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This article provides an introduction into some of the current techniques and assays utilised in Therapeutic Drug Monitoring (TDM) TDM is a multi disciplinary function that measures specific drugs at intervals to ensure a constant therapeutic concentration in a patient blood stream. The selection of an analytical technique for TDM involves a choice between immunoassay and chromatography technique. Once the methodology has been chosen, there are also numerous options available within these categories including FPIA, EMIT, KIMS, HPLC and nephelometric immunoassay. An overview of each method is given and it's processing of drugs. The future outlook in the methodology involved in TDM is also explored and discussed.
Therapeutic drug monitoring (TDM) is a multi disciplinary function that measures specific drugs at selected intervals to ensure a constant therapeutic concentration in a patient blood stream. (Ju-Seop Kang & Min Hoe Lee) The response to most drug concentrations is therapeutic, sub-therapeutic or toxic and the main objective of TDM is to optimize the response so the serum drug concentration is retained within the therapeutic range. When the clinical effect can be easily measured such as heart rate or blood pressure, adjusting the dose according to the response is adequate (D.J. Birkett et al). The practice of TDM is required if the drug meets the following criteria:
Has a narrow therapeutic range
If the level of drug in the plasma is directly proportional to the adverse toxic
If there is appropriate applications and systems available for the management of therapeutic drugs.
If the drug effect cannot be assessed by clinically observing the patient (Suthakaran and C.Adithan)
A list of commonly monitored drugs is given in table 1.
The advances in TDM have been assisted by the availability of immunoassay and chromatographic methods linked to detection methods. Both techniques meet the systemic requirements of sensitivity, precision and accuracy. Within both methods are many numerous options and will be further explored in this title. Ideally the analytical method chosen should distinguish between drug molecules and substances of similar composition, detect minute quantities, be easy to adapt within the laboratory and be unaffected by other drugs administrated. An overview of the current analytical techniques and future trends in TDM is emphasised in this title and its role in laboratory medicine.
NEPHLEOMETRIC IMMUNOASSAY AND its USE IN TDM
Immunoassays play a critical role in the monitoring of therapeutic drugs and a range of techniques in which the immunoassay can be existed exist. Nephleometric immunoassays are widely used for TDM and are based on the principle of hapten drug inhibition of immunoprecipitation. The precipitation is measured using nephelometric principles that measure the degree of light scattering produced. In some cases Turbidmetry principles can be applied to measure precipitation via the amount of transverse light.
In nephleometric immunoassays, if the drug molecule is a monovalent antigenic substance, a soluble immunocomplex is formed. However if the drug molecule is a multivalent antigenic substance, whereby two drug moieties are conjugated to a carrier protein, the conjugate reacts with the antibody to form an insoluble complex. The insoluble complex may compose of numerous antigens and antibodies, thus scattering the light. Therefore nephleometry of turbidmetry techniques are required to measure the reaction. In respect to this principle precipitation inhibition of a drug can be measured. The test sample (serum) is introduced to a fixed quantity of polyhaptenic antigen and anti drug antibody. The serum drug antigen competes with polyhaptenic antigen for binding to the anti drug antibody. Any free drug present in the sample inhibits the precipitation between the antibody and polyhaptenic antigen. Therefore the drug concentration ids indirectly proportional to the formation of precipitate which is quantified by a nephelometer. The more polyhaptenic antigen present, the more precipitate is formed until the maximum is encountered. Further addition of antigen causes a reduction in the amount of precipitate formed due to antigen excess. The use of nephelometric immunoassay for TDM is termed ''competitive'' due to the competitive binding for the sites on the antibody by the antigen. It also distinguishes the drug assay system from the conventional nephleometric immunoassay for proteins.
Variations of this assay exist including:
The use of saliva or CSF may be used as an alternative to serum. Both alternative matrixes contain less light scattering molecules and so a larger volume of sample is used in order to compensate.
Turbidmetric methods may also be applied to quantitative immunoprecipation . turbidmetric analysis is preformed at a lower wavelength and similarly detects immunoprecipation like nephelometric techniques.
End point analysis of immunoprecipitaion is commonly employed, however rate analysis is also applicable. Addition of formaldehyde blocks further precipitation and is utilised in end point analysis.
Agglutination inhibition immunoassay can also be detected by nephelometric immunoassay systems in which the drug or hapten is directly linked onto the surface of the particle and is generally suitable for low serum drug concentration while precipitation inhibition detects concentration above 1ug/ml
If homologus and heterologus drug concentrations are utilized for antibody and polyhaptenic antigen preparations, sensitivity and specificity may be increased.
Polyclonal and monoclonal antibodies may be employed in this assay. The use of monoclonal antibodies removes any interference caused by antibody cross reactivity. Choosing a hybrid cell with the most desirable antibody is difficult and therefore is most likely to be less sensitive than the use of polyclonal antibodies
Overall the nephelometric immunoassay is an excellent assay system for TDM. Advantages over other assay systems include its simplicity, speed and low cost. It is a homogenous method that requires no separation steps or isotopes. Only two reagents are required in limited amounts as if the antibody to antigen ratio is not optimum, the sensitivity is decreased. This is due to the formation of less precipitate in the absence of drug. In the presence of a drug, inhibition is less efficient. The sensitivity of the assay depends on antibody hapten binding, however it yields high specificity. Therefore nephelometric precipitation inhibition immunoassays are a novel technique in the clinical practice of TDM.
(Takaski Nishikawa Vol 1, 1984_)
FLUORESCENCE POLARIZATION IMMUNOASSAY AND its USE IN TDM
Fluorescence polarization immunoassay(FPIA) is a widely used 2 step homogenous assay that is conducted in the solution phase and is based on a rise in fluorescence polarization due to the binding of the fluorescent labelled antigen with antibody. The first step of the immunoassay involves the incubation of the serum sample with none labelled anti drug antibody. If the patient sample contains drug molecules, immune complexes will form between antibody and antigen. The second stage of this assay involves the addition of a flourscein labelled antigen (tracer) into the mixture(.Jacqueline Stanley 2002) The purpose of the flourscein tracer is to bind on any available sites on the drug specific antibody for detection purposes. If the first stage occurred in which the anti drug antibody formed a complex with the drug from the sample, less or no antigen binding sites will be available for the tracer to bind to. Consequently a higher proportion of the flourscein tracer is unbound in the solution. If the sample contains no drug an antigen, Step 1 does not occur and the anti drug antibodies will bind the flourscein antigen tracer. In this assay the degree of polarization is indirectly proportional to the concentration of drug present. (: Chris Maragos '2009)
IMAGE TAKEN FROM www.mdpi.com/journal/toxins
Fluorescence polarization is calculated to determine the concentration of drug present. Fluorscein labelled molecules rotate when they interact with polarised light. Larger complexes rotate less then smaller complexes and therefore remain in the light path. When the large immune complex is labelled with a fluorescent tracer, it is easily detected once present in the light path. If no drug was present in the sample, the availability of binding sites on the antibody entices the fluorscein tracer to bind, restricting it's motion resulting in a higher degree of polarisation, Thus it is easy to identify that polarization is indirectly proportional to the concentration of drug present. The benefit of utilising FPIA in TDM includes the elimination of processed to separate bound and free labels, an indicator that this assay is time efficient. An unique feature of this assay is that the label used is a flurophore and the analytical signal involves the measurement of the fluorescent polarization. ( Jacqueline Stanley 2002)
A standard curve is constructed to determine the concentration of drug present and is easily reproducible due to the stability of the reagents utilized and the simplicity of the method. However FPIA has some limitations and is prone to interference from light scattering and endogenous fluorescent tracers in the samples. To help overcome these limitations variations on the technique is employed including:
Use of a long wavelength label
The fluorscein tracers utilized produce adequate signals, however light scattering events can interfere with these signals. The use of a long wavelength label permits extended fluorescence relaxation times which may be more sensitive for the detection of high molecular weight antigens on drugs.
Use of CE-LIF
The use of capillary electrophoresis with laser induced fluorescence detection enhances the sensitivity of this method. This competitive FPIA separates free and antibody bound tracers and utilizes LIFP as a detection system.( David S. Smith & Sergei A 2008)
Overall FPIA has proven to be a time and cost effective, accurate and sensitive technique in TDM and remains one of the most promising methods in this clinical field.
ENZYME MULTIPLIED IMMUNOASSAY TECHNIQUE AND its USE IN TDM
Enzyme Multiplied Immunoassay Technique (EMIT) is an advanced version of the general immunoassay technique utilising an enzyme as a marker. EMIT is a 2 stage assay that qualitatively detects the presence of drugs in urines and quantitatively detects the presence of drugs in serum.( David S. Smith & Sergei A )Both the competitive and non-competitive forms of this assay are homogenous binding based that rapidly analyze microgram quantities of drug in a sample. in the competitive assay, the patient sample is incubated with anti drug antibodies. Antibody antigen reactions occur if there is any drug present in the sample. The number of unbound sites indirectly correlates with the drug concentration present. The second step involves the addition of an enzyme labelled specific drug which will bind to available binding sites on the antibody inactivating the enzyme. A enzyme widely used in EMIT assays is Glucose 6 Phosphate Dehydrogenase which primarily oxidises the substrate added (Glucose 6 Phosphate). The co-factor NAD+ is also reduced to NADH by the active enzyme. Any enzyme drug conjugate that is unbound remains active, therefore only in this case , can the oxidation of NAD+ to NADH occur. An increase in absorbance photometrically @ 340nm correlates with the amount of NADH produced. (Jacqueline Stanley 2002)
A non competitive format of this assay also exists, where by drug specific antibodies are added in excess to the sample resulting in antigen antibody interactions if the drug is present. A fixed amount of enzyme drug conjugate is then added which occupy any unbound sites present on the antibody. The active enzyme that is unbound oxidised NAD+ to NADH indicating presence of free enzyme conjugate and subsequently drug molecules present. (chemistry.hull.ac.uk/)
EMIT technology is becoming increasingly popular as a method to monitor therapeutic drug levels. Drugs monitored using this technique includes anti asthmatic drugs, anti epileptic drugs and cardio active drugs. Radioimmunoassay work on the same principle as competitive EMIT with the exception of the use of a radio isotope as a marker. Gamma radiation is emitted from the marker leading to a high level of sensitivity and specificity. As it uses radio isotopes it is not the most cost effective in today's modern environment.
MICROPARTICLE IMMUNOASSAY AND its USE IN TDM
Microparticle agglutination technology uses latex microparticles and plays a leading role in TDM in the quantitative measurement of carbarbapenzaine, phenytoin, theophylline and phenybarbital. Kinetic movement of microparticles in solution (KIMS) is a homogenous assay and is based on the principle of competitive binding between microparticles via covalent coupling. When free drug exists in the patient sample, it will bind to the antibody present. As a result the microparticle antigen complex fail to bind with the antibody and the formation of a particle aggregate does not occur. Micro particles in solution fail to scatter light causing a low absorbance reading. If the patient sample is negative for the drug, the micro particle drug complex binds to the antibodies. The complex that is formed upon binding blocks the transmitted light and causes light scattering resulting in increasing absorbance readings. Hence the degree of light scattering is inversely related to the concentration of drug present.
Light scattering spectroscopy improves the sensitivity and quantitation of particle based immunoassays, thus making KIMS a highly sensitive and accurate technique in TDM. Its popularity has developed throughout the years for many reasons. Reagents required for this assay are in expensive and have high stability. KIMS is a universal assay and can be preformed on a variety of analyzers. The assay has minimal interference as a change of absorbance is measured as a function of time while absorbance readings of interfering substances do not alter with time.( Frederick P. Smith, Sotiris A. Athanaselis)
CHROMATOGRAPHY AND its USE IN TDM
For many years liquid chromatography has been linked to detection systems and its application in TDM is becoming incredibility popular. Liquid chromatography was initially employed in response to difficulties arising in Gas Chromatography (G.C) due to heat instability and non specific adsorption on surfaces. High Performance Liquid chromatography is the main chromatography technique utilized for TDM. Thin Layer Chromatography (T.L.C) and Gas Chromatography are other alternatives, however have limitations that suppress their use in TDM. A derivatization step must be performed for highly polar and thermo liable drugs for G.C to be successful. TLC has a poor detection limit and is unable to detect low concentration of drug present. HPLC has revolutionized the monitoring of TDM with rapid speed and sensitivity of analysis and can separate a wider variety of drugs compared to GC and TLC. For this reason, HPLC is considered the most widely adaptable chromatographic technique when coupled with UV detection and Mass Spectrophotometry for TDM.( Phyllis R. Brown, Eli Grushka)
BASIC PRINCIPLES IN HPLC
HPLC is a separation technique performed in the mobile phase in which a sample is broken down into its basic constituents. HPLC is a separation technique that employs distribution differences of a compound over a stationary and mobile phase. The stationary phase is composed of a thin layer created on the surface of fine particles and the mobile phase flows over the fine particles while carrying the sample. Each component in the analyse moves through the column at a different speed depending on solubility in the phases and on the molecule size. As a result the sample components move at different paces over the stationary phase becoming separated from one another. Drugs that are localised in the mobile phase migrate faster as to those that are situated in the stationary phase. The drug molecules are eluted off the column by gradient elution. Gradient elution refers to the steady change of the eluent composition and strength over the run of the column. As the drug molecules elute of HPLC is linked to a detection system to detect the quantity of drug present in the sample. Detection systems include mass spectrophotometry and UV detection. (Mahmoud A. Alabdalla Journal of Clinical Forensic Medicine)
DETECTION SYSTEMS USED IN HPLC FOR TDM
Detection of HPLC with a diode array ultraviolet detector has proved to be a sustainable application system in the identification after HPLC analysis. The use of UV detection allows the online possession the compounds UV spectra. These detection system absorb light in the range of 180-350nm. UV light transmitted passes through a sensor and from that to the photoelectric cell. This output is modified to appear on the potentiometric recorder. By placing a monochromatoer between and light source and the cell, a specific wavelength is created for the detection , thus improving the detectors specificity. A wide band light source can also be used as an alternative method. In this case the light from the cell is optically dispersed and allowed to fall on the diode array.( Mahmoud A. Alabdalla Journal of Clinical Forensic Medicine)
HPLC can also be coupled to a mass spectrophotometer as a detection method. Mass spectrophotometry (MS) elucidates the chemical structure of a drug. Sensitivity of this technique is observed as it can detect low drug concentration in a sample. Specificity of this method can be futher enhanced by Tandem mass spectrophotometric analysis. This involves multiple steps of mass spectrophotometry. This is accomplished by separating individual mass spectrometer elements in space or by separating MS phases in time. (FranckÂ Saint-Marcoux et al)
FUTURE TRENDS IN TDM METHODOLOGY
AGILENT'S 1200 HPLC MICRO CHIP
Agilent's 1200 HPLC micro chip technology combines microfliudics with an easy use interface that confines the HPLC procedure tot his dynamic chip. The micro chip technology integrates analytical columns, micro cuvette connections and a metal coated electro spray tip into the chip to function as a regular HPLC analyzer. The compact chip reduces peak dispersion for a complete sensitive and precise technique. The microchip comes complete with an integrated LC system into sample enrichment and separation column. The operation of the chip is well defined and manageable upon insertion into the Agilent interface which mounts onto the mass spectrophotometer. The built in auto sampler loads the samples and the sample is moves into the trapping column by the mobile phase. Gradient flow from the pump moves the sample from the trapped column to the separation column. The drug is separated the same as the convention methods however reduced peak dispersion does produce better separation efficiency than the conventional method. This form of technology is currently in use in the United States but has not developed outside of the U.S(http://www.agilent.com)
This is the latest application on the market for the treatment and monitoring of drugs associated with metabolic disorders. The PhyzioType system utilizes DNA markers from several genes coupled with biostatisical knowledge to predict a patient's risk of developing adverse drug reactions. (Kristen k. Reynolds & Roland Valdes)
AMPLICHIP CYP450 TEST
The Amplichip CYP450 Test is a new technology that has revolutionised the TDM of anti psychotic drugs. This test has been approved by the FDA in 2006 but is not currently in use in laboratories in Ireland. This test is used for the analysis of CYP2D6 and CYP2C19 genes, both of which have an influence in drug metabolism. The function of this test is to identify a patient genotype so their phenotype is calculated. Based on the patient phenotype, a clinician determines the type of therapeutic strategy he/she will commence (Kristen k. Reynolds & Roland Valdes)
This paper illustrates the increasing role of immunoassay and chromatography techniques in the clinical laboratory routine monitoring of therapeutic drugs. Before an analytical technique is introduced into TDM it must meet the requirements of sensitivity, accuracy and specificity needed for most TDM applications. The methodology of TDM in today's clinical setting revolves around the use of immunoassays and chromatography techniques. A range of immunoassays was discussed revolving around their principle and advantages and limitations. The majority of immunoassays utilised in the TDM are homogenous based for rapid analysis and efficient turn around time for drug monitoring. Most immunoassays involved in TDM are based on the same principle of competitive binding for antibody. The factor that distinguishes each immunoassay is the detection methods used. Detection methods discussed in this reviewed include nephelometric techniques, flourscein labels, enzyme labels and the use of micro particles. Each method relies on different detection principles as discussed, however characteristics common to all methods include accuracy, sensitivity and specificity. The methodologies discussed also are time and cost efficient, both essential in laboratory assays.
Chromatographic techniques are also discussed with HPLC providing the most impact to TDM. Gas and thin layer chromatography are other chromatographic techniques, however neither can be utilised in TDM due to the limitations both techniques hold against TDM. . HPLC is a rapid sensitive method for the quantitation of drugs in a sample and for this reason is the most widely adaptable chromatographic technique applied in TDM. Like all chromatographic techniques drugs are separated based on the interaction of the drug with the stationary phase which determines the elution time. Detection methods primarily used are UV detection and mass spectrophotometry
The final thought on this overview of TDM was an insight into the future of its methodology and applications .Future and approved methods are discussed given a brief outline on each. The constant development of methodologies and techniques in this area of TDM are ongoing constantly keeping the area of TDM one of the most fastest and interesting in clinical medicine.