear Preparation and

Staining Machine with independent sampling
system.
 Operates at 120 smears per hour
 Ultrasound cleaning of the spreader ensures no
carryover of cells
 Supports both Single and Double Staining protocols
 Barcode printing ensures smears can be transferred
to the DM-96 Automated Microscope without the
need for subsequent labelling
SPECIFICATIONS

 Throughput: Up to 120 samples per hour

 Principles: Smearing: Intelligent wedge
method adjusted for spreader blade angle,
spreader speed, and consistent blood volume
is used
 Principle of Staining: Single stain or double
stain, individual reagent dispensing
PocH 100i
 KEY BENEFITS
 Ideal for ALL POC testing locations, the pocH-100i is ideal where space is
limited
 Integrates with other POC analysers for a total POC testing platform for
the patient
 Can be used by non laboratory based operators ensuring system suitability
for the full spectrum of the point of care testing market
 Safe data entry and full audit trail and system security in line with CPA
recommendations
 Guarantees operator safety from contamination from aerosols or droplets
and no moving piercer exposed to operator ensuring safer working
environment
 Providing a laboratory comparable result from a POC machine, reduced
turnaround time for patients
TECHNICAL FEATURES
 Specifically designed POC
testing analyser providing
laboratory quality results
 User friendly, easy to
operate, touchscreen with
easy to use icon driven
intuitive software
 With operator and patient ID
 Closed vial sampling
 Provides 19 hematology
parameters Including 3 part
WBC Differential with ANC