The HLA system comprises a group of genes crucial for the immune system, forming the major histocompatibility complex (MHC) on nearly all cells except red blood cells. HLA typing is essential for identifying compatible tissue and organ donors, particularly for transplants, as it helps ensure a close match between donor and recipient, thereby reducing the risk of transplant rejection. Techniques for HLA typing include serological methods and DNA-based procedures that enhance the accuracy of matching in transplantation scenarios.

1. What Is the HLA System?
The HLA system refers to a group of related genes that play an
important role in the immune system.
Together, the proteins made from these genes form something
called the major histocompatibility complex (MHC). These
proteins are attached to almost all of the cells of our body
(excluding red blood cells).

2. HLA stands for human leukocyte antigen
HLA typing is a kind of genetic test used to identify certain
individual variations in a person’s immune system.
The process is critical for identifying which people can safely
donate bone marrow, cord blood or an organ to a person who
needs a transplant.
HLA typing is also sometimes called HLA matching.

3. PURPOSE OF HLA TYPING
• The most common reason for HLA typing is to help determine
which people can provide the safest tissue transplants (
solid organ or hematopoietic stem cell transplantation).
• Potential tissue recipients must have the typing, as must anyone
who might potentially want to donate tissue.
• HLA type included in a bone marrow registry, for stem cell
transplantation.
• HLA typing may also be performed on terminally ill or recently
deceased people who will be serving as organ donors.
• The best possible donors have HLAs that closely match the
HLA patterns of the recipient.

4. CONDITIONS THAT REQUIRING TRANSPLANTATION
 There are many different health conditions that may need to be
treated through a transplant.
 For example, various types of blood cancers and genetic blood
disorders are treated through stem cell transplantation (taken
either from the bone marrow or from the peripheral blood).
 A solid organ transplant might be necessary for any essential
organ that has become severely damaged.
 This might happen through trauma, infection, autoimmune
disease, genetic illness, toxins, or many other disease processes.
 For example, one might need a kidney, liver or lung transplant if
one’s own organs are very functioning very poorly.

5. THE PROCESS OF HLA TYPING
HLA typing assesses the particular HLA genes that have inherited
Because there are a number of different HLA genes, as well as different
variations of these genes, there are very many different possible color
combinations that together make up your specific HLA type.
HLA typing also usually includes testing for antibodies targeted to specific
HLA proteins. Antibodies are made by part of the immune system.
If a person already has an antibody against an HLA protein it may attack that
protein if it is transplanted. This may cause the transplant to fail.
So generally, shouldn’t receive a transplant from someone if already have an
antibody against one of their HLA proteins.

6. CONT…….
HLA typing also often includes something called lymphocyte
cross matching. Lymphocytes are a type of immune cell.
Lymphocyte cross matching checks to see if the recipient has an
antibody against a protein on the donor’s lymphocytes.
If so, that person generally shouldn’t receive a transplant from
that particular person. These people are at high risk of a
transplant that won’t be successful.

7. Is HLA Typing the Same as Blood Typing?
No. HLA is much more complicated than blood typing because
there are many more HLA markers that make a person’s cells
unique.
There are only eight basic blood types, and many people can
safely receive more than one type of blood (depending on their
type).
To receive only blood from a person, do not need to be an HLA
match, because HLA is not present on red blood cells.
To receive a solid organ transplant, the recipient must have a
compatible blood type with the donor, as well as the best HLA
match possible.
For stem cell donations, one needs a very strong HLA match, but
blood type is not as important as it is for solid organ transplants.

8. How Are HLA Genes Inherited?
Because the HLA genes are located close together on DNA, they are usually
inherited as a group.
HLA type is composed of the set of HLA genes you inherited from mother
and the HLA genes you inherited from father.
Biological parents always share half of their HLA proteins with their children.
This is also called a “half match.” Conversely, a child always is a half match
with their parents.
The set of HLA alleles found on one chromosome is called haplotype.
Determination of haplotype is important for identification of HLA identical
siblings because sharing of antigen from different haplotypes is common.

9. HLA TYPING IMPORTANT ROLE
The primary target of immune responses to allogeneic transplants
Critical for response to antigenic stimuli
Implicated in genetic susceptibility to auto immune disease.

10. METHODS OF HLA TYPING
SEROLOGICAL
(Micro cytotoxicity test)
MIXED
LYMPHOCYTE TEST
MOLECULAR
METHODS

11. SEROLOGICAL TEST
Potential donor′s and recipient's WBC′s added to the different
wells of microtitre plates
Antibodies specific for HLA class I and II added
After incubation complement is added
Cytotoxicity is assessed by uptake or exclusion of dye

12. SEROLOGY
HLA Typing is done serologically by MICROCYTOTOXICITY
(microlymphocytotoxicity) which tests for complement
mediated lysis of peripheral blood lymphocytes with a
standard set of typing sera.
Viable peripheral blood lymphocyte are obtained by
discontinuous density gradient centrifugation using Ficol/
Tryosil or Ficol / Sodium Metrizoate at a density of 1.077 at
19⁰C-22⁰C

13. Microlymphocytotoxic test
3 stages
• Viable lymphocyte are incubated with HLA specific antibodies. If
the specific antigen is present on the cell the antibody is bound.
• Rabbit serum as a source of complement is added, incubate. If
antibody is bound HLA antigen on the cell surface it activates the
complement which damages the cell membrane making it
permeable to vital stains.
• Results are visualized by adding dye usually a flurochrome eg.
Ethidium Bromide although both trypan blue and Eosin Y have
been used .

14. • If the reaction has takes place the EB enters the cell and binds to
the DNA
• For ease double staining is normally used. A cocktail of Ethidium
Bromide and Acridine Orange, quenched using Bovine
Hemoglobin to allow simultaneous visualization of both living
and dead cells.
• White blood cells from potential donors and the recipient are
added to separate wells of a microtiter plate. The example depicts
the reaction of donor and recipient cells with a single antibody
directed against an HLA-A antigen.
• The reaction sequence shows that if the antigen is present on the
lymphocytes, addition of complement will cause them to become
porous and unable to exclude the added dye.

15. Presence or absence of
various MHC alleles
determined by antibody
mediated cytotoxicity

17. Cont…….
• Test is left for 10 minutes and then read using an inverted
fluorescent microscope.
• A mixture of T and B lymphocytes can be used for HLA class I
typing.
• B lymphocytes are required for HLA class II typing by
serology. (Normal population 85-90% to T and 10-15% B
cells)
• This can be achieve using a number of methods

18. In the past neuraminidase treated sheep red blood cell resetting
and nylon wool have been used.
Immunomagnetic bead separation is the current method of choice.
It utilizes polystyrene microspheres with a magnet able core
coated in monoclonal antibody for a HLA class II b chain
monomorphic epitope. Positive selection

19. N

21. MIXED LYMPHOCYTE REACTION
o It has been observed the lymphocytes from one donor, when
cultured with lymphocytes from an unrelated donor, are
stimulated to proliferate.
o It has been established that this proliferation is primarily due to
a disparity in the Class II MHC (DR) antigens and T cells of
one individual interact with allogeneic class-II MHC antigens
bearding cells (B cells, dendritic cells, Langerhans cells, etc…)
this reactivity was termed with mixed leukocyte reaction
(MLR) and has been used for studying the degree of
histocompatibility.

22. In this test, the lymphocyte (responder cells) are mixed with
irradiated or mitomycin C treated leukocytes from the recipient,
containing B-lymphocytes and monocytes (stimulator cells).
The responder T cells will recognize the foreign class II antigens
found on the donor and undergo transformation (DNA synthesis and
enlargement: blastogenesis) and proliferation (mitogenesis) occurs
The T cells that respond to foreign Class II antigens are typically
CD4+ TH-1 type cells
These changes are recorded by the addition of radioactive (tritiated,
3H)

24. NN

25. Molecular HLA typing
DNA based procedures has increased the accuracy of HLA typing
and lead to the identification of serologically undetected alleles and
many subtypes of serological specificities.
DNA based methods of type for HLA alleles have therefore
focused in the analysis of nucleotide variation occurring in both
exon 2 and 3 of class I genes and exon-2 of class II genes.

26. PCR BASED METHODS/ THREE CATEGORIES
 Polymorphism is identified directly as part of the PCR process,
although there are post amplification steps e. g (SSP)
 Product containing internally located polymorphism that can be
identified by a second technique e. g PCR sequence specific
oligonucleotide probing (SSOP), PCR- RFLP,PCR followed by
sequencing.
 Conformational analysis

27. STEPS OF
MOLECULAR
CLONING

28. DNA EXTRACTION
Genomic DNA extracted from nucleated cells; the purity of the
DNA extracted may be an important factor for successful results
GENE AMPLIFICATION/PCR
The specificity of the amplification can be locus specific e. g (HLA-
A, HLA-B, HLA-DRB1), group specific… e.g. (DRB1-01, DRB1-
02) Or allele specific DRB1-0401, DRB1-0402
The primers are selected to amplify a single allele or a group of
alleles
For PCR, the specificity is determined by the sequence of the
primers and amplification condition. Most typing scheme require
conditions the avoid the co-amplification of pseudogene.

29. Amplification of exon 2 (approximately 270bp ) of HLA class II
is sufficient to achieve the highest resolution level.
For typing HLA class I, both exon 2 and 3 and the intervening
intron (fragment longer than 900bp ) are amplified by single pair
of primers.
Detection of sequence polymorphism that define the alleles
• Sequence specific primers (SSP) group and alleles specific
primers
• Hybridization with sequence specific oligonucleotide probes
(SSOP)
• Sequence based typing (SSP)

30. Sequence specific priming
• The procedure relies on the
specificity of primer extension that
is matched or mismatched with the
template at its 3’end.
• A combination of 2 primers
designed for each of 2 polymorphic
sequence motifsin cis allows the
identification of an allele or a group
of alleles that are characterized by
these 2 motifs.
• The method is rapid and ideally suited
for small numbers of samples. Because very few sequences are
absolutely allele specific, SSP combine several primer to
discriminate a particular allele unambiguously.

31. Hybridization with sequence specific oligonucleotide probes (SSOP)
The procedure relies on the locus-specific
amplification of the genomic DNA segment
comprising the polymorphic sites of HLA alleles.
Amplified DNA is then immobilized on a
solid support, usually a nylon membrane,
and then hybridized with a battery of
(SSOP) (direct hybridization).
Fluorochromes are linked with the probes to
allow their detection by chemiluminescence.
Alternatively, SSO probes can be immobilized
on a solid support, for example color-coded microspheres,
and hybridized with labeled PCR product (reverse hybridization).
The higher the number of probes the better the resolution level. Usually 50-
100 probes per locus are used for intermediate/high resolution typing,
however, only the probe completely matched with the target sequence
amplified will hybridize and give a positive signal.

32. SEQUENCE BASED HLA TYPING
• Sequence based HLA typing involves determining the nucleotide
sequence of an amplified segment of HLA gene
• SBT presents the advantages over the other procedure because of
the relatively fast (24-48 hours), high level resolution
• More reliable and specific method

33. CONFORMATIONALANALYSIS
Different mutation generates specific conformational changes in
PCR products, these are identified by electrophoresis analysis.
E.g.….. heteroduplex analysis, single strand conformation
polymorphism (SSCP), denaturing gradient gel electrophoresis
(DGGE), temperature gradient gel electrophoresis (TGGE)

35. CLINICAL SIGNIFICANCE OF HLA TYPING
In organ transplantation
In transfusion therapy
Disease association
Disputed paternity
In cancer prevention
Anthropological studies

36. What is Transplant rejection?
• Transplant rejection is process in which the
transplanted tissue (of donor) is rejected by
the recipient's immune system and may result
in fatal illness if not treated/removed early.

37. Based on genetic relationship between donor and
recipient there are four types of Grafting methods:
1. Autografts are grafts in which the donor and
recipient is the same individual.
2. Isografts are grafts between the donor and
recipient of the same genotype.
3. Allografts are those in which the donor is of the
same species but of a different genotype.
4. Xenografts are those in which the donor is of a
different species from that of the recipient.

39. • For any successful tissue transplant without
immunological rejection, matched Major
Histocompatibility Locus Antigens (HLA)
between the donor and recipient are of
paramount importance.
• The greater the genetic disparity between
donor and recipient in HLA system, the
stronger and more rapid will be the rejection
reaction.

40. • All types of grafts have been performed in
human beings but xenografts have been found
to be rejected invariably due to genetic
disparity.
• Most commonly practiced grafting are:
- Skin grafting
- Kidney
- Bone marrow transplantation.

41. Exceptions in Rejections:
• Cornea transplants are rarely rejected because
the cornea has no blood supply.
• Also, transplants from one identical twin to
another are almost never rejected.

42. Graft versus Host Reaction
• In some cases esp. when the transplanted
tissue is bone marrow, a peculiar illness arises
besides rejection of the transplanted bone
marrow called GRAFT Vs HOST REACTION.
• The intensity of GVH reaction depends upon
the extent of genetic disparity between the
donor and recipient.

43. • The clinical features of Graft Vs Host reaction
include:
- Fever,
- Weight Loss,
- Anaemia,
- Dermatitis,
- Diarrhoea,
- Intestinal Malabsorption,
- Pneumonia,
- Hepatosplenomegaly.

44. MECHANISMS OF GRAFT REJECTION
• Except for autografts and isografts, an immune
response against allografts is
inevitable/unavoidable.
• The development of immunosuppressive
drugs has made the survival of allografts in
recipients possible.
• Rejection of allografts involves both cell-
mediated and humoral immunity.

45. 1. CELL-MEDIATED IMMUNE REACTIONS
• Mainly responsible for graft rejection and are
mediated by T cells (mainly by cytotoxic T
cells).
• T cells attack the graft and destroy it.

46. 2. HUMORAL IMMUNE REACTIONS
• In addition to the cell-mediated immune
reactions, humoral antibodies cause certain
rejection reactions.

47. TYPES OF REJECTION REACTIONS
• Based on the underlying mechanism and time
period, rejection reactions are classified into 3
types:
1- Hyperacute Rejection
2- Acute Rejection
3- Chronic Rejection

48. 1. HYPERACUTE REJECTION
• Hyperacute rejection appears within minutes
to hours of placing the transplant and destroys
it.
• It is mediated by Humoral system antibodies.
• This type of rejection is seen when a recipient
is given the wrong type of blood.
• For example, when a person is given type A
blood when he or she is type B.

49. 2. ACUTE REJECTION
• This usually becomes evident within a few
days to a few months of transplantation.
• Acute graft rejection may be mediated by
cellular or humoral mechanisms.

50. 3. CHRONIC REJECTION
• Chronic rejection may develop slowly over a
period of months to a year or so.
• The underlying mechanisms of chronic
rejection may be immunologic or ischaemic.
• Patients with chronic rejection of renal
transplant show progressive deterioration in
renal function as seen by rising serum
creatinine levels.

51. How to overcome transplant rejection?
• Tissue typing is done to prevent transplant rejection
• Here both the organ donor and the person who is
receiving the organ tissue typing is done to ensure that
the organ or tissue is as similar as possible to the tissues
of the recipient.
• The more similar the antigens are between the donor and
recipient, the less likely that the organ will be rejected.
• No match is usually 100 percent identical.
• No two people, except identical twins, have identical
tissue antigens.

52. AIDS
(Acquired Immunodeficiency
Syndrome )

53. 08/29/2024
Definition
Definition of AIDS is a syndrome caused by
infection with the human immunodificiency
virus (HIV) with ensuing compromise of the
body's immune system.

54. Mode of transmission
• AIDS can transmit by body fluid,
vaginal secretions, amniotic fluid and
breast milk.
• Blood product also can transmit to
recipient by blood transfusion.

55. IV injection drug
use
Sexual relationship
with HIV infected
Person who
received HIV
infected blood
Baby breastfeed
with HIV-infected
mother
Frequency exposure
to blood
Frequency exposure
to body fluid
Etiology

56. Clinical Manifestations
Fever Cough Chest pain
Skin rashed
Loss of
appetite
Diarrhea
Fatigue Tubercolusis Weight loss

57. Laboratory Investigation
1. Blood test such as
CBC and WBC.
2. ELISA ( Enzyme-
linked immunosorbent
assay ) test used to
detect HIV infection.

58. Complications
1. Kaposi's sarcoma.
2. Lymphomas.
3. Wasting syndrome.
4. Neurological
complications.
5. Kidney disease.

59. Management
Medical management
1. Antiretroviral drugs. (trx to fight the HIV
infection and slow down the spread of the
virus)
2. Antifungal cream Ciclopirox may help
eradicate HIV.
3. Antiemetics drug to treat the symptoms.
4. Antipyretics drug to treat the symptoms such
as PCM

60. Nursing management.
1. Notification.
2. Encourage patient to have protected sex such
as use a condom. HIV can spread by having
unprotected sex.
3. Encourage patient to do proper hygiene.
4. Advice patient to do not give blood donation.
5. Educate patient for risk factors for acquiring
AIDS diseases.

61. Health Education
1. Avoid from sharing needles and
toothbrushes.
2. Do not reuse condom.
3. Reduce the number of sexual partner to one.
4. Avoid donating blood, plasma, body organs
or sperm.
5. Encourage patient to have fluid intake to
avoid dehydration.

62. Hypersensitivity
• Exaggerated or Inappropriate
immune response that is
harmful to the host

63. Atopy:
• Atopy is the term for the genetic trait to have a
predisposition for localized anaphylaxis
• Atopic individuals have higher levels of IgE and Eosinophills

64. Allergens
• Allergens are nonparasite antigens that can stimulate a
type I hypersensitivity response.
• Allergens bind to IgE and trigger degranulation of
chemical mediators.

65. Hypersensitivity Reactions
• Immediate hypersensitivity refers to antibody
mediated reactions – symptoms develop within
minutes to hours
• Delayed hypersensitivity refers to cell mediated
immunity, symptoms not observed for 24 to 48
hours.

66. Four Classifications
• Type I (Immediate) Hypersensitivity
• Type II (cytotoxic) hypersensitivity
• Type III (immune complex mediated) hypersensitivity
• Type IV (delayed) hypersensitivity

68. Type I Hypersensitivity
(Anaphylactic) Reactions/Allergy
– Occur within minutes of exposure to antigen
– Antigens combine with IgE antibodies
– IgE binds to mast cells and basophils, causing
them to undergo degranulation and release
several mediators:
• Histamine
• Prostaglandins
• Leukotrienes
• Anaphylactic shock

69. Type I (Immediate) Hypersensitivity

70. Mast Cells and the Allergic Response

71. Mast Cells and the Allergic Response

72. Type I (Immediate) Hypersensitivity

73. Examples of type 1
• Allergic asthma
• Allergic conjunctivitis
• Allergic rhinitis
• Anaphylaxis
• Angioedema
• Urticaria (Hives)

74. Type II Hypersensitivity (Cytotoxic)
Reactions/antibody-dependent
– Involve activation of complement by IgG or IgM
binding to an antigenic cell.
– Antigenic cell is lysed
– Transfusion reactions:
• ABO Blood group system: Type O is universal
donor. Incompatible donor cells are lysed as
they enter bloodstream.

75. Hemolytic disease of the newborn is caused by type II
hypersensitivity reactions
When an Rh- mother carries an Rh+ fetus

76. Examples of type II
• Autoimmune hemolytic anemia
• Thrombocytopenia
• Erythroblastosis fetalis

77. Type III Hypersensitivity (Immune Complex)
Reactions
– Involve reactions against soluble antigens circulating in
serum.
– Antibody-Antigen immune complexes are deposited in
organs, activate complement, and cause inflammatory
damage.
• Glomerulonephritis: Inflammatory kidney damage.
– Occurs with slightly high antigen-antibody ratio is
present.

78. Type III (immune complex mediated) Hypersensitivity

79. Immune Complex Mediated Hypersensitivity

80. Examples of type III
• Serum sickness
• Arthus reaction
• Systemic lupus erythematosus (SLE)
• Post streptococcal Glomerulonephritis
• IgA nephropathy
• Rheumatoid arthritis

81. Hypersensitivity Type III Reactions
Systemic Reactions
Local Reactions
 Arthus Reaction:
Inflammation caused by
the deposition of immune
complexes at a localized
site.
Clinical Manifestation is :
Hypersensitivity
Pneumonitis
 Serum Sickness:
Systemic inflammatory
response to deposited
immune complexes at many
areas of body.
Clinical Manifestation is :
Fever, Urticaria, Artheralgia,
Eosinophila, Spleenomegally,
and Lymph adenopathy

82. Type IV (delayed) Hypersensitivity
• Used to describe the signs and symptoms associated with a
cell mediated immune response.
• Results from reactions involving T lymphocytes.
• Characteristics of this phenomenon are:
– Delayed, taking 12 hours to develop.
– Causes accumulation of lymphs and macrophages.
– Reaction is not mediated by histamine.
– Antibodies are not involved in the reaction.

83. Type IV Mechanism
APC resident in the skin
process antigen and
migrate to regional lymph
nodes where they activate
T cells
Sensitised T cells migrate
back to the the skin where
they produce cytokines
which attract macrophages
which cause tissue damage

84. Examples of Type IV
• Contact dermatitis
• Mantoux test
• Chronic transplant rejection

85. • An injection of tuberculin beneath the skin causes
reaction in individual exposed to tuberculosis or
tuberculosis vaccine
• Used to diagnose contact with antigens of
M. tuberculosis
» No response when individual not infected or
vaccinated
» Red, hard swelling develops in individuals
previously infected or immunized
The tuberculin response:
85

86. A positive tuberculin test

87. • Cell-mediated immune response
• Results in an intensely irritating skin rash
• Triggered by chemically modified skin proteins that the
body regards as foreign
• Acellular, fluid-filled blisters develop in severe cases
Contact Dermatitis
87

88. Contact dermatitis

90. Classification of Hypersensitivity
90

93. Thank you