automation in hematology

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Automation in Hematology

Ruel Bartholomeo B. Maguad, RMT, RN


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Introduction

Hematological diagnosis has developed from ratherphilosophical approaches in Ancient Greece to theapplication of logical principles in investigating humanblood within the last 2 centuries.

The 20th

century was a time of industrial standardization,and thus, a period in which established proceduresoperated by humans were perfected in hematology.

Charles A. Spencers Trunnion ModelMicroscope c. 1855


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Introduction

In the 21stcentury, modern computers and networktechniques are being integrated into a complex instrumentlinked to each other mechanically and logically by controlsystems based on algorithms.

This will be illustrated by 4 modern analysis systems.These diagnostic tools are not only of high importance forthe treatment of patients but also for the screening of bloodand blood products.


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Introduction

A state of health is characterized by homeostasis within thecellular components and plasma and a normal relationshipbetween solid and fluid components.

Diseases are characterized by changes in individual blood

parameters.It is therefore of greatest interest to be able to easily andrapidly measure these parameters at anytime with highprecision and accuracy to allow for a precise diagnosis.

Cell-dyn 3700


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History of Blood Testing

Inspection of blood was a basic principle of diagnosis fromancient times till the 16thcentury.

Blood inspection was an investigation of blood bled fromarteries and was called Hemoscopyor Hematoscopy; the

color and structure of the blood was investigated.


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Karl Vierordt(1818-1884)

Quantitative determination of individual cellularcomponents was first enabled in 1852 by the work of KarlVierordt, a physiologist from Tubingen, Germany.

He developed the 1st

counting method, in which a specificblood volume were smeared onto a slide.

The slide was covered with a glass grid and ALL 4.6-5.8million erythrocytes per microliter were counted.


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NeubauerIn 1924, Neubauer published his net structure; this led tothe manual cell counts which are still taken as GOLDSTANDARD in some areas.

Both venous and capillary blood was used as test materialfor the determination of WBC, RBC, and platelets.

Blood was isolated under optimized pre-analyticalconditions; coagulation was prevented with the dipotassiumsalt of ethelynediaminetetraacetic acid. (EDTA:1 mg/ml of

blood)


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Samples were prepared according to fixed procedures andthen counted in the Neubauer Counting Chamber:

The precision and accuracy were highly dependent on thenumber of counted cells, and at a reasonable level of effort,were subject to fluctuations of up to 10%.


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Hemocytometer Counting Chambers:


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Other models aside from the Neubauer CountingChamber:

1. Burker

2. Fuchs-Rosenthal

3. Thoma4. Schilling

5. Turk

These are only used routinely for special tests, such asexamining CSF.

The individual counting chambers differ in the number ofsquares and their marking and separation (course oflines)


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Moldovan Capillary Method

The 1ststep towards automation was made in 1934 withthe Moldovan Capillary Method.

Moldovan described the first instrument that could be

described as a flow cytometer, although the term flowcytometer was not coined until much later.

The instrument consist of a glass capillary tube mounted ona microscope stage.

Initially, Moldovan used narrow tubes but found that cells

tend to block them.When wider tubes were used, cells were not deliveredreproducibly and errors in detection and measurement wereobserved.


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Coulter Model A

The actual breakthrough in the development ofhematological instruments suitable for routine work wasachieved by Wallace Coulter in 1956 with his patent HighSpeed Automatic Blood Cell Counter.

The First Commercial Version of the Coulter Counter


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The Coulter Model A

The Coulter Model A was presented at the U.S. NationalElectronics Conference in Chicago as the FIRST NON-OPTICAL MACHINE for counting blood cells.

Coulter cell counters are based on the principle ofImpedance measurement, and for the first time enabled tocount much higher cell numbers than the known manualmethods.

There was still along way to go for the first semi-automatic

machines for routine work.

Coulter Counter Model F


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The Coulter Principle


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The most important conditions for achieving a goodautomation 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 developedexamination with established reliability and validity asdetermined by repeated evaluation of the method and

results.


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The Road to Standardization...


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The Road to Standardization...


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The International Council for Standardization in Hematology

(ICSH)

With this aim in view, the ICSH was founded in 1963 at thecongress of the European Society for Hematology byWallace Coulter and other leading scientists who worked inspecial committees on the standardization of requirementsfor the methods.


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History of Blood TestingInternational Council for Standardization in Hematology

(ICSH)According to the ICSH, the following are important steps toconsider on the standardization of requirements inautomation:The optimization of the detector, with respect to the pulse

volume and the reliable discrimination between genuineparticles and background electrical noise.Correction of counting errors from spontaneously lysedcells during passage through the capillary opening;optimization of the diluents, avoiding cell distortion.Attainment of constant volume flow in the suspensionpassing in real time through the opening.

Avoidance of particle recirculation after passing throughthe opening.Optimization of the materials for the machine lines;development of reference counting instruments andcalibrators.

These improvements were implemented and have led to thepowerful analytical instruments which are currentlyavailable.


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Modern Automated Systems

What is automation?

The use of a machine designed to follow a predeterminedsequence of individual operations repeatedly andautomatically.

Blood count parameters have been measured automaticallyfor more than 40 years. A variety of fully automatedinstruments are available.


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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 countthat emphasizes the size, shape, special characteristics andnumbers of the solid components of blood.


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Depending on the instrument, it may be possible to analyzecells from body fluids and bone marrow.

All instruments can process the test in either the manual orautomatic mode.

Cell-Dyn RubyAuto Mode


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The 4 Modern Analysis Systems:

Also known as The 4 General Principles of InstrumentOperation (IHFF... I Have Few Friends)

Impedance Measurement


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High Frequency Measurement


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Forward Scatter (FSC)


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Flow Cytometry


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Remember: I Have Few Friends...

I = Impedance Measurement

H = High Frequency Measurement

F = Forward ScatterF = Flow Cytometry


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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 openingbetween two electrodes.

Cells have lower conductivity than the diluent.

The resulting electrical impulse is proportional to the cellvolume.

The sum of the impulses from all cells in a fixedmeasurement volume is evaluated with a histogram.


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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 particlesenter the orifice of a Coulter counter in close succession.These are usually based on the concept of a sensing zonewithin which two or more particles cannot be counted

separately.How was this remedied?

This was remedied through Hydrodynamic Focusing


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Hydrodynamic Focusing

The principle of hydrodynamic focusing was an importantdevelopment in Impedance Measurement.

This almost totally prevented coincidence and gave a clean

Gaussian Curve.

It also improved the separation of small erythrocytes andplatelets.

This measurement is volume based so that no specialcalibration by the user is needed.

Gaussian Curve


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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 sizeand isolates the individual cells.

The cells are then passed through the electric field like astring of pearls and cannot be washed back by turbulencein the area of measurement.


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The Cell-Dyn 3700 employs Impedance Measurement withHydrodynamic Focusing


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High Frequency Measurement provides an analysis of theinternal structures of the cells.

Either a special reagent is added, or the measurements areperformed on leukocytes after completion of erythrocytelysis.

The cells are exposed to a high frequency field, and animpedance measurement is performed at the same time.

Frequency-the number of complete alternations per secondof 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 frommature cells using a special software: codename ACAS, andmuch additional information of the cell is provided.


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ACAS: Adaptive Cluster Analysis System

What do you mean by Cluster Analysis?

Cluster analysisis the task of assigning a set of objects

into groups (called clusters) so that the objects in thesame cluster are more similar (in some sense or another)

to each other than to those in other clusters.Cluster analysisis a main task of explorative data mining,and a common technique for statistical data analysis used

in many fields, including machine learning, patternrecognition, image analysis, information retrieval, andbioinformatics.


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Cluster Analysis

The result of a cluster analysis shown as the coloringof the squares into three clusters.


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ACAS: Adaptive Clustering Analysis System

It is a special software owned by Sysmex that is highlysensitive (detection of as few as 1% blasts).

Its excellent sensitivity results in peace of mind reliability

for the White Blood Cell Differential.


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ACAS: Adaptive Clustering Analysis SystemIt employs fluorescence staining of peripheral blood cells.

The outstanding cluster resolution and separation ofabnormal blood cells by fluorescence staining in the

Sysmex XT-series reduce limitations and potentialinaccuracies known from optical hematology analyzersmeasuring scattered light intensity at different angles only.

With their high nuclear activity, immature or antibodyproducing cells show a much higher fluorescence intensitythan normal cells. They are easily distinguishable in theDIFF scattergram. Atypical lymphocytes are clearlydetected based on their characteristic high fluorescenceintensity.


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ACAS: Adaptive Clustering Analysis System

A DIFF Scattergram employing ACAS


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ACAS: Adaptive Cluster Analysis SystemA surfactant causes complete lysis of red blood cells andplatelets and introduces pores in the membranes of whiteblood cells.

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

In the DIFF channel, the Sysmex XT-series differentiateswhite blood cells into four types and additionallydetermines immature granulocytes. This results in a highlevel of sensitivity and specificity.

The lytic destruction of erythrocytes, which may behave aslyse-resistant in manual or other automated procedures,results in low fluorescence intensity signals from the nucleiresidues. Thus, interference with the leukocyte differentialis prevented.


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Leukocyte quantification and differentiation of basophils areperformed in a separate channel.

In the WBC/Baso channel all cells except for basophils areshrunk under the influence of the specific reagent.

The usually small population of basophils is reliably

detected and counted in this rare-event channel.

WBC/Basoscattergram


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Both the DIFF- and the WBC/Baso channel utilize theSysmex proprietary Adaptive Cluster Analysis System(ACAS) instead of conventionally fixed orfloating discriminators to separate the populations.

In every clinical laboratory a precise leukocyte differentialneeds to be determined even for highly pathological oraged 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.


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The Sysmex XT series employs High Frequency Measurementwith ACAS software


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The measurement is performed with erythrocytes andplatelets transformed to a spherical form (spherederythrocytes) or treated with a surface active diluent tooptimize their shape.

There are 3 preconditions for exact reproducible scatter

signals when measuring and evaluating erythrocytes andplatelets:

1. Formation of isovolumetric spheres or optimization ofisovolumetric spheres

2. A monochromatic light source (pertaining to a singlewavelength of light)

3. Isolation of the blood cells in the measurement cell byhydrodynamic focusing


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The cell preparation is performed in isotonic solutions

Lauryl Sulfate (surfactant) is used to form isovolumetricspheres.

Glutaraldehyde (fixative) is used to fixed the erythrocytes.

The light source is a laser diode with a defined wavelength.


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How are individual cells differentiated and isolated?

This is achieved by differential staining with fluorescentdyes for RNA and DNA.

The cells are not only isolated individually but alsoseparated by the detection of different contents ofDNA/RNA.


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In a normal patient or donor sample, counting lasts forexactly 10 seconds.

During this time, about 50,000 individual erythrocytes andabout 3,000 platelets are counted simultaneously andmorphologically evaluated.


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Measurement Technology of Reticulocyte Analysis

Reticulocytes are determined from EDTA whole blood bybringing a blood aliquot in contact with a specificchromogen, new methylene blue or its derivatives. Such asOxazin 750.


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Measurement Technology of Reticulocyte Analysis

After staining of the so-called reticulo-granular filamentousmaterial (presumably ribosomal RNA), the cell suspensionis 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 thecombination of measurement signals.


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Hemascreen 22: Fully automated hematology analyzer22 parameter 5-part Differential Leukocyte Count

This new measuring system, with Multi-Element-Forward-Scattering (MEFS) laser technology, allow direct measurementof blood cells, with a high level of sensitivity as well asspecificity.


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Flow Cytometry

What is Flow Cytometry?

(Mosbys definition)

A technique in which cells suspended in a fluid flow one at atime through a focus of exciting light, which is scattered inpatterns characteristic to the cells and their components.

The cells are often labeled with fluorescent markers so thatlight is first absorbed and then emitted at alteredfrequencies.

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


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Flow Cytometry

Therefore, Flow Cytometry is a laser-based, biophysicaltechnology employed in cell counting, sorting, biomarkerdetection 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 beingmentioned.

It allows simultaneous multi-parametric analysis of thephysical and characteristics of up to thousands of particlesper second.

It is routinely used in the diagnosis of health disorders,especially blood cancers, but has many other applications inbasic research, clinical practice and clinical trials.


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Flow Cytometry

Principles: (Note how it employs all other principlesmentioned)

A beam of light (usually laser light) of a single wavelengthis directed onto a hydrodynamically focused stream ofliquid.

A number of detectors are aimed at the point where thestream passes through the light beam: one in line with the

light beam (Forward Scatter or FSC) and severalperpendicular to it (Side Scatter or SSC) and one or morefluorescence detectors.


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Flow Cytometry

Principle:


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Flow Cytometry

Principles:

Each suspended particle from 0.2 to 150 micrometerspassing through the beam scatters the ray and fluorescentchemicals found in the particle or attached to the particlemay be excited into emitting light at a longer wavelength

than the light source.


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Flow Cytometry

Principles:

This combination of scattered and fluorescent light is pickedup by the detectors (a.k.a. photomultipliers), and, byanalyzing fluctuations in brightness at each detector (onefor each fluorescent emission peak), it is possible to derive

various types of information about the physical andchemical structure of each individual particle.

Photomultipliers-a device

use in many radiationdetection applications thatconverts low levels of lightinto electrical pulses.


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Flow Cytometry

Principles:


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Flow Cytometry

Some flow cytometers on the market have eliminated theneed for fluorescence and use only light scatter formeasurement.

Other flow cytometers form images of each cellsfluorescence, scattered light, and transmitted light.


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Flow Cytometry

Modern flow cytometers are able to analyze severalthousand particles every second and can actively separateand isolate particles having specified properties. (ClusterAnalysis)

A flow cytometer is similar to a microscope, except thatinstead of producing an image of the cell, flow-cytometryoffers high-throughput automated quantification of setparameters for a large number of cells.


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Flow Cytometry

High-Throughput Automated Quantification of Set Parameters


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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 thatthey pass single filethrough the light beamfor sensing.


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Flow Cytometry

A measuring system

-commonly used are measurement of impedance and opticalsystems

-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 lightsignals


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Flow Cytometry

A Detector and Analogue to Digital Conversion (ADC)system

- Which generates FSC and SSC as well as fluorescencesignals from light into electrical signals that can beprocessed by a computer.


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Flow Cytometry

An amplification system (linear or logarithmic)

A computer for analysis


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Flow Cytometry

Acquisition

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

Acquisition is mediated by a computer physically connectedto the flow cytometer and the software which handles thedigital interface with the cytometer.

The software is capable of adjusting parameters for thesample being tested and also assists in displaying initialsample information while acquiring sample data to insurethat parameters are set correctly.


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Flow Cytometry

Current Flow CytometersModern instruments usually have multiple lasers andfluorescence detectors.

The current record for a commercial instrument is 4 lasers

and 18 fluorescence detectors.

Increasing the number of lasers and detectors allow formultiple antibody labeling, and can more precisely identifya target population by their phenotype markers.

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


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Flow Cytometry


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Conclusion


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Conclusion

The automation of hematological analytical

instruments has reached a high level. Results onthe composition of blood and its cellularcomponents can be provided very rapidly and

more extensively and with better accuracy andprecision 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 willremain indispensable for further development


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Conclusion

One could mention a practical example. For an operationon a 300 g premature baby, it is essential to know theplatelet count. The only available analytical material is acapillary tube with 20 l whole blood. This problem may besolved by returning to platelet counting in the Neubauerchamber a technique which has been regarded a long

time to be apparently antiquated. Counting all squares inthe middle chamber may lead to an acceptable reduction inthe error, which is unacceptable under normal conditions.This underlines that only the interaction between thesophisticated instruments of medical engineering and ourtalent for improvisation give acceptable results inapparently hopeless situations.


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Thank You Very Much

automation in hematology

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