Although significant progress has been made to improve blood safety and efficiency, laboratories still face workforce challenges, and the lack of global standards and better quality controls in blood management and haemovigilance pose a threat to patient safety. To help address these challenges, a symposium was hosted at the 2011 American Association of Blood Banks Annual Meeting with key opinion leaders from the transfusion medicine departments of the Cleveland Clinic, Children’s Hospital Los Angeles and USC Medical Center.
The attending experts broadly agreed that the key to safe, efficient blood management boils down to the ‘5Rs’: ensuring the Right patient gets the Right donor unit at the Right time, the Right way and for the Right reason. Presentations at the symposium discussed how the transfusion laboratory can deliver on the 5Rs through improvements in blood management and haemovigilance practices.
by Scott Saccal
Improving blood safety
Laboratories strive to protect patients’ health and deliver safe blood and blood components to the right person at the right time, but they are under constant pressure to do more with less – including fewer skilled laboratory technicians and scarcer financial resources. To help in meeting these demands, blood bank laboratories are increasingly employing automation. For example, many blood banks are standardising across instrument platforms and implementing testing technologies such as Column Agglutination (CAT). These testing methods are easier to use, and help reduce the opportunity for error and variation among both technologists and tests because they provide stable and clear endpoints that deliver accurate, objective and consistent results.
Automation helps minimise the labor-intensive, time-consuming manual tests that require specialised skills and significant experience to master, such as patient and unit typing, antibody screening and cross-matching. Automation also increases the capacity of technologists so they can focus on priorities such as time-sensitive emergency situations, difficult patient workups and quality-improvement processes. For example, computerised physician order entries have been found to reduce errors related to labour-intensive tasks by 50 percent, and all errors in general by 80 percent [1,2]. Ultimately, automated testing can increase a lab’s capacity, potentially allowing it to serve more patients while helping it operate more efficiently.
New approaches to load management and haemovigilance
Quality controls help ensure the safety of the patient at the end of the bloodline by helping to assure that from the moment a donation is made, the right blood and blood products are delivered to the right patient at the right time, and in the right way for the right reason. While great strides have been made to implement quality controls for the highest level of patient safety, there remains much work to be done.
Across the globe, a total of 106 countries have national guidelines on blood management, yet no universal safety standards exist. Further, broken system links and human errors due to distraction, fatigue and inattention account for approximately 70 percent of lab errors and cause catastrophic consequences such as the inappropriate administration of blood and/or adverse reactions [3]. Giving the wrong donor unit or giving an inappropriate transfusion can lead to serious complications, disease transmission and even fatal haemolytic reactions [4].
To combat the high incidence of laboratory errors, many hospitals and clinics are appointing Transfusion Safety Officers (TSO), to oversee work outside of the laboratory to improve patient safety during transfusions [5]. The increase in demand for blood and blood components suggests that additional measures will be needed to promote transfusion safety across departments, oversee institution-wide haemovigilance and error and accident reporting, provide education on transfusion reactions, implement guidelines, perform safety training and identify new technology for enhanced safety.
Additionally, hospitals and healthcare institutions around the world are developing ways to meet new standards – both by instituting their own rigorous policies, and by understanding and implementing the guidelines from organisations that oversee the safety of transfusions. The UK’s Serious Hazard of Transfusion programme and the U.S. Centers for Disease Control (CDC) National Healthcare Safety Network (NHSN) suggest voluntary reporting structures to create a reliable source of information for the medical and scientific community about blood transfusion issues, including warning facilities about adverse events that could be systemic.
Industry groups also are striving to improve patient care and safety while maximising healthcare system efficiencies. For example, AABB collaborates with the U.S. Department of Health and Human Services on biovigilance activities, including programmes directed at a variety of different domains such as donor haemovigilance and transfusion recipient haemovigilance. Through the collaboration, the organisations are gathering and analysing data to help find trends and establish best practices for safer, more efficient transfusions and transplants [6]. Similarly, the International Society of Blood Transfusion (ISBT) and the European Haemovigilance Network (EHN) began a working group in 2004 focused on creating a common set of definitions for issues in the field, which would enable global benchmarking and is intended ultimately to increase the safety of blood donors and recipients around the world [7].
Shared commitment to patient safety
Protecting the precious life of a patient who will receive a unit of blood remains the focus of blood bankers everywhere. As the pressures and demands rise, labs are finding new ways to be efficient and haemovigilant, while never losing sight of the real person at the end of the bloodline. Despite the continued shortage of highly skilled technologists and scientists entering the laboratory science workforce, blood bankers are utilising automation and best practices to improve transfusion testing, and implementing new approaches to blood management and haemovigilance to deliver on the 5Rs of blood safety. Protecting the safety of patients through efficient blood management and haemovigilance is a commitment all of us share as part of the transfusion medicine community.
References
1. Bates DW et al. Effect of computerized order entry and a team intervention on prevention of serious medication errors. Journal of the American Medical Association 1998; 280: 1211-1212
2. Bates DW et al. The impact of computerized order entry on medication error prevention. Journal of American Medical Informatics Association 1999; 6: 313-332.
3. Kaplan HS. Getting the right blood to the right patient: the contribution of near-miss event reporting and barrier analysis. Transfusion Clinique et Biologique 2005; 2: 380-384.
4. Shulman Ira A. ‘Assuring That The Right Patient Gets The Right Donor Unit, At The Right Time, The Right Way, And For The Right Reason.’ Slide 6. AABB annual meeting. San Diego, CA. Ortho Clinical Diagnostics Blood Management Symposium. October 24, 2011
5. Davey Richard J. ‘The Safety of the Blood Supply.’ Food and Drug Administration Division of Blood Applications webinar. Available at: http://www.fda.gov/downloads/AboutFDA/Transparency/Basics/UCM245738.pdf. Accessed October 20, 2011.
6. Biovigilance – AABB program — http://www.aabb.org/programs/biovigilance/Pages/default.aspx
7. Haemovigilance – ISBT — http://www.isbtweb.org/fileadmin/user_upload/WP_on_Haemovigilance/ISBT_StandardSurveillanceDOCO_2008__3_.pdf
The author
Scott Saccal
Worldwide Marketing Director
Transfusion Medicine
Ortho Clinical Diagnostics
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
Methods for the diagnosis of blood-borne parasitic infections have stagnated in the last 20–30 years. However, recently, there has been a tremendous effort to focus research on the development of newer diagnostic methods focusing on serological, molecular, and proteomic approaches. This article examines the various diagnostic tools that are being used in clinical laboratories, optimized in reference laboratories and employed in mass screening programmes.
by A. Ricciardi and Dr M. Ndao
Blood-borne protozoans are the causative agents of some of the world’s most devastating and prevalent parasitic infections. This group of pathogens includes members of the Trypanosoma, Leishmania, Plasmodium, Toxoplasma, and Babesia genera. Most of these infections, with the exception of toxoplasmosis and babesiosis, have always been described as being tropical or subtropical. However, the increase in international travel as well as the arrival of new immigrants has made some of these tropical diseases realities in developed countries as well. In addition, infection via contaminated blood (transfusions and organ transplants) has become a major problem. Clearly, the transmission of blood-borne protozoans is boundless and the actual number of cases is underestimated. Quick diagnosis has always been a priority in order to determine the appropriate treatment and prevent fatalities. In addition, now more than ever, advances in diagnostics can help prevent transmission and provide active surveillance. Currently, diagnostic and reference laboratories use an array of techniques including microscopy, serological assays, and molecular assays. Here, the advantages and disadvantages of the methods will be discussed.
Toxoplasmosis
Toxoplasmosis, caused by Toxoplasma gondii, has a worldwide distribution. In immunocompetent individuals, more than 80% of primary Toxoplasma infections are asymptomatic [1]. Toxoplasmosis becomes a problem when an individual is immunocompromised or during pregnancy. Diagnosis of toxoplasmosis varies according to the immune status of the patient.
Diagnosis of immunocompetent individuals relies on serology. Early antibody responses can be detected via methods such as the dye test, immunofluorescent assay, and agglutination test whereas later IgG titres are detected by enzyme-linked immunosorbent assay (ELISA). For many years, the Sabin-Feldman dye test was the gold standard diagnostic technique due to its sensitivity and specificity. In recent years, few laboratories have continued to use this method and rather focused on newer techniques such as indirect immunofluorescent antibody tests, hemagglutination tests, capture ELISAs, and immunosorbent agglutination assays (ISAGAs). Serological assays lack the capacity to differentiate between recent and older infections; IgM levels can persist for over two years [2]. In order to determine whether an infection is recent, avidity ELISA is performed. This assay verifies IgG avidity and is based on the concept that as the immune response progresses, an immunoglobulin’s affinity for a specific antigen will increase [3].
Diagnosis of Toxoplasma infection during pregnancy is crucial in order to prevent congenital toxoplasmosis. Prenatal diagnosis involves performing real-time polymerase chain reaction (PCR) using amniotic fluid. The PCRs used often target the B1 gene of the parasite [1]. Upon delivery, PCR is performed on either the placenta or the cord blood serum in order to detect parasites. ISAGAs are also often performed. If the tests are positive, cord blood samples at one week of life are sent to a reference laboratory [1]. Follow up serology is again performed at one month and then every two to three months. There have been recent advances in the field of toxoplasmosis post-natal diagnosis. An ELISA assay that measures interferon-gamma levels upon stimulation of whole blood cells with Toxoplasma crude antigens has been developed. This method has proven to be both sensitive and specific [4].
In the case of immunocompromised patients, a quick diagnosis is essential because the infection can be fatal. Diagnosis relies on detecting parasites either by PCR or microscopy. Microscopic examination of Giemsa-stained tissues or smears is the quickest and most inexpensive method for diagnosing toxoplasmosis. However, poor sensitivity is the major pitfall of this method. PCR can also be performed on blood or cerebral spinal fluid (CSF) samples in order to detect parasite DNA. However, the degree of sensitivity attained by the PCRs is questionable and requires further investigations [1].
Leishmaniasis
Protozoans of the Leishmania genus are transmitted to humans via sand fly bites. Visceral leishmaniasis (VL), which is a lethal infection if left untreated, can also be transmitted by blood transfusions, organ transplants, and sharing of needles among intravenous drug users.
Direct parasitological methods, such as microscopy and cultures, are the gold standard methods when diagnosing VL. These methods have high specificity, but varying sensitivity. Direct detection of parasites is performed by microscopic examination of aspirates from spleen, bone marrow, or lymph nodes [5]. Using spleen samples increases sensitivity, but the procedure to obtain the aspirates risks internal bleeding. Parasite culturing from aspirates is widely used by reference laboratories.
Extensive research on the development of Leishmania serological assays has uncovered a myriad of candidate diagnostic antigens. The most promising antigens were the kinesin-related proteins. From this group, rK39 was the most tested antigen [6–8]. The rK39 antigen has been used to develop an immunochromatographic strip test (ICT)-based rapid diagnostic test which is advantageous for mass screening in endemic areas. This test requires a drop of peripheral blood and can be completed in approximately fifteen minutes [7]. Although the rK39 ICT rapid test was quite successful in Asia, it was often unable to detect Leishmania infections in African patients [5]. Additionally, rapid diagnostic tests still need standardization in order to become a regular practice in clinical laboratories.
PCR is the main molecular tool for Leishmania diagnosis due to its high sensitivity and reliability. Different PCR target sequences that are commonly used include ribosomal RNA genes, kinetoplast DNA, mini-exon derived RNA, internal transcribed spacer regions, etc., [5]. Quantitative PCR is useful because it allows for the quantification of parasites as well as species typing. Furthermore, this technique can be used to monitor treatment efficiency. Unfortunately, equipment requirements as well as the high cost limit the use of PCR for mass screening purposes in the field. The introduction of loop-mediated isothermal amplification (LAMP) could facilitate the use of molecular techniques for diagnostics. LAMP is highly specific, carried out under isothermal conditions, quick, and requires less complicated equipment (5). Moreover, reagents can be kept at room temperature, and there are no post-PCR steps. Assessment of drug treatment can also be carried out through the use of nucleic acid sequence based amplification (NASBA) which amplifies RNA sequences under isothermal conditions. Coupled to oligochromatography, NASBA can be used to monitor the progression from active disease to cure [9].
Chagas Disease (American Trypanosomiasis)
Chagas disease is the result of an infection with the blood-borne protozoan Trypanosoma cruzi. The parasite is transmitted by the triatomine bug. The second most important mode of transmission is via contaminated blood. This includes blood transfusions, organ transplants, and congenital transmission.
During the acute stage of Chagas disease, parasites can be observed in the blood. For this reason, diagnosis is carried out by direct microscopic viewing of Giemsa-stained thin and thick blood smears [10]. Parasites may also be detected through the use of hemocultures. In Chagas endemic areas, xenodiagnosis may be performed. This method involves allowing the naïve triatomine bug to take a blood-meal from the patient, and then analysing the bug for the presence of trypanosomes. It is believed that with continued research, molecular methods will eventually replace indirect diagnostic techniques such as blood cultures and xenodiagnosis [10]. However, molecular tests need to be standardized for routine clinical practice.
During the chronic stage of Chagas disease, diagnosis relies on serology; however, these tests often yield results that are difficult to interpret [10]. Commonly used, standardized serological assays include indirect immunofluorescence (IIF), indirect hemagglutination (IHA), and ELISA. IIF and IHA are commonly used due to their good sensitivity; however, their results are operator-dependent, and there is a lack of studies which analyse their reproducibility [10]. Currently, the immunoblot and radioimmunoprecipitation assays are in the process of being standardized. Both tests showed promise in early studies. A great deal of work is also being focused on the development and standardization of molecular methods such as PCR, which could be useful in monitoring chronic phase, reactivation, and treatment response.
As previously mentioned, disease transmission can also occur from mother to child, leading to congenital Chagas. Screening of neonates can be performed via direct methods, such as microscopy, or PCR using venous or cord blood samples from the newborn. These tests have very high sensitivity when performed during the first month of life [10]. Serological analysis may also be performed.
Sleeping Sickness (African Trypanosomiasis)
Trypanosoma brucei is the causative agent of African trypanosomiasis, and it is transmitted via the bite of the tsetse fly. During the first stage of the disease, parasites can be found circulating in the peripheral blood. The second stage is marked by parasites crossing the blood-brain barrier and infecting the central nervous system (CNS). The parasitic subspecies dictates geographic distribution, prognosis, and diagnosis.
T. b. gambiense causes West African trypanosomiasis, which is a slow progressing disease and is characterized by low parasite loads [11]. Definite diagnosis is carried out by microscopic observation of blood, lymph node aspirate, or CSF for the presence of parasites. In the field, the card agglutination test for trypanosomiasis (CATT/T. b. gambiense) has been widely used since its development in 1978 (12). Whole blood is used, and the assay directly detects T. b. gambiense specific antibodies. CATT/T. b. gambiense is cheap, quick, and highly sensitive. However, the test can give rise to false positives in individuals who are co-infected with malaria [12]. Although CATT/T. b. gambiense is the most sensitive, similar tests such as micro-CATT and LATEX/ T. b. gambiense can also be used. If these assays generate positive results, they need to be confirmed by microscopy or other molecular methods.
T. b. rhodesiense causes East African trypanosomiasis, which progresses quickly and is characterized by high parasite loads (11). For this subspecies, there is no diagnostic equivalent to the CATT/T. b. gambiense. However, diagnosis by microscopic observations of thick and thin smears is simple due to the elevated parasite load associated with T. b. rhodesiense.
Microscopy is the most practical technique to be used in rural areas. However, microscopy requires adequately qualified personnel in order to prevent misdiagnosis. Molecular methods would substantially improve the diagnosis of African trypanosomiasis. PCR techniques have been developed to screen the CSF of patients. The discovery of the SRA gene in T. b. rhodesiense has proven to be a breakthrough for the promotion of PCR techniques. Reactions targeting this gene have the potential to identify a single trypanosome [11]. There has also been the introduction of fluorescence in-situ hybridization in combination with peptide nucleic acid probes aimed towards ribosomal RNA. However, these tools for diagnosis are new and require further optimization. Extensive research is being focused on standardizing molecular techniques and rendering them more accessible. The use of LAMP is a step forward in improving molecular
approaches [11].
Future research needs to focus on the improvement of molecular diagnostic techniques. Currently, second stage infections are diagnosed by microscopic observation of CSF. Research is being conducted to test various cytokines and antibodies as biomarkers for CNS infection [11].
Malaria
Malaria is the most important parasitic infection in the world due to its high mortality. The causative agents, parasites of the Plasmodium genus, are transmitted by Anopheles mosquitoes. Quick diagnosis is essential in order to determine the appropriate treatment as well as to prevent further transmission.
Microscopy is the gold standard for laboratory diagnosis. This method involves detecting parasites in Giemsa-stained thick and thin blood smears. However, microscopic results are operator-dependent, thereby causing the sensitivity to vary. A great deal of effort has been focused on developing rapid diagnostic tests (RDTs) which can be used in the field. These tests can supplement microscopy, but they cannot replace it yet. Current RDTs are serology based and use three different Plasmodium antigens: Plasmodium histidine-rich protein, Plasmodium lactate dehydrogenase, or Plasmodium aldolase [13]. These tests are quick, easy to perform, and require minimal patient samples. However, they are not specific for species such as P. malariae, P. ovale, and P. knowlesi. Furthermore, false positives may be observed due to cross-reactions in patients with Schistosoma mekongi or rheumatoid factor [14]. In addition, the tests inefficiently detect P. falciparum infections from South America, as this species does not produce the common histidine-rich proteins [15].
Currently, there are no commercially available molecular assays. Although some reference and government laboratories have developed their own molecular assays, their availability is limited. LAMP is currently in the spotlight. Poon et al. developed a LAMP test which detected the target sequence of P. falciparum 18S ribosomal RNA gene [16]. They stated that the price of this test was one tenth that of a conventional PCR. Recently, LAMP was further simplified in the form of a card test. It was used in combination with DNA filter paper and melting curve analysis. This system was shown to be highly specific and sensitive [17]. Improvement of the LAMP technique should be geared towards the development of rapid diagnostic tests which could potentially be used in the field.
Babesiosis
Babesiosis is caused by parasites belonging to the Babesia genus that are spread by certain ticks commonly found in North America. The parasites infect red blood cells (RBCs), and consequently cause hemolytic anemia. The disease can be fatal in splenectomy patients, immunocompromised individuals, and the elderly. Diagnosis is complicated by the symptoms’ resemblance to other tick-borne illnesses.
The gold standard of babesiosis diagnosis relies on detecting the parasites in the patients’ RBCs. This is achieved by microscopic observation of thick and thin blood smears. Babesia infections can be easily mistaken for P. falciparum infections [18]. Additionally, false negatives are common in immunocompetent individuals whose parasitemia can be lower than 1% [18]. Samples are often sent to reference laboratories in order to confirm ambiguous results. IFFs are used to detect anti-babesial IgM and IgG [18]. They are sensitive, specific, and reliable. ELISAs and immunoblots, although not standardized, can be performed to confirm the IFF results. However, compared to IFFs, Babesia detecting ELISAs require higher concentrations of antigen and have varying sensitivity [18]. Future research on babesiosis diagnosis is aimed at developing multiplex PCR assays that will be able to detect several tick-borne infections. PCR assays have the potential to yield positive results from 100µl blood samples containing as little as three parasites; demonstrating the incredible advantage that molecular techniques could contribute to diagnosis of this parasitic disease [18].
Proteomics
Dr Momar Ndao’s laboratory focuses on the improvement and advancement of diagnosis. Through our work, we hope to encourage the development of proteomic strategies for the diagnosis of parasitic infections. Mass-spectrometry platforms are the future of proteomics, and they can be used to identify biomarkers from biological fluids. Some techniques that can be used to analyse protein expression include matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (MALDI-TOF MS), surface-enhanced laser desorption ionization time-of-flight mass-spectrometry (SELDI-TOF MS), liquid chromatography combined with mass-spectrometry, isotope-coded affinity tags, and isobaric tags for relative and absolute quantification [19]. When SELDI is used, samples are directly spotted onto chemically active ProteinChip Array surfaces which can be chosen based on specific chemical and biological properties. With MALDI, samples are mixed with the matrix component prior to loading on a chip. These proteomic platforms can be useful in identifying biomarkers that are indicative of a specific pathophysiological state. Currently, members of our laboratory are using both SELDI and MALDI techniques extensively to identify biomarkers of blood borne parasites.
Summary
Quick and correct diagnosis of parasitic infections is crucial to avoid deaths and further disease transmission. Diagnostic methods include parasitological techniques, such as microscopy and culturing, serological assays, and molecular tests [Table 1]. Although several serological and molecular diagnostic tools are being tested and used by certain reference laboratories, results are always confirmed by microscopy which remains the gold standard. Many newer assays have not been standardized yet, thus, forcing diagnosticians to rely on microscopic observations. Unfortunately, the evolution of diagnosis in the field of parasitology has been slow to progress. Fortunately, in recent years, several groups have focused their research on the improvement of diagnostics. Current research emphasizes the development and optimization of molecular techniques such as PCR and LAMP. Additional work must concentrate on rendering molecular diagnostics more accessible. Although relatively new at the moment, proteomic platforms seem to be the future of diagnosis. These new techniques can identify biomarkers which can categorize susceptible individuals, distinguish between the different stages of an infection, and monitor whether treatments lead to cure. Diagnostic research has made much progression, however, there is still a lot of work to be done and improvements to be made. In order to better the diagnosis of blood-borne parasitic infections, research plus communication is the answer.
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The authors
Alessandra Ricciardi, BSc
National Reference Centre for Parasitology, Research Institute of the McGill University Health Center, Montreal, Canada
Momar Ndao, DVM, MSc, PhD
National Reference Centre for Parasitology at the Montreal General Hospital, Montreal, Quebec, Canada
E-mail: momar.ndao@mcgill.ca
March 2026
Prins Hendrikstraat 1
5611HH Eindhoven
The Netherlands
info@clinlabint.com
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