HLA and  non HLA antibodies in organ  Transplantation
J. Martorell. Servei Immunologia H. Clínic, IDIBAPS

HLA and	non HLA antibodies	in organ Transplant
-Sensitizing elements	-Autoantibodies (Non polymorphic)	- Kidney, Pancreas
-Broadness	-Tissue Specific Antigens (Endothelia)	- Liver
-Specificity (CREG)	¿Non Polymorphic or Polymorphic?	- Heart, Lung
-Title and Removal
-Isotype (IgG/IgM)	(Click on Table)

Techniques: CDC, Flow Cytometry on beats, ELISA, Flow Cytometry on cells

How to find a donor with a negative crossmatch for highly sensitized receptors?

 

Why are antibodies so important?:
"Hyperacute rejection of kidney allografts, associated with pre-existing
humoral antibodies against donor cells"
Kissmeyer-Nielsen F, et Al. Lancet 1966.

HLA antibodies

Sensitizing elements: 
- Pregnancy		1/4 
- Blood transfusion	1/4 
- Previous transplant  3/4 

Broadness of Panel Reacting Antibodies (PRA), expressed in % of positive panel cells: 
- Indicates the percentage of donors that will give a positive Cross-Match.

Specificity:
- Indicates the HLA specificities that should be avoided in the donor to avoid a positive 
Cross-Match and a hyperacute rejection.
- A receptor never has antibodies against its HLA specificities.
- Antibodies to specificities that belong to the same CREG to the ones expressed
 in the receptor are infrequent. 					

CREG (Cross Reacting Group):
HLA specificities that share epitops 
- Epitop or antigenic determinant : Distinct sites to were an antibody can bind.
- Is frequent to find receptors with antibodies against several HLA specificities that belong 
to the same CREG (A3-A11-A1; A2-A28; B5-35). 		(Slide 1)

Title: Last positive of a serial dilution of a sera :
Provides an approximate idea about the relative number of antibody molecules 
present per ml of sera or about its affinity for antigen.

Antibodies removal:
- Although low title antibodies can be removed by Protein A columns: 
	        - A rapid rebound of antibodies occurs in less than a week, usually in 24 hr.
	- High title antibodies can not be removed by Protein A columns.

Isotype (IgG/IgM)
	- Aloantibodies are usually IgG but can be IgM.
	- Autoantibodies are usually IgM but can be IgG.
- IgM can be inactivated by DTT (Dithiothreitol), that destroys -S-S- bonds
 Non HLA antibodies

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Non HLA antibodies

Autoantibodies (to non polymorphic antigens)
- Some patients present antibodies against non polymorphic antigens expressed 
on lymphocytes, and other cell membranes.
- Autoantibodies are not harmful to the graft.
- Are more frequent in patients with other autoimmune disease:
-Systemic Lupus Erithematosus, -Primary Biliar Cirrhosis, -Rheumatoid Arthritis...
- Can be detected by an auto-Cross-Match.
- Does not react with the purified HLA antigens used as target when 
	- ELISA and HLA coated beats FC are used for antibody screening.
- Are usually IgM and in consequence inactivated by DTT.
	- Not all antibodies destroyed by DTT are autoantibodies.
- Those reacting only with B cells could be anti-surface-IgG.

Tissue specific antigens (Non HLA) . (Polymorphic or Non Polymorphic).
In sera screening and crossmatching tests, the lymphocytes are used 
as representative target cells, because they have a good expression of the HLA antigens.  
In some rejections episodes, it has been described the presence, of antibodies 
reacting only with tissue specific antigens, expressed in endothelial cells and monocytes 
but not on lymphocytes membrane.  
- At least some of them had been proved to be against non polymorphic antigens.
	- Others seem to be polymorphic.
- Those tissue specific antigens could be the reason for some rejections seen in patients 
with negative lymphocyte crossmatch.
	- Are more frequent in retransplant patients.
- The prognostic value of those antibodies pretransplant is under discussion. 

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In Organ Transplantation

Crossmatch in kidney and pancreas Transplantation.
Crossmatch is the test that detects the presence or not in the receptor of antibodies 
against the antigens of a particular receptor.
A positive crossmatch had been considered an absolute contraindication for kidney 
transplant. Some special cases deserves some comments:

Historic Positive, Current Negative:
- The Cross-Match that is positive with historic sera but negative with the current sera 
had been considered a contraindication for transplantation since 1966 to 1980, 
on this year several papers reported a good survival in this kind of patients. 
- Today the issue is controversial and many centers consider that transplantation 
under this circumstances can have 1 year graft survival rate significantly lower than 
in the historic negative current negative cases (20 % less in our experience). 

T cell negative, B cell positive:
- Many center had described that graft survival in case of T cell negative, 
B cell positive crossmatch is identical to the found in the T and B negative crossmatch.
- Others by the contrary have found the presence of anti HLA-class-II antibodies 
reacting only with B cells in rejected kidneys. For some authors anti-HLA-class-II 
antibodies are related with higher incidence of rejection episodes. 

 Preliminary / Final 
- The final crossmatch should include receptor serum collected in the previous 48 hr 
and previous positive sera from at least the last 2 years, specially the pick samples.
- Some centers perform routinely a preliminary Cross-Match screening with the last 
available sera from sensitized receptors against all the ABO compatible donors, 
this helps to easily exclude positives from receptor selection process.

How to find a donor with a negative crossmatch for highly sensitized receptors?: 
	- HLA type the donor and the receptor.
- Find a donor with as much HLA-A and B identities as possible with the receptor: 
Remember: antibodies never react against receptor HLA specificities 
and infrequently react with specificities that belong to the same CREG.
	- Use the preliminary crossmatch policy.
	- Amplify the donor pool by establishing organ exchange agreements. 

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Crossmatch in Liver Transplantation.
- A positive crossmatch is not considered a contraindication for Liver transplantation 
for many groups, provided that liver is not susceptible to hiperacute rejection.
- But a positive crossmatch is considered a prognostic factor for more frequent, 
and more intense, graft rejection episodes. This group of high risk receptors 
would benefit from more aggressive immunosuppression protocols. 
	

Crossmatch in Heart and Lung Transplantation.
- 1 year heart survival in the T cell positive crossmatch group has been described 
to be as low as 28 % in comparison with the 73 % in the T cell negative crossmatch.
- Due to the short cold isquemia time supported by heart sometimes 
is not logistically feasible to have a pretrasplant crossmatch.
- Many centers had established the following policy:
	   - Extensive antibody screening in heart receptor candidates.
- If PRA is negative: pretransplant crossmatch is recommended but not mandatory.
- If PRA is Positive: pretransplant crossmatch is mandatory.
 Techniques for HLA antibodies screening and crossmatching:

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   TECHNIQUES FOR DETECTION OF ANTI-HLA ANTIBODIES

Techniques and Criteria

Screening (PRA)	CDC	Flow Cytometry on beats	ELISA
Cross-Matching	CDC	Flow Cytometry on cells

 

Techniques	Antibodies Detected	Target of Antibodies	"Cross- Match"
Cytotoxicity	Cytotoxic IgG & IgM  (1)	Ag. Lymphocyte membrane	Yes
Flow C Cells	All IgG (2)	Ag. Lymphocyte membrane	Yes
Flow C Beats	All IgG (2)	HLA-I and/or HLA-II
ELISA	All IgG (2)	HLA-I and/or HLA-II

(1) IgM can be inactivated using Dithiotreitol (DTT).
(2) Optionally IgM can be identified by using an anti-IgM as second antibody.

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For years the most used technique has been:
Complement Dependent Cytotoxicity (CDC): Detects the capacity of receptor 
antibodies to kill donor (or panel) cells in the presence of rabbit complement
Has several variants: 
- NIH-Standart: the most frequent in Europe; 
- AHG that uses anti-human globulin and washes, the most frequent in USA;
- Extended incubation time: to increase sensitivity.
Recently other techniques had appeared:			(Slide 2)	(Slide 3)

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Flow Cytometry (FC) on cells :
- Uses donor cells as target. 
- Antibodies are identified with an anti-human IgG antibody labeled with FITC.
- The careful selection of negative and positive controls is crucial.
- To have clear rules to establish threshold of positivity is essential.  
	- Used normally for cross-match. Too time consuming to be used for screening.
										(Slide 2)	(Slide 3)

Flow Cytometry (FC) on beats :
- Uses latex beats with attached HLA molecules. 
- Antibodies are identified with an anti-human IgG antibody labeled with FITC.
- Can distinguish anti-HLA from other kinds of antibodies.
	- Can easily distinguish anti-HLA-Class-I from anti-HLA-Class-II antibodies.
- Can be done in 2 steps: one to detect antibodies an the other to define specificity.
	- It can not yet be used for Cross-Matching.
										(Slide 2)	(Slide 3)

ELISA: (Enzyme linked immunosorbend assay)
- Uses plastic wells with attached HLA molecules. 
- Antibodies are identified with an anti-human IgG antibody labeled with  a 
color changing enzyme (Alkaline Phosphatase or Peroxidase). 
- Can distinguish anti-HLA-Class-I from anti-HLA-Class-II antibodies. 
- Can distinguish anti-HLA from other kinds of antibodies.
- Can be done in 2 steps: one to detect antibodies an the other to define specificity.
	- It can not yet be used for Cross-Matching.
										(Slide 2)	(Slide 3)
It is generally accepted that Flow cytometry detects all the antibodies detected by 
cytotoxicity and others that are not cytotoxic. 

ELISA technique have some discrepancies in the sense of CDC + / ELISA - 
and in the sense CDC - / ELISA +, in 3 to 10 % of sera, the prognostic value 
of this discrepancies is not yet well established. 
Specially striking are the discrepancies between FC and ELISA provided that 
according to some studies, 13 % of ELISA - sera are FC +.

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DISCUSSION BOARD PANEL DE DISCUSION