AUTOMATED CELL COUNTING

INSTRUMENTATION AND POINT

OF CARE TESTING

Principles of Instrumentation

Electronic Impedance

Also low voltage direct current resistance

Most common method used

Detection and measurement of changes in electrical

resistance produced by cells as the cross a small aperture

Blood cells are suspended in an electrically conductive

diluent

Two electrodes establish an electric current

External electrode located in blood suspension

Internal electrode located within a hollow glass tube

containing the aperture

When cell pass thru the aperture

Causes electric impedance

Measurable voltage pulses

Number of pulses equal number of cells

Size of voltage pulse proportional to size of cell

Pulses collected and sorted according to amplitude by pulse

height analyzers

Data plotted on histogram

Reflects volume distribution of cells

• X axis- size

• Y axis- cell number

May use proprietary lytic systems to allow separation and

quantitation of WBC into several populations

Factors which may affect measurements in

impedance instruments

Aperture diameter

RBC platelet aperture diameter smaller than WBC

aperture

Protein build up

Carryover

Minimized by internal cleaning systems

Coincident passage of more than one cell at a time

Large pulses and coincident passage loss

Coincident correction completed by machine before output

of results

Orientation of the cell in the aperture and

deformability of RBC

Recirculation of RBC back to sensing zone

Solved by sweep flow mechanisms

Abnormal pulses automatically cancelled out

Hydrodynamic focusing

Sample stream is surrounded by sheath fluid as it

passes thru the central axis of the aperture

Narrows the stream of cells to a single file (laminar flow) for

passage thru sensing zone

Eliminates data above and below the focus points

Eliminates recirculation of blood cells

Allows for greater accuracy and resolution of blood

cells

Radiofrequency

High frequency sine wave

High voltage electromagnetic current between

electrodes detects size of cells based on cell density

Pulse size proportional to interior density

As current passes thru cells, it is attenuated by N:C ratio,

nuclear density, and cytoplasmic granulation

Impedance and conductivity can be plotted

against each other

Scatterplot

Cell populations in clusters, number of dots representing

the concentration of the cells.

Allows separation of WBCs into five part differentials

Also known as VCS technology when combined with

lasers to characterize surface shape and reflectivity

of cell

Low frequency: Volume

High frequency: conductivity

Conductivity signal corrected for cell volume

Known as opacity

Light:scatter

Optical scatter

Flow cytometry

May be used as a primary methodology or in

combination with other principles

Cells are diluted and hydrodynamically focused thru

a quarts crystal past a focused light source

Usually a tungsten-halogen lamp or helium neon laser

Laser lights are emitted in single wavelength

Low divergence or spread

Interference to laser beam allows for enumeration and

differentiation of cellular element

Scattered light is converted into electrical signals by

photodetectors

Photodiodes- proportional

Photomultiplier tubes- magnifies weak signals

Filters and mirrors separate wavelengths and

present the wavelength to photodetectors

Lenses with blocker bars prevent nonscattered light

from entering detectors

Forward angle light scatter

Cell volume or size

Side scatter

90 degree light scatter

Results from reflection and refraction of light from

structures inside the cell

Forward low angle and forward high angle scatter

Correlate with cell volume and internal complexity

Differential analysis

COMMON CELL COUNTING

INSTRUMENTS

Hematology analyzers:

Hydraulics

Aspirating unit

Dispensers

Diluters

Mixing chambers

Aperture baths

Glow cells

hemoglobinometer

Pneumatics

Vacuums and pressures for moving valves and samples thru

the hydraulic systems

Electrical system

Controls operational sequence and computing circuitry for

data processing

Instruments will vary from each other

Sample handling

Computer functions

Start ups and shutdown

Internal diagnostics

QC

Maintenance

Calculations

Data storage

Delta checks

Critical values

flags

Coulter instrument

Uses VCS technology

Simultaneous measurement of volume, conductivity and

light scatter provides statistical accuracy

Onyx series

Complete CBC with 3 part differential

STKS and MAXM series

Complete CBC with five part differential

Reticulocyte analysis possible using off line sample

preparation

GEN S, LH 750

On line reticulocyte counts

can perform CD4/CD8 counts

DXH 800•Flow Cytometric Digital Morphology (FCDM) providing 10 times more

data per sample allowing reduced review rates

•multi-angle scatter technology, onboard reagents and new advanced

algorithms

RBC, WBC and Hemoglobin determined directly

Samples are aspirated and divided into two chambers

RBC aperture chamber

2-20 fl- platelets

36 fL- RBC

WBC aperture chamber

Diluent lyses RBCs to free hemoglobin

WBC counted by impedance

• Signals are sent to analyzer for correction and digital

conversion

• 35-90 fL- lymphocytes

• 90-160 fL- mononuclears

• 160-450 fL-Granulocytes

Fluid is delivered to hemoglobinometer for hg concentration

• Trasmittance read at 525 nm

Repeat counts must fall within specified tolerance limits to

be acceptable by the instrument

Allows for good reproducibility

Prevents errors resulting from aperture obstruction and

statistical outliers

Pulse height is measured and categorized

256 channels for WBC and RBC

64 channels for platelets

Histograms are then created

Computerized algorithms allows for flagging

Cell populations overlap

Distinct cell population does not exist

R1 flag

Excess signals at the lower threshold region of the WBC

histogram

High takeoff of histogram

• May indicate nucleated RBCs, clumped platelets, unlysed

RBCs

R2 flag

Loss of valley between lymphocyte and mononuclear

region owing to overlap or insufficient separation

Types of flags

User defined

Primary set for distributional abnormalities

User sets reference ranges and programs instrument to flag

parameters as high and low

Instrument specific

Primary suspect flags for morphologic abnormalities

Possible presence of abnormal cells triggered when cell

population falls outside expected regions

• Or when statistical limitations are exceeded

Calculated values

RBC indices

HCT

RDW

Sysmex Instrumentation

WBC, RBC, Hb, Hct, and platelet counts are

measured directly

Three hydraulic systems

WBC channel

RBC/platelet channel

Sheath stream with hydrodynamic focusing

Hct determined directly based on pulse height generated

by RBCs

Hemoglobin

Hb converted to oxyhemoglobin combining with Na lauryl

sulfate forming a hemichrome molecule

Measured at 555nm

Uses floating threshold to discriminate between cell

populations

Adjusted discrimination levels after particle size

distribution curve is made

Enables differentiation between cells of almost similar

sizes

All other parameters are calculated

K-4500 and KX 21

Provides CBC with 3 part differential

SE-9000/9500

Uses four detection chambers for WBC

Yields 5 part differentials

DC/RF detection system

Granulocytes further analyzed to determine immature cells

• Differential shrinkage and lysis method

Flaggings

Positive and negative flags

XE-2100

Fully automated reticulocyte counts

sysmex

CELL-DYN instrumentation

Abbott laboratories

WBC and differential derived from patented

multiangle polarized scatter separation

Hydrodynamically focused sample stream is directed

through a quartz flow past an argon ion laser

Scattered light is measured at multiple angles

0 degree forward scatter for size

90 degree orthogonal scatter for cellular complexity

90 degree depolarized light scatter for granules

Simply a 90 degree orthogonal light passed thru a polarizer

Scatter information is gathered from the different angles

and presented as scatterplots

May show different combinations

Lobularity vs complexity

Separates mononuclear cells from polymorphonuclear cells

Size vs complexity

Differentiates mono population

Cells falling below lymphocyte clusters are excluded

Granularity vs lobularity

Differentiates poly population

Eosinophils scatter more polarized light

Cell dyne

CELL DYN 1700

CELL DYN 4000

Three channels

Optical channel for WBC and differential

An additional impedance channel is present for comparison

Impedance channel for RBC/Platelets

Hb channel

WBC, RBC, platelets, Hb measured directly

CELL DYN Emerald

CELL DYN Ruby

CELL DYN Sapphire

Bayer Instrumentation

Uses Unified Fluids Circuit (UFC)

Four channels

RBC/platelet channel

Cells isovolumetrically sphered to eliminate optical noise

Flow cytometric light scattering

Differential scatter

• Low angle (2-3 degrees) for size

• High angle (5-15 degrees) for complexity

Eliminates the adverse effect of varied levels of hemoglobin

on RBC deformability

Mie theory of light scattering of dielectric spheres

• Used to plot scatter intensity signals from two angles

RBC volume (y axis) vs Hb concentration (x axis)

Hb Channel

Determined using modified cyanmethemoglobin

Peroxidase

RBC lysed and WBC stained for peroxidase

H2O2 + 4 chloro-1-naphthol + cellular peroxidase= dark

precipitate

Passes thru sheath stream flow cell

Tungsten halogen darkfield optics used to measure:

• X axis: Absorbance (peroxidase content)

• Y axis: Forward scatter (size)

WBC count taken from optical signals in the channel

• Simply used as internal control

Basophil lobularity

Cells treated with non ionic surfactant in acidic solution.

• Basophils resistant to lysis

• All other cells are left with nuclei

Uses two angle scatter

• X axis: high angle, nuclear complexity

• Y axis: low angle, cell size

Cluster analysis differentiates and quantifies cell populations

• Basophils: above horizontal threshold

• Lysed cells cluster into:

Neutrophils: right of x axis

Mononuclears: Left of X axis

Blast cells: below mononuclears

Advia 120

Advia 2120

Automated reticulocyte counts

Provides higher precision

Most employ optical scatter or flow cytometry

Pretreatment of sample with fluorescent dyes or nucleic

acid stains

Sysmex R3000/3500

Stand alone reticulocyte counter using a flow cell

Uses Auramine 0

Forward scatter: size

Side fluorescence: RNA content

• Low, mid, high fluorescence: inversely proportional with

reticulocyte maturity

• Immature reticulocyte fraction: ratio of immature retics to

total RBC

CELL DYN 3500R

Uses MAPPS technology

Offline RBC staining with new methylene blue N

CELL DYN 4000

MAPPS technology with fluorescent detection

Capable of fully automated random access reticulocyte

staining

Uses CD4K530

Proprietary membrane permeable dye that emits green light

Beckman Coulter

Offline preparation for STKS and MAXM

Uses new methylene blue

Reticulocytes have greater optical scatter and greater

opacity

Bayer ADVIA

Uses Ozanine 750

Utilizes three detectors

Low angle

High angle

Absorbance

Scattergrams:

High angle vs absorption

• Absorption scatter reflects amount of staining

• Sum of absorption equals IRF

Low angle vs high angle

Volume vs hemoglobin concentration

• Provides several unique reticulocyte indices

MCVr, CHCMr, RDWr, HDWr, CHr, CHDWr

Useful in evaluation EPO response

• CHr: hemoglobin content of reticulocyte calculated as

product of colume and Hgb concentration

Useful in early diagnosis of IDA in children

LIMITATIONS AND

INTERFERENCE

Calibration

Process of correcting an instrument for analytical

bias

Performed by using

Reference methods

Reference materials

Commercial calibrators

Performed:

Upon initial installation

Every 6 months

After major repair or changes

As required by the manufacturer

Cyanmethemoglobin

Remains the only standard in hematology for calibration

and quality control

Whole blood calibration largely replaced by

commercial calibrators which has been assayed

against reference methods

Calibrator bias due to differences in stability and preserved

cell suspensions

Always verify against reference methods or review

QC data

Instrument Limitations

Limitations are related to methodology employed

Inability to distinguish cells reliably from other

particles or cell fragments of the same size

Non lysed cells may yield inaccurate results

Automated data may be not be released

Failure of internal checks

Flaggings

Ensure that manual reviews are done

Sample Limitation

Cold agglutinins

Incterus

Lipemia

Hemolysis

Nucleated RBC

Platelet clumps

High WBC counts

Sample age

POINT OF CARE TESTING

Point of Care Testing (POCT)

Diagnostic testing done near the site of patient care

Physician’s office

Emergency room

Nurse stations

Special units

Testing site neutrality

It does not matter where testing is done or who performs

the tests so long as it follows same regulatory requirements

CLIA Test classification

Waived

Simple test with insignificant risk of erroneous result

Moderate complexity

High complexity

Most POCT are waived tests with few moderate

complexity tests

Popular due to ease of operation and rapid results

Advantages

Fast results which speed up patient management

Direct patient samples are used with minimal delay

Disadvantages

Cost per test

Oversight of POCT

Appropriate use

Proper method selection

Quality assurance

Personnel training

Needs definite policy and procedures

Quality Assurance

Accuracy and precision

Most have simple QC and calibration process

Minimal handling and procedural steps

Hemoglobin

Measured by modified hemoglobinometers

Hemocue

Utilizes small cuvette with lysing agent and reagents to

form Hb azide

Measured photometrically

Major disadvantage: blood cell contamination with tissue

juice

Stat-Site M meter

Reflectance photometry

Test card composed of molded plastic with fluid well

containing impregnated pads

Hb-Quick

Measures hemoglobin spectrophotometrically

Hematocrit

Centrifuge based microhematocrits

Conductivity based

Measures resistance of RBC to electrical conduction

Affected by low protein levels and elevated sodium levels

Cell counts

QBC II

Measuring device using fluorescence

Ichor Hematology Analyzer