automation in haematology bernard
TRANSCRIPT
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