Automation in Haematology

Dr. Bernard Natukunda

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

Semi-automated instruments

Require some steps, as dilution of blood samples Often measure only a small number of variables
Fully automated instruments

Require 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-based

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, also bigger cells found there i.e. monocytes, eosinophils, and neutrophils Statistical sampling error

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

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

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 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 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 laboratorys review rate changes by 1% (= 2,000 differentials a year), you pay or save almost $ 10,000

Initial 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 supply Photometric voltage Permit instrument to stabilize/equilibrate Let all components reach proper temperature Set in any parameters that may be required Ranges and temperatures

Calibration
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.

Calibration
You 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 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

Check the reagent containers for:

Sufficient quantity Not beyond expiration date No precipitates, turbidity, particulate matter, or unusual colour Proper connections between the instrument and the reagent containers

Ensure the Instrument is Functioning Properly

Check the waste container for: Sufficient capacity Proper connections Perform daily startup In addition to verifying daily startup results, verify acceptable: Reproducibility Carryover Control Results

Quality control
Purpose of Quality Control (QC)

Assures proper functionality of instrumentation Means of assuring accuracy of unknowns Monitoring the Integrity of the Calibration
- When controls begin to show evidence of unusual trends - When controls exceed the manufacturers defined acceptable limits

Data 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

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

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

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 IV

Histograms showing the size distribution of white cells, red cells and platelets.
Sizing is based on impedance technology.

Optical method

Laser light is used A diluted blood specimen passes in a steady stream through which a beam of laser light is focused As each cell passes through the sensing zone of the flow cell, it scatters the focused light Scattered light is detected by a photodetector and converted to an electric impulse The number of impulses generated is directly proportional to the number of cells passing through the sensing zone in a specific period of time

Optical 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 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 size 10 : indicator of cell structure and complexity 90 polarized: indicates nuclear lobularity 90 depolarized: differentiates eosinophils

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

Trends

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 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 measurements

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

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

Haemoglobin 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 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 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

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 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)

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 count

The total WBC count is determined in whole blood in

which RBCs have been lysed.

Fully automated multichannel instruments perform WBC

counting by
Impedance Light scattering Or both.

Automated 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 counting

3-part differential

usually cont

Granulocytes or large cells Lymphocytes or small cells Monocytes(mononuclear cells) or (middle cells)
5-part classify cells to

Neutrophils Eosinophils Basophils Lymphocytes Monocytes

Differential cell counting

A sixth category designated large unstained cells include cells

larger than normal and lack the peroxidase activity this include
Atypical lymphocytes Various other abnormal cells
Other counters identify 7 categories including

Large immature cells (composed of blasts and immature granulocytes) Atypical lymphocytes (including blast cells)

Differential cell counting

Analysis

may be dependant on:

Volume of the cell Other physical characteristics of the cells Sometimes the activity of cellular enzymes such as peroxidase
Technologies used

Light scattering and absorbance Impedance measurement

Automated differential

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

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 samples Flagging of abnormal samples

Platelet 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 include

MPV PDW Plateletcrit = MPV x platelet count.

Reticulocyte count
An 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

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

Flow Cytometry (forward light scatter, side light scatter,

side fluorescence) for: WBC differential, NRBC, reticulocytes, optical platelets

Radio frequency (RF) and direct current (DC) resistance

for: Immature granulocytes

HEMOGLOBIN MEASUREMENT
Red cells are lysed, hemoglobin is converted into sodium lauryl sulfate (SLS)-methemoglobin: Short reaction time
Absorbance at 555 nm is measured, and concentration of hemoglobin is calculated Good correlation with reference method

RED CELL AND PLATELET COUNTS

Red cells and platelets are both counted by the electric

impedance method.
In samples with large platelets or small RBCs or RBC

fragments, platelet counts by the light scattering method are used instead.

WBC/BASO CHANNEL
RBCs are lysed, degranulation of basophils is selectively suppressed Forward and side scatter are used to obtain a WBC and basophil count

4-DIFFERENTIAL CHANNEL
Red cells are lysed, DNA and RNA of WBCs are stained with fluorescent dye Side fluorescence and side scatter are used to obtain a four-part differential

IMMATURE 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 CHANNEL

Red cells are lysed, a lysing agent which disrupts the cell membranes of MATURE WBCs only is used. Analysis by radiofrequency and direct current impedance detection

IMMATURE 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 DIFF AND THE IMI CHANNEL