alleles expressed by the donor and those expressed
by the recipient. For this reason, laboratory evalua-
tion for transplantation begins with pre-transplant
HLA typing of the recipient and the intended donor
(Table 9.4; Milford and Amaç 1997). Clinical ex-
perience has shown that the HLA antigens most
important in allograft rejection are HLA-A, HLA-B
(class I histocompatibility molecules), and HLA-DR
(class II histocompatibility molecule) so these are the
antigens that are typed.
HLA typing consists of the identification of the
allelic form of the HLA antigens of interest. Until
recently, this has been done primarily by phenotyp-
ing of lymphocytes using the microlymphocytotoxic-
ity assay. In that method, lymphocytes isolated from
the blood of the person being studied are distributed
among the wells in a tissue typing tray. Each well of
the tray contains an antibody of known specificity.
After a period of incubation, complement is added
and the mixture incubated further. In the wells
containing antibody specific for an expressed HLA
antigen, the antibody binds the antigen, the comple-
ment cascade is activated, and the cells killed. The
extent of lymphocyte killing is determined by
fluorescence microscopy; viable cells stain one color
with a fluorescent dye that is retained only in living
cells and killed cells stain another color with a
second fluorescent dye that is only taken up by dead
cells. The HLA type is assigned based on the speci-
ficities of the antibodies in the wells in which
lymphocyte killing occurs. More recently, molecu-
lar diagnostic methods for HLA typing have become
available. These methods are based on determina-
tion of the base sequence in the region of DNA
coding for the respective HLA antigen. The
techniques most commonly applied are allele-specific
oligonucleotide hybridization and sequence specific
PCR amplification (Bunce
et al.
1997; see Chapter
10 for a discussion of molecular diagnosis).
Molecular diagnostic HLA typing has many advan-
tages over the phenotyping method including better
standardization and availability of the typing rea-
gents and much greater ease of study performance
and interpretation. Also, phenotyping failures due to
down-regulation of HLA expression or lymphocy-
topenia are avoiding using molecular diagnostic
techniques as are uncertainties that arise in the
phenotyping of individuals who are homozygous at
an HLA locus. For all these reasons, molecular
diagnostic HLA typing is supplanting HLA
phenotyping.
The presence of pre-existing recipient antibodies
to donor HLA antigens is tested using any of a
number of variants of the microlymphocytotoxicity
assay. Serum from the recipient serves as the
antibody source and the donor is the source of the
lymphocytes; hence the designation, lymphocyte
crossmatch. The detection of anti-HLA antibodies
by standard lymphocyte crossmatch is an exclusion-
ary histo-incompatibility because such antibodies
cause hyperacute rejection. The clinical significance
of anti-HLA antibodies revealed by sensitive variants
of the lymphocyte crossmatch (such as the flow
cytometry crossmatch) is less clear so their detection
is only a relative contraindication to transplantation.
Hyperacute rejection can also be caused by naturally
occurring antibodies to the A and B antigens of the
ABO blood group system so ABO blood group
typing of the donor and recipient is performed as
part of the pretransplant evaluation.
While a patient is awaiting transplantation, B
cell presensitization of a candidate allograft recipient
is assessed periodically with an antibody screen.
This is especially important for candidates who have
Tissue Injury
9-9
Table 9.4
Laboratory Evaluation in Tissue Transplantation
Tissue
Pre-transplant
Post-transplant
Solid organ
histocompatibility testing
monitoring for rejection
HLA matching
organ function
antibody screen
organ injury markers
lymphocyte crossmatch
monitoring of immunosuppressive agents
blood group crossmatch
Red cells
compatibility testing
if a transfusion reaction is suspected
blood group matching
tests of hemoglobin release
antibody screen
red cell injury markers
(red cell crossmatch)
direct antiglobulin test
evaluation of alloantibody
