Automation in Haematology 5-1

Automation In Haematology
Dr Mrs Eyiuche D Ezigbo

Thrombosis & Haemostasis Unit
Department of Medical Laboratory Sciences University Of
Nigeria
Enugu Campus
Outline
• Automated haematology Whole
Blood Cell Analyzers
• Basic Principles
• Result outputs-Histograms
• Problem-Solving –
Troubleshooting
Dr E D Ezigbo.Thrombosis &
Haemostasis Unit. UNEC
Objective:
At the end of this lesson, the students will be able to:
• Describe the general characteristics of automated haematology
analyzers
• Descripe the principles for performing cell counts eg. (Impedance

and light scatter)
• Discuss the detection of errors (flagging) and the remedial actions

with automated haematology analyzers
• Describe key aspects of automated haematology analyzers, including

histogram use and interpretation
What is Automated Hematology?
• Is the performance of Haematology Lab investigations

by using an Automated Analyzer as opposed to manual
procedures.
• There are various Hematology analyzers depending on

operating principles and the parameters they
performanaly
Why Automated Hematology?/Advantages of
Automated Analyzers
• They significantly increase the number of patients to be analysed,
making more efficient use of laboratory resources.
• They give an estimate of many variables which are manually not

possible and produce data with increased reliability, precision and
accuracy
• The automation in haematology is efficient, lacks inter-observer

variability and size distribution errors
• Data can be stored in automated analysers
• They analyze and produce results within a very short time.
 Help
To evaluate symptoms such as: weakness,
fatigue, bruising, fever, or weight loss
To diagnose conditions: anemia, infection
To diagnose diseases of the blood :
leukemia etc
To monitor the response to some types of
drug or radiation treatment
What do Automated Analyzers Perform?
 Counting of WBCs, RBCs and Platelets

 Measurement of Hemoglobin
 Calculation of Hematological Indices
(Absolute Values)
 Some can perform Differential coun3-
part and 5 part)
 Some can also indicate abnormalities of
RBCs, Platelets and WBCs (Flags)
CBC/FBC Performed on an Automated
Hematology Cell Analyzer
Well mixed EDTA sample is used
CBC is a group of tests (WBC, RBC, Hgb, Hct, Red Cell
Indices, Platelet Count, and automated differential)
Tests are performed simultaneously (usually in less than a
minute)
When the performance limits of an automated hematology
analyzer are exceeded, a manual method of cell counting
and blood smear review must occur
Manual cell count
Some Automated Haematology Whole
Blood Analyzers
The output of an automated haematology analyzer
Advantages Disadvantages
• Speed with efficient • Flagging of a laboratory test
handling of ,argr result demands labouriouus
intensive Manual examination of
number of samples
a blood smear/film
• Accuracy and • Comments on red blood cell
precision in morphology cannot be
quantitative blood generated.
tests • Platelet clumps are counted as
• Ability to perform single giving a false reduced
count.
multiple tests on a • Erroneously increase or decrease
single platform results due to interfering factors
• Significant reduction • Expensive with high running
of labour cost..
requirements
Types of automated Haematology Analysers
based on the WBC differentials
Three parts: Differentiates cells into three categories
1. Granulocytes
2. Lymphocytes
3. Monocytes/mixed cells
Five parts: Differentiate cells into the five basic leukocyte types
1. Neutophhils
2. Eosinophils
3. Basophils
4. Lymphocytes
5. Monocytes
Seven parts: In addition are able to distinguish

1. Nucleated RBCs
2. Abnormal and atypical cells and immature
Dr E D Ezigbo.Thrombosis
& cells
Automated CBC: Basic Technology
Cell counting and sizing (WBC,RBC,PLT)
• Electrical impedance method
• Radiofrequency
• Optical Scatter(with or without
cytochemistry)
Haemoglobin: spectrophotometric method

• Cyanmethaemoglobin
• Cyanide free Haemoglobin
Electrical Impedance or low-voltage
direct current (DC) resistance:Coulter
principle 1st develped in 1950´s
A stream of cells in suspension passes through a small aperture
across which an electrical current is applied. Each cell that passes
alters the electrical impedance and can thus be counted and sized.
Histograms showing the size distribution of white cells, red cells and
platelets. Sizing is based on impedance technology
The coulter principle
• The poorly conductive blood cells are suspended in a
conductive diluent
• The diluent is passed through an elastic filed created between
two electrodes
• The liquid passes through a small aperture
• The passage of each particle through the aperture
momentarily increases the impedance (resistance) of the
electrical path between the electrodes
• The increase in impedance creates a pulse that can be
measured.
• the number of pulses= blood count
• the amplitude(height) of the pulse=volume of cell
Radiofrequency (RF) or alternating current
resistance.
• Low-voltage DC impedance may be used in conjunction
with RF resistance or resistance to a high-voltage
electromagnetic current flowing between both
electrodes simultaneously.
• Although the total volume of the cell is proportional to

the change in DC, the cell interior density is
proportional to pulse height or change in the RF signal.
Radiofrequency probe
• VCS ( volume, conductivity, scatter)
technology by coulter
• Radio frequency probe with impedance

Sysmex
Beckman Coulter VCS technology
Volume Measurement:
As opposed to using light loss to

estimate cell size, VCS utilizes the
Coulter Principle of (DC)
Impedance to physically measure
the volume that the entire cell
displaces in an isotonic diluent.
This method accurately sizes all
cell types regardless of their
orientation in the light path.
CONDUCTIVITY MEASUREMENT:
Alternating current in the radio frequency (RF)

range short circuits the bipolar lipid layer of a
cell’s membrane, allowing the energy to penetrate
the cell.
This powerful probe is used to collect information
about the internal structure of the cell, including
chemical composition and nuclear volume.
SCATTER MEASUREMENT:
When a cell is struck by the coherent light of a LASER
beam, the scattered light spreads out in all directions.
Using a proprietary new detector, light scatter at angles
between 10 and 70 deg. are collected to obtain
information about cellular granularity, nuclear
lobularity and cell surface structure by the VCS
instrument.
Optical scatter: and/or absorption (with or without
cytochemistry)
• This may be used as the primary methodology or in combination with
other methods.
• In optical scatter systems (flow cytometers), a hydrodynamically

focused sample stream is directed through a quartz flow cell past a
focused light soure . Fig 1
• The light source is generally a tungsten-halogen lamp or a helium-

neon laser (light amplification stimulated emission of radiation).
• Laser light, termed monochromatic light because it is emitted at a

single wavelength, differs from brightfield light in its intensity,
coherence (i.e., it travels in phase), and low divergence or spread.
• These characteristics allow for detecting interference in the laser
beam and enable the enumeration and differentiation
Dr E D Ezigbo.Thrombosis & of cell types.
Figure 1 Dr E D Ezigbo.Thrombosis &
• 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

processes of absorption, diffraction (bending around
corners or the surface of a cell), refraction (bending
because of a change in speed), and reflection (backward
scatter of rays caused by an obstruction).
• The detection of scattered rays and their conversion

into electrical signals is accomplished by
photodetectors (photodiodes and photomultiplier tubes)
at specific angles..
• Forward-angle light scatter (0 degrees) correlates
with cell volume, primarily because of diffraction of
light.
• Orthogonal light scatter (90 degrees), or side scatter,

results from refraction and reflection of light from
larger structures inside the cell and correlates with
degree of internal complexity.
• Forward low-angle scatter (2 to 3 degrees) and

forward high-angle scatter (5 to 15 degrees) also
correlate with cell volume and refractive index or with
internal complexity.
• Differential scatter is the combination of this low-

angle and high-angle forward light scatter .
Optical Detection Principle(summary)
• 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 is focused
• As each cell passes through sensing zone of flow cell, it
scatters focused lights
• Scattered light is detected by a photodetector and converted
to an electrical pulse
• Number of pulses generated is directly proportional to the
number of cells passing through the sensing zone in a
specific time period
Haemoglobin:
Hb concentration is measured automatically by a modification of
the manual (HiCN) method. To reduce the toxicity of HiCN some
systems replace it with a non-toxic material Na- lauryl sulphate
RBC count:
The RBCs are counted automatically by two methods
Aperture impedance: where cells are counted as they pass in a
stream through an aperture.
Or by light scattering technology. The precision of electronic

counting for RBCs is much better than the manual count, and it is
available in a fraction of the time. This made the use of RBC
indices of more clinical relevance
PCV and red cell indices
Pulse height analysis allows either the PCV or
the MCV to be determined.
MCV=PCV(haematocrit)% x10
RBC in millions/ul
Normal values: male & female
82-97fl
Increased in : Macrocytosis
Decreased in: Microcytosis
MCH= mean cell haemoglobin
MCV= haemoglobin g/dl x10

RBC in millions/ul

27-32pg(pico grams)
increased: hyperchromic
Decreased: hypochromic
MCHC= Mean cell Hb Concentration
MCHC= haemoglobin/dlx 100

haematocrit%

30-34g/dl
Increased: hyperchromic
Decreased: hypochromic
Red cell distribution width (RDW-SD)
• RDW is an actual measurement of the width of
the erythrocyte distribution curve
• It is a measurement of Anisocytosithe
s(presence of more than one population of cells)
• May increase before MCV becomes abnormal
Reference values
Female: 36.4-46.3fl
.
Male: 35.1-43.9fl
It increases in many types of anaemias to indicate

the variation in red cell sizes
RDW-CV
The coefficient of variation( CV) is defined as the
% ratio of the standard deviation (x),
To the mean (µ)
CV = x/µ
Sometimes known as relative standard deviation
Reference values
Female: 11.7-14.4%
Male: 11.6-14.4%
.
Total WBC count

The total WBC count is determined in whole blood in which red cells
have been lysed. Fully automated multichannel instruments perform
WBC counting by either impedance or light scattering both.
Reticulocyte count
An automated retic count can be performed using the fact that various
fluoro-chromes combine with the RNA of the reticulocytes.
Fluorescent cells can then be enumerated using a flow cytometer. An
automated retic counter also permits the assessment of retic maturity
since the more immature reticulocytes have more RNA fluoresce more
strongly than the immature retics found normally in PB.
Histograms as seen on an Automated
Haematology Analyser: General Histogram
Characteristics
Graphic representations of cell frequencies (Y- axis) versus
cell sizes (X-axis) in femtoliters (fL)
Provide information about erythrocyte, leukocyte, and

platelet frequency and their distributions about the mean,
and also depict the presence of subpopulations
Provide means of comparing sizes of patient’s cells with

normal populations
Shifts in one direction or the other can be of diagnostic

importance Dr E D Ezigbo.Thrombosis &
Normal Histogram
WBC: Trimodal distribution with individual peaks and valleys at
specific regions
LYMPHS = 35-90 fL MONOS = 90-160 fL GRANS = 160-
450 fL
RBC: Unimodal, > 36 fL

PLT: Unimodal, 2-20 fL (fitted 0-70 fL)
All curves normally start and end at the baseline

All curves normally represent Gaussian distributions
X-Axis: Cell size in femtoliters (fL)

Y-Axis: # of cells
WBC/Coulter Histogram as a
Quality Control Tool
Abnormality / Indicator Probable Cause Comment
WBC histogram (lymph peak) does not Giant platelets, nRBC, Plt Review smear,
start at baseline clumping correct for nRBC
Elevation of left portion of granulocyte Left Shift Review smear
peak
Elevation of right portion of Neutrophilia Review smear
granulocyte peak
Trail extending downward at extreme
left, or lymph peak not starting at nRBC, Plt clumping, unlysed Review smear and
baseline RBC, cryoproteins, parasites for nRBC
Peak to left of lymph peak or lymph nRBC Review smear &

peak widening towards left correct nRBC
Atypical lymphs, blasts, plasma
Widening of lymph peak to right cells, hairy cells, eosinophilia, Review smear
basophilia
Monocytosis, plasma cells,
Wider mono peak eosinophilia, basophilia, Review smear
blasts
RBC/Coulter Histogram as a
Review smear
Left of curve does not touch baseline Schistocytes and extremely FBC and Platelet
small red cells
histogram
Transfused cells, therapeutic

Bimodal peak response Review Smear
Right portion of curve extended Red cell autoagglutination Review FBC &
Smear
Left shift of curve Microcytes Review smear &

FBC
Right shift of curve Macrocytes Review smear &

FBC
Platelet/Coulter Histogram as a
Peak or spike at left end of Cytoplasmic fragments Review smear

histogram (2-8 Fl)
Review smear +
FBC
Spike towards right end of Schistocytes, ( MCV & 
histogram microcytes, giant RDW)
platelets
( MPV & 
PDW)
Bimodal peak Cytoplasmic fragments Review smear

Knowledge Check
WBC Clumping
1. What results are affected?
2. What resolutions may be attempted to obtain

reliable results?
Knowledge Check Answer
WBC Clumping
1. What results are affected?
Answer:
1. False decrease in WBC
What resolutions may be attempted to obtain reliable
results?
Answers:
Redraw another EDTA specimen and review the results

Collect citrated blood for CBC or CBC/PLT (multiply WBC
and PLT by 1.11 to correct for anticoagulant dilution)
Incubate blood 37 ºC for 10-30 minutes and reanalyze
Estimate WBC from smear and perform manual differential
Perform a manual count to verify
Append appropriate comment to the result, “WBC Clumps”
END of Slides

Automation in Haematology 5-1

Uploaded by

Copyright:

Available Formats

You might also like

Automation in Haematology 5-1

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Automation in Haematology 5-1

Uploaded by

Copyright:

Available Formats

Automation In Haematology

Dr Mrs Eyiuche D Ezigbo

• Descripe the principles for performing cell counts eg. (Impedance

• Discuss the detection of errors (flagging) and the remedial actions

• Describe key aspects of automated haematology analyzers, including

• Is the performance of Haematology Lab investigations

• There are various Hematology analyzers depending on

• They give an estimate of many variables which are manually not

• The automation in haematology is efficient, lacks inter-observer

• Data can be stored in automated analysers

• They analyze and produce results within a very short time.

 Counting of WBCs, RBCs and Platelets

Seven parts: In addition are able to distinguish

Haemoglobin: spectrophotometric method

• Although the total volume of the cell is proportional to

• Radio frequency probe with impedance

As opposed to using light loss to

Alternating current in the radio frequency (RF)

• In optical scatter systems (flow cytometers), a hydrodynamically

• The light source is generally a tungsten-halogen lamp or a helium-

• Laser light, termed monochromatic light because it is emitted at a

• Light scatter results from the interaction between the

• The detection of scattered rays and their conversion

• Orthogonal light scatter (90 degrees), or side scatter,

• Forward low-angle scatter (2 to 3 degrees) and

• Differential scatter is the combination of this low-

Or by light scattering technology. The precision of electronic

MCV= haemoglobin g/dl x10

Normal values: male & female

MCHC= haemoglobin/dlx 100

Normal values: male & female

It increases in many types of anaemias to indicate

Total WBC count

Provide information about erythrocyte, leukocyte, and

Provide means of comparing sizes of patient’s cells with

Shifts in one direction or the other can be of diagnostic

RBC: Unimodal, > 36 fL

All curves normally start and end at the baseline

X-Axis: Cell size in femtoliters (fL)

Peak to left of lymph peak or lymph nRBC Review smear &

Transfused cells, therapeutic

Left shift of curve Microcytes Review smear &

Right shift of curve Macrocytes Review smear &

Peak or spike at left end of Cytoplasmic fragments Review smear

Bimodal peak Cytoplasmic fragments Review smear

1. What results are affected?

2. What resolutions may be attempted to obtain

1. What results are affected?

1. False decrease in WBC

Redraw another EDTA specimen and review the results

You might also like