automation in haematology bernard

Automation in Haematology

Automation in Haematology

Dr. Bernard Natukunda

Why should you use automation?Why should you use automation?

High volume: The Core Laboratory may report well over 450,000 CBCs and 200,000 differentials a year

Many parameters measured

Wide range of clinical applications

Automated techniques of blood counting

Automated techniques of blood counting

Semi-automated instrumentsRequire some steps, as dilution of blood samplesOften measure only a small number of variables

Fully automated instrumentsRequire only that an appropriate blood sample is presented to

the instrument.They can measure 8-20 variables including some new

parameters which do not have any equivalent in manual methods.

Fully automated Morphology-basedFully automated Morphology-based

Disadvantages of manual cell counting

Disadvantages of manual cell counting

Cell identification errors in manual counting:– mostly associated with distinguishing lymphocytes from monocytes; bands from segmented forms and abnormal cells (variant lymphocytes from blasts)– lymphocytes overestimated, monocytes underestimated

Slide cell distribution error:– increased cell concentration along edges, alsobigger cells found there i.e. monocytes, eosinophils, and neutrophils

Statistical sampling error

Advantages of automated cell counters

Advantages of automated cell counters

They have a high level of precision for cell counting and cell sizing greatly superior to that of the manual technology

The results are generally accurate

Aberrant results consequent on unusual characteristics of blood are “flagged” for subsequent review

Advantages of automated cell counters

Advantages of automated cell counters

Objective (no inter-observer variability) No slide distribution error Eliminate statistical variations associated with manual count based

on high number of cells counted Many parameters not available from a manual count, e.g. MCV,

and RDW More efficient and cost effective than manual method:

– Some cell counters can process 120-150 samples per hour– CAP assumes 11 minutes for a manual cell count

Automated cell counters can provide

Automated cell counters can provide

CBC: WBC, RBC, Hgb, Hct, PLT, RBC indices WBC Differential: 5 “normal” white cell types RDW, PDW, MPV Reticulocyte count Nucleated red cell count

All automated cell counters are screening devices. Abnormalities must be verified by a blood film, staining and scanning by an expert observer

Review processReview process

Whenever the automated cell counter flags a specimen, the technologist (or an automated system) has to• Retrieve the tube

• Make a slide

• Stain the slide

• Review the slide

• Either release the results from the cell counter (“scan”) or perform a manual differential

Importance of the review rateImportance of the review rate

If it takes a technologist 10 minutes to prepare, label, stain, review, count, and release results from a slide,and

If a technologist costs $ 25.00 per hour; it will cost $ 4.00 to make a slide.

If the laboratory’s review rate changes by 1% (= 2,000 differentials a year), you pay or save almost $ 10,000

Initial set up of instrumentInitial set up of instrument Check instrument for visual damage Check for any loose parts or connections Make sure all computer boards are properly sealed Check the socket to verify proper voltage outlet Plug power cord into (voltage stabilizer) electrical supply Confirm the correct voltage on instrument

Main power supplyPhotometric voltage

Permit instrument to stabilize/equilibrateLet all components reach proper temperature

Set in any parameters that may be requiredRanges and temperatures

CalibrationCalibration Calibration fine tunes your haematology analyzer and

provides the most accurate results possible. In the normal process of tracking data for an extended

period of time, your laboratory can make a specific decision to recalibrate a given parameter. Never adjust to a specific value for an individual sample.

For best performance, calibrate all the CBC parameters. The WBC differential is calibrated at the factory. They do not require calibration in the laboratory.

CalibrationCalibrationYou should calibrate your instrument:

At installation

After the replacement of any component that involves dilution characteristics or the primary measurements (such as the apertures)

When advised to do so by your service representative

Daily maintenanceDaily maintenance

Daily cleaning Background counts Electronic checks Compare open and closed mode sampling (using a

normal patient sample) Run controls

Ensure the Instrument is Functioning Properly

Ensure the Instrument is Functioning Properly

Check the reagent containers for:Sufficient quantityNot beyond expiration dateNo precipitates, turbidity, particulate matter, or unusual colourProper connections between the instrument and the reagent containers

Ensure the Instrument is Functioning Properly

Ensure the Instrument is Functioning Properly

Check the waste container for:Sufficient capacity Proper connections

Perform daily startupIn addition to verifying daily startup results, verifyacceptable:

ReproducibilityCarryoverControl Results

Quality controlQuality control

Purpose of Quality Control (QC)Assures proper functionality of instrumentationMeans of assuring accuracy of unknownsMonitoring the Integrity of the Calibration- When controls begin to show evidence of unusual trends- When controls exceed the manufacturer’s defined acceptable limits

Data reviewData review

A review of instrument data, such as background, control, and blood sample results, is helpful in detecting problems.

Sometimes a questionable blood sample result is the only symptom of subtle reagent or pneumatic problems.

Principles of automated cell counters

Principles of automated cell counters

Impedance (conductivity) system (Coulter) Optical system (H*1) Both impedance and optical Selective lysis (e.g. lysis of red cells and counting of

white cells) Special stains

Electrical impedance method The “Coulter Principle”

Electrical impedance method The “Coulter Principle”

Cell counting and sizing is based on the detection and measurement of changes in electrical impedance (resistance) produced by a particle as it passes through a small aperture

Particles such as blood cells are nonconductive but are suspended in an electrically conductive diluent

As a dilute suspension of cells is drawn through the aperture, the passage of each individual cell momentarily increases the impedance (resistance) of the electrical path between two electrodes that are located on each side of the aperture

Diagram illustrating the Coulter Principle

Diagram illustrating the Coulter Principle

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 of Coulter S Plus IVHistograms of Coulter S Plus IV

Histograms showing the size distribution of white cells, red cells and platelets.

Sizing is based on impedance technology.

Optical methodOptical method Laser light is used

• A diluted blood specimen passes in a steady streamthrough which a beam of laser light is focused• As each cell passes through the sensing zone of the flowcell, it scatters the focused light• Scattered light is detected by a photodetector andconverted to an electric impulse• The number of impulses generated is directly proportionalto the number of cells passing through the sensing zone ina specific period of time

Optical methodOptical method The application of light scatter means that as a single cell passes

across a laser light beam, the light will be reflected and scattered.

The patterns of scatter are measured at various angles.

Scattered light provides information about cell structure, shape, and reflectivity.

These characteristics can be used to differentiate the various types of blood cells and to produce scatter plots with a five-part differential

Light scatteringLight scattering Cells counted as passed through focused beam of light (LASER)

Sum of diffraction(bending around corners), refraction (bending due to change in speed) and reflection (light rays turned back by obstruction)

Multi angle polarized scatter separation (M.A.P.S.S)

0° : indicator of cell size10° : indicator of cell structure and complexity90° polarized: indicates nuclear lobularity90° depolarized: differentiates eosinophils

Types of Haematology Analyzers

Types of Haematology Analyzers

Smaller instruments: Measure WBCs, RBCs, Hgb, Hct, MCV, MCH, MCHC, and PLTs)

Advanced cell counters: Add:– Red cell morphology information, RDW– Mean platelet volume– Leukocyte differential

TrendsTrends Current trends include attempts to incorporate as many analysis

parameters as possible into one instrument platform, in order to minimize the need to run a single sample on multiple instruments (e.g CD4 counts, smear preparation)

Such instruments are being incorporated into highly automated combined chemistry/haematology laboratories, where samples are automatically sorted, aliquoted, and brought to the appropriate instrument by a robotic track system

Haematology AnalyzersHaematology Analyzers

Abbot (http:www//abbott.com):– Cell-Dyn

Siemens [Bayer] (http:www//bayerdiag.com):– Advia

Beckman-Coulter (http:www//beckmancoulter.com):– STKS – Gen-S

Sysmex (http:www//Sysmex.com):– SE

Laboratory measurementsLaboratory measurements

Hb concentrationHb concentration

• Hb is measured automatically by a modification of the manual (HiCN) method.

• To reduce toxicity of HiCN some systems replace it by a non-toxic material Na- lauryl sulphate.

Haemoglobin measurementHaemoglobin measurement

Sample is diluted with Cyanmethemoglobin reagent• Potassium ferricyanide in the reagent converts the hemoglobin iron from the ferrous state (Fe++) to the ferric state (Fe+++) to form methemoglobin, which then combines with potassium cyanide to form the stable cyanmethemoglobin

A photodetector reads color intensity at 546 nm

Optical density of the solution is proportional to the concentration of haemoglobin

RBC countRBC countThe 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 an electronic counting for RBCs is much better than the manual count, and it is available in a fraction of time.

This made the use of RBC indices of more clinical relevance.

Reliability of electronic counters

Reliability of electronic counters

They are precise but care should be taken so that they are also accurate.

Some problems which could be faced:Two cells passing through the orifice at the same time, counted as one cell.RBC agglutination(clump of cells)Counting bubbles or other particles as cells.

PCV and red cell indicesPCV and red cell indices

Pulse height analysis allows either the PCV or the MCV to be determined.

MCV=PCV/RBC count MCH= Hb/RBC count MCHC= Hb/PCV MCH & MCHC are derived parameters.

Red cell distribution width (RDW)Red cell distribution width (RDW) Automated instruments produce volume distribution

histograms which allow the presence of more than one population of cells to be appreciated.

Most instruments produce a quantitative measurement of variation in cell volume, an equivalent of the microscopic assessment of the degree of anisocytosis. This is known as the RDW.

Total WBC countTotal WBC count

The total WBC count is determined in whole blood in which RBCs have been lysed.

Fully automated multichannel instruments perform WBC counting by

ImpedanceLight scatteringOr both.

Automated differential countAutomated differential count

Automated differential counters which are available now generally use flow cytometry incorporated into a full blood counter rather than being standard alone differential counters

Automated counters provide a three-part or five- to seven-part differential count.

Differential cell countingDifferential cell counting 3-part differential usually cont

Granulocytes or large cellsLymphocytes or small cellsMonocytes(mononuclear cells) or (middle cells)

5-part classify cells toNeutrophilsEosinophilsBasophilsLymphocytes Monocytes

Differential cell countingDifferential cell counting

A sixth category designated “large unstained cells” include cells larger than normal and lack the peroxidase activity this include

Atypical lymphocytesVarious other abnormal cells

Other counters identify 7 categories includingLarge immature cells (composed of blasts and immature granulocytes)Atypical lymphocytes (including blast cells)

Differential cell countingDifferential cell countingAnalysis may be dependant on:

Volume of the cellOther physical characteristics of the cellsSometimes the activity of cellular enzymes such as peroxidase

Technologies usedLight scattering and absorbanceImpedance measurement

Automated differential counters employing flow cytometry classify far more cells than is possible with a manual differential count.

Accuracy in blood cell counting

Accuracy in blood cell counting

The accuracy of automated counters is less impressive than their precision.

In general automated differential counters are favourable to the manual in 2 conditions

Examination of normal blood samplesFlagging of abnormal samples

Platelet countPlatelet count

Platelets can be counted in whole blood using the same technologies of electrical or electro-optical detection as are employed for RBCs.

Other parameters includeMPVPDWPlateletcrit = MPV x platelet count.

Reticulocyte countReticulocyte countAn automated reticulocyte 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 flowcytometer.

An automated reticulocyte counter also permits the assessment of reticulocyte maturity since the more immature reticulocytes have more RNAfluoresce more strongly than the mature ones normally found in peripheral blood.

The Sysmex XE-2100 The Sysmex XE-2100

The Sysmex XE-2100 The Sysmex XE-2100 • Uses both impedance and flow cytometry• Throughput: 150 samples/hour• Provides 32 parameters, including reticulocyte and NRBC

counts• Uses an optical fluorescent platelet count when the

impedance count may be unreliable• Unique features:

– Can enumerate, not just flag for, immature granulocytes (metamyelocytes, myelocytes, promyelocytes)– Immature platelet fraction

The Sysmex XE-2100 The Sysmex XE-2100

Flow Cytometry (forward light scatter, side light scatter, side fluorescence) for: WBC differential, NRBC, reticulocytes, optical platelets

Radio frequency (RF) and direct current (DC) resistancefor: Immature granulocytes

HEMOGLOBIN MEASUREMENT

• Red cells are lysed, hemoglobin is converted intosodium lauryl sulfate (SLS)-methemoglobin:– Short reaction time

• Absorbance at 555 nm is measured, and concentrationof hemoglobin is calculated– Good correlation with reference method

RED CELL AND PLATELET COUNTS

Red cells and platelets are both counted by the electricimpedance method.

In samples with large platelets or small RBCs or RBCfragments, platelet counts by the light scatteringmethod are used instead.

WBC/BASO CHANNEL

• RBCs are lysed,degranulation ofbasophils isselectivelysuppressed

• Forward and sidescatter are used toobtain a WBC and basophilcount

4-DIFFERENTIAL CHANNEL4-DIFFERENTIAL CHANNEL

• Red cells are lysed,DNA and RNA ofWBCs are stainedwith fluorescent dye• Side fluorescenceand side scatter areused to obtain afour-part differential

IMMATURE GRANULOCYTE COUNTIMMATURE GRANULOCYTE COUNT

Includes promyelocytes, metamyelocytes, myelocytes,but NOT bands or blasts

A lysing reagent causes disruption of mature WBC membranes, while immature myeloid cells with low membrane lipid content remain intact.

IMI CHANNELIMI CHANNEL Red cells are lysed,

a lysing agent whichdisrupts the cellmembranes ofMATURE WBCs only is used.• Analysis by radiofrequency and direct currentimpedance detection

IMMATURE GRANULOCYTE COUNTIMMATURE GRANULOCYTE COUNT

• Correlation coefficient with visual count: 0.81

• Prediction of infection:– At 90% specificity, sensitivity of 35 - 40%– At 90% sensitivity, specificity of 20%– Better predictor of sepsis than WBC; comparable to ANC

ABNORMAL CELLS IN THE DIFFAND THE IMI CHANNEL

ABNORMAL CELLS IN THE DIFFAND THE IMI CHANNEL