Page 1948 - Hematology_ Basic Principles and Practice ( PDFDrive )

1728 Part XI Transfusion Medicine

indicate a provisional assignment, and this has been discontinued, HUMAN LEUKOCYTE ANTIGEN TYPING IN
but it is occasionally added to HLA-C antigen nomenclature to CLINICAL HEMATOLOGY AND DETERMINATION
distinguish it from complement genes; DP and DW also maintained
this letter to reinforce the dependency of their immune identification OF COMPATIBILITY HLA TYPING
predominantly through cellular techniques. Finally, HLA-Bw4 and
HLA-Bw6 retain the w to emphasize that the public epitopes are Originally HLA typing was done primarily in support of transplanta-
shared by several HLA-B and some HLA-A antigens. More recently, tion or transfusion needs with the purpose of identifying histocompat-
a bridge between immunologic and molecular nomenclature has been ibility through the best match between donor and recipient.
proposed whereby HLA antigens that encompass a single gene Allosensitization of recipients previously exposed to heterologous cell
product can be assigned a two-digit numeric extension corresponding products is tested by identifying alloreactive antibodies in serum, and
to the molecular nomenclature for that allele. 108 crossmatch procedures are performed to grade compatibility of candi-
date donor-recipient pairs. In addition, HLA testing has been applied
to identify links between a given disease and the genetic makeup of its
SEQUENCE-DEFINED ALLELIC NOMENCLATURE carriers. Strong associations are exemplified by birdshot uveitis (a
112
113
disease occurring exclusively in HLA-A29 individuals), type 1 dia-
The 10th International Histocompatibility Workshop recommended betes and other autoimmune diseases, 114–116 or long-term survival of
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in 1987 a sequence-based nomenclature to describe alleles not dis- HIV-infected individuals. These studies evaluated the role that
108
tinguishable by immunologic methods. Since then, the number genetic background may have contributed over environmental
of HLA alleles has rapidly increased. As of January 2015, a total factors. 112,117 HLA associations are thought to be caused by the dif-
of 13,023 alleles for HLA exist. This is a drastic increase from the ferential ability of distinct alleles to present immunogenic epitopes 113,115
original numbers established in 2003. Other designations are sum- or by the close linkage to the HLA class III region where potent
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marized in Table 113.2. HLA designates molecules belonging to immunomodulators such as tumor necrosis factor-α are located. It
the human MHC followed by the locus (-A, -B, etc.). Alleles are has also been suggested that HLA associations may be predictors of
60
then identified after an asterisk (*). Each HLA allele name has a immune responsiveness of cancer to immune therapy. However,
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unique number corresponding to up to four sets of digits separated such associations have remained quite difficult to reproduce. HLA
by colons. The length of the allele designation is dependent on the typing is requested for enrollment of patients into and interpretation
24
sequence of the allele and that of its nearest relative. All alleles receive of immunization protocols, because specific HLA-epitope combina-
at least a four digit name that corresponds to the first two sets of tions require high-resolution typing. 22,120
digits, longer names are only assigned when necessary. The digits Serologic testing takes advantage of fetomaternal sensitization. In
before the first colon describe the type, which often corresponds mammals, the progeny carries a full haplotype of paternal origin, and
to the serologic antigen carried by an allotype. The next set of pregnant women may develop antibodies against the paternal haplo-
digits are used to list the subtypes, numbers being assigned in the type. Maternal serum samples are collected at term and characterized
order that the DNA sequences have been determined. Alleles whose by testing their ability to kill HLA-bearing repository cell lines of
numbers differ in the two sets of digits must differ in one or more known phenotype in the presence of complement (complement-
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nucleotide substitutions that change the amino acid sequence of the dependent cytotoxicity [CDC]). CDC is used for HLA typing by
encoded protein. Alleles that differ only by synonymous nucleotide exposing circulating cells expressing HLA class I (most cells) and class
substitutions (also called silent or noncoding substitutions) within II (predominantly B cells) antigens from the individual (to be typed
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the coding sequence are distinguished by the use of the third set to previously characterized sera or monoclonal antibodies). Con-
of digits. Alleles that only differ by sequence polymorphisms in the versely, already typed repository cell lines are used in CDC to identify
introns or in the 5′ or 3′ untranslated regions that flank the exons alloantibodies in sera of sensitized individuals. The fraction of cell
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and introns are distinguished by the use of the fourth set of digits. lines killed by the sera roughly grades the intensity of allosensitization
A four-digit number is used when the first two digits refer to the or panel reactive antibody (PRA) reactivity. Some antibodies activate
original serologic family (e.g., HLA-A2 serologically would be HLA- complement and kill with poor efficiency, known as the cytotoxicity-
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A*02). Often numbers are missing because an original assignment negative absorption-positive (CYNAP) phenomenon. As judged by
was revoked (e.g., this is why there is no HLA-A*24:01). When cytotoxicity testing, CYNAP may underestimate allosensitization. By
silent mutations are identified (variation in nucleotide sequence modifying CDC with the addition of antihuman antibodies suitable
that does not translate into changes in amino acid sequence), the for complement activation, CYNAP can be circumvented (augmented
name of the allele remains identical, but another two digits are CDC). In this case, however, relatively innocuous antibodies can
added to designate a variant that has no functional significance. New cause overestimation of clinically relevant allosensitization. 124
sequences are submitted to European Molecular Biology Labora- Other methods identify alloantibodies, including immobilization
tory (EMBL; www.ebi.ac.uk/Submissions/index.html), GenBank of HLA molecules on solid surface to capture soluble antibodies and
(www.ncbi.nlm.nih.gov/Genbank/index.html), or DNA Data Bank flow cytometry using a spectrum of microbeads loaded with known
of Japan (DDBJ; http://www.ddbj.nig.ac.jp/submission-e.html). HLA alleles. 125–129 An interlaboratory comparison of serum screening
Requirements for new allele naming were described by Marsh for HLA-antibody determination suggested that enzyme-linked
4
et al. The WHO committee also made recommendations about immunosorbent assay and flow cytometry yield higher PRA activity
naming alleles with aberrant expression such as HLA-G isoforms values compared with CDC or augmented CDC. 125,127 However, the
and KIR. Some alleles are identifiable at the genomic level but are study suggested a lack of consistency among participant laboratories,
not translated into protein (pseudogenes). These are indicated by the leaving the question of which method most accurately defines clini-
addition of an N (for null) following the numerical designation of the cally relevant allosensitization unsolved, and a panel of various
allele. Mutations inducing a reduction of expression are marked by methods may be most informative. 72
L (low expression). An S denotes alleles expressed as soluble secreted CDC is declining in interest in the United States because most
molecules, such as HLA-B*44:02:01:02S characterized by an intronic laboratories are switching to easier-to-handle and higher-resolution
variant that disallows the expression of the transmembrane domain of molecular methods. However, immunologic methods remain valu-
the HLA molecule and is therefore only in soluble form. Differential able to characterize functional aspects of HLA because molecular
splicing of HLA-G leads to production of membrane-bound and methods cannot define whether an HLA allele is expressed nor, at
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soluble forms, which are respectively denoted by a lowercase m or least until recently, grade allosensitization. Thus it is likely that
s before HLA. Limited cytoplasmic expression is denoted by C and immunologic methods will continue to complement molecular
aberrant expression by A. Finally, KIR polymorphism will be clas- methods in the future. 130
sified by a new system that is in preparation. 4,97,110 A nomenclature The usefulness of conventional serologic typing of HLA antigens
system for cytokine polymorphism has not yet been developed. 111 has been limited by the availability of allele-specific sera. Most