Featured Articles

Therapeutic drug monitoring of methadone

, 26 August 2020/in Featured Articles /by 3wmedia

Methadone maintenance therapy is central to the treatment of opiate dependence. Assessment of adherence is essential to ensure success and to prevent misuse of prescribed medications. A variety of specimen types can be tested for methadone and its main metabolite using a number of different analytical methods. The benefits and limitation associated with each are discussed.

by Dr Elizabeth Fox and Dr Deepak Chandrajay

Introduction
Opiate dependence is an important problem worldwide. In the UK, individuals seeking help with their addiction are referred to substance misuse services where they are usually offered methadone or buprenorphine substitution therapy [1]. Methadone is a synthetic opioid with pharmacological actions similar to opiates mediated through the mu receptor. Treatment is initiated at a dose of 10–40 mg daily and gradually increased by 10–20 mg weekly. The usual maintenance dose is 60–120 mg daily, but some clients require a higher dose for symptomatic relief [2]. Its long half-life allows for a once-daily dosing schedule and the accumulation in the body means that steady-state plasma concentrations are easily achieved after repeated administration.
Methadone reduces or eliminates withdrawal symptoms and helps the subject reach a drug-free state in a controlled way. There is evidence of reduced illicit opiate misuse, criminal activity and mortality when patients are on maintenance therapy [2, 3]. Injecting behaviours and incidence of HIV infection are also reduced [4].
Methadone is a lipid soluble drug with an oral bioavailability of approximately 95%. It is metabolized by cytochrome P-450 (CYP) enzymes and demethylated to 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). Both parent drug and metabolite are excreted in the urine and can also be detected in blood, oral fluid, sweat and hair. Co-administration of CYP enzyme inducing drugs such as rifampicin, phenytoin, and zidovudine can precipitate opiate withdrawal symptoms. Fluoxetine and fluvoxamine can inhibit CYP enzymes and have an opposite effect on methadone metabolism [5].
In contrast to most other forms of therapeutic drug monitoring where blood concentration is maintained within a narrow therapeutic window, methadone is monitored almost exclusively to confirm adherence with the treatment regimen. The client may seek to falsify the drug test to feign adherence when the drug is actually being sold to others, or simply to mask illicit drug use. Such individuals will submit a specimen spiked with methadone mixture, therefore effective methods for methadone testing should use matrices which are resistant to tampering and/or include measures to detect falsified samples. Absence of EDDP from a methadone-positive urine sample strongly suggests that it has been spiked with medication. Measurement of urine creatinine will identify samples which have been diluted or substituted, for example with tea. Methadone mixture is green so a green tinge to urine should raise suspicions of sample spiking. The temperature and pH of fresh urine specimens can be recorded to assess reliability. Salivary IgG is useful to confirm integrity of oral fluid specimens.

Analytical methods used for methadone testing
The key analytical methods used to measure methadone and EDDP are immunoassay, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). The benefits and limitations of each are summarized in Table 1. Immunoassay is rapid, high throughput when automated and low cost. Sensitivity is determined by the detection cut-off concentration of the test kit and specificity by the specific antibody used in the kit. Point-of-care test (POCT) kits are available for use with urine and oral fluid in the clinic and require little expertise or training. POCT offers the significant advantage of producing instant results that can be discussed during the consultation. Laboratory-based immunoassays can be run on multichannel clinical chemistry analysers and do not require additional staff training. All immunoassay-based techniques are prone to interference from unrelated compounds due to cross-reaction with the specific antibody. Cross reactivity data are available from the manufacturer and should be borne in mind when interpreting results. False-positive methadone results have been documented with diphenhydramine, doxylamine and phenothiazines [6]. Immunoassay-based tests, whether designed for POCT or laboratory use, are sold as screening tests. The manufacturers recommend the confirmation of positive results using an alternative methodology such as LC-MS/MS or GC-MS. That said, not all laboratories and substance misuse clinics routinely confirm methadone and EDDP positive results. UK Department of Health guidance on adherence testing recommends only that positive screen tests are confirmed ‘if appropriate’[1].

Mass spectrometric techniques offer the best possible sensitivity and specificity and are considered the ‘gold standard’. Test menus are user-defined which allows simultaneous detection of methadone and EDDP and any other drug as required. Disadvantages of these techniques are that they require expensive specialist instrumentation, labour intensive sample preparation, and complex data interpretation. Turnaround times can be lengthy and they are not amenable to POCT. LC-MS/MS methods require considerably less sample preparation than GC-MS and are now used in many clinical laboratories. A few labs including our own have adopted LC-MS/MS for first-line drug screening of urine and oral fluid specimens.

Specimen types for methadone testing
The main sample types are summarized in Table 2.

Urine
Regular urine testing is the most commonly used means of confirming adherence with methadone prescription. The advantages of urine are that both methadone and EDDP are readily detected and collection is easy and non-invasive. Presence of EDDP provides proof that methadone has been ingested and not spiked into the sample. A disadvantage of urine is that it is easy to manipulate. A sample of donor urine, if the donor is taking methadone, submitted in place of the patient’s own is difficult to recognize. Supervised collection is not always desirable as it is an invasion of privacy and subjects may suffer from ‘shy bladder’. The concentrations of methadone and EDDP do not correlate well with dose (because of the variability of untimed urine samples), so qualitative urine analysis is only suitable for confirming use.

Commercially available methadone and EDDP immunoassays typically have a fixed cut-off or detection threshold of between 100 and 300 µg/L. Multidrug panel tests usually include either methadone or EDDP. Choosing a test which specifically detects EDDP will minimize the chance that a spiked urine is passed off as positive. An estimated 4% of methadone-positive samples submitted to our laboratory lack detectable EDDP; a methadone-only assay would not identify these specimens (unpublished observation). Our approach to urine testing is to measure both methadone and EDDP by LC-MS/MS in all samples. The testing strategy in an increasing number of substance misuse clinics is to use POCT as a first-line test, then to refer suspicious or disputed samples to the lab for confirmation. A laboratory immunoassay would offer no further information for samples that have already been tested at the point of care and could theoretically suffer the same interference.

Oral fluid
An alternative matrix for methadone testing is oral fluid. The advantage of oral fluid is that collection is simple, easily observable and can be done in the consultation room. POCT devices are available for instant results or samples can be sent for laboratory analysis. Both immunoassay and mass spectrometric analytical methods are available. The amount of methadone and EDDP present in oral fluid is dependent upon salivary pH. Methadone is a basic drug and under acidic conditions it becomes ionized and ‘trapped’ in the saliva. Unstimulated saliva is more acidic than stimulated saliva so false negatives can be avoided by asking the subject to abstain from eating, drinking or chewing for 10 minutes prior to collection. A recent study involving subjects on daily methadone doses found that the concentration of methadone in saliva correlated poorly with dose and that EDDP was below detection in 12% of samples [7]. However, methadone was readily detectable in all samples suggesting that oral fluid is a useful specimen for confirming adherence. Oral fluid methadone does not reflect the plasma concentration so would not be useful for assessing dose adequacy. Contamination with methadone from the oral cavity is a problem and absence of EDDP, if measured, should be interpreted with caution as it does not necessarily equate to sample adulteration.

Blood
The main advantage of using blood to monitor methadone therapy is that it’s virtually impossible to falsify the sample. Plasma concentration correlates with methadone dose but the concentrations at which therapeutic effect is achieved have not been well defined. Several studies have suggested target concentrations; other studies have found no correlation between plasma concentration and either heroin use or opiate withdrawal symptoms [8]. A further study suggested that the pharmacodynamics of methadone can be altered by the presence of other drugs therefore altering the relationship between plasma methadone and effect [9]. There is debate in the literature as to whether plasma concentration is any more useful than daily dose for predicting response to treatment [10]. Given the polypharmacy present in the majority of subjects receiving methadone, routine use of plasma methadone to titrate dose is likely to need further evaluation. Intravenous drug users tend to have poor venous access so collecting samples may be challenging. Methods using dried blood-spot samples to circumvent this problem have been described but skin contamination with methadone is likely to be an issue [11]. Blood is not the ideal specimen to assess use of other substances because of the very short detection window, so additional testing may be required. In conclusion, blood testing is best reserved for difficult cases where knowledge of the plasma concentration may be helpful.

Other matrices
Monitoring of methadone therapy using sweat analysis has been evaluated. Patches are typically worn for up to 7 days then dispatched to the laboratory for analysis. They are tamper-evident and claim to be difficult to adulterate. Large inter- and intra-individual variations in sweat methadone concentration have been observed and there is only a weak correlation between patch concentration and dose. Sweat testing is, however, useful for detecting exposure to other substances so may be applicable to some cases. Hair analysis can be used to retrospectively confirm adherence with methadone treatment but is not useful for real-time assessment.

Concluding remarks
The current trend is for substance misuse services to perform methadone adherence testing in the clinic and refer samples to the laboratory for confirmation where necessary. Substance misuse clinic personnel are not laboratory scientists, therefore a key role of the laboratory that performs confirmatory testing is to develop a good working relationship and ensure all aspects of testing are fully understood.

References
1. Department of Health (England) and the devolved administrations. Drug Misuse and Dependence: UK Guidelines on Clinical Management. London: Department of Health (England), the Scottish Government, Welsh Assembly Government and Northern Ireland Executive. 2007; www.nta.nhs.uk/uploads/clinical_guidelines_2007.pdf.
2. National Institute for Health and Clinical Excellence. Methadone and buprenorphine for the management of opioid dependence. Technology appraisal guidance 114. NICE 2007; http://guidance.nice.org.uk/TA114.
3. Advisory Council on the Misuse of Drugs. Reducing drug-related deaths: a report by the Advisory Council on the Misuse of Drugs. ACMD, Home Office 2000; ISBN 0-11-341239-8.
4. NTORS, The National Treatment Outcome Research study. 2001; http://webarchive.nationalarchives.gov.uk/+/www.dh.gov.uk/en/publicationsandstatistics/publications/publicationspolicyandguidance/dh_4084908.
5. McCance-Katz EF, Sullivan L and Nallani S. Drug interactions of clinical importance among the opioids, methadone and buprenorphine and other frequently prescribed medications: a review. Am J Addict. 2010; 19(1): 4–16.
6. Lancelin F, Kraoul L, Flatischler N, Brovedani-Rousset S, Piketty ML. False-positive results in the detection of methadone in urines of patients treated with psychotropic substances. Clin Chem. 2005; 51(11): 2176–2177.
7. Gray TR, Dams R, Choo RE, Jones HE, Heustis MA. Methadone disposition in oral fluid during pharmacotherapy for opioid-dependence. Forensic Sci Int. 2011; 206 (1–3): 98–102.
8. Shiu JR, Ensom MHH. Dosing and monitoring of methadone in pregnancy: literature review. Can J Hosp Pharm. 2012; 65(5): 380–386.
9. Kharasch ED, Walker A, Whittington D, Hoffer C, Sheffels Beynek P. Methadone metabolism and clearance are induced by nelfinavir despite inhibition of cytochrome P4503A (CYP3A) activity. Drug Alcohol Depend. 2009; 101(3): 158–168.
10. Hallinan R, Ray J, Byrne A, Agho K, Attia J. Therapeutic thresholds in methadone maintenance treatment: a receiver operating characteristic analysis. Drug Alcohol Depend. 2006; 81(2): 129–136.
11. Saracino MA, Marcheselli C, Somaini L, Pieri MC, Gerra G, et al. A novel test using dried blood spots for the chromatographic assay of methadone. Anal Bioanal Chem. 2012; 404(2): 503–511.

The authors
Liz Fox* PhD, FRCPath and Deepak Chandrajay MBBS, MRCP
Specialist Laboratory Medicine, St James’s University Hospital, Leeds, UK
*Corresponding author
E-mail: Elizabeth.fox@leedsth.nhs.uk

Measurement traceability of Mindray CL-2000i Chemiluminescence Immunoassay System

, 26 August 2020/in Featured Articles /by 3wmedia

Establishing metrological traceability of measurement is essential to improve the accuracy and comparability of measurement results. With increasing recognition of the importance of traceability, some regulatory policies have been applied to enforce its implementation. Technology advancement also provides more tools for improving measurement traceability. During the assay development on the Mindray CL-2000i Chemiluminescence Immunoassay System, well recognized highest reference methods or reference materials were used in assigning the values of master calibrators; the accuracy of product calibrators was guaranteed through an unbroken metrological traceability chain.

by Xiang Yu and Ke Li

Introduction
With the advancement in automation over the past 20 years, most of the immunoassays have been shifted from traditional manual assays to fully automatic systems leading to an overall improvement of the quality of measurements. The accuracy and comparability of testing results have been emphasized, since they are the keys to defining and using common clinical decision values and reference intervals, following constant standards and practice guidelines, pooling data from different studies based on different analytical systems to facilitate clinical research.

One critical mechanism to improve the accuracy and comparability of clinical testing results is to make the testing results traceable to higher reference materials or methods in calibration hierarchy. Briefly, the testing results should have metrological traceability. The general principles and features have been described in the document of the International Organization for Standardization (ISO) 17511:2003 [1].

Ideally, results produced by different routine methods for the same measurand should be metrologically traceable to the highest level of calibration hierarchy – the International System of Units (SI units), with an estimated measurement uncertainty. However, only a limited number of analytes, including some metabolites, electrolytes, steroid hormones, has reference materials available with traceability to the SI unit. Most of the clinical analytes still have no primary and secondary reference measurement procedures and are not traceable to the SI unit. They are not well defined and have only traceability to an international conventional standard or manufacturers’ internal standard, such as tumour markers and viral antigens [2].

The EU directive on in vitro diagnostic devices (IVDD) enacted in 1998 stated “The traceability of values assigned to calibrators and/or control materials must be assured through available reference measurement procedures and/or available reference materials of a higher order” [3]. Therefore, for all the IVD analytical system (reagents), manufacturers must ensure their products are standardized against available reference materials or methods in order to be distributed in the EU market.

Traceability chain and value assignment procedure on Mindray CL-2000i System
Mindray CL-2000i system is a closed system composed of a fully automatic immunoanalyser, related reagents and calibrators. The calibration hierarchy was established and documented strictly based on EN ISO 17511:2003 [1]. Mindray’s traceability procedure is indicated in figure 1, ensuring the establishment of metrological traceability between the testing results and the highest standard available. Based on the characteristics of different analytes, three major traceability chains have been used: traceable to an SI unit, traceable to an international conventional calibrator, and traceable to manufacturers’ selected procedure.

Measurements traceable to the SI unit
If the chemical and physical properties of an analyte are well defined, there should be a primary reference measurement procedure with the measurement traceable to the SI unit (mole). CL-2000i total T3, total T4, progesterone, testosterone and estradiol are traceable to this highest level of calibration hierarchy. Mindray has performed the traceability of the above measurements in collaboration with the Reference Institute for Bioanalytics (RfB), a German reference laboratory certified by the Joint Committee for Traceability in Laboratory Medicine (JCTLM) [4]. Thirty Mindray master calibrators at different levels covering the whole detection range were assigned values for each analyte at RfB with the reference measurement procedure of Isotope dilution mass spectrometry (ID-MS). The calibrator values with uncertainty were then applied to define the values of Mindray working calibrators and product calibrators, and the metrological traceability between the testing results of CL-2000i end-users’ routine measurement procedure and the SI unit was finally established. The assays that are traceable to the SI unit are indicated in Table 1.

Measurements traceable to an international conventional calibrator
The reference materials, such as WHO standards and some national standard materials are defined by convention or consensus, without traceability to the SI unit; the assigned values are in arbitrary units (e.g. WHO international unit). Most of assays for tumour markers, hormones, and viral antigen/antibody of the CL-2000i system are traceable to this kind of reference materials, indicated in Table 1.

Measurements traceable to manufacturers’ selected procedure
For analytes that are either not traceable to the SI unit, or for which no reference method and reference material are available, a commercial certified measurement procedure with traceability, high accuracy and analytical specificity was selected for Mindray master calibrator value assignment; the measurement accuracy of the Mindray routine measurement procedure is ensured and also indicated in Table 1.

Principle of the traceability of Mindray CL-2000i end-user’s measurement results
The immunoanalyser is calibrated by measuring three levels of product calibrators and relative light units (RLUs) generated. The corresponding concentration of each calibrator was used to adjust the master calibration curve stored in the barcode of each lot of reagents.

The value of end-user’s product calibrators and the master curve stored in the barcode are both defined by the Mindray routine measurement procedure that is calibrated by Mindray working calibrators in the manufacturer’s laboratory. The working calibrators have roughly 12 concentration levels and have the same matrix as the end-user’s product calibrators.

It is the Mindray standard measurement procedure that determines the values of Mindray working calibrators. The Mindray standard measurement procedure makes use of the Mindray standard CL-2000i automatic immunoassay analyser, standard reagents, and Mindray master calibrators. Mindray master calibrators are composed of a series of human serum at different concentration levels. They are stored at -70°C and represent the highest accepted standard available.

The values of the Mindray master calibrators are fixed, and the measurement standard established by the Mindray standard measurement procedure is preferably not variable and should be kept as consistent as possible. On the other hand, the value of working calibrators and end-user’s product calibrators can be flexible within a certain range. The assigned values of calibrators will be adjusted according to the results of internal QC and method comparison so as to ensure the traceability between the reference and end-user’s routine measurement procedures.

Discussion
We have made our best efforts for the traceability of the Mindray CL-2000i system, eventhough the implementation of traceability is challenging, especially the traceability in immunoassays.

Firstly, majority of analytes lack a primary reference measurement procedure and thus are not traceable to the SI units. The chemistry and physical properties of these analytes still require more accurate definition.

Secondly, the international conventional calibrators have played an important role in harmonizing testing results. However, there are still some issues with using the international standards, such as the long term stability of WHO standards, the matrix effect, the difference between different generations of the standards, and difference between the source of the standards and the real sample in the clinic.

Thirdly, some of the analytes have neither reference materials nor reference methods available, and are only traceable to manufacturers’ in-house standards. The harmonization of clinical results could not be fully implemented [5].

References
1. ISO 17511:2003. In vitro diagnostic medical devices –measurement of quantities in biological samples – metrological traceability of values assigned to calibrators and control materials. Geneva, Switzerland: ISO
2. Database of higher-order reference materials and reference measurement methods/procedures. http://www.bipm.org/en/committees/jc/jctlm/jctlm-db
3. Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in vitro diagnostic medical devices. Off J Eur Union 7 December 1998; L 331:1–37.
4. JCTLM: Joint Committee for Traceability in Laboratory Medicine. http://www.bipm.org/en/committees/jc/jctlm/
5. Danni L. Meany and Daniel W. Chan Comparability of tumor marker immunoassays: still an important issue for clinical diagnostics? Clin Chem Lab Med 2008; 46(5):575–576.

The authors
Xiang Yu*, MSc and Ke Li, PhD
Immunoassay Department, Shenzhen
Mindray Bio-Medical Electronics Co. Ltd., Nanshan, Shenzhen, 518057 China

*Corresponding author
Email: yuxiang@mindray.com

HIT Testing In Minutes

, 26 August 2020/in Featured Articles /by 3wmedia

Monitoring BK virus in kidney transplant patients

, 26 August 2020/in Featured Articles /by 3wmedia

A new, benchtop molecular analyser allows renal transplant centres to monitor urine and serum BK viral loads in house, permitting earlier diagnosis and management of BK virus associated nephropathy (BKVAN) in renal transplant recipients.

BK virus infection
Named after the renal transplant patient (with initials B.K.) that it was first isolated from in 1971, BK virus (BKV) is a Polyomavirus, characterized by its nonenveloped, icosahedral capsid and its circular, double-stranded DNA genome. Although BKV is prevalent around the world, estimated to have infected more than 80% of the global population, infection with the virus is usually asymptomatic or associated with only mild respiratory tract symptoms in healthy individuals [1]. Following primary infection, which typically occurs in early childhood, the virus persists in a latent form in the kidneys and urinary tract of its host [2].

Reactivation of BKV can occur in immunocompromised and immunosuppressed individuals [3]. In most cases reactivation of the virus is benign but it can pose a particular challenge for renal transplant patients. In such cases immunosuppression can cause a lytic BKV infection that results in viruria in 30-50% and viremia in 13-22% of renal transplant recipients [4]. BKV infection is one of the most common viral complications to affect renal allografts [5]. It can lead to BKV associated nephropathy (BKVAN) in up to 10% of renal transplant recipients, and is associated with graft failure in 15-80% of affected patients [3,6-9].

Not every latent infection leads to viral reactivation and BKVAN in renal transplant patients. In addition to immunosuppression, other risk factors, such as intragraft inflammation and host-specific immunity have been suggested [3]. The progression of BKVAN may occur without obvious signs or symptoms, other than raised serum creatinine, and so it is often misdiagnosed [9].

Management of BKVAN
Treatment of BKV infection and BKVAN in renal transplant patients usually involves a reduction or modification of immunosuppressive therapy. It is generally agreed that early diagnosis and treatment are extremely important to prevent damage to the allograft [3,9]. At a later stage of infection, when intragraft inflammation has developed, reduction of immunosuppression may not be effective and may even be detrimental to the allograft [3]. For this reason, frequent monitoring of BKV in renal transplant patients, to detect early onset BKV infection, is recommended to ensure timely intervention [3].

Confirmation of BKVAN is performed by histological examination of an allograft biopsy sample. However, clinical intervention is often based on the presence of viral replication as a surrogate marker and early indicator of BKV infection. For this reason, non-invasive urine and blood tests have value in screening for BKV reactivation, monitoring the clinical course of infection, or monitoring response to therapy [3].

Urine cytology has been used to screen for BKV reactivation in renal transplant recipients. However, since the virus may be shed in the urine of healthy individuals, quantitative results are required for this method to have diagnostic value [3]. Furthermore, accurate interpretation of cytology results requires training and expertise as it is often difficult to distinguish BKV from other viral infections [9].

Recently, molecular techniques for the detection and quantification of BKV in blood and urine have become available. Such methods offer greater specificity for BKV and provide a valuable tool for identifying patients at risk of BKVAN before renal function deteriorates.

Monitoring BK viral loads
Quantitative measurements of BK viral load in urine and blood by molecular techniques are useful for monitoring the course of BKV infection [9] and for predicting the development of BKVAN [4,7,10,11]. Viral reactivation can be detected in the urine several weeks before the virus is detected in the blood, and viremia can be detected months before histological evidence of BKVAN is present [3]. Monitoring BKV loads in the urine and serum or plasma of renal transplant recipients, therefore, may be valuable in identifying those at risk of developing BKVAN, allowing further investigation and early intervention if necessary [3,9]. Such measurements are also valuable in monitoring response to therapy [3,9].

Although suggested BKV load thresholds for quantitative molecular measurements vary, and laboratories are encouraged to establish their own cut off values for the purpose of clinical management [9], BK viral loads of greater than 10,000 copies/mL in blood [6,11-14] and greater than 10 million copies/mL in urine are considered predictive for BKVAN [6,11,12].

Current guidelines recommend screening for BKV in the serum or plasma of kidney transplant patients monthly for the first 3-6 months after transplantation, and then every 3 months up to one year post-transplantation [15]. These guidelines also recommend that patients are screened for BKV if there is an unexplained rise in serum creatinine or following treatment for acute rejection [15].

Faster quantification of BKV
Due to the specialist nature of BKV testing and the resources and expertise required to perform BKV measurements by urine cytology or nucleic acid testing, many centres are required to send samples to a reference laboratory for analysis. Some laboratories have adopted in-house polymerase chain reaction (PCR) BKV assays. These can be labour intensive, variable in terms of specificity for BKV, and may require further confirmatory testing on positive samples, which can cause significant delays and can potentially impact patient management.

A new molecular method is now available that can reduce the turnaround time for quantitative BKV results significantly and provide the high specificity required for making important clinical decisions about the management of renal transplant patients. The Diasorin Liaison® Iam benchtop instrument, with its small footprint and ease of operation, offers a cost effective and scalable solution for laboratories servicing renal transplant centres. Demonstrating no cross reactivity with other significant pathogens, including JCV, the Diasorin Iam BKV assay provides reliable, quantitative results on the same day as sample receipt [16].

The Iam BKV assay uses loop-mediated isothermal amplification (Diasorin Q-LAMP) to measure BKV DNA in urine, plasma or serum. Unlike conventional LAMP technology, Diasorin Q-LAMP is a rapid, real-time, fluorescent technique that allows quantitative analysis of individual or multiple targets in a single reaction [16].

Q-LAMP is based on the recognition of multiple primer binding regions on the target nucleic acid and amplification of the target sequence, which is facilitated by polymerase with strand displacement activity. Quantification is achieved through the use of fluorophore-labelled primers and an observed decrease in fluorescence during amplification of the target sequence, together with known calibrators. The Diasorin Iam BKV assay is a duplex Q-LAMP assay, designed to recognize a consensus sequence common to all known BKV subtypes. Integral controls provide verification of the efficiency of the extraction process and demonstrate the absence of inhibitors [16].

The Iam BKV Q-LAMP assay fits easily into daily laboratory routines. Once samples are prepared and loaded onto the Liaison® Iam instrument, no operator intervention is required during an assay run, allowing staff to walk away until the routine is completed and the result is displayed. The Iam BKV assay is extremely sensitive, with a limit of detection (defined as that concentration of BK virus with a 95% probability of detection by probit analysis) of 450 cps/mL in plasma (95% Confidence Interval 350 – 770 cps/mL) and 540 cps/mL in urine (95% CI 440 – 780 cps/mL) [16]. The BKV primers used represent all known BKV subtypes (Ia, Ib-1, IB-2, Ic, II,III and IV) and show no significant homology with a range of pathogens, including SV-40 virus and Herpes viruses, or cross reactivity with the closely-related Polyomavirus, JCV [16].

Improved management of renal transplant patients
The DiaSorin Iam BKV assay for the detection and quantification of BKV has been in use at the 975-bed Westmead Hospital in Sydney since July, 2013. Westmead Hospital is a major teaching hospital for Sydney University and one of Australia’s largest centres for post-graduate training to specialist level in all fields. The Department of Renal Medicine and Transplantation and the Centre for Transplant and Renal Research work closely with the Centre for Infectious Diseases and Microbiology (CIDM), which is part of Pathology West, a leading public pathology service in New South Wales. The focus of the Transplant and Renal Research Group is to improve the lives of people with end-organ failure through transplantation. It also aims to reduce the number of people requiring dialysis by preventing the progression of chronic renal disease.

Senior Hospital Scientist at the Westmead CIDM laboratory, Dr Neisha Jeoffreys, comments, “BKV is an important pathogen in renal transplant patients. It can cause serious complications and so early detection of viral reactivation and accurate monitoring of viral loads is a vital aspect of patient management.”

The Westmead CIDM laboratory provides a BKV testing service to the hospital’s renal transplant outpatient clinics as well as other specialist clinics associated with the centre. They also test samples from other pathology groups in their reference capacity. Dr Jeoffreys explains, “Renal transplant patients are tested routinely using the Iam BKV assay at 1, 2, 3, 6, 9 and 12 months post transplantation. Patients that test positive for BKV are tested more frequently, every 2-4 weeks.”

“”The role of the quantitative Iam BKV assay is to determine if the patient is likely to develop BKVAN, which may lead to premature graft loss. Patients with high BKV levels will have their immunosuppression regime modified in order to reduce BKV levels while preventing graft rejection. Ongoing monitoring of BK viral load then assists the renal physicians to ensure the right amount of immunosuppression is delivered to reduce the risk of BKVAN and maintain a healthy graft. Quantitative results allow the physicians to determine the appropriate point at which to modify the treatment.”

Previously, the laboratory used a qualitative in-house conventional PCR method for the detection of BKV followed by monthly quantification of viral loads in BKV-positive patients using a commercially available real-time PCR assay.

“We feel that the Iam BKV assay enables us to provide a better service for our renal specialists,” Dr Jeoffreys continues. “We like the scalability of the Liaison® Iam instrument. We have 3 instruments, which provide the flexibility to perform 1 or up to 21 samples at the same time, optimizing reagent usage. This has allowed us to provide faster turnaround of results as we can now perform quantitative assays immediately and several times a week. The Liaison® Iam method has also helped to improve workflows as it is fast and easy to perform, with less hands-on time than our previous methods, which makes it more cost effective.”

“It is hoped that the rapid quantitative results provided by the Iam BKV assay will allow our renal physicians to respond more quickly to high or escalating BK viral loads,” Dr Jeoffreys concludes. “This will ultimately reduce the rate of graft loss due to BKVAN and allow for better patient management with reduced immunosuppression.”

The Iam BKV assay was the first assay to become available on the Diasorin Liaison® Iam instrument. The growing portfolio of tests available on this platform includes assays for Varicella zoster virus, Parvovirus B19 and Toxoplasma gondii.

^{Dr Neisha Jeoffreys is Senior Hospital Scientist at the Centre for Infectious Diseases and Microbiology (CIDM) based at Westmead Hospital, part of the Pathology West Institute of Clinical Pathology and Medical Research (ICPMR).}

For further information, please contact Tiffany Page, Global Marketing Manager, Molecular Infectious Disease, DiaSorin, tiffany.page@ie.diasorin.com
www.diasorin.com.

References
1. Goudsmit, J. et al. J. Med. Virol. 10, 91–99 (1982).
2. Shinohara T, Matsuda M, Cheng SH, et al. J Med Virol. 1993;41:301-305.
3. Babel, N, Volk, H and Reinke, P (2011). Nat. Rev. Nephrol. 7: 399–406
4. Hirsch, H. H. et al. J. Med. 347, 488–496 (2002).
5. Ramos, E., Drachenberg, C. B., Wali, R. & Hirsch, H. H. Transplantation 87, 621–630 (2009).
6. Hirsch, H. H. et al. Polyomavirus-associated nephropathy in renal transplantation: interdisciplinary analyses and recommendations. Transplantation 79, 1277–1286 (2005).
7. Brennan, D. C. et al. Am. J. Transplant. 5, 582–594 (2005).
8. Hirsch HH. Clin Infect Dis. 2005;41:354-360.
9. Bechert, CJ, Schnadig, VJ, Payne, DA and Dong, J. (2010) Monitoring of BK Viral Load in Renal Allograft Recipients by Real-Time PCR Assays. Am J Clin Pathol 133:242-250.
10. Babel, N. et al. Transplantation 88, 89–95 (2009).
11. Dadhania, D. et al. Transplantation 86, 521–528 (2008).
12. Costa, C. et al. Nephrol. Dial. Transplant. 23, 3333–3336 (2008).
13. Hirsch HH, Steiger J. Polyomavirus BK. Lancet Infect Dis. 2003;3:611-623.
14. Ding R, Medeiros M, Dadhania D, et al. Transplantation. 2002;74:987-994.
15. KDIGO, Am. J. Transplant. 9 (Suppl. 3), S44–S58 (2009).
16. Diasorin Iam BKV assay Instructions for Use, BKV-524-02,EN 12/12.

Quantitative proteomics: closing the gap between biomarker discovery and validation – the rate-limiting step

, 26 August 2020/in Featured Articles /by 3wmedia

Many potential cancer biomarkers have been identified, however, the transfer of these biomarkers from discovery to clinical practice is still a process filled with pitfalls and limitations. To prove clinical utility and performance, these candidate biomarkers need to be reproducible, specific, sensitive, and validated using large sample cohorts, all of which require development of robust and highly sensitive multiplex protein assays.

by Dr G. M. Mustafa, Prof. J. R. Petersen, Prof. L. Denner Prof. F. A. Hussain and Prof. C. Elferink

Background
Although remarkable scientific and technological advances in medicine have been achieved, cancer incidences are still increasing worldwide with the ratio of deaths to new cancer cases remaining relatively unchanged at 49% [1]. The ability of physicians to effectively treat and cure cancer is directly dependent on their ability to detect cancer at an early stage. When cancer develops in an area of the body, such as an organ, as long as it hasn’t spread (metastasized), it may respond well to treatment and in some cases a cure may be possible. Many disease states, especially various types of cancer, can be better diagnosed by the aid of biomarkers, a key element of modern diagnostics and the value of which continuing to increase in modern medicine. As indicators of biological status, biomarkers, whether detected in blood, urine, or tissue, can be useful for the clinical management of various diseases. The changes in biomarker concentration have the potential to guide therapy in disease progression, prognosis and potentially be used to identify the stage of the cancer. Overall, protein biomarkers are needed to help our understanding of cancer biology and to improve our ability to diagnose, monitor and treat cancers.

Biomarker research
One of the goals of biomarker research is to discover and validate markers that can be used in clinical research applications such as patient stratification, diagnosis and therapeutic monitoring, or in pharmaceutical development to fully characterize the behaviour and efficacy of candidate drugs. The identification of prognostic, predictive or pharmacodynamic biomarkers can be carried out using genomic or proteomic technologies. Proteomic profiling of body fluids, such as serum, has potential as a sensitive diagnostic tool for early cancer detection. Serum provides a rich sample for diagnostic analyses because the expression and release of proteins (potential biomarkers) into the bloodstream occurs in response to specific physiological states. As human plasma has a 1010 dynamic range of proteins [2] with 22 proteins being responsible for 99% of the proteins identified, one of the challenges working with such a complex biological fluid is the difficulty in identifying medium and low abundance proteins. Thus, sample enrichment is a very important step in the discovery phase.

Biomarker validation – the rate-limiting step
Discovery platforms typically result in an extensive hit-list of candidate biomarkers. Important analytical and clinical hurdles must be overcome to allow the most promising protein biomarker candidates to advance into clinical validation studies. Over the last several years, many proteins have been proposed as potential biomarkers for various cancers without further evaluation of their clinical utility. The lack of follow-up is due, to a large extent, to the lack of an efficient technology for reproducible and accurate verification of these proteins as biomarkers in a specific disease state. Without proper validation, the identification of biomarkers is of minimal utility. To show clinical utility, biomarkers must be validated using a reliable assay with an independent cohort in a prospective or longitudinal study. A search of the scientific literature clearly indicates that most published biomarkers are inadequate for replacing an existing clinical test or that they are only useful for detecting disease in an advanced stage, where treatment success and/or survival rates are low. Traditionally, validation has been performed with immunoassays which require antibodies that are often unavailable or of poor quality. In addition the immunoassays may not adequately identify or differentiate between post-translational modifications. The process of generating antibodies is also time consuming and costly and is therefore not a practical solution for screening the hit-list of proteins derived from discovery.

Targeted mass spectrometry for multiplexed protein quantitation
A targeted mass-spectrometry-based antibody-free platform for multiplexed protein quantitation fills the well-recognized gap between early biomarker discovery and final clinical assay. Stable-isotope-dilution multiple-reaction-monitoring mass spectrometry (SID-MRM-MS) is a novel technique whereby the quantification of a protein can be calculated from isotopically labelled peptide standards of known concentrations that correspond to the protein of interest [3]. With two to five unique peptides per protein, the concentration of the protein of interest can be accurately determined. Further, the technique can be multiplexed to allow for the simultaneous measurement of many proteins. We have validated this approach using serum samples to identify biomarkers in hepatocellular carcinoma and are confident that it also has the potential for biomarker discovery in other cancers [4]. This powerful workflow opens up new possibilities for biomarker research that may lead to faster, more robust, and improved clinical assays. Targeted proteomics workflows based on SRM and MRM on triple quadruple mass spectrometry platforms show the potential for rapid verification of biomarker candidates in plasma by using heavy isotope-labeled internal standards. This approach has the selectivity, reproducibility, and sensitivity for a range of multiplexed protein assays and has the potential for quantifying protein isoforms in addition to posttranslational modifications for which good quality antibodies often do not exist. The ability to rapidly quantify proteins in a highly multiplexed manner using MRM with internal standard peptides closes the gap between discovery and validation in the biomarker pipeline, which is the rate-limiting step in the biomarker world. Using a triple quadruple mass spectrometer, the first mass analyser (Q1) is set to only transmit the parent weight of a peptide from the target protein, the collision energy is then optimized to produce a diagnostic charged fragment of a peptide fragment in the second mass analyser (Q2), and the third mass analyser (Q3) is set to only transmit and identify this diagnostic peptide fragment (Fig. 1). The key strength of this work flow is that the protein assay development for hundreds of proteins can occur on the order of a month. Once identified, it just takes months to validate the low to sub-ng/ml concentration of candidate biomarkers in hundreds of blood samples. No target-specific reagents, such as antibodies, are needed and only a very small amount of sample (<500ng) is required for quantitation. In addition all potential biomarkers can be quantified in a single assay that is more accurate, rapid, and cost-effective. The capacity of SRM for multiplexed, high-throughput analysis, together with its sensitivity and quantitation, positions SRM as a promising application in medical screening [5, 6]. Summary
The type of clinical proteomics described here has important direct ‘bedside’ applications. We can foresee a future in which the physician will use these proteomic analyses at many points in the management of disease and drug discovery.

References
1. Yeom YI, Kim SY, Lee HG, et al. Cancer biomarkers in ’omics age. Biochip Journal 2008; 2(3): 160–174.
2. Anderson L. Candidate-based proteomics in the search for biomarkers of cardiovascular disease. J Physiol. 2005; 15: 23–60.
3. Lange V, Picotti P, Domon B, Aebersold R. Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol. 2008; 4: 222.
4. Mustafa MG, Petersen JR, Ju H, Cicalese L, et al. Biomarker discovery for early detection of hepatocellular carcinoma in hepatitis C-infected patients. Mol Cell Proteomics 2013; 12(12): 3640–3652
5. Keshishian H, Addona TA, Burgess M, et al. Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution. Mol Cell Proteomics (2007); 6(12): 2212–2229
6. Zhao Y, Jia W, Sun W, et al. Combination of improved 18O incorporation and multiple reaction monitoring: a universal strategy for absolute quantitative verification of serum candidate biomarkers of liver cancer. J Proteome Res. (2010); 9(6): 3319–3327

The authors
Gul M. Mustafa*¹ PhD, John R. Petersen²PhD, Larry Denner³ PhD, Feroze A. Hussain⁴ MD, and Cornelis Elferink¹ PhD
¹ Department of Pharmacology, University of Texas Medical Branch, Galveston, Texas, USA
² Victory Lakes Clinical Laboratory, Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA
³ Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas, USA
⁴ Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas, USA