A goal of clinical proteomics is to find a disease indicator (biomarker) to identify the presence of, or monitor, a disease. It may be surprising that approximately one-third of all cancer cases could be effectively treated if detected at an early enough stage. As a heterogeneous disease, cancer evolves via multiple pathways and is a culmination of a variety of genetic, molecular and clinical events. Given that there is significant variation in the risk of developing cancer and that early detection often results in increased survival, developing technologies capable of identifying patients at highest risk and detecting tumours in the earliest stages of development is a pressing need.
by Dr Gul M. Mustafa, Prof. Cornelis Elferink and Prof. John R. Petersen
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, ranking sixth among cancers in incidence worldwide and is the 3rd leading cause of cancer death. Despite some significant improvements in diagnosis and treatment of human liver diseases over the last decade, the HCC mortality rate has not changed to any extent. Currently there are approximately 20,000 new case in the US annually with millions world-wide [1]. The projected rise in the new HCC cases in the US and the world is mainly due to latent hepatitis C virus (HCV) infections in the general population, accounting for approximately 80% of HCC cases several decades after initial infection. The less than 5% survival rate of patients with HCC is primarily due to the disease eluding early detection and diagnosis, when options for effective treatment still remain. Surveillance of patients at highest risk for developing HCC, notably patients with cirrhosis, would benefit greatly from a biomarker assay capable of accurately detecting HCC in its earliest stages when it is still possible to intervene. One of the most widely used markers for HCC is alpha fetoprotein (AFP) although it is non-specific, providing low sensitivity and poor specificity, especially for early detection of HCC [2]. The false-negative rate with AFP level can be 40% for tumours < 3 cm in diameter. More reliable methods such as triple phase Computed Tomography (CT) imaging and liver biopsies exist, but these are expensive and not conducive to long-term surveillance. Therefore, the identification of superior biomarkers will be of huge clinical significance to
at-risk populations.
The ideal biomarker for this type of application would be one where HCC is detected with a high sensitivity and specificity in easily obtained biological samples in a non-invasive, or minimally invasive, manner. Blood represents the best source for detection of HCC related biomarkers, as every cell in the body leaves a record of its physiological state by the products it sheds into the blood, either as a waste or as a signal to neighbouring cells. What some may view as cellular refuse in is reality a diagnostic gold mine. Because of its easy accessibility from patients on a regular basis and because it is in contact with all the tissues in the body, it is an excellent choice for a proteomics approach as it may reveal when changes, such as development of HCC, occur. The systematic analysis of the whole serum or plasma proteome may thus provide a functional meaning to the information provided by genome expression studies. Expression of proteins, their isoforms or post-translational modifications, can be detected by proteomic analysis and these data can provide precious information to better understand the pathologic/molecular basis of HCC [3]. Proteomic analysis may also allow monitoring of the course of the disease process from cirrhosis to HCC, eventually leading to earlier diagnosis which is essential in determining the best course of treatment options and possible outcomes. In addition to earlier diagnosis proteomic analysis may also be useful in measuring the efficacy/progress of treatment or detecting tumour reoccurrence both of which are missing in HCC treatment.
Proteomics analysis
Proteomics analysis is currently considered to be the best tool for the global evaluation of protein expression, and has been widely applied in the analysis of diseases, especially cancer research. For us the approach was to compare the serum/plasma protein profile from patients infected with HCV against the sera from patients with confirmed HCC. Proteins found to be consistently altered between the two patient populations can then be identified and further characterised to determine if they can be used as biomarkers of HCC. While on the surface this sounds simple, due the complexity of the proteome and the wide dynamic concentration range (9 orders of magnitude from pg/mL to mg/mL) of constituent protein/peptide species it is an extremely challenging task. Because the serum/plasma proteome is predominated by high abundance proteins such as albumin and immunoglobulins, extensive fractionation prior to analysis is required. To reduce the few over-represented (i.e. abundant) proteins, without losing any valuable information, existing fractionation methodologies often discard the high abundance carrier proteins, such as albumin, and thus fail to capture the information associated with this valuable resource. We have used aptamer-based technology (Bio-Rad) a technology that reduces the dynamic range and thus retains the complexity of the serum peptidome, in contrast to strategies that just deplete carrier proteins.
Quantitative protein expression profiling
Because proteins entering the blood from surrounding tissue are much less abundant, it is this fraction that is likely to contain most of the undiscovered biomarkers. Quantitative protein expression profiling is a crucial part of proteomics, and such profiling requires methods that are able to efficiently provide accurate and reproducible differential expression values for proteins in two or more biological samples. Thousands of different protein species present in the biological fluid or tissue must be separated, identified and characterised, which cannot be accomplished by a single experimental approach. An effective approach is two-dimensional differential in gel electrophoresis (2D-DIGE) and mass spectrometry [4]. While two-dimensional electrophoresis (2DE) has been widely used for proteomics research, the inter-gel variation along with excessive time/labour costs are major problems. Two-dimensional differential in gel electrophoresis (2D-DIGE) is a modification of 2DE and is considered as one of the most significant advances in quantitative proteomics. Using 2D-DIGE, two samples that are to be compared are pre-labelled with mass- and charge-matched fluorescent cyanine dyes and co-separated on the same 2D gel. The use of internal standards in every gel minimises problems associated with technical variability. Moreover with the great sensitivity and dynamic range that is afforded by the fluorescent dyes, 2D-DIGE can give greater accuracy of quantitation than traditional silver staining. The data captured from these gels using the Imagers, such as the Typhoon trio, along with and proprietary (Decyder) software can be configured to give inter-gel and intra-gel statistical analysis providing both a quantitative and qualitative analysis. We and others are using this approach to identify differentially expressed proteins for differential expression between the pre-cancerous and cancerous patient groups.
Stable isotope labelling
Another technique that can be useful in the analysis of the whole serum proteome is stable isotope labeling using O16/O18. This is a quantitative proteomic technique that distinguishes individual peptides during LC-MS/MS on the basis of a 4 Dalton m/z change after differential O16/O18 labelling that takes place at the C-terminal carboxyl group of tryptic fragments [5]. It is then possible to determine the ratio of individual protein expression levels between the two samples. Alternatively it is possible to use O16/O18 stable isotope labeling to determine the differential expression between two patient groups. In this way the low molecular weight serum peptidome (<20kDa), suspected of harbouring metabolites and degradation products reflecting HCC, can also be interrogated
Selected reaction monitoring
Selected reaction monitoring (SRM), which is used to monitor a precursor and its product ion m/z, is another powerful proteomic tool using tandem mass spectrometry to monitor target peptides within a complex protein digest. The specificity and sensitivity of the approach, as well as its capability to multiplex the measurement of many analytes in parallel, renders it amenable to biomarker discovery and validation proteomics. Using the selectivity of multiple stages of mass selection of tandem mass spectrometers, these targeted SRM assays are the mass spectrometry equivalent of a Western blot. An advantage of using a targeted mass spectrometry-based assay over a traditional Western blot is that it does not rely on the creation of highly selective immunoaffinity reagents. Thus, targeted SRM assays using heavy isotope-labelled internal standards can be multiplexed in quantitative assays that can be directly applicable to clinical settings. A targeted proteomics workflow based on SRM on a triple Quadrupole mass spectrometry platform shows the potential of fast verification of biomarker candidates reducing the gap between discovery and validation in the biomarker pipeline. Although useful, due diligence needs to be exercised in developing and validating SRM assays.
Sample handling
Biomarker research necessitates a clear, rational framework. Technologically, the platform needs to be able to detect low abundant plasma/serum proteins and reproducibly measure them in a high throughput manner. Conceptually, the choice of the technological platform and availability of quality samples should be part of an overall study design that integrates basic and clinical research. Sample preparation is an important and very critical part of clinical proteomics as the collection, sample handling and storage can have a significant impact on the integrity of the proteins being detected. It is so important that a standard operating procedure outlining the steps that should be followed in collecting and storing clinical samples was recently published [6]. In addition to a standardised collection procedure, biological samples need to be carefully chosen based on well-established guidelines either for candidate discovery in the form of controls and the disease being detected or for validation of the candidate biomarkers using well characterised samples.
Most importantly, the samples should be representative of the target population and directly address the clinical question. A conceptual structure of a biomarker study can be provided in the form of sequential phases, each having clear objectives and predefined goals [Figure 1]. Furthermore, guidelines for reporting the outcome of biomarker studies are critical to adequately assess the quality of the research, interpretation and generalisation of the results. By being attentive to and applying these considerations, biomarker research should become more efficient and lead to biomarkers that are translatable into the clinical arena.
Aknowledgements
This research was supported by a pilot grant from the Clinical Translational Sciences Award (5UL1RR029876) and the Mary Gibb Jones endowment.
References
1. Kim WR. The burden of hepatitis C in the United States. Hepatology 2002; 36: 30-34.
2. Sterling RK, Wright EC, Morgan TR, Seeff LB, Hoefs JC, Di Bisceglie AM, Dienstag JL, Lok AS. Frequency of elevated hepatocellular carcinoma (HCC) biomarkers in patients with advanced hepatitis C. Am J Gastroenterol 2012; 107(1): 64-74.
3. Maria P, Laura ML, Antonio RA, Jose LM, Javier B, Ruben C, Jordi M and Manuel de la Mata. Proteomic analysis for developing new biomarkers of hepatocellular carcinoma. World J Hepatol 2010; 2(3): 127-135.
4. Sun W, Xing B, Sun Y, Du X, Lu M, Hao C, Lu Z, Mi W, Wu S, Wei H, Gao X, Zhu Y, Jiang Y, Qian X, He F. Proteome analysis of hepatocellular carcinoma by two-dimensional difference gel electrophoresis: novel protein markers in hepatocellular carcinoma tissues. Mol Cell Proteomics 2007; 6(10): 1798-808.
5. Miyagi M, Rao KC. Proteolytic 18O-labeling strategies for quantitative proteomics. Mass Spectrom Rev 2007; 26(1):121-36.
6. Tuck MK et al. Standard operating procedures for serum and plasma collection: early detection research network consensus statement standard operating procedure integration working group. J Proteome Res 2009; 1: 113-117.
The authors
Gul M. Mustafa, Ph.D. Postdoctoral Fellow, Department of Pharmacology
Cornelis Elferink, Ph.D., Professor, Department of Pharmacology, Director Sealy Center Environmental Health and Medicine
John R. Petersen, Ph.D., Professor and Director Victory Lakes Clinical Laboratory, Department of Pathology,
University of Texas Medical Branch
301 University Boulevard
Galveston, Texas 77555, USA
e-mail: jrpeters@utmb.edu
We have investigated the effects of three (Lepirudin, Argatroban and Bivalirudin) direct thrombin inhibitors (DTI) on routine and dedicated assays.
We found routine tests to be non-discriminative between concentrations of different DTI. The dedicated Hemoclot assay showed identical lineair increases for all three DTI.
We conclude that a dedicated calibrated assay based on a diluted thrombin time (Hemoclot) appears to be the most suitable assay for monitoring purpose.
by Dr Joyce Curvers, Dr Volkher Scharnhorst and Dr Daan van de Kerkhof
Clinical background
The use of direct thrombin inhibitors (DTIs) for prophylactic or therapeutic anticoagulation is increasing due to their predictable bioavailability, short half life and limited interaction with other medication [1-5]. The current idea is that the newer anticoagulants should not require laboratory monitoring because of these advantages. However, although monitoring of anticoagulant therapy may not be required for ‘standard’ patients, patients with an increased bleeding risk, specific co-medication (such as amiodarone or bridging therapy with coumarins), or a deviant body mass or water homeostasis (e.g. neonates, during pregnancy, the obese, the elderly, in renal insufficiency, oedema, cardiac disease) may still require occasional blood analysis. In addition when the compliance or effectiveness of the anticoagulants is doubted, measurement of the coagulation status can be crucial for the correct treatment of a patient. Since DTIs interfere with the central clotting enzyme thrombin, almost every coagulation assay is affected by its presence in blood. This also accounts for routinely used assays such as the aPTT or PT (and INR) [6].
Up to date, there is no consensus on how oral or intravenous administrable DTI should be monitored and specifically which assay should ideally be used [6,7]. In this study we performed an in vitro study in which we investigated the effect of increasing concentration levels of three DTIs: lepirudin, bivalirudin and argatroban in six plasma pools on aPTT, PT, TT and on dedicated DTI-assays (Hemoclot from Hyphen BioMed and Ecarin Clotting Time from STAGO) on a coagulation analyser (STA-R Evolution, Roche).
Materials and methods
Six different pools (N>20 samples per pool) were collected from residual plasma from patients with aPTT and PT values within reference limits (assuming that patients did not take any anticoagulant medication based on their normal aPTT and PT values).
Argatroban (Arganova, Mitsubishi Pharma, lot PF41977, 100 mg/mL) and lepirudin (Refludan, Pharmion, lot 24661611L, 50mg) were provided by the local hospital pharmacy. Bivalirudin (Angiox or angiomax, The Medicines company, lot 1574697, 250 mg) was a kind gift from the Medicines Company. All DTIs were diluted with saline (0,9% NaCl) to 5 g/L. These stock solutions were spiked into the pooled plasmas (N=6) to reach final concentrations of 1, 2, 3, 4 and 5 mg/L. Therapeutic doses of DTI are currently advised at 2 mg/L (according to package leaflet). Different plasma pools with each different concentration of different DTIs were frozen in triplicates at <-70˚C until time of measurement.
Clotting times in the aPTT, prothrombin time (PT) and thrombin time (TT) as well as the dedicated assays Hemoclot (a diluted TT) and the Ecarin Clotting Time (ECT) were recorded.
Results
For all thrombin inhibitors investigated here, the fold increase compared to no DTI in six pools measured in routine tests (aPTT, PT and thrombin time) are shown in Figure 1. The aPTT shows a non-linear concentration-response relationship with a more gradual increase at higher DTI concentrations resulting in a limited sensitivity of the assay in this range. The concentration-response relationship for the PT was linear but with different sensitivities for the different DTIs. The low sensitivity was found especially for bivalirudin and lepuridin with respectively a maximum 2- and 3-fold increase in PT coagulation time at 5 mg/L. The thrombin time also showed a linear concentration-response relationship, with a high increase in coagulation time as function of concentration, especially for lepuridin, exceeding the maximum installed measuring range (i.e. 240 sec) of the STA-R evolution.
Figure 2 shows the data for the dedicated thrombin inhibitor tests. Similar results as for the PT were observed for the ECT, also with respect to the differences between different direct thrombin inhibitors. Lepirudin showed an increase in ratio up to 5-fold baseline value in the ECT. The increase in the Hemoclot was linear for all DTIs with similar increase as a function of concentration measured.
Conclusion
Concluding, dedicated DTI assays overcome the drawbacks of routine assays such as the PT, aPTT or TT, in which the ability to discriminate between different concentrations is insufficient. This would suggest that monitoring DTIs using the aPTT is obsolete. We have shown that dose-response curves of DTIs in dedicated assays such as the Hemoclot and ECT are acceptable. Moreover, they can be applied in a routine setting, have short turn around times and can be used to distinguish inappropriate from appropriate dosing without the necessity of reanalysis after dilution. Given that a calibrator is included in the assay kit and the test gives similar result for different DTI formulations, the Hemoclot assay appears to be the most suitable assay for monitoring purposes (apparent in this study). As more new oral thrombin inhibitors such as dabigatran etexilate find their way into troutine practice, dedicated assays may aid the clinician in better decision making concerning anticoagulant therapy, especially in certain groups of patients in need of monitoring. However, research is needed to properly determine therapeutic and prophylactic concentration ranges, with calibrated dedicated DTI assays.
Current status
The administration of (oral and) intravenous direct thrombin inhibitors is increasing, since more applications are becoming available. The pharmaceutical companies pay little attention to the fact that, in certain situations, indication of the concentration is warranted.
We are currently validating a calibrated assay based on a diluted thrombin time for use in our laboratory (and clinic), as are several other laboratories nation-wide.
Future prospects
Up to now little is known about interference of different anticoagulants combined with DTI (e.g. during bridging therapy) and the effects on the different dedicated assays. Future research will show the value of the different DTI assays in monitoring patients in order to distinguish proper dosing from under dosage or over dosage.
Moreover, standardisation and calibration of (present and new) dedicated assays for the measurement of DTI is a major issue of concern. Therefore we are currently conducting research in which a comparison of coagulation assay results with actual concentrations of the different DTI (measured with LCMSMS) is investigated.
Notification
Part of this publication is included in a manuscript that will be published in the American Journal of Clinical Pathology.
References
1. Di Nisio M, Middeldorp S, Buller HR. Direct thrombin inhibitors. N Engl J Med 2005; 353: 1028-1040.
2. Stone GW, Witzenbichler B, Guagliumi G et al. HORIZONS-AMI Trial Investigators. Bivalirudin during primary PCI in acute myocardial infarction. N Engl J Med 2008; 358: 2218-2230.
3. Mehran R, Lansky AJ, Witzenbichler B et al. HORIZONS-AMI Trial Investigators. Bivalirudin in patients undergoing primary angioplasty for acute myocardial infarction (HORIZONS-AMI): 1-year results of a randomised controlled trial. Lancet 2009; 374: 1149-1159.
4. Connolly SJ, Ezekowitz MD, Yusuf S et al. RE-LY steering committee and investigators Dabigatran versus warfarin in patients with atrial fibrillation. N Engl J Med 2009; 361: 1139-1151. Erratum in: N Engl J Med 2010 Nov 4;363(19):1877
5. Schulman S, Kearon C, Kakkar AK et al. for the RE-COVER study group. Dabigatran versus warfarin in the treatment of acute venous thromboembolism. N Engl J Med 2009; 361: 2342-2352.
6. Gosslin RC, Dager WE, King JH et al. Effect of direct thrombin inhibitors, bivalirudin, lepirudin and argatroban, on prothrombin time and INR values. Am J Clin Pathol 2004; 121: 593-599.
7. Van Ryn J, Stangier J, Haertter S et al. Dabigatran etexilate – a novel, reversible, oral direct thrombin inhibitor: interpretation of coagulation assays and reversal of anticoagulant activity. Thromb Haemost 2010; 103: 1116-1127.
The authors
Joyce Curvers PhD, Volkher Scharnhorst PhD and Daan van de Kerkhof PhD
Clinical Laboratory
Catharina Hospital Eindhoven
Eindhoven
The Netherlands
Non-invasive prenatal testing (NIPT) based on cell free fetal DNA (cffDNA) circulating in maternal blood is moving rapidly forward. With promises of improved safety, earlier detection and easier access to tests, NIPT has the potential to bring many positive benefits to prenatal care. Here we discuss the recent developments in this area.
by Dr Melissa Hill, Dr Angela Barrett, Dr Helen White and Professor Lyn Chitty
Non-invasive testing using cell free fetal DNA
Prenatal diagnosis of genetic conditions or aneuploidy has traditionally required invasive diagnostic tests [chorionic villus sampling (CVS) and amniocentesis] which carry a small but significant risk of miscarriage of around 1% and can only be safely conducted after 11 weeks in pregnancy. In 1997 Lo and colleagues identified the presence of cell free fetal DNA (cffDNA) in maternal plasma and in doing so opened the door to a safer approach to prenatal diagnosis whereby non-invasive prenatal testing (NIPT) of fetal genetic material could be performed using a maternal blood test [1].
The cffDNA is an attractive target for prenatal testing. In addition to avoiding the risk of miscarriage it is anticipated that NIPT will be available early in pregnancy as cffDNA can be detected from 4 to 5 weeks with sufficient levels for analysis by 7 to 9 weeks. The cffDNA emanates from trophoblast cells in the placenta and is pregnancy specific as it is cleared from the circulation within 30 minutes of delivery. It is now also evident that the whole fetal genome is represented in the maternal plasma, suggesting that tests for many genetic conditions will be possible [2].
The major barrier to developing specific prenatal tests based on cffDNA has been the relative concentration of the fetal material. The cffDNA represents only a small proportion (around 10%) of the cell free DNA that is present in the maternal circulation, as the vast majority is maternal in origin. As a result, it is difficult to determine what genetic information is specific to the fetus against the large background of maternal cell free DNA.
For this reason NIPT was initially limited to the identification of alleles present in the fetus but not in the mother because they were inherited from the father or because they arose de novo. These tests include fetal sex determination, which uses targets on the Y chromosome, fetal rhesus genotyping in Rhesus D (RhD) negative mothers, and paternally inherited or de novo autosomal dominant single gene disorders. All of which can be conducted using relatively straightforward molecular techniques. More recently, new technologies such as digital PCR and massively parallel sequencing (MPS) have allowed researchers to develop NIPT for single gene disorders where parents have the same mutations and for aneuploidies, both of which need to take into account the presence of the mother’s allele or chromosomes.
Early clinical successes with non-invasive testing
The two early success stories for NIPT have been fetal rhesus genotyping and fetal sex determination, which are performed using real-time quantitative PCR. Analysis of cffDNA in the plasma of RhD-negative pregnant women who have a past history of haemolytic disease of the newborn or have elevated levels of Anti-D antibodies has been used clinically to determine the fetal RHD status for almost a decade. Large scale validation studies demonstrate high specificity and sensitivity and fetal RHD typing of all RhD-negative pregnant women has the potential to become routine clinical practice in the next few years.
Similarly, NIPT for fetal sex determination is increasingly offered as standard genetic care for women with pregnancies at risk of genetic conditions that primarily affect a particular sex. Fetal sex determination informs the need for genetic diagnosis, allowing up to 50% of carriers to avoid unnecessary invasive testing, and is important for guiding pregnancy management in some conditions. The test has been shown to be reliable when performed after 7 weeks gestation [3], and clinical utility was demonstrated in a recent UK audit as only 32.9% of women subsequently underwent invasive testing [4]. Importantly for implementation, NIPT is viewed positively by women who have had the test and has been shown to be cost neutral compared to invasive testing, which means women can have the clinical benefits of the test at no extra cost to health services [4].
NIPT for single gene disorders
The first use of cffDNA for the diagnosis of a single gene disorder was for the autosomal dominant condition myotonic dystrophy [5]. NIPT has also been possible for other paternally inherited autosomal dominant disorders such as early onset primary dystonia and Huntington’s disease. NIPT for the autosomal dominant condition achondroplasia, which commonly occurs as a de novo mutation, has also been successful. NIPT for autosomal recessive or maternally transmitted autosomal dominant disorders is more difficult due to the need to distinguish between the maternal and fetal free DNA. Exclusion of the paternal mutation is possible in autosomal recessive conditions where the parents carry different mutations. If the paternal allele is detected, there is a 50% risk that the fetus will inherit the disorder, and invasive testing would be recommended. If the paternal allele is not detected invasive testing is not required.
It is now clear that NIPT can be successfully applied to recessive conditions where parents carry the same mutation using digital PCR, which allows high copy number counting and quantification of alleles. Digital PCR requires the dilution of template DNA to an average concentration of less than one molecule per well, and hundreds to thousands of replicates of a PCR reaction are analysed. The mutation status of the fetus is predicted with an approach known as relative mutation dosage (RMD), which is based on the premise that if both the woman and the fetus are heterozygous there will be an allelic balance between the wild type and normal alleles; if the fetus is homozygous for either the mutant or the wild type allele there will be an over-representation of one or the other [6]. Another approach to identify single gene disorders non-invasively that we may see more of in the future is MPS, which has been used to determine the inheritance of two different β-thalassemia alleles [2].
NIPT for aneuploidies
Prenatal screening and diagnosis for fetal aneuploidy is offered routinely to all pregnant women in many countries to detect trisomy 21 (Down’s syndrome), trisomy 18 (Edwards syndrome) and trisomy 13 (Patau syndrome). The extensive scale of current prenatal screening programmes for these conditions means that success in developing accurate NIPT for aneuploidies will transform antenatal care. Several approaches have been explored including fetal specific epigenetic markers and SNP based methods. The most promising approach to date utilises MPS, which allows large scale single molecule counting to detect the increase in the number of sequences that result from the trisomic chromosome.
The first successful proof-of-principle studies utilising MPS to detect fetal aneuploidy from maternal plasma were published in 2008 [7,8]. Using this methodology, millions of short DNA sequences are generated from genomic locations. The sequences are then compared with the known human genome sequence, to establish how many sequences have been derived from each chromosome. For example, by comparing the total number of uniquely mapped chromosome 21 sequences obtained from a cfDNA sample with the number obtained from a normal genomic DNA sample, very small increases in the amount of chromosome 21 can be detected in the cfDNA sample if the fetus carries an additional chromosome 21.
Several large scale validation studies have subsequently demonstrated high levels of sensitivity (100%) and specificity (98–99%) using MPS. Efforts to decrease costs and increase throughput have seen many groups use multiplexing of patient samples into samples libraries that are run on one lane of the sequencing platform (2–12 patients per lane). Another strategy to decrease costs has been the use of targeted or ‘chromosome-selective’ MPS approaches where the sequencing assay is targeted to non-polymorphic loci on specific chromosomes such as 18 and 21 [10]. Studies with targeted MPS also show the potential for greater accuracy with the use of a novel bioinformatic algorithim (FORTE) that considers the proportion of specific cffDNA in the samples and accounts for the prior risk of trisomy (taken from published data on maternal and fetal gestational age related risks) to predict the likelihood of fetal trisomy for each patient [9].
Following the success of the validation studies, NIPT for aneuploidy is being offered through commercial providers in some countries (Sequenom, BGI, Berry Genomics, Aria, LifeCodexx). It is not yet clear, however, how these new tests will be introduced more widely into antenatal care. At present the small false positive rate means that the test is considered an “advanced screening test” that should be confirmed by invasive testing. Other considerations for implementation include the cost of the technology, the gestational limits of the test and the structure of existing screening programmes. All of these factors will impact on whether NIPT is introduced as a replacement for invasive testing or as an adjunct to current screening tests offered to all women.
Conclusions
NIPT is rapidly bringing about dramatic changes to antenatal care. Fetal sex determination and RhD genotyping are now available as clinical services in a number of countries. Testing is already possible for some single gene disorders, and new technologies such as digital PCR and MPS are allowing the challenges of testing for recessive conditions and aneuploidies to be met. Successful implementation, however, will require more than the development of laboratory tests and we must consider ethical issues, research stakeholder views and assess implementation strategies to ensure NIPT is offered in a way that best meets women’s needs. For this reason studies such as the RAPID programme in the UK (www.rapid.nhs.uk) that look at all aspects of test development and implementation are important.
Notification
This article summarises a recent review published in Best Practice in Clinical and Obstetric Gynaecology: Hill M, Barrett AN, White H, Chitty LS. Uses of cell free fetal DNA in maternal circulation. Best Pract Res Clin Obstet Gynaecol. 2012 Apr 27 [Epub ahead of print].
References
1. Lo YM, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum. Lancet. 1997; 350: 485–487.
2. Lo Y, Chan K, Sun H, et al. Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci Transl Med. 2010; 2: 61ra91.
3. Devaney SA, Palomaki GE, Scott JA, et al. Noninvasive fetal sex determination using cell-free fetal DNA: a systematic review and meta-analysis. JAMA. 2011; 306: 627–636.
4. Hill M, Lewis C, Jenkins L, Allen S, Elles R, Chitty LS. Implementing non-invasive prenatal fetal sex determination using cell free fetal DNA in the United Kingdom. Expet Opin Biol Ther. 2012; Suppl 1: S119–126.
5. Amicucci P, Gennarelli M, Novelli G, et al. Prenatal diagnosis of myotonic dystrophy using fetal DNA obtained from maternal plasma. Clin Chem. 2000; 46: 301–302.
6. Lun FM, Tsui NB, Chan KC, et al. Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma. Proc Natl Acad Sci U S A. 2008; 105: 19920–19925.
7. Fan HC, Blumenfeld YJ, Chitkara U, et al. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci U S A. 2008; 105: 16266–16271.
8. Chiu RW, Chan KC, Gao Y, et al. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Natl Acad Sci U S A. 2008; 105: 20458–20463.
9. Sparks AB, Struble CA, Wang ET, et al. Optimized Non-invasive evaluation of fetal aneuploidy risk using cell-free DNA from maternal blood. Am J Obstet Gynecol. 2012; 206: 319.
The authors
Melissa Hill PhD1, Angela Barrett PhD1,
Lyn Chitty MRCOG, PhD1* and
Helen White PhD2
1 Clinical and Molecular Genetics Unit, UCL Institute of Child Health and Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK
2 National Genetics Reference Laboratory (Wessex), Salisbury District Hospital, Salisbury, UK
*Corresponding author
e-mail: l.chitty@ucl.ac.uk
Together with HIV/AIDS and TB, malaria is one of the major public health challenges of the developing world. Prompt diagnosis is a priority. Rapid diagnostic tests are readily available, quick to yield results and can be effectively used in resource-limited settings.
by Meghna Patel
Malaria is a tropical disease caused by parasites of the genus Plasmodium and transmitted by Anopheles mosquitoes. Being endemic in more than 100 countries, half the world’s population is at risk for malaria. Children are at particular risk, accounting for most malaria deaths globally [1]. Each year roughly 250 million people are infected and nearly a million people die from the disease [2]. Malaria causes significant morbidity and mortality, particularly in resource-poor regions. Sub-Saharan Africa is the hardest hit region in the world and parts of Asia and Latin America also face significant malaria epidemics [3]. Four major species of malarial parasite infect humans: Plasmodium falciparum, P. vivax, P. ovale and P. malariae. The first two species cause the most infections worldwide. On the continent of Africa, P. falciparum malaria predominates, whereas in parts of Asia and Latin America, P. vivax is more prevalent. Two other species, P. ovale and P. malariae, are also capable of causing human disease. A fifth species, Plasmodium knowlesi, is found in Southeast Asia; it mainly causes malaria in simians but it can also infect humans.
Since malaria is preventable and treatable, such high incidences point to inappropriate management of the condition in some cases, with incorrect or inefficient diagnosis and/or treatment. Rapid and accurate diagnosis of malaria before treatment is essential for effective and timely treatment of patients and to minimise the spread of drug resistance and thus the requirement of more expensive drugs, frequently unaffordable for resource-poor countries [4]. This review discusses the currently available techniques for malaria diagnosis
focusing on rapid diagnostic tests (RDT).
Diagnosis
As in other pathological conditions malarial diagnosis is based on clinical investigations and pathological laboratory analysis. Diagnosis based on clinical symptoms is the least expensive, most commonly used method in resource poor conditions. However, the overlapping of malaria symptoms with other tropical diseases impairs its specificity and therefore encourages the indiscriminate use of anti-malarials for managing febrile conditions in endemic areas.
Laboratory diagnosis of malaria includes identifying malarial parasites or their antigens/products in patient blood. Although this may seem simple, diagnostic efficacy depends on various factors such as stage and forms of the various malarial species, endemicity of different species, density of parasitaemia etc.
In the laboratory, malaria is diagnosed using different techniques e.g. conventional microscopic diagnosis by examining stained thin and thick peripheral blood smears, other concentration techniques, e.g. quantitative buffy coat (QBC), rapid diagnostic tests and molecular diagnostic methods, such as PCR. The pros and cons of these methods have also been described, chiefly related to sensitivity, specificity, accuracy, precision, time consumed, cost-effectiveness, labour intensiveness, the need for skilled microscopists etc.
Malaria is conventionally diagnosed by microscopic examination of stained blood films using Giemsa, Wright’s or Field’s stains [5]. Even though microscopic examination is considered to be the gold standard method, the most important limitation is its relatively low sensitivity, thus the generation of false negative results, particulary when microscopy is carried out using a low quality microscope and/or by less experienced personnel, and with low parasitaemias as in asymptomatic malaria. Furthermore the technique is laborious and not really suitable for remote rural settings, with no electricity or health facility resources.
The QBC technique was designed to enhance microscopic detection of malaria parasites [6]. This technique utilises micro-haematocrit tubes, fluorescent dyes and an appropriate fluorescence microscope for detection. Although simple, reliable and user-friendly, QBC is not widely applicable as it is costly, requires specialised instrumentation and is far from ideal for determining species and numbers of parasites.
Serological methods to diagnose malaria usually target antibodies against asexual blood stage malarial parasites. Immunofluorescence antibody testing (IFA) has proved a reliable serological test for malaria [7]. Although IFA is sensitive and specific, it is time-consuming and subjective. Furthermore the reliability greatly depends on the use of standardised reagents, in turn dependent on the expertise of laboratory workers.
Recent developments in malaria diagnosis suggest the use of PCR-based techniques. These techniques have proven to be one of the most specific and sensitive diagnostic methods, especially in malaria cases with low parasitaemia or mixed infections [8]. PCR was found to be more sensitive than QBC and some RDTs [9,10]. Compared with the gold standard method for malaria diagnosis, PCR has exhibited higher sensitivity and specificity [8]. Moreover, PCR can also help detect drug-resistant parasites, and is compatible with automation so that large numbers of samples can be processed. Some modified PCR methods e.g., nested PCR, real-time PCR and reverse transcription PCR are reliable and appear to be useful second-line techniques. Although PCR appears to offer the paramount sensitivity and specificity, its adoption in labs is limited due to the complex methodology, high cost and the demand for specialised instruments, the complex quality control and the difficulty of recruiting trained technicians especially in resource-poor conditions.
As the majority of malaria cases are found in countries where cost-effectiveness is an especially important factor and the ease of diagnostic test performance and training of personnel are also major considerations, new technology has given due attention to these points and utilised techniques that comply with diagnostic need without being very demanding. This has mainly resulted in the
development of RDTs.
Rapid diagnostic tests
RDT are largely based on the principle of immunochromatograpy, in which either monoclonal or polyclonal antibodies against the parasite antigen are immobilised to capture the parasite antigens from the peripheral blood. Currently, immunochromatographic tests target the histidine-rich protein-II of P. falciparum, a pan-malarial Plasmodium aldolase and the parasite-specific
lactate dehydrogenase.
Histidine-rich protein II of P. falciparum (PfHRP-II) is a water soluble protein that is produced by the asexual stages and young gametocytes of P. falciparum. It is abundantly expressed on the red cell membrane surface [11].
Parasite lactate dehydrogenase (pLDH) is a soluble glycolytic enzyme produced by the asexual and sexual stages of the live malarial parasites [9]. It is present in and released from the parasite-infected erythrocytes. It has been found in all four major species causing malaria in humans as their respective isoforms.
Plasmodium aldolase is an enzyme of the glycolytic pathway expressed by sexual and asexual stages of malaria parasites. RDTs have been developed in different test formats such as dipstick, card, well and cassette. The test procedure varies between different test kits. In general, the blood sample is mixed with a buffer solution that contains a haemolysing compound and a specific antibody that is labelled with a visually detectable marker such as colloidal gold. If the target antigen is present in the blood, a labelled antigen-antibody complex is formed and it migrates forward in the test strip and is captured at the test line. It is essential to include a control line to check on test validity. A washing buffer is then added to clear the background for easy
visualisation of the coloured lines.
RDTs are available in kit form with all the necessary reagents so they can be utilised even in remote places by less skilled personnel to generate results within a short period of time, usually within 15-20 minutes.
WHO recommended a few desirable characteristics for RDTs regarding their accuracy and sensitivity (WHO/MAL/2000.1091). According to this RDTs should be at least as accurate as results derived from microscopy performed by an average technician under routine field conditions, the sensitivity should be above 95% compared to microscopy, and the detection of parasitaemia should be such that levels of 100 parasites /µL (0.002% parasitaemia) should be detected reliably with a sensitivity of 100%. One product received U.S. FDA clearance in June 2007.
Today most RDTs have achieved this goal for P. falciparum, but not for other species. Roughly, RDT sensitivity declines at parasite densities < 500/µL blood for P. falciparum and < 5,000/µL blood for P. vivax [12]. RDT consumption, especially in developing countries, has increased over the past few years.
SPAN diagnostics offers RDTs i.e. ParaHIT-Total and ParaHIT-f in both dip stick, as well as in device format, for rapid and reliable diagnosis of malaria. ParaHIT-f is intended to diagnose malaria caused by P. falciparum with the use of P. falciparum specific HRP-II, wheareas ParaHIT-Total explores HRP-II and pan malarial species specific aldolase, as separate lines to screen malaria and for
differential determination of P. falciparum.
References
1. WHO, World Malaria Report 2010; December 2010.
2. WHO 10 facts on malaria
3. CDC, Malaria
4. Barnish G et al. Newer drug combinations for malaria. BMJ 2004; 328: 1511–1512
5. Warhurst DC et al. Laboratory diagnosis of malaria. J Clin Pathol 1996; 49: 533-538
6. Clendennen TE 3rd et al. QBC and Giemsa stained thick blood films: diagnostic performance of laboratory technologists. Trans R Soc Trop Med Hyg 1995; 89: 183-184
7. She RC et al. Comparison of immune fluorescence antibody testing and two enzyme immunoassays in the serologic diagnosis of malaria. J Travel Med 2007; 14: 105-111
8. Morassin B et al. One year’s experience with the polymerase chain reaction as a routine method for the diagnosis of imported malaria. Am J Trop Med Hyg 2002; 66: 503- 508
9. Makler MT et al. A review of practical techniques for the diagnosis of malaria. Ann Trop Med Parasitol 1998; 92: 419-433
10. Rakotonirina H et al. Accuracy and reliability of malaria diagnostic techniques for guiding febrile outpatient treatment in malaria-endemic countries. Am J Trop Med Hyg 2008; 78: 217-221
11. Rock EP et al. Comparative analysis of the Plasmodium falciparum histidine-rich proteins HRP1, HRP2 and HRP3 in malaria diagnosis of diverse origin. Parasitology 1987; 95: 209–227.
12. Wongsrichanalai C et al. A Review of Malaria Diagnostic Tools: Microscopy and Rapid Diagnostic Test (RDT). Am J Trop Med Hyg 2007; 77: 119–12.
The author
Meghna Patel
SPAN Diagnostics Ltd
Udhna, Surat, India
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