Autoimmune hemolytic anemia (AIHA)

Sacha Zeerleder, MD PhD

Internist – hematologist

Department of Hematology Academic Medical Center Amsterdam

Department of Immunopathology, Sanquin Research, Amsterdam

s.s.zeerleder@amc.uva.nl

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