The HLA system , Immunolgy, Graft versus Host Reaction

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

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 T he 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 . T o 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. 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

11. 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 19 ⁰ C -22 ⁰C

12. 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 .

13. 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.

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

15. 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

16. 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 s election

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18. MIXED LYMPHOCYTE REACTION It has been observed the lymphocytes from one donor, when cultured with lymphocytes from an unrelated donor, are stimulated to proliferate. 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.

19. 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)

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21. 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.

22. 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

23. 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.

24. 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)

25. 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.

26. 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.

27. 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

28. CONFORMATIONAL ANALYSIS 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)

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

30. 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.

31. 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.

32. 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.

33. 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 .

34. 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.

35. 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.

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

37. 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.

38. 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.

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

40. 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

41. 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.

42. 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.

43. 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.

44. 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.

45. AIDS (Acquired Immunodeficiency Syndrome )

46. 8/8/2024 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.

47. 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.

48. 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 E tiology

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

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

51. Complications Kaposi's sarcoma. Lymphomas. Wasting syndrome. Neurological complications. Kidney disease.

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

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

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

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

56. 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

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

58. 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.

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

60. 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

61. Type I ( Immediate ) Hypersensitivity

62. Mast Cells and the Allergic Response

63. Mast Cells and the Allergic Response

64. Type I ( Immediate ) Hypersensitivity

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

66. 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.

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

68. Examples of type II Autoimmune hemolytic anemia Thrombocytopenia Erythroblastosis fetalis

69. 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.

70. Type III ( immune complex mediated ) Hypersensitivity

71. Immune Complex Mediated Hypersensitivity

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

73. 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

74. 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.

75. 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

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

77. 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

78. A positive tuberculin test

79. 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

80. C ontact dermatitis

81. Classification of Hypersensitivity 90

82. Thank you