Chemistry Instrumentation SOLO 1

Automation

• the process by which analytical instruments perform multiple tests with minimal involvement of an analyst

• controlled operation of an appartus, process, or system by mechanical or electronic devices with minimal human intervention

total laboratory automation

refers to a comprehensive, automated system encompassing the preanalytic, analytic, and postanalytic phases of testing

Automation advantages

• assists technologists in test performance

• assesses results of the tests performed

• reduces or eliminates monotonous and repetitive tasks

single channel analysis

• each specimen is subjected to a single process so that only results ofr a single analyte are produced

• ex. osmo

multiple-channel analysis

• each specimen is subjected to multiple analytical processes so that a set of test results is obtained on a single specimen

• ex. CMP

parallel analysis

• all specimens are subjected to a series of analytical processes at the same time and in a parallel fashion

• centrifugal analyzers use discreet pipetting to load aliquots of specimen and reagents sequentially in to discreet chambers

• the specimens are analyzed in parallel

analyzer set up for parallel analysis

either

• a multiple specimen/single chemistry mode (running the same test on several specimens)

• single specimen/multiple chemistry mode (running multiple tests in parallel on one specimen)

sequential analysis

• each specimen enters the analytical process one after another

• each result or set of results emerges in th same order as specimens are entered

batch analysis

• many specimens are grouped in the same analytical session

• only one analyte assay is ran at a time

• different assays may be ran on an analyzer by swithcing reagents

random access analysis

• most common configuration of analyzers

• any specimen, by comman to the processing system, is analyzed by any available process in or out of sequence with other specimens

• each specimen may be analyzed for a different selection of tests

What is the error rate of human entry/misreading of specimen numbers?

1 in 300

What is the error rate of barcode misreading specimen labels?

1 in 1 million

Liquid level sensors

• minimizes splatter

continuous flow

• sample is aspirated though the smaple probe into a stream of flowing liquid, whereby it is transported to analytical stations in the instrument

• peristaltic pumps

discete anlysis

• samplee is aspirated by sample probe and delivered, often with reagent, through the same orifice into a reaction cup or another container

• positie liquid displacement pipettes

fixed volume pipettes

used for delivery of samples and reagents in analyzers that perform a single assay or small variety of tests

selectable-volume pipettes

• allow for the selection of a limited number of predetermined volumes

• used in systems that allow many different applications

carry-over

• transport of a quantity of analyte or reagent from one specimen reaction into a subsequent one

• adequate flush-specimen ratio and wash stations reduce

• in discrete systems, caused by pipetting system

dispoable sample probe tips eliminate:

• contamination of one sample by another inside the probe

• carryover of one specimen into the specimen in the next cup

closed analyzers

can only use reagents from a particular manufacturer

open analyzers

able to use in-house reagents or reagents purchased from suppliers different from the analyzer’s manufacturer

factors that affect the chemical reaction:

• the vesssel in which the reaction occurs (RV)

• the cuvet in which the reaction is monitored

• the timing of the reaction

• mixing and transport of reactants

• thermal conditioning of fluids

bidirectional interface

analyzer can receive data from the patient sample barcode and also transmit results to the LIS in electronic formats

auto-verification

automatically releasing results that have no associated problems

A tray of clear sample cups sits on a lab bench top for an extended period of time. Which one of the following analytes might be affected?

bilirubin

manufacturer-recommended sequence of events

• preventative maintenance

• reagent loading

• calibration

• QC

• operating instrument

• evaluating diagnostic messages

immunoassay

• an assay based on the reaction of an antibody that is specific for an antigen

• most commmonly used IgG

immunoassay label

• a substance with measurable property attached to an antigen, antibody, or binding substance

• easiest way to identify an assay

radioactive labels

• first assay developed

• labels used are radioactive isotopes

• no longer used due to safety

enzyme labels

• Ex: ALP, horseradish peroxidase, Beta- galactosidase

• used for antigen or antibody and react with ligand

• an enzyme substrate is added to produce a detectable color change

• conversion of substrate is proportional to the quantity of antigen

enzyme label requirements

• high amount of stability

• extreme specificity

• abscense from the antibody or antigen

• no alteration by inhibitor with the system

fluorescnet labels (fluorochromes/phores)

• compouds that absorb radiant energy of one wavelength and emit radiant enery of a longer wavelength

• generally emitted light is detected at an angle of 90* from the path of excitation light using a fluorometer or modified spectrometer

• fluorescence polarization immunoassay ises polarized light to excite label

stokes shift

the difference between the excitation wavelength and emission wavelength

Luminescent labels

• unitilizes label to emit a photon of light as the result of an electrical, biochemical, or chemical reaction

• examples

  • luminol

  • acridium ester

competitive immunoassay

• reactants are either simultaneously or sequentially mized together

simultaneous method immunoassay

• labeled antigen and unlabeled antigen simultaneously compete with the antibody

• the avidity of the antibody for labeled and unlabeled antigen must be the same

• bound label is inversely proportional to the concentration of unlabeled antigen

sequential method immunoassay

• step 1: unlabeled antigen from the patient is mixed with excess antibody; binding is allowed to reach equillibrium

• step 2: labeled antigen is the added sequentially and allowed to equilibriate; after separation the bound label is measured and is used to calculate the unlabeled antigen concentration

noncompetitive immunoassay (excess reagent, two-site, or sandwich assay)

• immobilized, unlabeled antibody captures the antigen of interest

• a washing removes unreacted molecules

• the labeled detector antibody (conjugate) is added

• another washing removes free labeled detector antibody

• the signal from the bound labeled antibody is proportional to the antigen captured

homogenous assays

• does not require a physical separation of the free and bound labeled analyte

• the activity of expression of the label depends on whether the labeled reactant is free or bound

• it does not require a physical separation step such as washing

heterogenous assay

• uses a physical separations step to separate free and bound labeled analyte

• the labeled, unbound analyte is separated or washed away and the remaining labeled bound analyte is measured

solid phase

• in heterogenous assays physical separation is achieved by interaction with this

• solid particles, beads, trays, or tubes onto which antibody or antigen is absorbed

• used to immobilize reagent antibody or antigen in order to separate free from bound labeled reactant after washing

adsorption

• most common separation technique is the use of paramagnetic particles that can be quickly immobilized to a solid phase by application of a magnetic field

• separation is accomplished by wash steps that occur while the magnetic particles are immobilized by a magnet

Adsorption technique

• unbound constituents and labels are removed by aspiration

• the magnet is removed

• additional reagents are added to produce an alaytical signal

Enzyme linked immunosorbent assay (ELISA)

• heterogenous immunoassay with enzyme label

• use solid phase as separation technique

ELISA formats

• competitive assay using labeled Ag

• competitive assay using labeled Ab

• noncompetitive assay using labeled Ag

• noncompetitive assay using labeled Ab

Enzyme Multiplied Immunoassay Technique (EMIT)

• homogenous assay

• unlabeled Ag competes with labeled Ag for Ab binding sites

• as the concentration of unlabeled Ag increases, less enzyme labeled Ag can bind to Ab

• enzyme is catabolically active when labled Ag is free

calibration

• links the analytical signal with the concentration of analyte

• purpose of curve is to establish a relationship between concentration of analyte and magnitude of signal given by measuring device

interpolation

• connecting the points on the calibration plot to form the line or curve of best fit

• establishes an expected signal range of concentrations of analyte that fall between the lowest and highest on the calibrator

analyte measurement range (AMR)

defines the lowest to highest measurable quantities

if the signal is below the AMR…

the result is typically reported as less than the lower end of the AMR or less than the lowes calibrator used in the clinical laboratory

if the signal is above the AMR…

• the result may be reported as greater than the upper limit of the AMR or greater than the highest calibration used in the lab

• sample may be diluted to bring analyte to concentration within AMR and reanalyzed (the multiplied by dilution factor)

hook effect

excess antigen binds to free labeled antibody, prohibiting the labeled antibody to bind to the capture anitbody

human anti-mouse antibody

are found in individuals who are exposed to mouse antigens and produce FP

heterophile antibodies

are formed in patients who have autoimmune diseases and other disorders and produce FP

point of care testing

analytical patient testing activities provided within the institution but performed outside the physical facilities of the clinical lab

point of care testing locations

• emergency departments, operating rooms, ICUs

• outpatient clinics, physician offices

• urgent care, nursing homes, pharmacies

• counseling centers, ambulances

main advantages of POCT

• smaller sample volumes

• more efficient work flow

• recued TAT

• reduced cost of overall patient care

• improved patient outcomes with immediate result access

disadvantages of POCT

• more costly

• less accuracy

• central lab and POCT results not always comparable

• result integration to EMR challenging

• numerous operators to train

• difficulties with documentation of test results, billing, regulatory compliance

• maintenace of QC and quality assurance of test results difficult

• lot numbers, reagent and control expiration can be problematic

CLIA categories of testing

• waived tests

• moderate complexity tests

• high complexity tests

• non-medical lab tests

CLIA certificate types

• certificate of waiver(waived tests)

• certificate of registration(moderate/high tests)

• certificate of PPMPs(waived tests outside lab+microscopic)

• certificate of compliance(inspection)

• certificate of accreditation(billing)

key components of POCT design

• operator interface

• bar code identification system

• sample and reagent delivery mechanisms

• reaction cell

• sensor

• control and communications system

• data management and storage

• manufacturing requirements

connectivity

provides the ability to transmit patient results to the medical record