Rapid and/or high-throughput genotyping for human red blood cell, platelet and leukocyte antigens, and forensic applications

Abstract

Background

Traditionally, transfusion medicine, platelet and human leukocyte antigen (HLA) typing, and forensic medicine relied on serologic studies.

Methods

In recent years, molecular testing on nucleic acids has been increasingly applied to these areas. Although conventional molecular diagnostic methods such as PCR-sequence-specific priming, PCR-restriction fragment-length polymorphism, PCR-single-strand conformation polymorphism, sequence-based typing, and DNA fingerprinting have been shown to perform well, their use is limited by long turnaround times, high cost, labor-intensiveness, the need for special technical skills, and/or the high risk of amplicon contamination. With advance of fast and/or high-throughput methods and platforms that often combine amplification and detection, a new era of molecular genotyping is emerging in these fields dominated by serology for a century. As new targets, short tandem repeats, mitochondrial DNA and Y-chromosome sequences were introduced for forensic applications. This article reviews the current status of the application of rapid and/or high-throughput genotyping methods to these areas.

Results

The results are already promising with real-time PCR, pyrosequencing, microarrays, and mass spectrometry and show high concordance rates with classic serologic and earlier manual molecular diagnostic methods. Exploration of other emerging methodologies will likely further enhance the diagnostic utility of molecular testing in these areas.

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.