Molecular Protocols in Transfusion Medicine

By Gregory A. Denomme, Maria Rios and Marion E. Reid

This essential methods manual for immunohematologists (or hematologists and immunohematologists) provides information on genes that encode antigens on red blood cells, platelets and neutrophils. The book begins by covering general concepts in molecular biology and specific protocols such as DNA preparation, PCR-RFLP and allele-specific PCR. Information on the erythrocyte, platelet and neutrophil antigen systems and the molecular basis of polymorphisms are presented clearly in a gene facts sheet format. Database accession numbers and useful adjuncts such as Request forms, worksheets for PCR/enzyme digests also serve to benefit the user. The information is clearly presented and easily accessible and is complemented by the excellent diagrams and tabular material. This book is invaluable for both new and experienced researchers in the field and other related disciplines.

• Essential for hematologists and those involoved in tissue typing and the study of human genetic polymorphisms
• Presents clearly and concisely the information on a particular variant and the technique used to detect it
• Organized by antigen and provides sequences of polymorphisms and primers
• Details the general concepts and critical information on genes, their products, and sources of relevant nucleic acids
• Includes protocols that allow investigators to set up assays with minimal effort (protocols include primers, reagents, reaction conditions, sizes of amplified products, restriction fragment digests, and the relevant safety information)
• Provides information that helps interpret results in clinical settings
• Contains additional sources of information (e.g., key references, web site addresses, glossary, Database accession numbers, request forms, and worksheets for PCR/enzyme digests)

• Language: English
• Publisher: Academic Press
• Release date: Apr 27, 2000
• ISBN: 9780080536903

Book preview

cell

Part 1

Introduction

1.1 Background

Since the development of the polymerase chain reaction (PCR) (Mullis and Faloona 1987), a plethora of applications using this technique have been reported. The majority of genes encoding either proteins that carry blood group antigens or transferases that attach blood group-specified carbohydrates have been cloned and the molecular bases associated with many cell surface markers on red blood cells (RBCs), platelets, and neutrophils have been determined. Thus, it is possible to perform molecular analysis for these inherited alleles. Such analyses include allele-specific PCR amplification (AS-PCR), and PCR amplification followed by restriction enzyme digestion (PCR-restriction fragment length polymorphism (RFLP)).

There are 25 red cell blood group systems, consisting of over 200 antigens, which are recognized by the International Society of Blood Transfusion (ISBT) (Daniels et al 1995, 1996, 1999). Genes encoding 21 of these systems have been cloned and sequenced. The molecular basis that gives rise to many of the polymorphisms that result in blood group antigens and phenotypes are known (Reid and Lomas-Francis 1996; Reid and Yazdanbakhsh 1998; Avent 1997).

There are 14 human platelet antigen systems that are expressed on seven glycoproteins. Six of the seven genes have been cloned and sequenced (Warkentin and Smith 1997; Santoso and Kiefel 1998).

There are eight neutrophil systems consisting of at least 11 antigens. Three of six genes have been cloned and sequenced. These antigens are not related to human leukocyte antigens (HLA) expressed on neutrophils.

A wide variety of molecular events generate blood group antigen diversity (Reid and Rios 1999; Alberts et al 1994). These include:

• missense mutation (by far, the most common)

• nonsense mutation

• mutation of motifs involved in transcription

• alternative splicing

• deletion of a gene, exon(s) or nucleotide(s)

• insertion of an exon(s) or nucleotide(s)

• alternate initiation

• chromosome translocation

• single crossover

• gene conversion

• other gene rearrangements

To provide a comprehensive clinical service, the following skills are desirable when interpreting results of molecular analyses:

• a basic knowledge of the principles and mechanisms of molecular techniques

• an understanding of gene structure, function, and expression

• an understanding of blood group antigens and the serological techniques used to characterize their expression

• an ability to correlate results to the clinical problem being addressed

1.2 Aim, Scope and Purpose of the Book

The aim of this book is to provide general principles, generic protocols for basic techniques, and specific protocols for genotyping red cell, platelet and neutrophil blood groups in clinical or research transfusion medicine laboratories. The protocols we provide for specific alleles have been shown by us to be robust and reproducible in a clinical setting. For other alleles, protocols are not given but relevant information is provided on the relevant gene figure or in a table. The book is designed in a convenient, easy-to-use format with pertinent references and protocols for performing molecular analyses.

We have made the assumption that the reader has a basic understanding of the genetic code, and of the pathway from a gene to its product (Figure 1.1). Such information is found in molecular biology textbooks (Alberts et al 1994) and review articles on blood group genetics (Reid and Rios 1999; Avent 1997; Daniels 1999; Warkentin and Smith 1997; Stroncek 1997). Details for the design of primers, and gene sequence analysis, are also beyond the scope of this book. Description of these and other more complex techniques can be found elsewhere (Sambrook et al 1989; Ausubel et al 1997).

Figure 1.1 From cell to PCR RFLP.

1.3 Organization of the Book

The book is divided into seven parts. Part 1 provides a brief overview of the background, aims and scope of the contents; Part 2 gives general considerations and principles for molecular analyses; Part 3 gives molecular protocols including preparation and storage of DNA, preparation of gels and reagents, PCR-RFLP, AS-PCR and quality control; Parts 4–6 give terminology, clinical applications, facts sheets, and molecular protocols; Part 7 contains Uniform Resource Locator (URL) addresses and a brief description of databases that are available through the Internet, a list of definitions, and examples of worksheets and