Post on 12-Aug-2020
Autoimmune hemolytic anemia Autoimmune hemolytic anemia (AIHA)(AIHA)
Sacha Zeerleder
Department of Hematology
Academic Medical Center Amsterdam
Masja de Haas
Department of Red Blood Cell Serology
Sanquin Diagnostics
BelangenverklaringIn overeenstemming met de regels van de Inspectie van de Gezondheidszorg (IGZ)
Naam: Sacha Zeerleder
Organisatie: Academic Medical Center/Sanquin Research
☐
�
Ik heb geen 'potentiële' belangenverstrengeling
Ik heb de volgende mogelijke belangenverstrengelingen:�
Type van verstrengeling / financieel belang Naam van commercieel bedrijf
Ontvangst van subsidie(s)/research ondersteuning: Unrestricted Grant Viropharma
Ontvangst van honoraria of adviseursfee:
Lid van een commercieel gesponsord ‘speakersbureau’:
Financiële belangen in een bedrijf (aandelen of opties):
Andere ondersteuning (gelieve te specificeren):
Wetenschappelijke adviesraad:
Ik heb de volgende mogelijke belangenverstrengelingen:
BelangenverklaringIn overeenstemming met de regels van de Inspectie van de Gezondheidszorg (IGZ)
Naam: Masja de Haas
Organisatie: Sanquin
�
☐
Ik heb geen 'potentiële' belangenverstrengeling
Ik heb de volgende mogelijke belangenverstrengelingen:☐
Type van verstrengeling / financieel belang Naam van commercieel bedrijf
Ontvangst van subsidie(s)/research ondersteuning:
Ontvangst van honoraria of adviseursfee:
Lid van een commercieel gesponsord ‘speakersbureau’:
Financiële belangen in een bedrijf (aandelen of opties):
Andere ondersteuning (gelieve te specificeren):
Wetenschappelijke adviesraad:
Ik heb de volgende mogelijke belangenverstrengelingen:
Autoimmune hemolytic anemia (AIHA)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 AIHAPathogenesis 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
Ineffective antigen presentation• Immature DCs• Decreased co-stimulatory help by T-
cells• Oxidative changes in RBC antigen structure?
Complement system• Altered expression of CR1• Decreased experssion membran-
boud complement regulators (CD59)
cells
Functional B/T cell abnormalities• Polyclonal lymphocyte activation• Alteration of cytokine profile• Dysregulation of T-Regs
AIHAAIHA
Laboratory analyses:
• Hemolysis (LDH �, haptoglobin �, hyperbilirubinemia�)
• Positive Coombs
• (reticulocytosis)
Diagnosis AIHADiagnosis AIHA
AIHA is characterized by an increased breakdown of red blood cells (RBC) due to autoantibodies (auto-Ab’s) with/without complement activation.
• (reticulocytosis)
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 than the specificity of the autoantibody
Isotype autoantibody:
determines complement activation and Fc gamma receptor binding
Complement activation:
• IgM: most efficient
• IgG1, IgG3: efficient
• IgG2, IgA: weak
AIHA: biological activity of autoantibodiesAIHA: biological activity of autoantibodies
• IgG2, IgA: weak
• IgG4: no complement activation
Fc-gamma receptors
• IgG1, IgG3 �FcgRI (CD64)
• IgG1, IgG3 (low affinity, if complexed medium affinity ) �FcgRIIa
C1rC1s
C1rC1s
AIHA: complement activationAIHA: complement activation
C1rC1s
C1rC1s
C4
C2
AIHA: complement activationAIHA: complement activation
C1rC1s
C2a C4b
C3c
AIHA: complement activationAIHA: complement activation
C3
C3bC3b C3d
C3c
FI
C3d
C3c
AIHA: complement activationAIHA: complement activation
C3b
C6C7
C8C5b
C5
Complement activation
RBC-lysis
AIHA AIHA –– destruction of autologous RBCsdestruction of autologous RBCs
Fc-gamma receptor
CR receptordepositie
C3dC3c
C3d
IgG C3c C3d
positive positive
AIHA: direct Coombs (antiglobuline test)AIHA: direct Coombs (antiglobuline test)
positive positive
R
G
C3dC3c
C3d
AIHA: direct Coombs (antiglobuline test)AIHA: direct Coombs (antiglobuline test)
IgG (IgA) IgM C3c C3d
positive
AutoAuto--antibodies and complementantibodies and complement
Direct Coombs: • C3d+++• IgM/G/A neg.
Direct Coombs: • C3d++500 800IgG
A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B120
20
40
60
80
hemolysisIgMIgG
sample
AU
Significant proportion C3d++ due to IgM!
• C3d++• IgG ++• IgM/A neg.
E. Meulenbroek, MS in preparation
A2 A3 A4 A5 A6 A7 A8 A9A10 A11 A12 B1 B2 B3 B4 B5 B6 B7 B8 B9B10 B11 B12
0
100
200
300
400
500
0
200
400
600
800IgG
IgMC3 deposition
fractions
AU
IgG
/ IgM C
3 M
FI
Cold antibodies
• mostly IgM
• binding at <30°C
30°°°°
37°°°°C
binding
lysis
30°°°°
37°°°°C
binding
lysis
AIHA: classificationAIHA: classification
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
binding 30°°°°C
binding
Cold Abs: agglutination vs hemolysisCold Abs: agglutination vs hemolysis
C1rC1s
C3b
C6C7C8C5b
C5
C4
C2
Autoantibodies leading to intravascular hemolysisAutoantibodies leading to intravascular hemolysis
16°°°°C30°°°°C CC
Max
imal
bin
din
g
� antibodies potentially leading to intravascular hemolysis
� mostly IgM
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 18 20 22 24 26 28 30 32 34 38 40 Temp (°°°°C)14128 10
Warm autoantibodies:
• Idiopathic (primary)
• Secondary: CLL, lymphoma, SLE
Cold autoantibodies
• Idiopathic (primary)
Rare
Incidence 1:80‘000
>40 years, peak >70
Very rare
AIHA: classificationAIHA: classification
18% will develop overt
lymphoma in the
future
18% will develop overt
lymphoma in the
future
• Idiopathic (primary)
• Secondary: mycoplasma infection, viral infections (EBV), lymphoproliferative diseases
Paroxysmal cold hemoglobinuria
• Idiopathic (primary)
• Secondary: viral infections, syphilis
Mixed warm and cold autoantibodies
>50 years
Very rare
Children
Cholangiocarcinoma (Klatskintumor) � staging laparoscopy
Pre-operative screening:
• A positive, CcDee
• direct Coombs 4+, anti-IgG negative, anti-IgA negative, anti-IgM negative, anti-C3c negative, anti-C3d positive
AIHA with cold autoAIHA with cold auto--Abs: female patient born 1956 Abs: female patient born 1956
Surgeon calls the hematologist on duty
• Blood type and screen immediately “clots”
• What does that mean? Dangerous?
• I want to perform the laparascopy asap!
AIHA with warm autoAIHA with warm auto--Abs: female patient born 1924 Abs: female patient born 1924
Chronic lymphatic leukemia (Rai III), del17p (poor prognosis)
� Treatment with chlorambucil, achievement of very good partial remission
IH: direct Coombs negative
2009 (07): Non-STEMI infarction
Lab: Hb 5.2 mmol/l, 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-WASIH: - Coombs 3+; IgG 3+, IgA/M neg, C3bc neg, C3d pos, NS-WAS
- alloantibodies: anti-Jk(a)
����Transfusion 3 RBC concentrates (RhD neg, Kell neg)
2009 (08): Non-STEMI infarction
Lab: Hb 3.9 mmol/L, Lc 1.8x109/l, Tc 44x109/l; LDH 457
U/L, haptoglobin <0.02 g/L
IH: “everything is positive”
� Transfusion
� Decrease the production of autoantibodies
• Corticosteroids
• plus immunosuppressive therapy
• Monoclonal antibodies (antiCD20)
• Danazol
ImmunoImmuno--supressivssupressivs
splenectomysplenectomy
AIHA with warm autoantibodiesAIHA with warm autoantibodies -- treatmenttreatment
• Danazol
� inhibit the breakdown/removal of RBCs
• intravenous gammaglobulines
• splenectomy
� Treatment of the underlying disease
rituximabrituximab
corticosteroidscorticosteroids
IVIGIVIG
Transfusion Transfusion –– AIHA AIHA
Analyst calls: “everything is positive….”
� Type: RBC coated with autoantibodies
Is there really a need for transfusion (vital indication)?
� are there clinical signs of hypoxia??
� Is there time for extensive IH analyses?
� Screen: patient serum reacts with test RBCs
Hence: T&S not possible (needs time!)
Important: prevention of alloantibody formation
�Patients with alloantibodies have an increased risk to develop additional alloantibodies
Transfusion can exacerbate hemolysis!
AIHA AIHA –– transfusion: recommendation 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 autoAIHA with WA auto--AbsAbs–– steroids + cytotoxic agentssteroids + 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 neededGehrs 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 WATreatment AIHA with WA--AbsAbs–– splenectomysplenectomy
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 dosageCoon 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 WATreatment AIHA with WA--Abs Abs –– gammaglobulinsgammaglobulins
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 WATreatment AIHA with WA--Abs Abs –– antianti--CD20CD20
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
Retrospective studies: CR 20-75%Shanafelt et al 2003, Narat et al. 2005, D’Arena et al. 2006, D’Arena et al. 2006, Peñalver et al. 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 et al. 2012
Dose: 375 mg/m2, 1x/week, 4 gifts (low dose: 100 mg/m2, 1x/week, 4 times)
difficult…….
Treatment AIHA with cold autoTreatment AIHA with cold auto--Abs Abs
fortunately: mostly mild anemia
“keep-it warm”
Treat onderlying disease
If indicated: transfusion (op 37°°°°C)
• Steroids: less effective as compared to warm auto-AbDacie 1992
• Cytostatic drugs
Cyclophosphamide/chlorambucil: successful in some published case reportsPetz 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...
• Steroids: less effective as compared to warm auto-Ab Dacie 1992
• Cytostatic drugs
Cyclophosphamide/chloorambucil: successful in some published case reportsPetz 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)
AIHA with cold antibodies AIHA with cold antibodies -- treatmenttreatment
• 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
Hemolysis Hemolysis -- complement inhibitioncomplement 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
C5
C6C7
C8C5b
Eculizumab
C1C1--esterase inhibitor: complement inhibitionesterase inhibitor: complement inhibition
control cetor EDTA0
20
40
60
80
100
1200.1% serum20 U/ml Cetor
% C
3 d
epo
sitio
n
serum (S) S+cetor S+anti-C5 healthy0
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)
C3b
C3b C3dC3c
C3d
C1-esterase remmer
control cetor EDTA(n=2) (n=6) (n=6)
serum (S) S+cetor S+anti-C5 healthy
Wouter et al. Blood 2013
AHIA: take home message AHIA: take home message
• Rare disease -often associated with lymfoproliferative diseases
• Positive direct Coombs-test plus signs of hemolysis
• Isotype determines the biological activity of autoantibodies (IgM, IgG1, IgG3)
� complement deposition mostly due to IgM (often no IgM detectable)
• Classification based on binding characteristics of the autoantibody
� cold AS: bind at <30°C vs warm AS: 37°C
• Transfusion: Rhesus Kell compatible, ev. as far as possible antigen-compatible
• AIHA warm auto-Abs:
� 1e line: steroids
� 2e line: splenectomy, cytoxic drugs, Anti-CD20,
• AIHA with cold auto-Abs
� “Keep it warm”, anti-CD20 (±±±±fludarabin)
Hospital laboratoryphysician
DAT pos
hemolysis!
Transfusion!
AHIA: take home message AHIA: take home message
Reference laboratory