drug assays TDM

why do we do TDM

why do we do TDM

1. drug has narrow therapeutic range (optimal therapeutic effect by a dose near that which elicits toxic effects)

2. relationship between dose and plasma conc is unpredictable, but plasma conc is related to efficacy

3. non linear PK parameters or steep dose-response curve

4. toxicity associated with significant and irreversible outcomes

others: unsure of compliance, renal disease, drug interference on plasma levels, suspected toxicity or abuse, difficulty interpreting clinical evidence of therapeutic or toxic effects

List specimen types + identify which are most common

most common

• venous blood (#2)

• serum (#1)

• plasma

• urine

less common:

• arterial blood

• capillary blood

• cerebrospinal fluid

• feces

• aspirites

• calculi

• tissue and cells

• other fluids (synovial)

what is the diff btwn serum and plasma

preparation of plasma requires anticoagulant

preparation of serum requires blood clotting- leads to loss of fibrinogen/other clotting factors

List the types of tubes and what they are used to sample

Plain tube (no anticoag)- general (serum)

Plain tube w/ SST gel- general (serum)

EDTA anticoag- lipids, red cell analysis, whole blood analysis (plasma)

lithium heparin anticoagulant- general (plasma)

flouride oxalate- glucose, lactate, alcohol (plasma)

trace element- zinc, copper (serum)

heparinized syringe- arterial blood sampling

purpose of centrifugation

Most lab tests require serum or plasma which requires centrifugation to seperate blood cells from serum

these samples have no cells and are much cleaner to permit photometric based analysis

pre analytical factors affecting TDM

dose accuracy

timing of sample

physiological changes in pt

handling/storage of specimen

purpose of semi purification or sample preperation prior to analysis

removes potentially interfering substances

common types of sample preperation

Ultrafiltration

protein precipitation

liquid liquid excraction

solid phase extraction

chromatography

centrifugation

what techniques require semi purification or sample prep prior to analysis

many chromatographic techniques + measurement of free drug level

clinical utility for monitoring free drugs mainly applys to

phenytoin, valproic acid, carbamazepine

mycophenolic acid mofetil and certain protease inhibitors

When and why to use free drug measurement

measurement of serum conc for highly protein bound drugs may be misleading

uremia, liver disease, hypoalbumenia- significantly affects free drug conc but serum conc doesnt change

drug-drug int may cause disproportionate increase in free drug conc

elderly pts have increased free drug due to hypoalbumenia

How do we measure free drugs (technique wise)

Use ultrafiltration to seperate free drug from bound drug + use a assay with good sensitivity

why not to do free drug measurement

less accurate and reproduciable

time and resource intensive- more complicated sample prep and susceptibility to changes in equipment for processing, temperature, pH and dilution

assumed to be similar to total drug levels when the free drug fraction does not change significantly btwn individuals (NOT TRUE)

immunoassay or chromatographic method: which is less labor intesive/fastor turn around time

immunoassay

immunoassay or chromatographic method: which has lack of specificity for parent drug vs metabolite and also cross reactivity is an issue

immunoassay

What things can interfere with immunoassay results

metabolites

similar drugs

bilirubin, lipemia, hemoglobin, paraproteins, heterophillic Abs

immunoassay or chromatographic method: can analyze multiple drugs in a single assay

Chromatographic- specifically liquid chromatography tandem MS

What is a MAJOR concern with immunoassay

Ab specificity

what is the most common analytical method

immunoassay

immunoassay sample requirement

low, <100microL

sample types used in immunoassay

mainly serum and plasma

immunoassay or chromatographic method: which is rapid and sensitive

immunoassay

polyclonal vs monoclonal Abs

polyclonal- population of Abs recognizing a target Ag at several epitopes

monoclonal- single Ab protein recognizing a target Ag at a single epitope

enzymatic label types used in immunoassay

colorimetric

flourescence

luminescence

main enzymes for enzymatic labeled immunoassay

alkaline phosphatase

horseradish peroxidase

examples of assays which use enzymatic labels (vs non enzymatic)

CEDIA, EMIT, FPIA

examples of non enzymatic labels used in immunoassay

particle, radioactive, flourescense

examples of assays which use non enzymatic labels

ELISA, RIA

What is the point of the labels in immunoassays

provide a means by which free antigens or antigen/Ab complexes can be detected

Drug assays are which type of immunoassay

competitive

Competitive vs Immunometric immunoassays

competitive measures small molecules while immunometric measures large molecules

competitive uses a single antibody but competes with labelled analogue of target compound while immunometric uses two Abs and forms sandwich with target

separation technique used in RIA

Immunoprecipiation- Ag + mAb bind, then polyclonal Abs bind to mAB whcih forms immune complexes that can be removed by centrifugation which can allow seperation from unbound/unlabelled drug

steps involved in competitve immunoassay

Add labeled drug
Add sample containing drug and incubant

if low drug conc= high signal

if high drug conc= low signal

explain EMIT

Competition btwn sample Ag + labeled Ag occurs

If labeled Ag wins competition and binds Ab there will be no enzymatic rxn

If sample Ag wins and binds Ab, enzyme is free to cause rxn creating chromagen product

explain PETINA

Conjugate (drug + high MW carrier) can bind to Ab and form complexes which cause turbidity signal by forming light scattering aggregates

drug competes with the conjugate and if it wins it binds to Ab and decreases turbidity signal by inhibiting aggregate formation

explain competitive binding assay

Uses MMP (magnetic microparticles) coated with antidrug antibodies and enzyme labeled drug competed with drug in the sample for binding

following removal of unbound enzyme labelled drug and unbound unlabelled drug, the enzyme from the enzyme labelled drug can convert stable dioxetane phosphate to unstable dioxetane with decomposes to emit light

chromatagraphy sample requirement

variable

chromatography sample type

serum, plasma, blood

what is the purpose of chromatography

seperate compounds for MS measurements

explain gas chromatography

Gas pushes solutes thru a column which volatile substances can detach and attach as the temp of the system is increased

compounds separate based on volatility of the individual compounds (related to mass, structure, charge)

explain liquid chromatography

liquid solvent pushes solutes thru the column

separation of compounds depends on polarity (related to mass, charge and/or structure)

Gas chromatography detectors

nitrogen phosphorous

electron capture

flame ionization

thermal conductivity

mass spectrometry

differences in gas chromatography and liquid chromatography

Gas- volatile compounds while liquid is volatile and non volatile

Gas may require derivatization but liquid does not

Gas usually small molecules (<500Da), liquid usually has less restraints

Gas usually requires excration from biological fluids

Gas chromotography and ionization usually works under high pressure vaacuum, liquid is atmospheric

Ionization methods are diff: gas is electron ionization or chemical ionization, liquid is electrospray, APCI, APPI, MALDI

liquid can have multiplexing

liquid chromatography detector types

Ultraviolet

Flourescence

Conductivity

Refractive index

Mass Spectrometry

LC MS is the preferred method for which drugs

TCAs, antiretrovirals, immunosuppresants

Therapeutic Ranges are based on…

population data from drug therapy trials and RCTs

application of population based ranges to an individual therefore assumes similairty to target pop

Challenges in applying therapeutic ranges

Difference in metabolism, physiology and/or underlying disease process can alter these relationships leading to unexpected and undesirable results

Application of a therapeutic range defined for a drug typically used in combo where the individual TDM situation is not the same drug combo used in the original study where the range was defined

analytical factors impacting TDM results

Method selectivity for target drug vs metabolites

Method reproducibility compared to the therapeutic range

availability of pure standard reference materials

interfering substances in the sample

characterustics of protein binding

Reversible - except for alkylating agents

Differs across drugs

ratio of bound to free is stable- decrease in free results in dissociation of bound to restore equilibrium

acid drugs bind to albumin, basic to AAG

Filtration devices used to measure free drugs

ultrafiltration (most common)

ultracentrifugation

equilibrium dialysis

Free measurements are independant or dependant of changes in protein binding and is pharmacologically active form

Independant

Explain the concept of antibody interferences

Have capture antibodies which the labelled analyte analogue and analyte compete for binding (normal)

Can get heterophillic Abs (abs to the abs) which binds to the capture Abs and causes false +

list endogenous/exogenous factors which affect digoxin immunoassays (cross rxtivity)

Natural medicines- oleander leaves, chinese medicines: Chan Su, Lu-Shen-Wan

K sparing diuretics- eplenorone, potassium canreoate

Digoxin metabolites

Digibind/DigiFab

Issues with blood collection from young children

Uses micro containers and capillary sampling

many pre analytical issues: hemolysis, inadequate volume, improper ratio between blood and anticoagulant

Impact of tubes containing gel

phenytoin, phenobarb, carbamazepinem lidocaine, quinidine, TCAs conc may be lowered

What drugs can or can’t be measured with Tubes containing no anticoag and no gel

all - including free drugs

what drugs can or can’t be measured with the heparin (lithium, sodium, ammonium) tube

Lithium heparin should not be used for Li measurement

Aminoglycosides should not be measured on heparinized plasma if EMIT method

what drugs can or cant be measured with the EDTA tube

preferred for immunosuppressants (whole blood)

mycophenolic acid (plasma)

Correct order for collecting tubes

Blood Culture tubes
Citrate tubes

SST

Heparin tubes

EDTA tubes

Flouride tubes

Carbemazepine interferences in immunoassays

hydroxyzine, cetirizine

carbamazepine 10,11 epoxide

oxcarbezepine metabolite

Phenytoin interferences immunoassays

HPPH and glucuronide conjugate

fosphenytoin and metabolite in uremia

Phenobarbital interferences immunoassays

high amounts of: amobarbital, butabarbital, secobarbital, phenytoin

What is cross reactivity

Occurs when Abs recognize structurally related drugs/compounds

inactive drug metabolites can also cross react

ways to validate drug assays

Trueness- linear range, limit of detection, limit of quantification, analytical specificity- interferences and cross reactivity

Precision- reproducibility, repeatability