Page 2384 - Williams Hematology ( PDFDrive )

2354 Part XIII: Transfusion Medicine Chapter 137: Human Leukocyte and Platelet Antigens 2355

The last field defines alleles that differ by sequence polymorphisms in more often than would be predicted if HLA loci were in equilibrium. At
introns or in the 5′ or 3′ untranslated regions that flank the exons and equilibrium, the frequency of an allele at one locus is independent of the
introns. In addition, there are suffixes that are used to describe expres- frequencies of alleles at linked loci. For example, the gene frequency of
sion status. Null alleles (not expressed) are identified by the suffix “N.” HLA-A1 is 0.145 and that of HLA-B8 is 0.1 in North American whites.
Low surface expression is represented by “L.” Secreted molecules not Given no preferential association between A1 and B8, then the hap-
present at the surface are assigned “S.” lotype frequency would be 0.0145 (0.145 × 0.1). However, population
studies demonstrate that the actual frequency of the HLA-A1, B8 hap-
18
INHERITANCE OF MAJOR lotype is 0.0726. The degree of linkage disequilibrium is defined as the
HISTOCOMPATIBILITY COMPLEX ANTIGENS observed frequency minus its expected frequency, 0.0581 in this exam-
ple. Although particular alleles found in linkage disequilibria differ for
The genes of the MHC demonstrate more polymorphism than any other various racial groups, all racial groups display significant disequilibria.
genetic system; that is, many alleles exist for each locus. Each individual, Different races and ethnic groups can vary greatly in the frequency with
however, has one allele for each locus per chromosome, and therefore, which HLA antigens are found. 19
encodes two HLA antigens per locus. The identification of each HLA
antigen of an individual is called a phenotype. Because HLA genes are HUMAN LEUKOCYTE ANTIGEN TYPING
closely linked, recombination within the MHC is rare (≤1%), and a Tissue typing for HLA antigens can be performed by various methods
complete set of HLA genes usually is inherited from each parent as a using serologic, cellular, and molecular technologies. The most frequent
unit. The genes inherited from each parent are referred to as a haplo- procedures used in the clinical setting are serologic and molecular.
type. Maternal and paternal haplotypes can be identified through fam- Cellular assays such as the mixed lymphocyte reaction and the primed
ily studies. Identification of both haplotypes of an individual provides lymphocyte test were common prior to the widespread adoption of
the genotype. Family studies consist of typing for the HLA-A, HLA-B, DNA methods. Compared to DNA techniques, cellular methods are
HLA-C, HLA-DR, and HLA-DQ antigens to identify haplotypes and to labor-intensive and require the use of radioisotopes; they are mainly
rule out genetic recombination within the MHC. Because HLA genes used in research laboratories.
are inherited together on a single chromosome, four combinations of
maternal and paternal haplotypes are possible provided no recombina- Serology
tion occurs (Fig. 137–2). The microlymphocytotoxicity complement-dependent cytotoxicity (CDC)
test has been a fundamental procedure for defining HLA antigens for
Linkage Disequilibrium more than 30 years, although it has been supplanted by molecular typ-
20
Because the MHC is so highly polymorphic, the probability that any ing methods. In this assay, a suspension of lymphocytes is incubated
two unrelated individuals are HLA identical is extremely low. However, with human alloantisera or monoclonal antibody in a microtiter tray.
21
the system exhibits a phenomenon known as linkage disequilibrium. Rabbit serum is added as a source of complement. Cell death is induced
That is, HLA alleles are inherited together on the same chromosome when antibody binds to antigen on the cell surface and the complement
cascade activated. Death is visualized microscopically by the uptake
of vital dye or by immunofluorescence. Panels used to determine a
patient’s HLA type consist of two to four antisera that recognize the
Father Mother
same specificity, which requires approximately 150 different reagents for
class I antigens and 80 to 150 for class II antigens. Antisera are usually
obtained from multiparous women, multiply transfused patients, and
from patients who have rejected allografts. Monoclonal antibodies are
ab cd
also commercially available for many HLA specificities. Serology for
class II (DR and DQ) antigens requires enrichment for B lymphocytes,
which can be accomplished with antibody or immunomagnetic bead
reagents.

Molecular Human Leukocyte Antigen Typing
The development of the polymerase chain reaction (PCR) revolu-
22
tionized the approach to HLA typing. Several DNA-based methods are
commonly accepted for HLA typing. These include sequence-based
typing, sequence-specific primer (SSP) amplification and sequence-
23
Child #1 #2 #3 #4 #5 specific oligonucleotide (SSO) probe hybridization. All of these meth-
ac ac ad bc bd ods involve amplification of genomic DNA from selected portions of
HLA genes with oligonucleotide primer pairs. Generally exons 2 and
Code: Haplotype: 3 of class I and exon 2 of class II genes are amplified. These exons encode
a A1, B8, Cw1, DR17, DR52, DQ2 most of the polymorphisms of the classes I and II molecules. Molecular
b A2, B44, Cw2, DR11, DR52, DQ7 HLA typing is primarily of clinical interest in marrow/blood stem cell
c A3, B7, Cw3, DR15, DR51, DQ6 transplantation.
d A11, B55, Cw3, DR4, DR53, DQ8 The advent of “next-generation sequencing” (NGS) strategies
has proven useful to high-throughput sequencing of HLA genes. NGS
Figure 137–2. Pedigree representing inheritance of HLA antigens. methods also overcome limitations of Sanger-based methods, includ-
Each of the four parental haplotypes is coded by a letter: a and b rep- ing combination ambiguities that result from heterozygous samples
resent paternal haplotypes; c and d represent maternal haplotypes. Each
child inherits one paternal and one maternal haplotype such that four in diploid genomes or between alleles where sequence varies outside
combinations are possible. the target region (i.e., exons 2 and 3). NGS methods are described as

Kaushansky_chapter 137_p2353-2364.indd 2355 9/21/15 3:49 PM