AUTOMATION IN HEMATOLOGY
The man who started it all
Wallace H. Coulter (1913 –1998)
Inventor of the first automated
analyzer for counting and sizing cells
based on his famous ‘Coulter Principle’
Dr. Ajit Kumar Singh
PGT MD (lab medicine)
CNCI (Kolkata)

Necessity for Automation
Cell counts
Dx of Hemoglobinopathies
Immunophenotyping
Dx of Leukemias & Lymphomas
Coagulation Abnormalities.

Automation
Advantages
 Speed & Efficient Handling
 Accuracy & Precision
 Multiple Tests on Single
Platform
 Significant Reduction of
labor.
Disadvantages
 Flagging
 RBC Morphology
 Erroneous results
 Expensive

Types of Automated Hematology
Analyzers
Semi-automated
analyzers
 Measures only few
parameters
Some steps like
dilution of
 blood is carried out
manually
Fully automated
analyzers
 Measures multiple
parameters.
 Requires only
anticoagulated blood
samples.

Components of a cell counter
 HYDRAULICS
 Aspirating unit.
 Dispensers.
 Diluters.
 Mixing chambers.
 Aperture bath.
 Hemoglobinometer.
 PNEUMATICS
 Vacuums & Pressures for operating valves.
 ELECTRICALS
 Analyzers & Computing circuitary.

XN-550
 Compact 6-part differential analyzer
 Throughput of 70 samples per hour
 Single sample analysis in closed mode
 Fully integrated IPU including LCD color touchscreen
 Only 25 µL aspiration volume in whole blood mode
 More than just CBC + DIFF – added clinical values available

Principles of working of an automated
blood analyzer
 Electrical Impedance.
 Light Scatter.
 Fluorescence.
 Light Absorption.
 Electrical Conductivity.

Electrical impedance
 Cell counting & sizing is based on the Coulter principle - detection &
measurement of changes in electrical impedance (resistance) produced by a
blood cell as it passes through an electrical field.
 Blood cells are poor conductors of electricity but are suspended in an
electrically conductive diluent.
 2 chambers filled with a conductive buffered electrolyte solution separated
by a glass tube having a small aperture.
 A DC current is generated between two electrolytes.

Electrical impedance
 As a cell passes through the aperture, flow of current is impeded and a
voltage pulse is generated.
 The no: of pulses indicate the no: of the blood cells.
 The amplitude (height) of each pulse is proportional to the cell volume.
 The requisite condition for cell counting by this method is high dilution of
sample.

RBC RBC Count
•MCV
•Size distribution histogram
•RDW
•Hematocrit
•MCH
•MCHC
WBC o Total Count
o 3 part differential
Lymphocyte
Mononuclear cells
Granulocyte
Platelets o Platelet count
o Platelet histograms giving
MPV
PDW

Optical light scatter
 Each cell flows in a single line through a flow cell.
 A LASER device is focused on the flow cell.
 As LASER light beam strikes a cell, it is scattered in various directions.
 Photodetectors capture the light.
 Forward Scatter Light (FALS) ∝ to cell size.
 Side Scatter Light (SS) (90°) corresponds to nuclear complexity & granularity
of cytoplasm.
 Used to distinguish between granulocytes, lymphocytes & monocytes.

Optical light scatter

Variables measured by using OPTICAL LIGHT
SCATTER
 RBC Count
 The 5 part differential
 Neutrophils
 Eosinophils
 Basophils
 Lymphocytes
 Monocytes
 Mean Cell Volume

Fluorescence flow cytometry (FFC)
Fluorescence flow cytometry (FFC) is used to analyze physiological and chemical
properties of cells. It can also be used to analyze other biological particles in
urinalysis analyzers. It provides:
 Information about cell size and structure
 Information about a cell’s interior
In flow cytometry, we examine cells and particles while they are flowing through
a very narrow flow cell.

Fluorescence flow cytometry (FFC)
 First a blood sample is aspirated and proportioned, then diluted to a pre-set ratio
and labelled with a proprietary fluorescence marker that binds specifically to
nucleic acids.
 Next the sample is transported into the flow cell. The sample is illuminated by a
semiconductor laser beam, which can separate the cells using three different
signals:
 forward-scattered light (forward scatter or FSC)
 side-scattered light (side scatter or SSC)
 side-fluorescence light (side fluorescence or SFL).

Fluorescence flow cytometry (FFC)
 The intensity of the forward scatter indicates the cell volume. The side
scatter provides information about the internal cell structure and its content,
such as nucleus and granules. The side fluorescence indicates the amount of
nucleic acids present in the cell.
 Cells with similar physical and chemical properties form a cluster in a graph
known as a scattergram.

Fluorescence flow cytometry (FFC)
 The principle of fluorescence flow cytometry is used in different analysers for
haematology and urinalysis. For blood cell counts we use fluorescence flow
cytometry, e.g. for the WBC and differential, for NRBC counting and
reticulocyte measurement.
 In urinalysis analysers, fluorescence technology is also used for counting
bacteria, red blood cells, white blood cells and other elements.

What is flow cytometry
 Flow – cell in motion
 Cyto – cell
 Metry – measure
 Measuring property of cell while in a fluid stream
The fluorescence can then be measured to determine the amount and type of
cells present in a sample. Up to thousands of particles per second can be
analysed as they pass through the liquid stream.
A beam of laser light is directed at a hydrodynamically-focused stream of fluid
that carries the cells. Several detectors are carefully placed around the stream,
at the point where the fluid passes through the light beam.

What is flow cytometry
 One of these detectors is in line with the light beam and is used to measure
Forward Scatter or FSC. Another detector is placed perpendicular to the
stream and is used to measure Side Scatter (SSC).
 Since fluorescent labels are used to detect the different cells or components,
fluorescent detectors are also in place. The suspended particles or cells,
which may range in size from 0.2 to 150μm, pass through the beam of light
and scatter the light beams.
 The fluorescently labelled cell components are excited by the laser and emit
light at a longer wavelength than the light source.

Flow Cytometry
 Measures multiple cellular & fluorescent properties of cells when they flow as
a single cell suspension through a laser beam.
 Provides the following information about a cell:
 • Cell size (forward scatter)
 • Internal complexity or granularity (side scatter)
 • Relative fluorescence intensity

Components of Flow Cytometry
 Fluidics (The Flow System)
 The sample is injected into a stream of sheath fluid within the flow chamber.
 They are forced into the center of the stream forming a single file by the principle of
HYDRODYNAMIC FOCUSING.
 ‘Only 1 cell or particle can pass through the LASER Beam@ a given moment.’
 The sample pressure is always > than the sheath pressure ensuring a high flow rate, thus allowing
more cells to enter the stream@a given moment.
• High Flow rate used for immunophenotyping analysis of cells.
• Low Flow rate used for DNA Analysis.

Components of Flow Cytometry
 Optics
 Following cell delivery, a light source like the Argon- ion LASER is required to excite the cells.
 When light from a Laser Beam intersects a cell at the ‘interrogation point’, 2 events occur -
 Light Scattering
 Fluorescence (Emission of Light )
 Light Scattered in the forward direction is detected in Forward Scatter Channel ∝ to cell size and
that
 scattered@90° to axis of Laser path is detected in Side Scatter Channel ∝ to granularity of cell.
 The cells tagged with fluorescence emit a momentary pulse of fluorescence.
 A system of optical mirrors and filters then direct the specified wavelengths of light to the
designated photodetectors.

Components of Flow Cytometry
 Electronics
 The photodetectors - photodiodes and photomultiplier tubes convert the optical signals (photons)
to corresponding electronic signals(electrons).
The electronic signal produced is proportional to the amount of light striking a cell.
 The electric current travels to the amplifier and is converted to a voltage pulse
 The voltage pulse is assigned a digital value representing a channel by the Analog-to Digital
Converter (ADC) .
 The channel no: is transferred to the computer which displays it to the appropriate position on the
data plot.

Components of Flow Cytometry

Common Applications of Flow Cytometry
1. Leukemias and lymphomas Immunophenotyping (evaluation of cell surface
markers),diagnosis,
detection of minimal residual disease, and to identify
prognostically important subgroups.
2. Paroxysmal nocturnal
hemoglobinuria
Deficiency of CD 55 and CD 59.
3. Hematopoietic stem cell
transplantation
Enumeration of CD34+ stem cells.
4. Feto -maternal hemorrhage Detection and quantitation
of foetal hemoglobin in maternal blood sample.
5. Anemias Reticulocyte count.
6. Human immunodeficiency virus
infection
For enumeration of CD4+ lymphocytes
7. Histocompatibility cross

Data Analysis
 Data is collected and stored in the computer – can be displayed in various
 formats.
 Parameters – Forward Scatter, Side scatter, emitted fluorescence.
 Data plots :
 Single Parameter – Histogram
 Two Parameters – Dot Plot

sodium lauryl sulphate (SLS) detection
method
 Hemoglobin is a routine diagnostic parameter in each blood count. The
method recommended by the ICSH (International Committee for
Standardization in Hematology) for measuring hemoglobin concentration is
the cyan-methemoglobin method.
 SLS hemoglobin detection method uses cyanide-free sodium lauryl sulphate
(SLS). The reagent lyses red blood cells and white blood cells in the sample.
The chemical reaction begins by altering the globin and then oxidising the
heme group.
 Now the SLS’ hydrophilic groups can bind to the heme group and form a
stable, colored complex (SLS-HGB), which is analyzed using a photometric
method.

sodium lauryl sulphate (SLS) detection
method
 An LED sends out monochromatic light and by moving through the mixture
light is absorbed by the SLS-HGB complexes. The absorbance is measured by a
photo sensor and is proportional to the hemoglobin concentration of the
sample.
 Absorption photometric methods are usually influenced by the turbidity of the
sample itself. In blood samples, turbidity can be caused due to lipaemia or
leucocytosis. By using the SLS-HGB method these interferences can be
minimised due to the effect of the reagent.

PLT-F channel
 The new PLT-F method is based on a Fluorocell fluorescent dye (oxazine), an
extended counting volume, and an extended counting time.
 Compared with the PLT-O method, platelets are more clearly distinguished
from other blood cells using the difference in forward scattered light and the
fluorescence intensity.
 The fluorescence marker specifically labels platelets and no other blood cells,
which minimises interferences and is one reason for the extremely good
correlation with the CD41/CD61 immune flow cytometry method. Another
reason is the high measurement accuracy in the low concentration range
since the PLT-F channel analyses a 5-fold larger sample volume of the
aspirated sample compared to the DC detection measurement.

PLT-F channel
 The IPF supports quick and efficient differential diagnosis of
thrombocytopenia as it initially suggests whether its cause is in the bone
marrow or in the peripheral blood.
 The membranes of the platelets are perforated by the lysing reagent but they
remain largely intact during this process. Subsequently, the fluorescence
marker specifically labels the RNA inside the platelets, avoiding interferences
with other cells or fragments of similar size.
 Using the forward scattered light and the fluorescence signal, the platelets
are separated from red blood cells and white blood cells.

PLT-F channel
 The PLT-F channel also allows the rapid and fully automated quantification of
the immature platelet fraction (IPF and IPF#). Immature platelets can be
separated from the mature platelets since they are more reactive and contain
more RNA than mature ones. This is reflected by increased fluorescence
signals, which are inversely proportional to the degree of maturity of the
platelets.

WBC differential channel
 Analysing white blood cell differentials consists of a cytochemical reaction of
the cells with a reagent set, followed by fluorescence flow cytometric
analysis.
 The WBC differential channel provides counts of 10 white blood cell
subpopulations including immature granulocytes (IG) as well as flag
information in cases of abnormalities.
 The specially developed lysis reagent initially perforates the cell membranes
while leaving the cells largely intact. The fluorescence marker labels the
intracellular nucleic acids (mostly RNA) in the second step. The composition
of these two reagents effects a mild reaction with the blood cells, so that
almost all of the blood cells’ structure remains intact.
 Thus, optimal separation is achieved, particularly of lymphocytes and
monocytes.

WBC differential channel
 The prepared sample is then analysed using fluorescence flow cytometry. The
measurement signals related to side scatter (SSC) and side fluorescence (SFL)
are analysed and depicted in a scattergram.
 Cells with similar cytochemical properties fall within the same area in the
scattergram and can be separated using an advanced software algorithm.

WBC differential channel

Dr . AJIT KUMAR SINGH
PGT, MD(Lab medicine)
CNCI KOLKATA
Thank you…

Thank you..