First Movement Practicale: Fine-tuning the Instrument of Epitope-Based Histocompatibility

1
First Movement Practicale Fine-tuning the
Instrumentof Epitope-Based Histocompatibility

• Rene Duquesnoy
• University of Pittsburgh Medical Center

2
Lecture Outline

• Immunogenicity and antigenicity of epitopes
• CDRs and antigen-antibody interactions
• HLAMatchmaker analysis of antibody reactivity
  with Luminex single allele panels
• Commonly recognized class II epitopes
• Epitope immunogenicity
• HLAMatchmaker and platelet transfusions

3
Identification of Structurally Defined HLA
Epitopes

• Empirical approach
• Look for correlations between antibody reactivity
  patterns and the presence of distinct polymorphic
  amino acid residues in different sequence
  positions on reactive alleles
• Conduct absorption/elution of sera with selected
  alleles
• HLAMatchmaker
• Epitope structure is based on three-dimensional
  modeling of different antigen-antibody complexes,
  molecular contact points between epitope and
  paratope and the contribution of important
  residues to epitope functionality
• Consider the immunogenetic relationship between
  antibody producer and immunizer concept of
  immunogenicity versus antigenicity
• Determine if the antibody reactivity patterns
  correspond to HLAMatchmaker-predicted epitope
  mismatches

4
Important Consideration
Differentiate between Immunogenicity of
epitopes (induction of specific
antibodies)  and Antigenicity of epitopes
(reactivity with antibodies)
5
HLA Mismatch Immunogenicity

• Mismatched HLA antigens have different epitope
  loads
• HLA epitopes have different degrees of
  immunogenicity
• A better understanding of HLA immunogenicity will
  permit a permissible mismatch strategy for
  non-sensitized transplant patients

6
Antigenicity

• Reactivity of epitopes with specific antibodies
• Serum screening methods
• Complement-dependent lymphocytotoxicity CDC, AHG
• Antigen-binding assays Elisa, Flow cytometry,
  Luminex with single antigens
• Technique-dependent antibody reactivity

7
How Do Antibodies React with Antigens?
8
Six Complementarity-Determining Regions of the
Antibody Combining Site

• CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3
  represent the binding face or paratope which
  contacts the structural epitope comprising 15-25
  amino acids

9
Crystal Structure of HLA-A1-MAGE-A1 Complex with
Fab-Hyb3
Hulsmeyer et al. J. Biol. Chem., 2005. 280
2972-2980, 2005
10
T-Cell Receptor-HLA Complex
TCR
a
b
HLA
11
Six Complementarity-Determining Regions of the
Antibody Combining Site

• CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3
  represent the binding face or paratope which
  contacts the structural epitope comprising 15-25
  amino acids
• The antibody specificity is often determined by a
  centrally located loop (CDR-H3) that binds to the
  functional epitope a configuration of 3-6 amino
  acids in the structural epitope
• Eplets may define these functional epitopes
• Other CDRs serve as contact sites to stabilize
  binding to antigen (they play a role in the
  affinity of antibody)

12
Noncovalent Forces in Antigen-Antibody
Complexing
Electrostatic forces Attraction between opposite charges
Hydrogen bonds Hydrogen shared between electronegative atoms (N,O)
Van der Waals forces Fluctuations in electron clouds around molecules oppositely polarize neighboring atoms
Hydrophobic forces Hydrophobic groups pack together to exclude water molecules
13
The Reaction between Antibody and Antigen Depends
on the Binding Force
Binding Force CDR Contact Site Reaction
Too low No SpecCDR No Eplet Negative
Borderline Is SpecCDR insufficient? Eplet No binding?
Intermediate SpecCDR is sufficient or requires a 2nd CDR Eplet 2nd Contact Site Binding only
Strong SpecCDR 2nd CDR more CDR? Eplet 2nd Contact Site more? Binding and conformational change of Fc to expose C1q binding site
14
Complementarity Determining Regions
15
Structural Basis of a HLA-B51 Mismatch
Seen by A2,A68 B27,B44
Seen by A2,A68 B35,B44
Polymorphic Residues on B51
16
B51 for A2,A68 B27,B44
B51 for A2,A68 B35,B44
?
?
How do antibodies react with structurally defined
epitopes?
17
HLAMatchmaker-Based Analysis of Human Monoclonal
Antibody Reactivity Demonstrates the Importance
of an Additional Contact Site for Specific
Recognition of Triplet-Defined Epitopes

• Rene J. Duquesnoy, Arend Mulder, Medhat Askar,
  Marcelo Fernandez-Vina and Frans H.J. Claas
• Human Immunology 66 749-761 2005

18
Antibody Producer A2,A68 B7,B27
Cw2,Cw7 Immunizer A3 (pregnancy) Triplets
62Qe,142mI,144tKr,151aHe,163dT

• Two examples of mAbs
• OK2H12 anti-62Qe
• OK4F9 anti-142mI

19
Reactivity of mAb OK2H12 with 62Qe-carrying
alleles
20
56G is the only residue shared between HLA-A3 and
62Qe-carrying alleles that react with OK2H12
21
Shared Polymorphic Amino Acids in the
62Qe-Defined Specificity Site
Critical Second Contact Site
Antibody Specificity Site
22
Shared Polymorphic Amino Acids in the
62Qe-Defined Specificity Site
Critical Second Contact Site
Antibody Specificity Site
Distance between 56G and 62Q is 11 Angstroms,
sufficient for contact by two different CDRs
23
Shared Polymorphic Amino Acids in the
62Qe-Defined Specificity Site
Critical Second Contact Site is a Self Residue
Antibody Specificity Site
Distance between 56G and 62Q is 11 Angstroms,
sufficient for contact by two different CDRs
24
Immunizing HLA-A3 Triplotype62Qe,142mI,144tKr,15
1aHe,163dT

• Two examples of mAbs
• OK2H12 anti-62Qe
• OK4F9 anti-142mI

25
Reactivity of mAb OK4F9 with 142mI-Carrying
Alleles
Lymphocytotoxicity
Antigen-Binding
Allele
Triplets shared with HLA-A3
N
Pos Rx
Rx Strength
Flow Beads
Elisa
62Qe,
142mI
,144tKr,151aHe,163dT
A0301
79
99
7.6


62Qe,
142mI
,144tKr
A0101
48
100
7.4


62Qe,
142mI
,144tKr
A1101
68
97
7.8


142mI
,151aHe
A2601
37
97
7.5


A2902
142mI
16
100
8.0


62Qe,
142mI
A3001
13
100
8.0


62Qe,
142mI
A3002
12
92
6.5
nd
nd
62Qe,
142mI
A3101
29
97
7.6


A3301
142mI
13
92
7.3


142mI
A3303
12
75
6.3
nd

142mI
,151aHe
A3401
19
100
8.0

nd
142mI
,151aHe
A3402
7
100
8.0
nd

142mI
,151aHe
A6601
8
100
8.0


62Qe,
142mI
A7401
5
100
8.0


142mI
A2301
17
0
1.0
Neg
Neg
142mI
,144tKr
A2402
78
8
1.6
nd
Neg
142mI
,144tKr
A2403
4
25
1.7
Neg
Neg
142mI
,144tKr
A2407
5
0
1.0
nd
nd
142mI
,151aHe
A2501
9
0
1.0
Neg
Neg
142mI
62Qe,
A3201
22
0
1.0
Neg
Neg
A2/A28
none
67
6
1.3
Neg
Neg
26
The sequence 79G,80T,81L,82R,83G is shared
between HLA-A3 and the 142mI-carrying alleles
that react with OK4F9
27
Locations of 142mI-Defined Specificity and the
79GTLRG-Defined Critical Secondary Contact Sites
on A0301
Crtical Second Contact Site (Self Sequence)
Antibody Specificity Site
Distance between 142I and 82R is 13
Angstroms, Sufficient for contact by two
different CDRs
28
Residue Polymorphisms and Reactivity of
Bw6-Reactive SFR8-B6 Monoclonal Antibody
Lutz et al. J. Immunol. 153 4099, 1994 Studied
the effect of single amino acid substitutions by
mutagenesis of HLA-B7 molecules

• Antibody Specificity site 80N, 82R, 83G
• Critical contact site 90A (about 11 Angstroms
  away)
• Permissive substitutions in 62 positions!

29
Many Residue Substitutions Do Not Affect
Reactivity With Antibody
30
Permissive Residue Substitutions that Do Not
Inhibit Reactivity of the 62Qe-Specific mAb
31
Locations of Permissive Polymorphic Residues for
62Qe-Specific mAb Reactivity
56G
62Qe
32
Complement-Dependent Cytotoxic AntibodyversusN
on-Cytotoxic (Binding Only) Antibody
33
Serological Analysis of the HLA-A10 Complex

• Monospecific antisera against the HLA-A10
  splits A25 and A26 (7th Int. Workshop)

Typing Serum Reaction with A25 Reaction with A26
Anti-A25 Yes No
Anti-A26 No Yes
34
Serological Analysis of the HLA-A10 Complex
Duquesnoy and Schindler Tissue Antigens 7
65-73, 1976Hackbarth and Duquesnoy Transplant.
Proc. 9 43-45, 1977

• Antisera against the HLA-A10 splits A25 and A26
• Absorption studies

Serum Reaction with A25 Reaction with A26
Anti-A25 Cytotoxicity No Cytotoxicity Only Binding
Anti-A26 No Cytotoxicity Only Binding Cytotoxicity
Can HLAMatchmaker explain this?
35
Anti-A25 Serum
Serum HLA-type Ab Producer Ag Unshared Triplets
Jun A1,A3B7.B8 A25 Cytotoxic 80rIAlr, 150tAHe,156W, 183A, 193Av
A26 Only Binding 150tAHe,156W,183A, 193Av
Immunizing Antigen
36
Anti-A26 Serum
Serum HLA-type Ab producer Ag Unshared Triplets
Mich A3,A23B7,B13 A26 Cytotoxic 76An, 90D, 150tAHe, 156W,163R,183A,193Av
A25 Only Binding 90D,150tAHe,156W, 163R,183A,193Av
Immunizing Antigen
37
Anti-A10 Sera
Serum HLA-type Ab Producer Ag Unshared Triplets
Sand A1,A24B7,B44 A25 Cytotoxic 66rNv, 150tAHe,156W, 183A, 193Av
A26 Cytotoxic 66rNv, 150tAHe,156W, 183A, 193Av
Elli A1.A32B13,B64 A26 Cytotoxic 150tAHe,156W
A25 Cytotoxic 150tAHe,156W
Immunizing Antigen
38
Structural Basis of Cytotoxic Antibody Reactivity
against A25 and A26
Anti-A26
Anti-A25
Critical Second Contact Site 76An
Critical Second Contact Site 82aLr
Antibody Specificity Site 150tAhe
Antibody Specificity Site 150tAhe
Immunizing A26 antigen
Immunizing A25 antigen
The Critical Second Contact Site is a Non-Self
Sequence and is Necessary for Complement Binding
39
Emerging Concepts

• The HLA antibody specificity site consists of a
  cluster of few polymorphic amino acids on the
  molecular surface
• This site would be contacted by CDR-H3
• Antibody reactivity may require a second contact
  site which is about 7-15 Angstroms away from the
  antibody specificity site
• This site would be contacted by a different CDR

40
Interpretations of Negative Reactions of a Serum
with HLA Antibodies

1. The mismatched HLA antigen has a different
   epitope not recognized by patient antibody
   Acceptable mismatches are identified through
   sharing of epitopes not recognized by patients
   antibodies

41
Interpretations of Negative Reactions of a Serum
with HLA Antibodies

1. The mismatched HLA antigen has a different
   epitope not recognized by patient antibody
   Acceptable mismatches are identified through
   sharing of epitopes not recognized by patients
   antibodies
2. The mismatched HLA antigen carries the antibody
   specificity site but lacks the Critical Contact
   Site (CCS) for antibody binding Acceptable
   mismatches are more difficult to identify

42
Interpretations of Negative Reactions of a Serum
with HLA Antibodies

1. The mismatched HLA antigen has a different
   epitope not recognized by patient antibody
   Acceptable mismatches are identified through
   sharing of epitopes not recognized by patients
   antibodies
2. The mismatched HLA antigen carries the antibody
   specificity site but lacks the Critical Contact
   Site (CCS) for antibody binding Acceptable
   mismatches are more difficult to identify
3. Hidden polymorphisms may affect the conformation
   of surface residues in the antibody specificity
   site and/or critical second contact site

43
HLAMatchmaker Analysis of Antibody Reactivity
Patterns with Luminex Single HLA Alleles
Example Screening for antibodies against HLA-DP
44
Anti- HLA-DP Reactivity of Serum Group B Pt 43,
Patient types as DPB10201,-
45
Anti- HLA-DP Reactivity of Serum Group B Pt 43
Patient types as DPB10201,-
Questions Do the antibodies react with DPB or
DPA or both ? What are the antibody specificities
? Which DP antigens are acceptable mismatches
? Which DP antigens are unacceptable?
46
HLAMatchmaker Analysis of Anti- HLA-DP Reactivity
of Serum Group B Pt 43 (Step 1)
47
HLAMatchmaker Analysis of Anti- HLA-DP Reactivity
of Serum Group B Pt 43 (Step 2)
48
HLAMatchmaker Analysis of Anti- HLA-DP Reactivity
of Serum Group B Pt 43 (Step 3)
49
HLAMatchmaker Analysis of Anti- HLA-DP Reactivity
of Serum Group B Pt 43 (Step 4)
50
Molecular Locations of Positions 84 and 87
51
84DE87AV Corresponds to an Antibody-Defined
Epitope
52
HLAMatchmaker-Predicted Unacceptable DPB Alleles
53
HLAMatchmaker-Predicted Acceptable DPB Alleles
54
HLAMatchmaker Determination of Unacceptable DPA
Alleles
55
Predominant Eplets Reacting with anti-HLA-DP
Antibodies
55DE is similar to the 57DE eplet on DR11 All
55DE-reactive sera reacted also with DR11
56
Retransplant Candidates Have Donor-Specific
Antibodies that React with Structurally Defined
HLA-DR,DQ,DP Epitopes
Duquesnoy et al. Transplant immunology,
18352-360, 2008
57
Three Groups of HLA-Sensitized Patients

• A. Sensitizing tissue is absent
• Previous transplant has been removed
• Prior transfusion and pregnancy
• B. Sensitizing tissue is present
• Transplanted organ still in place
  No evidence of pre-transplant
  sensitization
• C. Combination of A and B

58
Incidence of Anti-HLA-DR, -DQ and -DP Antibodies
in HLA Class II Sensitized Patients with or
without a Transplant Present
p0.001 plt0.0001
59
Effect of eplet mismatching and the Incidence of
antibodies to donor DRB1, DRB3, DRB4 and DRB5
mismatches
60
Eplet mismatching and the incidence of
donor-specific anti-HLA-DQ antibodies
61
Frequencies of antibodies to donor DQB eplet
mismatches
62
Frequencies of antibodies to donor DQA eplet
mismatches
63
The Antibody Response to an HLA Mismatch is
Restricted to a Small Number of Epitopes
64
SERUM ANALYSIS AFTER TRANSPLANT NEPHRECTOMY
REVEALS RESTRICTED ANTIBODY SPECIFICITY PATTERNS
AGAINST STRUCTURALLY DEFINED HLA CLASS I
MISMATCHES
Adeyi et al. Transplant Immunology, 14 53-62,
2005
65
Thirty Patients With Rejected Kidney Transplants
Underwent Allograft Nephrectomy
100
80
PRA
60
40
20
0
Before Tx Pre-AlloNx.
Post-AlloNx
Adeyi et al. Transplant Immunology, 14 53-62,
2005
66
Donor Epitopes   A3 62Qe,66rNv,70aQs,76
Vd,80gTl,144tKr,149aAh,151aHe,163dT A31
56R,62Qe,66rNv,74iD,76Vd,80gTl,193Av   B55
131S   B63 45Ma,66rNm,70aSa,74Y,131S  
Adeyi et al. Transplant Immunology, 14 53-62,
2005
67
Donor Epitopes (serum-reactive epitopes are shown
in underlined bold font)   A3
62Qe,66rNv,70aQs,76Vd,80gTl,144tKr,149aAh,151aHe,1
63dT A31 56R,62Qe,66rNv,74iD,76Vd,80gTl,193Av  
B55 131S   B63 45Ma,66rNm,70aSa,74Y,131
S  
Adeyi et al. Transplant Immunology, 14 53-62,
2005
68
Donor Epitopes (serum-reactive epitopes are shown
in underlined bold font)   A3
62Qe,66rNv,70aQs,76Vd,80gTl,144tKr,149aAh,151aHe,1
63dT A31 56R,62Qe,66rNv,74iD,76Vd,80gTl,193Av  
B55 131S   B63 45Ma,66rNm,70aSa,74Y,131
S  
Unacceptable epitopes identify unacceptable
antigens e.g. 45Ma is present on B13, B46,
B57,B62, B63. B75, B76, B76
Adeyi et al. Transplant Immunology, 14 53-62,
2005
69
Donor Epitopes B13 41T,45Ma,76En,80rTa,82aLr,14
4tQl,163E 82aLr is expressed by all
Bw4-associated HLA-B antigens A23, A24, A25 and
A32 144tQl is unique for HLA-B13 After six
months the anti-76En and anti-82aLr antibodies
have disappeared, Only anti-144tQl antibodies
are detected
Adeyi et al. Transplant Immunology, 14 53-62,
2005
70
Relative Immunogenicity of Eplets

• How often do mismatched eplets induce specific
  antibodies?

14th International HLA Workshop Project
Analysis of donor-specific antibodies in
patients whose rejected kidney transplants had
been removed
71
Preliminary Results Eplet Immunogenicity in 62
Cases
Duquesnoy, RJ and Claas FHJ Progress Report of
14th International Histocompatibility Workshop
Project on the Structural Basis of HLA
Compatibility, Tissue Antigens, 69 (Suppl. 1)
1-5, 2007
72
15th International Workshop Project on Epitope
Immunogenicity

• Analyze post-allograft nephrectomy sera for
  antibodies against donor class I and class II
  epitopes
• Serum screening with single alleles (Luminex) and
  by CDC
• So far, 40 laboratories worldwide will contribute
  informative cases
• For more information go to
• http//www.15ihiws.org/project.php?n13

73
HLA Mismatch Immunogenicity

• Mismatched HLA antigens have different epitope
  loads
• HLA epitopes have different degrees of
  immunogenicity
• A better understanding of HLA immunogenicity will
  permit a permissible mismatch strategy for
  non-sensitized transplant patients

74
Use of HLAMatchmaker in Platelet Transfusions
75
HLAMatchmaker-Driven Analysis of Responses to HLA
Typed Platelet Transfusions in Alloimmunized
Thrombocytopenic Patients

• Ashok Nambiar, Rene J. Duquesnoy, Sharon Adams,
  Yingdong Zhao, Jaime Oblitas, Susan Leitman,
  David Stroncek and Francesco Marincola
•   Department of Transfusion Medicine, National
  Institutes of Health

Blood 107 1690-1697, 2006
76
T R A N S F U S I O N P R A C T I C
E Structural epitope matching for
HLA-alloimmunized thrombocytopenic patients a
new strategy to provide more effective platelet
transfusion support? Rene J. Duquesnoy
Transfusion, 48 221-227, February 2008
77
Alloimmunization-Induced Refractoriness to Random
Donor Platelet Transfusions

• Antibody reactivity with
• HLA Class I antigens
• Platelet-specific antigens
• Other antigens (MICA?, HLA class II?, blood
  groups)
• Refractory patients
• Hematological defects (e.g. aplastic anemia)
• Malignancies (e.g. leukemia)
• Highly sensitized liver transplant candidates

78
Alloimmunization-Induced Refractoriness to Random
Donor Platelet Transfusions

• Antibody reactivity with
• HLA Class I antigens
• Platelet-specific antigens
• Other antigens (MICA?, HLA class II?, blood
  groups)
• Refractory patients
• Hematological defects (e.g. aplastic anemia)
• Malignancies (e.g. leukemia)
• Highly sensitized liver transplant candidates

79
Treatment of HLA Alloimmunization-Induced
Refractoriness

• Platelet transfusions from HLA matched donors
  (Yankee et al. N Eng J Med 2811208, 1969)
• Platelet transfusions from donors mismatched for
  cross-reacting HLA antigens (Duquesnoy et al.
  Amer J Hematol 2 219, 1977)

80
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81
Serological Cross-Reactivity Between HLA Antigens

• HLA antigens carry private and public
  epitopes
• Cross-Reacting Groups (CREGs) of HLA antigens
  share the same public epitope
• Rodey and Fuller Crit Rev Immunol 7229, 1987

82
Duquesnoy et al. American Journal of Hematology.
2219-226, 1977
83
Duquesnoy et al. American Journal of Hematology.
2219-226, 1977
84
Responses to Platelet Transfusions with Different
HLA Match Grades
Duquesnoy et al. American Journal of Hematology.
2219-226, 1977
85
Limitations of the Cross-Reactivity Based HLA
Match Grade System

• Many BX matches are unacceptable epitope
  mismatches
• A and BU match groups may have incompatible
  epitopes revealed by 4-digit DNA typing
• CREG matching considers only HLA-A and HLA-B how
  important is HLA-C?

86
Many BX Matches Have Incompatible Epitopes

• Cross-Reacting Matches and Bw4/Bw6 mismatches
• Eplet differences between Cross-Reacting Antigens

Effect of BW4/6 incompatibility on responses to
BX matched platelet transfusions Patients 1-8
lower increments Patients 9-21 comparable
increments
McElligott et al Blood 59 971, 1982
87
Eplet Differences Between Cross-Reacting
Antigens Example A2 CREG
88
Limitations of the Cross-Reactivity Based HLA
Match Grade System

• Many BX matches are unacceptable epitope
  mismatches
• A and BU match groups may have incompatible
  epitopes revealed by 4-digit DNA typing
• CREG matching considers only HLA-A and HLA-B how
  important is HLA-C?

89
Eplet Differences Between 4-Digit Alleles
90
Limitations of the Cross-Reactivity Based HLA
Match Grade System

• Many BX matches are unacceptable epitope
  mismatches
• A and BU match groups may have incompatible
  epitopes revealed by 4-digit DNA typing
• CREG matching considers only HLA-A and HLA-B.
  How important is HLA-C?
• Not Important (Duquesnoy et al Transplant. Proc.
  9 1827, 1977)
• Low expression on platelets (Mueller-Eckhart et
  al Tissue Antigens 16 91, 1980)
• Important for some patients (Saito et al
  Transfusion 42 302, 2002)

91
HLA-C Eplets in Positions 1-193
92
Example of Luminex Screen for Antibodies Specific
for HLA-C (1)
What is the cut-off point to distinguish between
positive and negative reactions?
93
Example of Luminex Screen for Antibodies Specific
for HLA-C (2)
Antibody react with Cw0102 of donor Reactivity
with donors Cw0701? What HLA-C epitopes are
recognized?
Do HLAMatchmaker analysis What are the mismatched
eplets?
94
Example of Luminex Screen for Antibodies Specific
for HLA-C (3)
These are the eplets on HLA-C alleles of donor
and the panel Cw1203 has no mismatched eplets
for this patient One or more eplets on donors
Cw0102 must react with antibody Any reactivity
with 65QNR of donors Cw0701?
95
Example of Luminex Screen for Antibodies Specific
for HLA-C (4)
No reactivity with 65QNR of donors Cw0701 No
reactivity with the eplets of the negative
alleles Cw0202, Cw0401, Cw0501, Cw1502,
Cw1701 and Cw1801 Record these negative alleles
in the HLAMatchmaker program
96
Example of Luminex Screen for Antibodies Specific
for HLA-C (5)
The 77TVS eplet of donors Cw0102 is present on
all reactive alleles 77TVS is an unacceptable
mismatch
97
Example of Luminex Screen for Antibodies Specific
for HLA-C (6)
98
Proposed HLA Epitope-Based Matching Protocol
(steps 1 and 2)

• Perform HLA-A, B, C typing of patients and donors
  by DNA methods at the high-resolution (4-digit
  allele) level.
• Screen patient sera with HLA typed panel
• Complement-dependent methods direct and/or
  antiglobulin-augmented lymphocytotoxicity
• Antigen-binding assays such as Luminex, Flow
  Cytometry and ELISA preferably with single HLA
  class I alleles
• HLAMatchmaker-based analysis of serum reactivity
  pattern to identify acceptable mismatches

99
Proposed HLA Epitope-Based Matching Protocol
(step 3)

• 3. Conduct a platelet donor search
• Establish a computerized platelet donor registry
  that incorporates an HLAMatchmaker-based search
  engine
• Enter the HLA type of the patient and the
  non-reactive mismatched alleles in this database
  and the computer will generate a list of donors
  with matches and acceptable mismatches at the
  eplet level
• No need for platelet cross-match testing for HLA
  incompatibility

100
Proposed HLA Epitope-Based Matching Protocol
(step 4)

• 4. Evaluate the outcome of the platelet
  transfusion, if increment is low then
• Determine whether serum reactivity patterns have
  improperly been interpreted in terms of HLA
  mismatch acceptability
• Look for antibodies against platelet-specific
  antigens and blood groups, or autoimmune
  phenomena and drug reactions
• Consider clinical conditions such as
  coagulopathy, infection and hepatosplenomegaly

101
Prevention or Delay of HLA Alloimmunization

• HLAMatchmaker-based selection of apheresis
  platelets with minimal numbers of mismatched
  eplets
• From existing inventories of stored platelets
• Do a computer search for compatible platelet
  donor
• Avoid immunogenic eplets
• Leukoreduction of platelet preparations prior to
  transfusion

102
Prevention or Delay of HLA Alloimmunization

• HLAMatchmaker-based selection of apheresis
  platelets with minimal numbers of mismatched
  eplets
• From existing inventories of stored platelets
• Do a computer search for compatible platelet
  donor
• Avoid immunogenic eplets
• Leukoreduction of platelet preparations prior to
  transfusion

103
Prevention or Delay of HLA Alloimmunization

• HLAMatchmaker-based selection of apheresis
  platelets with minimal numbers of mismatched
  eplets
• From existing inventories of stored platelets
• Do a computer search for compatible platelet
  donor
• Avoid immunogenic eplets
• Leukoreduction of platelet preparations prior to
  transfusion

104
Conclusions

• The serological cross-reacting antigen matching
  system introduced in 1977 should be replaced by a
  system that incorporates modern concepts of
  epitope reactivity with antibody.
• The proposed HLA epitope matching protocol is
  expected to benefit platelet transfusion outcome
  and increase the number of compatible donors for
  refractory patients.

105
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106
If you would like to discuss HLAMatchmaker in
Pittsburgh, come visit me in my office
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on the Allegheny River