Autoimmune Autoimmune Hemolytic AnemiasHemolytic Anemias
Donald R. Branch, Ph.D.Donald R. Branch, Ph.D.ScientistScientist
Research & DevelopmentResearch & DevelopmentCanadian Blood ServicesCanadian Blood Services
Toronto, Ontario CANADAToronto, Ontario CANADA
[email protected]@utoronto.ca
Lecture OutlineLecture Outline Diagnosis of hemolytic anemiasDiagnosis of hemolytic anemias Immune hemolytic anemiasImmune hemolytic anemias Classification of AIHAsClassification of AIHAs Serological diagnosis of specific AIHAsSerological diagnosis of specific AIHAs Selection of blood for transfusing patients Selection of blood for transfusing patients
with AIHAswith AIHAs Transfusion of patients with AIHAsTransfusion of patients with AIHAs
No Need to be Terrified
Diagnosis of Hemolytic AnemiaDiagnosis of Hemolytic Anemia
Shortened red cell survival (Shortened red cell survival (<<100 days)100 days) Structural, Hb variant, drug oxidant, Structural, Hb variant, drug oxidant,
immuneimmune Can be acute (intravascular hemolysis)Can be acute (intravascular hemolysis) Extravascular hemolysis Extravascular hemolysis Can be life threateningCan be life threatening
What is Hemolytic Anemia?
Tests to Diagnose AIHA Tests to Diagnose AIHA
Hemoglobin and hematocritHemoglobin and hematocrit Rule-out bleeding/hereditary causes – red cell morphology Rule-out bleeding/hereditary causes – red cell morphology
and blood filmand blood film Reticulocyte countReticulocyte count BilirubinBilirubin Serum haptoglobinSerum haptoglobin LDHLDH Other tests (hemoglobinemia, hemosiderin)Other tests (hemoglobinemia, hemosiderin) DAT!DAT! Cooperation between blood bank, Cooperation between blood bank,
hematology and urinalysis depts.hematology and urinalysis depts.
Agglutinated Cells
Increased Reticuloctye Count
SpherocytosisSpherocytosis
Neutrophil Erythrophagocytosis
Immune Hemolytic AnemiasImmune Hemolytic Anemias
DIRECT DIRECT ANTIGLOBULINANTIGLOBULIN
TESTTESTTest for IgG and/or complement coating the patient’s red blood cells
Classification of Immune Classification of Immune Hemolytic AnemiasHemolytic Anemias
AlloimmuneAlloimmuneHTRHTRDHTRDHTRHDFNHDFN
Autoimmune hemolytic anemias (AIHAs)Autoimmune hemolytic anemias (AIHAs)DAT-positiveDAT-positiveDAT-negativeDAT-negative
•• Drug-inducedDrug-inducedAutoimmuneAutoimmuneDrug-adsorptionDrug-adsorptionImmune-complexImmune-complex
•• Bystander Immune CytolysisBystander Immune CytolysisSickle cell hemolytic transfusion reaction syndromeSickle cell hemolytic transfusion reaction syndrome Reactive hemolysisReactive hemolysis
Classification of Classification of AIHAsAIHAs
Warm Antibody AIHA (warm AIHA)Warm Antibody AIHA (warm AIHA) Cold Antibody AIHA (cold agglutininCold Antibody AIHA (cold agglutinin syndrome; CAS)syndrome; CAS) Paroxysmal Cold Hemoglobinuria (PCH)Paroxysmal Cold Hemoglobinuria (PCH) Combined Cold and Warm (“Mixed AIHA”)Combined Cold and Warm (“Mixed AIHA”) Atypical AIHAAtypical AIHA
AIHA with a Negative DATAIHA with a Negative DATWarm Antibody AIHA Caused by IgM orWarm Antibody AIHA Caused by IgM or
IgA AutoantibodiesIgA Autoantibodies Drug-Induced AIHA (drug-induced immuneDrug-Induced AIHA (drug-induced immune HA)HA)
Serological Diagnosis of AIHASerological Diagnosis of AIHA
Warm Antibody AIHA and DATWarm Antibody AIHA and DAT
70% of all AIHA are warm type70% of all AIHA are warm type ~~80% of warm AIHA prefer oldest RBCs80% of warm AIHA prefer oldest RBCs ~20% have no preference for age of RBCs~20% have no preference for age of RBCs
Associated with IgG, (IgA), (IgM) autoantibodies:Associated with IgG, (IgA), (IgM) autoantibodies:DAT: IgG + C (67%) DAT: IgG + C (67%)
IgG only (20%) IgG only (20%) C only (13%)C only (13%)
Must use a polyspecific antiglobulin reagent for DATMust use a polyspecific antiglobulin reagent for DAT
Criteria for Diagnosis of Cold Antibody Criteria for Diagnosis of Cold Antibody AIHAAIHA
1.1. Clinical findings indicative of acquired Clinical findings indicative of acquired hemolytic anemia.hemolytic anemia.
2.2. Positive direct antiglobulin test with anti-Positive direct antiglobulin test with anti-C3d C3d ONLYONLY!!
3.3. Serum antibody optimally reactive at 4C Serum antibody optimally reactive at 4C but can react up to 30C, usually anti-I/i but can react up to 30C, usually anti-I/i specificityspecificity
Mixed AIHA
Criteria for Diagnosis of Mixed Criteria for Diagnosis of Mixed AIHAAIHA
1.1. Evidence indicative of acquired hemolytic anemia.Evidence indicative of acquired hemolytic anemia.2.2. Severe hemolysis – intravascular/extravascularSevere hemolysis – intravascular/extravascular3.3. DAT positive with DAT positive with BOTHBOTH IgG and C3d. IgG and C3d.4.4. Serum contains an IgG antibody reactive at 37C Serum contains an IgG antibody reactive at 37C
indistinguishable from warm autoantibody.indistinguishable from warm autoantibody.5.5. Serum Serum ALSOALSO contains contains LOW TITERLOW TITER antibody antibody
reactive up to 30C (usually 37C) that is optimally reactive up to 30C (usually 37C) that is optimally reactive at 4C and is IgM; often shows anti-I/i.reactive at 4C and is IgM; often shows anti-I/i.
6.6. Eluate contains IgG antibody having same specificity as Eluate contains IgG antibody having same specificity as serum antibody.serum antibody.
7.7. RESPONDSRESPONDS remarkably remarkably WELL WELL to steroid therapy.to steroid therapy.
(Shulman IA, Branch DR, et al. Autoimmune hemolytic anemia with both coldand warm autoantibodies. JAMA 253:1746-1748, 1985)
Paroxysmal Cold Hemoglobinuria Paroxysmal Cold Hemoglobinuria (PCH)(PCH)
Incidence: May be quite common in young Incidence: May be quite common in young children.children.
Acute onset of severe hemolysis with Acute onset of severe hemolysis with hemoglobinemia and hemoglobinuria.hemoglobinemia and hemoglobinuria.(hemoglobinuria very common).(hemoglobinuria very common).
Erythrophagocytosis by neutrophils is Erythrophagocytosis by neutrophils is common and is distinctive.common and is distinctive.
Donath-Landsteiner test is diagnostic.Donath-Landsteiner test is diagnostic. Patients have a stormy course but good Patients have a stormy course but good
ultimate prognosis.ultimate prognosis.
Erythrophagocytosis in PCHErythrophagocytosis in PCH RBC rosetting around neutrophils andRBC rosetting around neutrophils and erythrophagocytosis by neutrophils, rather erythrophagocytosis by neutrophils, rather than monocytes, are prominent findings in than monocytes, are prominent findings in PCH.PCH. Rarely seen in other forms of immuneRarely seen in other forms of immune hemolysis, where monocytes are usually hemolysis, where monocytes are usually involved.involved. May be first clue to diagnosis. May be first clue to diagnosis.
The Donath-Landsteiner The Donath-Landsteiner TestTest
Characteristics of Typical D-L AntibodiesCharacteristics of Typical D-L Antibodies
Biphasic HemolysinBiphasic Hemolysin IgG Immunoglobulin ClassIgG Immunoglobulin Class Anti-P SpecificityAnti-P Specificity May also react by IATMay also react by IAT
pP + complementP
DAT-negative AIHADAT-negative AIHA RareRare Often severe - fatalOften severe - fatal Sometimes anti-IgA/anti-IgM usefulSometimes anti-IgA/anti-IgM useful Mononuclear phagocyte assay may be Mononuclear phagocyte assay may be
useful – useful – use patient monocytesuse patient monocytes Often responds to steroid treatmentOften responds to steroid treatment
Criteria for Diagnosis of DAT-Criteria for Diagnosis of DAT-negative AIHAnegative AIHA
1.1. Evidence indicative of acquired hemolytic Evidence indicative of acquired hemolytic anemia.anemia.
2.2. DAT negative with anti-IgG/anti-C3dDAT negative with anti-IgG/anti-C3d3.3. No unexpected antibodies detectable in No unexpected antibodies detectable in
serum or eluate (sometimes antibody can serum or eluate (sometimes antibody can be detected in eluate)be detected in eluate)
4.4. Responds to prednisone treatmentResponds to prednisone treatment
Specificity of Warm Specificity of Warm AutoantibodiesAutoantibodies
Anti-Rh-like specificityAnti-Rh-like specificity Anti-e or anti-E (Anti-e or anti-E (SIMPLE SPECIFICITY)SIMPLE SPECIFICITY)
Anti-pdl – does not react with –D- or RhAnti-pdl – does not react with –D- or Rhnull null
Anti-dl – does not react with RhAnti-dl – does not react with Rhnullnull
Relative Rh specificity – titration studiesRelative Rh specificity – titration studies Type I or Type II AutoantibodiesType I or Type II Autoantibodies Other blood groups – Kell, Gerbich (high Other blood groups – Kell, Gerbich (high
frequency antigens)frequency antigens)
Not Usually Necessary – Academic Exercise
Relative Specificity
A Guide to Transfusion A Guide to Transfusion of Patients with of Patients with
Autoimmune Autoimmune Hemolytic AnemiaHemolytic Anemia
No Need to Hide When a Patient Presents with AIHA
Important PrinciplesImportant Principles
Indications for transfusion are not Indications for transfusion are not significantly different than for similarly significantly different than for similarly anemic patients without AIHA.anemic patients without AIHA.
Specialized laboratory procedures are Specialized laboratory procedures are necessary. necessary. Critical to look for Critical to look for alloantibodiesalloantibodies
Communication between laboratory Communication between laboratory personnel and clinicians is critical.personnel and clinicians is critical.
Provision of Safe Blood
Risks of Transfusion in AIHARisks of Transfusion in AIHA
Autoantibody will cause shortened Autoantibody will cause shortened survival of transfused RBC.survival of transfused RBC.
Alloantibodies may be present in Alloantibodies may be present in addition to the autoantibody. addition to the autoantibody. Must Must Be Identified and Antigen-Negative Be Identified and Antigen-Negative Blood Given!Blood Given!
Risks caused by the increase in RBC Risks caused by the increase in RBC mass as a result of transfusion.mass as a result of transfusion.
Blood Transfusion in Blood Transfusion in Autoimmune Hemolytic Autoimmune Hemolytic
AnemiaAnemia Blood should never be denied a Blood should never be denied a patient with a justifiable needpatient with a justifiable need,, even though the compatibility testeven though the compatibility test may be strongly positive.may be strongly positive. Probably the most common Probably the most common mistake is reluctance to mistake is reluctance to transfuse even those patients transfuse even those patients with severe anemia.with severe anemia.
Communication Between Clinician Communication Between Clinician and Transfusion Serviceand Transfusion Service
Responsibilities of Transfusion ServiceResponsibilities of Transfusion Service Initiate communication.Initiate communication. Indicate extent of compatibility testing Indicate extent of compatibility testing
performed, e.g., auto- or alloadsorption.performed, e.g., auto- or alloadsorption. Clinician should be assured that, after Clinician should be assured that, after
appropriate compatibility testing, an acute HTR appropriate compatibility testing, an acute HTR is unlikely.is unlikely.
Indicate that RBCs will provide temporary Indicate that RBCs will provide temporary benefit even if they do not survive normally benefit even if they do not survive normally because of the patient’s autoantibody.because of the patient’s autoantibody.
Communication Between Clinician Communication Between Clinician and Transfusion Serviceand Transfusion Service
Responsibilities of ClinicianResponsibilities of ClinicianIndicate urgency of situation.Indicate urgency of situation.Understand principles of compatibility Understand principles of compatibility
testing.testing.Seek assurance that appropriate Seek assurance that appropriate
compatibility testing is to be compatibility testing is to be performed.performed.
Indications for TransfusionIndications for Transfusion A common mistake is reluctance to transfuse A common mistake is reluctance to transfuse
patients with AIHA.patients with AIHA. If appropriate compatibility procedures are If appropriate compatibility procedures are
performed, survival of transfused RBCs is performed, survival of transfused RBCs is generally about as good as that of the patient’s generally about as good as that of the patient’s own RBCs. own RBCs. Alloantibodies MUST be ruled out!Alloantibodies MUST be ruled out!
Significant temporary benefit is to be expected.Significant temporary benefit is to be expected. Patients should not be denied transfusion because Patients should not be denied transfusion because
of an RBC autoantibody.of an RBC autoantibody.
American Journal of Clinical Pathology 1982; 78(2):161-167
ABO and Rh
IgG auto - Treat cells with ZZAP to remove autoantibody and retypeIgM auto – warm everything to 37C and retype or ZZAP treat and retype
“Spontaneous” Agglutination
(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/ % OF SERA REFERENCE #SERA TESTED WITH ALLOABS •Morel 8/20 40 •Branch and Petz 5/14 36•Wallhermfechtel et al 19/125 15•Laine and Beattie 41/109 38•James et al 13/41 32•Issitt et al (alloadsorptions) 13/34 38•Issitt et al (autoadsorptions) 5/41 12•Leger and Garratty 105/263 40 _______
•TOTALS: 209/ 647 32%
(Branch DR, Petz LD: Detecting alloantibodies in patients with autoantibodies. Transfusion 39:6-10, 1999) (editorial)
Adsorption ProceduresAdsorption Procedures Warm autoadsorption Warm autoadsorption using ZZAPusing ZZAP is the optimal is the optimal
procedure for alloantibody detection.procedure for alloantibody detection. One should obtain adequate volumes of patient’s One should obtain adequate volumes of patient’s
RBCs to perform the procedure.RBCs to perform the procedure. RBCs should be retained for subsequent procedures.RBCs should be retained for subsequent procedures. The number of ZZAP autoadsorptions needed is The number of ZZAP autoadsorptions needed is
variable depending on the strength of the IAT and variable depending on the strength of the IAT and the ability to reduce the DAT.the ability to reduce the DAT.
Usually 2 adsorptions is sufficientUsually 2 adsorptions is sufficient Autoadsorption is not reliable in a recently Autoadsorption is not reliable in a recently
transfused patient. transfused patient.
ZZAP!!!!!!!
Allogeneic AdsorptionAllogeneic Adsorption
1.1. If patient’s RBCs are not available for If patient’s RBCs are not available for autoadsoprtion.autoadsoprtion.
2.2. If the patient has been transfused If the patient has been transfused recently, and if the patient’s pre-recently, and if the patient’s pre-transfusion RBCs are not available for transfusion RBCs are not available for autoadsorption.autoadsorption.
Selection of Red Cells for Selection of Red Cells for Allogeneic AdsorptionAllogeneic Adsorption
R1R1R1R1 R2R2R2R2 rrrr One cell Jk(a-)One cell Jk(a-) Another cell Jk(b-)Another cell Jk(b-) Use ZZAPUse ZZAP to denature other important to denature other important
antigensantigens
Allogeneic AdsorptionAllogeneic Adsorption Advance planning is important !Advance planning is important ! Large aliquots of appropriately Large aliquots of appropriately
phenotyped RBC should be obtained, phenotyped RBC should be obtained, treated with ZZAPtreated with ZZAP, and stored at 4, and stored at 4ooC for C for up to 2 months.up to 2 months.
If such procedures are infrequent, If such procedures are infrequent, cryopreservation may be preferable.cryopreservation may be preferable.
Phenotyping patient may make it feasible Phenotyping patient may make it feasible to use fewer cells in adsorptions.to use fewer cells in adsorptions.
Single-Cell Allogeneic Adsorption
Cold Antibody AIHACold Antibody AIHA Perform compatibility tests at 37Perform compatibility tests at 37ooC.C. In a small percentage of patients, cold In a small percentage of patients, cold
autoadsorption autoadsorption using ZZAPusing ZZAP may be may be necessary.necessary.
Allogeneic adsorptions can also be Allogeneic adsorptions can also be performed, but are rarely necessary.performed, but are rarely necessary.
Use of a blood warmer may be Use of a blood warmer may be necessary and the patient’s room kept necessary and the patient’s room kept at a higher temperature.at a higher temperature.
Mixed AIHAMixed AIHA Administer corticosteroids as soon as Administer corticosteroids as soon as
possible. possible. Can be immediately effective Can be immediately effective without need for transfusion.without need for transfusion.
Compatibility testing the same as for warm Compatibility testing the same as for warm antibody AIHA.antibody AIHA.
Rule out clinically significant alloantibodies.Rule out clinically significant alloantibodies.
PCHPCH Compatibility test will appear compatible Compatibility test will appear compatible but RBC survival is not likely to be betterbut RBC survival is not likely to be better than that of the patient’s own RBC.than that of the patient’s own RBC. Autoantibody specificity generally is anti-Autoantibody specificity generally is anti- P, but it is usually inadvisable to wait for P, but it is usually inadvisable to wait for antigen-negative RBC from rare donor antigen-negative RBC from rare donor files.files.
George GarrattyLarry Petz
Don Branch
The Three Amigos
AcknowledgementsAcknowledgements Larry PetzLarry Petz Phyllis MorelPhyllis Morel George GarrattyGeorge Garratty Jean-Michel TurcJean-Michel Turc Canadian Blood ServicesCanadian Blood Services