automationinhematologypart1-160820102053.pptx

AUTOANALYSER
BY
Dr.Girish Pandey

I N D E X
I. Necessity for Automation.
II. Advantages & Disadvantagesof Automation.
III. Types of Automated HematologyAnalyzers.
IV. Principles involved in Automation.
V. Pentra ES 60 HaematologyAnalyzer.
VI. Pentra DF Nexus HaematologyAnalyzer.
I. Histograms.
II. Flags
III. Quality Control

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

Inventor of the first automated analyzer forcounting and sizing cells
based on his famous ‘Coulter Principle’
The man who started it all-
Wallace H. Coulter (1913 –1998)

Automation
Advantages Disadvantages
⚫ Speed & Efficient Handling
⚫ Accuracy & Precision
⚫ MultipleTests on Single Platform
⚫ Significant Reductionof labor.
⚫ Flagging
⚫ RBC Morphology
⚫ Erroneous results
⚫ Expensive

I N D E X
III. Typesof Automated Hematology Analyzers.
VI. Pentra DF Nexus HaematologyAnalyzer.
I. Histograms.
II. Flags

Types of Automated Hematology Analyzers
Semi-automated Fullyautomated analyzers
analyzers
Measures only few parameters
Some steps like dilution of
blood iscarried out manually
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.

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
Coulterprinciple - detection &
measurement of changes in electrical
impedance (resistance) produced by a blood
cell as it passes through an electrical field.
 Blood cells are poorconductors of electricity
but are suspended in an electrically
conductivediluent.
 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 avoltagepulse 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 requisitecondition forcell
counting by this method is high
dilutionof sample

Variables measured by using el ctrical impedance
RBC •RBC Count
e•MCV
•Size distribution histogram
•RDW
•Hematocrit
•MCH
•MCHC
WBC •Total Count
•3 part differential
 Lymphocyte
 Mononuclear cells
 Granulocyte
Platelets •Platelet count
•Platelet histograms giving
 MPV
PDW

Optical light scatter
Each cell flows in a single line througha flowcell.
A LASER device is focused on the flowcell.
As LASER light beam strikes a cell, it is scattered in variousdirections.
Photodetectorscapture the light.
Forward Scatter Light (FALS) 𝖺 tocell size.
Side Scatter Light (SS) (90°) corresponds to nuclearcomplexity &
granularityof cytoplasm.
Used todistinguish betweengranulocytes, lymphocytes & monocytes.

Variables measured by using OPTICAL LIGHT SCATTER
• RBC Count
• The 5 partdifferential
 Neutrophils
 Eosinophils
 Basophils
 Lymphocytes
 Monocytes
• Mean Cell Volume

Flow Cytometry
Measures multiplecellular & fluorescent
properties of cellswhen they flow as a
single cell suspension through a laser
beam.
Provides the following information
aboutacell:
• Cell size (forward scatter)
• Internal complexity orgranularity (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.
⚫ Theyare forced into the centerof 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
cellsto enter the stream@agiven moment.
⚫ High Flow rate used for immunophenotyping analysis
of cells.
⚫ Low Flowrate used for DNA Analysis.

⚫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 acell
at the ‘interrogation point’, 2 events occur -
 LightScattering
 Fluorescence (Emissionof Light )
⚫ Light Scattered in the forward direction is
detected in Forward Scatter Channel 𝖺 to cell
sizeand that scattered@90° to axisof Laser path
is detected in Side Scatter Channel 𝖺 to
granularity of cell.
⚫ The cells tagged with fluorescence emita
momentary pulse of fluorescence.
⚫ A system of optical mirrors and filters then
direct the specified wavelengths of light to the
designated photodetectors.

⚫Electronics
⚫ The photodetectors - photodiodes and
⚫ photomultiplier tubes convert the optical
signals (photons) to corresponding electronic
signals(electrons).
⚫ Theelectronicsignal produced is proportional
to the amount of lightstriking acell.
⚫ Theelectric currenttravels to the amplifier
and is converted to avoltage pulse
⚫ Thevoltage 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.

Data Analysis
⚫ Data is collected and stored in thecomputer – can bedisplayed in various
formats.
⚫ Parameters – Forward Scatter, Side scatter, emitted fluorescence.
⚫ Dataplots –
Single Parameter – Histogram
Two Parameters – Dot Plot

Gating
A boundary that can beset to restricttheanalysis toa specific population within
thesample.
Could be
Inclusive – Selection of events that fall within the boundary.
Exclusive - Selectionof events that fall outside the boundary.
Dataselected by the gate is then displayed in subsequentplots.

Sorting
Consists of collecting cells of interest
(defined through criteriaof sizeand fluorescence)
for
furtheranalysis
(microscopy /functional/ chemical analysis)

Common Applications of Flow Cytometry
1. Leukemias and lympomas Immunophenotyping (evaluation of cell surface
markers),diagnosis,
detectionof minimal residual disease, and to identify
prognostically important subgroups.
2. Paroxysmal nocturnal
hemoglobinuria
Deficiencyof 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
matching

Estimation of Reticulocyte Count
⚫ Estimation based upon uptakeof variousdyes and fluorochromes by the RNA of
reticulocytes.
⚫ The flourescent cells areenumerated using a flow cytometer.
⚫ Variousdyes used are –
 Auramine O
 Thiazole Orange
 CD4K 530
 Oxazine 750
 New Methylene Blue.
 Flow cytometry allows classification of reticulocytes into 3 maturation stages –
 Low Fluorescence Reticulocytes (LFR).
 Middle Fluorescence Reticulocytes (MFR).
 High Fluorescence Reticulocytes (HFR).

Estimation of reticulocyte count – Reticulocyte parameters
 Immature Reticulocyte Fraction ( IRF) / Reticulocyte Maturation
Index.
 The immature reticulocyte fraction (IRF) is calculatedas
 Sum of immature reticulocytes (MFR & HFR)
 Earlysensitive markerof erythropoiesis.
 Early identification of marrow regeneration in patients undergoing
BMT/chemotherapy.
 Reticulocyte Hemoglobin Equivalent (RET-He) / Reticulocyte
Hemoglobin Concentration (CHr)
 Gives the Hbcontent of freshly produced RBCs.
 Early detection of Fedeficiency anemia.
 Monitoring of erythropoietin & Fe therapy
.
 Mean reticulocyteVolume (MCVr)
 ↑ rapidly following Fe therapy in subjectswith depleted Festores.
 ↓ rapidly with development of iron deficient erythropoiesis.

Other Methods
⚫PEROXIDASE based cell counts.
 Myeloperoxidase is used tocount neutrophils.
 Lymphocytesare notstained.
⚫FLUORESCENCE based cell counts.
 Forreticulocyteand plateletcount.
 Best fordetecting immatureplatelets.
⚫IMMUNOLOGICAL based cell counts.
 Accurate plateletcountusing CD41/CD61 Abs

I N D E X
V. Haematology Analyzer.
I. Histograms.
II. Flags

Parameters
26 Parameters in C B C mode + 5 DIFF mode
12 parameters in C B C mode
26 parameters in 5 DIFF mode
=
12 parameters (CBC mode)
+
14 parameters (5 W B C sub-populations & ALY, LIC)
W B C (White Blood Cell)
R B C (Red Blood Cell)
H G B (Hemoglobin Concentration)
H C T (Hematocrit)
M C V (Mean Corpuscular Volume)
M C H (Mean Corpuscular Hemoglobin)
M C H C (Mean Corpuscular Hemoglobin Concentration)
R D W (Red Distribution Width)
P L T (Platelets)
PCT (Plateletcrit)
M P V (Mean Platelet Volume)
P D W (Platelets Distribution Width)
W B C (White Blood Cell)
R B C (Red Blood Cell)
H G B (Hemoglobin Concentration)
H C T (Hematocrit)
M C V (Mean Corpuscular Volume)
M C H (Mean Corpuscular Hemoglobin) CBC
M C H C (Mean Corpuscular Hemoglobin Concentration)
R D W (Red Distribution Width)
P L T (Platelets)
PCT (Plateletcrit)
M P V (Mean Platelet Volume)
P D W (Platelets Distribution Width)
L Y M (Lymphocytes) in % and 
M O N (Monocytes) in % and 
N E U (Neutrophils) in % and  5 W B C sub-populations
EOS (Eosinophils) in % and 
B A S (Basophils) in % and 
A L Y (Atypical Lymphocytes) in % and 
LIC (Large Immature Cells) in % and 

Micro-sampling MDSS (Multi-Distribution Sampling System)
Micro-sampling:
30 μL in CBC modeand 53 μL in CBC+DIFF modeareaspired
Ideal for pediatric,oncologyorgeriatricsampletypesor
wheneverasmall samplevolume is required
Theremainingvolumemay beused foradditional analysissuch
assedimentationrate, smear…Itwill avoid to punctureagain the
patient
Blood split into precisealiquots
Aliquotsdistributeddirectlyintopre-heated analysischambers
with a synchronized tangential flow of diluent for appropriate
dilutions withoutviscosityproblem
Perfect mixing and homogenizationof blood with
reagents.
No sampling shear-valve to distribute blood sample in
all appropriate chambers: No maintenance, noclogging
Immediate CBC+DIFF test selectionwithouta cleaning
cycle in between.

Estimation of Hemoglobin
⚫ Sample isdiluted with Drabkin’sreagent.
⚫ Potassium ferricyanide in the reagentconverts Hb Fe from
ferrous (Fe2+) to Ferric (Fe3+) to form methemoglobin.
⚫ This methemoglobin combineswith potassiumcyanide to form
thestablecyanmethemoglobin.
⚫ Hbconcentration is measured bya photodetectorwhich reads
absorbanceof cyanmethemoglobinat 540 nm.
⚫ Cyanide freereagents likesodium lauryl sulphate arealso used

HGB measurement
Spectrophotometry
ABX Lysebio : cyanide-free!
The newest developed reagent for RBC lysisand determination of HGB.
Advantage
It does not contain cyanide.
Itcould be thrown with the regularwastes (depending of the national regulations).
How does itwork ?
By action of lysis agent, contained in the reagent, hemoglobin is released.
All the heme iron is oxidized and stabilized.
Oxidation resulting complexes are measured through theoptical part of the first dilution
chamber by spectrophotometryata wavelengthof 550nm.
Result = Absorbency valuex coef. of calibration.

WBC and DIFFERENTIAL count
WBC/BAS count
 

Electronic Impedance Variation Principle
Differentiation between the BAS and the otherWBC is obtained by the useof the ABX
BASOLYSE II reagentwith it specific lysing action.
Nucleus of WBC populations arecounted between the
electronic thresholds from 0 to <BA2>.
BAS arecounted between theelectronic thresholds
<BA2> and <BA3>.
Results
WBC = Numberof cells counted within a specified amount of time pervolume x WBC calibration
coefficient.
BAS = Numberof cells counted within a specified amountof time pervolume x WBC calibration
coefficient in a percentageas the total numberof leukocytes (BAS and WBC nuclei).

LMNE count
Step 1: Cytochemistry
25 μL of whole blood is delivered into the LMNE chamber in a tangential flow of the reagent ABX Eosinofix.
The blood sample is incubated at a regulated temperature with ABX Eosinofix during 12 seconds.
- It lysesRBC
- It stains EOS cytoplasm, granulesand nuclei withaspecificdyeagent : Chlorazol Black
- It stabilizesWBC in theiroriginal state : 48 hourpost-drawstability
Then, the sample is diluted in a currentconductor diluent.

LMNE count
Step 2: Flowcytometry
The prepared sample is injected through the flow cytometer: DHSS : Double Hydrodynamic Sequential System
1 - Focused flow for impedancemeasurement
Cellvolume measurement:
Thedilution isaspirated through acalibrated aperture.
Twoelectrodes are placed on each side of the aperture.
Electric current passes through the electrodes continuously.
When a cell passes through the aperture, electric resistance (or impedance)
between the 2 electrodes increases proportionately with cell volume.
2- Focused flow for optical detection
Analysisof the internal cellularstructure by measuring light absorbency
of cells.

•
•
•
•
Lymphocytes
Monocytes
Neutrophils
Eosinophils
LMNE count
Step 3: Resultsareobtained and displayed in LMNE matrix
LMNE matrix is obtained from:
- IMPEDANCE measurement
- OPTICAL detection
4 sub-populations are perfectly separated
becauseof the high definition system:
Thequalityof the resolution allows the counting
of 2 additional sub-populations:
• Large Immature Cells (LIC) : myelocytes, promyelocytes, large blasts.
• Atypical Lymphocytes (ALY) : large lymphocytes, activated lymphocytes, blasts.
BAS are removed in proportion to LMN populations.

I N D E X
PART 1
VI. Pentra DF Nexus Haematology Analyzer.
PART 2
I. Histograms.
II. Flags

Sampling principles for RBC & platelets

Sampling principles for WBC Count & Hb measurement

Sampling principles for Basophil count

Sampling principles for Differential Count

Measurement of Parameters
Directly Measured Derived from
Histogram
Calculated
Hemoglobin Mean Cell Volume (MCV) Hematocrit
RBC Count Red Cell Distribution
Width (RDW)
Mean Cell Hemoglobin
(MCH)
WBC Count Differential Leukocyte
Count (DLC)
Mean Cell Hemoglobin
Concentration (MCHC)
Platelet Count Platelet Distribution
Width
Reticulocyte Count

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