Prenatal screening and diagnosis is important for early detection and therapy of neonates affected by genetic disorders. Chip-based capillary electrophoresis can analyse small sample volumes quickly and easily. This technique may be used for prenatal as well as pre-implantation genetic diagnosis, where sample volumes are tiny and difficult to obtain. It could resolve many difficulties in current testing methods, which are slow and require large sample volumes.

by Dr Hua Hu and Dr Zhiqing Liang

Potential of chip-based capillary electrophoresis for rapid diagnosis of genetic disorders
Genetic diseases often lead to disability or mortality. As they are difficult to cure, prenatal screening and diagnosis is important. Many techniques are used to detect such disorders, such as gel electrophoresis, reverse dot blot methodology, allele-specific oligonucleotide probes (ASO), real-time PCR, mass-spectrometry (MS), sequencing and so on. These methods are accurate for determining the gene mutation, but have low sensitivity and are time-consuming.

Based on the common capillary electrophoresis, chip-based capillary electrophoresis uses ‘lab-on-a-chip’ technology. It can implement the sample introduction, reaction, separation and detection, and is a multifunctional, rapid, high performance, low volume, miniature analytical apparatus. The apparatus is not only low volume but also the automatic mode of operation reduces detection time of detection and experimental artefacts. This technique may be used for prenatal and pre-implantation genetic diagnosis where samples are tiny and difficult to obtain. Moreover, the rapid detection time can reduce parent anxiety.

We have optimised the detection beam path, separation gel and laser device of our chip-based capillary electrophoresis system in order to increase the sensitivity, resolution and stability. The improvement of the chip-based electrophoresis detector over the conventional confocal set-up was in the use of a holed reflecting mirror instead of a dichroic mirror, which significantly eliminated the effect of reflected laser light on the fluorescence detection. Moreover, the lasers were focused onto a very small spot (5 μm diameter) giving higher intensity light, thus giving higher efficiency fluorescence excitation. Additionally, the focused spot was much smaller than the width of the separation channel (60 μm), so avoiding the illumination of rough capillary side walls which can cause scattering of laser light, therefore increasing sensitivity. To further facilitate the optical alignment, a CCD imaging system was installed above the poly (methyl methacrylate) (PMMA) chip, confocal with the laser-induced fluorescence (LIF) detector. Also a dynamic movie of the electrophoretic process could be recorded using the CCD imaging system [Figure 1]. The device exhibited good reproducibility and is suitable for high-throughput applications.

We employed dual channel sample detection, with the two different wavelengths, for example Cy3 and Cy5. A standard substance was labelled with the fluorescent Cy3 label and the test sample was labelled the Cy5 fluorophore. By comparing the fluorescence of the Cy3 and Cy5 samples, we can deduce the size of the test sample and ascertain the genotype [Figure 2]. Our results show that chip-based capillary electrophoresis is an accurate, rapid and highly sensitive detection method suitable for prenatal diagnosis.

The advantages and limitations of this technique for prenatal diagnosis of β-thalassemia
Thalassemia is the commonest genetic disorder worldwide, especially in the Mediterranean and Asia. Severe thalassemia is associated with high mortality. Prenatal screening for thalassemia is important to prevent the spread of the condition and to give at-risk couples the option of avoiding an affected child. Thus the prenatal screening of the carrier parent and affected fetus is strongly recommended in developing countries where the treatment of affected patients is expensive and may not available.

Chip-based capillary electrophoresis may be promising for the prenatal and pre-implantation genetic diagnosis of β-thalassemia, where high sensitivity is vital as sample volume is limited and difficult to obtain. Sensitivity studies showed the chip-based capillary electrophoresis system was capable of detecting 1 ng of genomic DNA (1 ng/μl), and had a linear range of detection of 1–50 ng/μl. The detection system requires only 1 μl of sample at 0.04 nM. We utilised chip-based capillary electrophoresis and developed rapid assays for prenatal diagnosis of β-thalassemia. Chip-based capillary electrophoresis decreased the analysis time for genotyping to 200 s. The separation time is shorter than gel electrophoresis and capillary electrophoresis, which accelerates the prenatal diagnosis. Moreover we detected the size ladder and samples simultaneously by a dual-channel detection system, which are labelled with Cy3 and Cy5 fluorescence, respectively, to improve the detection precision.

We compared three methods including agarose gel, polyacrylamide gel and chip-based capillary electrophoresis. Traditional agarose gel electrophoresis and ethidium bromide staining is cheap but is potentially harmful to health,  and the sieving capability of agarose gel is limited to 50 bp, which makes it difficult to separate multiple PCR products. Polyacrylamide gel has a higher separation capability but is time-consuming. Chip-based capillary electrophoresis is faster and can discriminate 4–10bp differences, something that is not possible using traditional gel electrophoresis [1], making it convenient for primer design.  Reverse dot blotting is easy and quick but requires large amounts of DNA (about 2 μl of 0.1 μg/μl DNA), and the process is cumbersome, requiring more than 7 hours. Chip-based capillary electrophoresis is sensitive and only requires about 1ng/μl DNA which is important for precious sample detection. Only 3.5 hours are needed, including the procedure of extracting DNA, PCR, purification and detection, which reduces the time for awaiting the result and eases the anxiety of patients. It allows 10 bp resolution and takes only a few seconds. These results show that chip-based capillary electrophoresis is a quick, sensitive detection system with improved resolution. The sensitive characteristics of chip-based capillary electrophoresis provide obvious advantages over slab-gel electrophoresis and capillary electrophoresis for biomedical and clinical applications.

Although chip-based capillary electrophoresis has tremendous potential in disease diagnostics, its wider use has been limited by the size and cost of the instrumentation. Most reports of chip-based capillary electrophoresis have used glass or silica as the base materials for chip fabrication. We utilised the polymer substrate PMMA which made the chips less expensive and easier to make. The cost of analysis of one sample was about 10$ (75¥), which is identical to reverse dot blotting and acceptable to most patients. There are chip-based capillary electrophoresis designed with integrated circuit chips which include inexpensive portable systems, complementary metal-oxide-semiconductor chips and low-cost components [2]. This instrument is powered and controlled using a universal serial bus interface to a laptop computer which can readily analyse the DNA produced by a standard medical diagnostic protocol. The improvement of chip-based capillary electrophoresis will facilitate its wide use in prenatal diagnosis.

Possible future applications
Most genetic diseases are diagnosed by invasive prenatal testing, which carries a high risk of abortion, infection and other complications. Fetal cells and cell free fetal DNA [3] in the maternal circulation were discovered nine years ago and can be used for non-invasive prenatal fetal sex determination, and diagnosis of chromosome 21 trisomy and RhD. At present targeted massively parallel sequencing of maternal plasma is used for non-invasive prenatal diagnosis of β-thalassemia [4]. However, it is not applied widely, partly because of sample rarity and technological complexity. The diagnostic reliability of circulating DNA analysis depends on the fractional concentration of the targeted DNA, the analytical sensitivity, and the specificity. Maternal plasma includes maternal and fetal DNA of which the fraction ranges from a few percent or lower early in pregnancy and increases with gestational age [5]. Hence non-invasive prenatal testing of the fetal genome generally takes place after at least 13 weeks of pregnancy. In addition, some cases require re-testing because of too low fetal DNA content in the first blood sample.

The discrimination of single-nucleotide difference between circulating DNA samples is technically challenging and demands the adoption of highly sensitive and specific analytical systems. Chip-based capillary electrophoresis can analyse small samples speedily and conveniently. We constructed the platform of chip-based capillary electrophoresis to detect the point mutations and achieved prenatal diagnosis of β-thalassemia quickly by detecting fetal DNA in maternal plasma. This chip-based capillary electrophoresis detection system is capable of the non-invasive prenatal diagnosis of β-thalassemia. Its use would facilitate prenatal diagnosis of the genetic disorder rapidly and sensitively. This method has high sensitivity, high-speed and high throughputs, and is very suited for prenatal diagnosis.

Most genetic diseases are a result of point-mutations and DNA fragment deletions. The main DNA defect of β-thalassemia is a point-mutation. However, the gene defects of α-thalassemia include point-mutations and klenow fragment deletions. In the clinic, point-mutations are generally detected by reverse dot blotting or sequencing, and the deletion detected by Gap-PCR and agarose gel electrophoresis [6]. These techniques often require two different detection methods and equipment, and are labour intensive and time-consuming. Combining chip-based capillary electrophoresis with multiplex allele specific PCR, results in the sensitive and reliable detection of the point mutation. This method can also be used to separate and detect the PCR products of different length arising due to deletion events. Therefore, using chip-based capillary electrophoresis, we may detect not only point-mutations but also deletions, and so can simultaneously detect α-thalassemia and β-thalassemia, which is rapid and convenient.

Conclusion
In future we may use the detection system for non-invasive prenatal analysis of circulating DNA and pre-implantation genetic diagnosis. Moreover, the system could also be used for the detection of α-thalassemia and β-thalassemia together. This could resolve many problems associated with traditional methods of genetic analysis, which are slow and require larger sample volumes and so are not suited for prenatal diagnosis.

References
1. Tabe Y, Kawase Y, Miyake K, Satoh N, Aritaka N, Isobe Y, et al. Identification of Bcl-2/IgH fusion sequences using real-time PCR and chip-based microcapillary electrophoresis. Clin Chem Lab Med 2011; 49(5): 809–815.
2. Behnam M, Kaigala GV, Khorasani M, Martel S, Elliott DG, Backhouse CJ, et al. Integrated circuit-based instrumentation for microchip capillary electrophoresis. IET Nanobiotechnol 2010; 4(3): 91–101.
3. Y M Dennis Lo, Noemi Corbetta, Paul F, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997; 350: 485–487.
4. Lam KW, Jiang P, Liao GJ, Chan KC, Leung TY, Chiu RW, Lo YM. Noninvasive prenatal diagnosis of monogenic diseases by targeted massively parallel sequencing of maternal plasma: application to β thalassemia. Clin Chem 2012; 15.
5. Palomaki GE, Deciu C, Kloza EM, et al. DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: an international collaborative study. Genet. Med 2012; 14: 296–305.
6. Rosnah B, Rosline H, Zaidah AW, Noor Haslina MN, Marini R, Shafini MY, et al. Detection of common deletional alpha-thalassemia spectrum by molecular technique in Kelantan, Northeastern Malaysia. ISRN Hematol. 2012; 462969.

The authors
Hua Hu PhD and Zhiqing Liang PhD*
Departments of Obstetrics and Gynecology
Southwest Hospital
Third Military Medical University,
Chongqing, China 400038

*Corresponding author:
e-mail: zhiq.lzliang@gmail.com

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