ReviewRapid and/or high-throughput genotyping for human red blood cell, platelet and leukocyte antigens, and forensic applications
Introduction
During the past 15 to 20 years, our understanding of the molecular biology of genes has grown rapidly, owing to the advance and application of technologies like DNA sequencing, polymerase chain reaction (PCR), and molecular cloning. The completion of the Human Genome Project has not only considerably increased our knowledge of the human genome but also revolutionized genomic screening techniques. Newer platforms of low-cost, high-throughput and rapid genotyping methods allow for practicable molecular testing in clinical settings. Traditionally, transfusion medicine, tissue typing, and forensic medicine relied on serologic studies. Work with earlier molecular diagnostic methods such as PCR-sequence-specific priming (PCR-SSP), PCR-restriction fragment-length polymorphism (PCR-RFLP), PCR-single-strand conformation polymorphism (PCR-SSCP), sequence-based typing (SBT), and DNA fingerprinting has proved the principle that molecular testing can be successfully applied to blood group, platelet and human leukocyte antigen (HLA) typing, and forensic medicine. In general, these early methods are relatively easy to set up, as they do not require expensive instrumentation up front. They are particularly well suited for small laboratories with relatively low workload. However, use of these methods often requires skilled technical staff (i.e., they are costly in training) and they are highly demanding in technologist time due to the large number of manual steps involved (i.e., they are labor-intensive). These methods also may be costly in maintaining quality control and they carry a high risk of amplicon contamination. While widespread use of molecular testing with traditional methods in clinical settings has been hampered by these limitations, advances in new platforms of molecular testing with real-time (quantitative) PCR and pyrosequencing that can combine amplification and detection allow molecular genotyping for entering fields dominated by serology for more than a century. Although microarrays (‘DNA chips’) are slower, they have the advantage of being able to perform simultaneous genotyping for an almost unlimited number of antigens. This article will review the current status of the application of rapid and high-capacity genotyping methods to these areas of interest.
Section snippets
Background
Since the discovery of the ABO blood group system and its clinical implications by Landsteiner in 1900 [1], serological testing has been serving the clinical needs of transfusion medicine. Serology testing, however, is not without its limitations. One problem is the limited availability of antisera, the other is the technical difficulty in serotyping when cells are coated with molecules such as immunoglobulins or when a patient is recently transfused. With the availability of genetic
Background
As is the case for blood group serotyping, serological methods also are limited by the availability of specific antisera against various human platelet antigens types. So far 24 platelet-specific alloantigens have been identified by antisera, of which 12 are grouped in 6 bi-allelic systems (HPA-1, -2, -3, -4, -5, -15). The molecular basis is known for 22 of the 24 serologically defined antigens. Genotyping for HPAs can help to resolve difficult cases of serotyping in general. However, it is
Background
In contrast to transfusion medicine, HLA typing is one of the few areas that adopted molecular testing early on. Molecular testing has been proven to be superior over serotyping, and the recognized contribution of high resolution HLA genotyping to the success of bone marrow transplantation makes it even a higher priority. At the present, commonly used molecular diagnostic methods for HLA typing include PCR-SSO (sequence-specific oligo hybridization), PCR-SSP and SBT. Of these, PCR-SSO produces
Background
In the past, serologic testing such as blood group typing has played a significant role in forensic medicine. However, molecular diagnostic tests such as the so-called DNA fingerprinting for identity/paternity testing [47] entered early this field and conventional serological tests are being fast replaced or complemented by nucleic acid tests in forensic laboratories [48], [49], [50], [51]. There are some special issues and technical challenges facing forensic DNA testing. One such issue is
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Routine use of DNA testing for red cell antigens in blood centres
2011, Transfusion and Apheresis ScienceCitation Excerpt :Applications differ by method and platforms, also the ways the results are detected, by the number of genotypes/alleles that can be included, by their ability to be completely automated, their suitability for high-throughput testing and the accreditation for in vitro diagnosis. Further the costs of each method differs substantially [25–27]. Today conventional PCR applications are predominantly SSP-PCRs (sequence specific primers; allele specific primers).
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2010, Critical Reviews in Oncology/HematologyCitation Excerpt :Moreover, HPA genotyping could improve the success of PLT transfusion [133]. Although RBC, PLT and white blood cell genotyping is a hot issue in TM and several genomic approaches have been successfully proposed [134], proteomics could rapidly grow as a serious competitor due to its complete automation, high-throughputness, extreme rapidity of the multiplexed analysis, cost-effectiveness, very high sensitivity and specificity. Indeed, although the initial purchase of mass spectrometric equipment is at this moment rather costly, the running costs for assays like the one presented by Wu and Csako can be very low (3.5 cents per genotype) [134].
Future of Molecular Testing for Red Blood Cell Antigens
2010, Clinics in Laboratory MedicineCitation Excerpt :In general, there are two common methods for detection of products using real-time PCR: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA (SYBR Green [Applied Biosystems, Foster City, CA, USA]); and (2) sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter that permits detection only after hybridization of the probe with its complementary DNA target (TaqMan [Applied Biosystems, Foster City, CA, USA]). The first has been used in combination with various instruments (eg, Lightcycler [Roche, Indianapolis, IN, USA] and ABI 7500 [Applied Biosystems, Foster City, CA, USA] to detect RH 1-5 [D, C/c, E/e]; JK1/2; and KEL1/2).5 Some other medium throughput assays are real-time PCR with melting-curve analysis and pyrosequencing.
Pyrosequencing
2009, Molecular Diagnostics: Second Edition