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 fl36 fL-

platelets 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
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

Instrument specific

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

Differentiates mono population Cells falling below lymphocyte clusters are excluded Differentiates poly population Eosinophils scatter more polarized light

Size vs complexity

Granularity vs lobularity

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 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

Passes thru sheath stream flow cell

Basophil lobularity

Cells treated with non ionic surfactant in acidic solution.

Basophils resistant to lysis All other cells are left with nuclei X axis: high angle, nuclear complexity Y axis: low angle, cell size Basophils: above horizontal threshold Lysed cells cluster into:

Uses two angle scatter

Cluster analysis differentiates and quantifies cell populations

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 Proprietary membrane permeable dye that emits green light

Uses CD4K530

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

High angle vs absorption Absorption scatter reflects amount of staining Sum of absorption equals IRF Low angle vs high angle

Scattergrams:

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

Physicians 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