Errors Requesting appropriate tests Writing prescription Reading prescription Sample collection Sampling times environmental factors Drug interferences.

ErrorsRequesting appropriate testsWriting prescriptionReading prescriptionSample collectionSampling timesenvironmental factorsDrug interferencesPatient identification Sample transferTechnician errorsInstrumental errorsMethod limitationsData entry mistakesInterpretation of results

How can we trust

1-Chemiluminescence is the best known method of immunoassay and

2-Automation systems are the best solutions to reduce errors

and

Combination of these two allow us to get accurate, precise and reliable results from laboratories

Which parameters are important in evaluating a method

• Signal / noise

• User safety

• Sensitivity

• Specificity

• Incubation time

• Ease of carrying out

Hormone measurement

• The Problem– very small amounts of hormone in a very complex mixture

• Pre-immunoassay– complex and insensitive methods (chemical methods, whole

animal or tissue bioassay)

– insensitive

– imprecise

– inaccurate

• Immunoassay– first described in 1960

– very rapid expansion since early 1970s

– advantages (simplicity, speed, precision, accuracy, sensitivity)

Relative sensitivities of tests (approx)

Usual operating range [Ab] or [Ag]

precipitationimmunoelectrophoresisdouble/radial diffusion

10 g/ml - 1 mg/ml

immunofluorescence 0.1 - 10 g/ml

ELISA (colour) 0.1 - 10 ng/ml (chemiluminescence) 0.01 - 10 ng/ml

radioimmunoassay 0.01 - 10 ng/ml

In fluorescence this can lead to difficulties with fluorescers with a small Stokes shift. Fluorescence may not be easy to resolve from the exciting wavelength.

Another problem is associated with scattering of the incident light to the detector, especially when samples are somewhat turbid.

Why ELISA cant be fully automated

• Can you tell the difference between how many marks are in each box?

400 360Sensitivity of Absorbance Measurements

Spectrophotometry: Luminescence

Sensitivity of Luminescence

Measurements

• Can you tell the difference between how many marks are in each box?

0 40

Spectrophotometry: Luminescence

Immunoassay Signal Generation

10-14 10-15 10-16 10-17 10-18 10-19

ENZYME COLOUR DETECTION

ENZYME FLUORESCENCE DETECTION

TIME RESOLVED FLUORESCENCE DETECTION

RADIOACTIVITY (I125)

CHEMILUMINESCENCE DETECTION

Minimum Detection Limit (moles of tracer)

Det

ecti

on

Me

tho

d

Sensitivities of different tracers

VITROS ECi DETECTION SYSTEM Vitros ECi

What you want

• Accuracy

• Precision

• Speed

• Sensitivity

• Specificity

Immunoassays:

• Radioimmunoassay (RIA, IRMA)

• Elisa (enzyme linked immunosorbant assay)

• Immunofluorometric assay

• Immunluminometric assay

Immunoassays basics

(virus?)

Physical Concepts in immunoassays:

• Radioactivity

• Spectrophotometery

• Fluorescence

• Luminescence

• The wavelength of light determines how it interacts with matter.

• We use these interactions as a probe to obtain chemical information about samples.

• Spectrophotometry is the use of “light” in chemical measurements

Spectrophotometry

400 nm 750 nm

IRUV

IR typically referred to by wavenumber (10-12,500 cm-1)

Blue - OrangeBlue-green - RedGreen - Purple

Fluorescence

FITC + UV light

Phosphorescence

Chemiluminescence

Bioluminescence

Antigen

HRP label

2nd Antibody

Immunometric Assay on Streptavidin Coated Well

Substrate

Light

1st Antibody

Biotin tagSolid PhasePlastic Well

Streptavidin

Biotin spread evenly over well

Ferritin Antibody: Passive Adsorption

Ferritin Antibody: Biotinylated on Vitros Coated Well

Basic requirements for immunoassay

• Standards

• Specific antibodies

• Labelled antigen or antibody

• Separation system

• Quality control

Advantages of 2-site immunometric assays

• Increased sensitivity

• Increased precision

• Better specificity

• Greater assay range

• Shorter assay times

Disadvantages of 2 site immumetric assays

• Need for large quantities of pure antibody (monoclonal antibodies usually employed)

• 2 antibody binding sites required (limit range of analysis)

• High dose “hook” effect• Need for multiple washing steps• Non specific interference due to heterophyllic

antibodies

Advantages of isotopic labels

• Simple coupling reactions

• Label properties do not alter on coupling

• No background signal

• Efficient/convenient detection systems

• No additional cost to detect signal

• Very useful for research assays

Non isotopic labels - advantages

• No radioactivity– safety aspects– disposal

• Extended life of label• Speed of detection• Ease of automation• Theoretical increase in sensitivity• Possibility of homogeneous assays• Simple/safe label preparation

Non isotopic labels - disadvantages

• Safety aspects of labels/substrates

• Serum/buffer effects

• Extra manipulations in detection

• Inefficient detection in some cases

• No recounting possible in some systems

• Limitation of separation system

• “Dedicated” instruments

• Commercial pressures

Hormone assay in the future

• Dominated by immunoassay techniques• ? GCMS

• Increased sensitivity• Better automation

– computers– robotics– non isotopic labels

• Near patient testing devices

Immunoassay automation

• Non isotopic labels

• Microprocessor power

• Improved robotics

• Better antibodies faster reaction times

Immunoassay Analysers

• Immunological reagents

• Precision usually good

• Wide variations in sensitivity, specificity and accuracy between analysers

• Careful definition of assay requirements

• Whether any one analyser will satisfy all requirements

Types of automated system

• Work simplified batch systems

• Automated batch systems

• Total automation (black box) - random access

• Portable (bedside biochemistry) instruments

Intellicheck Technology – advanced instrument intelligence - monitors and verifies:

Patient sample qualitydetects short samples, clots, bubbles, viscosity

Sub-system performance Sample & reagent volumes in reaction well Reports issues to the operator

Advantages of automation

Single Use Disposable Tip Sampling

No sample carryover

Bubble, Clot, Low & High viscosity detection

Auto dilution facility using Disposable Tip

Ensures sample integrity

Advantages of automation

Intellicheck - Sample Metering Verification Patented Pressure Level-Sensing

technology Bubble, Clot & viscosity detection

Normal Aspiration

Abnormal

Aspirate bubble detected

Pressure+ve/-ve

Intellicheck™ TechnologySample + Reagent Verification

Verification of correct sample and assay reagent volume dispensed into the Microwell

Level sensing technology

Eliminates the potential for a misreported result by not processing an assay when an exception is detected

Detection with automatic recovery

Ensures result integrity

Aspirate Probe

Level sense detection extension integrated with Aspirate Probe

Dispense Probe

Sample +

Reagent

Well Wash Probe Assembly

Microwell

Advantages of automation

• Increased precision

• Work simplification

• Versatility

• Less contact with samples

• Rapid turnaround time

Disadvantages of automation

• Lack of reagent choice

• Total reliance on manufacturer

• Lack of range of analytes

• Little use for “research” assay

• Increased cost