Automation in hematology
Until Slide 23
Hematology Automation
Two General Principles for cell counting :
 Electronic impedance (resistance): called the Coulter Principle.
 Light scattering.
Electronic Resistance (Impedance)
• Blood is diluted in an isotonic electrolyte solution which conduct
electricity very well, while blood cells are non-conductive.
• Cells passing through the aperture will displace its own volume of
isotonic solution and increase the (resistance) because of their nonconductivity between the two electrodes located on each side of the
aperture .
• This electrical resistance is represented by a pulse, each pulse means
a cell, sum of these pulses equals the total cell count.
• Pulse height is directly proportional to the cell size.
Impedance Technology Hematology Counters
 Based on the Coulter Principle
(electrical resistance) principle.
 Blood cells are nonconductive to
electricity,
so when they pass through an electrical
field they will increase the electrical
impedance (resistance).
Electronic Resistance (Impedance)
A sample of blood (EDTA tube) is placed in an analyzer and the cells
are sorted according to size, granularity, and shape by using
Electronic impedance OR Light scattering.
Light scattering
Cells counted as passed through
focused beam of light( LASER).
The amount of light scattered is
proportional to the volume of the cell.
Multi angle scatter separation
indicates
•cell size.
•Cell structure and complexity.
•Nuclear lobularity
Diluted blood sample is aspirated into the counter and divided into 2
portions;
• First part will be enforced toward the RBC chamber in which red
blood cells and platelets are counted and sized
• The second portion will be moved towards the WBC chamber,
where it is diluted with a red blood lysing reagent .so that red
blood cells will not be counted or interfere with white blood cells.
Examples of Haematology analyzers:
Sysmex, Abott ,Beckman Coulter
• 3 parameters differential : Granulocytes,
Lymphocytes and Monocytes.
• 5 Parameters differential :Lymphocytes, Monocytes,
Neutrophils, Eosinophils, and Basophils.
• Nucleated red blood cell counts and immature
granulocytes are emerging as sixth and seventh
parameters
Sysmex xs 1000i
Sysmex Instrumentation
How data are reported
Principles of Measurement
Direct Measurement:
RBC – (Impedance),
WBC – (Impedance),
Platelets – (Impedance), (2-30 fl)
Hgb – SLS-Hb (555 nm) ( sodium lauryl
sulfate)
HCT – cumulative pulse height
detection
Indirect Measurement:
MCV, MCHC, and MCH (calculations)
RDW and MPV (CV of respective
histograms)
WBC differential
Impedance ( CD)/RF detection
employs differential lysis
Flags
RF ( Radio frequency)and DC (Direct
Current) Detection method
• Simultaneous application
of DC and RF produce
information on cell size
and internal composition
WBC differential plot generated by all analyzers
shows a similar cell distribution pattern
Red Cell Histogram
• Represents the relationship between RBCs size
and number
Normal red cell histogram displays cells form (36- 360 ) fl
If RBCs are larger than normal
If RBCs are smaller than normal
If the curve is bimodal
shift to the right
shift to the left
2 population of RBCs
Platelets Histogram
The share of platelets >12 fL in the
total platelet number is presented in %.
The standard range is 15–35%. An
increase of the parameter may be an
indication for :
platelet aggregates, microerythrocytes
and giant platelets.
WBC Histogram
Instrument calibration
• Calibration provides the most accurate results possible.
• For best performance, calibrate all the CBC parameters.
• The WBC differential is calibrated at the factory. They
do not require calibration in the laboratory.
When to Calibrate
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)
• As a routine once or twice a year
(Daily Maintenance)
• Turn on the analyzer. On initial start up, the instrument will perform selfchecks
• If all the self-checks is satisfactory, then the instrument is ready for
analysis.
• If an error message is displayed on the analyzer screen , an alarm will
sound.
The following corrective action will need to be taken before analysis can
go ahead:
• Silence the alarm.
• Press Help to display the error message
• Press OK and the relevant corrective action will be automatically performed
• Run the internal QC after start up procedure is completed.
Weekly maintenance
• Check the quality control chart for evidence of drift.
• Check the daily averages of (MCV, MCH and MCHC) for any drift or
sudden change outside an established 2SD.
• Clean the orifice and cell with a fine brush and flush several times
with diluents. Never attempt to clear the orifice with a sharp device
such as a needle or blade.
•
•
•
•
Check seals to determine the possibility of leakage.
Check tubing.
Check stock of reagents, diluents, and disposables.
In a special logbook record the dates of all maintenance checks,
replacements of components, servicing by manufacturer's agent,
recalibrations, and other necessary information.
•
Problem Solving – Troubleshooting
• An instrument problem is differentiated from a
specimen-related problem by running a control.
• If the control results are acceptable, the problem is
probably specimen-related. Check for:
– clots
– hemolysis
– lipemia
Sysmex xs-1000i - Principles of measurement
Direct measurement :
• RBC, Platelet: Sheath flow , impedance.
• HCT: Cumulative pulse height
• WBC : Platelet: Sheath flow , impedance.
• Hg: Sodium Lauryl Sulfate (555 nm)
Indirect measurement :
• MCV,MCH and MCHC : Calculations
MCV=(PCV/RBC)*10
MCHC=(Hb/PCV%)*100
MCH=(Hb/RBC)*10
• WBC differential: Fluorescent Flow Cytometry.
Throughput Single Sample Mode:
• 60 samples/hour (max.)
• Auto Sampler Mode: 53 semples/ hour (max.)
Sample Volumes : 20μL
Data Storage : 10,000 samples
Histograms
• RBC, PLT, and WBC
plotted on histogram
• X-Axis
– Cell size in
femtoliters (fL)
• Y-Axis
– # of cells