Drug Conc Measurements 1

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-- --- used can affect the tx range

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analytical method

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steps in pharm analysis

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sample collection

sample prep

sample analysis

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sample analysis includes

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chromatography

immunoassays

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sample analysis:

ex of chromatography

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HPLC

GC

TLC

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sample analysis

ex of immunoassays

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RIA

EMIT

ELISA

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blood sample is collected into tube containing ------

after centrifugation, the fluid component is ------

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anticoagulant

plasm

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eg of anticoagulant

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heparin

EDTA

citrate

oxylates

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blood sample is collected in -- tube which contains plastic barriers to separate the fluid and cellular components.

blood allowed to clot and then centrifuged. results in fluid called ----

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SST

serum

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t/f

results are significantly different btw plasma and serum

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f

similar

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most assays performed on

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serum

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anticoagulants in the tube could displace the drug from -- ---- sites

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protein binding

(e.g. quinidine, warfarin)

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saliva collection

t/f

used frequently

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f

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saliva is noninvasive/invasive

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noninvasive

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salivary conc approximate --- drug conc

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free

(not total drug conc)

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saliva collection advantages for AED

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painless

noninvasive

untrained personnel can be taught the collection process

performed at home

sample mailed to the clinical lab or clinic

saliva drug conc could be collected immediately postictally or with the onset of an AE ("real-time" conc)

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immunoassays:

--- (specific) for compound measured

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antibody

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immunoassays:

----- that can be labeld for quantitation

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antigen

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labes for immunoassays:

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radioisotope

enzyme or enzyme substrate

fluorophore

chromophore

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immunoassays:

a --- binding assay can then be developed

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competitive

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heterogenous immunoassays:

requires --- step prior to measurement

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separation

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immunoassays:

no change to label w/ binding to ---

--- and --- are heterogenous

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antibody

RIA

MEIA

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homogeneous immunoassays:

doesn't require ---

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separation step prior to measurement (automation)

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homogeneous

rapid/slow turnaround

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rapid

(STAT labs)

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homo immunoassays:

measurable change in the label w/ . . .

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binding to antibody

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which assays are homo immunoassays:

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EMIT

FPIA

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primary determinant of sensitivity and specificity of the immunoassay is the

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nature of the drug-protein interaction

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immunoassays:

defined by affiity of drug for protein

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sensitivity

(ability to bind to even tiny amounts of drug)

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immunoassays:

defined by cross affinity of other compounds inthe biological matrix

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specificity

(does it bind to anything w/ specific characteristics)

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radioimmunoassays

homo/hetero-geneous assay

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heterogeneous

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radioimmunoassays

competitive/noncompetitive formats

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both

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radioimmunoassays

competitive: what competes

when?

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radiolabeled and unlabeled compete for binding sites on a limited amount of a specific drug-directed antibody

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radioimmunoassays

the amount of label directly correlates w/ the amount of antigen present

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sandwich (noncompetitive) format

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radioimmunoassays

the amount of antigen is inversely correlated w/ the amount of label

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competitive

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enzyme radioimmunoassays

what's rarely used

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RIA's

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enzyme radioimmunoassays

what's commonly used

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EIA

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advantages of enzyme radioimmunoassays

more sensitive than

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EMIT

FPIA

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enzyme radioimmunoassays

inexpensive/expensive

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less expensive

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enzyme radioimmunoassays

disadvantages:

-- consuming

--activity

limited ----- stability

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time

radioactivity

limited reagent stablity

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enzyme radioimmunoassays

types of EIAs

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EMIT

ELISA

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emit is hetero/homogenerous

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homogeneous

doesn't require separion of the free and antibody bound enzyme lable

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enzyme radioimmunoassays

whichis a heterogenous method

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ELISA

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enzyme radioimmunoassays

drug -- bound to an enzyme competes w/ free drug for antibody against drug

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covalently

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enzyme radioimmunoassays

the amount of drug-enzyme conjugate bound to antibody is a fx of the amount of --- ---- present in the system

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free drug

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EMIT

---/----- mixed w/ pt sample

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antibody/substrate

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EMIT

drug in pt sample binds to ---

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antibody

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EMIT

when is enzyme label drug acitve

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when not bound

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EMIT

more active = more ----

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absorbance

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EMIT

serum drug conc is --- to amount of active enzyme

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proprotional

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advantages of EMIT

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simple

specific

sensitive

rapid

can be automated

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difference btw elisa and emit

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emit not all drug-enzyme conjugates are inactivate, which limits drug sensitivity

w/ elisa there separation of antibody-bound and free labeled drug (heterogenous)

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immunoassays: FPIA

homogenouse assay:

rxn carried out in a ---- soln

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single

(doesn't require a wash step)

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basic prinicple of immunoassays: FPIA

------labeled drug competes w/ unlabedled drug for binding to antibody raised against the drug

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fluorescein

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fpia used for

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to provide accurate and sensitive measurement of tx drugs, and drugs of abuse, toxicology and some hormones

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fpia uses 3 key concepts to measure specific anlytes in homogenouse format

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fluorescense

rotation of molecules

polarized light

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rotation of molecules in soln:

larger molecules rotate more slowly in soln that do -----molecules

what's this used for

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smaller

used to distinguish btw smaller antigen-fluorescein molecule, AgF, which rotates rapidly, and teh larger Ab-AgF complexes, whcih rotate slowly in soln

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polarized

--- polarization technology distinguishes antigen-fluorescein (AgF) label from antibody bound-antigen fluorescein (Ab-Ag) by their different fluorescence polarization properties when exposed to polarized light (light waves that are only present in a single plane of space)

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fluorescense

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free labeled drug

level of polarized fluorescen light emitted is high/low

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low

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antibody-bound labeled drug

level of polarized fluorescent light emitted is high/low

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high

so if pt's sample contains a high conc of drug, it displaces most of the labeld drug so the polarized fluorescent light emitted is low

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advantages of fpia

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rapid turnover times

good sensitivity

ease of operation (automation)

available to quantitate a wide variety of drugs

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disadvantage of fpia

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interference

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advantages of immunoassays

---/--- fast, practical (automation)

long reagent -----

small/large sample vol

no ------

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EMIT/FPIA

shelf-life

small

radioactivity

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disadvantages of immunoassays

less sensitive than ---

more --- to perform

-----

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RIA

expensive

interference

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microparticle enzyme immunoassay - MEIA

uses isolation of antibody/antigen complexes on a solid phase surface of --- --- called ----

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small beads

microparticles

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MEIA has been widely adapted to automate the measurement of large molecules such as markers associated w/

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cardiac

fertility

ca

metablic

hepatitis

thyroid testing

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components of MEIA

---- antibody solid phase

antibody-enzyme ----

enzyme ------

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microparticle-antibody solid phase

antibody-enzyme conjugate

enzyme substrate

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microparticle antibody solid phase:

-- microparticles that are coated w/ --- bind specific analyte being measured

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latex

antibody

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antibody enzyme conjugate

--- phosphatase enzyme conjugated to antibody

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alkaline

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enzyme substrate

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fluorescnet 4-methyl umbelliferone phosphate (MUP)

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MEIA uses a --- format

so the amount of analyte is ------- to the amount of signal

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noncompetitive

directly proportional

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chemiluminescent magnetic immunoassay CMIA or CLIA

analytes are labeled w/ --- --- counpounds that produce light when combined w/ a trigger reagent

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chemiluminescent

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CMIA or CLIA

method very similar to ----, though chemilum rxn offers high/low sensitivity and -- of measurement

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MEIA

high

ease

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CMIA or CLIA

uses a noncompetitive/competitive sandwich format that yields results that are directly proportional to the amount of analyte

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noncompetitve

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special considerations of immunoasays

assay interference can either be --- or ---

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cross reactivity

physiologic interferance

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x reactivity r/t --- specificity

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antibody

may x-react w structurally similar compounds

(aminoglycosides gentamicin and netilmicin)

(degradation products - vanco)

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physiologic interference include

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bilirubin, protein

no common

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to ensure quality

include samples of known -- --- conc w/ every analysis run

qc samples are processed along w/ -- ----

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drug/metabolite

unknown samples

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two measures of quality in the lab

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accuracy

precision

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the closer a measured value is to the true value

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accuracy

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the closer measured values are to each other when the same sample is measure multiple times

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precision