HLA TYPING AND CROSS MATCH

SHASHI ANAND
 “Histo” is a Greek word that means “tissue”.
 Histocompatibility refers to the quality or of being
histo or tissue compatible.
 This term is used in transplantation to describe the
ability of a donor’s tissue or organ to be accepted by
a recipient.
 There is a group of genes present in all
animals called the major histocompatibility
complex (MHC).
 In humans the MHC is called human

leukocyte antigen (HLA).

 The more HLA antigens shared between a

recipient and donor, the better the potential

outcome of the transplantation.
 For bone marrow transplantation, a near-

perfect match is required whereas for kidney

transplantation, a lesser match can result in
a successful, functioning transplant.
  Human Leukocyte Antigens are the human
form of MHC and are proteins located on the
surface of white blood cells and other tissues
in the body.
 HLA antigens play an important role in the

immune system’s defense against invaders

such as bacteria, viruses and parasites.
 In transplantation, when the donor’s HLA is

different from the recipient’s, the immune

system of the recipient will recognize the
donor’s HLA antigens as foreign.
 This causes rejection of the transplanted

tissue or organ.
•Transplantation is a procedure (or set of
procedures) in which diseased and/or
nonfunctioning organs, tissue or bone marrow is
replaced with the same obtained from another
healthy person.

•Transplants between identical twins will not be

rejected because all the genes (antigens) are
identical.
 Human Leukocyte Antigen (HLA)
 The HLA system is the name given to the

human MHC, which was first described by

Jean Dausset in 1952 after observing the
development of alloantibody to leukocytes
following blood transfusions
 The HLA system comprises a group of cell-

surface antigen-presenting proteins encoded

by a region on the short arm of chromosome
6 and is divided into class I and class II
molecules.
 Humans have three class I HLA (A, B, C) that
are present on all nucleated cells and six class
II HLA (DPA1, DPB1, DQA1, DQB1, DRA, DRB1)
that are present only on antigen-presenting
cells and lymphocytes.
 Class I HLA presents intracellular antigens

while class II HLA present extracellular

antigens. HLA are highly polymorphic with
almost 6000 HLA Class I and over 1500 HLA
Class II alleles having been identified
 Three of the seven heterodimers (HLA-A, -B,

and -DRB1) contribute to the majority of the

immunogenicity of mismatched antigens and
therefore traditional HLA-typing methods
have primarily focused on these alleles.
 HLA typing is used to identify the best donor
for a transplant recipient.
 In the best case scenario the donor will have

the exact HLA antigens as the recipient. The

risk of transplant rejection is lessened for
well-matched donor-recipient pairs.
 Certain diseases are associated with

particular HLA types. For example, a person

with ankylosing spondylitis (a disease like
rheumatoid arthritis) is likely to have HLA-
B27 antigens. HLA typing can help a
physician differentiate ankylosing spondylitis
from other disorders.
HLA testing is performed on a sample of blood.
 An individual’s HLA typing can be

determined by testing the HLA proteins on

the surface of white blood cells or by testing
DNA from the same cells.
The blood can be separated into components
such as red blood cells, white blood cells,
platelets and plasma or serum.
 After blood clots, the fluid portion is called

serum.
 HLA typing is performed on the white blood

cells known as lymphocytes

 There are 2 specific types of antibody testing
that are performed:
 Cross Match: testing for antibodies specific

against a particular donor

 Panel-reactive Antibody (PRA): testing the

patient's blood for antibodies against many

different HLA antigens

In transplantation, anti-HLA antibodies can attack donor HLA

antigens on the surface of the transplanted tissue and injure the
organ.
 A cross match is a laboratory test performed
before transplantation to detect antibodies in
the recipient’s blood that may destroy the
transplanted organ.
 The test is performed with the white blood

cells (lymphocytes) from the potential donor

and the serum (the straw yellow-colored fluid
formed when blood clots) of the patient.
 In cases where lymphocytes from a potential

donor are attacked by the patient’s

antibodies, transplantation with this donor
may not occur and another will be sought.
  A PRA test can be performed many different
ways. Basically, we take known HLA proteins
and react them with a patient’s serum. If the
patient has antibodies to a particular HLA
protein, the antibodies will bind. We then add
other special proteins to allow us to detect
that the antibody is bound to the HLA
protein.
 By determining which HLA proteins a patient
reacts to, we can determine if a potential
patient/donor combination is likely to have a
good or bad outcome.
 Anti-HLA antibodies can be triggered by blood
transfusion, multiple pregnancies and by
transplantation. Lymphocytes present in
transfused blood are the source of the
immunization causing anti-HLA antibody
development.
 Since a person can develop antibodies after a
transfusion, it is important for a transplant
candidate to submit a blood sample to the HLA
laboratory approximately 14 days after each
transfusion. The serum will be tested to
determine whether anti-HLA antibodies have
been produced.
 If anti-HLA antibodies were produced as a
response to the transfusion, the HLA specificity
can be defined and avoided in selection of a
suitable organ donor.
  There are 3 different HLA class I antigens: A, B
and C. There are several HLA class II antigens but
the most commonly tested are: DR and DQ. There
are many different antigens within each group;
for example there are at least 21 different HLA-A
antigens.
 HLA antigens are named with the letter of the
locus followed by a number designation unique
for a particular antigen, i.e. A1 or A24. Each
person inherits one A, B, C, DR and DQ antigen
from each parent. Thus, we have 2 HLA-A
antigens, 2 HLA-B antigens and so on.
 In addition, the number of possible combinations
of HLA-A with -B and -C and –DR and –DQ
makes billions of different HLA types possible.
 The best potential for an HLA match is within
the family. Each person inherits one A, B, C,
DR and DQ antigen from each parent. Thus,
we have 2 HLA-A antigens, two HLA-B
antigens and so on.
 The number of different HLA combinations is

limited to the HLA types of the parents. A

typical HLA typing would be expressed as:
A11, 32; B35, 44; DR1, 7; DQ5, 9. In this
case, A11; B35; DR1; DQ5 may be inherited
from the mother and A32; B44; DR7; DQ9
from the father.
 The combination of HLA-A, B, DR and DQ
antigens inherited from each parent is called a
haplotype. Each parent has 2 HLA haplotypes one
of which will be inherited by each child. For all
children sharing the same parents, there will only
be 4 possible combinations of HLA antigens or
haplotypes. The odds of finding “matches”
between siblings greatly increases compared to
the general population because we have a 1-in-4
chance of being identical to each sibling.
 When an unrelated donor is sought the number
of potential HLA combinations becomes so
enormous that finding the patient’s exact HLA
type may seem like searching for a needle in a
haystack.
 The A, B and O blood groups and HLA genetic
systems are not inherited together. Just as
the gene for eye color is separate from the
gene that indicates blood group, so are your
genes different for HLA compared to blood
group genes.
 In kidney transplantation, the donor and
recipient must be of the same ABO blood
group.
 For bone marrow transplantation, the blood
group of the donor is not important. In fact,
after bone marrow transplantation, the
patient will have the donor’s blood type. 
Tissue Typing for Transplantation
Tissue typing to prevent transplant rejection includes both HLA and ABO
matching.
Traditionally this was done serologically by taking lymphocytes from a patient
and incubating them with selected specific antiserum. Complement and dye (try
pan blue) were then added. If antibodies reacted specifically with the
lymphocyte the cell would be damaged and take up the dye.
Because of the high number of transplant rejections with tissues that were
apparently matched it was realized that serology isn't specific enough to
guarantee a match. Tissue typing today, especially for bone marrow
transplantation, is done by sequencing HLA genes of the donor and the
recipient.
Overview of the complex relationship between dendritic cells
and effector T and B cells.Immature DC (MDC and PDC)
maturate in response to appropriate stimuli (e.g. microbial
products, TLR ligands). Mature DC secretes immunoregulatory
cytokines (including IFN-α and IL-12] and with cell-cell contact,
modulates effector cell response including NK cells, B and T cells
as well as providing a positive feedback to DC to initiate ongoing
activation and maturation. Activated effector cells could in turn
modulate DC activation, maturation, and survival as well as
enhancing other effector cell functions through the production of
cytokines (IFN-γ) and/or via cell-cell contact.
DC play a critical role in the initiation and regulation of adaptive T cell
responses, the maintenance of central and peripheral tolerance in normal
steady-state and hence are essential in regulating immune responses in
solid organ and cellular transplantation. DC have dual roles in organ
transplantation. They are responsible for allorecognition and presentation
of foreign antigens to T cells, which may initiate allograft rejection; but are
also involved in the promotion of transplant tolerance.
DC encounter foreign antigens such as donor antigens (in solid organ
transplantation), bacteria and tumour antigens resulting in the secretion
of cytokines (e.g. IFN) and activation of natural killer (NK) cells,
macrophages and eosinophils. Following antigen capture and
processing,

DC undergo maturation and migrate to secondary lymphoid tissues

where they present processed antigen/peptide coupled to major
histocompatibility complexes (MHC) to T cells, allowing for selection and
expansion of antigen-specific cluster designation (CD)4+ T-helper cells.

These CD4+ T-helper cells subsequently amplify the immune responses

by regulating antigen-specific (e.g. CD8+ cytotoxic T cells, B cells), and
antigen non-specific (e.g. macrophages, NK cells, and eosinophils)
effector cells.