INSTRUMENTATION IN HEMATOLOGY

General Principles of Automated

Blood Cell Analysis
 Electronic impedance
- Developed by coulter in the 1950’s
- Low-voltage direct current (DC) resistance
- Most common methodology used
 Optical scatter
- Introduced by Technicon Instruments Corporation in 1960’s
 Radio frequency
- Alternating current resistance
- A modification used in conjunction with DC electronic
impedance
 Laser optical counters
- Ortho Clinical Diagnostics Inc, in the 1970’s
Electronic Impedance
 Based on detection and measurement of changes in
electrical resistance produced by cells as they traverse
a small aperture
 Based on OHM’S LAW: Voltage = current x resistance
 Cells are suspended in an electrically conductive diluent
such as saline
 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 will change the conductance
Electronic Impedance
 The change in electrical resistance, or the size of the pulse
generated by the cell is proportional to its volume

 The cell count is determined by the number of pulses

generated
 THRESHOLDS
➢ A voltage limit with which a pulse is compared

➢ Both a LOWER and UPPER threshold can be set

➢ Increasing the LOWER THRESHOLD → eliminates

unwanted SMALL PULSES such as debris

➢ UPPER THRESHOLD → eliminates LARGE PULSES

➢ By manipulating the upper and lower thresholds

✓ it is possible to produce a “window”
✓ specific particle size range
Instrument Components
APERTURE TUBE
A 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 (orifice)
 Contains an internal electrode as well as a
tube that may be used to deliver
electrolyte solution into the aperture tube
Instrument Components
Stopcock Assembly
 Connects the aperture tube to the
manometer
 Itcontains two stopcocks (vacuum control
and filling or flushing)
 When both stopcocks are opened, the
aperture can be filled and flushed with
electrolyte solution.
 Contains two inlets:
• One from vacuum pump
• From reservoir of electrolyte solution
Instrument Components
Aperture Current Electrodes
There are two platinum electrodes
Internal electrode (positive)
External electrode (negative)
Function is to generate current across
the aperture
Current polarity alternates with each
count
Instrument Components
Oscilloscope
On the front of the instrument
Displays a visual representation of
voltage pulses caused by cells as
they pass through the critical volume
A visual guide to the size and
number of particles being counted
Instrument Components
Threshold Dials
One or two threshold controls are
located in front of the instrument
Control the upper and lower
threshold
Marked in units up to 100
Instrument Components
Amplification Switch
Referred to as the attenuation switch
Located in front of the instrument
and controls the first amplification
pulse signal
Instrument Components
Digital Read out
A five-digit numerical display which
records cells as they are counted
An audible click is emitted as each
thousand is counted
Instrument Components
Debris Monitor
Consist of a 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 the count
Reagents and Supplies
1. Electrolyte
 Bufferred isotonic salt solution that may
contain one or more preservatives
 Must be particle free
 Used to dilute cells and to flush the
instrument
2. Lysing Reagent
 A detergent solution
 Used to lyse erythrocytes by dissolving
their stroma when leukocyte counts are to
be performed
Reagents and Supplies
3. Cleaning agent
 Used at regular intervals to remove
protein build up from the aperture
tube and electrodes
4. Sample Container
 May range from small glass beakers to
plastic vials manufactured for this
purpose.
 Must be particle free and their internal
surfaces do not react or attract cells
Electronic Impedance
 Separatechannels are used for WBC and
RBC counting
 The Wbc’s are counted in a dilution in
which the RBC’s have been lysed
 In the RBC channel, the dilution is great
enough to make the number of WBC’s
negligible and not a source of
interference.
 Mostly, platelets and RBCs generally, are
counted together separated by pulse
heights
Electronic Impedance
Coincidence
 More than one cell arriving at the orifice
at a time
 Position of the cell with respect to the
center of the orifice may make the cell
appear larger and create an artificially
large pulse
Recirculation
 Recirculating back of cells into the
sensing zone
HISTOGRAMS

❖X-AXIS → cell size (femtoliters → fL)

❖Y-AXIS → # of cells
WBC HISTOGRAM

❖WBC’s: 35-450 fL
❖RBC’s are lysed during counting: HYPOTONIC
SOLUTIONS
❖1st peak: lymphocytes/small cells (35-90 fL)
❖2nd peak: monocytes/medium cells (90-160 fL)
❖3rd peak: granulocytes (160-450 fL)
RBC HISTOGRAM

❖RBC’s: 36-360 fL
❖WBC’s are lysed during counting: ISOTONIC
SOLUTIONS
❖Normal RBC histogram: single peak between 70-110 fL
(correlate with MCV)
❖Derived from RBC histogram: MCV and RDW
PLATELET HISTOGRAM

❖Platelets: 2-20 fL
❖Counted together with RBC’s
❖Derived from platelet histogram: PCV and PDW
Electronic Impedance
Differential Analysis
Coulter counter S-Plus series
Internal flags such as region (R)
codes, backlighting of values, or
instrument data rejection may occur
if the values or shapes of the curves
are not acceptable to the instrument
according to preset tolerances
Optical/Light Scatter
 Cells are counted as passed through focused
beam of light (laser)
 Sample of blood is diluted with an isotonic
diluent and then hydrodynamically focused
through a quartz flow cell past a focused light
source
 Laser/ monochromatic light is emitted as a
single wavelength
 INTERROGATION
- Light source needs to be focused on the
same point where cells are focused
Optical Scatter
 As cells pass through the sensing zone, light scatters in all
directions
 Photodetectors sense and collect the scattered rays at
different angles and then convert to data into an electrical
pulse
o PHOTODIODES
- Convert light photons to electronic signals proportional
in magnitude to the amount of light collected
o PHOTOMULTIPLIER
- Used to collect the weaker signals produced at a 90
degree-angle and multiply the photoelectron into
stronger signals
 The number of pulses generated is directly proportional to the
number of cells passing through the sensing zone
Optical Scatter
 Optical scatter measures patterns of light at various
angles:
• Forward light scatter (0 degree)
• Right angle scatter (90 degrees)
► Forward angle light scatter: cell volume
► Forward low angle light scatter (2-3 degrees): cell size or
volume
► Forwardhigh angle light scatter (5-15 degrees): relates to
the internal complexity or refractive index of cellular
components/ degree of structure inside the cell
► Orthogonal light scatter/ side scatter (90degrees):
combination of reflection and refraction and relates to
internal components/ complexity
 Median Angle Light Scatter (MALS)
 Defined as the light scatter information that is obtained in different
angles
 Light scatter in median angles is proportional to cell size, granularity,
surface properties and reflectance
o 90 degrees (polarized): nuclear lobularity
o 90 degrees (depolarized): granularity
o 10 degrees: cell structure and complexity
o 0 degrees: size
Multiple Angle Light Scatter
Flow Cytometry
 combines fluid dynamics, optics, laser science, high-
speed computers, and fluorochrome-conjugated
monoclonal antibodies (MAbs) that rapidly classify
groups of cells in heterogeneous mixtures
 The principle of flow cytometry is based on cells being
stained in suspension with an appropriate fluorochrome
- An immunologic reagent, a dye that stains a specific
component, or some other marker with specific
reactivity.
 Fluorescent dyes used in flow cytometry must bind or
react specifically with the cellular component of interest
(e.g., reticulocytes, peroxidase enzyme, DNA content).
Principles of Flow Cytometry
 A suspension of stained cells is pressurized
using gas and transported through plastic
tubing to a flow chamber within the
instrument
 In the flow chamber, the specimen is
injected through a needle into a stream of
physiologic saline called the sheath
 The sheath and specimen both exit the flow
chamber through a 75-µm orifice.
 This laminar flow design confines the cells to
the center of the saline sheath, with the cells
moving in single file
Principles of Flow Cytometry
 The stained cells then pass through the laser
beam
 The laser activates the dye and the cell
fluoresces
 Although the fluorescence is emitted throughout
a 360-degree circle, it is usually collected by
optical sensors located 90 degrees relative to
the laser beam.
 The fluorescence information is then transmitted
to a computer, which controls all decisions
regarding data collection, analysis, and cell
sorting.
 Flow cytometry performs fluorescence analysis
on single cells.
Principles of Flow Cytometry
 Both intrinsic and extrinsic properties of cells
can be analyzed
 Intrinsic properties include forward and right
angle light scatter → correlate with size and
granularity of a cell
 Extrinsic properties rely on the binding of
various probes to the cells
 The scattered passes through a variety of
filters and lenses and measured by
photomultiplier tubes which convert the light
signals into electronic signals
Principles of Flow Cytometry
 Light scattered along the axis of the laser
beam is forward scatter
 Light scattered perpendicular to the axis is
side scatter or orthogonal scatter
 Forward scatter: is roughly proportional to
cell size
 Side scattter: cytoplasmic granularity
 Granulocytes have a much larger side
scattered light signal than to lymphocytes.
 Cellular features measured by Flow
Cytometry
Cell size or volume
DNA content
Cytoplasmic Granularity
Cell-surface antigens
Intracellular enzymes
RNA content
 Emerging Clinical applications of Flow
Cytometry
Detection of small populations of cells
Determination of cell surface phenomena
Evaluation of leukocyte function
Evaluation of intracellular metabolism
Cytogenetics
Semen analysis
Detection of autoantibodies
Measurement of cytotoxicity
Analysis of platelet function
ERRORS IN AUTOMATION:

❖ INSTRUMENTAL ERRORS:
➢ Negative Errors → Excessive Lysis of RBC’s
➢ Positive Errors
✓ Bubbles caused by shaking
✓ Extraneous electrical pulses from improperly grounded equipments
✓ Aperture plugs
✓ **improper setting of aperture current (positive or negative)
ERRORS IN AUTOMATION:

❖ CAUSED BY NATURE OF SPECIMEN:

➢ Giant platelets: counted as RBC or WBC
➢ Leukocyte cytoplasmic fragment: counted as platelets or RBC’s
➢ Increased number of fragmented RBC’s: inaccurate RBC and platelet
counts
➢ Agglutination of cells: false decrease
➢ Agglutinated RBC and platelets: false increase WBC count
➢ Platelet satellitism: decreased platelet count
➢ RBC’s that resists lysis: increased WBC count
 POTENTIAL CAUSES OF ERRONOUS RESULTS WITH AUTOMATED CELL COUNTERS
PARAMETER SPURIOUS INCREASE SPURIOUS DECREASE
WBC COUNT Cryoglobulin, Cryofibrinogen Clotting
Heparin Smudge Cells
Monoclonal proteins Uremia + Immunodepressants
Nucleated RBC’s
PLATELET CLUMPING
Unlysed RBC’s
RBC COUNT Cryoglobulin, Cryofibrinogen Autogglutination
GIANT PLATELETS Clotting
High WBC count (>50,000/uL) Hemolysis (In Vitro)
Microcytic RBC’s
HEMOGLOBIN Cryoglobulin, Cryofibrinogen Clotting
Carboxyhemoglobin (>10%) Sulfhemoglobin
Hemolysis (in vitro)
Heparin
High WBC count (>50,000/uL)
Hyperbilirubinemia
Lipemia
Monoclonal proteins
HEMATOCRIT (automated) Cryoglobulin, Cryofibrinogen Autoagglutination
GIANT PLATELETS Clotting
High WBC count (>50,000/uL) Hemolysis
Hyperglycemia (>600 mg/dL) Microcytic RBC’s
HEMATOCRIT (microhematocrit) Hyponatremia EXCESS EDTA
Plasma Trapping Hemolysis
Hypernatremia
MCV Autoagglutination Cryoglobulins
High WBC count (>50,000/uL) Cryofibrinogen
Hyperglycemia Giant platelets
Reduced red cell Hemolysis (in vitro)
deformability Microcytic RBC’s
Swollen RBC’s
MCH High WBC count Falsely low Hgb
(>50,000/uL) Falsely High RBC
Falsely Elevated Hgb
Falsely low RBC
MCHC Autoagglutination High WBC count
Clotting (>50,000/uL)
Hemolysis (In vitro, in vivo) Falsely low Hgb
Falsely elevated Hgb Falsely High Hct
Falsely decreased Hct
PLATELETS Cryo, hemolysis, Micro RBC, Clotting
red cell inclusion, white cell Giant platelets
fragments Heparin
Platelet clumping
Platelet satellitosis
END