j.paul robinson, purdue university ee 520 lecture 2000.ppt page 1 biomedical technologies for blood...

J.Paul Robinson, Purdue University EE 520 lecture 2000.ppt

Biomedical technologies for blood cell measurements

Introduction to the terminology, types of measurements, capabilities of flow cytometry, uses & applications

• Comparison between flow cytometry and fluorescence microscopy• Scatter• Fluorescence• Sensitivity, precision of measurements, statistics,

populations•Speed, combinatorial measurements (multiparameter)


What can Flow Cytometry Do?

• Enumerate particles in suspension• Determine “biologicals” from “non-

biologicals”• Separate “live” from “dead” particles• Evaluate 105 to 5x106 particles/min• Measure particle-scatter as well as innate

fluorescence or 2o fluorescence• Sort single particles for subsequent analysis

Flow Cytometry Publications/year

YEARS

00

300300

600600

900900

12001200

15001500

18001800

21002100

2400

2700

0 13 2879

113223

480

611

811

940

1,078

1,232

1,494

1,855

2,713

2,332

2,445

1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1991 19921989 1990

Papers

1993 1994

2899

3345

Data taken from Medline search using the keywords: “flow Cytometry”

1995 1996


Papanicolaou1941 - originally studies the reproductive system of primates during the estrous cycle and observed changes in cells exfoliated from the female genital tract during the the cycle- mixed a series of stains to identify changes he observed- Developed for using quantitative cytology and morphology for the exfoliative cytologic diagnosis of cervical carcinoma in humans- developed sets of critical stains and interpretations


Gucker - 1947

• Developed a flow cytometer for detection of bacteria in aerosols

• Published paper in 1947 (work was done during WWII and was classified).

• Goal was rapid identification of airborne bacteria and spores used in biological warfare

• Instrument: Sheath of filtered air flowing through a dark-field flow illuminated chamber. Light source was a Ford headlamp, PMT detector (very early use of PMT)


P.J. Crossland-TaylorSheath Flow Principle

A Device for Counting Small Particles Suspended in a Fluid through a Tube

P.J. Crosland-TaylorBland-Sutton Institute of Pathology

Middlesex Hospital, London, W.1. June 17, 1952 Nature 171: 37-38, 1953

A Device for Counting Small Particles Suspended in a Fluid through a Tube

P.J. Crosland-TaylorBland-Sutton Institute of Pathology

Middlesex Hospital, London, W.1. June 17, 1952 Nature 171: 37-38, 1953

“Provided there is no turbulence, the wide column of particles will then be accelerated to form a narrow column surrounded by fluid of the same refractive index which in turn is enclosed in a tube which will not interfere with observation of its axial content.”


Wallace Coulter

Wallace Coulter - Coulter orifice - 1956 - (as early as 1948) - measured changes in electrical conductance as cells suspended in saline passed through

a small orifice

• Cells are relatively poor conductors• Blood is a suspension of cells in plasma which is a relatively

good conductor• Previously it was known that the cellular fraction of blood

could be estimated from the conductance of blood• As the ratio of cells to plasma increases the conductance of

blood decreases


The Coulter Principle•2 chambers filled with a conductive saline fluid are separated by a small orifice (100m or less)

•Thus, most of the resistance or impedance is now in the orifice.

•By connecting a constant DC current between 2 electrodes (one in each chamber), the impedance remains constant. If a cell passes through the orifice, it displaces an equivalent volume of saline and so increases the impedance.

Wallace Coulter - Coulter orifice - 1948-1956

Cell counter

vacuum

orifice

1998 photo© J.Paul Robinson


Instrument Components

Fluidics: Specimen, sorting, rate of data collection

Optics: Light source(s), detectors, spectral separation

Electronics: Control, pulse collection, pulse analysis, triggering, time delay, data display, gating, sort control, light and detector control

Data Analysis: Data display & analysis, multivariate/simultaneous solutions, identification of sort populations, quantitation


What are the principles?

• Hydrodynamically focused stream of particles

• Light scattered by a laser or arc lamp• Specific fluorescence detection• Electrostatic particle separation for

sorting• Multivariate data analysis capability


Richard SweetRichard Sweet developed the electrostatic ink-jet printer which was the principle used by Mack Fulwyler to create a cell-sorter.


Mack Fulwyler Mack Fulwyler - sorter 1965 - electronic cell volume 1965 -

at Los Alamos National Labs - this instrument separated cells based on electronic cell volume (same principle as the Coulter counter) and used electrostatic deflection to sort. The cells sorted were RBC because they observed a bimodal distribution of cell volume when counting cells - the sorting principle was based on that developed for the inkjet printer by Richard Sweet at Stanford in 1965.

Electronic Cell Volume

After determining that the bimodal distribution was artifactual, this group were able to sort neutrophils and lymphocytes from blood.


The mysterous red cell problem


Kamentsky

Kamensky’s first benchtop instrument the Cytograph. This measured scatter using a He-Ne laser. This particular instrument was a model prior to the fluorescence detection model.

He also built a fluidic cell-sorter to evaluate the cells identified in his RCS An RCS was sent to Stanford for use by Leonard Herzenberg . The unit was also the model for the Technicon D instrument guilt by Technicon.

1970 Model“Cytograph” currentlyat Purdue University



Hydrodynamics and Fluid Systems

• Cells are always in suspension • The usual fluid for cells is saline• The sheath fluid can be saline or

water• The sheath must be saline for sorting• Samples are driven either by

syringes or by pressure systems


Fluidics• Need to have cells in suspension flow in single

file through an illuminated volume• In most instruments, accomplished by injecting

sample into a sheath fluid as it passes through a small (50-300 µm) orifice

• When conditions are right, sample fluid flows in a central core that does not mix with the sheath fluid

• This is termed Laminar flow (Sheath Flow Principle)

• The introduction of a large volume into a small volume in such a way that it becomes “focused” along an axis is called Hydrodynamic Focusing

[RFM]


• Whether flow will be laminar can be determined from the Reynolds number

• When Re < 2300, flow is always laminar

• When Re > 2300, flow can be turbulent

Fluidics - Laminar Flow

Re d v

whered tube diameter

density of fluidv mean velocity of fluid

viscosity of fluid

[RFM]

V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3

Notice how the ink is focused into a tight stream as it is drawn into the tube under laminar flow conditions.

Notice also how the position of the inner ink stream is influenced by the position of the ink source.

[RFM]

Fluidics


Fluidics SystemsPositive Pressure Systems

• Based upon differential pressure between sample and sheath fluid. • Require balanced positive pressure via either air or nitrogen• Flow rate varies between 6-10 ms-1

+ + ++ + ++ + +

Positive Displacement Syringe Systems

• 1-2 ms-1 flow rate• Fixed volume (50 l or 100 l)• Absolute number calculations possible• Usually fully enclosed flow cells

100 l

Sample loop

Sample Waste

Flowcell3-way valve

Syringe


Syringe systems

• Bryte HS Cytometer

3 way valve

Syringe


Fluidics - Particle Orientation and Deformation

“a: Native human erythrocytes near the margin of the core stream of a short tube (orifice). The cells are uniformly oriented and elongated by the hydrodynamic forces of the inlet flow.

b: In the turbulent flow near the tube wall, the cells are deformed and disoriented in a very individual way. v>3 m/s.”

V. Kachel, et al. - MLM Chapt. 3[RFM]


Closed flow cells

Laser direction


Fluidics - Flow Chambers

H.B. Steen - MLM Chapt. 2

Flow through cuvette (sense in quartz)

[RFM]


Flow chamber blockage

A human hair blocks the flow cell channel. Complete disruption of the flow results.



The Elements of Flow Sorting

• Sample Preparation• Hardware Setup• Droplet formation• Timing• Coincidence - Purity and Efficiency• Sterile Sorting Concepts


488 nm laser

+-

Fluorescence Activated Cell SortingFluorescence Activated Cell Sorting

Charged Plates

Single cells sortedinto test tubes

FALS Sensor

Fluorescence detector

Purdue University Cytometry Laboratories


Droplet formation

T. Lindmo, D.C. Peters & R.G Sweet - MLM Chapt. 8

As liquid is ejected into air, it will form droplets. By vibrating the nozzle at a defined frequency, the size of these droplets and the position along the stream where they form can be controlled with great precision.

(Murphy)

Last Attached Droplet

Satelite droplet


Droplet break off

Video of the droplet formation in a sort stream from a Cytomation instrument. Source: Purdue CDROM vol 4, 1998

Video2.mpg


488 x 10-3

Laser power• One photon from a 488 nm argon laser has an energy of

E= 6.63x10-34 joule-seconds x 3x108

• To get 1 joule out of a 488 nm laser you need 2.45 x 1018 photons

• 1 watt (W) = 1 joule/second a 10 mW laser at 488 nm is putting out 2.45x1016 photons/sec

• UV Laser at 325 nm is putting out 1.63x1018 photons/sec• He-Ne laser at 653 nm is putting out 3.18x1018 photons/sec

E=h and E=hc/E=h and E=hc/

= 4.08x10-19 J

Shapiro p 77Shapiro p 77


Light Scatter• Materials scatter light at wavelengths at which they

do not absorb• If we consider the visible spectrum to be 350-850 nm

then small particles (< 1/10 ) scatter rather than absorb light

• For small particles (molecular up to sub micron) the Rayleigh scatter intensity at 0o and 180o are about the same

• For larger particles (i.e. size from 1/4 to tens of wavelengths) larger amounts of scatter occur in the forward not the side scatter direction - this is called Mie Scatter (after Gustav Mie) - this is how we come up with forward scatter be related to size



Optics for forward scatter

scatterdetector

iris

blocker

Laser beam

Stream in air or a round capillary


Brewster’s Angle• Brewster’s angle is the angle at which the reflected light is linearly polarized

normal to the plane incidence• At the end of the plasma tube, light can leave through a particular angle

(Brewster’s angle) and essentially be highly polarized• Maximum polarization occurs when the angle between reflected and transmitted

light is 90o

thus Ør + Øt = 90o

since sin (90-x) = cos x

Snell’s provides (sin Øi / cos Øi ) = n2/n1

Ør is Brewster’s angle


Ør = tan -1 (n2/n1)


Brewster’s Angle



Fluorescence• Excitation Spectrum

– Intensity of emission as a function of exciting wavelength

• Chromophores are components of molecules which absorb light

• They are generally aromatic rings

• The wavelength of absorption is related to the size of the chromophores

• Smaller chromophores, higher energy (shorter wavelength)


Fluorescence• Stokes Shift

– is the energy difference between the lowest energy peak of absorbance and the highest energy of emission

495 nm 520 nm

Stokes Shift is 25 nmFluoresceinmolecule

Flu

ores

cnec

e In

tens

ity

Wavelength


Properties of Fluorescent Molecules

Large extinction coefficient at the region of excitation High quantum yield Optimal excitation wavelength Photostability Excited-state lifetime Minimal perturbation by probe Dye molecules must be close to but below saturation

levels for optimum emission Fluorescence emission is longer than the exciting

wavelength The energy of the light increases with reduction of

wavelength


Fluorescence

Resonance Energy Transfer

Inte

nsi

ty

Wavelength

Absorbance

DONOR

Absorbance

Fluorescence Fluorescence

ACCEPTOR

Molecule 1 Molecule 2


Absorption• Basic quantum mechanics requires that molecules

absorb energy as quanta (photons) based upon a criteria specific for each molecular structure

• Absorption of a photon raises the molecule from ground state to an excited state

• Total energy is the sum of all components (electronic, vibrational, rotational, translations, spin orientation energies) (vibrational energies are quite small)

• The structure of the molecule dictates the likely-hood of absorption of energy to raise the energy state to an excited one



Mercury Arc Lamps

Arc

Lens

Lens




Arc Lamp Excitation Spectra

Irra

dia

nce

at

0.5

m (

mW

m-2

nm

-1)

Xe Lamp

Hg Lamp



Laser Power & NoiseLight Amplification by Stimulated Emission of

Radiation

• Laser light is coherent and monochromatic (same frequency and wavelength)

• This means the emitted radiation is in phase with and propagating in the same direction as the stimulating radiation

• ION lasers use electromagnetic energy to produce and confine the ionized gas plasma which serves as the lasing medium.

• Lasers can be continuous wave (CW) or pulsed (where flashlamps provide the pulse)

• Laser efficiency is variable - argon ion lasers are about 0.01% efficient (1 W needs 10KW power)



Lasers


Goals of Light Collection

• Maximum signal, minimum noise• Maximum area of collection• Inexpensive system if possible• Easy alignment• Reduced heat generation• Reduced power requirement


Optical Collection systems

He-Cd Laser Argon LaserHe-Ne Laser



Interference in Thin Films

• Small amounts of incident light are reflected at the interface between two material of different RI

• Thickness of the material will alter the constructive or destructive interference patterns - increasing or decreasing certain wavelengths

• Optical filters can thus be created that “interfere” with the normal transmission of light



Interference and Diffraction: Gratings

• Diffraction essentially describes a departure from theoretical geometric optics

• Thus a sharp objet casts an alternating shadow of light and dark “patterns” because of interference

• Diffraction is the component that limits resolution



Interference filters

• They are composed of transparent glass or quartz substrate on which multiple thin layers of dielectric material, sometimes separated by spacer layers .

• Permit great selectivity.


Optical Filters

Dichroic Filter/Mirror at 45 deg

Reflected light

Transmitted LightLight Source

• Interference filters: Dichroic, Dielectric, reflective filters…….reflect the unwanted wavelengths

• Absorptive filters: Colour glass filters…..absorb the unwanted wavelengths


Transmitted LightTransmitted LightLight SourceLight Source

520 nm Long Pass Filter520 nm Long Pass Filter

>520 nm >520 nm LightLight

Transmitted LightTransmitted LightLight SourceLight Source

575 nm Short Pass Filter575 nm Short Pass Filter

<575 nm <575 nm LightLight

Standard Long and Short Pass Standard Long and Short Pass FiltersFilters

Standard Band Pass FiltersStandard Band Pass Filters

Transmitted LightWhite Light Source

630 nm BandPass Filter

620 -640 nm Light620 -640 nm Light


Transmission determination

• Constructive and destructive interference occurs between reflections from various layers

• Transmission determined by :– thickness of the dielectric layers– number of these layers – angle of incidence light on the filters

Optical DesignOptical Design

PMT 1

PMT 2

PMT 5

PMT 4

DichroicFilters

BandpassFilters

Laser

Flow cell

PMT 3

Scatter

Sensor

Sample


PMT• Produce current at their anodes when photons impinge upon

their light-sensitive cathodes• Require external powersource• Their gain is as high as 107 electrons out per photon in• Noise can be generated from thermionic emission of electrons

- this is called “dark current”• If very low levels of signal are available, PMTs are often cooled

to reduce heat effects• Spectral response of PMTs is determined by the composition of

the photocathode• Bi-alkali PMTs have peak sensitivity at 400 nm• Multialkali PMTs extend to 750 nm • Gallium Arsenide (GaAs) cathodes operate from 300-850 nm

(very costly and have lower gain)

Signal Detection - PMTs

Cathode Anode

Dynodes

Photons in

AmplifiedSignal Out

EndWindow

• Requires Current on dynodes• Is light sensitive• Sensitive to specific wavelengths• Can be end`(shown) or side window PMTs

Secondary emission


Diode Vs PMT• Scatter detectors are frequently diode

detectors

Back of Elite forward scatter detector showing the preamp

Front view of Elite forward scatter detector showing the beam-dump and video camera signal collector (laser beam is superimposed)

Sample stream




Review of Electronics• Reactance like resistance provides an impediment to the flow of

current, but unlike resistance is dependent on the frequency of the current

• If a DC current is applied to a capacitor a transient current flows but stops when the potential difference between the conductors equals the potential of the source

• The capacitance measured in Farads (F) is equal to the amount of charge on either electrode in Coulombs divided by the potential difference between the electrodes in volts - 1 Farad = 1 coulomb/volt

• DC current will not flow “through” a capacitor - AC current will and the higher the frequency the better the conduction

• In a circuit that contains both inductance and capacitance, one cancels the other out

• The combined effect of resistance, inductive reactance and capacitive reactance is referred to as impedance (Z) of the circuit

• Impedance is not the sum of resistance and reactance• z=(R2+(Xl-Xc)2)½ (Xl = inductive reactance, Xc = capacitive reactance)


Linear and Log circuits

• Linear circuits• Logarithmic circuits• Dynamic range• Fluorescence compensation


Why use linear amps?• The problem with compensation is that it needs to be

performed on linear data, not logarithmic data. Thus, either the entire electronics must be built in linear electronics, which requires at least 16 bit A-D converters, or a supplementary system must be inserted between the preamp and the display.

• We need the dynamic range for immunologic type markers, but we can’t calculate the compensation easily using log amps - certainly not without complex math.

• Flow cytometers amplify signals to values ranging between 0-10V before performing a digital conversion.

• Assuming this, with 4 decades and a maximum signal of 10 V we have:

10 100 1000 10000

1 100mv 10mv 1mv

Factor reduction

pulse output


How many bits?

• Assume we convert linear analog signals using an 8 bit ADC - we have 256 channels of range (2n) (28-256) corresponding to the range 0-10 V

• Channels difference is 10/256=40mV per channel

0 50 100 150 200 250

10V1V

100mV

Channels


Ideal log amp

0 50 100 150 200 250

10 V1 V

100 mV

0 50 100 150 200 250

10 V1 mV

Channels

Linear

Log

1 V100 mV10 mVLog amp


Log amps & dynamic range

Compare the data plotted on a linear scale (above) and a 4 decade log scale (below). The date are identical, except for the scale of the x axis. Note the data compacted at the lower end of the the linear scale are expanded in the log scale.


Data AcquisitionData Acquisition

• Each measurement from each detector is referred to as a “variable” or in flow parlance a “parameter”

• Data are acquired as a “list” of the values for each “parameter” (variable) for each “event” (cell)

[RFM]


Data Acquisition - Listmode

Data Acquisition - Listmode

Event Param1FS

Param2SS

Param3FITC

Param4PE

1 50 100 80 90

2 55 110 150 95

3 110 60 80 30

[RFM]


Data Presentation Formats

• Histogram• Dot plot• Contour plot• 3D plots• Dot plot with projection• Overviews (multiple histograms)


FITC Fluorescence

Mo1

CD4 CD8

CD8

CD45

leu11a

CD20 Tube

ID


Data Analysis

• Frequency Distributions• Gaussian distribution• Normal distributions• Statistics• Skewness and Kurtosis


Coefficient of Variation

Crucial in establishing:• Alignment• Fluidic stability• Staining of cells

MEAN

CV=3.0

CV=3.0

%CV Definition = St.Dev x 100MEAN


Precision - C.V.• Precision: CV• Sensitivity• MESF Units (Mean Equivalent Soluble Fluorescein)

• Accuracy and Linearity• Noise• Background• Laser noise• Shapiro’s 7th Law of Flow Shapiro’s 7th Law of Flow

Cytometry:Cytometry:

““No Data Analysis Technique Can No Data Analysis Technique Can Make Make

Good Data Out of Bad Data!!!”Good Data Out of Bad Data!!!”


One parameter frequency One parameter frequency histogramhistogram

establish regions and calculate coefficient of variation (cv)establish regions and calculate coefficient of variation (cv)cv = stdev/mean of half peakcv = stdev/mean of half peak

# of events for# of events forparticular particular parameterparameter


Histogram AnalysisNormalized Subtraction

• Very accurate• Assumption that control & test histogram are same shape• Match region finds best amplitude of control to match test histogram

False Negatives

Match region


Kolmogorov-SmirnovK-S Test

Flu

ores

cnec

e In

tens

ity

Channel Number

Cum

ulat

ive

Fre

quen

cy D

istr

ibut

ion

50

100

0 50 100 50 100

A good technique for estimating the differences between histograms


Histogram AnalysisIntegration

• Very subjective analysis• Not easily automated• Not good for weakly fluorescent signals

False PositivesFalse Negatives

Fre

quen

cy“Positive” histogram


Histogram AnalysisAccumulative Subtraction

• Very accurate• Assumption that control & test histogram are same shape• Match region finds best amplitude of control to match test histogram

Negative ControlActualNegatives

TestN

umbe

r of

Eve

nts

Cum

ulat

ive

Eve

nts

ActualPositives


Histogram OverlaysHistogram Overlays


Density Dot Plot Density Dot Plot Contour PlotContour Plot

Color of dots can give indicationColor of dots can give indication Identify subpopulationsIdentify subpopulationsof frequency of eventsof frequency of events with proper contour lineswith proper contour lines


log

PE Back gate

Forward gate

1P Fluorescence 2P Fluorescence 2P Scatter


Isometric Plot Isometric Plot 3 3 Parameter Parameter

- simulated surface is created- simulated surface is created - 3 parameter data- 3 parameter data- # of particles used as 3rd parameter- # of particles used as 3rd parameter - 3-D space- 3-D space

Multi-color studies generate a lot of data

1 2 3 4 5 6 7 8 9 10

3 color4 color 5 color

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+

Log Fluorescence

QUADSTATS

Log

Flu

ores

cenc

e

++

-- +-

-+


ANegativePositive

Decision Tree in Acute Leukemia

HLA-DR

TCD13,33

CD19

TdT

CD10

CD20

Mu

B,T

AMLL AML

T-ALL

AML-M3

AUL

?

PRE-BI

PRE-BII

PRE-BIII

PRE-BIVPRE-BV

CD13,33

From Duque et al, Clin.Immunol.News.

j.paul robinson, purdue university ee 520 lecture 2000.ppt page 1 biomedical technologies for blood...

Documents