Automation in Hematology

Ruel Bartholomeo B. Maguad, RMT, RN

Introduction
Hematological diagnosis has developed from rather
philosophical approaches in Ancient Greece to the
application of logical principles in investigating human
blood within the last 2 centuries.
The 20
th
century was a time of industrial standardization,
and thus, a period in which established procedures
operated by humans were perfected in hematology.
Charles A. Spencers Trunnion Model
Microscope c. 1855
Introduction
In the 21
st
century, modern computers and network
techniques are being integrated into a complex instrument
linked to each other mechanically and logically by control
systems based on algorithms.
This will be illustrated by 4 modern analysis systems.
These diagnostic tools are not only of high importance for
the treatment of patients but also for the screening of blood
and blood products.
Introduction
A state of health is characterized by homeostasis within the
cellular components and plasma and a normal relationship
between solid and fluid components.
Diseases are characterized by changes in individual blood
parameters.
It is therefore of greatest interest to be able to easily and
rapidly measure these parameters at anytime with high
precision and accuracy to allow for a precise diagnosis.

Cell-dyn 3700
History of Blood Testing
Inspection of blood was a basic principle of diagnosis from
ancient times till the 16
th
century.
Blood inspection was an investigation of blood bled from
arteries and was called Hemoscopy or Hematoscopy; the
color and structure of the blood was investigated.

History of Blood Testing
Karl Vierordt
(1818-1884)



Quantitative determination of individual cellular
components was first enabled in 1852 by the work of Karl
Vierordt, a physiologist from Tubingen, Germany.
He developed the 1
st
counting method, in which a specific
blood volume were smeared onto a slide.
The slide was covered with a glass grid and ALL 4.6-5.8
million erythrocytes per microliter were counted.
History of Blood Testing
Neubauer
In 1924, Neubauer published his net structure; this led to
the manual cell counts which are still taken as GOLD
STANDARD in some areas.
Both venous and capillary blood was used as test material
for the determination of WBC, RBC, and platelets.
Blood was isolated under optimized pre-analytical
conditions; coagulation was prevented with the dipotassium
salt of ethelynediaminetetraacetic acid. (EDTA:1 mg/ml of
blood)
History of Blood Testing
Samples were prepared according to fixed procedures and
then counted in the Neubauer Counting Chamber:









The precision and accuracy were highly dependent on the
number of counted cells, and at a reasonable level of effort,
were subject to fluctuations of up to 10%.
History of Blood Testing
Hemocytometer Counting Chambers:

History of Blood Testing
Other models aside from the Neubauer Counting
Chamber:
1. Burker
2. Fuchs-Rosenthal
3. Thoma
4. Schilling
5. Turk
These are only used routinely for special tests, such as
examining CSF.
The individual counting chambers differ in the number of
squares and their marking and separation (course of
lines)

History of Blood Testing
Moldovan Capillary Method
The 1
st
step towards automation was made in 1934 with
the Moldovan Capillary Method.
Moldovan described the first instrument that could be
described as a flow cytometer, although the term flow
cytometer was not coined until much later.
The instrument consist of a glass capillary tube mounted on
a microscope stage.
Initially, Moldovan used narrow tubes but found that cells
tend to block them.
When wider tubes were used, cells were not delivered
reproducibly and errors in detection and measurement were
observed.
History of Blood Testing
Coulter Model A
The actual breakthrough in the development of
hematological instruments suitable for routine work was
achieved by Wallace Coulter in 1956 with his patent High
Speed Automatic Blood Cell Counter.

The First Commercial Version of the Coulter Counter
History of Blood Testing
The Coulter Model A
The Coulter Model A was presented at the U.S. National
Electronics Conference in Chicago as the FIRST NON-
OPTICAL MACHINE for counting blood cells.
Coulter cell counters are based on the principle of
Impedance measurement, and for the first time enabled to
count much higher cell numbers than the known manual
methods.
There was still along way to go for the first semi-automatic
machines for routine work.
Coulter Counter Model F
History of Blood Testing
The Coulter Principle
As a particle passes through the aperture, it creates a
resistance. The bigger the particle, the more the resistance,
the greater the voltage. Each voltage spike is directly
proportional to the size of the cell. Today every modern
hematology analyzer depends in some way on the Coulter
Principle.

What is impedance then?
It is a form of electrical resistance observed in an
alternating current that is analogous to the classic electric
resistance that occurs in a direct current circuit.

History of Blood Testing
The Coulter Principle
History of Blood Testing
The most important conditions for achieving a good
automation were:
1. The standardization of the methods
2. The elimination of methodological errors

What do you mean by standardization?
A standardized test is any empirically developed
examination with established reliability and validity as
determined by repeated evaluation of the method and
results.
History of Blood Testing
The Road to Standardization...
History of Blood Testing
The Road to Standardization...
History of Blood Testing
The International Council for Standardization in Hematology
(ICSH)





With this aim in view, the ICSH was founded in 1963 at the
congress of the European Society for Hematology by
Wallace Coulter and other leading scientists who worked in
special committees on the standardization of requirements
for the methods.


History of Blood Testing
International Council for Standardization in Hematology
(ICSH)
According to the ICSH, the following are important steps to
consider on the standardization of requirements in
automation:
The optimization of the detector, with respect to the pulse
volume and the reliable discrimination between genuine
particles and background electrical noise.
Correction of counting errors from spontaneously lysed
cells during passage through the capillary opening;
optimization of the diluents, avoiding cell distortion.
Attainment of constant volume flow in the suspension
passing in real time through the opening.
Avoidance of particle recirculation after passing through
the opening.
Optimization of the materials for the machine lines;
development of reference counting instruments and
calibrators.
These improvements were implemented and have led to the
powerful analytical instruments which are currently
available.

Modern Automated Systems
What is automation?
The use of a machine designed to follow a predetermined
sequence of individual operations repeatedly and
automatically.
Blood count parameters have been measured automatically
for more than 40 years. A variety of fully automated
instruments are available.


Modern Automated Systems
All instruments determine the:
1. Hemogram
2. The differential blood count
3. The reticulocyte analysis in EDTA whole blood

Hemogram-a written or graphic record of a differential blood count
that emphasizes the size, shape, special characteristics and
numbers of the solid components of blood.
Modern Automated Systems
Depending on the instrument, it may be possible to analyze
cells from body fluids and bone marrow.
All instruments can process the test in either the manual or
automatic mode.
Cell-Dyn Ruby
Auto Mode
Modern Automated Systems
The 4 Modern Analysis Systems:
Also known as The 4 General Principles of Instrument
Operation (IHFF... I Have Few Friends)
Impedance Measurement
Modern Automated Systems
High Frequency Measurement

Modern Automated Systems
Forward Scatter (FSC)

Modern Automated Systems
Flow Cytometry

Modern Automated Systems
Remember: I Have Few Friends...
I = Impedance Measurement
H = High Frequency Measurement
F = Forward Scatter
F = Flow Cytometry
Impedance Measurement

The first principle of measurement
Applies the Coulter Principle
This is based on the measurement of changes in resistance
during cell passage through a small defined opening
between two electrodes.
Cells have lower conductivity than the diluent.
The resulting electrical impulse is proportional to the cell
volume.
The sum of the impulses from all cells in a fixed
measurement volume is evaluated with a histogram.


Impedance Measurement
Impedance Measurement
Problems encountered in Impedance measurement:
Connecting Cables
Environmental Noise
Instrumentation Limitations
Counting by Coincidence
What does counting by coincidence mean?
It is the count loss that occurs when two or more particles
enter the orifice of a Coulter counter in close succession.
These are usually based on the concept of a sensing zone
within which two or more particles cannot be counted
separately.
How was this remedied?
This was remedied through Hydrodynamic Focusing
Impedance Measurement
Hydrodynamic Focusing
The principle of hydrodynamic focusing was an important
development in Impedance Measurement.
This almost totally prevented coincidence and gave a clean
Gaussian Curve.





It also improved the separation of small erythrocytes and
platelets.
This measurement is volume based so that no special
calibration by the user is needed.

Gaussian Curve
Impedance Measurement
Hydrodynamic Focusing

It eliminates the need for calibration.
Calibration-the process of measuring or calibrating against
an established standards.
The sample stream is coated with a coat stream fluid
(sheath stream).
This reduces the diameter of the sample stream to cell size
and isolates the individual cells.
The cells are then passed through the electric field like a
string of pearls and cannot be washed back by turbulence
in the area of measurement.
Impedance Measurement
Hydrodynamic Focusing

Impedance Measurement
Hydrodynamic Focusing
Impedance Measurement
The Cell-Dyn 3700 employs Impedance Measurement with
Hydrodynamic Focusing
High Frequency Measurement
High Frequency Measurement provides an analysis of the
internal structures of the cells.
Either a special reagent is added, or the measurements are
performed on leukocytes after completion of erythrocyte
lysis.
The cells are exposed to a high frequency field, and an
impedance measurement is performed at the same time.
Frequency-the number of complete alternations per second
of an alternating current.
The high frequency impedance impulse depends on the
internal structures and volumes of the cells.
In this way, immature granulocytes are separated from
mature cells using a special software: codename ACAS, and
much additional information of the cell is provided.
High Frequency Measurement
ACAS: Adaptive Cluster Analysis System

What do you mean by Cluster Analysis?
Cluster analysis is the task of assigning a set of objects
into groups (called clusters) so that the objects in the
same cluster are more similar (in some sense or another)
to each other than to those in other clusters.
Cluster analysis is a main task of explorative data mining,
and a common technique for statistical data analysis used
in many fields, including machine learning, pattern
recognition, image analysis, information retrieval, and
bioinformatics.

High Frequency Measurement
ACAS: Adaptive Cluster Analysis System
Cluster Analysis

The result of a cluster analysis shown as the coloring
of the squares into three clusters.
High Frequency Measurement
ACAS: Adaptive Clustering Analysis System
It is a special software owned by Sysmex that is highly
sensitive (detection of as few as 1% blasts).
Its excellent sensitivity results in peace of mind reliability
for the White Blood Cell Differential.

High Frequency Measurement
ACAS: Adaptive Clustering Analysis System
It employs fluorescence staining of peripheral blood cells.

The outstanding cluster resolution and separation of
abnormal blood cells by fluorescence staining in the
Sysmex XT-series reduce limitations and potential
inaccuracies known from optical hematology analyzers
measuring scattered light intensity at different angles only.

With their high nuclear activity, immature or antibody
producing cells show a much higher fluorescence intensity
than normal cells. They are easily distinguishable in the
DIFF scattergram. Atypical lymphocytes are clearly
detected based on their characteristic high fluorescence
intensity.

High Frequency Measurement
ACAS: Adaptive Clustering Analysis System

A DIFF Scattergram employing ACAS
High Frequency Measurement
ACAS: Adaptive Cluster Analysis System
A surfactant causes complete lysis of red blood cells and
platelets and introduces pores in the membranes of white
blood cells.

A second reagent specifically stains white blood cell nuclei
as well as other cellular components.

In the DIFF channel, the Sysmex XT-series differentiates
white blood cells into four types and additionally
determines immature granulocytes. This results in a high
level of sensitivity and specificity.

The lytic destruction of erythrocytes, which may behave as
lyse-resistant in manual or other automated procedures,
results in low fluorescence intensity signals from the nuclei
residues. Thus, interference with the leukocyte differential
is prevented.
High Frequency Measurement
ACAS: Adaptive Cluster Analysis System
Leukocyte quantification and differentiation of basophils are
performed in a separate channel.
In the WBC/Baso channel all cells except for basophils are
shrunk under the influence of the specific reagent.
The usually small population of basophils is reliably
detected and counted in this rare-event channel.

WBC/Baso
scattergram
High Frequency Measurement
ACAS: Adaptive Cluster Analysis System
Both the DIFF- and the WBC/Baso channel utilize the
Sysmex proprietary Adaptive Cluster Analysis System
(ACAS) instead of conventionally fixed or
floating discriminators to separate the populations.
In every clinical laboratory a precise leukocyte differential
needs to be determined even for highly pathological or
aged samples.
Differential results are obtained from samples even older
than 48 hours.
The Sysmex XT-series reduces the necessity for re-
collection of samples and repetition of analyses.

High Frequency Measurement
The Sysmex XT series employs High Frequency Measurement
with ACAS software
Forward Scatter (FSC)
The measurement is performed with erythrocytes and
platelets transformed to a spherical form (sphered
erythrocytes) or treated with a surface active diluent to
optimize their shape.
There are 3 preconditions for exact reproducible scatter
signals when measuring and evaluating erythrocytes and
platelets:
1. Formation of isovolumetric spheres or optimization of
isovolumetric spheres
2. A monochromatic light source (pertaining to a single
wavelength of light)
3. Isolation of the blood cells in the measurement cell by
hydrodynamic focusing

Forward Scatter (FSC)
The cell preparation is performed in isotonic solutions
Lauryl Sulfate (surfactant) is used to form isovolumetric
spheres.
Glutaraldehyde (fixative) is used to fixed the erythrocytes.
The light source is a laser diode with a defined wavelength.


Forward Scatter (FSC)
How are individual cells differentiated and isolated?
This is achieved by differential staining with fluorescent
dyes for RNA and DNA.
The cells are not only isolated individually but also
separated by the detection of different contents of
DNA/RNA.
Forward Scatter (FSC)
In a normal patient or donor sample, counting lasts for
exactly 10 seconds.
During this time, about 50,000 individual erythrocytes and
about 3,000 platelets are counted simultaneously and
morphologically evaluated.

Forward Scatter (FSC)
Measurement Technology of Reticulocyte Analysis
Reticulocytes are determined from EDTA whole blood by
bringing a blood aliquot in contact with a specific
chromogen, new methylene blue or its derivatives. Such as
Oxazin 750.
Forward Scatter (FSC)
Measurement Technology of Reticulocyte Analysis
After staining of the so-called reticulo-granular filamentous
material (presumably ribosomal RNA), the cell suspension
is measured in laser light.
Scatter properties are measured at high and low angles,
together with the absorption of the stained reticulocytes.
Comprehensive reticulocyte analysis is possible with the
combination of measurement signals.

Forward Scatter (FSC)
Hemascreen 22: Fully automated hematology analyzer
22 parameter 5-part Differential Leukocyte Count
This new measuring system, with Multi-Element-Forward-
Scattering (MEFS) laser technology, allow direct measurement
of blood cells, with a high level of sensitivity as well as
specificity.
Flow Cytometry
What is Flow Cytometry?
(Mosbys definition)
A technique in which cells suspended in a fluid flow one at a
time through a focus of exciting light, which is scattered in
patterns characteristic to the cells and their components.

The cells are often labeled with fluorescent markers so that
light is first absorbed and then emitted at altered
frequencies.

A sensor detecting the scattered or emitted light measures
the size and molecular characteristics of individual cells.


Flow Cytometry
Therefore, Flow Cytometry is a laser-based, biophysical
technology employed in cell counting, sorting, biomarker
detection and protein engineering.

Why Is flow cytometry the most important and most
commonly used principle of instrumentation at present?
It is a combination of the general principles being
mentioned.
It allows simultaneous multi-parametric analysis of the
physical and characteristics of up to thousands of particles
per second.
It is routinely used in the diagnosis of health disorders,
especially blood cancers, but has many other applications in
basic research, clinical practice and clinical trials.


Flow Cytometry
Principles: (Note how it employs all other principles
mentioned)

A beam of light (usually laser light) of a single wavelength
is directed onto a hydrodynamically focused stream of
liquid.

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 or FSC) and several
perpendicular to it (Side Scatter or SSC) and one or more
fluorescence detectors.

Flow Cytometry
Principle:
Flow Cytometry
Principles:
Each suspended particle from 0.2 to 150 micrometers
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.
Flow Cytometry
Principles:
This combination of scattered and fluorescent light is picked
up by the detectors (a.k.a. photomultipliers), and, by
analyzing fluctuations in brightness at each detector (one
for each fluorescent emission peak), it is possible to derive
various types of information about the physical and
chemical structure of each individual particle.
Photomultipliers-a device
use in many radiation
detection applications that
converts low levels of light
into electrical pulses.
Flow Cytometry
Principles:
Flow Cytometry
Some flow cytometers on the market have eliminated the
need for fluorescence and use only light scatter for
measurement.
Other flow cytometers form images of each cells
fluorescence, scattered light, and transmitted light.
Flow Cytometry

Modern flow cytometers are able to analyze several
thousand particles every second and can actively separate
and isolate particles having specified properties. (Cluster
Analysis)

A flow cytometer is similar to a microscope, except that
instead of producing an image of the cell, flow-cytometry
offers high-throughput automated quantification of set
parameters for a large number of cells.


Flow Cytometry
High-Throughput Automated Quantification of Set Parameters
Flow Cytometry
A Flow Cytometer has to have five main components:
A flow cell-liquid stream (sheath fluid)
-which carries and
aligns the cells so that
they pass single file
through the light beam
for sensing.
Flow Cytometry
A measuring system
-commonly used are measurement of impedance and optical
systems
-lamps (mercury and xenon)
-high power water-cooled lasers (argon, krypton, dye laser)
-low-power air-cooled lasers (argon @ 488 nm, red He-Ne @
633nm, green He-Ne, He-Cd @ UV range
-diode lasers (blue, green, red, yellow) resulting in light
signals
Flow Cytometry
A Detector and Analogue to Digital Conversion (ADC)
system
- Which generates FSC and SSC as well as fluorescence
signals from light into electrical signals that can be
processed by a computer.
Flow Cytometry
An amplification system (linear or logarithmic)
A computer for analysis
Flow Cytometry
Acquisition

The process of collecting data from samples using the flow
cytometer is termed as acquisition.

Acquisition is mediated by a computer physically connected
to the flow cytometer and the software which handles the
digital interface with the cytometer.

The software is capable of adjusting parameters for the
sample being tested and also assists in displaying initial
sample information while acquiring sample data to insure
that parameters are set correctly.

Flow Cytometry
Current Flow Cytometers
Modern instruments usually have multiple lasers and
fluorescence detectors.

The current record for a commercial instrument is 4 lasers
and 18 fluorescence detectors.

Increasing the number of lasers and detectors allow for
multiple antibody labeling, and can more precisely identify
a target population by their phenotype markers.

Certain instruments can even take digital images of
individual cells, allowing for the analysis of fluorescent
signal location within or on the surface of cells.


Flow Cytometry
Cell-Dyn Ruby utilizes Flow Cytometry with MAPSS Technology

Multiple Angle Polarized Scatter Separation (MAPSS)
Flow Cytometry
Conclusion
Conclusion
The automation of hematological analytical
instruments has reached a high level. Results on
the composition of blood and its cellular
components can be provided very rapidly and
more extensively and with better accuracy and
precision than ever before.

A scenario of this sort is, however, counteracted
by an essential characteristic of human beings
our talent for improvisation which is and will
remain indispensable for further development
Conclusion
One could mention a practical example. For an operation
on a 300 g premature baby, it is essential to know the
platelet count. The only available analytical material is a
capillary tube with 20 l whole blood. This problem may be
solved by returning to platelet counting in the Neubauer
chamber a technique which has been regarded a long
time to be apparently antiquated. Counting all squares in
the middle chamber may lead to an acceptable reduction in
the error, which is unacceptable under normal conditions.
This underlines that only the interaction between the
sophisticated instruments of medical engineering and our
talent for improvisation give acceptable results in
apparently hopeless situations.
Thank You Very Much