BB Lecture 13
Antibody Production
AUTOIMMUNE HEMOLYTIC ANEMIA
(AIHA)
 Defined as shortened RBC survival mediated by the
immune response, with the red cells particularly
destroyed by humoral antibody
 THREE CATEGORIES/ CLASSIFICATIONS:
1. Alloimmune Patient produces antibodies to foreign
or non-self RBC antigens; introduced
through transfusion, transplant or
pregnancy
2. Autoimmune Patient produces antibodies against
his/her own RBC antigens
3. Drug-induced Result of a patient’s antibody
production to a particular drug or drug
complex, with resulting damage to RBCs
 Proper recognition and investigation of both
alloantibodies and autoantibodies are important in both
diagnosis of immune hemolytic anemia and proper
selection of blood components for transfusion
Autoantibodies
 Antibodies that are directed against the individual’s own
RBCs
 Also called “Autoagglutinins”
 Most react with high-incidence RBC antigens
 Agglutinate, sensitize or lyse RBCs of most random
donors, as well as their own
 Some may readily attach to individual’s own RBCs without
necessarily causing RBC destruction
 Approximately 1 in 1,000 healthy blood donors will have
positive Direct Antiglobulin Test (DAT) but will have
normal hematocrit and be symptomatic
** Notice that there is a NORMAL FEEDBACK MECHANISM to
where the blue arrow is pointed at **
 Antibody production vs. Self-antigens occurs when
immune regulatory responses fail
□ Suppressor T cells induce tolerance to self-antigens -
by inhibiting B cell activity
□ Loss of suppressor T cell function
□ Autoantibody production
 Investigating autoantibodies:
□ In individuals with no proper history of blood
transfusion (BT), presence of a positive DAT, a
positive autocontrol or serum autoantibody
 May not be indicative of AIHA
 But may indicate presence of autoantibodies
 We must verify the presence of immune-
mediated RBC destruction; which is not always
accompanied with decreased Hb and Hct levels
dues to initial compensation
 RBC destruction causes:
□ Increase in Reticulocyte count
 NV: 0.5-1.5%
 AIHA: >3%
□ Increase in Lactate dehydrogenase (LDH)
 NV: 140-280 U/L
 ↑ unconjugated bilirubin
□ Decrease in Haptoglobin levels
 NV: 30-200 mg/dl
□ Mild decrease in Hb and Hct levels
COMPENSATED ANEMIA:
 Rate of RBC production = rate of RBC destruciton
UNCOMPENSATED ANEMIA:
 Rate of RBC destruction > RBC production
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BB Lecture 13
 Blood picture of Hemolytic anemia
□ Macrocytosis - Characterization of AUTOANTIBODIES in AIHA
evidence of young
cell population
□ Spherocytosis -
evidence of cell
membrane damage
□ With intravascular
RBC destruction,
hemoglobinemia
and hemoglobinuria
may occur

Confirming AIHA

Diagnostic Tests fo 1. DAT - using polyspecific and Warm reactive - 70%

Symptomatic Patients: monospecific antiglobulin reagents Cold reactive - 18%
Drug-induced - 12%
2. Characterization of autoantibodies
in the serum or eluate using standard
Ab detection and ID procesures Cold Reactive Autoantibodies
Other causes of  Hereditary spherocytosis
Hemolysis:  Hemoglobinopathies Characteristics of normal (Benign) Cold and Pathological Cold
 RBC enzyme defects Autoantibodies
Characteristic Normal (benign) Pathological
Thermal range Reactive ≤ 20-24℃ Reactive ≥ 30℃

Titer at 4℃ ≤ 62 ≥ 1,000
Affected vs Unaffected
Reactivity Marginally Strongly enhanced
 US population studies: enhanced with with albumin
albumin
□ 0.1% or 1 in 1000 of normal blood donors and
□ up to 15% of hospitalized patients (using
Common specificity Anti-I or anti-IH Anti-I
polyspecific antiglobulin reagent) have positive
DATs with no evidence of hemolytic anemia Capable of binding Yes (in vitro); IgM Yes (in vivo)
complement DAT: 0-1+ due to C3 DAT: 2-4+ due to C3
CONTRIBUTING FACTORS: Clinically significant No Yes
 Thermal amplitude of Ab reactivity
Associated with No Yes - may be
 IgG subclass of antibody
disease secondary to viral
 Amount of Ab bound to RBCs
infections of
 Ability of Ab to fix complement in vivo
M. pneumoniae
 Activity of individual’s macrophages
 Quantitative or qualitative change in band 3 and
proteins 4.1 and 4.2 in RBC membrane structure

Laboratory Tests Affected by COLD AUTOAGGLUTININS

Unafected vs Affected
 Some patients with hemolytic anemia may not show
autoantibodies by routine techniques
 Patients may have more IgG than normal on their RBCs,
but less than the amount detectable by routine
antiglobulin test
 Patient’s RBCs may be sensitized with anti-IgA or anti-IgM,
not routinely detected using commercial antiglobulin
reagents
1. ABO typing
 RBCs are heavily coated with cold agglutinins may directly
agglutinate and cause false-positive results
 Valid results can be achieved by washing cells once or
twice with NSS warmed to 37 ℃
 Cold autoantibody is eluted from the cells during warm
washing
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BB Lecture 13
Group O cells coated with cold autoAb: Anti-A Anti-B
Serum-suspended RBCs 1+ 1+ 3. Direct antiglobulin

Warm-washed, saline-suspended RBCs 0 0

 In vitro sensitization of RBCs is usually weak; ≤1
 With monospecific reagents, cells are reactive with anti-
 If more potent autoagglutinins are present, incubate C3d but not with IgG
patient’s whole blood sample at 37 ℃ prior to warm  A negative control of 6% albumin or saline is
washing recommended for DAT, especially when there are
□ Or use waterbath or heat block to 37 C for 15-30 discrepancies with ABO or Rh typing
minutes
 Warm incubation - elutes the cold agglutinin from the
patient’s RBCs, and warm washes remove it to prevent
from reattaching in vitro
□ May use thiol reagents if warm saline is not
effective
 Discrepancies in serum ABO (reverse) typing are resolved
if the cold reactive autoantibody is removed by
autoadsorption, and tests with A1, B, O and autologous
cells are repeated with atuoadsorbed serum 4. Antibody detection & Identification

Typical reactivity observed with patient serum containing a

2. Rh(D) typing clinically significant Alloantibody (anti-Fya) and Cold Agglutinin

 False-positive reactions were commonly seen with RCSs tested Standard Prewarmed Standard
polyclonal high-protein anti-S reagent antiglobulin antiglobulin antiglobulin
□ Anti-D and Rh control both positive testing w/ testing w/ testing w/
□ Invalid test Polyspecific Polyspecific Anti-IgG
 Valid results seen with the use of: AHG AHG
□ Combination of monoclonal & low-protein anti-D Fy(a+b-) 2+ 2+ 2+
reagents
□ Washing the cells with warm saline; thiol reagents Fy(a+b+) 2+ 2+ 2+
 Cold reactive IgM autoagglutinins can activate the
complement cascade in vitro
Fy(a-b+) 1+w 0 0
□ Complement binding to RBC surface lead to false-
positive reactions in weak D (antiglobulin) test
□ If cells from clotted sample and polyspecific Atuo control 1+w 0 0
antihuman serum are used
 The use of anti-IgG antiglobulin reagent or a sample
collected in EDTA is recommended when cold
 Cold agglutinins react best at 4℃ but are generally not
autoagglutinins are present
detected because routine antibody screening is no longer
performed at this temperature.
Anti-D Rh Control
 Antibodies reactive only at room temperature are
Immediate spin phase 0 0 considered to be clinically significant
Indirect antiglobulin phase 1+ 1+  Reactivity caused by cold autoagglutinins: can mask
(poly AHG) the presence of clinically significant alloantibodies
 Using anti-IgG antiglobulin reagents - will eliminate
Indirect antiglobulin phase 0 0
most problems with cold autoagglutinin reactivity in
(anti-IgG)
the IAT phase
RBCs collected in EDTA 0 0  To thoroughly investigate reactivity observed in
antiglobulin testing, it may be necessary to remove
the cold reacting autoantibodies by cold adsorption
procedures

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5. Compatibility testing Reactivity of Cold Agglutinins at 4℃ with ABO-Compatible

RBCs
 Discrepancies are noted because the most common RBC ANTI-I ANTI-i ANTI-H ANTI-IH ANTI-
encountered cold autoantibody (autoanti-I) is directed vs PHENOTYPES Prb
an antigen that is found on the RBCs of most random Group O Iadult 4+ 0 - 1+ 4+ 4+ 4+
donors and on most reagent RBCs
Group A2 Iadult 4+ 0 - 1+ 0 - 1+ 0 - 1+ 4+
 Perform using:
□ Prewarming serum Group A2 Iadult 4+ 0 - 1+ 2+ 2+ 4+
□ Autoadsorbed serum
Bombay Oh 4+ 0 - 1+ 0 0 - 2+ 4+
□ Allogenic adsorbed serum
Iadult
□ Anti-IG antiglobulin reagent
Group O iadult 0 - 1+ 4+ 4+ 0 - 1+ 4+

Group O icord 0 - 1+ 4+ 4+ 0 - 1+ 4+
Specificity of Cold Autoagglutinins
Group A1 iadult 0 - 1+ 4+ 0 - 1+ 0 4+

Group A2 iadult 0 - 1+ 4+ 2+ 0 - 2+ 4+
Anti-I, Anti-i Anti-H, Anti-IH
Bombay Oh 0 - 1+ 4+ 0 0 4+
 At birth, an infant’s RBCs  Group O and A2 cells iadult
express the i antigen react best because they Group O Iadult 4+ 1+ - 2+ 4+ 4+ 0
 As infant matures, Ag have the largest amount ficin-treated
expressed changes from i of H antigen
to I antigen  Group A1 and A1B cells  Other Cold reactive Autoagglutinins:
 Amount of I antigen react weakly because □ Anti-Pr
increases by age 2 they have the least H □ Anti-Gd
 Most cold reactive antigen □ Anti-Sdx (anti-Rx)
autoantibody have anti-I  Anti-IH is found more
specificity often in serum of A1 and
A1B individuals
 Agglutinates RBCs
Pathological Cold Autoagglutinins
that have both I Cold Hemagglutinin Disease
and H antigens (Idiopathic Cold AIHA)
 Group O and A2
cells also react best  Most cold autoagglutinins do not cause RBC destruction,
but in some patients they may cause HA in varying
Typical reactivity observed at 4℃with serum containing degrees
autoanti-I with Iadult and icord cells Chronic, Idopathic  No identifiable cause
Serum Serum Iadult cells icord cells
dilution Acute, Transient disorder  M. Pneumoniae infection
Benign Cold Neat 3+ 1+w  Infectious mononucleosis
Cold agglutinin syndrome  Other names:
1:2 1+ 0  Cold hemagglutinin
1:4 1+ 0 disease (CHD)
 Idiopathic AIHA
1:8 1+w 0  Comprises 18% of cases
Pathological Neat 4+ 3+ of AIHA
Cold  Considered a moderate
1:2 4+ 2+ chronic HA
 Produced by a cold
1:4 4+ 1+
autoAb that reacts at
1:8 3+ 0 4℃
 Also at 25-30 ℃
 IgM; activates
complement

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BB Lecture 13

Cold Agglutinin Syndrome Pathological Cold Autoagglutinins

Cold Autoantibodies Related to Infection
 Seen in older individuals; (Secondary Cold AIHA)
peak >50 y/o
 Almost always anti-I Offending cold autoAb: IgM w/ anti-I specificity
 Acrocyanosis of hands, feet
ears, nose; slight numbness  Episodes of cold AIHA often follow an upper respiratory
in the extremities infection (URI)
 Changes occur with □ For both primary and secondary
exposure to cold  50% of patients with M. pneumoniae pneumonia have
 Involves weakness, pallor, cold agglutinin levels >64
weight loss  CHD occurs in the 2nd or 3rd week of illness
 Patients with CHD live more □ Rapid onset of hemolytsis is seen
comfortably in warmer climates □ Pallor, Jaundice
 LABORATORY FINDINGS:  Resolution within 2-3 weeks when the infection subsides
□ Reticulocytosis □ Self-limiting hemolysis
□ Positive DAT - autoagglutination in whole blood  Cold agglutinins occus as an immunologic response to
samples mycoplasma antigen
□ Peripheral Smear:
 Agglutinated RBCs  TREATMENT for CHD:
 Polychromasia □ Generally unnecessary
 Anisopoikilocytsos □ Avoid the cold, keep warm, move to milder climate
□ Tolerate symptoms of (moderate) anemia, rather
than take drugs
□ Plasma exchange therapy in more severe cases
 Clinical Criteria for the DIAGNOSIS of CHD:  This is to maintain low levels of
□ Clinical signs of an acquired HA, with history of autoagglutinating antibodies
acrocyanosis and hemoglobinuria on exposure to
cold Paroxysmal Cold Hemoglobinuria (PCH)
□ (+) DAT - using polyspecific antihuman sera
□ (+) DAT - monospecific anti-C3 antisera  Least common type of AIHA
□ (-) DAT - using monospecific IgG antisera □ 1 - 2%
□ Presence of reactivity in patient’s serum due to cold  Often in children with prior viral illness
autoantibody □ Measles
□ Cold agglutinin titer of 1,000 or greater in saline at □ Mumps
4C with visible agglutination of anticoagulated blood □ Chickenpox
at room temperature □ IM
□ Ill-defined flu syndrome
 Selection of blood for Transfusion  Formerly associated with
□ Most patients with CHD do not require blood Treponema pallidum
transfusion □ However, this is no longer
□ Potent cold autoAb interfere with most routine tests reported due to effective
□ Method of choice: COLD AUTOADSORPTION treatment of syphilis
□ For patients undergoing surgical procedures that
use hypothermia (lowering of body temperature to
PCH involves:
22-30 C), blood can be warmed by approved blood
1. RBC destruction caused by: Cold autoanibodies
warmer, or perform procedure without
2. A Biphasic autohemolysin (IgG) that:
hypothermia
a) binds to patient’s RBCs at lower temp and
Not necessary for patients with benign cold
b) fixes complement


autoagglutinins
3. It is associated with Donath-Landsteiner Ab which is an
autoAb with anti-P specificity

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BB Lecture 13
 DONATH-LANDSTEINER TEST: used to confirm diagnosis
of PCH Reactions of a positive Donath-Landsteiner Test
 Hemolysis - occurs when sensitized RBCs circulate and Incubation Tubes 1 px Tubes 2 px Tubes 3
are exposed to 37C and sensitized cells undergo phases serum serum + normal
complement-mediated intravascular lysis (t A1, B1, C1) normal seum serum
(t A2, B2, C2) (t A3, B3, C3)
DONATH-LANDSTEINER TEST Ice bath ff by + + 0
37C (all A
tubes)
 Collection of fresh blood sample; maintained at 37C;
Ice bath only 0 0 0
serum separated
(all B tubes)
 Incubate 3 sets of 3 test tubed containing patient’s
37C (all C 0 0 0
serum at various temperatures with group O RBCs that
tubes)
express P antigen, and labeled:
 A1-A2-A3, B1-B2-B3, C1-C2-C3
 Paroxysmal or intermittent episodes of hemoglobinuria
 Tubes 1 and 2 of each set contain 10 drops of patient’s
occur open exposure to cold
serum
 Tubes 2 and 3 of each set contain 10 drops od fresh
Acute attacks  FF by sudden onset of:
nromal serum (complement source)
 Fever, shaking chills malaise
 One volume of 50% suspension of washed P+ RBCs is
 Abdominal cramps and back
added to each tube, and all tubes are mixed
pain
 Place the 3 A tubes in a melting ice bath for 30 mins,
IV hemolysis  Hemoglobinemia
and then for 1 hour at 37C (biphasic incubation)
 Hemoglobinuria
 Place the 3 B tubes in a melted ice
 Bilirubinemia
 Keep the 3 C tubes at 37C for 90 minutes
Severe and rapidly  Hb levels as low as 4-5 g/dl
 The rubes are then centrifuged and supernatant fluids
progressive anemia
are examined for hemolysis
Peripheral smear  Polychromasia
 NRBC
 Poikilocytosis
+/- Splenomegaly  Hyperbilirubinemia
 Renal insufficiency

 TREATMENT of PCH:
□ Protection from cold exposure in chronic forms

FACTORS PCH CHD

Patient Children & Elderly or middle-aged
population young adults adults
Pathogenesis Ff viral infection Idiopathic,
lymphoproliferative d/o; ff
M. pneumoniae infection
Clinical Hbinuria, acute Acrocyanosis;
features attacks upon autoagglutination of blood
exposure to at RT
cold (self-
limiting)
Severity of Acute and rapid Chronic and rarely severe
hemolysis
Site of Intravascular Extravascular/intravascular
TO INTERPRET A POSITIVE D-L TEST hemolysis
 If biphasic hemolysin is present, tubes 1C Autoantibody IgG (anti-P IgM (anti-I,i) monophasic
and/or 2C will be hemolyzed. class specificity;
biphasic
 All other tubes will not be hemolyzed.
hemolysis)
DAT 3-4+ 3-4+ monospecific C3 only
monospecific
C3 only
Thermal range Moderate High (up to 30°C-31°C)

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BB Lecture 13
(<20°C)  Peripheral smear:
Titer Moderate (<64) High (>1,000) □ Polychromasia
Donath- Positive Negative □ Macrocytosis
Landsteiner
□ NRBC
test result
Treatment Supportive (d/o Avoid cold □ Spherocytosis
terminates □ RBC fragmentation
when
underlying
illness resolves)
** d/o - disorder; ff - following
RBC Hemolysis

Warm Autoantibodies

 React best at 37 C
 Not found often in random population compared to cold
autoanti-I
 Comprises 70% of AIHA
 Mostly harmless antbodies, but these are
indistinguishable from the harmful ones
□ Should be reported
 IgG1 predominating (87%)
 Presence may alert physician to an underlying immune
□ Strength: IgG3 .> IgG1 > IgG2 > IgG4
process
 Nancy and Garratty: the strength of the DAT correlated
with the presence of multiple IgG subclasses on the RBCs,
Clinical Findings
which in turn correlated with the severity of hemolysis
 The spleen is 100x more efficient than the liver in
 Variable extent of anemia
removing IgG-sensitized RBCs (extravascular)
 Anemia of sufficient severity to require blood transfusion
 Macrophages have two biological receptors on their
in some
membranes:
□ Hb <7 g/dl are not uncommmon
1. Receptors for Fc fragments of IgG1 and IgG3
 Onset may be insidious
2. Receptors for C3b fragment of complement
 Triggers:
□ Infection, Trauma, Surgery, Pregnancy, Underlying
 Factors affecting the Activity of Macrophages:
disease
□ Subclass of IgG, especially IgG1 & IgG3
 Onset may be sudden and unexplained
□ Presence of complement (C3b) fragments
 Idiopathic or secondary to a pathologic disorder
□ Quantity of immunoglobunlin or complement
□ Number and activity of :
Diseases frequently associated w/ WAIHA
 Helper T cells (CD4)
 Suppressor T cells (CD8)
 RE neoplasms - CLL, HD, NHL, MF, MDS
 Collagen disease - SLE, RA
Serologic characteristics
 Infectious diseases
 Immunologic diseases
 Do not directly agglutinate saline-suspended RBCs after
 GI diseases
37C incubation
 Carcinoma (non-ovarian)
 May activate complement
 Pregnancy
 Usually enhanced by enzyme techniques
 CRF
 Both clinically significant alloantibodies and
autoantibodies react best at the indirect antiglobulin
Signs and Symptoms
phase
 More complicated and time-consuming procedures for
 Presenting complaints:
resolving the problems may have to be used
□ Pallor, weakness, dizziness, dyspnea, jaundice,
unexplained fever

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BB Lecture 13
Laboratory tests Affected
1. ABO typing
 Not usually affected since most warm autoantibodies are
not direct agglutinins
2. Rh(D) typing
 False-positive Rh typing - occurs when patient’s cells are
coated with immunoglobulin
 Monoclonal antisera - have low incidence of false-
positive test results
 It is not absolutely necessary to determine the correct
Weak D typing of patients with WAIHA since D negative
[Rh(-)] RBCs can be transfused, if needed
3. Direct Agglutination Test
 Positive in WAIHA
 As autoantibody is produced, it absorbs onto the antigen
of that defined specificity present on the patient’s own
RBCs
□ RBCs are coated with IgG w/ or w/o complement, or
the latter alone (..so w/o complement alone)
4. Antibody Detection and Identification
 There is a need to confirm that antibody coating the
patient’s cells is an autoantibody, with little effort needed
to determine its specificity
 The main goal of additional testing:
□ Is to detect and identify all clinically significant
alloantibodies that might be masked by the
autoantibody
5. Evaluation of Autoantibody
 Using patient’s medical history, an accurate history of
previous blood transfusions and pregnancies, diagnoses
and medications
 To identify specificity of warm reactive autoantibody, an
eluate prepared from the patient’s RBCs must be tested,
including patient’s serum with a panel of reagent RBCs
 Warm autoantibodies in serum - may show weak anti-e
specificity
 While the eluate - may show pan-agglutination of all
RBCs tested
 If patient has NO HISTORY w/in the past 3 months, with
no evidence of mixed-cell population, then antibody in
aluate is autoantibody
 WITH HISTORY or RECENT blood transfusions or
pregnancy, the serum may contain alloantibody +
autoantibody
6. Detection and Identification of Alloantibodies
 When autoantibody is detected in serum, it will typically
mask any alloantibodies present:
1. If autoantibody demonstrates a simple specificity,
such as anti-e
a) Test a panel of cells negtaive to the
corresponding Ag and positive for all common
clinically significatnt RBC antigens (Rh, Kell,
Duffy, Kidd, S and s) to rule out presence of
underlying alloantibodies
2. In patients with NO RECENT blood transfusion in the
last 3 months, determine the presence of RBCs
phenotype using either monoclonal antisera or
using patient’s RBC treated with agent known to
remove coating IgG
3. May need to determine patient’s phenotype using
reticulocyte harvesting procedure if autoadsorption
technique is not possible
4. If not possible to determine patient’s RBC by
phenotyping, differential allogenic adsorptions
a) Strength of reactivity in the indirect
antiglobulin phase of antibody screen or panel
is a good indicator of how many adsorptions
will be necessary
Things to consider when performing autoadsorption:
 Not recommended in those who have had blood
transfusion the past 3 months
 May be difficult to obtain sufficient autologous
cells for multiple adsorptions in severely anemic px
 Weakly reactive alloantibody - could be diluted
and missed if multiple adsorptions are done
 May absorb an alloantibody to a high-incidence
RBC antigen present on the allogenic cells, but not
in the patient’s own red cells
Selection of Blood for Transfusion
 Many patients with WAIHA never require blood
transfusions
□ This is only reserved for severe anemia or for certain
surgical procedures
 For simple specificity (anti-e), select donor units that are
negative for corresponding antigen
□ Finding compatible units for patients with broad
specificity warm autoagglutinin is difficult
Treatment
 Treat underlying disorder, if present
 Supportive (eg. Cardiovascular) measures are important
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BB Lecture 13
Blood transfusions are AVOIDED, in order to prevent

further/accelerated hemolysis; reserved for life-
threatening anemia
□ Volume of RBC transfused should be conservative
□ AIM: relief of symptoms, not restoring a normal Hct
TREATMENT
CORTICOSTEROID and IV  Initial high doses of 100 to 200
Ig mg maintained until Hct
stabilizes; withdrawn slowly over
2-4 months
 Mode of action:
 Reduction of Ab synthesis
 Altered Ab activity
 Alteration of macrophage
receptors for IgG and C3,
which reduces the
clearance of Ab-coated
RBCs
 Beneficial in 50-67% of WAIHA
 Danazol and Iv Ig - for
Prednisone-resistant cases
SPLENECTOMY  Needs clinical evaluation and
judgement
1. Failure of steroid therapy
2. Need for continuous high-
dose steroid maintenance
3. Complications of steroid
 Causes decreased production of
Ab and removes a potent site of
RBC damage and destruciton
 Beneficial in 60% of WAIHA, with
steroid dosage >15 mg/day to
maintain remission
IMMUNOSUPPRESSIVE  Last approach
DRUGS  Azathioprine (Imuran) and
Cyclophosphamide
 Interfere with antibody synthesis
by destroying dividing cells
 Adverse effects: infection,
infertility, risk of birth
 Defects, development of
malignancies
 Cyclosporin associated with renal
damage
 Rituximab - associated with
adverse effects with long-term
use
Mixed-Type Autoantibodies
 Antibody activity (not mere presence) with both warm
and cold components
 RARE
 Thermal amplitude of cold autoAb (IgM) key to
pathogenicity; warm is IgG type
 Extremely acute hemolysis and frequently require blood
transfusions
 Must include both cold and warm adsorptions to
completely remove autoagglutinins
 TREATMENT: Corticosteroids
IgM Warm Autoantibodies
 Patient’s RBCs are strongly coated with complement and
frequently cause discrepancies with ABO/Rh typing and
DAT due to spontaneous agglutination
□ Cannot do warm washing since Ab is warm
agglutinin
 Treatment of RBCs with 0.01M DTT - to remove enough
IgM for valid ABO/Rh and DAT results
 Flow cytometry - is ideal fo detecting IgM
 Poor prognosis - if anemia is sever (Hb <5 g/dl)
 TREATMENT: Plasma exchange, IVIG, Steroids, Blood
transfusions
DAT Negative AIHA
 Amount of IgG coating the red cells is lower than the
detectable limit of commercial IgG reagents
 May order “super Coombs” test or “DAT hemolytic
anemia workup”
 Progress with the following:
□ Cold saline/saline LISS wash - prior to DAT testing
 This is to detect low-affinity IgG lost if washing
done at room temp
□ Direct polybrene DAT
□ DAT testing - with anti-IgA and anti-IgM
□ DAT - by solid-phase or column agglutination
 Flow cytometry, ELISA, monocyte monolayer assays
(MMAs), concentrated eluated
Comparison of WARM and COLD AIHA
Factors Warm AIHA Cold AIHA
Optimal reaction >32 C <30 C
tempetature
Ig classification IgG IgG
Complement May bind complement Binds complement
activation
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Site of hemolysis Usually extravascular Extravascular/ Drug-Adsorption (Hapten) Mechanism
(no cell lysis) Intravascular (cell
lysis)  Drug binds firmly to proteins, including those of RBC
Frequency 70-75% of cases 16% of cases membrane
PCH (1-2%)  Good immunogens
Specificity Frequently broad Rh Ii system  Ab to Penicillin (often IgG)
specificity PCH autoanti-P  Cells from patients with (+) DAT - are usually coated with
IgG alone
 Patient’s serum and eluate are non-reactive with reagent

Drug-Induced Immune Hemolytic RBCs and random donor cells

Anemia (DIIHA)

 Suspected when:
□ Requested for diagnostic testing on a patient
suspected of hemolytic anemia
□ Unexpected results in routing testing
 Positive autologous control in antiglobulin
phase of Ab screening or compatibility testing  Ab screen - NEGATIVE
or (+) DAT  Crossmatches - compatible in all phases
 DIIHA is very rare (1 in 1 million) → that’s why we have to
rule out other potential cases first Cefotetan-induced hemolysis
 Drugs should be suspected as a possible explanation for □ Most frequently encountered DIIHA >50%
immune hemolysis or (+) DAT when there is no other □ IV lysis of RBCs; profound anemia 7-10 days
reason for the serologic and hematologic findings, and if □ Severe anemia (Hb ~ 4 g/dl)
the patient has a recent history of taking high doses of
drugs associated with DIIHA
Drug-dependent or Immune Complex
3 Mechanisms supporting the Unifying hypothesis “Innocent Bystander” Mechanism

1. Drug binds to RBC membrane and Ab reacts with the 1. Patient ingests the same drug after immunization
drug a) Or a drug bearing the same haptens
 Test drug-coated RBCs 2. Formation of a drug-antidrug complex
2. Drug complexes with drug Ab 3. Complement cascaded may be activated
 Testing in the presence of the drug 4. RBCs as “innocent bystanders”
3. Drug induces an autoimmune response but Ab is 5. Soluble drug-antidrug complex nonspecifically adsorbs
directed at the RBC membrane loosely to the RBC surface
 Test cannot differentiate between drug-induced or
idopathic Neoantigen formation
Drug interacts noncovalently with a specific membrane
Drug-related DATs component to form a new antigen determinant

Drug-dependent
 Require presence of drug
in order to react
Drug-independent
Membrane Modification
 Mimic warm autoAbs
Nonimmunologic Protein Adsorption
serologically
 But where the formation of
 Cephalosporins (eg Cephalothin - Keflin)
autoAb was stimulated by
□ both operate through
the drug
drug-adsorption and
are able to modify
RBCs so that plasma
proteins can bind to
the membrane

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BB Lecture 13
Serologic reactions observed with Drug-Induced Positive DATs Drug Penicillins IgG Strongly Often 3-45 of px
Adsorption Streptomyocin + (-) on large
Mechanism Ig on RBC Serum and Eluate react Cephalosppori doses of
with: ns PCN;
usually EV
Immune complex C3 and occ. Cells, only if serum was
Membrane Cephalosporins Many + - No
IgG and IgM incubated with drug modification plasma hemolysis;
prior to testing. proteins but 3%
may
Routine testing with
develop (+)
reagent RBC is negative DAT
Durg Adsorption IgG; rearly C3 Cells only if they are Methyldopa- Methyldopa IgG Strongly + 0.8%
coated with specific induced + develop
HA -
drug prior to testing.
WAIHA;
Routine testing with 15% DAT
reagent RBCs is
negative  TREATMENT:
Drug-independent IgG All “normal” RBCs. □ TOC: Discontinuation of the drug
Routine testing with □ Excellent prognosis - as long as the cause is
reagent RBCs is positive
recognized and drug is stopped
Membrane IgG, IgM, IgA, No cells tested.
Modification C3 Mechanism in
nonimmunologic
protein adsorption

Autoantibody Formation

 Alpha-methyldopa (aldomet) induces production of

autoantibody that recognizes RBC antigens
□ Ab produced serologically indistinguishable from
WAIHA
□ Positive DAT in 10-20%
□ Significant immune HA - in 0.5-1% usually after 6
months of intake; resolves with discontinuation
 MOA: Methyldopa; could either be:
□ Alteration of function of suppressor T cells
 Kirtland, Howitz, Mohler
□ Alteration of RBC membrane components  Always to CONSIDER:
 Worlledge, et al □ Obtain patient’s medical history
 Blood transfusions, pregnancies, medications,
** both leading to rpoduction of autoAbs ** diagnosis
□ Perform DAT - using RBC collected in EDTA
 Serology of Aldomet-Induced (+) DAT/IHA is identical to □ Screen patient’s serum for RBC alloantibodies
idiopathic WAIHA □ Prepare and test and eluate for RBC alloantibodies if
□ It is difficult to establish that Aldomet is the trigger px has been recently transfused
□ Unless if patient is unknown to have taken the drug,
then suspicion increased

Mechanisms leading to Development of Drug-Related Ab

Mechanism Prototype Ig class DAT Eluate Frequency
results of
Hemolysis
Immune Quinidine IgM/ IgG + Often Samll
complex Phenacetin (-) doses of
drugs may
cause
acute IV
hemolysis;
RF
common

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