Autoimmune Hemolytic

Anemias
Dr. Musa Alzahrani
MBBS, FRCPC, ABIM, MHSc
Assistant professor & consultant hematologist
Objectives
• Learn the pathophysiology of AIHA
• Understand the difference between direct and indirect Coomb’s tests
• Know the different types of drug induced AIHA
• Construct an approach to hemolysis
• Construct an approach to management of AIHA
Hemolysis
• Definition: increased rate of RBC destruction.
• Traditionally divided into intravascular and extravascular hemolysis.
• But this is not helpful! why?
• A more useful approach is to think of intrinsic vs extrinsic reasons.
Intravascular vs extravascular
• Intravascular hemolysis:
• Causes: (ABO incompatibility, G6PD deficiency, PNH, PCH, clostridial sepsis,
mechanical valve, severe burns)
• plasma free hemoglobin (hemoglobinemia)
• decreased haptoglobin (bound by free Hb)
• hemoglobinuria and hemosiderinuria
• chronically, may lead to iron deficiency

• Extravascular hemolysis (occurs in RES)

• positive direct antiglobulin test (DAT or direct Coombs’) if immune-mediated
• no hemoglobinemia, hemoglobinuria, hemosiderinuria
 Hemolytic anemia Autoimmune
hemolytic anemia

Hereditary
spherocytosis
or
elleptocytosis

G6PD or
pyruvate kinase
deficiency
TTP/HUS, DIC, Valve
hemolysis
Sickle cell
anemia
Thalassemia
AHA classification
• AHA is classified by the temperature at which autoantibodies bind
optimally to the patient RBCs.
• In adults, 80%-90% of AIHA are mediated by antibodies that bind to
RBCs at 37°C (warm autoantibodies).
• In the cold hemolytic anemias, the autoantibodies bind to RBCs at
temperatures <37°C (cold autoantibodies).
• Some patients have both warm and cold autoantibodies.
• Known as mixed AIHA.
Classification
• AIHA are further divided by the presence or absence of an underlying
related disease.
• When no underlying disease is recognized, the AIHA is termed
primary or idiopathic.
• Secondary cases are those in which the AIHA is a manifestation or
complication of an underlying disorder.
Antibody structure
Fc attaches to Fc receptor
IgG vs IgM
Antibodies structures
Complement fixation
Complement mediated hemolysis
Cold agglutination
Extravascular hemolysis
Extravascular hemolysis
Spherocytosis Loss of central
pallor

Central pallor
1/3rd
Pathophysiology

• Warm AHA
• The most common type of AIHA
• Is mediated by warm reactive autoantibodies of the IgG isotype.
• May or may not fix complement,
• but they do not cause direct agglutination of RBCs because of their small size.
• Hemolysis is mediated by Fc receptor–expressing macrophages,
primarily located in the spleen.
Cold AHA

• Cold-reactive autoantibodies bind optimally to RBCs at temperatures <37°C.

• Cold autoantibodies are typically of the IgM isotype
• Because of their large, pentameric structure, they are able to span the
distance between several RBCs to cause direct agglutination.
• They are able to fix complement.
• The consequence of complement fixation is clearance of C3b-coated cells
by attachment to complement receptors on macrophages, primarily in the
spleen, and Kupffer cells in the liver.
• Direct lysis by completion of the terminal complement sequence may also
occur.
Cold AIHA
• Cold autoantibodies are characteristic of AIHA associated with
• Mycoplasma infection
• Epstein-Barr virus.
• In the elderly: almost always associated with B-cell lymphoproliferative
disorders.
• It is caused by a monoclonal IgM antibody that binds to carbohydrate I
antigens or i antigens at temperatures below body temperature.
Paroxysmal Cold Hemoglobinuria (PCH)
• Cold reacting IgG (Donath-Landsteiner) autoantibodies, may cause
significant intravascular lysis of RBCs as a result of their ability to fix
complement.
• It is almost always idiopathic or happen after URTI.
• Rarely can be associated with congenital syphilis.
• In children: Donath-Landsteiner hemolytic anemia accounts for
almost one-third of AIHA cases.
• The target antigen of the autoantibodies is in the P blood group
system.
Drug-induced immune hemolytic anemia

• The clinical and laboratory features of drug-induced and idiopathic

AIHA are similar.
• so a careful history of drug exposure should be obtained.
• Huge list of culprit drugs!
• Second- and third-generation cephalosporins account for about 88%
of drug-induced immune hemolytic anemia.
Drug induced AIHA
• Drugs may cause immune hemolysis by three different mechanisms:
1. Induce formation of autoantibodies directed against the pt’s own RBC
antigens. (Anti RBC)
2. The hapten-drug adsorption mechanism (characterized by the presence of
antidrug antibodies in the blood).
• These antibodies bind only to RBC that are coated with tightly bound drug.
3. Antibodies form a three way complex (formed by the drug and a specific
RBC membrane antigen and an antibody).
• This is termed ternary or immune complex mechanism.
Hapten or drug adsorption mechanism

• Hapten or drug adsorption mechanism applies to drugs that bind

firmly to proteins on the RBC membrane.
• The classic setting is very high-dose penicillin therapy, but other drugs
such as cephalosporins and semisynthetic penicillins also are
implicated.
Ternary or immune complex mechanism: drug antibody-target
cell interaction

• The pathogenic antibody recognizes the drug only in combination

with a particular antigen on the RBC.
• The DAT is positive usually only for complement.
Autoantibody mechanism

• Several drugs, by unknown mechanisms, induce the formation of

autoantibodies reactive with RBCs in the absence of the instigating
drug.
• The most studied drugs in this category has been α-methyldopa and
levodopa.
• Pts with CLL treated with fludarabine may develop severe and
sometimes fatal autoimmune hemolysis.
Non-immunologic protein adsorption

• <5% of pts receiving cephalosporin antibiotics develop +DAT because

of nonspecific adsorption of plasma proteins to their RBC membranes.
• May occur within 1-2 days after the drug is started.
• Hemolysis does not occur.
• However it may complicate cross-match and DAT interpretation.
Clinical manifestations

• Several clinical features of AHA are common to both warm and cold-
antibody types.
• Patients may present with signs and symptoms of anemia (eg,
weakness, dizziness), jaundice, abdominal pain, and fever.
• Mild splenomegaly is common.
• Hepatomegaly and lymphadenopathy may be evident at presentation
depending on the etiology.
laboratory findings
• Anemia may vary from mild to severe, usually with either normocytic or macrocytic
cells.
• Reticulocytosis is usually present.
• Reticulocytopenia, however, may be present up to one-third of the time as a result
of:
• intercurrent folate deficiency
• infection
• Marrow infiltration by a neoplastic process.
• Indirect bilirubin and LDH are elevated to varying degrees, and the haptoglobin is
depressed.
• The blood smear often demonstrates spherocytes.
• Nucleated RBCs also may be present.
Cold AIHA
• Occasionally spurious marked elevations in the MCV and MCHC
measurements and decrease in the RBC count are observed
• Due to simultaneous passage of two or three agglutinated RBCs
through the aperture of the automated cell counter.
The anti-human
globulin can be
non specific:

polyspecific

Or specific:
Anti IgG
Or Anti C3b
DAT (Direct anti-globulin test)
• The DAT (Coombs test) is usually positive in AIHA but may be negative in some
patients.
• The threshold of detection of commercial antiglobulin reagents is:
• 200-500 antibody molecules per cell.
• However <100 molecules of IgG per cell may cause hemolysis.
• IgM are usually removed from RBCs during washing and usually are not detected.
• Most commercial reagents do not detect IgA.
• AIHA:
• 30%-40% will have only IgG
• 50% will have both IgG and C3
• and only approximately 10% will have C3 alone.
DAT
• The strength of the direct Coombs test maybe useful:
• +1 weak
• +2 intermediate
• +3 is strong
Management
• Starts with group and screen.
• Grouping is to identify: ABO and Rh systems
Antibody screen is the IAT (indirect
antiglobulin test)
Antibody identification panel
In AIHA: panagglutinin
Treatment

• Hemolysis could be life threatening.

• Transfusions can be lifesaving.
• Transfusion of RBCs in immune hemolytic anemia is often
problematic.
• Finding serocompatible donor blood is rarely possible because, in
most cases, the autoantibody is a panagglutinin.
• The difficult technical issue relates to detection of RBC alloantibodies
masked by the presence of the autoantibody.
Management
• Use the “least incompatible” blood for transfusion, but this is a
misnomer because all units will be serologically incompatible.
• Patients with a history of pregnancy, abortion, or prior transfusion are
at risk of an alloantibody.
• Consultation with the blood bank is important
• Hemolysis could be life-threatening, and delay in transfusion over
concerns about red cell incompatibility can lead to mortality.
• Transfused slowly while the patient is monitored carefully for signs of
a hemolytic transfusion reaction.
Warm AIHA
• Therapy is aimed at:
• decreasing the production of autoantibody.
• decreasing clearance of RBCs from the circulation.
• Glucocorticoids such as prednisone usually are the first-line treatment
• Remission can be achieved with prednisone at approximately 60-100 mg/d (or 1 mg/kg/d)
• Approximately two-thirds of adult pts respond to prednisone, with approximately 20% achieving
complete remission.
• Pulse steroids (eg, 1 g methylprednisolone intravenously) can be effective in some pts in whom
standard therapy has failed.
• If in remission the dose should be decreased by 10 mg/d each week until a dose of 30 mg/d is
reached.
• Subsequent dose reduction should then proceed more slowly (at 5 mg/d per week), with the goal
of either maintaining remission with prednisone at 20mg every other day or complete weaning of
prednisone if the DAT becomes negative
Second line therapies
• Splenectomy is often considered if:
• hemolysis remains severe for 2-3 weeks at prednisone doses of 1 mg/kg,
• if remission cannot be maintained on low doses of prednisone, or
• if the patient has intolerable adverse effects or contraindications to
glucocorticoids.
• Removing the spleen results in a reduced rate of clearance of IgG-
coated cells.
• Pts should receive: pneumococcal, H. influenzae, and meningococcal
vaccines before splenectomy.
• Approximately two-thirds of pts will have complete or partial remission
with splenectomy, but relapses are common.
Other options
• Rituximab:
• Standard-dose (375 mg/m2) and low-dose (100 mg/m2) monoclonal anti-
CD20 (rituximab) has been useful in refractory cases.
• Response rates range from 40% to 100%.
• Immunosuppressive drugs:
• such as cyclophosphamide, azathioprine, MMF, and cyclosporine, danazol,
have been used with variable response rates.
Cold AIHA
• Maintaining a warm environment may be all that is needed.
• Cold agglutinin disease usually does not respond to glucocorticoids.
• Recently, rituximab has demonstrated efficacy in treating cold agglutinin
disease, with response rates approaching 50%.
• Chlorambucil and cyclophosphamide have been beneficial in selected
cases.
• Chemotherapy is indicated if the disorder is associated with a
lymphoproliferative disorder.
• Splenectomy usually is not effective because cells typically are cleared
by intravascular hemolysis or hepatic Kupffer cells.
Drug induced AIHA:

• Removal of the offending agent is the principal treatment.

• May respond to steroids
• Typically drug induced hemolysis:
• occurs 7-10 days after the drug is started
• stops a few days after the drug is D/C’ed but can stay up to 2 weeks.
Clinical case
• A 68-y.o M admitted with c/o fatigue, SOB, wt loss and night sweats x 6 m.
• PMHx: diet-controlled diabetes.
• Meds: takes no medications.
• On examination: pallor, scleral icterus, axillary adenopathy and splenomegaly.
• Lab: Hb 80g/L and an MCV of 143 fL. LDH is elevated at 2,321 U/L (upper limit of
normal 420), indirect bilirubin at 2.1 mg/dL, and reticulocyte count at 13%.
• The peripheral blood smear shows agglutinated RBCs.
• The blood bank reports a direct Coombs test positive for complement (3+) but
negative for immunoglobulin G (IgG).
• Serum protein electrophoresis reveals a monoclonal IgM.
• Abdominal CT scan reveals splenomegaly and diffuse adenopathy.
Case resolution
• The pt has cold agglutinin disease, likely secondary to underlying
lymphoma. (IgM producing lymphoma: lymphoplasmacytic lymphoma
= LPL)
• The red cell count is artifactually low, and the MCV and MCHC are
falsely elevated secondary to red cell agglutination.
• Warming of the blood tube with immediate measurement and slide
preparation will minimize agglutination.
• The direct antiglobulin test is positive only for complement.
Thanks
• Questions?