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