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Unit 13 Investigation of a Positive
DAT and Immune Hemolysis
Terry Kotrla, MS, MT(ASCP)BB
Significance of Positive DAT
 Positive Direct Antiglobulin Test (DAT) does NOT mean that
RBCs will have shortened survival.
 Positive DAT without clinical problems occur in
1:1000-1:14,000 blood donors and 1-15% of hospital
patients.
 IgG and C3d Coating of RBCs
 Healthy individuals can have 5-90 molecules if IgG/RBC and 5-
40 C3d/RBC, this is below threshold of detection.
 DAT can detect 100-500 IgG/RBC and 400-1100 C3d/RBC
Causes of Positive DAT
 Autoantibodies
 Alloantibodies - HTR
 Passively acquired alloantibodies (plasma, derivatives)
 Maternal alloantibodies
 Nonspecifically adsorbed proteins or membrane
modification.
 Drug induced antibodies.
 Antibodies produced by passenger lymphocytes
 Complement activation due to bacterial infection,
autoantibodies or alloantibodies.
Autocontrol versus DAT
 Autocontrol (AC) run as part of antibody work up and is not
the same as DAT.
Autocontrol treated like screen or panel cells.
Add serum/plasma and enhancement reagent.
Incubated and taken to AHG.
May be positive due to testing procedure, may detect in-vitro
sensitization
 If AC positive perform DAT, may be negative.




 DAT cells are taken directly to AHG, no adding of serum or
enhancement, no incubation, detects IN-VIVO sensitization.
DAT
 Test freshly washed cells with antiglobulin reagents (anti-IgG and/or
anti-C3d) to detect IN-VIVO coated of RBCs.
 The 2 Fab sites on the antiglobulin molecule bind to the Fc portion of
sensitizing antibody or complement on 2 adjacent RBCs, bridging gap
and causing visible agglutination.
 Strength of agglutination proportional to amount of bound protein.
Reasons for Performing DAT
 Screen for clinically unexpected autoimmune phenomena
 Detect early manifestation of immune response to recent
transfusion.
 Assist in diagnosis of HDFN.
Collection of Blood Sample
 To verify in-vivo sensitization EDTA sample should be used.
 EDTA sample will provide RBCs for elution if necessary.
 If cold hemagglutinin suspected keep sample at 37C.
False Positive DAT
 Want to detect IN-VIVO sensitization not in-vitro.
 False positive most often associated with using refrigerated or
clotted samples.
 Any positive obtained on clotted sample should be confirmed
with EDTA sample.
 EDTA will be provide RBCs for elution if necessary.
Patient History
 Crucial to investigation.
 Is there:
 History of recent transfusion.
 Administration of drugs previously associated with immune
hemolysis.
 History of hematopoietic progenitor cell or organ
transplantation.
 Administration of IVIG or IV anti-D.
Serologic Investigation
 Perform DAT on RBCs with anti-IgG and anti-C3d to
characterize protein(s) coating RBCs.
 Test serum/plasma to detect and identify clinically significant
antibodies to red cell antigens, may have to distinguish autofrom allo-antibodies, if present.
 Prepare and test eluate from DAT positive RBCs to define
whether coating protein has red cell antibody specificity.
 Eluate from complement only coated cells should be tested if
clinical evidence of hemolysis.
 Concentrates IgG, if present, which may not be present in
patient serum/plasma.
Elution
 Removes antibody from sensitized RBCs and recovers
antibody in usable form.
 Many types of elution procedures available, no single method
is ideal in all situations.
 Thorough washing of the RBCs ESSENTIAL to ensure that
antibody detected in eluate is only RBC bound antibody.
 Last wash will detect
Elution (whenever DAT is positive)
Elution techniques “free” antibodies from the
sensitized red cells so that the antibodies can be
identified
Y
Y
Y
Positive DAT
Y
Y
Sensitized
RBC
Elution
Frees antibody
Antibody ID
Once antibody is freed from RBC test against panel to ID.
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Further Studies
 No further testing if:
 No unexpected antibodies present in serum/plasma.
 Only autoantibody detected in eluate.
 No recent transfusion
 Confirm specificity of alloantibody if present.
 If DAT positive but serum/plasma and elution studies are
negative suspect drug induced hemolysis – reference lab.
 If ABO incompatible components transfused test for anti-A
and anti-B.
 If patient an infant perform appropriate testing on maternal
sample and elution on cord cells.
Immune Hemolysis
 Causes shortened RBC survival due to immune reaction.
 Diagnosis of hemolysis :
  H&H
  reticulocytes
  LDH
  haptoglobin
  bilirubin
 Hemoglobinemia and/or hemoglobinuria may indicate acute
hemolysis.
Classification Hemolytic Anemia
 Autoimmune
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


Warm autoimmune hemolytic anemia (WAIHA)
Cold agglutinin syndrome (CAS)
Mixed type – both warm and cold autoantibodies present
Paroxysmal cold hemoglobinuria (PCH)
 Alloimmune
 Hemolytic transfusion reaction
 Hemolytic disease of the fetus and newborn
 Drug induced
 Drug dependent
 Drug independent
Warm Autoimmune Hemolytic Anemia
(WAIHA)
 Most common type of AIHA and most difficult problem
encountered in the transfusion service.
 DAT
 67% of the cases, RBCs are coated with both IgG and
complement.
 20% of the cases, RBCs are coated with IgG alone.
 3) 13% of the cases, RBCs are coated with complement alone.
WAIHA
 Serum/Plasma testing
 May have little free autoantibody, may all be on RBCs
 Once all antigen sites coated, detectable
 Approximately 50% of WAIHA will have autoantibody reactive
with ALL CELLS TESTED.
 DANGER – alloantibody may be present also
 Eluate
 Usually reactive with all cells tested.
 Negative reactions due to complement only OR presence of
drug induced antibody
WAIHA – Antibody Specificity
 May be very complex.
 Often initially directed against Rh antigen complex.
 Specificity against simple Rh or other blood group antigens
occasionally seen.
 If simple specificity, give antigen negative, if high frequency
or complex antibody, antigen negative not practical.
WAIHA – Autoadsorption - MEMORIZE
 If patient NOT recently transfused perform WARM autoadsorption.
 If RBCs heavily coated may need to pretreat with ZZAP to remove Ig.
 Incubate patient serum/plasma with patient RBCs.
 Antibody directed against patient RBCs
 Incubate at 37C for 60 minutes
 Will attach and be removed from serum/plasma
 Separate (harvest) adsorbed serum/plasma
 Test adsorbed serum/plasma against screen cells.
 Negative – autoantibody only
 Negative AND positive reactions – alloantibody present perform panel on
adsorbed serum and identify.
 All positive – procedure unsuccessful, repeat entire process with 1X
adsorbed serum.
Remove
serum and
test for
alloantibody
2
tubes
Wash x3 after
incubation
Centrifuge after
incubating; and
transfer serum to 2nd
tube of treated cells;
incubate and
centrifuge again
WAIHA
Blood Bank Investigation
Warm Autoadsorption
 To ensure that no alloantibodies are being masked by autoanibodies
Remove plasma
Patient plasma
37oC
1 hour
ZZAP
Repeat
Spin
Alloantibody
Autoantibody
Patient RBC
DAT neg
WAIHA – Transfusion Considerations
 Best to AVOID transfusing if possible.
 If transfusion essential give smallest volume of RBCs necessary to
maintain oxygen transport.
 If allo- plus autoantibody present blood must be negative for antigen to
which alloantibody is directed.
 Serologically compatible blood may not be found.
 Some facilities perform the following, be aware may lead to false sense
of security. MUST prove presence/absence of alloantibodies first!
 Crossmatch with adsorbed serum.
 Perform electronic crossmatch.
 Crossmatch with serum/plasma and select those units which react the
weakest.
WAIHA
Blood Bank Investigation
 Autoantibody may still be present
 Look for reaction strength patterns
 Issue “least incompatible” XM blood
Recipient's plasma
Donor's cells
1
Reaction
wk
2
2+
3
wk
WAIHA – Transfusion Considerations
 Patients with no apparent hemolysis do quite well.
 Difficulty is with pretransfusion testing.
 Survival of transfused RBCs is about the same as the patient’s
own RBCs.
 Patients with active hemolysis – challenging
 Transfused RBCs may be destroyed more rapidly.
 May increase hemoglobinemia and hemoglobinuria
 May cause DIC
 Transfusion is a clinical decision and should not be withheld
due to serological incompatility.
Cold Agglutinin Syndrome (CAS)
 Less common BUT most common type associated with cold
reactive autoantibodies.
 Accounts for approximately 16% of immune hemolysis.
 Occurs as acute or chronic form.
 Acute secondary to lymphproliferative diseases or Mycoplasma
pneumoniae infections.
 Chronic seen in elderly and may result in Raynaud’s phenomena
and hemoglobinuria if exposed to extreme cold.
 Often characterized by RT agglutination of EDTA cells to the
extent the sample appears clotted.
Cold Agglutinin Syndrome (CAS)
 DAT - Only complement is detected on the RBCs
 Why is complement the only protein present?
 Antibody is IgM, which in-vivo binds to RBCs in peripheral
circulation where temperature falls to 32C or less.
 IgM causes binding of complement in the cold.
 When RBCs return to warmer circulation IgM dissociates
leaving RBCs coated with complement only.
 Eluate will be negative as only complement is binding to the
RBC.
Cold Agglutinin Syndrome (CAS)
 Serum/Plasma
 IgM reactive antibody associated with immune hemolysis react
at > 30C and 60% have titers 1000> when tested at 4C.
 May show high thermal amplitude.
 Anti-I specificity, less commonly anti-i associated with IM.
 Antibody specificity NOT diagnostic as anti-I seen in healthy
patients and rarely reacts at titers above 64.
 Pre-warmed testing may eliminate reactivity.
 May cause false positive forward and reverse typing
 Forward – cells so heavily coated spontaneously agglutinate.
 Reverse cells have I antigen and will agglutinate.
CAS – Two Types of Autoadsorptions
 Rabbit Erythrocyte Stroma Test (REST)
 Cold auto adsorpiton
Rabbit Erythrocyte Stroma Test (REST)
 For patients who have been recently transfused.
 Rabbit cells have I antigen.
 Procedure
 Incubate patient serum/plasma with rabbit stroma at 4C.
 I antibody absorbed out.
 Remove (harvest) serum/plasma
 Test absorbed serum against screen cells.
 Use with caution: may adsorb out clinically significant
antibodies to D, E, Vel antigens and IgM antibodies regardless
of specificity.
Cold Autoadsorption Test
 CANNOT be performed on recently transfused patients.
 Collect EDTA sample, keep warm.
 Separate plasma from RBCs.
 Wash RBCs with warm saline.
 Add aliquot of plasma/serum to RBCs incubate at 4C for 1
hour.
 Harvest serum/plasma and test against screen cells.
 Negative – no alloantibody.
 Positive and negative – alloantibody present, run panel.
 All positive, unsuccessful, repeat with 1X adsorbed sample.
CAIHA
Blood Bank Investigation
Cold Autoabsorption
Spin
Alloantibody
 autoantibody
Supernatant used for
IgM
Warm
saline
Wash
Spin
Spin
Incubate patient’s
cell and serum @ 4oC
 Ab screen
 Reverse grouping cells
 Autocontrol
 Limitation: Will not remove a high titer cold agglutinin completely
CAS – Transfusion Considerations
 Rarely require transfusion.
 Use prewarmed technique for compatibility tests or use cold
autoadsorbed serum.
 Transfuse blood through blood warmer.
Warm versus Cold Autoantibodies
WARM
 Reacts at 37C
COLD
 Reacts at RT or below
 Broad antibody specificity
 Titer > 1000
 IgG and C3d coating RBCs
 Complement only coating RBCs
 Insidious to acute
 Anti-I most common
 Anemia severe
 Fever, jaundice frequent
 Intravascular not common
 Splenomegaly
 Hematomegaly
 Adenopathy
 Use great caution when transfusing.
 Often chronic anemia
 9-12 g/dL (less severe)
 Autoagglutination
 Hemoglobinuria, acrocyanosis and
raynaud’s with cold exposure
 No organomegaly
 Transfusion well tolerated, use
blood warmer
Mixed Type Autoantibodies
 One-third of patients with WAIHA have nonpathologic IgM
antibodies reactive at RT
 Further divided
 Patients with high-titer, high thermal amplitude IgM
 Patients with normal titer (<64), high thermal amplitude IgM
 Often present with hemolysis and complex serum reactivity
present in all phases of the testing.
 DAT
 Both IgG and C3d detectable.
 IgG due to warm autoantibody.
 C3d bound due to IgM autoantibody
Mixed Type Autoantibodies
 Serum/Plasma
 Cold – can have anti-I or-i, but often has no specificity
 Warm – serologically indistinguishable from WAIHA
 Eluate activity indistinguishable from WAIHA.
 Adsorptions – may be necessary to perform at both 37C
and 4C.
 Transfusion considerations identical to those described
for WAIHA.
Paroxysmal Cold Hemoglobinuria (PCH)
 DIFFERENT from Paroxysmal Nocturnal Hemoglobinuria in
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
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which hemolysis is caused by acid produced during sleep.
RAREST form of DAT positive AIHA.
Presents as acute transient condition secondary to viral
infections particularly in young children.
Caused by a biphasic hemolysin which induces hemolysis
after exposure to cold.
Results in hemoglobinuria and hemoglobinemia.
PCH – DAT and Eluate
 DAT
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Autoantibody is IgG which acts as cold agglutinin.
IgG binds to RBCs in colder parts of body.
Causes complement to be bound irreversibly.
IgG elutes off of RBCs in warmer parts of the body.
ONLY COMPLEMENT is detected.
 Eluate
 Since only complement coats cells eluate is negative.
 Antibody specificity
 Anti-P
 Not necessary to transfuse rare P negative blood.
PCH – Serum/Plasma Testing
 Autoantibody described as “biphasic hemolysin”.
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Binds to RBCs at low temperatures.
Binds complement.
As cells warm up hemolysis occurs.
Basis of Donath-Landsteiner test.
 Donath-Landsteiner
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Usually add fresh complement
Antibody binds at cold temperatures
Test is warmed to 37C.
As warming occurs complement is activated and lysis of RBCs
occurs.
Donath Landsteiner Test
30’@4ºC
60’@37 ºC
90’@4 ºC
90’@37 ºC
Patient
Serum
+
-
-
Patient Serum
+
-
-
-
-
-
Normal fresh serum
Normal Fresh
PCH – Donath Landsteiner Test
Drug Induced Hemolytic Anemia
 VERY RARE - 1 in a million chance.
 May induce formation of antibodies either against the drug or red cell
antigens.
 Drugs may act as haptens once firmly bound to RBC.
 Four theoretical mechanisms
 Drug adsorption
 Immune complex formation
 Autoantibody production
 Non-specific protein adsorption
 NOTE: Newest edition of Technical Manual mentions the following
categories but also categorizes by activity. Most references still contain
the categories listed above.
Drug Adsorption
 Approximately 3% of patients receiving large dose IV penicillin will
develop positive DAT.
 Less than 5% of these will develop hemolytic anemia.
 Mechanism
 Drug adsorbed onto RBC in-vivo, becomes a hapten.
 If patient develops antibodies to penicillin they will bind to penicillin on
RBC.
 Hemolysis occurs extravascularly.
Drug Adsorption – Lab Features
 DAT strongly positive due to IgG coating.
 Serum/Plasma
 Antibody screen negative unless alloantibodies
coincidentally present.
 High titer IgG penicillin antibodies present will only react with
drug coated RBCs in-vitro.
 Eluate reactive with drug coated RBCs only – reference lab.
 Hemolysis is subacute at onset but may become life-
threatening if not recognized in time.
 Discontinue penicillin.
Immune Complex Formation
 Drug binds with antibody in-vivo, in the plasma.
 Immune drug-antibody complex binds to RBC and initiates complement




activation.
Leads to INTRAVASCULAR hemolysis.
After RBC hemolyzed drug-antibody complex dissociates and attaches
to new RBC.
Small amounts of drug can results in acute hemolysis.
Piperacillin (synthetic penicillin), second and third generation
cephalosporins and anti-ceftriaxone have been implicated.
Immune Complex Formation
 DAT – only complement is present on RBCs.
 Serum/Plasma
 Antibody screen negative unless alloantibody present.
 Test serum against RBCs in presence of drug, may cause
sensitization, agglutination or hemolysis
 Eluate – negative, since only complement is coating the
RBCs.
Autoantibody Production
 Serological findings indistinguishable from those associated
with WAIHA.
 First case was alpha methyldopa, L-dopa has also been
implicated as well as other drugs closely related to alpha
methyldopa.
Autoantibody Production
 DAT is Positive in approximately 15% of patients receiving alpha
methyldopa.
 Only 0.5 - 1.0% of patients taking alpha-methyldopa develop hemolytic
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
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
anemia.
RBCs are usually coated only with IgG, but occasionally weak complement
coating is also present.
DAT usually becomes positive only after 3-6 months of therapy.
Development of a positive DAT is dose-dependent; approximately 36% of
patients taking 3 g of the drug daily develop a positive DAT compared with
11% of patients receiving 1 g per day.
Strength of the positive DAT becomes progressively weaker once alphamethyldopa therapy is discontinued.
 May take from 1 month to 2 years.
 Patients with hemolytic anemia due to alpha-methyldopa therapy improve within
the first week or so after drug therapy discontinued.
Autoantibody Production
 Serum/Plasma – reactive with all cells tested.
 Eluate reactive with all cells tested.
 KEY IS HISTORY – patient taking medication known to cause
autoantibody production.
Laboratory Investigation
 When patient has positive DAT history MUST include
medications.
 Perform DAT to classify coating protein.
 Perform elution if IgG coating detected.
 Test patient serum/plasma for unexpected antibodies.
 If drug antibodies suspected send to reference lab.
 Reference lab has ability to coat cells with drug and/or
introduce drug into test system.
References
 http://www.nzma.org.nz/journal/122-1301/3749/
 AABB Technical Manual 16th edition, 2008
End of Unit 13