ANTIHLA ANTIBODIES IN KIDNEY TRANSPLANTATION Rabat, July 2, 2005

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ANTI-HLA ANTIBODIESIN KIDNEY TRANSPLANTATIONRaba
t, July 2, 2005

• Domenico Adorno
• National Council of Researches
• Institute of Organ Transplantation and
  Immunocytology
• LAquila - Italy

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ANTI-HLA ALLOANTIBODIES
Any individual who has experienced sensitizing
events, such as transfusion, pregnancy or
previous transplant, is at risk for developing
anti-HLA antibodies. So, it is important to
detect and identify these antibodies prior to the
patient receiving an organ for transplant or
re-transplant.
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PRA(Panel Reactive Antibodies)
Pool of well-characterized panel cells in order
to detect alloantibody reactivity and specificity
in patients sera. The PRA represents the
percentage of positive cells.
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CROSSMATCHIN KIDNEY TRANSPLANTS
Negative Crossmatch Proceed to
transplant Positive Crossmatch
Controindication
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CDC CROSSMATCH IN KIDNEY TRANSPLANTS
Patel R., Terasaki P.I. N.Engl. J. Med. 1969
280 735

• Positive crossmatch high risk (80) of immediate
  graft loss.
• Prospective crossmatch if positive, transplant
  is not allowed.
• Some antibodies (not anti-HLA) are directed
  against donor lymphocytes but are not clinically
  relevant.

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CROSSMATCH (I)
1969. Standard-, Basic-, direct NIH-CDC. 1969.
Extended incubation of T and B CDC. 1970. Amos
(3 washes) and Amos-modified (1 wash) T and B
CDC washes eliminate anticomplementary
factors. 1972. AHG CDC T (Johnson A.H.)
the addition of human anti-globulin
identifies low titer antibodies as
well as antibodies that do not fix complement
in vitro. 1984. AHG CDC B (Gebel H.M.) as
B cells express surface immunoglobulin,
AHG could bind to these and activate the
complement false positive result. Using a
two-color immunofluorescent technique, B cells
are first marked with a fluorescent
anti-immunoglobulin which prevents binding
between AHG and surface immunoglobulins.
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CROSSMATCH (II)
1983. T and B Flow crossmatch (FCXM) (Garavoy)
after incubation with recipient serum, donor
lymphocytes are stained with a fluorochrome-conjug
ated secondary antibody (anti-human IgG or
IgM)

• Independent and simultaneous evaluation of
  anti-T and anti-B antibodies (marked with
  anti-CD3 and anti-CD19).
• Identification of the type of antibody (IgG,
  IgM).
• Identification of antibodies independently by
  complement activation.
• 25 - 250 times more sensitive than CDC.
• Semi-quantitative measure of antibody binding.

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CHARACTERIZATION OF ALLOANTIBODIES

• 1976. Positive crossmatch due to
  auto-antibodies (IgG or IgM) of no
• clinical rilevance
• - absorption of sera with autologous
  lymphocytes
• - absorption of sera with DTT
• Identification of anti-HLA specificity using
  microbeads covered
• with purified HLA antigens as a
  target, isolated from cell lines
• (Pei R.).
• Identification of anti-HLA specificity using
  soluble HLA antigens
• as a target, fixed in the solid phase
  of the ELISA (Zachary A.A.).
• 2003. Identification of anti-HLA antibody
  specificity using single
• antigens bound to microbeads and
  studied using Flow cytometry
• (Pei R.)
• - more sensitive
• - makes the analysis of
  anti-HLA antibodies easier in the
• presence of high PRA.

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TECHNIQUES FOR EVALUATINGANTI-HLA ANTIBODIES (I)
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TECHNIQUES FOR EVALUATINGANTI-HLA ANTIBODIES (II)
Analysis of HLA Presensitization (PRA)
By W.E. Herczyk 2003
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INCIDENCE OF ANTI-HLA SENSITIZATION (I)
ENHANCING CHARACTERIZATION OF PRE-SENSITIZATION
STATUS IN KIDNEY TRANSPLANT CANDIDATES USING
SENSITIVE TECHNIQUESA. Piazza, E. Poggi, G.
Ozzella, P.I. Monaco,C.U. Casciani, D.
AdornoTransplant 2001 Meeting, Chicago May
11-16 2001
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INCIDENCE OF ANTI-HLA SENSITIZATION (II)
FlowPRA Screening Results
Plt0.00001
P0.0367
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THE ROLE OFIg-M DONOR-SPECIFIC ANTIBODIES

• Few studies.
• Contrasting results.
• In the absence of definitive studies, these
  antibodies are not considered in clinical
  practice.

Our laboratory has found anti-HLA IgM antibodies
(identified using microbeads coated with single
HLA antigens) both pre- and post- transplant.
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THE ROLE OF NON ANTI-HLA ANTIBODIES

• The following have been associated with graft
  rejection
• Anti-phospholipid antibodies
• Anti-endothelium antibodies
• Organ-specific antibodies

These antibodies do not react with donor
lymphocytes and are not investigated in clinical
practice.
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ANTI-HLA ANTIBODIES The official position of
ASHI(approved on June 7th 2003)

• Positive crossmatches due to anti-HLA IgG
  antibodies reacting specifically with donor
  lymphocytes are clinically relevant.
• A positive CDC crossmatch is the strongest
  controindication to transplant.

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CLINICAL SIGNIFICANCE OF LOW TITER AND/OR
NONCOMPLEMENT-FIXING ANTIBODIES (CDC- , FCXM)
(I)

• Points open to discussion
• do these antibodies represent an unacceptable
  clinical risk ? Or in other words do they
  automatically cause antibody- mediated hyperacute
  or accelerated rejection ?
• do they affect an individuals chances of
  receiving a transplant ?

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CLINICAL SIGNIFICANCE OF LOW TITERAND/OR
NONCOMPLEMENT-FIXING ANTIBODIES(CDC- , FCXM)
(II)
Incidence - First
transplants 15 - Re-transplants
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Early Graft Loss (lt 3 months)
(HM Gebel et al. Am J of Transplant 2003 3 1488)
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CLINICAL SIGNIFICANCE OF LOW TITERAND/OR
NONCOMPLEMENT-FIXING ANTIBODIES(CDC- , FCXM)
(III)

• Conclusions
• low titer and/or noncomplement-fixing anti-HLA
  antibodies, only detectable by sensitive methods
  such as flow cytometry, can have significant
  clinical consequence.
• these antibodies appear to represent a
  significant risk factor that should be integrated
  into the patient assessment algorithm.

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POSITIVE CROSSMATCH FOR B-CELLS ONLY(CDC)

• Can be caused by anti-class II antibodies or low
  levels of anti-class I antibodies.
• Can be due to non-HLA antibodies.
• Can be the result of serum IgG binding to B cells
  in a non-specific manner by means of Fc receptors.

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THE CLINICAL SIGNIFICANCE OF A POSITIVE
CROSSMATCH FOR B-CELLS ONLY

• To interpret the crossmatch for B-cells in CDC
• Perform an auto-crossmatch
• Analyze serum by solid-phase assays documented to
  be more sensitive than CDC assay (Flow cytometry
  or ELISA)

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THE CLINICAL SIGNIFICANCE OFCURRENT NEGATIVE
HISTORIC POSITIVE (CNHP) CDC CROSSMATCH FOR
T-CELLS (I)

• Strategy currently adopted
• accurate immunological history of the patient
• serum testing every 3 months (EFI) or every month
  (ASHI) for PRA evaluations.

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THE CLINICAL SIGNIFICANCE OFCURRENT NEGATIVE
HISTORIC POSITIVE (CNHP) CDC CROSSMATCH FOR
T-CELLS (II)

• Evolution
• 1970 all historic sera were tested if only one
  of the sera tested was positive, then transplant
  was contraindicated.
• 1982 (Cardella) no difference in one year graft
  survival between CNHP and CNHN recipients.
• Many centers performed crossmatching only with
  current serum for sensitized patients without, in
  general, the onset of hyperacute rejection.

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THE CLINICAL SIGNIFICANCE OFCURRENT NEGATIVE
HISTORIC POSITIVE (CNHP) CDC CROSSMATCH FOR
T-CELLS (III)
(HM Gebel et al. Am J Transplant 2003 3 1488)
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THE CLINICAL SIGNIFICANCE OFCURRENT NEGATIVE
HISTORIC POSITIVE (CNHP) CDC CROSSMATCH FOR
T-CELLS (IV)

• The CNHP recipients showed an increased rate of
  antibody-mediated rejection in the first three
  months.
• However a significant proportion of CNHP
  recipients have been transplanted successfully.

Conclusion The cumulative published data suggests
that while a positive crossmatch with an
historical serum does not exclude
transplantation completely, it is nevertheless a
significant risk factor.
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EVALUATION OF RISK FOR ANTIBODY-MEDIATED
REJECTION OR EARLY GRAFT LOSSON THE BASIS OF
CROSSMATCH (I)
(HM Gebel et al. Am J Transplant 2003 3 1488)
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EVALUATION OF RISK FOR ANTIBODY-MEDIATED
REJECTION OR EARLY GRAFT LOSSON THE BASIS OF
CROSSMATCH (II) The official position of ASHI
(approved on June 7th 2003)

• High risk transplant is normally ruled out.
• If transplanted, patients require major
  pre-transplant intervention (IVIG
  plasmapheresis to modify risk),
  post- transplant additional treatment and
  accurate monitoring.
• Intermediate risk transplant is normally ruled
  out.
• If transplanted, patients may
  require augmented
• immunosuppression and accurate
  post-transplant monitoring.
• Negligible Risk transplant takes place with
  no changes in their normal practice.

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ANTIBODY-MEDIATED REJECTION

• Kissmeyer-Nielsen hyperacute rejection
  associated with pre-existing anti-HLA antibodies
  directed against donor cells.
• 1968. Patel R., Terasaki P.I. Hyperacute
  rejection associated with a positive crossmatch
  in kidney transplantation.
• 1970. The predominant role of T mediated
  rejection the post-transplant study of
  antibodies appears to be of little clinical
  relevance (antibodies are only a marker for
  T-mediated sensitization)
• 1990. Re-evaluation of rejection mediated by
  anti-HLA antibodies.

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ANTIBODY-MEDIATED REJECTION (AMR) (I)Consensus
Opinion from the Antibody Working
Group(Transplantation 2004 78 181)
AMR Antibody mediated rejection Hyperacute AMR
high titer of DSA alteration of graft function
and, nearly always, graft loss within 24 hours
post-transplant due to pre-formed
DSA. Accelerated AMR low titer of DSA rejection
occurs after 24 hours no response to standard
therapy. Acute AMR de novo production of DSA
or post-desensitization restart of DSA
production rejection from 1 week to 3-6 months
post-transplant no response to standard therapy.
Late or chronic AMR de novo production of
DSA usually 1 year post-transplant few symptoms
but progressive poor or no response to standard
therapy.
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AMR (II)
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AMR (III) Consensus Opinion from the Antibody
Working Group(Transplantation 2004 78 181)

• HYPERACUTE AMR
• high levels of pre-formed DSA rapidly attack
  graft endothelium and activate the complement
• endothelial cells are stimulated to secrete Von
  Willebrand factor which mediates platelet
  adhesion and aggregation
• platelet activation following exposure to
  subendothelial proteins
• thrombosis and vascular occlusion

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AMR (IV) Consensus Opinion from the Antibody
Working Group(Transplantation 2004 78 181)

• ACCELERATED-ACUTE AMR
• altered graft function similar to acute tubular
  necrosis
• biopsy shows inflammatory cells in the
  peritubular capillaries no tubulitis which is
  the most significant symptom of T-cell rejection
  no immunoglobulins are detectable C4d deposits
  present in peritubular capillaries.
• circulating DSA
• may coexist with cell-mediated rejection (CMR).

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AMR (V) Consensus Opinion from the Antibody
Working Group(Transplantation 2004 78 181)

• DELAYED or CHRONIC AMR
• circulating DSA
• C4d deposits in peritubular capillaries (not
  always)
• Histological findings of glomerulopathy
• May occur with CMR

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AMR DIAGNOSIS (I) Consensus Opinion from the
Antibody Working Group(Transplantation 2004 78
181)

• Altered graft function.
• Typical histological findings and peritubular C4d
  deposits.
• Presence of DSA.

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AMR DIAGNOSIS (II) Consensus Opinion from the
Antibody Working Group(Transplantation 2004 78
181)

• Histological findings
• Polymorphonucleate/macrophage infiltration.
• Presence of thrombi in capillaries and/or
  fibrinoid necrosis and/or acute tubular damage.

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AMR DIAGNOSIS (III) Consensus Opinion from the
Antibody Working Group(Transplantation 2004 78
181)
Immunopathological findings
Immunofluorescence reveals peritubular Cd4
deposits class 2 of the Banff classification
C4
C4a
C4b
C4d (binds stably to the tissue)
C4c
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AMR DIAGNOSIS (IV) Consensus Opinion from the
Antibody Working Group(Transplantation 2004 78
181)
Clinical situations at high risk with stable
graft function

• Presence of typical histological findings and DSA
  pre-clinical process, mediated by antibodies,
  which may or may not develop into AMR (preventive
  treatment aimed at eradicating DSA is useful).
• The absence of histological findings but the
  presence of DSA latent immunological response
  (if this occurs in the immediate post-operative
  period, therapy needs to be changed or increased).

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AMR THERAPY

• Treatment must be early and aggressive
• Anti-thymocyte globulins
• Mycophenolate Mofetil (MMF)
• Plasmapheresis
• Intravenous administration of Ig (IVIG)
• Immunoadsorbment with protein A with or without
  cyclophosphamides
• Anti-CD20

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PROTOCOLS FOR HYPERIMMUNE PATIENTS
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ACCEPTABLE MISMATCH PROGRAM(EUROTRANSPLANT 1985)

• (Hyperimmune patients PRA 85)
• Identification of acceptable mismatches (MMs) by
  analyzing the phenotype of negative panel donors
  or testing patient sera with one MM donors
  (20,000 blood donors typed for HLA).
• The crossmatch is forecasted to be negative and
  the kidney is sent to the transplant center.
• Crossmatch is performed on current and historical
  sera.

(FH Claas et al. Transplantation 2004 78 190)
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HLA MATCHMAKER(RJ Duquesnoy)

• A molecularly based algorithm developed to
  identify acceptable HLA antigens in hyperimmune
  patients without the need for extensive serum
  screening.
• Based on the concept that immunogenic epitopes
  are represented by amino acid triplets on exposed
  parts of HLA class I molecules, accessible to
  alloantibodies.
• In many cases this program permits the
  identification of mismatched HLA antigens that
  share all of their polymorphic triplets with the
  patients HLA antigens and, therefore, could be
  considered fully compatible.
• HLA Matchmaker provides an assessment of
  donor/recipient HLA compatibility at a structural
  level in contrast to conventional methods based
  on the counting of numbers of mismatched HLA
  antigens or CREGs.

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STUDY OF THE EPITOPES INVOLVED IN THE PRODUCTION
OF CLASS I ANTI-HLA ANTIBODIES IN KIDNEY
TRANSPLANTS

• A. Piazza, E. Poggi, L. Borrelli, A.
  Scornajenghi,
• G. Ozzella, P.I. Monaco e D. Adorno
• Istituto CNR Trapianti dOrgano Sezione di Roma

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THE TYPE OF HLA-A EPITOPES INVOLVED IN THE
PRODUCTION OF DS-Abs (I)

• 32 different epitopes (with a total of 76
  amino-acid residues)
• Highest frequency 127-K (Lys)9,2 116-D
  (Asp)7,9
• 62-G (Gly)/Q (Gln)6,6

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THE TYPE OF HLA-A EPITOPES INVOLVED IN THE
PRODUCTION OF DS-Abs (II)
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THE TYPE OF HLA-B EPITOPES INVOLVED IN THE
PRODUCTION OF DS-Abs (I)

• 16 different epitopes (with a total of 45
  amino-acid residues)
• Highest frequency 77-N (Asn)/S (Ser)17,8
  81-A (Ala)13,3
• 80-I (Ile)/N (Asn)11,1 82-L
  (Leu)11,1
• 83-R (Arg)11,1 163-E (Glu)/L
  (Leu)/T (Thr)11,1

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THE TYPE OF HLA-B EPITOPES INVOLVED IN THE
PRODUCTION OF DS-Abs (II)
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CLINICAL RELEVANCE OF ANTI-HLA ANTIBODIES
DEVELOPED AFTER KIDNEY TRANSPLANTATIONA.
Piazza, E. Poggi, L. Borrelli, G. Ozzella, P.I.
Monaco, D. Settesoldi, D. Fraboni, S.
Scornajenghi, C. Cortini, A. Iacona,C.U.
Casciani, D. Adorno9 Congresso Nazionale AIBT,
Pesaro 19-21 Settembre 2002
Preliminary data in A. PIAZZA, E. POGGI, L.
BORRELLI., S. SERVETTI , P.I. MONACO, O. BUONOMO,
M. VALERI, N. TORLONE, D. ADORNO and C.U.
CASCIANI. Impact of donor-specific antibodies on
chronic rejection occurrence and graft loss in
renal transplantation. Transplantation 2001 71
1106-1112. A. PIAZZA, E. POGGI, L. BORRELLI., M.
VALERI, O. BUONOMO, S. SERVETTI, C.U. CASCIANI,
D. ADORNO. Relevance of post-transplant HLA class
I and class II antibodies on renal graft outcome.
Transplant Proc 33(1-2) 478-480, 2001
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Anti-DSA Analysis using FlowPRA
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Anti-DSA and Transplant Outcome
P 0,0001
P 0,0001
Anti-DS Positive Patients
Anti-DS Negative Patients
Acute Rejection
Graft Loss
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CONTROL OF ANTIDONOR ANTIBODY PRODUCTION WITH
TACROLIMUS AND MYCOPHENOLATE MOFETIL IN RENAL
ALLOGRAFT RECIPIENTS WITH CHRONIC
REJECTIONT.P. Theruvath, S.L. Saidman, S.
Mauiyyedi, F.L. Delmonico, W.W. Williams, N.
Tolkoff-Rubin, A.B. Collins, R.B. Colvin, A.B.
Cosimiand M. PascualTransplantation 2001
71(1) 77-83
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Figure 2 Kinetics of circulating donor-specific
antibodies (DSA) and renal allograft function in
four patients with chronic humoral rejection
(CHR).
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SUPPRESSION OF ANTIDONOR ANTIBODY PRODUCTION BY
MYCOPHENOLATE MOFETIL IN KIDNEY TRANSPLANTED
PATIENTSA. Piazza, E. Poggi, O. Buonomo, F.
Pisani, S. Servetti,C.U. Casciani , D.
AdornoTransplant 2002 Meeting, Washington
April 26-May 1 2002
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Immunological and Clinical data
All before therapy switch All but
two before therapy switch


P0.0281
P0.0005
MMF-switched-group
Aza-group
MMF-group
Anti-donor antibodies
ARj
DFG
Viral infections
Graft failure
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PREDICTING KIDNEY GRAFT FAILUREBY HLA
ANTIBODIESA PROSPECTIVE TRIALPaul I.
Terasaki and Miyuki OzawaAm J Transplant 2004
4 438
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CONCLUSIONS (I)
INCIDENCE AND ROLE OF ANTIBODY IN GRAFT
INJURY Junchao Cai and Paul I. Terasaki Transplant
. Reviews 2004, 18 192-203
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CONCLUSIONS (II)

• . Antibody-mediated rejection in allograft
  recipients is a complicated pathological
  process..not yet completely clear.
• . The following factors may determine how fast
  rejection develops
• The level of donor-specific antibodies in
  blood..
• The capability of transplanted organ tissue
  repair this regeneration capability is tissue
  dependent.
• Immunosuppressive and anticoagulation therapy

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CONCLUSIONS (III)
. Alloantibodies, either preexisting or de novo
developed, are associated with hyperacute, acute,
and chronic rejection. ..Post-transplantation
antibody monitoring becomes extremely critical in
transplant clinics, not only because it can help
determine the extent of a patients humoral
response to allograft but also, and perhaps more
importantly, it will direct clinicians to
optimize immunosuppressive therapy.