webinar lsc basics nov15vp.ppt

11

HUMAN HEALTH • ENVIRONMENTAL HEALTH

© 2015 PerkinElmer

Radiometric Detection Optimize Your Counting Results

Valérie Putmans, M.Sc. | Scientific App. Support Sr. Spec.

Webinar, 13 November 2015

22

Radiometric Detection Steps

Basics on RadioactivitySample Preparation

Radioactivity Detection� Liquid Scintillation Counting Principle� Products offering (cocktails & vials & instruments)

Effect & Correction of QuenchLow Level Counting

33 © 2009 PerkinElmer

Radioactivity Basics

44

Periodic Table

55

(0,7202%)

(99,2743%)

66

What is radioactivity?

Example: 12C consists of 6p+ 6n° 6e- so 12C has an atomic weight of 12 (6p+ + 6n°). The 13C isotope has 7n°, and the 14C isotope has 8n°. Their atomic weight are 13 & 14.

RADIOACTIVITY = When the nucleus of an atom is unstable, it will decayinto a more stable form. During this process energetic particles are emitted (known as radiation) due to the disintegration of the radioactive element

p+ constant (# p+ = # e- for the charge balance), BUT the number of neutronsmight vary, and those possible forms are called "ISOTOPES"

The various forms (isotopes) of an element might be stable or unstable.

77

What type of radiation?

There are three ways for an unstable nucleus to decay.It may give out:

� An alpha particle ( α )� A beta particle ( β )� A gamma rays ( γ ) and/or X-ray

What is measured? � Number of disintegrations (or counts) per minute (or second)

DPM / CPM - DPS / CPS

� Ratio between D and C = "counting efficiency"

Tissue Aluminum Concrete Lead

++�

��

-

CPM

Eff.DPM =

32 = 15 p+ + 17 n° (32P)

32 = 16 p+ + 16 n° (32S)

β-

88

Beta Particle Radiation & Spectrum

Energy


Sample Preparation

1010

Why talking about Sample Preparation ?

Good sample preparation = Good LSC counting!

Aqueous samples Biological samples Solid Supports

(water/buffer or organic samples)

(tissues, organs, plant material, …) (filters, gels, …)

� Using the LSC cocktail specific to the sample nature assures the best efficiency and most reliable results

� The scope is to obtain a mixture...• liquid• as clear as possible• with the least interferences

... to assure that a maximum of radiations will reach the scintillating molecules

� The preparation of the sample will vary from sample to sample:

1111

Examples of Specific Needs

DirectCounting

� Addition of raw sample to a suitable cocktaile.g. Aqueous, Organic , Urine, Serum, Plasma, Water soluble protein

� Selection of cocktail < acids, alkali, double/triple bonds, halides, ... �!

1212

� Dissolution / chemical digestion of sample < solubilizere.g. Blood, Plasma, Serum, Feces, Homogenates & Bacteria


DirectCounting

Solubilization



2. Add solubilizer (Soluene-350 / Solvable) and heat at ~55ºC

1. Add sample to glass vial

3. Add peroxide to decolorize

4. Add LSC Cocktail

5. Dark adapt before counting

1313



DirectCounting

Solubilization

Combustion

� Oxidation / burning sample to convert the carbon and hydrogen to 14CO2 and 3H2O respectively from biological, environmental and industrial samples < oxidizer

e.g. for all of the above sample types without exception

� Addition of raw sample to a suitable cocktaile.g. Aqueous, Organic, Urine, Serum, Plasma, Water soluble protein


1414



DirectCounting

Solubilization

Combustion

FiltersCounting

� Dry / Wet / Solubilized filter (glass fiber, paper, membrane, ...)e.g. DNA, TCA precipitates, tritiated thymidine, receptor-ligand binding …, isolated or collected on a solid support

� + all users of radioactivity perform Swipe Tests �!



� Oxidation / burning sample to convert the carbon and hydrogen to 14CO2 and 3H2O respectively from biological, environmental and industrial samples < oxidizer

e.g. for all of the above sample types without exception

1515

Recommended Cocktail for Various Filters

Filter Type Filter-Count Ultima Gold F Ultima Gold MV Soluene-350 + Hionic-Fluor

Glass FiberDry X X XWet X XDissolved

Cellulose NitrateDry X XWet XDissolved X

Cullulose AcetateDry X X XWet X XDissolved X

Mixed Cellulose EstersDry X XWet XDissolved X

PVCDry X XWet XDissolved X

PolyacrylonitrileDry X X XWet X XDissolved

PolycarbonateDry X X XWet X XDissolved

TeflonDry X X XWet X XDissolved

NylonDry X X XWet X XDissolved

PETDry X X X XWet X X XDissolved

Normal PaperDry X X X XWet X X XDissolved

1616

Swipe Assays – Suggested Methods & Materials

� Aqueous accepting cocktaile.g. Ultima Gold, OptiFluor, InstaGel Plus

Sample Preparation� Dampen the filter with water/ethanol (1:1)� Wipe the test area (100 cm²)� Place the filter in a 20 mL LSC Vial and

add 1 mL water/ethanol� Add 10 mL cocktail and shake to form a

clear mixture� Allow to dark and temp adapt in the

counter for 15 minutes and count

PerkinElmer Application Note # 008365_01 (2008)

Swipe Materials LSC Cocktails

� Filters• Paper (absorbent)• Glass Fiber (GF/A, GF/B or GF/C)

� Cotton buds� Nylon disks

1717



Radioactivity Detection� Liquid Scintillation Counting Principle� Products offering (cocktail & vials & instruments)



Detection of Radioactivity by Liquid Scintillation Counting

1919

Liquid Scintillation Counting is used to measure the quantity of radioactivity coming from beta-emitting isotopes present in several types of samples

LSC requires specific “cocktails” to absorb the energy emitted by these radioisotopes and re-emit it as flashes of light

To accomplish these two actions (absorption and re-emission), cocktails contain two basic components:� the solvent� the phosphor(s) / “scintillator(s)”

What is LSC?

Many cocktails also contain detergents / surfactants to allow the mixing with aqueous samples (= microemulsion)

2020

Liquid Scintillation Cocktail

Scintillation Counting

Solvent

Emulsifier

Fluor

Classical like toluene, xylene pseudocumene, or safer alternatives like DIN, alkyl benzene or PXE

A detergent / surfactant type molecule that ensures proper mixing of aqueous samples

Fluorescent solutes (primary & secondary scintillators)

Components:

2121



Beta particles are emitted, which cause solvent molecules to become excited

excited solvent molecules

2222

What does a Cocktail look like?

Clear microemulsion ‘Milky” appearance 2-phase separation

GOOD

On standing

BAD

2323



The energy of the solvent molecules is transferred to the fluor molecules, which in turn emit light

excited fluor molecules

lightphotons


Liquid ScintillationCocktails & Vials

2525

Classical vs. Safer LSC Solvents

� Hazardous if swallowed� Hazardous by inhalation� Irritating to respiratory system, skin

and eyes� High vapor pressure� Low flash point (flammable liquid and

vapor)

Classical CocktailsToluene, Xylene, Pseudocumene

� Hazardous if swallowed� Lower toxicity� Irritating to skin and eyes� Low vapor pressure� High flash point (non-flammable)� (Biodegradable)

Newer Safer* Cocktails DIN, PXE, LAB

* why SAFER not just “safe”? NO chemical can be properly classified as “SAFE”

�� “safer than” the classical solvents such as Toluene, Xylene and Pseudocumene

2626

Sorting of Liquid Scintillation Cocktails

(1) Historically, the cocktails have been formulated with classical organic solvents. These are fairly flammable, irritating and hazardous. Later on, other organic solvents have been tested and adopted as safer alternatives (non-flammable, lower toxicity, biodegradable), with similar chemical characteristics for counting applications

(2) As explained earlier, many cocktails contain a surfactant, allowing the cocktail to be mixed with aqueous samples. They are called emulsifying cocktails. The ones which are purely organic are called lipophilic.

CLASSICAL(1) "SAFER"(1)

LIPOPHILIC (2)

Insta-Fluor Plus Ultima Gold FBetaPlate ScintOpti-Fluor OMicroScint-O / -EHigh Efficiency Mineral Oil Scintillator

EMULISFYING (2)

BioFuor Plus / Pico-Fluor Plus(former Pico-Fluor 15 / 40)Hionic-FluorInsta-Gel PlusFilter-CountFlo-Scint A / II / III

Ultima Gold / AB / LLT / MV / XROptiSafe HiSafe 2 / 3 / SuperMixOpti-FluorInstaSafe GelMicroScint PS / 20 / 40Ultima-Flo M / AF / AP

2727

How to choose a cocktail?

Beside the aspect on safety and the need to use a lipophilic or emulsifying cocktail, the cocktail selection depends on the sample(chemical composition, volume, pretreatment).

Ultima-Flo / Flo-Scint � flow scintillation counting coupled to HPLCHionic-Fluor � highly ionic solutions (high salts concentration)Insta-Gel Plus / InstaSafe Gel form a gel when mixed with large volumes of waterFilter-Count � dissolves some types of filtersMicroScint , SuperMix and BetaPlate Scint � microplates countingthe Ultima Gold range is the golden standard in scintillation cocktails and offersvariations adapted to specific assay conditions: AB (alpha/beta discrimination), F(dry filters), LLT (low level tritium), MV (mini-vials), XR (extra-large volumes)

�� The cocktail selection is NOT isotope-dependent �!

2828

What vials are best? Glass or Plastic ...

� optical clarity / good visibility

� chemically inert, suitable with solvents and aggressive reagents � lower background than glass (absence of

40K which is the largest contributor to background in glass vials)

� Polyethylene produced from petrochemicals is preferred since the raw materials contain minimal measurable background

� shatterproof and therefore safer in the laboratory

� combustible and therefore easier for waste disposal

2929

Beta counters / LS counters

Tri-Carb ( 4810TR / 4910TR / 5110TR / 6220 Quantulus GCT )

MicroBeta²

3030






Coping with Quench in Liquid Scintillation Counting

Tips and Techniques

3232

ββββ hννννradioactivemolecule

solventmolecule

fluormolecule

photomultipliertube

The Basic Liquid Scintillation Process

Chemical Quench

Color Quench

Physical Quench

Ensure that you have a homogeneous solution to avoid physical quench

XX

3333

Quenching in LSC (e.g. 3H)Co

unts

Spectralyzer Channels (keV)

Region A

Region B

CPM DPM

1 60,000 100,000

2 50,000 100,000

3 37,000 100,000

4 20,000 100,000

5 15,000 100,000

5 4 3 2 1

Quenched

Unquenched

�� "Figure of Merit" = (Eff)²/B FoM(B) > FoM(A)

3434

Quench Correction Methods for DPM Determination

Internal standard� Add known DPM to unknown sample to determine counting

efficiency� Extremely accurate but labor intensive and expensive

Use of Quench curves� Quench parameter SIS (< sample spectral endpoint)� For single isotope with good counting statistics

� Quench parameter tSIE (< external standard spectrum)

Manual Method

Automatic Methods

Direct DPM

Direct DPM� DPM extrapolation from sample CPM's counted in 6

energy windows (related to efficiency of 14C-unquenched standard efficiency in those windows)

� For single isotope with good counting statistics

3535

Determination of the QIP (SIS)

From the β−spectrum distribution we can calculate the average kinetic energy of all β-particles

Spectral Index of the Sample

Quench parameter (SIS) derived from sample spectrum (spectral endpoint)

Increasing Quench

High SIS-Value Low SIS-Value

3636

(gamma)

Quench parameter (tSIE) derived from an external γ-source spectrum

Determination of the QIP (tSIE)

e- (ejected electron)

E1 = v1

(scattered photon)

Spectral Index of the Transformed External standard spectrum (using the RST – Reverse Spectral Transform –technique)

Compton EffectSolvent Molecule

3737

What is a Quench Curve?

Quench Level (sample)

Quench Parameter (SIS or tSIE)

Coun

ting

Effic

ienc

y (%

)

� Low Quench� High Quench Parameter� High Counting Efficiency

� High Quench� Low Quench Parameter� Low Counting Efficiency

3838

What is a Quench Curve?

Quench Level (sample)

Quench Parameter (SIS or tSIE)

Coun

ting

Effic

ienc

y (%

)

� Low Quench� High Quench Parameter� High Counting Efficiency

� High Quench� Low Quench Parameter� Low Counting Efficiency

3939

How are DPM Calculated for Unknowns?

Tritium

0

10

20

30

40

50

60

70

0 200 400 600 800 1,000t-SIE

Efficiency (%)

1

t-SIE is 400

2

CPM is 10,000

3

From Curve

Efficiency = 42%

4

Using:CPM

EfficiencyDPM =

10,000

0.42DPM = = 23,809

CPM

EfficiencyDPM =

4040

tSIE Quench Curves for Tritium and Carbon-14

Tritium

Carbon-14

0

20

40

60

80

100

0 200 400 600 800 1,000

t-SIE

Efficiency (%)

External Standard (tSIE) Quench Curve

CPM

EfficiencyDPM =

4141





4242

What is Low Level LSC and how to count it?

Low level LSC is any counting where the sample activity approaches background

Typically, this means sample activities less than 50 CPM

The object is to lower the background for an increase in signal to noise (S/N) and thus increase both sensitivity and the accuracy of the count result

Increased S/N is achieved by several factors including � proper cocktail selection� count region optimization� shielding and electronic background discrimination (TR-LSC)� instrument settings

4343

Low Level LSC Counting : Background subtract or not?

Samples activity above 500 CPM

� background for 3H ~18 CPM� reported activity of 518 CPM

� error of reporting of about 3.6%

� errors of 5 (-10) % usually acceptable and considered accurate enough

No background subtract needed

Samples activity between 50 and 500 CPM

� background for 3H ~18 CPM� reported activity between 68 CPM

and 518 CPM

� reporting errors between 36% and 3.6%

� becomes unacceptable for lower sample activities

Use background subtraction for accurate results

4444

Detection Limits Working Expressions

LLD = DLpCi/L

2.22 VE (MDA)

T = Sample Count time in minutes 2.22 = conversion DPM to pCiB = background in CPMV = Volume of sample in litersE = CPM/DPM (fractional efficiency)

L = 4.65 B/T (Limit of Detection)D

4545

Low Level LSC CountingFactors that influence background reduction

Cocktail choice

Plastic vials

Minimize external cosmic influences(heavily shielded room or increased instrument shielding)

Optimize counting regions

Electronic background reduction (i.e. TR-LSC)

4646

Conclusion

... the key is to get the best combination, best protocol !

Any LSC cocktail will work with any counting, BUT ...

Poor sampling can only produce an inadequate sample.

A inadequately prepared sample can only produce unreliable results.

No amount of instrument sophistication can ever produce good results from a badly prepared sample, nor when the scintillation cocktail is not chosen well.


[email protected]

Questions

4848

http://www.perkinelmer.com/cocktails

4949

Assay Support Knowledge db (www.perkinelmer.com/ASKRAD)

5050

Thank You!

webinar lsc basics nov15vp.ppt

Documents