AUTOMATION IN HEMATOLOGY

(BLOOD CELL COUNTERS)

Varun Uppal
Ishwar Chand
 TIMELINE…..

Green, Ralph, and Sebastian Wachsmann-Hogiu. "Development, history, and future of automated cell counters." Clinics in laboratory medicine 35.1
(2015): 1-10
 AUTOMATION
Greek-automatos: self acting

Definition:
The process of following a pre determined process of operations with
little or no human labor, using specialized equipment and devices that
perform and control processes.
Why automation?
Space Economy
Increase work efficiency of lab
Manpower Economy
Time Economy

To generate high quality results Precision

Accuracy

Ensures cost-effectiveness
 Various Generations Of Coulter Counter

First Generation Fifth Generation

•Single channel DLC 5 Part Differential with
Derived 5 Differential
•Manual Dilutions
Actual 5 Part Differential
• Parameters Measured work station (All Above + Slide
one by one Maker +Slide Stainer).
•No DLC

Second Generation Third Generation Fourth Generation

•Double Channel •Double Channel
•Manual dilutions •Dilution inside the
•No DLC counter DLC 3 part differential
•Partial
DLC(lymphocyte%)
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.
GENERAL PRINCIPLES OF
AUTOMATED
BLOOD CELL ANALYSIS
 ELECTRONIC IMPEDANCE
Electronic impedance, or low-voltage direct current (DC) resistance,
developed by Coulter in the 1950s ,most common methodology used
 Based on the detection and measurement of changes in electrical
resistance produced by cells(or particles) as they traverse a small
aperture

• Cells suspended in an electrically conductive diluent such as saline are

pulled through an aperture (orifice) in a glass tube.
• In the counting chamber, or transducer assembly, low-frequency
electrical current is applied between an external electrode (suspended
in the cell dilution) and an internal electrode (housed inside the
aperture tube).
• Electrical resistance between the two electrodes, or impedance in the
current, occurs as the cells pass through the sensing aperture, causing
voltage pulses that are measurable.
 The aperture creates what is called a sensing zone. As a particle passes through
the aperture, a volume of electrolyte-containing solution equivalent to the
immersed volume of the particle is displaced from the sensing zone. This causes a
short-term change in the impedance across the aperture.

Each pulse is recorded as an oscillation

 the height of which is proportional to the
volume and size of the cell
 the total number of pulses gives us the total
count.
The data are plotted on a frequency
distribution graph, or volume distribution
histogram, with relative number on the y-
axis and volume on the x-axis. The
histogram produced depicts the volume
distribution of the cells counted.
 FACTORS AFFECTING VOLUME MEASUREMENTS IN
IMPEDANCE
 Aperture diameter:
• RBC/platelet aperture is smaller than the WBC aperture to increase
platelet counting sensitivity.
• On earlier systems, protein buildup occurred, decreasing the diameter of
the orifice, slowing the flow of cells, and increasing their relative electrical
resistance. Protein buildup results in lower cell counts, which result in
falsely elevated cell volumes required
Frequent manual aperture cleaning, but current instruments
incorporate burn circuits or other internal cleaning systems to prevent or
slow protein buildup.

 Carryover of cells from one sample to the next also is minimized by these
internal cleaning systems.

 Coincident passage of more than one cell at a time through the orifice
causes artificially large pulses, which results in falsely increased cell
volumes and falsely decreased cell counts
• Orientation of the cell in the center of
the aperture
• Deformability of the RBC, which may be
altered by decreased hemoglobin
content

• Recirculation of cells back into the

sensing zone creates erroneous pulses
and falsely elevates cell counts.
A backwash or sweep-flow mechanism
prevents recirculation of cells back into
the sensing zone
 HYDRODYNAMIC FOCUSING
Avoids many of the potential problems inherent in
a rigid aperture system.

• The sample stream is surrounded by a sheath

fluid as it passes through the central axis of the
aperture.
• Laminar flow allows the central sample stream
to narrow sufficiently to separate and align the
cells into single file for passage through the
sensing zone.

The outer sheath fluid:

 minimizes protein buildup and plugs,
 eliminates recirculation of cells back into the
sensing zone
 reduces pulse height irregularity because off-
center cell passage is prevented.
 coincident passage loss also is reduced because
blood cells line up one after another in the
direction of the flow
 RADIOFREQUENCY
• Whereas the impedance principle uses low -voltage DC current
to measure a cell ’s total volume, the radiofrequency method
uses high -voltage electromagnetic current (AC) to measure a
cell ’s nucleus.
• The alternating current in the radiofrequency range 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.
• Two different cell properties, such as low-voltage DC impedance
and RF resistance, can be plotted against each other to create a
two-dimensional distribution cytogram or scatterplot
 OPTICAL SCATTER
• In optical scatter systems (flow cytometers), a
hydrodynamically focused sample stream is directed
through a quartz flow cell past a focused light
source.
• The light source is generally a tungsten halogen lamp
or a helium-neon laser.
• Laser light, termed monochromatic light because it is
emitted at a single wavelength, differs from
brightfield light in its intensity, its coherence (i.e., it
travels in phase), and its low divergence or spread.

These characteristics allow for the detection of

interference in the laser beam and enable enumeration
and differentiation of cell types
• As the cells pass through the sensing zone
and interrupt the beam, light is scattered in
all directions.
• Light scatter results from the interaction
between the processes of absorption,
diffraction, refraction , and reflection.
• The detection of scattered rays and their
conversion into electrical signals is
accomplished by photodetectors
(photodiodes and photomultiplier tubes) at
specific angles.

• Analogue-to-digital converters change the

electronic pulses to digital signals for
computer analysis
 Forward-angle light scatter (FALS)
• Illuminating beam that has been bent to
a small angle from direction of the
original beam.
• It measures size or volume of cells
primarily because of diffraction of light

Side scatter (SSC)

• The illuminating beam that is scattered
by particle to an angle of 90 from the
illuminating beam
• results from refraction and reflection of
light from larger structures inside the cell
and correlates with degree of internal
complexity.
• It is sometimes referred to as a
granularity signal or an orthogonal light
scatter signal
FLUORESCENCE
• When fluorescent dyes are added to
the cells before their introduction into
the analyzer, they will stain certain cell
membrane and intracellular
structures.
• As these cells are passed through the
sensing zone they emit different
wavelengths of fluorescent light,
which vary according to properties of
the fluorochrome.
• Photodetectors collect and measure
the light in different wavelength
ranges (including blue, green, orange,
and red) and scatter wavelengths by
the use of specific optical filters, which
include band -pass, long - pass, and
short -pass filters.
• Cells are then categorized according to
their side -scattered light and
fluorescence –intensity characteristics
PRINCIPLE INSTRUMENTS
Hematology blood cell analyzers are produced by multiple manufacturers
 Abbott Laboratories (Abbott Park, IL);
 HORIBA Medical (Irvine, CA);
 Siemens Healthcare Diagnostics, Inc. (Deerfield, IL);
 Beckman Coulter, Inc. (Brea, CA);
 Sysmex Corporation (Kobe, Japan)

Hematology analyzers have some common basic components, including hydraulics, pneumatics, and
electrical systems.
The hydraulics system: an aspirating unit, dispensers, diluters, mixing chambers, aperture baths or
flow cells or both, and a hemoglobinometer.
The pneumatics system: generates the vacuums and pressures required for operating the valves and
moving the sample through the hydraulics system.
The electrical system: controls operational sequences of the total system and includes electronic
analyzers and computing circuitry for processing the data generate. A data
display unit receives information from the analyzer and prints results,
histograms, or cytograms.
BECKMAN COULTER
 REAGENTS INVOLVED
Diluent
ISOTON 4 diluent, are isotonic electrolyte solutions that:
• Dilute whole-blood samples.
• Stabilize cell membranes for accurate counting and sizing.
• Conducts aperture current.
• Rinse instrument components between analyses.
• Carry and focus the sample stream in the flow cell to direct the blood cells through the aperture.

CBC Lytic Reagent

LYSE S III Diff lytic reagent:
• Rapidly lyses erythrocytes (RBCs), freeing hemoglobin (Hgb) and reducing the size of cellular debris to a level that does not interfere with leukocyte (WBC)
count.
• Causes a substantial conversion of the Hgb to a stable cyanide-containing pigment, the absorbance of which is directly proportional to the Hgb concentration
over the clinical range.

LH Series PAK Reagent System

The Diff Lytic Reagent:
• Dilutes the blood samples
• Rapidly lyses erythocytes (RBCs)
• Reduces cellular debris to an insignificant level
The Diff Preservative:
• Maintains leukocyte (WBCs) in their near-natural state
The LH Series RETIC PAK Reagent Kit
• Retic A Retic Stain is a special solution of New Methylene blue dye. The dye precipitates the basophilic
RNA network found in the reticulocytes.
• Retic B Retic Clearing Solution is a hypotonic acid solution to clear hemoglobin from the cells without
removing the precipitated dye.

Cleaners
• LH Series Cleaner and COULTER CLENZ cleaning agent clean and rinse the internal surfaces of the
instrument components.
• Daily use prevents protein buildup and eliminates the need for routine aperture bleaching and blood
sampling valve maintenance.
A pump draws a maximum of 300 μL (LH 700 Series only) or
550 μL (LH 700 Series SlideMaker) of sample through the
needle and through the Blood Sampling Valve (BSV).
 DELIVERY
CBC
After the sample is aspirated:
• The center section of the BSV rotates and segments the sample
into two separate volumes.
• One volume of sample, 1.6 μL, is delivered with 10 mL of diluent to
the RBC bath. This dilution is used for RBC/Plt counting and
MCV/Plt sizing.
• The other volume, 28 μL, is delivered with 6 mL of diluent to the
WBC bath. This dilution is used to count WBC and develop Hgb.
• During delivery to the WBC bath, 1 mL of lytic reagent is added to
the dilution to lyse the red cells and convert Hgb

Differential (Diff) & retic

• During aspiration of the blood sample, blood is pulled through two
shear valves before it reaches the rear blood detector.
• These shear valves isolate the diff and retic sample segments for
processing.
 ANALYSIS
• In the RBC chamber, RBCs and platelets are counted and discriminated by electrical
impedance as the cells are pulled through each of three sensing apertures (50 μm
in diameter, 60 μm in length).
• WBCs are counted simultaneously by impedance in each of three sensing apertures
(100 μm in diameter, 75 μm in length).
• The WBC dilution is passed to the hemoglobinometer for determination of
hemoglobin concentration (light transmittance read at a wavelength of 525 nm).
• Electrical pulses generated in the counting cycles are sent to the analyzer for
editing, coincidence correction, and digital conversion.
• Two of the three counts obtained in the RBC and the WBC baths must match within
specified limits for the counts to be accepted by the instrument
• Pulse height is measured and categorized by pulse height analyzers; 256 channels
are used for WBC and RBC analysis, and 64 channels are used for platelet analysis.
• Volume-distribution histograms of WBC, RBC, and platelet populations are
generated
• In the WBC channel, a special lysing reagent causes differential shrinkage of
the leukocytes, which allows the different cells to be counted and
volumetrically sized based on their impedance. A WBC histogram is
constructed from the channelized data

• More recent Beckman Coulter instruments, the LH 700 Series Coulter’s

proprietary VCS (volume, conductivity, scatter) technology in a separate
channel to evaluate WBCs for the determination of a five-part differential.
• After RBCs are lysed and WBCs are treated with a stabilizing reagent to
maintain them in a near-native state, a hydrodynamically focused sample
stream is directed through the flowcell past the sensing zone
Volume
VCS utilizes the Coulter Principle of (DC) Impedance to physically measure the volume
that the entire cell displaces in an isotonic diluent. This method accurately sizes all cell
types regardless of their orientation in the light path.

Conductivity
Alternating current in the radio frequency (RF) range short circuits the bipolar lipid layer of a cell's
membrane, allowing the energy to penetrate the cell. This powerful probe is used to collect
information about cell size and internal structure, including chemical composition and nuclear
volume.

Scatter
When a cell is struck by the coherent light of a LASER beam, the scattered light spreads out in all
directions. Using a proprietary new detector, median angle light scatter (MALS) signals, are
collected to obtain information about cellular granularity, nuclear lobularity and cell
surface structure

Simultaneous Measurements:
VCS is the only single channel analysis that uses 3 independent energy sources to probe
approximately 8,192 cells in their near native state
Reticulocyte Analysis
• A supravital dye, New Methylene Blue, is incubated with whole-blood
samples. The dye precipitates the basophilic RNA network found in
reticulocytes. Hemoglobin and unbound stain are removed by adding a
clearing reagent, leaving clear spherical mature RBCs and darkly stained
reticulocytes.
• Stained reticulocytes are differentiated from mature red cells and other
cell populations by light scatter, direct current measurements, and opacity
characteristics
 DATAPLOT DEVELOPMENT
At the Workstation, raw data is analyzed to:
• Position each cell within a three-dimensional space
• Plot the data on a two-dimensional DataPlot
• Display and report results

• The Beckman Coulter AccuGate algorithm uses adaptive contouring methods designed for finding optimal
separation between overlapping clusters of data
• In the DataPlots, different colors represent different memberships (types of cells)
• DIFF:
Lymphocytes Blue
Neutrophils Purple
Eosinophils Orange
Monocytes Green
Basophils White
Non-white debris Red
• RETIC:
RBCs Red
Cell volume (VOL), determined by the low-frequency The Reticulocyte Data Plot shows mature red cells and
impedance measurement, is plotted on Reticulocytes. Cell volume is plotted
the Y-axis; rotated light scatter (RLS) is plotted on the X- on the Y-axis, and linear light scatter (LLS) is plotted on
axis. the X-axis.
The 3D DataPlot view classifies by density, light scatter and opacity.
 PARAMETERS
Parameters are either:
• Measured directly. • Derived From RBC or Plt Histogram

• Calculated
• Derived From WBC Histogram
RED CELL INDICES
• MCV :The counter provides MCV which is derived from the histogram (sum
of pulse height / sum of pulse).
• MCH: This is the average weight of the hemoglobin in picograms in a red
cell. It is a calculated value.
MCH =hemoglobin in pg/L / red cell count in milions/L
• MCHC :This indicates the average weight of hemoglobin as compared to the
cell size.
MCHC = (Hemoglobin in g/dL / HCT) x 100
• Hematocrit: Hematocrit (Hct) or (PCV) is the volume of the red cells as
compared to the volume of the whole blood sample
HCT(%) = (RBC X MCV ) /10
RDW: quantitative measurement of cell volume, an equivalent of
microscopic assessment of degree of anisocytosis
Can be expressed as RDW-SD or RDW-CV
RDW-SD is the arithmetic distribution Width RDW-CV is computed as
measured at the 20% Relative Height Level, RDW-CV (%) = 100 X δ/µ
taking Histogram peak as 100%.
The RBC Histogram of normal blood sample
crosses this 20% level twice. RBC-SD is
reported in Femtolitres
 PLATELET INDICES
• Mean platelet volume:
 The average volume of individual platelets derived from the Plt histogram.
 It represents the mean volume of the Plt population under the fitted Plt
curve multiplied by a calibration factor.
 This number is expressed in femtoliters.

• Platelet distribution width: measure of platelet anisocytosis(n=15-35%)

• Plateletcrit: product of MPV and platelet count

• Platelet large cell ratio(P-LCR):

Number of platelets falling above the 12 fl threshold on plt size histogram
divided by total number of platelets(9-14 fl)
 RETICULOCYTE PARAMETERS
• Retic Percent (RET%)
RET% is calculated as the ratio of reticulocytes to the total number of red
cells

• Retic Absolute Number (RET#)

Absolute number of reticulocytes computed from the reticulocyte percent
(RET%) multiplied by the RBC count.
Immature reticulocyte fraction (IRF) Mean reticulocyte volume (MRV)
The IRF parameter is an indication of new reticulocyte The MRV parameter is the average volume of all
synthesis. It is calculated from the RET% as reticulocytes (or the mean volume of all retic
the total number of reticulocyte events in the outermost light events). It is calculated from the RET% and reported in
scattering region, corresponding to femoliters (fL).
immature reticulocytes, relative to the total number of
reticulocytes and is reported as this ratio.
NRBC ENUMERATION
• Achieved through the combined use of impedance and VCS
technology and a proprietary algorithm.
• The first step in NRBC enumeration is the identification of
particles in the NRBC signature position in the differential data
plot. This information is generated from VCS analysis of the
cells.
• Once particles have been identified in this region, the LH 700
Series examines the far left region of the WBC histogram for
the presence of particles.
• If the VCS dataplot and the WBC histogram both indicate the
presence of NRBCs, then the combined information is further
evaluated for special data patterns -- such as small
lymphocytes, giant platelets, and aging blood.
• If the combined information from the VCS dataplot and the
WBC histogram are consistent with NRBCs, the NRBC count is
derived from the WBC histogram.
 HISTOGRAMS

• Rbc, wbc, platelet plotted on histograms

• X-axis: cell size in femtolitres

• Y-axis: # of cells

• Need?
Visual scanning of the histogram gives a good initial sense of the range, size,
shape, and other salient features of the cell morphology.
In addition, it is frequently used, along with the peripheral blood film, as an
aid in monitoring and interpreting abnormal morphological changes,
 RBC HISTOGRAM
Distribution of RBC population according to their size and their relative
number.
The Normal red cell distribution curve (Histogram) is Gaussian (bell shape)
and the peak of the curve should be within the normal MCV of 80.0-100.0 fL

The size ranges for RBC histograms are between 24 fL and 360 fL, the
instrument counts only those cells with volume sizes between 36 fL and 360
fL as red cells.
Two discriminators are used upper and lower
Lower Discriminator is placed between the platelets and RBCs
Red Cells Histogram
• (24- 36 fl ) flag may be due
1- RBCs fragments
2- WBC's fragments
3- Giant plts
4- Microcyte

• Shift to right :
- Leukemia
- Macrocytic anemia
- Megaloblastic anemia

• Shift to left :
- Microcytic anemia (IDA)
• Bimodal
- IDA, Megaloblastic anemia with transfusion.
-Sideroblastic anemia.

• Cold agglutination
PLATELET HISTOGRAM
Normal platelet histogram displays cells from (2-20 fl).
• (0-2)
• Air Bubbles
• Dust
• Electronic and Electrical noise
• Over 20 fL
• Microcyte
• Scishtocyte
• WBC's fragments
• Giant Plts
• Clumped plts
WBC HISTOGRAM
• 3-part differential usually counts
• Granulocytes or large cells
• Lymphocytes or small cells
• Monocytes(mononuclear cells) or (middle cells)
• 5-part classify cells to
• Neutrophils
• Eosinophils
• Basophils
• Lymphocytes
• Monocytes
• A sixth category designated “large unstained cells” include cells larger than normal and lacking
the peroxidase activity which include
• Atypical lymphocytes
• Various other abnormal cells.
• Other counters identifies 7 categories including
• Large immature cells(composed of blasts and immature granulocytes)
• Atypical lymphocytes(including blast cells).
Histogram provides Percent and Absolute
Count for these three populations
Small Cell Ratio [SCR]/Small Cell Count [SCC]
Mixed Cell Ratio [MCR]/Middle or Mixed Cell Count [MCC]
Large Cell Ratio [LCR]/Large Cell Count [LCC]
 SYSMEX INSTRUMENTATION
• Provide complete RBC, platelet, and WBC analysis with three-part differential; the larger XT-1800i
(SF-3000 and SE-9000) that performs a CBC with five-part differential; and the XE series and the
newest XN series that also provide a fully automated reticulocyte count
• Two unique features enhance the impedance technology:
 in the RBC/platelet channel, a sheathed stream with hydrodynamic focusing is used to direct cells
through the aperture, which reduces coincident passage, particle volume distortion, and
recirculation of blood cells around the aperture; and
 in the WBC and RBC/platelet channels, floating thresholds are used to discriminate each cell
population
-This floating threshold circuitry allows for discrimination of cell populations on a sample-by-
sample basis
• The platelet analysis on the XN also utilizes a fluorescent count, in addition to the impedance
count and optical count, called the PLT-F, performed by optical measurement.
• In the hemoglobinflow cell, hemoglobin is oxidized and binds to sodium lauryl sulfate (SLS)
forming a stable SLS–hemoglobin complex, which is measured photometrically at 555 nm
• The SE-9000/9500 uses four detection chambers to analyze WBCs and obtain a five-part differential:
the DIFF, IMI (immature myeloid information), EO, and BASO chambers by low-frequency DC and high-
frequency current(DC/RF detection method)
• In the XN-1000, fluorescent flow cytometry is used for the WBC count, WBC differential, and
enumeration of nucleated RBCs.
• The XE-5000, the XT-2000i and the XN series determines the reticulocyte count and IRF by measuring
forward scatter and side fluorescence.
• RET-He (reticulocyte hemoglobin equivalent) that measures the hemoglobin content of the
reticulocytes.It uses a proprietary polymethine dye to fluorescently stain the reticulocyte nucleic acids.
 ABBOTT INSTRUMENTATION
• Instruments include the smaller CELL-DYN Emerald, which provides CBC with three-part differential, and the larger CELL-
DYN Sapphire and the midrange CELL-DYN Ruby,provide a CBC with five-part differential and random fully automated
reticulocyte analysis
• A unique von Behrens plate is located in the RBC/platelet counting chamber to minimize the effect of recirculating cells.
• The platelet analysis is based on a two-dimensional optical platelet count using fluorescent technology, the same
technology used for direct nucleated RBC counting by adding a red fluorescence to the sample to stain nucleated red
cells.
• The WBC count and differential are derived from the optical channel using CELL-DYN’s patented multiangle polarized
scatter separation (MAPSS) technology with three-color fluorescent technology
• Scattered light is measured at multiple angles:
- 0-degree forward light scatter measurement is used for determination of cell volume,
- 90-degree orthogonal light scatter measurement is used for determination of cellular lobularity,
- 7-degree narrow-angle scatter measurement is used to correlate with cellular complexity,
-90-degree depolarized light scatter measurement is used for evaluation of cellular granularity
• The CELL-DYN Sapphire uses MAPSS technology to allow fully automated, random access reticulocyte testing.
• The RBCs are stained with a proprietary membrane-permeable fluorescent dye (CD4K530) that binds stoichiometrically
to nucleic acid and emits green light as the cells, in a sheath-stream, pass through a flow cell by an argon ion laser.
 SIEMENS HEALTHCARE DIAGNOSTICS INSTRUMENTATION
• Siemens Healthcare Diagnostics Inc. manufactures the ADVIA 2120 and 2120i provide a complete hemogram and
WBC differential, with fully automated reticulocyte count
• Four independent measurement channels are used in determining the hemogram and differential: RBC/platelet
channel, hemoglobin channel, and peroxidase (PEROX) and basophil lobularity(BASO) channels for WBC and
differential data
• RBCs and platelets are isovolumetrically sphered before entering the flow cell to eliminate optical orientation noise.
• Hemoglobin distribution width (HDW), an analogous to RDW index, is calculated as the SD of the RBC hemoglobin
concentration histogram. The reference interval for HDW is 2.2 to 3.2 g/dL.
• Cell hemoglobin concentration mean (CHCM), analogous to MCHC, is derived from cell-by-cell direct measures of
hemoglobin concentration
• The reticulocyte reagent isovolumetrically spheres the RBCs and stains the reticulocytes with oxazine 750, a nucleic
acid–binding dye.
• Unique reticulocyte indices (MCVr, CHCMr, RDWr, HDWr, CHr, and CHDWr) are provided.
• The CHr or reticulocyte hemoglobin content of each cell is calculated as the product of the cell volume and the cell
hemoglobin concentration
QUALITY ASSURANCE
 PRE ANALYTICAL
Sample:
ICSH recommends dipotassium EDTA at concentration of 1.50-2.20mg/ml of
blood
EDTA in excess of 2mg/ml:
• Decrease in PCV by centrifugation
• Increase in MCHC
• Platelets swell and disintegrate leading to artificial high counts
• Leuco-agglutination affecting both neutrophils and lymphocytes
PHYSIOLOGICAL VARIATIONS

A. Pregnancy
increased erythropoeitic activity accompanied with increase in plasma volume & progressive decrease in Hb,
Hct & RBC. Moderate leukocytosis is common
B. Elderly
anemia becomes more common in those older than 70 years
C. Posture
Small but significant alteration in plasma volume with increase in Hb & Hct from lying to sitting especially in
women
D. Diurnal & seasonal variations
E. Altitude
Reduced plasma volume with increase in Hb & Hct
 ANALYTICAL

Instrument selection
 CALIBRATION
• Calibration, or the process of electronically correcting
an instrument for analytical bias (numerical difference
from the “true” value), may be accomplished by
appropriate use of reference methods, reference
materials, or commercially prepared calibrators
• USE OF CALIBRATOR
 Installation
 Change of reagent lot
 Instrument maintenance
 Replacement of major component or repair of instrument
 Installation of new software
 QC shows a shift or trend when no new change has
occurred

 The S-CAL calibrator kit is an acceptable alternative to the whole-blood reference method of
calibration.
 S-CAL calibrator is traceable to reference methods and materials
 CONTROLS
• Substances used in routine practice for checking the performance of an
analytical process (or instrument).
• They may or may not have a pre-assigned value.
• Even though some control preparations have assigned values, they should not
be used as calibrators or standards because:
• Assigned values are only approximations
• Controls are stable for a limited time only
 COULTER 5C Cell control monitors the CBC and differential (Diff) parameters.
Range from high, normal and low values for the common CBC parameters

 LATRON primer prepares the tubing and instrument components for the LATRON control.
 LATRON control monitors the performance of the volume, conductivity and light scatter
measurements.

 Retic-C cell control monitors the reticulocyte (Retic) parameters.

 Lin-C linearity control identifies the reportable range of the instrument's CBC parameters
 INTERNAL QUALITY CONTROL
• IQC is based on monitoring the haematology test procedures that are
performed in the laboratory and includes measurements on specially
prepared materials and repeated measurements on routine specimens,
together with daily statistical analysis of the data.
• IQC is primarily a demonstration of precision.
• It includes:
1.Duplicate tests on patient specimens
2.Check tests
3.Delta test
4.Patients data
5.Correlation check
6.Control & Control charts
XB Analysis
• Studies (Bull 1974, Koepke 1981) indicate that the red cell indices (MCV,
MCH, and MCHC) of patient populations are stable over time. This stability
characteristic of the indices is the basis of a quality-control technique called
XB Analysis
• Population means (target values) are established by analyzing as large a
sample as possible, at least 250, but ideally 1,000 blood samples.
• Once the target values have been established, the XB Analysis can be
applied using quite small batches from the patient population.
• The hematology system is considered "in control" when the batch means are
within established limits of the target values
XM Analysis
• XM Analysis is a quality-control method that uses an Exponentially
Weighted Moving Average (EWMA) of CBC, Diff, NRBC and
Reticulocyte Parameters and compares them with known target
values, to monitor instrument performance.
 EXTERNAL QUALITY CONTROL(EQA)
• The term EQA, also known as proficiency testing (PT), was adopted in 1979
by a WHO working group on Quality Assurance of Health Laboratories.
• It is defined as:
“a system whereby a set of reagents and techniques are assessed by an
external source and the results of the testing laboratory are compared with
those of an approved reference laboratory or agency.”

The objective is to achieve between-laboratory and between-method

comparability.
EQA complements internal quality control
Even after adequate internal control, errors arise which are only
detectable by objective external assessment
EQAS IN OUR
DEPARTMENT
 Advantages
1.Large Number of Specimens can be analyzed in short period of time

2. Most of the methods performed in Automation are generally

Reliable
 Accurate
Precise
3. Statistical Error is very less as compared to Manual
method because large number of cells are counted.

% Error Manual Electronic

Method Method
RBC +11% +1-2%
WBC +16% +2-3%
Platelets +22% +3-4%
4.Variety of Information from a single Aspirated
sample can be obtained.

Instrument Measured parameters

K- 1000 18
Pentra 23
LH 700 series 25
5.Less amount of blood is required for analysis.

Instrument Blood Aspirated

K – 1000 100µL

LH 700 300µL

Capillary Mode 20µL

6. Although the Various type of Auto analyzers are Expensive but in long run,
they prove to be cost effective because the amount of the reagent and
specimen required can be as low as 300µL and 50µL respectively.

7.Internal and External Quality Control programs can be implemented

efficiently and effectively by using auto analyzers.

8.In case of Fully Automated Analyzers the Laboratory Staff members don't
come in contact with specimens and reagents
[Bio hazardous material] and hence working with auto analyzer is very safe.
9. Automation allows the laboratories to process much larger
workload without a comparable increase in number of staff
members.
 Flagging of the Abnormal Specimen
 Automatic Wash Cycle to reduce carry over
 Automatic double/triple counting for checking precision and accuracy
 Automatic checking for the Reagents.
 Automatic Threshold limit changes for platelets/ microcytes.
 Connectivity with the printer reduces the clerical work.
 LIMITATIONS

Instrumental specimen
 Instrumental

Biohazardous
Laser
Waste
Hazards
Electrical
Hazards
• A common limitation of impedance methods is an instrument’s inability to distinguish
cells reliably from other particles or cell fragments of the same volume
e.g. Cell fragments may be counted as platelets in specimens from chemotherapy-
treated patients with increased WBC fragility
RBCs containing variant hemoglobins such as Hb S or Hb C are often resistant to lysis,
and the unlysed cells can be falsely counted as nucleated RBCs and interfere in the
hemoglobin reaction

• Suppression of automated data, particularly WBC differential data, may occur when
internal instrument checks fail or cast doubt on the validity of the data
NEW PARAMETERS & CLINICAL UTILITY