Therapeutic Drug Monitoring:

1
Therapeutic Drug Monitoring

• Assays the science

2
Therapeutic Drug MonitoringAssays - the science

• Terminology
• Standard deviation
• Coefficient of variation
• Types of assays
• Advantages, limitations and disadvantages for
  each assay
• Range of sample concentrations

3
In the beginning

• 1950
• Gas liquid chromotography (GLC) - antiepileptics
• Bioassays antibiotics
• Radio-immuno assay (RIA) digoxin
• 1960
• High pressure (later changed to performance)
  liquid chromotography antiarrthymics,
  theophylline, many others as time went on) (HPLC)
• 1970
• Enzyme multiplier immunoassay technique (EMIT)
• Fluorescence polarization immunoassay (FPIA)

4
Assay terminology -1

• Accuracy how close is the value to the actual
  value
• Precision - Reliability / Reproducibility
• How close should subsequent runs of an assay be
  to each other to be considered equivalent?
• Standard deviation
• Mean 2 SD 95 of values
• Mean 3 SD 99 of values
• Coefficient of variation
• Strive for gt 10

5
Standard deviation

• A measure of the average difference of the
  individual values from the mean
• This is an intermediate step in determining
  reliability
• SD S(xi mean)2
• v n - 1

6
Standard deviation problem

• An assay measures values of 10, 11, 9, 12 and 13
  (ignore units for now)
• What are the mean and SD?
• What is the range for 99 of values?
• What is the range for 95 of values?

7
Coefficient of variance

• Standard deviation expressed as a percentage of
  the mean
• Relative variation
• CV (SD/mean)100
• Used to compare the precision between two assays
  (the assay with the smallest CV is the more
  precise)
• What is the 95 and 99 CV for the previous
  problem?

8
Inaccurate and ImpreciseNot valid and not
reliable
9
Inaccurate, but preciseNot valid, but reliable
10
Accurate and preciseValid and reliable
11
Assay terminology - 3

• Sensitivity
• Ability of an assay to accurately quantitate low
  drug concentrations
• Usually the lowest concentration in an assay can
  differentiate from zero
• Specificity
• Ability of an assay to differentiate the drug in
  question from similar or like substances
• Need to have assay run with patient specimens,
  not just healthy volunteers
• Predictive performance
• Mean error measures bias and can be positive or
  negative
• Root mean square error measures reliability and
  the smaller the better
• Used to compare two different assays

12
Assay Methodologies

• Immunoassays
• RIA
• EIA
• FIA (TDx is this)
• Chromotography
• GLC
• HPLC
• Photometry
• Bioassay

13
MethodologiesImmunoassays - 1

• Theory labeled (reagent) and unlabeled (patient)
  drug compete for binding sites on a specific
  antibody
• The antibody-drug complex changes the signal from
  the signal from the known quantity of labeled drug

14
MethodologiesImmunoassays - 2

• Advantages
• Simple
• Reliable
• Specific
• Automated
• Safe (low radiation)

15
MethodologiesImmunoassays - 3

• RIA highly sensitive (picograms)
• Short shelf life (increases costs)
• Nuclear waste
• Cross reactivity
• Used for digoxin and cyclosporine
• EMIT concentration is proportional to absorbance
  of light
• Rapid turn around time
• Sensitive and stable
• Background interference from endogenous
  substances
• Not great at high concentrations
• FIA (TDx assay) concentration inversely
  proportional to polarization
• Sensitive, stable, simple, automated
• Background interference from endogenous
  substances
• AG, CBZ, PYT, Pb, vancomycin, theophylline

16
MethodologiesChromatography

• GLC
• Not used clinically
• Labor intensive extraction and derivation
• HPLC
• Advantages precise, specific, useful for
  multiple drug assays
• Unique feature metabolite identification
• Labor intensive less automated
• Antiretrovirals, TCAs, CAM, antiarrhythmics

17
Antimicrobial TDM Assays
18
Therapeutic Drug MonitoringAssays - the science

• Terminology
• Standard deviation
• Coefficient of variation
• Types of assays
• Advantages, limitations and disadvantages for
  each assay
• Range of sample concentrations