Autoimmune hemolytic anemia (AIHA)
Sacha Zeerleder, MD PhD
Internist – hematologist
Department of Hematology Academic Medical Center Amsterdam
Department of Immunopathology, Sanquin Research, Amsterdam
Autoimmune hemolytic anemia (AIHA)
AIHA is characterized by an increased breakdown of red blood cells (RBC) due to autoantibodies (auto-Ab’s) with/without complement activation.
Pathogenesis AIHA
AIHA is characterized by an increased breakdown of red blood cells (RBC) due to autoantibodies (auto-Ab’s) with/without complement activation.
RBC antigens • Rh-related polypeptides • Molecular mimicry • Oxidative changes in RBC antigen structure?
Complement system • Altered expression of CR1 • Decreased experssion membran- boud complement regulators (CD59)
Ineffective antigen presentation • Immature DCs • Decreased co-stimulatory help by T- cells
Functional B/T cell abnormalities • Polyclonal lymphocyte activation • Alteration of cytokine profile • Dysregulation of T-Regs
AIHA
Laboratory analyses:
• Hemolysis (LDH , haptoglobine , hyperbilirubinemia)
• Positive Coombs
• (reticulocytosis)
Diagnosis AIHA
Specificity of autoantibodies:
• Rhesus antigen (50% in AHIA with warm autoantibodies)
• I/i antigen (AHIA cold antibodies)
• P-antigen (paroxsysomal cold hemoglobinuria)
But: biological activity of the autoantibody determines the clinical activity rather then than the specificity of the autoantibody
AIHA is characterized by an increased breakdown of red blood cells (RBC) due to autoantibodies (auto-Ab’s) with/without complement activation.
Isotype autoantibody:
determines complement activation and Fc gamma receptor binding
Complement activation:
• IgM: most efficient
• IgG1, IgG3: efficient
• IgG2, IgA: weak
• IgG4: no complement activation
Fc-gamma receptors
• IgG1, IgG3 FcgRI (CD64)
• IgG (low affinity) FcgRIIa
AIHA: biological activity of autoantibodies
C1r C1s
C1r C1s
AIHA: complement activation
C1r C1s
C2a C4b
C4
C2
AIHA: complement activation
C3
C3b C3b C3d
C3c
FI
C3d
C3c
AIHA: complement activation
C3b
C6 C7
C8 C5b
C5
AIHA: complement activation
Complement activation
RBC-lysis
Fc-gamma receptor
CR receptor depositie
AIHA – destruction of autologous RBCs
C3d C3c
C3d
IgG C3c C3d
positive positive
R
G
AIHA: direct Coombs (antiglobuline test)
C3d C3c
C3d
AIHA: direct Coombs (antiglobuline test)
IgG (IgA) IgM C3c C3d
positive
Cold antibodies
• mostly IgM
• binding at <30°C
Warm antibodies
• Mostly IgG
• optimal binding at 37°C (rarely IgM)
Biphasic hemolysins
• mostly IgG, binding optimal in the cold, complement activation 37°C
30°C
37°C
binding
lysis
AIHA: classification
Warm autoantibodies:
• Idiopathic (primary)
• Secondary: CLL, lymphoma, SLE
Cold autoantibodies
• Idiopathic (primary)
• Secondary: mycoplasma infection, viral infections (EBV), lymphoproliferative diseases
Paroxysmal cold hemoglobinuria
• Idiopathic (primary)
• Secondary: viral infections, syphilis
Mixed warm and cold autoantibodies
Rare
Incidence 1:80‘000
>40 years, peak >70
Very rare
>50 years
Very rare
Children
AIHA: classification
18% will develop overt
lymphoma in the
future
AHIA with warm autoantibodies
2008: Chronic lymphatic leukemia (Rai III), del17p (poor prognosis)
Treatment with chlorambucil, achievement of very good partial remission
IH: direct Coombs negative
05/2009: Non-STEMI infarction
Lab: Hb 6.9 g/dl, Lc 4.5x109/l (slightly increased lymph), Tc 230x109/l;
LDH 667 U/L, bilirubin 27 umol/l, haptoglobin <0.02 g/L
IH: - Coombs 3+; IgG 3+, IgA/M neg, C3bc neg, C3d pos, NS-WAS
- alloantibodies: anti-Jk(a)
Diagnosis?
What to do next?
Transfusion
Decrease the production of autoantibodies
• Corticosteroids
•plus immunosuppressive therapy
•Monoclonal antibodies (antiCD20)
• Danazol
inhibit the breakdown/removal of RBCs
• intravenous gammaglobulines
•splenectomy
Treatment of the underlying disease
rituximab
corticosteroids
Immuno-supressivs
splenectomy
IVIG
AIHA with warm autoantibodies - treatment
Transfusion – AHIA
Analyst calls: “everything is positive….”
Type: RBC coated with autoantibodies
Screen: patient serum reacts with test RBCs
Hence: T&S not possible (needs time!)
Is there really a need for transfusion (vital indication)?
are there clinical signs of hypoxia??
Is there time for extensive IH analyses?
Important: prevention of alloantibody formation
Patients with alloantibodies have an increased risk to develop additional alloantibodies
Transfusion can exacerbate hemolysis!
AIHA – transfusion: recommendation
• Compatible for alloantibodies (and complement-binding antibodies)
Negative for the respective antigen
• Prevention of alloantibody formation
Blood product as far as possible compatible with the recipient antigens
• Compatible for specific autoantibodies (in case of fulminant hemolysis)
• Transfusion: slow! Check vital signs as well as hemolysis parameters
laboratory procedures may take
hours..(absorption techniques etc)
Selection blood product
Select compatible for Rh (phenotype) and K
If possible: Kidd > Duffy > S > s
AIHA with WA auto-Abs– steroids + cytotoxic agents
Steroids:
2 important effects: decrease
• RBC removal (breakdown) in the spleen (decrease of the density of Fcg-receptors on MP)
• production of autoantibodies
in approx. 60-70% of the patients achieve a remission (10-15% CR), frequently a
maintaining dose of steroids is needed Gehrs et al. 2002, Pirofsky et al. 1975, Murphy et al. 1976, Zupanska et al. 1981
In case there is - no response on steroids
- prednisolon maintenance dose >15-20 mg/d
- side effects steroids
combination with cytotyoxic drugs
• Cyclophosphamide (100 mg/d vs pulse regime 50mg/kg bw over 4 days)
Murphy et al. 1976, Zupanska et al.1981, Panceri et al. 1992, Silva et al. 1994, Moyo et al 2002
• Azathioprine (100-150 mg/dag)
Pirofsky 1975, Worlledge et al. 1968
Treatment AIHA with WA-Abs– splenectomy
Mechanism:
• Reduce removal of RBCs in the spleen (+ reduction of autoantibody formation) Allgood et al. 1967, Habibi et al 1974
Difficult to predict which patients will benefit from splenectomy
50% shows improvement of anemia (in 2 weeks)
20% show long-time remissions
in 50% of the patients in remission: reduction of the prednisolon dosage Coon 1985, Chertkow et al. 1956, Allgood e al. 1967, King et al. 2005, Pirofsky 1974
Elective splenectomy – laparoscopy
CAVE: Vaccination!! (H. influenzae, Str. meningitidis, Str. pneumoniae)
But: mortality after splenectomy ~1.3% (children 1.7%) Bisharat et al. 2001, Collins et al. 1992, Katkhouda et al. 1998
Treatment AIHA with WA-Abs – gammaglobulins
Mechanism:
• decrease de removal of RBCs in de spleen
Indication:
• Treatment of refractory AIHA with WA
• additional to the basic therapy in severe AIHA (improvement of the recovery of RBC transfusions)
Dosage
• 1g/kg bw during 2 days or 0.4g/kg during 5 days
40% temporary improvement of anemia (however: no longstanding CR) Flores et al. 1993, Macintyre et al. 1985, Bussel et al. 1986, Majer et al. 1988, Bjorkholm et al. 1993
Treatment AIHA with WA-Abs – anti-CD20
Retrospective studies: CR 20-75% Shanafelt et al 2003, Narat et al. 2005, D’Arena et al. 2006, D’Arena et al. 2006, Peñalver 2010
Prospective studies:
• 2 studies with CR>85%, 3 studies with CR>60%, 1 study CR 40% Zja et al. 2003, Zecca et al. 2003, Gupta et al. 2002, Quartier et al 2001, Trape et al. 2003, Barcellini 2012
Dose: 375 mg/m2, 1x/week, 4 gifts (low dose: 100 mg/m2, 1x/week, 4 times)
Anti-CD20 (Rituximab):
• Chimeric (mouse/human) antibody recognizing CD20
• CD20 is expressed on all B-cells (pre-pre-B cells) except on plasma cells
Mechanism: decrease the production of autoantibodies by specifically eliminating B-cells
Efficacy: not clear; convincing concept, little data from RCT, publication bias
Treatment AIHA with WA Abs
steroids
Steroids + immunopressivs/
cytotoxic medication
Anti-CD20 Rituximab
splenectomy
Gamma-globuline
Transfusion Treatment underlying
disease
08/2009:
Female patient (77 yrs) diagnosed with cholangio carcinoma (Klatskintumor) – admission for staging laparoscopy
Preoperative screening:
• A positief, CcDee
• directe Coombs 4+, anti-IgG negative, anti-IgA negative, anti-IgM weak positive, anti-C3c negative, anti-C3d positive
Patient with AIHA with cold auto-Abs
What is your advice???
Cold Abs: agglutination vs hemolysis
C1r C1s
C3b
C6 C7
C8 C5b
C5
C4
C2
Autoantibodies leading to intravascular hemolysis
antibodies potentially leading to intravascular hemolysis
mostly IgM, rarely IgA
Laboratory: Do auto-Abs have the potential to induce hemolysis (in-vitro)?
Hemolysis of RBCs
without additives: dangerous
with additives : potentially dangerous
(increasing susceptibility to in-vitro hemolysis)
16°C 30°C C
16 18 20 22 24 26 28 30 32 34 38 40 Temp (°C) 14 12 8 10
Max
imal
bin
din
g
difficult…….
fortunately: mostly mild anemia
Treatment AIHA with cold auto-Abs
Treat onderlying disease
If indicated: transfusion (op 37°C)
AIHA with cold antibodies – keep it warm
keep perioperative temperature around 37°C
administration of blood products at 37°C.
AIHA with cold antibodies - treatment
• Steroids: less effective as compared to warm auto-Ab Dacie 1992
• Cytostatica
Cyclophosphamide/chloorambucil: successful in some published case reports Petz 2001, Gehrs et al 2002, Worlledge et al. 1982
• Splenectomy: not effective (Case reports: splenectomy effective in AHIA with cold hemolysins)
McCurdy et al, 1958, Dacie 1992
• Gammaglobulines: case reports reporting beneficial effects...
• Anti-CD20 (Rituximab)
- Retrospective studies: 2 studies (n=9/52): CR 10%, PR 50-60%
- Prospective studies:
3 studies (n=9/20/27): CR 5% -16%, PR 33-50%
1 study (n=29): Rituximab + fludarabine; CR 21%, PR 55%
41% grade III-IV toxicity! Schoellkopf et al 2006, , Berentsen et al 2004, Berentsen et al 2010, Peñalver et al 2010, Barcellini et al 2012
Response rate 40-50%, complete response rare, frequently relapse
AIHA treatment - plasmapheresis
• Not a routine treatment- might be applied in fulminant hemolysis
(ultimum refugium)
• case reports on successful plasmapheresis in AIHA (warm &cold auto-Abs)
• Publication bias!!!
Cold auto-Abs: decrease in anti-IgM titer up to 30% (!), sometimes
followed by HD steroids/cyclophosphamide
Warm auto-Abs: decent effects
• “compassionate need” in case of potentially fatal hemolysis
• Combination with immmunosuppression essential
CAVE:
no FFP in case of complement activation and consumption
Strong cold auto-Abs: perform procedure at 37°C
McCarthy 1999, Nydegger 1990, Silva 1994, Petz 2004
C3b
C5
C6 C7
C8 C5b
Hemolysis - complement inhibition
Eculizumab:
• inhibition activation C5
• effective in PNH: less hemolysis, increase Hb
• part of the PNH patients: C3 deposition on RBC
stay dependent on RBC transfusion
Eculizumab
C3b
C3b C3d
C3c
C3d
C1-esterase inhibitor: complement inhibition
C1-esterase remmer
control cetor EDTA
0
20
40
60
80
100
120
(n=2) (n=6) (n=6)
0.1% serum20 U/ml Cetor
% C
3 d
ep
os
itio
n
serum (S) S+cetor S+anti-C5 healthy
0
20
40
60
80
100
% ly
sis
Patients: n=6; healthy controls: n=4 (16% serum) • C1-inhibitor (20 U/ml) • anti-C5 (100 μg/ml)
C1-inhibitor in AIHA
0 2 4 6 8 10 12 14 16 18 20 220123456789
10
500
1000
1500
2000V/R V/R V/R
##
LD
H (
U/L
)H
b (
g/d
l)
3.7 g/dl
MP
days
3 RBC concentrates
65-year old female patient with a relapse of
a marginal zone B-NHL
Lab:
• Coombs: positive C3d, neg IgM, IgG, IgA
• auto anti-I
• non- specific warm autoantibodies (IgM)
• allo anti-Wr(a)
3 packed cells
C1-inhibitor
Dose C1-inhibitor:
6000 – 4000 – 2000 -1000 U every 12 hrs (according to: Caliezi et al. 2002)
14 15 16 17 18 19 20 21 22
0
1
2
3
days
DA
T
Complement deposition
AHIA: take home message
AIHA: rare disease
• often associated with lymfoproliferative diseases
• older patients
Positive direct Coombs-test plus signs of hemolysis
Isotype determines the biological activity of autoantibodies (IgM, IgG1, IgG3)
• Complement activation, affinity Fc-gamma receptors
Classification based on binding characteristics of the autoantibody
• cold AS: bind at <30°C vs warm AS: 37°C
• CAVE: IgM antibodies binding at 30°C inducing hemolysis at 37°C (hemolysins)
treatment:
Transfusion:
• Rhesus Kell compatible
• ev. as far as possible antigen-compatible
AIHA warm auto-Abs:
• 1e line: steroids
• 2e line:
Anti-CD20, splenectomy, cytoxic drugs
AIHA with cold auto-Abs
• Keep it warm
• anti-CD20
• Ev. plasmapheresis
AHIA: take home message
Hospital laboratory physician
Reference laboratory
DAT pos
hemolysis!
Transfusion!
AHIA: take home message
Treatment AHIA
• Steroids
• Anti-CD20 (rituximab)
• Azathioprine, cyclophosphamide
• plasmapheresis
• Splenectomy
• gammaglobulines
• splenectomy
• steroids
• complement inhibitors
• plasmapheresis