imun-transplant-rom8

Imunitate de transplant

Transplant

• Definitii– Autogrefa: transfer de tesuturi de la – la

acelasi organism (piele)– Singenic (isogrefa): transplant intre gemeni

identici (univitelini)– Allogrefa: transplant intre indivizi diferiti ai

aceleiasi specii– Xenogrefa: transplant de la o specie la alta

Compatibilitate de Transplant

• Pentru a creste sansa de supravietuire a transplantului:– Cel mai important: compatibilitate ABO– Absenta Ac citotoxici preformati impotriva

Ag HLA ale donatorului– Compatibilitate HLA, in particular pentru

locii D

Disorders of MHCMajor Histocompatibility Complex

• Transplantation workups– Transplant recipient must be blood group antigen (ABO)

compatible with the donor and not have any preformed anti-HLA (cytotoxic) antibodies in the blood

• Compatibility in both areas prevents hyperacute transplantation reactions

• Normally, these antibodies should not be present unless the recipient has had a blood transfusion in the past or has been pregnant and had a fetal-maternal bleed during delivery and been exposed to paternal antigens on the fetal cells

VIROLOGICAL ASSESSMENT

Both donor and recipient are tested for: VHB, VHD, VHC, HIV 1/2, CMV, EBV, HSV 1 si 2, VZV, HTLV 1/2 , rubella virus, toxoplasma gondii and chlamydia.

MethodsIndirect diagnostic tests (serological) Direct diagnostic tests, molecular biology tests (PCR, RT-PCR).

HLA

• Determines the fate of transplantation.

• Plays a role in the control of cellular interactions resposible for both cellular and humoral immune responses.

• Is associated with a variety of diseases.

IMUNOGENETICA 1. Cross- match - CDC - ELISA

2. HLA Typing by molecular biology methods – PCR SSOP- sequence-specific oligonucleotide probe hybridization (medium resolution ) SSP – sequence-specific primers (high resolution)

SBT – allele SEQR (the highest available resolution)

3. Anti-HLA antibody detection and identification - AHG CDC - ELISA


• Lymphocyte crossmatch– Used to screen recipient serum for anti-HLA

antibodies• Recipient’s serum, complement and donor B lymphocytes

are mixed together in a test tube. Lysis of donor lymphocytes is indicative of cytotoxic antibodies in the recipient’s serum directed against donor lymphocytes

• The identity of these antibodies must then be determined in order to find a suitable donor who is negative for the corresponding HLA antigen(s).

Sample of cells or tissue

Combine DNA with sequence-specific primer fix for each allele

Amplify by PCR

DNA

80ng for Class I

40 ng for Class II

Importance of DNA Quality

100 ng Genomic DNA 1% Agarose Gel

R C T G G T C A T R

A C T G G T C A T A Allele 1G C T G G T C A T G Allele 2R C T G G T C A T R Allele 1+2

G C T G G T C A T A Allele 3A C T G G T C A T G Allele 4R C T G G T C A T R Allele 3+4

Allele 1+2 = 3+4Allele 1+2 = 3+4

SBT for ALL HLA Typing Requirements: Resolving Heterozygous Ambiguities HR Typing

How are heterozygous ambiguities identified by Assign-SBT ?

The sequence of the test sample(R040901046[SDRB1) is identical

(MM=0) to the sequencesof:

DRB1*030101+150101DRB1*0319+1505

Assign-SBT Resolves AmbiguitiesSequences are arranged in “layers”…Master sequence

Patient result

Types of Transplants

• Corneal– Best graft survival rate since the cornea is

avascular and the lymphatic drainage from the eye is not as well developed as in other tissues

– Associated with transmission of prions-Creukfeld-Jacob disease-Transmissible spongiform encephalopathy; also has been associated with amoebic transmission (granulomatous amebic encephalitis)


• Renal– Between living donors with a 2 haplotype match = 90-

95% 5 year survival

– With a 1 haplotype match = 80% 5 year survival

– Cadaver transplants between unrelated donors is the most common type of transplantation. Similar statistics to 1 haplotype match when the recipient receives multiple blood transfusions prior to the surgery (induces tolerance to the allograft) and is placed on immunosuppressive therapy


• Liver – In adults with chronic active hepatitis or

cirrhosis– In children with biliary atresia– 1 year survival rate is slightly greater than 90%


• Cardiac transplantation– In adults, used in patients with chronic ischemic

heart disease and congestive cardiomyopathy– In children, endocardial fibroelastosis is the

usual indication– Endomyocardial biopsies are the best means of

diagnosing allograft rejection– Approximately 80% of transplants survive 1 year


• Bone marrow transplants– Used in the treatment of aplastic anemia, leukemia

and immunodeficiencies– Goal is to infuse donor marrow containing

pluripotential hematopoietic stem cells that will eventually repopulate the lymphoid, erythroid, myeloid, and megakaryocytic series in the recipient.

– GVH occurs in almost 2/3rds of cases– Increased incidence of CMV pneumonitis

Transplant Rejection

• The chance of a sibling in a family having another sibling with 0, 1, or 2 haplotype match is:– 25% - 0 haplotype match– 25% - 2 haplotype match– 50% - 1 haplotype match– However, a 2 haplotype match is rarely achieved due to

crossovers between the individual loci during meiosis when homologous chromosomes line up close to each other


• Three types of transplant rejections

– Hyperacute rejection

– Acute rejection

– Chronic rejection


• Hyperacute rejection:– occurs within minutes of attaching the allograft to the

recipient’s blood supply– Due to the presence of an ABO mismatch or preformed

cytotoxic antibodies in the host against foreign HLA antigens in the donor tissue (example; a blood group A recipient would have anti-B IgM antibodies and would react against a group B donor heart)

• Hyperacute rejection is rare because ABO and anti-HLA cytotoxic antibody screening is performed prior to the surgery


• Acute rejection– Most common type of rejection encountered– Usually occurs within the first 3 months of the transplantation– Involves cell-mediated and antibody-mediated reactions.

Cell-mediated has the greatest role in rejection– The type II antibody-mediated hypersensitivity produces a

necrotizing vasculitis with subsequent vessel damage and intravascular thrombosis


• Acute rejection– Vessel events can occur over a period of time

leading to fibrosis and vessel lumen obliteration

– The cell-mediated component involves cytotoxic T cells producing extensive interstitial infiltrate in the graft with edema and damage to the tissue (Type IV hypersensitivity)

– Can be reversible with immunosuppressive drugs such as cyclosporin A, corticosteroids, and OKT3.


• Chronic rejection– Irreversible– Occur over a period of months to years– Extensive fibrosis and loss of organ structure

characterize the histologic findings in the transplant– Activated macrophages release growth factors that

stimulate fibroblasts to deposit collagen– There is also chronic ischemia secondary to antibody-

mediated damage to the vessels

Type IV Hypersensitivity– Cytotoxic T cells interact with class I antigens on nucleated cells– If the antigens are altered (virally infected cells, neoplastic cells) or the cell is

foreign to the host (transplant), the cytotoxic T cells will attach to the cell membrane, release perforins and destroy the cell.

• Examples: Acute and chronic transplant rejections; destruction of hepatocytes infected by hepatitis B virus


• Cyclosporin A inhibits CD4 helper T cell release of interleukin-2 (blocks calcineurin) which stimulates the proliferation of cytotoxic and helper T cells

• Corticosteroids inhibit macrophage production of interleukin-1 and tumor necrosis factor and are cytotoxic to immature cortical derived thymocytes

• OKT3 is a monoclonal antibody preparation that attaches to the CD3 antigen receptor of T cells, blocking their reaction with the graft

ID/CC A 45 year old male with refractory acute myeloid leukemia is brought to the emergency room with fever, a generalized rash, jaundice, right upper quadrant pain, severe diarrhea, and dyspnea; two months ago, he underwent an apparently uncomplicated bone marrow transplantation.

HPI Prior to the transplant, he received radiotherapy and chemotherapy as well as broad-spectrum antibiotics

PE VS: normal blood pressure. PE: cachexia; moderate dehydration; 2+ jaundice; violaceous and erythematous macules as well as papules and bullae with scale formation over extremities

Labs Elevated IgE level. CBC/PBS: falling blood counts; relative eosinophilia. Elevated direct serum bilirubin and transaminases, no infectious agents on stool exam

Graft versus Host Reactions

• Potential complication in bone marrow and liver transplants and in blood transfusions administered to patients with T cell immunodeficiency

• Donor lymphocytes produce interleukin-2 • -->activation of NK cells (primary effector cells in

acute GVH reactions)-->lymphokine-activated NK cells are called LAKs and produce extensive epithelial cell necrosis in the biliary tract (jaundice), skin (maculopapular rash), and GI tract (diarrhea)

Graft versus Host Reactions

• May progress into chronic GVH which is marked by the presence of extensive fibrosis

• To lessen the risk of GVH, donor tissue is pretreated with anti-thymocyte globulin to remove donor T cells.

• Cyclosporin A is used also

Transplant complications

• Immunosuppressive therapy has increased the incidence of:– Cervical cancer– Malignant lymphomas (immunoblastic)– Basal and squamous cell carcinomas of the skin

• Squamous cell CA is the most common overall malignancy

• Other complications include infection and bone marrow suppression

Antibody Monitoring System

ELISA assay designed to detect donor reactive IgG antibodies in

recipient sera

Used for Immunological monitoring of donor-specific

HLA alloantibodies in transplant patients that may

lead to early graft loss or chronic rejection

• Retrospective Crossmatch

• Prospective Crossmatch

• Post-transplant

Immunological Monitoring

• Detects only HLA donor specific antibodies

• IgG specific - will not detect IgM

(autolymphocytotoxic) antibodies

• Detects non-complement binding antibodies

• Detects Class II specific HLA antibodies in

presence of strong Class I antibody

1st step: lysate preparation

takes about 15 minutes after isolation of cells

LYSATE PREPARATION

2nd step: ELISA

takes about 3 to 4 hrs - depending on number of donors

NegativeControl

LysateControl

PositiveControl

Class I Class II

RecipientSamples


• Mixed Lymphocyte Reaction (MLR)– Utilized to test for class II antigen (D loci)

match between the recipient and donor– Functional lymphocytes from the recipient and

previously irradiated (killed) donor lymphocytes are mixed together in a test tube with tritiated thymidine.

– A baseline radioactive count is obtained before the donor lymphocytes are added to the tube.


• Mixed Lymphocyte reaction (MLR)– If the recipient’s lymphocytes have different D

antigens than those located on the donor lymphocytes, they become activated which increases the radioactive count in the test tube over the baseline reading

– This reaction evaluates the potential for recipient rejection of the donor graft, but does not provide information on whether the graft will reject the host


• Mixed lymphocyte Reaction (MLR)– A modified test to evaluate the risk of a GVH

reaction is to irradiate (kill) the recipient’s lymphocytes and to allow the functional donor lymphocytes an opportunity to react against the host’s HLA D loci

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