2. 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
3. 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.
5. 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
6. 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
7. 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
8. 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
9. 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
10. 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 laboratory’s review rate changes by 1% (= 2,000
differentials a year), you pay or save almost $ 10,000
11. 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
12. 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.
13. 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
14. Daily maintenance
Daily cleaning
Background counts
Electronic checks
Compare open and closed mode sampling (using a
normal patient sample)
Run controls
15. 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
16. 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
17. 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 manufacturer’s defined acceptable limits
18. 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.
19. 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
20. 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
21. 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.
22.
23. Histograms of Coulter S Plus IV
Histograms showing the size
distribution of white cells, red
cells and platelets.
Sizing is based on impedance
technology.
24. 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
25. 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
26. 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
27.
28. 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
29. 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
32. 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 non-
toxic material Na- lauryl sulphate.
33. 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
34. 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.
35.
36. 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.
37. 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.
38. 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.
39. 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.
40.
41. 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.
42. 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
43. 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)
44. 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.
45. 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
46. 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.
47.
48. 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.
50. 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
51. 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
52. 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
53. 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.
54. 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
55. 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
56. 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.
57. 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
58. 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
