Autoimmune
Hemolytic Anemias
Donald R. Branch, Ph.D.
Scientist
Research & Development
Canadian Blood Services
Toronto, Ontario CANADA
don.branch@utoronto.ca
Lecture Outline
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Diagnosis of hemolytic anemias
Immune hemolytic anemias
Classification of AIHAs
Serological diagnosis of specific AIHAs
Selection of blood for transfusing patients
with AIHAs
Transfusion of patients with AIHAs
No Need to be Terrified
Diagnosis of Hemolytic Anemia
What is Hemolytic Anemia?
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Shortened red cell survival (<100 days)
Structural, Hb variant, drug oxidant,
immune
Can be acute (intravascular hemolysis)
Extravascular hemolysis
Can be life threatening
Tests to Diagnose AIHA
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Hemoglobin and hematocrit
Rule-out bleeding/hereditary causes – red cell morphology
and blood film
Reticulocyte count
Bilirubin
Serum haptoglobin
LDH
Other tests (hemoglobinemia, hemosiderin)
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DAT!
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 Cooperation
between blood bank,
hematology and urinalysis depts.
Agglutinated Cells
Increased Reticuloctye Count
Spherocytosis
Neutrophil Erythrophagocytosis
Immune Hemolytic Anemias
DIRECT
ANTIGLOBULIN
TEST
Test for IgG and/or complement coating the patient’s red blood cells
Classification of Immune
Hemolytic Anemias
Alloimmune
HTR
DHTR
HDFN
 Autoimmune hemolytic anemias (AIHAs)
DAT-positive
DAT-negative
• Drug-induced
Autoimmune
Drug-adsorption
Immune-complex
• Bystander Immune Cytolysis
Sickle cell hemolytic transfusion reaction syndrome
Reactive hemolysis
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Classification of
AIHAs
 Warm Antibody AIHA (warm AIHA)
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Cold Antibody AIHA (cold agglutinin
syndrome; CAS)
Paroxysmal Cold Hemoglobinuria (PCH)
Combined Cold and Warm (“Mixed AIHA”)
Atypical AIHA
AIHA with a Negative DAT
Warm Antibody AIHA Caused by IgM or
IgA Autoantibodies
Drug-Induced AIHA (drug-induced immune
HA)
Serological Diagnosis of AIHA
Warm Antibody AIHA and DAT
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70% of all AIHA are warm type
~80% of warm AIHA prefer oldest RBCs
 ~20% have no preference for age of RBCs
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Associated with IgG, (IgA), (IgM) autoantibodies:
DAT: IgG + C (67%)
IgG only (20%)
C only (13%)
Must use a polyspecific antiglobulin reagent for DAT
Criteria for Diagnosis of Cold Antibody
AIHA
1.
2.
3.
Clinical findings indicative of acquired
hemolytic anemia.
Positive direct antiglobulin test with antiC3d ONLY!
Serum antibody optimally reactive at 4C
but can react up to 30C, usually anti-I/i
specificity
Mixed AIHA
Criteria for Diagnosis of Mixed
AIHA
1.
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4.
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7.
Evidence indicative of acquired hemolytic anemia.
Severe hemolysis – intravascular/extravascular
DAT positive with BOTH IgG and C3d.
Serum contains an IgG antibody reactive at 37C
indistinguishable from warm autoantibody.
Serum ALSO contains LOW TITER antibody
reactive up to 30C (usually 37C) that is optimally
reactive at 4C and is IgM; often shows anti-I/i.
Eluate contains IgG antibody having same specificity as
serum antibody.
RESPONDS remarkably WELL to steroid therapy.
(Shulman IA, Branch DR, et al. Autoimmune hemolytic anemia with both cold
and warm autoantibodies. JAMA 253:1746-1748, 1985)
Paroxysmal Cold Hemoglobinuria
(PCH)
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Incidence: May be quite common in young
children.
Acute onset of severe hemolysis with
hemoglobinemia and hemoglobinuria.
(hemoglobinuria very common).
Erythrophagocytosis by neutrophils is
common and is distinctive.
Donath-Landsteiner test is diagnostic.
Patients have a stormy course but good
ultimate prognosis.
Erythrophagocytosis in PCH
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RBC rosetting around neutrophils and
erythrophagocytosis by neutrophils, rather
than monocytes, are prominent findings in
PCH.
Rarely seen in other forms of immune
hemolysis, where monocytes are usually
involved.
May be first clue to diagnosis.
The Donath-Landsteiner
Test
Characteristics of Typical D-L Antibodies
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Biphasic Hemolysin
IgG Immunoglobulin Class
Anti-P Specificity
May also react by IAT
P
P + complement
p
DAT-negative AIHA
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Rare
Often severe - fatal
Sometimes anti-IgA/anti-IgM useful
Mononuclear phagocyte assay may be
useful – use patient monocytes
Often responds to steroid treatment
Criteria for Diagnosis of DATnegative AIHA
1.
2.
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4.
Evidence indicative of acquired hemolytic
anemia.
DAT negative with anti-IgG/anti-C3d
No unexpected antibodies detectable in
serum or eluate (sometimes antibody can
be detected in eluate)
Responds to prednisone treatment
Specificity of Warm
Autoantibodies
Not Usually Necessary – Academic Exercise
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Anti-Rh-like specificity
Anti-e or anti-E (SIMPLE SPECIFICITY)
Anti-pdl – does not react with –D- or Rhnull
Anti-dl – does not react with Rhnull
Relative Rh specificity – titration studies
Type I or Type II Autoantibodies
Other blood groups – Kell, Gerbich (high
frequency antigens)
Relative Specificity
A Guide to Transfusion
of Patients with
Autoimmune
Hemolytic Anemia
No Need to Hide When a Patient Presents with AIHA
Important Principles
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Indications for transfusion are not
significantly different than for similarly
anemic patients without AIHA.
Specialized laboratory procedures are
necessary. Critical to look for
alloantibodies
Communication between laboratory
personnel and clinicians is critical.
Provision of Safe Blood
Risks of Transfusion in AIHA
Autoantibody will cause shortened
survival of transfused RBC.
 Alloantibodies may be present in
addition to the autoantibody. Must
Be Identified and Antigen-Negative
Blood Given!
 Risks caused by the increase in RBC
mass as a result of transfusion.
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Blood Transfusion in
Autoimmune Hemolytic
Anemia
Blood should never be denied a
patient with a justifiable need,
even though the compatibility test
may be strongly positive.
 Probably the most common
mistake is reluctance to
transfuse even those patients
with severe anemia.
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Communication Between Clinician
and Transfusion Service
Responsibilities of Transfusion Service
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Initiate communication.
Indicate extent of compatibility testing
performed, e.g., auto- or alloadsorption.
Clinician should be assured that, after
appropriate compatibility testing, an acute HTR
is unlikely.
Indicate that RBCs will provide temporary
benefit even if they do not survive normally
because of the patient’s autoantibody.
Communication Between Clinician
and Transfusion Service
Responsibilities of Clinician
 Indicate
urgency of situation.
 Understand principles of compatibility
testing.
 Seek assurance that appropriate
compatibility testing is to be
performed.
Indications for Transfusion
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A common mistake is reluctance to transfuse
patients with AIHA.
If appropriate compatibility procedures are
performed, survival of transfused RBCs is
generally about as good as that of the patient’s
own RBCs. Alloantibodies MUST be ruled out!
Significant temporary benefit is to be expected.
Patients should not be denied transfusion because
of an RBC autoantibody.
American Journal of Clinical Pathology 1982; 78(2):161-167
ABO and Rh
“Spontaneous” Agglutination
IgG auto - Treat cells with ZZAP to remove autoantibody and retype
IgM auto – warm everything to 37C and retype or ZZAP treat and retype
(Branch DR, Petz LD. A new reagent (ZZAP) having multiple applications in
immunohematology. Am J Clin Pathol. 78:161-167, 1982)
RBC ALLOANTIBODIES IN PATIENTS
WITH WARM AUTOANTIBODIES
#ANTIBODIES/
REFERENCE
#SERA TESTED
•Morel
8/20
•Branch and Petz
5/14
•Wallhermfechtel et al
19/125
•Laine and Beattie
41/109
•James et al
13/41
•Issitt et al (alloadsorptions)
13/34
•Issitt et al (autoadsorptions)
5/41
•Leger and Garratty
105/263
•TOTALS:
209/ 647
% OF SERA
WITH ALLOABS
40
36
15
38
32
38
12
40 _______
32%
(Branch DR, Petz LD: Detecting alloantibodies in patients with autoantibodies. Transfusion 39:610, 1999) (editorial)
Adsorption Procedures
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Warm autoadsorption using ZZAP is the optimal
procedure for alloantibody detection.
One should obtain adequate volumes of patient’s
RBCs to perform the procedure.
 RBCs should be retained for subsequent procedures.
 The number of ZZAP autoadsorptions needed is
variable depending on the strength of the IAT and
the ability to reduce the DAT.
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Usually 2 adsorptions is sufficient
Autoadsorption is not reliable in a recently
transfused patient.
ZZAP!!!!!!!
Allogeneic Adsorption
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If patient’s RBCs are not available for
autoadsoprtion.
If the patient has been transfused
recently, and if the patient’s pretransfusion RBCs are not available for
autoadsorption.
Selection of Red Cells for
Allogeneic Adsorption
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R1R1
R2R2
rr
One cell Jk(a-)
Another cell Jk(b-)
Use ZZAP to denature other important
antigens
Allogeneic Adsorption
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Advance planning is important !
Large aliquots of appropriately
phenotyped RBC should be obtained,
treated with ZZAP, and stored at 4oC for
up to 2 months.
If such procedures are infrequent,
cryopreservation may be preferable.
Phenotyping patient may make it feasible
to use fewer cells in adsorptions.
Single-Cell Allogeneic Adsorption
Cold Antibody AIHA
Perform compatibility tests at 37oC.
 In a small percentage of patients, cold
autoadsorption using ZZAP may be
necessary.
 Allogeneic adsorptions can also be
performed, but are rarely necessary.
 Use of a blood warmer may be
necessary and the patient’s room kept
at a higher temperature.
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Mixed AIHA
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Administer corticosteroids as soon as
possible. Can be immediately effective
without need for transfusion.
Compatibility testing the same as for warm
antibody AIHA.
Rule out clinically significant alloantibodies.
PCH
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Compatibility test will appear compatible
but RBC survival is not likely to be better
than that of the patient’s own RBC.
Autoantibody specificity generally is antiP, but it is usually inadvisable to wait for
antigen-negative RBC from rare donor
files.
The Three Amigos
Larry Petz
George Garratty
Don Branch
Acknowledgements
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Larry Petz
Phyllis Morel
George Garratty
Jean-Michel Turc
Canadian Blood Services
don.branch@utoronto.ca