Significance of HLA in Transfusion and

Transplantation

Terry Aly

Clinical Pathology Conference

February 8, 2008

Main Topics• HLA System Overview• HLA and Transfusion

– Alloimmunization• Refractoriness to platelets• Graft rejection

– Transfusion-Associated Graft-Versus-Host Disease (TA-GVHD)

– Transfusion-Related Acute Lung Injury (TRALI)

• HLA and Transplantation– Bone marrow– Cord blood– Solid organ

The Human Leukocyte Antigen (HLA) System

• Essential to immune function: HLA molecules present peptide antigens to the immune system (T-cells)

• Important for self versus non-self distinction

HLA Class I Monitors Inside of the Cell

CN

CR

Tapasin

Dr. Brian Freed

HLA Class II Monitors Outside of Cell

DM monitors peptide

specificity for DR

Peptides

Extra-cellular Proteins

DMDr. Brian Freed

32

•Binds 8-10mers•Expressed on mostNucleated cells

•Presents CytosolicProteins to CD8+ T cells

•Binds 13-25mers•Expressed on APCs,Macs, B cells, activatedT cells

•Presents VesicularProteins to CD4+ T cells

Class I Class II

HLA Class I and II Molecules Have a Distinct Structure and Function

BDC

Antigen

APCAPC

Endocytosis

T Cell Receptor

Peptide

MHC II

CD4+

T cellCD4CD4++

T cellT cell

T cell Recognition of Antigen on an APC

Teaching slides: www.barbaradaviscenter.com

Antigen–presenting cells (APCs): monocytes, macrophages, dendritic cells, B cells

HumoralVersusCellularImmune

Response

Class I (1.1 Mb)

Class II (2.2 Mb)

Class III (0.7 Mb) Complement & cytokines

HLA-DQB1*0302HLA-DRB1*0401

HLA-DRB1*0401Allele:

Haplotype:

Genotype:

J. Noble

Gene low high resolution typing

“subtype”=01

HLA-DQB1*0302

HLA-DRB1*0301 DRB1*02

HLA-DRB1*04

HLA-DQB1*0201

Why care about HLA type for transfusions?

• Alloimmunization with increased risk for platelet refractoriness and humoral transplant rejection– Recipient can make antibody against donor HLA

antigen, most common

• Transfusion-Related Acute Lung Injury– donor HLA antibodies react against recipient antigens

• Transfusion-Associated Graft-Vs-Host Disease

Alloimmunization• Alloimmunization: Development of

antibodies after exposure to non-self human antigen – HLA on leukocytes or platelet specific antigens– May occur with transfusions (PRBC>platelet

because PRBC contain more leukocytes), pregnancies, or transplants

Alloimmunization

Refractoriness to platelets*

Humoraltransplant rejection

Antigen exposure associated with leukocytes in transfusion, pregnancy, or transplantation

ResolvesSpontaneously(Transfusion Ab resolve

more than pregnancy Ab)

*Antibodies can react with Class I HLA (most common), ABH antigens, or platelet antigens that exist on surface of platelets

How to prevent alloimmunization?Trial to Reduce Alloimmunization to Platelets (TRAP) study

• Leukoreduction (LR) effect– Removes 99.9% of leukocytes, leaving < 5*106 per unit

– Only 19% of AML patients receiving leukoreduced platelets developed lymphocytotoxic antibodies vs. 45%* receiving unmodified platelets

*alloimmunization rate likely higher: current methods detect more alloantibodies than previously

The Trial to Reduce Alloimmunization to Platelets Study Group. N Engl J Med. 1997.

How to prevent alloimmunization?

• UV irradiation was equally effective in TRAP study (prevents lymphocytes from proliferating or stimulating), but blood banks typically use Gamma instead of UV b/c can penetrate plastic bags

• LR or irradiation decreased alloimmunization from 13% to 3-4% in previously unexposed patients

The Trial to Reduce Alloimmunization to Platelets Study Group. N Engl J Med. 1997.

16%10%8%

Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. The Trial to Reduce Alloimmunization to Platelets Study Group. N Engl J Med. 1997.

UVB-PC: UV B-irradiated, pooled platelet concentrates, random donorsF-PC: Filtered pooled platelet concentrates, random donors F-AP: Filtered apheresis, single random donor

Who needs alloimmunization prevention?

• Give LR or irradiated blood products to patients who are:– chronically transfused (20-70% become

refractory)– immunoincompetent– pre- and post-transplant

• All transplant patients? Include kidney?• Time-frame?

– everyone??

Prestorage Universal Leukoreduction in Canada

• Universal prestorage leukoreduction (ULR) of red cell and platelet products has been performed in Canada since August 1999 (and UK too)

Seftel MD, Growe GH, Petraszko T, et al. Universal prestorage leukoreduction in Canada decreases platelet alloimmunization and refractoriness. Blood. 2004 Jan 1;103(1):333-9.

Prestorage Universal Leukoreduction (ULR)—Canada study

• Retrospective analysis of 13,902 platelet transfusions in 617 patients undergoing chemotherapy for ALL or stem cell transplantation before (n=315) and after (n=302) ULR instituted

• Alloimmunization was significantly reduced (19% to 7%, P <.001) in the post-ULR group.

• Alloimmune platelet refractoriness was similarly reduced (14% to 4%, P <.001).

Prestorage Universal Leukoreduction (ULR)—UK study

• ULR reduced TA-GVHD rate in immunocompetent individuals from 13 prior to ULR to 0 after ULR

• ULR reduced post-transfusion purpura rate from 10.3 per year to 2.3 per year

• Alloimmunization was significantly reduced (19% to 7%, P <.001) in the post-ULR group.

• Alloimmune platelet refractoriness was similarly reduced (14% to 4%, P <.001).

Refractoriness

• Repeated failure to achieve the expected increment in platelet count after 2 or more platelet transfusions

• % Maximum increment = ([pretransfusion count – posttransfusion count in platelets/µL] X blood volume in mL) ÷ (number of platelets transfused, ie, number of U X 6 X 105)

Slichter SJ. Evidence-Based Platelet Transfusion Guidelines Hematology 2007

Refractoriness

• Can be due to:• Alloimmunization to HLA or platelet antigen• ABO mismatch• Non-immune factors such as insufficient

dosing, poor platelet quality, hypersplenism, accelerated consumption, sepsis, DIC, GVHD, and bleeding

Slichter SJ. Evidence-Based Platelet Transfusion Guidelines Hematology 2007

Management of Refractoriness

• Stage 1: Check dosing • Stage II: Try ABO matched platelets <48 hours

old• Stage III: If urgent, give cross-matched platelets.

Screen for anti-HLA and anti-platelet antibodies– Antibody-mediated or not? Solid-phase assay– HLA antibody mediated or not? Panel reactive antibody

(PRA) as general screen for HLA Ab positivity.– If positive, single antigen testing with microparticle

beads to ID which antigen(s) to avoid in donors

Slichter SJ. Evidence-Based Platelet Transfusion Guidelines Hematology 2007

Table 1   --  Refractoriness and alloimmunization rates after transfusing ABO matched versus mismatched platelets

            New antibodies

Platelet transfusions Enrolled Female patients Possible prior sensitization[a] Platelet transfusions[b] Platelet refractoriness[c] Anti A/B[d] Anti-HLA Platelet-specific

ABO matched 13 10 (77%) 9 (69%) 7 (5–9) 1 (8%) 0 1 (8%) 1 (8%)

ABO mismatched 13 2 (15%) 4 (31%) 9 (4–30) 9 (69%) 7 (54%) 5 (38%) 4 (31%)

P value         0.001      

Data from Carr R, Hutton JL, Jenkins JA, et al. Transfusion of ABO-mismatched platelets leads to early platelet refractoriness. Br J Haematol 1990;75:408–13

ABO matching prevents platelet refractoriness

Problems with HLA Typing and Matching

• HLA matching requires the availability of large numbers of HLA-typed donors. A registry of about 18 000–25 000 HLA-typed people is needed to provide at least five HLA-A and HLA-B matched donors for 80% of white patients.

• HLA-matched platelets requires the recruitment of specific donors, such platelets can only be obtained by apheresis

• Expensive

K Takahashi, T Juji and H Miyazaki, Determination of an appropriate size of unrelated donor pool to be registered for HLA-matched platelet transfusion, Transfusion, 1987.

Problem with cross-matching to find compatible platelets

• Limited to units available for testing

• 5 day life to platelets, future transfusions will require re-crossmatching

Antibody Specificity Prediction

The antibody specificity prediction (ASP) method of donor selection for refractory alloimmunized patients

appears as effective as HLA matching or crossmatching. Far more donors are identified in a

file of HLA-typed donors by the ASP method than by HLA matching, and this indicates that the ASP

method provides important advantages regarding the availability of compatible platelet components.

Percentage of platelet recovery is similar for HLA-matched, cross-matched, and antibody specificity prediction method

matched donor platelets

Petz LD. Selecting donors of platelets for refractory patients on the basis of HLA antibody specificity. Transfusion. 2000.

PRA=panel reactive antibodies

Cross-Reactive Groups

• Systems have been developed to match donor and recipient by assigning HLA-A and HLA-B antigens with shared public epitopes to clusters called cross-reactive groups (CREGs).

• When one or two mismatches of HLA-A or HLA-B antigens in CREGs is permitted, a pool of 1000–3000 donors will meet the transfusion needs of most white patients

HLA Matchmaker

• Based on the principle that short three-amino acid sequences characterize polymorphic sites of the HLA molecules, and are the critical components of allo-sensitizing epitopes

• Retrospective study has shown that platelets selected with this algorithm result in higher post-transfusion count rises than those selected using traditional HLA matching strategies

A Nambiar, RJ Duquesnoy and S Adams et al., HLAMatchmaker-driven analysis of responses to HLA-typed platelet transfusions in alloimmunized thrombocytopenic patients, Blood 107 (2006), pp. 1680–1687.

Clinimmune

• Testing available for:

• Clinical Transplantation- solid organ and hematopoietic stem cell

• Platelet Transfusion support

Clinimmune• HLA-A, B, Cw, DRB, DQB1, DPB1 loci

– Low to high resolution testing for HLA-A, B, Cw, DRB1,DRB3/4/5, DQB1

– Intermediate to high resolution testing for HLA-DPB1 – Serological and molecular testing

• HLA Antibody Detection – PRA=Panel Reactive Antibody, including HLA Specificity

Analysis • Luminex • ELISA • Complement Dependent Cytotoxicity, including AHG (anti-human

globulin) – Titration/Quantitation studies for pre-transplant immunotherapy

protocols – Crossmatching for transplant compatibility

• Flow cytometry • Complement Dependent Cytotoxicity, including AHG (anti-human

globulin)

From Clinimmune website

Other options to treat refractoriness?

• For highly alloimmunized patients and those with rare HLA types, finding compatible platelets can be difficult.

• Several immune-modulatory therapies tried: intravenous immune globulin; cyclosporin A; vinblastine; staphyloccal protein A; removal of HLA antigens with citric acid.

• Despite anecdotal positive outcomes, these strategies are usually not successful or practical

Transfusion-associated graft-vs-host disease

• Depends on – immune system function in recipient – lymphocytes in transfused component– degree of HLA similarity between donor and

recipient• Donor lymphocytes not recognized as foreign if

HLA similar• More common with related donors

– Especially if donors homozygous for shared HLA haplotype with recipient

Transfusion-associated graft-vs-host disease

• Caused by persistent chimerism after blood transfusion– Transfused lymphocytes avoid destruction by host

immune system, proliferate, and attack host organs– Causes skin rash (histo “satellite dyskeratosis”), fever,

liver dysfunction, diarrhea, bone marrow dysplasia– Occurs 4-30 days post-transfusion, >90% fatal– In 1980, incidence in patients with hematologic cancers

or lymphoproliferative disorders 0.1-1%, now lower due to leukoreduction and irradiation; more rare if not immunocompromised

www.up-to-date.com/TA-GVHD

TA-GVHD more common in immunocompromised patients receiving directed donor HLA-matched products,

but can occur in immunocompetent individuals and can be random donor

with high HLA similiarity

For example……

• Common “ancestral” haplotype–DR3, B8, A1 haplotype–In 9% of chromosomes in the

Caucasian population, and 18% of chromosomes in Caucasians with diabetes (Alper 2006)

–Diabetic population at risk for kidney transplantation

DQB1

HLA-A

HLA-F

25.2 Mb

A B A B B B A B B B A A A B A B A B A B B B A B A B A B A B B B A B A B A A A B B B B B A A B BA A B B A A A A A A A A - - A B A A A B A B A A A B A B A A B B B B A A A A A B A A A A A B A AA B B B B B B B B B B B - - B B B B B B B B B B A B B B B B B B A B B B B B B B B B B B A B B BA B A A A B A B A B A B A B A A A B A A A B A A B B A A A B A A A B A B A A A A A B A B A B A BA B A A B B A B B B B B A B A B A A A A A A A B B B B B A B A A A B A B B B B B B B A B A B A BB B B B B B B B B B B B B B B B A B B B B B A B A B A B A A B B A B B B B B B B B B B B A B A BA B A A A B A B B B B B B B B B B B B B A A B B A A A B A B A B B B A A A A A A A B B B B B A BA B B B A B B B A B B B A B B B A A A B B B A A A B B B A B B B B B A B A B B B A B A B A B A BB B B B A B A A A B - - B B B B A B B B B B A B A B A B A A B B B B A B B B B B B B A B B B A BA B B B A B B B A B A A A A A B B B B B B B A B A B A A B B B B A B B B B B B B A A A B B B A BB B A B B B A B B B - - B B B B B B B B A B A B B B A B A B B B B B A B B B A B B B B B A B A BB B A B A A A B A A A A A A A B A B A A A B A A A B A A A B B B A B A B B B A B A A A A A B A AA A A A A A A B A A B B A A A A A A A B A A A A A A A A A A A A A B A A A A A A A A A B A A A BA A A B A B A A A A A A A B A B A A A B A A A A A A A B A A A A A A A B A A A B A A A B A A A AB B A A A B A A A A A A - - A B A A A A A A A B A A A B A A A B A B A A A B B B A B A A A A A BA B - - A B A A A A - - - - B B B B B B A B A B B B A B A B A B A B A B A B A B B B A B A B B BA B A B A A B B A B A B - - A A B B B B B B A B B B B B B B A A A A A B A A A B A B A A B B A BB B A B B B A A A A A A A B B B A A B B B B B B A B B B A B B B A B A B A B A B B B A B A B B BA A A B A A A A B B A B B B A B A A A A A A A B A A A A A B A A A B A B B B - - A B A B A A A AA A - - A A A A A A A A A A A B A A A A A A A B A A A A A A A A A A A B A A A A A A A A A A A AA A B B B B B B B B B B B B A B B B B B B B B B A B B B B B B B B B B B B B B B B B B B B B B BA A A A B B B B A B A B A B A A A B A B A A A B A B A B A B A A B B A A A B A B A B A B A B A BA A B B B B B B A B A B B B A B A B A B A A A B A B A B A B A B B B A B B B A B B B A B B B B BA A B B B B A B A A A B - - A B A B A B A A A B - - A B A B A A A B A B A A A A A B A B A B A BA A A A A A A A A A A A A A A A A B A B A A A A A A A B A A A B A A A B A B A B A A A B A B A AB B A A A A A A A B A A A A A B A B A B A A A B A B A B A A A B A A A B A B A B A A A B A B A BB B B B A B B B B B B B B B B B A B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B BB B B B B B A B A A - - B B B B B B B B B B B B A B B B B B B B A B B B B B B B B B B B B B B BA A A A A A A A A A A A A A A A A A A A A A A A A A A B A B A B A A A A A A A A A A A A A A A AA B A B B B B B A B - - - - A B A B B B B B A B B B B B B B B B A B A B A B A A A B A B B B B BB B - - B B B B B B A B B B A B A B A B A B A B A B B B - - A B B B A B B B B B B B B B B B A BA A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A AA A - - A B A B A B A B A A B B A A A B B B A B B B A B B B B B A B A B B B A B A B A A A A B BA B - - A B B B B B A B A B B B A B A B A B A B A B B B A A B B A B B B B B A B A B A B B B A BB B B B B B B B B B B B B B B B B B B B B B A A A B A B B B B B A B A B B B B B B B B B A B B BA B - - A A A B A B - - - - A B A B A A A B A A A B A B A A A B A B B B A A A B A B A A A B A A

24 DR3B8A1 (8.1) Homozygous IndividualsColumn=individual; Row=SNP; Yellow=Homozygous region for 8.1 allelesGray=non-8.1 allele; White=8.1 allele; Dark Gray Bar=1st SNP homozygous for non-8.1 alleles

32.8 Mb

7.6 Mbconserved

telomere

Illumina 2:

My thesis work

Case study• 31 yo F receives kidney transplant from her father,

matched for all but one antigen, DR7• 2 wks later, she needs PRBC transfusion

– Family wants her to receive directed donor transfusion from father

• Should a directed donor transfusion from her father be permitted?

• If so, should it be leukoreduced or irradiated?• If not, should a random donor blood product be leukoreduced

or irradiated?• Should the guidelines for kidney transplant patients differ from

those of heart, lung or bone marrow transplant patients?• What if patient is known to have one HLA antibody formed

against transplant?

Follow-up Information

• Received directed donor routine PRBC given from father (not leukoreduced or irradiated)

• Humoral transplant rejection diagnosed

• PRA antibody negative prior to transfusion; DR7 antibody detected post-transfusion

HLA and TransplantationBone marrow

• HLA class I and II matching High resolution

• Aim for at least 9/10 HLA alleles matching

HLA and TransplantationCord Blood

• Low resolution HLA class I match; high-resolution class II HLA match

• Safer than bone marrow transplant because cord blood induces less reaction

• Aim for at least 4 out of 6 matching for transplantation

HLA and TransplantationSolid organ

• Lung: Class II HLA match• Heart: Class I HLA match on case-by-case basis,

no class I• Liver: No HLA typing indicated• Kidney: Class I and II HLA matching but O.K if

only 1 or 2 out of 6 matching HLA alleles because prognosis depends more on timing of transplant; prefer negative cross-matching strategy