automated hematology analyser for labora

Automated blood cell counters
Principle, Uses ,Advantages,
Disadvantages ,Quality control

What do Automated Analyzers Perform?
• Counting of WBCs, RBCs and Platelets
• Measurement of Hemoglobin
• Calculation of Hematological Indices (Absolute
Values)
• Some can perform Differential counts (3 part and
5 part)
• Some can also indicate abnormalities of RBCs,
Platelets and WBCs (Flags)

Advantages
• Automation provides both greater accuracy and
greater precision than manual method.
• These analyzers typically provide the eight standard
hematology parameters (complete blood count
[CBC] plus a three-part or five part differential
leucocyte counts in less than one minute on 100µl
of whole blood.
• Automation thus allows for more efficient workload
management and more timely diagnosis and
treatment of disease.

Disadvantages
• Limitations in the methodology
• Many systems are impractical for small numbers of
Samples
• Back-up procedures must be available in case of
instrument failures
• Automated systems are expensive to purchase and
Maintain
Regular maintenance requires technicians and service
engineers
• Tests that are not required are also run

CELL-COUNTING TECHNOLOGIES
1) Electrical-Impedance Cell Counting
Blood cell counting and sizing uses low -voltage
DC current (resistance to cell volume)

external
electrode
internal
electrode
aperture
vacuum
Impedance..
The number of pulses = blood cell count
The amplitude (height) of the pulse = Volume of cell
The poorly conductive blood cells
are suspended in a conductive
diluent which passes through an
electric field created between two
electrodes.

Computing and generation of graph
• Each pulse is recorded
as an oscillation , the
height of which is
proportional to the
volume and size of the
cell
• These oscillations are
rearranged according to
volume interval to form
a histogram.

2) Radiofrequency conductivity
High-voltage electromagnetic current (AC) short -
circuits the bipolar lipid layer of a cell ’s membrane,
allowing the energy to penetrate the cell.
This enables the collection of information
proportional to cell size and internal structure,
including chemical and physical composition and
nuclear volume

3) Optical Light-Scatter Cell Counting
• A laser beam (monochromatic)or tungsten-
halogen light beam is directed at a stream of blood
cells passing through a narrow channel
• When the light beam strikes a cell, the beam is
scattered at an angle.
• The speed and angle of scatter of the beam of light
(index of refraction of the cell)differ according to the
cell type and is influenced by the shape and volume
of the cell

Optical Light-Scatter Cell Counting
Sensors detect how much light is scattered and how much of the beam is
absorbed by the cell
Combination of electrical impedance and light-scatter

4) Optical absorbance
• Based on a cytochemical reaction using the
intracellular myeloperoxidase enzyme of the
leukocytes
• Absorbance of white light from a tungsten light source
is a measure that is proportional to the intensity of
the peroxidase reaction
• Neutrophils, monocytes, and eosinophils are
peroxidase positive
• Lymphocytes and basophils are peroxidase negative

5) Fluorescence
Fluorescent dyes stain cell membrane and
intracellular structures
As they pass through the sensing zone they emit
different wavelengths of fluorescent light
Immunophenotype (cell surface antigens using
labeled antibodies), DNA (nucleated red cells), and
RNA (reticulocytes) content, and other cellular
characteristics

automated hematology analyser for labora

88 µl sample volume
Individual proportions for
each application

Overview
One system. Three different measurement techniques
• Sheath Fluid DC (Direct Current) Detection Method :
Impedance based counting of RBCs and PLTs.
Hydrodynamic focusing of RBCs and PLTs using a sheath fluid.
Includes cumulative pulse height detection for HCT measurement.
• SLS haemoglobin method
Cyanide free, photometric HGB measurement
• Fluorescence flow cytometry
Separation and counting of various WBC and nucleated blood cells.

SLS Haemoglobin Method
• Lysis of RBC and other cells by SLS (Sodium lauryl
sulfate )
• Sodium lauryl sulfate is used as a deteregent
• RBCs are lysed to release haemoglobin
• Fe 2+ is oxidized to a trivalent Fe 3+ state
• Hydrophilic group of SLS interacts with Fe³
• A stable reaction product is formed
• Photochemical properties are used for photometric
measurement at 555 nm

Sheath Flow DC (Direct Current) Detection
Method
Measurement of RBCs and PLTs on the
RBC/PLT channel
• 4 µl of whole blood diluted with Cellpack and
ejected from nozzle tip.
• Cells undergo hydrodynamic focusing.
• A sheath fluid promotes the formation of a
stream of
individual cells.

• All blood cells pass through
the center of the aperture
• Across the aperture an
electrical current is applied
• Slight resistance changes are
caused, which are seen as
electrical pulses
• The pulse height accurately
reflects the blood cell volume

• Cell-specific resistance signals are sensitively
detected.
• Data are translated/visualized into histograms.
• Cell number and cell size are reflected.
• RBC count and PLT count are provided.

• Result output - RBC histogram
• Histogram reflects number and cell size (cell
volume) of RBCs.
• Gaussian normal distribution.
• Lower (LD) and upper discriminator (UD)
indicate limits.

Result output. RBC and PLT histograms

Coulter principle of cell counting based on
Electronic impedance
• Two electrodes are placed on each side of the aperture
• Electric current passes through the electrodes continuously.
• The poorly conductive blood cells are suspended in a conductive diluent
(liquid).
• The diluent is passed through an electric field created between two
electrodes.
• The liquid passes through a small aperture (hole).
• The passage of each particle through the aperture momentarily increases
the impedance (resistance) of the electrical path between the electrodes.
• The increase in impedance creates a pulse that can be measured.
• The number of pulses = blood cell count
• The amplitude (height) of the pulse = Volume of cell

Possible causes of Curve not
starting at baseline :
Platelet clumps
Giant platelets
RBC fragments
Microcytes

The RDW is a quantitative measure of how
variable the size of the individual red cells is
(anisocytosis)
RDW-SD and RDW-CV

Multiple peak of RBC Histogram

Calculation of RBC indices
Parameter Meaning Calculation Unit
MCV
mean corpuscular
volume
Size
or volume of one
average RBC
= HCT x 10
RBC
fl
MCH
mean corpuscular
haemoglobin
Haemoglobin
content in
one average RBC
= Hb x 10
RBC pg
MCHC
mean corpuscular
HGB concentration
Average
haemoglobin
concentration
within
RBCs
= Hb × 100
HCT g/dl

• Hematocrit is determined by Pulse height
determination method

Platelet Parameters
• MPV –Mean Platelet Volume
»MPV = PCT[%]/ PLT count
»Reference range: 8 –12 fl
»Besides P-LCR, the MPV might indicate increased
platelet production.
»Parameter used for the control of platelet
generation
• Platelet distribution width (PDW) measures
volume variability in platelet size

Platelet Parameters
• P-LCR –Platelet Large Cell Ratio
»Indicates platelets larger than 12 fl
»Reference range: 15 –35 fl
»High P-LCR values might implicate and increase in
immature platelets.
»In addition, changes in the P-LCR can be a sign for
‒PLT clumps
‒Giant PLT
‒Microerythrocytosis

Three-Part Differential count
3 distinct size classifications using Electrical Impedance

Five-Part Differentials
• VCS (Volume, Conductivity and light Scatter) technology -Beckman
Coulter series
o Volume (V): Measured using electrical impedance
o Conductivity (C): Analyzes the ability of a cell to conduct an
electric current, revealing internal structures like the nucleus and
cytoplasm.
o Scatter (S): Measures the intensity of light scattered by a cell at
different angles, reflecting cell surface variations
• Multi-angle polarized light-scatter separation (MAPSS):Abbott
Diagnostics markets the Cell-Dyn series-light-scatter pattern
measured from four specific angles

Fluorescence Flow Cytometry
• Fluorescence is the emission of
light by a substance that has
absorbed light.
• Sub-cellular structures are
specifically labeled with
fluorescent dyes.
• Cells are irradiated with a
beam of a semiconductor laser.
• Cells emit light according to
the fluorescent marker.

Principle ： Flow Cytometry
WNR, WDF, WPC, RET, PLT-F channel
SSC provides information on cell complexity
and intracellular structures
Three dimensional signal detection

Three dimensional signal detection
Emitted light is analysed according to three different
dimensions:
• Forward Scattered Light (FSC) provides information on
cell size
• Side Scattered Light (SSC) provides information about
the internal cell structure and its content, such as
nucleus and granules
• Side Fluorescent Light (SFL) reflects type and amount
of nucleic acids present in the cell

WNR channel
• Lysercell WNR:
‒Acidic lysis and perforation reagent.
‒Causes hemolysis of the red blood cells.
‒Perforates the cell membrane of the white blood cells,
‒Causes a reduction of the cell size of white blood cells (except basophils).
‒WBCs (except basophils) will form a single population.
‒NRBCs are degraded, except their nucleus
• Fluorocell WNR:
- Penetrates the cell membrane
‒Marks nucleic acids & cell organelles of
White blood cells and Nucleated red blood cells.

Reagent reactions ： WNR channel
Reagent:
・ Lysercell WNR
・ Fluorocell WNR
WNR Scattergram
SFL
FSC
BASO
WBC
NRBC
Debris
Parameters:
WBC, NRBC, BASO
Flag:
PLT Clumps?, NRBC Present,
WBC Abnormal Scattergram

WDF Channel
• Lysercell WDF:
‒Lysercell WDF causes haemolysis of the red blood cells and platelets.
‒It perforates the cell membrane of the WBCs, depending on the cell
characteristics of each white blood cell
• Fluorocell WDF:
‒Labeling of nucleic acids & cell organelles of
‒White blood cells and
‒other nucleated cells.
‒Fluorescence intensity varies among
‒Different types of WBCs.
‒Types and amount of nucleic acids.

Reagent:
・ Lysercell WDF
・ Fluorocell WDF
WDF Scattergram
SSC
SFL
EO
NEUT
LYMPH
Debris
MONO IG
Blast
Abn_Lymph
Parameters:
NEUT, LYMPH, MONO, EO, IG
Flag:
Atypical lymph?, Blast/Abn Lymph?,
IG present, PLT Clumps?,
WBC Abnormal Scattergram
Reagent reactions ： WDF channel
Atypical lymph

Ningombam A, Acharya S, Sarkar A, Kumar K, Chopra A. Scattergram
patterns of hematological malignancies on sysmex XN-series analyzer. J
Appl Hematol 2021;12:83-9

Daily Start-Up Procedures*
• Daily cleaning
• Background counts
• Electronic checks
• Check calibration
• Run controls
• Compare open and closed mode
sampling (use a normal patient sample)
* Check instrument manual for
procedures
Must be
within
specified limits

Calibration
Calibration is required :
• at installation and every 12 months
• when control materials reflect an unusual trend or
shift, or are outside of the laboratory’s acceptable
limits, and other means of assessing and correcting
unacceptable control values fail to identify and correct
the problem.
• When service troubleshooting and/or parts
replacement has prompted a significant change in
range of control values

Quality Control Methods
• stabilised material
(Commercial)
• Patient replicates
• Delta checks

Stabilised Material
• Commercially available
• Known values (assayed only)
• Can be run over time
• Analyse low, normal and high control
• Results stored in the instrument computer
Monitored with Levey-Jennings graphs
– Easily illustrates trends and shifts

QC Method: Patient Replicates
• Previously analysed patient sample
• Easily obtained
• Cost effective
• Results and samples readily available

QC Method: Delta Checks
• Compare a patient’s own leukocyte, haemoglobin, MCV, and
platelet values with previous results
– If difference between the two is greater than
laboratory-set limits, current result is flagged for
review

• Accuracy
– Value obtained is the closest to the correct value or the
true value
• Precision
– Relates to reproducibility or how close the test results are
to one another when performed repeatedly
Cont’d.,

Lab 1 Lab 2 Lab 3 Lab 4
10.0 9.0 10.3 10.0
9.0 9.0 10.0 10.0
11.2 9.0 9.6 10.1
11.1 9.0 10.0 10.0
9.3 9.0 10.3 10.1
8.9 9.0 10.4 10.0
9.1 9.0 9.8 9.9
Sample with Hb 10 g/dl

10.0
9.0
11.2
11.1
9.3
8.9
9.1
9.0
9.0
9.0
9.0
9.0
9.0
9.0
10.3
10.0
9.6
10.0
10.3
10.4
9.8
10.0
10.0
10.1
10.0
10.1
10.0
9.9
Accuracy and Precision

Quality Control
• Purpose of QC
– Assures proper functionality of instrumentation
– Means of assuring accuracy of unknowns
– Monitors the integrity of the calibration
• When controls begin to show evidence of unusual
trends
• When controls exceed the vendor’s defined acceptable
limits

• A single measurement on the
same sample repeated each day
for a period 20–30 days
• Convenient to use QC material
Precision (Between Batch, Long Term)

Mean = average of data =
65
Sum of all data divided by the total number
of data points
= (X1+X2+X3+….XN)/N
Example:
8+9+7+7+9+8 =48 (Sum)
= Sum/number of data points = 48/6 =8
MEAN = 8

Standard Deviation (SD)
66
Standard Deviation (SD) = is a measure of how
much the data varies around the MEAN
SD =

Coefficient of Variation (CV)
67
CV is SD expressed as a proportion of the mean
CV = (SD / Mean) x 100
CV is expressed as a percent (%)

Defining QC ranges
69
• QC range limits are defined by SD values
• Typically an acceptable range is established
using +/- 2 Standard Deviations (SD) around
the MEAN
• Statistically this covers 95% of the expected
values

Standard deviation
 Measure of the variability in a data
set (precision measurement )
 It is the square root of the variance
The First SD
 Represents 68% of the values that
will cluster above or below the
mean
The Second SD
 Represents 95.5% of values falling
above or below the mean
The Third SD
 Represents 99.7% of values above or
below the mean
Gaussian Curve
Quality Control Terms - Cont’d.,

71
Things that increase your CVH
–Day to day instrument differences
–Electrical and power quality
–Different persons operating the instrument
–QC material preparation
– Reagent Quality

SMILE 72
Increasing CV
Jan 2.1
Feb 2.3
Mar 2.2
Apr 2.4
May 2.5
Jun 2.8
Jul 2.9
Aug 3.1
Sep 3.2
Oct 3.4
Nov 2.5
Dec 2.3
CVH 2.64
1 2 3 4 5 6 7 8 9 10 11 12
0
0.5
1
1.5
2
2.5
3
3.5
4
12 month plot with
increasing CVH
%CV
h
Month
Monitor CVH to alert for problems

Levey-Jennings Chart (QC Chart)

Shift Trend
Shift and Trend
• Change of reagent lot
• Major instrument
maintenance
• Faulty Calibration
• Malfunction of analyser
• Gradual accumulation of
debris in sample/reagent
tubing
• Gradual deterioration of
control materials
• Gradual deterioration of
reagent

When a Control is Outside its Expected
Range
Ensure the control
– Material was mixed and warmed properly
• If not, mix it according to the package insert
– Identification information was entered correctly
• If using the numeric keypad, verify you typed the
correct information
– Setup information (assigned values and expected
ranges) matches the control package insert for
the current lot number being used
• If they do not match, change the control’s
information to match the package insert
1
￫

When a Control is Outside its Expected
Range
If any of the problems existed, rerun the
control; otherwise, proceed to the next step
2

What should be done if QC Results are
Unacceptable?
Verify instrument functioning
Check for shifts and trends
Troubleshoot
Repeat the assay

Troubleshoot “OUT OF
CONTROL” VALUES

Correct procedure for running QC
• Remove the vial from
the refrigerator and
equilibrate with room
temperature (15 – 30
°C) for at least 15
minutes before use.
• Roll each vial between
the palms of your hands
for 15 seconds

• Holding the vial from end
to end between the thumb
and forefinger, invert the
vial 20 times
• Analyse the QC material on
the instrument as per SOP
• Subsequent analyses
during this test period may
be performed by inverting
the vial 5 times prior to
instrument analysis.
• Return the vial to the
refrigerator (2 – 8 °C) for
storage

Rule Criteria
Error
type
Action
12s One control measurement exceeding 2 SD of control limits
either above or below the mean
RE/SE Warning
13s Single control measurement exceed the 3 SD of control
limits either above or below the mean
RE Rejection
22s
2 consecutive control measurements exceed 2 SD of control
limits on the same side of mean (within run/across run)
SE Rejection
R4s One control level above 2 SD and another control level below
2 SD in the same run
RE Rejection
41s
4th consecutive control measurement exceeding 1 SD on the
same side of the mean(within run/across run) SE Rejection
10x
10 consecutive measurements on the same side of the mean
(within run/across run) SE Rejection
Westgard Multirule

• 41s rule detects small systematic error; very few
applications
• 10x rule Detects very small errors
• Preferred Rules - 13s , 22s , R4s in hematology
Westgard Rule Application in Cell Counters

• External quality control or external assessment scheme (EQAS)
or Proficiency Testing program (PT)
• Process of controlling the accuracy of an analytical method by
inter-laboratory comparisons
• Objective evaluation by an external agency (international/
national / regional basis)
• Samples are sent to the laboratory from a provider at specified
time intervals. They are tested along with patient samples
Proficiency Testing Program (EQA)

• The results are returned to the provider and the provider determines if the
results are acceptable
• The EQAS coordinator gathers all the results and groups them (peer groups)
according to the laboratories analytical methods, analyzers or any other
criteria
• Target value (consensus mean) and its total variation (expressed as standard
deviation)
• Comparative performance with other labs
• PT will not detect all problems in the laboratory
Proficiency Testing Program (EQA)

automated hematology analyser for labora

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