Trojan Antibodies and Transplant Malware: Improbable Sensitizations

1
Trojan Antibodies and Transplant Malware
Improbable Sensitizations Hemolytic
Passenger Lymphocyte Syndromes 
Canadian Blood Services Transfusion Medicine
Resident Topic Teaching Session
Tuesday, October 5th 2010, 1200 100
pm Conference Dial-In 1-866-752-7690, Passcode
1775282 www.transfusionmedicine.ca

• Christine Cserti-Gazdewich, MD FRCPC
• Assistant Professor, University of Toronto
• Departments of Laboratory Hematology (Pathology)
  Clinical Hematology (Medicine)
• Blood Transfusion Laboratory, University Health
  Network
• Toronto, Ontario, Canada
• office 416-340-5390, pager 416-790-9597, email
  Christine.Cserti_at_uhn.on.ca

NO CONFLICTS OF INTEREST. FUNDING FOR RESEARCH
FROM PUBLIC FOUNDATIONS ONLY.
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Princess Margaret Hospital
Ontario Regional Blood Coordinators
Toronto General Hospital
Toronto Western Hospital
University Health Network
provincial blood management
Holland Ortho Arthritic Centre
Sunnybrook Hospital
Womens College Hospital
Canadian Blood Services
Sunnybrook Health Sciences Centre
national blood provider
St Michaels Hospital
Ontario Transfusion Coordinators
Hospital for Sick Children
provincial blood conservation
Mount Sinai Hospital
Trillium Gift of Life Network
TRANSFUSION MEDICINE COLLABORATIVE
provincial organ network
University of Toronto teaching hospitals
Hospital Transfusion Services
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Goals

• Re-orient to sensitization biology in transfusion
  medicine
• Appreciate what is known about hemolytic
  passenger lymphocyte syndromes (PLS) in
  transplantation
• Note some twists in ABO-incompatible stem cell
  transplantation
• Consider new insights into accommodation

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It All Starts With This One Tube
crossmatch sample
group reserve
blood group
group screen
D.A.T.
plasma
cross type
type and cross
indirect Coomb's red cell antibody screen
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ABO Types in the General Population

• A donor plasma
• anti-B IgM

• O donor plasma
• anti-A,B IgG
• anti-A anti-B IgG IgM

• AB donor plasma
• no anti-A or anti-B

• B donor plasma
• anti-A IgM

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30 blood group antigen systems (ABO, Rh, )
containing structures that are naturally
polymorphic (278 antigens 38 in
collections/series)
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Who Are The Antibody-Makers Anyway?

• anyone whose immune system has seen a foreign red
  cell at any other time in their lives
• the parous (85 of child-bearing age females)
• feto-maternal hemorrhage (FMH), especially at
  parturition
• any transfusion history
• up to 1-3 of the healthy donor population
• up to 1/3rd of those with a hospital admission
  history
• any tissue grafts or transplants (solid organs,
  hematopoietic progenitor stem cells),
  inevitably contaminated with passenger
  erythrocytes

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Antibody Makers

• 13 of people are antibody makers
• difference between -makers and -others appears,
  at least in part, to be absence vs integrity of
  T-reg-suppressive activity

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Guy (Gal) Walks In The Door What Are The
Chances?
INTRINSIC SEROCONVERSION RISK
HOW RECENT HOW DURABLE THE ANTIBODY IS
EXPOSURE
X
X
1 5 of the population has a positive red cell
antibody screen
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Targets for Graft versus Host Attack

• HLA Antigens (Platelets, Tissues) ie- GVHD
• Red Cell Antigens ie- Passenger Lymphocyte
  Syndrome (PLS)

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Diversity (Polymorphisms) Set Up The Tissue
Incompatibility Fight
extensive variations therein
ALLOIMMUNIZATION
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Diversity (Polymorphisms) Set Up The RBC
Incompatibility Fight
Kell K vs k, others
ABO
Duffy Fya vs Fyb
Rh(D)
Kidd Jka vs Jkb
subtle (eg SNP) variations therein
complete absence or presence
etc MNS, P, Lutheran, Lewis, Diego, Scianna,
Dombrock, Colton, LW, Chido/Rodgers, Gerbich,
Cromer, Knops, Indian
other Rh(CE) antigens C vs c E vs e
ISOIMMUNIZATION
ALLOIMMUNIZATION
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The Vocabulary of Immune Violence

• MAJOR vs MINOR incompatibility
• ACTIVE vs PASSIVE immunity

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Directions of Incompatibility

• MAJOR
• incoming donors
• cellular antigens are the incompatible target
• host-source immunity mounts the attack
• PRODUCT REJECTION
• by a fighting recipient
• eg. ABO incompatible RBC mistransfusion
• A into O donor A cells destroyed by host O
  plasma
• eg. acute rejection of organs

• MINOR
• donor-source immunity mounts the attack
• hosts cellular antigens are the incompatible
  target
• HOST REJECTION
• by a fighting donor
• eg. GVHD from a solid organ transplant, a
  transfusion, or a BMT
• eg. TRALI

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Active vs Passive Immunity

• ACTIVE
• renewable humoral (via cellular) fighting power

• PASSIVE
• finite (humorally contained) fighting power

n ? 8
n x
most forms of MINOR incompatibility
all forms of MAJOR incompatibility
some forms of MINOR incompatibility
passenger donor plasma cell
recipient plasma cell
graft
product
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GVHD What does it take?

• living passenger lymphocytes
• proliferative stimulus something recognizably
  different in the host to attack
• invulnerability some way for the passengers to
  get away with it
• host too immune suppressed
• host immune oblivious (eg. homozygous
  haploidentical donor indistinguishable from self)

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Passenger Lymphocyte Syndromes
passenger donor plasma cell
graft
host RBC

• minor incompatibility scenario of donor-origin
  (graft-associated) alloimmune hemolytic anemia
• of host RBCS

aka. HUMORAL GRAFT VERSUS HOST DISEASE FOR THE
BLOOD BANK
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PLS The Implicated RBC Antigen Spectrum (Usual
Victims)
555-fold

• ABO gt Rh gt minor RBC antigens
• probability of minor discrepancy
• 25 for ABO vs lt15 for Rh
• presence of preformed Ab or primary historical
  sensitizations
• 100 for ABO vs 0.3 for Rh(D)
• Rh usually from donors who have been
  alloimmunized previously
• prior donor alloimmunization almost necessary

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First Reports of Anti-RBC PLS

• 1964 1st hemolytic ABO PLS Marchioro, NYAS
• 1985 1st hemolytic anti-D PLS Swanson,
  Transfusion
• 1990 1st multiorgan (L, K) PLS Ramsey, aaBB

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PLS Lymphoid Load Spectrum (Usual
Aggressors)
(heart-) lung
kidney
HPC PB gt BM
small bowel, pancreas
liver

• dead or alive
• graft (usually) unaffected
• at least 10 6 10 7

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Timing For Serologically Expressive Chimerism

• median onset 9 days (5 17)
• median duration 4.5 weeks (2 23)
• undetectable by 3 months
• the records
• fastest onset 12 h
• most delayed onset 45 d
• longest persistence 2 years for Ab (2 mo for
  HA)

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The Role of Incidental HLA Matching (graft
stealth by tolerance of inadvertently
HLA-similar passengers)

• borne out in TA- SOT- GVHD literature
• anti-RBC PLS may also be more likely with
  inadvertent HLA matching

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Things That May Make it Worse

• density of vulnerable antigen
• group A recipients
• use of non-B-cell suppressive IST
• cyclosporine
• pro-hemolytic complement-fixing capabilities of
  antibody
• inflammation / cytokines
• TNF-a IL-1 (conditioning or ischemic injuries)
• co-stimulatory interactions CD40 - CD154, CD28 -
  B7
• IL-2 RaCD25-dependent clonal expansion with Th1
  CTL response
• overly receptive RES/MPS

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Things That May Make it Better (or
Preventatively Subdue it?)

• post- (or pre-?) transplant graft irradiation
• reducing immunosuppression de-repression
  revenge
• evolution towards non-hemolytic antibody
  properties
• promotion of activity of inhibitory FcR
• non-hemolytic red cell antigen loss
• senescence of passenger lymphocytes
• Ag-Ab excesses
• ? negative feedback to memory B cells

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Transfusion Care in PLS

• issuing antigen-negative blood, compatible with
  respect to graft-origin antibodies, and not just
  the same blood type as the host
• failure to do so Incorrect Blood Component
  Transfused (IBCT) medical error event /- ABO
  mis-transfusion
• top-up transfusions or therapeutic red cell
  exchange (TREX)

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Is There a Role for Apheresis?
Red cell replacement of removed plasma (hybrid
TPRE)
Whole blood removal eg. manual red cell
exchange, replacing with pRBC
plasma/NS/albumin (reconstituted whole blood)
Therapeutic red cell exchange (TREx)
Top-up transfusion
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When Sh/(C)ould Therapeutic Plasma Exchange Be
Offered?

• removal of pathologic antibody
• IgM gt IgG
• high titer gt low titer
• detox of RBC breakdown products
• for extreme cases with intravascular hemolysis
• plasma free hemoglobin ? danger thresholds ?
• massive acute hemolysis (MAH)
• free plasma hemoglobinemia gt 60 µmol/L
• gt 387 mg/dL (N 0.5 5 mg/dL)
• stromal factors DIC

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Other Management Options

• rituximab
• graft irradiation
• splenectomy
• cytotoxic agents, prednisone adjustments
• combination therapy RBC/TREX, IST, apheresis,
  splenectomy

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A Passenger Lymphocyte Traceback Story

• Why does my D patient now have anti-D after
  transplant?
• -passive sensitization?
• -partial D with alloimmunization?
• -active (passenger lymphocyte-mediated)
  sensitization?

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case 2
case I
case 3
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Case 1 The good news ABO compatibility
Donor A Negative
Recipient A Positive
The bad news the seeming Rh(D)-compatibility
isnt
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Case 1

• 66F presented for a R SLT for COPD
• group screen pre-op
• A, Rh(D)
• screen negative amid a history of A transfusions
• group screen POD17 in the pre-transfusion
  investigations of her new hemolytic anemia
• A, Rh(D)
• screen anti-D anti-C
• DAT anti-D anti-C

D
e
C
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Case 1 Traceback of Donor

• elderly multiparous female
• group A, Rh(D)-negative (rr)
• screen

Anti-D
Anti-C
Anti-E
D
C
E
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RECIPIENT
Serologically C D e c- E-
D
e
Weiner designation R1 R1 (17 of population)
C
Anti-C
Anti-D
Anti-E
D
E
C
DONOR
e
Serologically c e C- D- E-
c
passenger lymphocyte
Weiner designation r r (15 of population)
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therapeutic plasma exchange
36 - 40
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Case 1 Indirect Attributable Mortality Due to
PLS

• POD 21, Hb 6.0 g/dL
• massive AMI (demand-ischemic with rAF)
• grade IV EF
• left with refractory CHF
• cardiac dysrhythmiae fluid imbalances
• died day 330 post transplant
• 10 months later
• 113 days after last detectable anti-D

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Case 2 Recipient of the Contralateral Donors
Lung

• 43 y old female presented for L SLT 9/28/07
• A, R1 r , negative pre-transplant screen
• lifelong transfusion-free history
• screened monthly due to Case 1 patient

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Case 2 Novel Serological Course
serologically positive
120
159
acute rejection
TPE
173
259
266
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Case 3 It happened again!
RECIPIENT 3 ?
DONOR 2 ?
anti-C anti-D anti-V
O, R1 R1 ( CC DD ee )
O, r r ( cc dd ee )
anti-C 0 ? anti-D

• 30 year old male presented for DLT for CF
• RBC antibody screen negative pre-operatively
• RBC antibody screen POD 23
• anti-D on IAT
• anti-D anti-C on eluate of DAT
• no hemolysis
• ongoing persistence at follow-ups (gt 6 mo)

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Why Did anti-D PLS Happen So Often In Our Series?

• expected
• (13 risk of D- to D) x (2 risk of anti-D) x
  (100 transfer) 0.3
• observed
• 3/92 or 3 95 CI 1 9
• 100 transfer did indeed occur based on lookback
• 3 donors were found to be D-sensitized
• of a denominator of 12 D- donors, rate was 25
  6 57, 95 CI
• (gt12x higher than expected )

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Update 10 Sequential Cases at the UHN Over 3.87
Years of Surveillance (q 25 wk)
Implicated organ Donor antibodies Cognate recipient attack Significant hemolysis?
Lung Anti- C, D, E C, D yes
Lung Anti- C, D, E D no
Lung Anti- C, D, V C, D no
Liver Anti- B B yes
Lung Anti- B, Jka, N B, Jka, N yes
Liver Anti- D D yes (day 798)
Liver Anti- B B yes
Liver Anti- A A yes
Liver Anti- A (IgM) A yes
Lung Anti-A A yes
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The Only Prospective Post-Solid Organ Tranplant
Sensitization Surveillance Study in the
Literature
aaBB Abstracts 2007

• 27 of the ABOi KTs developed DAT
• checked q 10d x 3 checks post-operatively

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Types Of HPCT ABO Incompatibility
R D Type
O O O A B A B AB AB AB Major
A B AB AB AB O O O A B Minor
A B B A Both
O A B AB O A B AB None
75 random combinations (assuming equal
prevalence of types) are incompatible (12/16)
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Major Incompatibility 5-ways non-O into O, or
AB into A or B
recipient
donor
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Minor Incompatibility 5-waysO into non-O, or A
or B into AB
donor
recipient
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All Possible Players(or the Worst Case
Scenario) Bidirectional Incompatibility 2-ways
A to B or B to A
donor
recipient
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Donor Distributions By Recipient
R D Type Ptype specific
O O O O A B AB O Major Major Major None 55 major 45 identical
A A A A A O B AB None Minor Both Major 40 compatible 45 minor 11 bidirectional 4 major
B B B B B O A AB None Minor Both Major 11 compatible 45 minor 40 bidirectional 4 major
AB AB AB AB AB O A B None Minor Minor Minor 4 compatible 96 minor

• PROBABILITIES
• given
• O 0.45
• A 0.40
• B 0.11
• AB 0.04

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Probability of HPCT ABO Incompatibility Among
MUDs
Recipient Major Minor Bidirectional Compatible
O 24.75 n/a n/a 18.00
A 1.60 18.00 4.4 16.00
B 0.44 4.95 4.4 1.21
AB n/a 3.84 n/a 0.16
P total 26.8 26.8 8.8 37.6
62.5
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Quick Summary of Vagaries of ABOi in HPCT

• MAJOR INCOMPATIBLE HPCT
• multi-lineage marrow aplasia/hypoplasia
• delayed (red cell) engraftment / pure red cell
  aplasia (PRCA)
• INCOMING
• DONOR CELLS
• FOUGHT OFF
• (acute hemolysis of passive contaminant, delayed
  establishment of active production)

• MINOR INCOMPATIBLE HPCT
• delayed,
• potentially severe hemolytic anemia
• HISTORICAL
• RECIPIENT CELLS FOUGHT OFF
• (acute hemolysis of recipient cells by passive
  antibody, delayed hemolysis of recipient cells by
  active antibody)

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ABOi HPCT and Increased RBC Alloimmunization?

• Described once by de la Rubia et al in 2001, in a
  transplant cohort of 217 transplant patients, of
  whom 8 had developed non-ABO RBC antibodies
• ABO blood group incompatibility (p 0.005) and
  patient's age (p 0.02) were the only two
  variables significantly associated with the
  development of RBC alloantibodies
• Took a peak (in 2006 while at BIDMC) at 90
  consecutive transplant patients, of whom 8 had
  non-ABO RBC antibodies

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ABO-identical MUD transplants, n 6
ABO-identical MRD transplants, n 44
ABO-incompatible MUD transplants, n 12 (odds
3X higher for ABO-incompatible HPCTs among MUD vs
MRD)
MRD transplants, n 72 (4X as many MRDs as MUDs)
MUD transplants, n 18
ABO-incompatible MRD transplants, n 28
ABO-incompatible transplants, n 40 (28 MRD, 12
MUD 30)
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patients post- ABO identical HPCT, n 50 (0 0
new alloimmunizations)
patients post- ABO incompatible HPCT, n 40 (6
15 new alloimmunizations)
2/2 of historically alloimmunized patients
experienced antibody persistence
0/1 of historically alloimmunized patients
experienced antibody persistence
p0.006 (2-tailed Fishers exact test)
1
1
1
1
10 antibodies
1
1
1
3
8 alloimmunized patients
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Minor Not So Innocent, Yet Again
21
ABO minor incompatible HPC transplants
15
ABO major incompatible HPC transplants

• red cell sensitization (n8)
• 1/3rd of ABO minor incompatible HPC transplants
• 1/15th of ABO major incompatible HPC transplants

(7)
(1)
Suggests that antibody formation is not only more
frequently observed in ABO-mismatched cases, but
specifically in the minor incompatibility
scenario.
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The Discovery of Non-Hemolytic Red Cell Antigen
Loss
Y
D

• after transfusion of Ag cells to Ab host
• hemolytic clearance of DAT allogeneic cells
• non-hemolytic persistence of DAT cells
• non-hemolytic transformation of Ag-, DAT- cells

R
Y
Y
D
R
Y
D
Y
R
Y
D
R
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How Does Loss of Ag Happen Without Losing the RBC?

• no single mechanism found yet
• ? synthetic feedback suppression of antigen
• ? post-expression tear-away
• ? partial alteration of antigen
• ? benign antibody binding
• you dont need a spleen, but it wont happen
  ex-vivo
• need a liver with Fc?RIII
• need to simultaneously cross-link antibodies that
  have separate specificities for parts of the same
  target antigen

D
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A Natural Mechanism, Neglected Because of Its
Silence

• CR1 (CD35) on RBCs have been known to transfer
  immune complexes to Kupffer cells of the liver
  without undergoing hemolysis!
• CR1 also present in B cells, myeloid cells
• Unnatural clinical precedents, good and bad
• organ accommodation
• CD20 CLL cells becoming CD20- and thus escaping
  the effects of rituximab (anti-CD20)

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CR1 (CD35) Biology Expression Implications
genotype / /- -/-
phenotype high moderate low/absent
Normal range 50 1200/cell
20-fold natural variation in healthy people
Low expression lt200 / cell

• C3b/C4b complement receptor CR1 CD35, chr
  1q32 within RCA (regulators of complement
  activation) family
• On red blood cells
• Primary function limits activation of
  complement pathway
• Binds complement cleavage products C3b and C4b,
  thus acting as a cofactor to inactivate them to
  iC3b and iC4b
• Secondary function provide a removal/clearance
  function
• Immune complexes (IC) bind to C3b, which is then
  trafficked on CR1 of RBCs (and other cells) to
  the liver and spleen for removal by macrophages
• On B-cells and macrophages
• Pro-inflammatory cytokine release in response to
  immune complexes?

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Final Aphorisms

• just because something isnt major incompatible,
  it doesnt mean its compatible enough
• sometimes minor is anything but
• unexpected antibodies for a specific
  self-alloantigen, at a time when de novo antibody
  production is assumed to be stymied, tell us
  interesting stories
• knowing more about what controls the difference
  between harmful versus harmless sensitizations
  has vast implications for us in troubleshooting
  our way through transfusion medicine, transplant
  immunobiology, biologic therapy