HEMATOLOGY

AUTOMATION
Donna Therese M. Taguinod, RMT, MPH
Limitations of Manual Cell Counting
1. It is subjective, labor-intensive and statistically
unreliable (only 100-200 cells are counted)
2. It is imprecise with reported Coefficients of
Variation (CV) ranging from 30-110%
3. Experience is needed to make technically
adequate smears consistently.
4. Non-uniform distribution of WBCs over the
smear (large WBCs near the edges,
lymphocyte are scattered throughout)
Limitations of Manual Cell Counting
5. Non-uniform distribution of RBCs (small
crowded RBCs at the thick edge, large flat cells
without central pallor at the feathered edge)
6. Cell identification errors
ü Lymphocytes from monocytes
ü Bands from segmented forms
ü Variant lymphocytes from blasts
 Automated Analyzers
Ø are medical laboratory instruments designed to
measure different chemicals and other
characteristics in a number of biological
samples quickly, with minimal human
assistance.
Ø Note:
Automation of laboratory testing DOES NOT
remove the need for human expertise.
Results must still be evaluated by Medical
Technologists and other qualified clinical
laboratory professionals.
 BENEFITS OF AUTOMATION
1. Reduction of medical errors
2. Improved safety for laboratory staff
3. Faster turn-around-time of results
4. Partially alleviating the impending shortage of
skilled laboratory staff
 Evolution of Automated Cell
Counters
A. First half of the 20th century
• For Blood cell counts (RBC, WBC, Platelets) - use
of counting chamber (hemocytometer)
• Hemoglobin concentration – analyzed
colorimetrically (cyanmethemoglobin method)
• Hematocrit – measured by high speed
centrifugation of a column of blood (Wintrobe tube
or microhematocrit tube)
• WBC differential count – by stained blood smear
 Evolution of Automated Cell
Counters
B. 1932
• MCV, MCH, MCHC was developed by Wintrobe.

C. 1934
• First step towards automation with the Moldavan
capillary method
• A suspension of blood cells was illuminated in a
dark field and counted in an optical chamber which
converted the light impulses into electrical
impulses.
 Evolution of Automated Cell
Counters
D. 1956
• Actual breakthrough in the development of
hematological instruments suitable for routine work.
• High speed automatic blood cell counter was
developed by Wallace Coulter.
 Evolution of Automated Cell
Counters
E. At present
• Modern versions of analyzers are capable of
measuring 10 or more parameters.
v Electronically measured are Hemoglobin, RBC count,
MCV, Platelet count, WBC count, Mean Platelet Volume
(MPV)
v Calculated are Hematocrit, MCH, MCHC, RDW, PDW
and plateletcrit
v WBC differentials (relative or percent absolute count)
Ø The differentials maybe three-part (granulocytes,
lymphocytes, monocytes) or five-part ( neutrophils,
lymphocytes, monocytes, eosinophils, basophils)
 Evolution of Automated Cell
Counters
E. At present
• Enumeration of nucleated RBC and reticulocytes.
• May also include verification systems.
1. Reviews past patient results (delta check)
2. Instrument flagging
v These ‘flags’ are warnings generated and displayed by
the machine to alert the laboratory personnel that the
machine has detected some abnormality in cell
population or distribution that needs attention.
v Some examples of these flags include blast flag, atypical
flag, flag for discrepancy of the two WBC counts,
nucleated RBC (NRBC) flag, flag for immature
granulocytes, etc.
 Evolution of Automated Cell
Counters
E. At present
• Mean Platelet Volume (MPV) is determined from
platelet histogram curve.
v Reference Range : 6.5-12 fL
• Red Cell distribution Width (RDW) provides an
estimate of RBC anisocytosis.
v Computed as: RDW = (A-B) ÷ (A+B) x k
v Reference Range : 8.5-14.5%
 AUTOMATED ANALYZERS
ADVANTAGES DISADVANTAGES
1. Rapid, objective and 1. Spurious results may also be
statistically significant (8000 produced.
or more cells are counted) 2. For impedance based
2. More efficient counters –may not
3. Samples can be processed distinguish tiny platelet
singly, in batches or clumps and nucleated RBCs
continuously. (120-150 (platelet clumps for
samples per hour) WBC/RBC; nRBC for
4. Reproducible absolute lymphocytes))
leukocyte counts 3. Large or unidentifiable
atypical cells, toxic immature
neutrophils and markedly
reactive lymphocytes may be
misclassified.
 BASIC COMPONENTS OF MOST
HEMATOLOGY ANALYZERS
HYDRAULICS
-aspirating unit -aperture baths
-hemoglobinometer -dispensers
-dilutors
-mixing chambers
 BASIC COMPONENTS OF MOST
HEMATOLOGY ANALYZERS
PNEUMATICS
-vacuums
-pressure for operating valves and moving
the sample through the system
 BASIC COMPONENTS OF MOST
HEMATOLOGY ANALYZERS
PNEUMATICS
-vacuums
-pressure for operating valves and moving
the sample through the system
BASIC COMPONENTS OF MOST
HEMATOLOGY ANALYZERS
ELECTRICAL SYSTEMS
-Electronic analyzers
-computing circuitry for processing data
METHODS OF CELL COUNTING
MANUAL :
• NEUBAUER HAEMOCYTOMETER
• FUCHS ROSENTHAL COUNTING
CHAMBER
• SPEIRS LEVY COUNTING CHAMBER

AUTOMATED SYSTEM
 INSTRUMENT PRINCIPLES
q An automated analyzer aspirates whole blood,
divides it, dilutes it, mixes it and analyzes it for
hemoglobin and cell characteristics.
q Basic principles of operation are the ff:
1. Electrical impedance 5. Hydrodynamic
2. Radiofrequency focusing
3. Optical Scatter 6. Use of flow cells
4. VCS Technology 7. Multiple Angle
(Volume, Conductivity Polarized Scatter
& Scatter) Separation (MAPPS)
ELECTRICAL
IMPEDANCE
 ELECTRICAL IMPEDANCE
METHOD:
• a.k.a. Coulter Principle, Electronic Resistance,
Low Voltage direct current resistance, Coulter
Principle
• Developed by Coulter in the 1950’s
• The first principle of measurement
• MOST COMMON METHODOLOGY USED
• EXAMPLE: COULTER COUNTER AND
SYSMEX
COULTER COUNTER

SYSMEX
HEMATOLOGY
ANALYZER
 ELECTRICAL IMPEDANCE
METHOD:
• Cells are sized and counted by detecting and
measuring changes in electrical impedance
(resistance) when a particle passes through a
small aperture between two electrodes.
 ELECTRICAL IMPEDANCE
METHOD:
Utilizes non-conductive properties of blood cells:
1. As blood cell passes through orifice of aperture it
displaces its own volume
2. Increased resistance between electrodes results in
an electrical pulse
3. RBCs and Platelets counted together, separated
by pulse heights
4. Hydrodynamic focusing forces cells to pass single
file through sensing zone
ELECTRICAL IMPEDANCE
METHOD:
Ø Two electrodes
establish an electrical
current:
1. The external electrode
is located in the blood
cell suspension.
2. The second electrode
is the internal electrode
and is located in the
glass hollow tube,
which contains the
aperture.
 PRINCIPLE OF OPERATION:
• A blood sample is diluted in electrolyte such as
saline, a good conductor of electrical current,
and the cells are pulled through an aperture by
creating a vacuum. The cells are relatively poor
conductor of electric current.
• Low-frequency electrical current is applied to
the external electrode and the internal
electrode. A measured volume of blood
suspension is pulled through the aperture.
 PRINCIPLE OF OPERATION:
• DC current is applied between the two
electrodes.
• Differences in the electrical resistance between
the two electrodes occur as the cells pass
through the aperture, causing changes in
voltage pulses that are amplified and counted.
 PRINCIPLE OF OPERATION:
• Because the size of the voltage pulse is
directly proportional to the size of the cell,
voltage pulses caused by cells of a specific size
can be amplified, discriminated and counted
through the threshold circuits.
• THRESHOLD CIRCUIT- used to discriminate
between particle sizes accurately. It is a voltage
limit with which a pulse is being compared
RBC = 36-60 f MONOCYTES= 90 –160 fl
PLATELETS=2-20 fL GRANULOCYTES= 160-450 fL
LYMPHOCYTES=35-90 fL
OSCILOSCOPE: where voltage
pulses are displayed.
 OHM’S LAW

VOLTAGE= CURRENT X RESISTANT

1.HEIGHT OF THE PULSE is directly proportional to

the size of cell
2.NUMBER OF VOLTAGE PULSES is directly
proportional to number of cells
 ELECTRICAL IMPEDANCE
METHOD:
§ The number of impulses is proportional to the
number of cells counted.
§ The size of the voltage pulse is directly
proportional to the volume or size of the cell.
§ The pulses are channelized by their height or
amplitude, and data are collected and plotted
on a volume/frequency distribution histogram.
Ø The resulting distribution curves are automatically
discriminated according to the lower (platelets) or
higher values (leukocytes) and then depicted
graphically.
 Sources of errors for electrical
impedance method:
A. INSTRUMENT ERRORS
1. POSITIVE ERROR
a. BUBBLES
b. EXTRANEOUS ELECTRICAL PULSES
c.. APERTURE PLUGS- most common
problem
2. NEGATIVE ERROR
a. EXCESSIVE LYSING OF RBC
3. EITHER POSITIVE OR NEGATIVE ERROR
a. IMPROPER SETTING OF APERTURE
CURRENT
 Sources of errors for electrical
impedance method:
B. PHYSIOLOGICAL ERROR
üGiant platelets may be counted sa RBCs and
WBCs
üFragment of Leucocyte cytoplasm(seen
during leukemia therapy) may be counted sa
platelets and RBCs
üIncreased number of schistocytes may make
inaccurate RBCs and platelet count
üAgglutination of RBCs, WBCs and platelets
make inaccurate count for each of the respective
cell counts
üPlatelet satellitism (induced by EDTA )
ü Abnormal red cells(sickle cells,
codocytes/target cells, extremely hypochromic
cells, Nucleated RBCs) tend to resist lysis which
may result in high WBC count.
**PRESENCE OF NRBC=
__________________
CORRECT WBC COUNT IF:
1. ADULT ________________________
2. Neonates ______________________
CORRECTED WBC CT. =uncorrected WBC count x 100_
100 +NRBC
Sample problem:
13 reticulocytes where seen by Mr. Kyle, a
Medical Lab ScIentist at East Avenue
Med.Center whlle performing a WBC diff.
count on smear of peripheral blood from a
newborn patient diagnosed with sepsis.
The SYSMEX ANALYZER reported a WBC
count of 17,000/cu.mm. What should Mr.
Kyle do ?
 Sources of errors for electrical
impedance method:
C. OTHER ERRORS
1. COINCIDENCE – 2 or more cells are
passing through the aperture** (CHECK THE DEFINITION KAY
KOPKE)

2. NON AXIAL PASSAGE/RECIRCULATION

OF CELLS- cells are “swirling” back into the
sensing zone
 OPERATION IN ELECTRICAL
IMPEDANCE INSTRUMENTS
REAGENTS AND SUPPLIES
1. ELECTROLYTE (SALINE)
Buffered isotonic salt solution that may contain one or more
preservatives
Preservative: AZIDE (not used today because of explosion
hazards)
Purpose: TO DILUTE AND TO FLUSH THE INSTRUMENT
2. DETERGENT SOLUTION
LYSING AGENT
Purpose: Lyse the RBCs by dissolvoing their stroma when
leukocyte counts are perfomed
 OPERATION IN ELECTRICAL
IMPEDANCE INSTRUMENTS
3. CLEANING AGENTS
•Purpose: remover protein build up from the
aperture tube and electrodes
•Ex: diluted hypochlorite solutions and
detergents

4. SAMPLE CONTAINER
•Small glass beakers to plastic vials.
•Should be particle free and that their inter
surfaces not react with or attract cells
 SPECIMEN DILUTION IN E.I.
INSTRUMENTS
Dilutions are done:
• Manually
• Automatic dilutors
WBC COUNTING
• 1:500 dilution (40 ul blood in 20 ml of
electrolyte/saline)
RBC COUNTING
• 1:50,000 dilution (200 ul of the 1:500
dilution of blood in a 20 ml electrolyte/saline)
 ELECTRICAL IMPEDANCE
REPORT
• COMPLETE BLOOD COUNT
• SCATTERGRAM
• HISTOGRAM
• FLAGGING
 A. COMPLETE BLOOD COUNT
(CBC)
-RBC count
-WBC count
-WBC differential
-Hemoglobin
-Hematocrit
-Platelet count
 ELECTRICAL IMPEDANCE
METHOD:
§ Hemoglobin Concentration
Ø is obtained from the WBC dilution. Sample is diluted
with cyanmethemoglobin reagent.
Ø 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.
Ø A photodetector reads color intensity at 546 nm
Ø Optical density of the solution is proportional to the
concentration of hemoglobin
 ELECTRICAL IMPEDANCE
METHOD:
§ Hematocrit
Ø The hematocrit is calculated as the sum of all
impulses from the directly measured cell volume.
Ø The HCT is computed from the RBC count and
the MCV and calculated from the following
formula:
%HCT = (RBC count x MCV) ÷ 10
 ELECTRICAL IMPEDANCE
METHOD:
§ RBCs and WBCs
Ø are counted in duplicate and triplicate.
Ø Each of the duplicated counts must agree within
standardized range of deviation from each other.
§ Platelets
Ø are counted in duplicate and triplicate in the RBC
aperture bath.
Ø Particles ranging from 2 to 20 fL ( 1 fL= 10-15/L= 1
mm3) in the RBC bath sorted as platelets and
plotted as a platelet histogram.
 RED BLOOD CELL
• RBC size: 36-60 fl
ü SHIFT TO THE RIGHT- If RBC is larger than normal
ü SHIFT TO THE LEFT-If RBC is smaller than normal
ü BIMODAL CURVE-two population of red blood cell **
• Characteristic of RBC diluting fluid: SHOULD ALWAYS BE
ISOTONIC**- to facilitate counting and prevent lysis of Red
blood cells.
• Dilution of Diluting fluid: 1:6250 **
• RBC HISTOGRAM:
ü X axis: SIZE/VOLUME
ü Y axis: RELATIVE NUMBER
• DERIVED FROM RBC HISTOGRAM: MCV, RDW
• Calculated parameters: MCH, MCHC, HCT
 PLATELET
• Platelet size: 2-20 fl
• Charac of diluting fluid: SHOULD ALWAYS BE
ISOTONIC
• Dilution of diluting fluid: 1:6250
• THE ACTUAL COUNTING TAKES PLACE IN THE RBC
APERTURE
• PLATELET HISTOGRAM:
ü X axis: SIZE/VOLUME
ü Y axis: RELATIVE NUMBER
• DERIVED FROM PLATELET HISTOGRAM: MPV,
PDW(Steininger) or MPV,PLATELET COUNT(Brown)
 Total WHITE BLOOD CELL

• WBC size: LYMPHOCYTE(35-90 fl) ,

MONOCYTE(90-160 fl), GRANULOCYTES(160-450
fl)
ü SHIFT TO THE LEFT: presence of immature
granulocytic cells
ü SHIFT TO THE RIGHT: increased number of
hypersegmented neutrophils
• Characteristic of WBC diluting fluid:
HYPOTONIC(WEAK ACID) – to lyse RBCs except
lysed resistant RBCs (NRBCs**)
• Dilution: 1:251
 ELECTRICAL IMPEDANCE
METHOD:
§ RBC indices
Ø are computed parameters commonly obtained from
automated cell counters.
Ø The MCV is determined directly from the voltage-
pulse heights from the RBC count or histogram curve.
Ø The MCH is computed from the MCV and the MCHC and
calculated from the following formula ( note that 1 µg= 1
pg)
MCH (pg) = ( MCV x MCHC) ÷ 100 or
MCH (pg) = (Hgb x 10) ÷ RBC count
Ø The MCHC is computed from the HGB and HCT and
calculated from the following formula:
MCHC (%) = (Hgb ÷ Hct) x 100
 B. BLOOD CELL HISTOGRAMS
Ø Are graphic representations of cell frequencies
versus size.
Ø Provides information about RBC, WBC & platelet
frequency; distribution about the mean and
presence of subpopulations.
Ø The relative number is on the y-axis; the size or
volume is on the x-axis.
Ø The volume, given in mm3 of fL, is plotted against
the relative frequency for platelets, WBCs and
RBCs
 B. BLOOD CELL HISTOGRAMS
• It is provided by many high volume
instruments(mainly in ELECTRICAL IMPEDANCE
INSTRUMENTS) to provide SIZE DISTRIBUTION
OF THE DIFFERENT CELL POPULATIONS
• The volume/size given in cu.mm or fl of cells are
plotted against the relative number for platelets,
RBCs and WBCs.
 RBC Histogram:
• Represents the cell
counted in the RBC
dilution, ranging from 36 to
360 fL
• Normal RBC histogram: a
single peak should be found
normally between 70 and 110
fL, and the peak should
coincide with the MCV.
• Abnormal RBC histogram:
result when the MCV of the
curve falls outside of the
normal range of 80 to 100fL or
when the RDW is > 14.5
 WBC Histogram:
• Provide a count and plot of
cells in the WBC aperture
bath larger than 45 fL.
• Normal WBC histograms
have three distribution
peaks.
1. The first peak, ranging from 45 to
90 fL, represents a small
mononuclear population of cells
( i.e. lymphocytes)
2. The second peak, ranging from
90 to 160 fL, represents a minor
population of larger mononuclear
cells (i.e. monocytes)
3. The third peak or the major
population, ranges from 160 to
450 fL, represents normal mature
types of granulocytes.
 Platelet Histogram:

§ Represents cells in the

RBC counting baths
that are in the size
range of 2 to 20 fL
§ Atypical platelet
histograms can result
in some disorders
when large platelets
are present.
NORMAL WBC HISTOGRAM have
THREE DISTRIBUTION PEAKS:
1. FIRST PEAK-small mononuclear
cell(LYMPHOCYTES)which can be
categorized as SMALL CELLS
2. SECOND PEAK- large mononuclear cell
(MONOCYTES )which can be categorized
as MEDIUM CELLS
3. THIRD PEAK- polymorphonuclear cells(
GRANULOCYTES-NEB) which can be
categorized as LARGE CELLS
 WBC DIFFERENTIAL COUNT:
**THREE PART DIFFERENTIAL:
LYMPHOCYTE,MONOCYTE,GRANULOCYTE

**FIVE PART DIFFERENTIAL:

LYMPHOCYTE, MONOCYTE, NEUTROPHIL,
EOSINOPHIL, BASOPHIL
HEMOGLOBIN DETERMINATION
_____________________: CYANMETHEMOGLOBIN METHOD
Uses: MODIFIED DRABKIN’S REAGENT
1.__________________-converts Hemoglobin to methemoglobin (Hi)

2._____________-converts methemoglobin to cyanmethemoglobin (HiCN)

**to reduce toxicity to cyanide it has been replaces by Na-lauryl sulphate
3. ___________________improve the lysis of red cells and decreases turbidity
due to presence of abnormal protein such as lipoprotein.
4._____________________- shortens conversion time from 10-15 mins into 3
minutes and prevent precipitation of abnormal globulins(MM and WM)

**CYANMETHEMOGLOBIN is measured at______.

**OD of the solution is directly proportional to the Hemoglobin concentration
**This method measures all forms of hemoglobin except:
________________________
 C. FLAGGING
vABNORMAL WBC HISTOGRAM PATTERNS
vREGION CODE ”R “ FLAGS / ERROR FLAGS-
signal irregularities in the WBC distribution .
R1-warns of increased interference in the area left of
the lymphocyte peak at approx. 35 fl (due to sickled
RBCs, NRBCs,clumped/giant platelets)
R2-warns of excessive overlap of cell populations
lymphocyte/ mononuclear cell boundary(due to
Atypical lymphocytes, Blast, plasma cells)
R3- warns of excessive of cell population at the
granuclocyte/ mononuclear cell boundary at approx.
160 fl (due to bands, metamyelocyte)
R4-warning due to extension of cell distribution past
the upper end of the WBC threshold at approx 450 fl
(most commonly occur if granulocyte is very
high)
 C. FLAGGING
RM-error code for MULTIPLE REGION OVERLAP
H- when a parameter value is higher than the set normal
limit.
L-when parameter value is lower than the set normal limit.

Others:
@ :data is outside the linearity limit
* :data is doubtful
+ or - :data is outside the reference limit
---- :data does nota appear due to analysis error or
abnormal sample
++++ :data exceeds display limit
 RADIOFREQUENCY
§ Radiofrequency (RF) resistance is a high-voltage
electromagnetic current flowing between the electrodes to
detect the size of cells based on the cellular density.
§ RF is a high-frequency pulsating sine wave.
§ Conductivity or RF measurements provide information
about the internal characteristics of the cell.
Ø When exposed to high-frequency current, the cell wall acts as a
conductor.
Ø As the current passes through the cell, measurable changes are
detected.
§ The cell interior density or nuclear volume is directly
proportional to pulse size or a change in RF resistance.
Ø The nuclear to cytoplasmic ratio, nuclear density, and cytoplasmic
granulation are determined.
 OPACITY
§ Is a mathematical equation derived from the
ratio of RF and DC.
§ It represents conductivity related to cellular
density and the internal structure of cells.
§ Nuclear cells have low density.
§ Cells with a higher nuclear-to-cytoplasmic ratio
have low opacity.
§ WBCs that have more cytoplasm have higher
opacity.
 OPTICAL LIGHT
SCATTERING/DETECTION
 OPTICAL LIGHT
SCATTERING/DETECTION
OPTICAL SCATTER SYSTEM = FLOW
CYTOMETERS
•Uses laser or non laser light
•It is a technology that analyzes thousands of
cells individually and rapidly for various physical
or other properties.
•Used to obtain quantitative measurements of
multiple individual cellular properties.

EXAMPLE: TECHNICON H ANALYZER**

Take note that in Technicon H system: A
ratio of signals is used to determine the
degree of LOBULARITY OF THE NUCLEI.
This information used to indicate degree of
left shift present(LOBULARITY INDEX)

TECHNICHON H
ANALYZER
 OPTICAL LIGHT
SCATTERING/DETECTION
• Cells are detected and counted as they pass
through a focused BEAM OF LIGHT .
• Instruments using light scatter methodology that
are capable of making multiple measurements of
individual cells processed in a flowing fluid are
termed “flow cytometers”
 OPTICAL LIGHT
SCATTERING/DETECTION
• Lights sources:
üTUNGSTEN HALOGEN LAMP
üHELIUM NEON LASER
ü*TAKE NOTE: LASER LIGHT is the most
common light source used in FLOW
CYTOMETERS because of properties of
intensity, stability and monochromatism.
 PRINCIPLE OF OPERATION:
§ A sample of blood is diluted
with isotonic diluents and
then hydrodynamically
focused through a quartz
flow cell.
§ Cells pass through a flow
cell on which a beam of
light is focused.
§ The light source is a laser
light that is light
amplification by stimulated
emission of radiation.
Ø Laser light or
monochromatic light is
emitted as a single
wavelength.
 PRINCIPLE OF OPERATION:
§ As the cell passes
through the sensing
zone, light is scattered in
all directions.
§ Photodetectors sense
and collect the scattered
rays at different angles.
§ These data are then
converted to an electric
pulse.
Ø The number of pulses
generated is directly
proportional to the
number of cells passing
through the sensing zone.
§ The patterns of light are measured
at various angles:
1. LOW ANGLE FORWARD LIGHT SCATTER (2-
10 deg) – CELL SIZE/VOLUME
2. SIDE/RIGHT ANGLE LIGHT SCATTER (90
deg)- CELL GRANULARITY/INTERNAL
STRUCTURE
THREE INDEPENDENT PROCESSES
IN OPTICAL SCATTER:
1.DIFFRACTION-bending of light around the
corners using small angles
2.REFRACTION-bending of light because of
change in speed using intermediate angles
3.REFLECTION-light rays turned back by
obstruction using large angles.
 OPTICAL SCATTER
§ Cell counts, size, cell structure, shape, and
reflectivity are determined by the analysis of the
scatter light data.
Ø Forward angle light scatter (0 degree) is diffracted
light which relates to volume.
Ø Forward low angle light scatter (2 to 3 degrees)
relates to cell size or volume.
Ø Forward high-angle scatter (5 to 15 degrees)
relates to the internal complexity or refractive index
of cellular components.
Ø Orthogonal light scatter (90 degrees) or side scatter
is a combination of reflection and refraction and
relates to internal components.
 SCATTERPLOTS
Ø Are graphic representations
of 2 or more measurable
characteristics of cells.
Ø The 3-dimensional plots use
conductivity and light scatter
to visualize & analyze cell
data.
Ø It provides information about
population abnormalities &
subpopulation of cells.
Ø Prominent cell populations
viewed by scatterplots
include lymphocytes,
granulocytes and
monocytes.
 VCS Technology (Volume,
Conductivity, and Scatter)
§Low-frequency current measures
volume, while high frequency
current measures changes in
conductivity
§Light from the laser bouncing off
white cells characterizes the
surface shape and reflectivity of
each cell.
§This technology differentiates
white cell characteristics.
§VCS: single channel that
analyzes approximately 8,000 cells
in a near-native condition.
 VCS Technology (Volume,
Conductivity, and Scatter)
Volume (V)
§The cell volume is measured by
electrical impedance using low
frequency direct current.
§To ensure accuracy, Coulter has
incorporated pulse editing and
sweep flow technology.
§This technology allows the cells
that are being counted to line up
in a single file to ensure size
measurement integrity and to
prevent cells from being counted
twice.
 VCS Technology (Volume,
Conductivity, and Scatter)
Conductivity (C)
§Radio frequency
penetrates the cell which
generates the data points of
cell size and cell internal
structure.
§Conductivity is measured
by using high-frequency
electromagnetic current for
nuclear and granular
constituents
 VCS Technology (Volume,
Conductivity, and Scatter)
Scatter (S)
§Mid-angle scatter detected
by a beam of laser light
which generates data about
cellular granularity and cell
surface structure.
§Conductivity is influenced by
the internal structures of the
cell such as the nuclear-to-
cytoplasm ratio and the
cytoplasmic granular content.
 VCS Technology (Volume,
Conductivity, and Scatter)
§ A monochromatic
helium:neon laser is the
light source to measure light
scatter for surface structure,
shape, and granularity.
§ Forward angle light scatter
is affected by cell shape,
surface characteristics, and
cytoplasmic granular
content.
§ The enumeration of relative
percentage and the absolute
number of each five cells are
displayed in a scatter plot.
 Hydrodynamic Focusing
§ This is a technique that
narrows the stream of
diluted cells to single file,
while passing through
the detection aperture.
§ After passing through the
aperture, the cells are
then directed away from
the back of the aperture.
§ This process eliminates
the recirculation of cells
and the counting of cells
twice.
 Flow Cytometry
§ It uses a laser light source to measure the physical or
antigenic characteristics of cells, viral particles, DNA
fragments, bacteria and latex beads as they pass
through a beam of laser light.
§ Each cell can be measured for the intensity of
scattered light, including cell size and density, nuclear
complexity, cell granularity and the staining capacity of
dye in each cell.
§ It is a core component of hematology cell counters
and the technology used to differentiate white blood
cells.
Ø used to help characterize acute and chronic leukemia and
lymphomas.
Ø Reticulocyte counts can be counted by this principle.
§ As each particle passes single-file through a laser light
source, it produces a characteristic light pattern that is
measured by multiple detectors for scattered light
(forward and 90 degrees) and fluorescent light (if the cell
is stained with a fluorochrome).
Fluorescein isothionate (FITC) – the most common
fluorochrome used
 Flow Cytometry
§ Application
1. Immunophenotyping
2. Diagnosis and staging of leukemia/lephma
3. Lymphocyte screening panel: AIDS patiemts
4. DNA content analysis
5. RNA content
6. Enzyme studies
7. Fetal cell enueration
 Flow Cytometry
Use of Flow Cells:
§ Flow cells are composed of quartz rather than
glass and provide a better atmosphere in which to
measure cellular qualities.
§ Light does not bend and UV light can pass through
the flow cell.
§ The flow cells measure cell volume, internal
content, and cell surface, shape, and reflectivity.
 Flow Cytometry
Median Angle Light Scatter (MALS)
§ Is defined as the light scatter information that is
obtained between the 10-degree and the 70-degree
angles.
§ Light scatter in the median angles is proportional to
cell size, granularity, surface properties and
reflectance.
 Flow Cytometry
Multiple Angle Polarized Scatter Separation
(MAPPS)
§ Each cell is analyzed through detection of scattered
laser light or flow cytometry and is subjected to
various angles to separate cells into cell populations.
§ A cell suspension is prepared with a diluent, which
maintains the WBCs in their native state, which is
then passed through an air-cooled Argon ion laser
light source.
§ The WBC count and differential are derived from this
patented optical channel.
§ A unique design of Cell DYNE
 Flow Cytometry
Multiple Angle Polarized Scatter Separation
(MAPPS)
• The four degrees of
separation are:
ü 0 degrees (low angle)
measures cell size.
ü 10 degrees (wide
angle) measures cell
complexity.
ü 90 degrees
(Orthogonal) measures
cell lobularity
ü 90 degrees depolarized
(90D) measure cellular
granularity.
 Flow Cytometry
Multiple Angle Polarized Scatter Separation
(MAPPS)
ØNeutrophils and eosinophils
are separated from
mononuclear cells by
plotting 90 light scatter
data, which are on the y-
axis, and wide-angle
forward light scatter data,
on the x-axis.
ØEosinophils are separated
from the neutrophil Ø The lymphocyte, monocyte, and
basophil populations can be
population by the
separated by plotting low angle
eosinophils’ ability to
forward light scatter on the y-axis
depolarize the polarized and wide-angle forward light scatter
light scatter. on the x-axis
 DATA REPORTING
§ CBC is reported numerically.
§ Differential count is numerically recorded and
graphically displayed.
Ø Scatterplots/scattergrams place a specific cell on a
grid identification system. Provide colorful imaging
of normal and abnormal cells.
Ø Histograms measures size threshold of cells
compared with the normal data for each of the cells.
 Common Errors in Automation:
ERROR
Missing § Any WBC or RBC count grossly out of the
parameter(s) normal range must be treated with suspicion
and the sample must be repeated.
§ The “rule of three” (i.e. three times the Hgb
value should agree +/- 3 % with Hct value). If
it does not, the sample must be repeated.
§ Any one of the indices being “singly” put of
range must be considered suspicious.
Carryover from § Carryover results from incomplete removal of
high to low WBC all WBC from the counting chamber between
counts can amount counts.
to a 2% to 3% ü If the ratio of successive counts exceeds
3:1, the second count may be in error by
error.
as much as 5%.
ü It may be necessary to repeat any low
WBC count that follows a high count.
 Common Errors in Automation:
ERROR
Increased WBC § Increased Hgb values because of
counts of increased cellular turbidity in the WBC
>50,000/mm3 counting baths.
§ MCV and HCT may also be increased
because the number of WBCs is high
enough to produce error when sized and
counted in the RBC aperture bath.
High patient § high MCV and Hct with a low MCHC.
glucose
concentrations of
>400 mg/dL result
in intracellular
hyperosmolality in
RBCs
 Common Errors in Automation:
ERROR
Cold agglutinins in § Falsely high MCV, low RBC counts and
high titer very high MCHCs.
ü A good clue for this problem is to look
for RBC clumping in a thin area on the
blood smear.
ü Warming the blood or diluent to 37 C
may eliminate this problem.
Very high plasma
§falsely increase the Hgb, MCH and MCHC.
lipid levels resulting
in lipemic plasma
may produce
turbidity in the
WBC aperture bath
 VARIOUS INSTRUMENTS:
BRAND Description
§ Beckman- Coulter Gen S, Coulter LH series, UniCel DxH
Coulter Coulter Analysis System
• Uses VCS technology
• LH series includes nRBC enumeration
• Retic is stained with NMB and analyzed by VCS.
(Immature retic fraction , mean retic volume,
RET%, Retic#)
§ Abott Cell-Dyn system (Cell-Dyn Ruby, Cell-Dyn
Sapphire)
§ Uses 3 independent measurement
technologies:
1. Optical channel for WBC & differential
2. Impedance channel for RBC and Platelets
3. Hgb channel for hgb
§ Uses MAPSS
 VARIOUS INSTRUMENTS:
BRAND Description
§ Sysmex XT, XE , XS and XN series
• Uses electrical impedance, hydrodynamic focusing
and fluorescent flow cytometry with light scatter
• Uses cyanide-free reagent for hgb determination
(555 nm)
• Platelets are measured by impedance and
fluorescent optical (PLT-O)
• Stromatolyser-NR for nRBC enumeration
• XE-5000 and XN has body fluid mode
§ CellaVision • CellaVision DM96
Automated • Is an automated cell identification technology
Digital Cell that locates cells and pre-classifies and
Morphology characterizes WBC and RBC images.
 VARIOUS INSTRUMENTS:
BRAND Description
§ Advia Technicon Series, Advia 120/2120 from Siemens
Healthcare Dx.
• Uses Unifluidics technology, flow cytometry
• Platelets are measured through volume (size)
and by refractive index (density)
• CSF analysis
§ Mindray BC series
• Uses electrical impedance, flow cytometry and
light scattering
§ ABX Pentra
• Uses impedance and cytochemistry
Pictures of Instruments:
 QUALITY ASSURANCE AND
QUALITY CONTROL
§ Provide guidelines for instrument calibration,
calibration verification, instrument validation
and QC.
§ Calibration procedures are established by the
manufacturer to ensure accuracy and precision.
v Must be performed at initial installation
v Should be verified every 6 months
§ Calibration verification should be performed
after part replacement or instrument repair.
 QUALITY CONTROL
§ QC involves the use of stabilized control
material with established values to verify that
the instrument is running properly.
§ It monitors and checks the analyzer’s
performance. (accuracy and precision)
§ Usually three level of controls are used.
§ It should be run for the following reasons:
1. when the reagent lot number has been changed
2. After calibration
3. After an unusual shift or trend in patient results
4. After maintenance.
LABORATORY
INFORMATION
SYSTEMS
(LIS)
 Computer Basics
§ Central Processing Unit (CPU)
§ Random Access Memory (RAM)
§ Peripheral devices external to the CPU
ü Data Storages Devices (CD, floppy disks, USB,
etc.)
ü Hard Drives
ü External Drives
ü Softwares (OS, application programs, etc.)
 LIS:
§ A collection of data that is structured using one of
several different database architectures:
Ø One common approach is the relational database, a
collection of data items organized as a set of tables
from which data can be accessed or reassembled in
many different ways without having to reorganize the
database tables.
Ø The standard user and application program interface to
a relational database is the structured query language
(SQL). SQL statements are used both for interactive
queries for information from a relational database and
for gathering data for reports.
Ø In addition to being relatively easy to create and
access, a relational database has the important
advantage of being easy to expand.
 Laboratory Information System
§ Computer Technology:
1. Specimen processing
2. Inventory control
3. Quality control
4. Online monitoring
5. Data entry on patient’s charts
6. Data interpretation
 LIS: Functions
1. Pre-analysis
Ø Patient registration (if not received from external
system)
Ø Test ordering
Ø Customized requisitions (e.g., outreach clients)
Ø Phlebotomy draw lists
Ø Bar-coded collection labels and aliquot labels
Ø Specimen tracking/racking system
 LIS: Functions
2. Analysis
Ø Instrument worklist (via interface and automatic
download)
Ø Manual worklist
Ø Manual results entry
Ø Automated results entry via interface
Ø Result validation and manual or automatic release
Ø Quality control
 LIS: Functions
3. Post-analysis
Ø Requisition-based patient reports (final, partial)
Ø Cumulative patient reports
Ø Corrected report
Ø Results inquiry
Ø Electronic reporting to external interfaced systems,
e.g., HIS, billing
 LIS: Functions
4. Management
Ø Pending (incomplete) list
Ø Turnaround time reports
Ø Workload statistics
Ø Ad hoc report writer
Ø HIS and instrument integrity monitoring tools
LIS role in ADT (registration) and order
processing.
The LIS exchanges information with other systems via an
interface engine (see text). Note that the LIS receives
patient demographics and admission/discharge/transfer
information (from the registration system) and electronic
test orders for inpatients (from HIS) and outpatients (from
EMR). It also sends patient results to a variety of
systems.
Overview: Example of facilities and
locations that interact with the LIS
bidirectionally (to exchange orders and
results – purple shading) or
unidirectionally (to receive results only
– yellow shading).