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Immune Hemolytic Anemias
Author: Erin Tretter, MBA, MT(ASCP)
Reviewer: Kathy W. Jones, MS, MLS(ASCP)CM
Course Instructions

Please proceed through the course by clicking on the blue arrows or text links. Use the table of contents to monitor your progress. Your progress will be
saved automatically as you proceed through the course, and you may later continue where you left off even if you use a different computer. You may
encounter practice questions within the course, which are not graded or recorded.
Course Info

This course carries the following continuing education credits:

P.A.C.E. Contact Hours: 2.00 hour﴾s﴿

Course Number: 578‐022‐20
Florida Board of Clinical Laboratory Science CE ‐ General ﴾Blood Banking / Immunohematology﴿: 2.00 hour﴾s﴿
Course Number: 694787

Level of instruction: Intermediate

Intended audience: Clinical laboratory technologists, technicians, and pathologists. This course is also appropriate for clinical laboratory science
students and pathology residents.

Author information: Erin Tretter, MBA, MT﴾ASCP﴿, is currently the Rapid Response Laboratory Supervisor at Paoli Hospital. Erin received her Masters in
Business Administration from Florida Institute of Technology where she is a member of the Phi Kappa Phi Honor’s Society. She received her BS in Medical
Technology from California University of Pennsylvania and has experience as a Generalist, including Blood Bank, Hematology and Chemistry. Erin is
currently the Blood Bank Clinical Instructor for the Clinical Laboratory Science Program at St. Christopher’s Hospital for Children and has 7 years
experience teaching immunohematology concepts and laboratory procedures to laboratory science students. She has also taught Blood Bank in the
Clinical Laboratory Technician program at the Community College of Philadelphia.

Reviewer information: Kathy W. Jones, MS, MLS﴾ASCP﴿CM is currently an Associate Professor in the Medical Laboratory Science Program at Auburn
University Montgomery where she is responsible for the program courses in Clinical Hematology and Clinical Immunology. She holds a BS degree in
Biology as well as a Master’s degree in Adult Education from Troy University. She received her Medical Laboratory Science certificate from St. Margaret’s
Hospital School of Medical Technology and will celebrate her 40th year in Laboratory Science this year. Before she became a full‐time educator, she served
in various roles in the clinical laboratory which included five years as a Hematology Supervisor.
 Immune Hemolytic Anemias
Introduction

Immune hemolysis is defined as the shortened survival of red blood cells

﴾RBCs﴿ resulting from an immune reaction. Hemolysis may not result in
anemia if bone marrow compensation is sufficient. There are many causes
of hemolysis other than immune reactions. The diagnosis of hemolytic
anemia depends on the clinical findings and laboratory data such as:
Increased reticulocyte count
Abnormal RBC morphologies
Elevated unconjugated ﴾indirect﴿ bilirubin
Reduced or absent serum haptoglobin levels

The serologic findings in the blood bank help to determine if the

hemolysis is immune‐mediated and what type of immune hemolytic
anemia may be present. This is important since treatment options vary
with each type.

Immune Hemolytic Anemias

Direct Antiglobulin Test ﴾DAT﴿

The DAT is a serologic procedure that is used to detect in vivo binding of

IgG antibody and/or complement on the red cells ﴾in vivo sensitization of
RBCs﴿. It is used primarily for the detection and differential diagnosis of
various forms of immune hemolysis including autoimmune hemolytic
anemia, hemolytic transfusion reactions, drug‐induced hemolysis, and
hemolytic disease of the newborn ﴾HDN﴿.
The procedure involves washing the patient's red cells to remove residual
plasma proteins and then testing the washed cells with antiglobulin
reagent. Depending upon the antiglobulin reagent used, a positive test
means that either IgG and/or complement components were bound to the
red cells in vivo, or inside of the body.
Initial testing is often done using polyspecific antihuman globulin ﴾AHG﴿
that contains both anti‐IgG and at least anti‐C3d ﴾In some preparations,
anti‐C3b and other anti‐complement activity may also be present﴿.
Once reactivity is noted with polyspecific AHG, monospecific reagents
may be used to differentiate between IgG and complement. This extended
testing is useful in suspected cases of paroxysmal cold hemoglobinuria,
cold hemagglutinin disease, warm autoimmune hemolytic anemia, and
drug‐induced hemolytic anemia. However, the use of anti‐C3 would not
be required for diagnosing HDN as a positive DAT would always be the
result of the production of IgG antibody and would not involve
complement.

Immune Hemolytic Anemias

Classification of Hemolytic Anemias

There are three types of immune hemolytic anemias:

1. Alloimmune hemolytic anemia occurs when the immune system produces antibody against foreign or non‐self antigens.
2. Autoimmune hemolytic anemia occurs when the immune system fails to distinguish the difference between self and non‐self antigens.
3. Drug‐induced hemolytic anemia occurs when drugs stimulate the formation of antibodies against the drug itself or against intrinsic RBC
antigens.

Alloimmune Hemolytic Anemia

Alloimmune Hemolytic Anemia

Alloimmune hemolytic anemia occurs when the immune system produces

antibody against foreign or non‐self antigens. Examples include:
Hemolytic transfusion reactions
Hemolytic disease of the newborn ﴾HDN﴿

Hemolytic transfusion reactions occur immediately or within days of a Red

Alloimmune hemolytic anemia occurs when the immune system produces
antibody against foreign or non‐self antigens. Examples include:
Hemolytic transfusion reactions
Hemolytic disease of the newborn ﴾HDN﴿

Hemolytic transfusion reactions occur immediately or within days of a Red

Blood Cell transfusion. The transfusion recipient is exposed to a foreign red
cell antigen on the donor cells, which stimulates an immune response. The
transfused red cells are sensitized in vivo causing a positive direct
antiglobulin test ﴾DAT﴿. Antibodies involved are usually of the IgG
classification.
In HDN, the mother is sensitized to a foreign antigen on the fetal red cells.
Sensitization usually occurs through a feto‐maternal hemorrhage. The
mother will produce an antibody to the foreign red cell antigen. If the
antibody is an IgG antibody, it may cross the placenta and bind to fetal cells,
causing red cell destruction.
Other MediaLab courses address these topics in more detail. More
information can also be found by referring to the references listed by clicking
the "More Info" button below.

More Info

Autoimmune Hemolytic Anemias

Autoimmune Hemolytic Anemia ﴾AIHA﴿: Introduction

Autoantibodies are immunoglobulins directed against self antigens. Most autoantibodies react with high‐incidence red cell antigens. Recognition of these
antibodies is important during serologic testing.
Autoantibodies may complicate routine testing by causing ABO and Rh typing problems.
AIHA is usually confirmed by a positive direct antiglobulin test ﴾DAT﴿ and demonstration of the autoantibody in the plasma or eluate.
AIHA is classified as either cold‐reactive or warm‐reactive. The table above lists different types of AIHA and the antibody classes involved. These will be discussed
in detail on the following pages.
Autoimmune Hemolytic Anemia Antibody Type Complement
Association
Warm Autoimmune Hemolytic Anemia IgG Sometimes
﴾WAIHA﴿ autoantibodies
Cold Agglutinin Syndrome ﴾CAS﴿ IgM Yes
autoantibodies
Mixed‐Type ﴾Serological features of Both IgM and IgG Yes
both warm and cold autoantibodies﴿ autoantibodies

Paroxysmal Cold Hemoglobinuria IgG biphasic Yes

﴾PCH﴿ hemolysin

Autoimmune Hemolytic Anemias

Symptoms of Autoimmune Hemolytic Anemias

A complete patient history ﴾including diagnosis, medications, and transfusion

history﴿ can provide beneficial information to aid in the identification of the
causative autoantibody.
Many symptoms are possible as a result of an autoimmune hemolytic
anemia. Some common symptoms, which can present as mild to severe,
include:
Fatigue
Jaundice
Shortness of breath
Hemoglobinemia
Hemoglobinuria
Splenomegaly

Autoimmune Hemolytic Anemias

Testing for Autoantibodies

The serum/plasma reactivity can be indicative of the type of autoantibody present. The tests/procedures listed in the table below can be used to detect the
presence of an autoantibody and differentiate between the types of autoantibodies.
 Autoimmune Hemolytic Anemias
Testing for Autoantibodies

The serum/plasma reactivity can be indicative of the type of autoantibody present. The tests/procedures listed in the table below can be used to detect the
presence of an autoantibody and differentiate between the types of autoantibodies.
Test Use Result Comments
Antibody Determine the If there is The phase of reactivity can determine if it is a cold‐
Screen antibody reactivity with all reactive or warm‐reactive autoantibody. If there is a
present in cells tested, specificity noted ﴾eg. PCH antibody with P antigen
serum/plasma including an specificity﴿, a more specific determination of the
autocontrol, an autoantibody can be made.
autoantibody is
likely.
Direct Detect in‐vivo Usually positive if By using monospecific antiglobulin reagents ﴾IgG and
Antiglobulin sensitization an autoantibody is Complement AHG Reagents﴿, can differentiate between
Test ﴾DAT﴿ of red blood present IgM and IgG antibodies and determine if complement is
cells involved
Elution Remove If warm‐reactive, Red cells must be thoroughly washed with normal saline,
antibody from IgG buffered saline, or low ionic strength saline ﴾LISS﴿ prior to
sensitized cells autoantibodies are an elution procedure to ensure that the antibody
to determine present, the eluate recovered represents only that antibody bound to red
antibody will be reactive cells, not “free” antibody in the plasma or serum.
specificity with all cells

Autoimmune Hemolytic Anemias

Possible Serological and Laboratory Presentations

Destruction of red cells ﴾hemolysis﴿ may be intravascular ﴾within blood

vessels﴿ or extravascular ﴾outside of blood vessels﴿. Complement causes
intravascular hemolysis, which can lead to hemoglobinemia ﴾free hemoglobin
in the serum/plasma﴿ and hemoglobinuria ﴾free hemoglobin in the urine﴿.
Phagocytosis of red cells by macrophages facilitates extravascular hemolysis,
which causes increase in serum bilirubin.
Potential serological/laboratory findings encountered when an immune‐
mediated anemia is present include:
Decreased hemoglobin and hematocrit
Spherocytosis
Reticulocytosis
Possible compensatory increase in nucleated red blood cells
Elevated bilirubin and lactate dehydrogenase ﴾LD﴿ levels
Decreased haptoglobin Hover over the image to zoom in.
Positive DAT due to IgG and/or complement sensitization
Positive antibody screen

Autoimmune Hemolytic Anemias

Ungraded Practice Question
True or False: A direct antiglobulin test ﴾DAT﴿ is usually negative in cases of autoimmune hemolytic anemia.

Select true or false

True

False

Autoimmune Hemolytic Anemias

Ungraded Practice Question
True or False: A direct antiglobulin test ﴾DAT﴿ is usually negative in cases of autoimmune hemolytic anemia.

Select true or false

True

False
Select true or false

True

False

Feedback

The correct answer is false. The DAT will be positive in autoimmune hemolytic anemias, since it is detecting the presence of in vivo bound autoantibodies on a
patient's red blood cells. The autoantibody that has sensitized the patient's red blood cells is causing the red blood cell destruction.

Autoimmune Hemolytic Anemias:

Cold Autoimmune Hemolytic Anemia
Cold Autoimmune Hemolytic
Anemia

Cold‐reactive autoantibodies may be encountered in serologic testing. Cold autoantibodies are generally not clinically significant but they can cause difficulties
during ABO/Rh typing and antibody detection.
Benign cold autoantibodies are IgM immunoglobulins present in the plasma at 4°C. They have been known to react at room temperature, on occasion, and can
activate complement as well. These autoantibodies usually have a low titer; typically less than 1:64. Common benign autoantibodies include Anti‐I, Anti‐i, and
Anti‐IH.
Pathologic cold autoantibodies are known to react at much broader thermal ranges. Antibodies that cause disease usually bind to red cell antigens at 30‐32°C.
These antibodies are found in cold hemagglutinin disease ﴾often secondary to infection﴿ and in paroxysmal cold hemoglobinuria ﴾PCH﴿.

Autoimmune Hemolytic Anemias:

Cold Hemagglutinin Disease ﴾CHD﴿ Cold Autoimmune Hemolytic
Anemia

Cold hemagglutinin disease ﴾CHD﴿ or cold agglutinin syndrome ﴾CAS﴿

represents about 18% of autoimmune hemolytic anemia cases. A cold
autoantibody that reacts between 4°C and 30°C can cause a moderate,
chronic anemia. The antibody involved is usually IgM, which activates
complement. Antibody specificity is usually anti‐I.
CHD is predominantly in individuals over 50 years of age. Symptoms
generally occur in the winter months and include acrocyanosis of the hands,
feet, ears and nose and numbness in the extremities. Cold weather activates
the cold autoantibody. It agglutinates red cells in the capillaries of the
extremities and fixes complement, causing hemolysis. Patients may exhibit
hemoglobinuria, weakness, pallor, and weight loss. Patients may also exhibit
jaundice and Raynauds disease. Most patients with CHD live more
comfortably in warmer climates. Hover over the image to zoom in.

Laboratory findings include a positive direct antiglobulin test ﴾DAT﴿ with

complement only and reticulocytosis. The peripheral smear may demonstrate
agglutinated red blood cells ﴾RBCs﴿, as indicated by the arrows in the upper
image on the right, and/or polychromatophilic RBCs, as indicated by the
arrows in the lower image on the right. Agglutination of red cells may cause
difficulties when performing a CBC analysis.
Most patients with CHD do not require transfusion, but when they do,
challenges present during pretransfusion testing, including ABO
discrepancies or masking of alloantibodies.
CHD can also occur secondary to infection, including Mycoplasma
pneumoniae infection, where the cold autoantibody is anti‐I and infectious
mononucleosis, where the cold autoantibody in most cases is anti‐i . Usually
the hemolytic episode is resolved when the infection subsides.

Hover over the image to zoom in.

Autoimmune Hemolytic Anemias:

Paroxysmal Cold Hemoglobinuria ﴾PCH﴿ Cold Autoimmune Hemolytic
Anemia

PCH is the least common type of AIHA. It's usually seen in children who have
had viral infections such as measles, mumps, chickenpox, or infectious
mononucleosis. It can also occurs idiopathically in adults. In PCH, a biphasic
hemolysin causes red cell destruction. An IgG cold autoantibody reacts with
red cells in cold areas of the body such as the extremities when the individual
is exposed to cold. Complement binds irreversibly to the red cells. When the
cells circulate to warmer areas of the body, the cells undergo complement‐
PCH is the least common type of AIHA. It's usually seen in children who have
had viral infections such as measles, mumps, chickenpox, or infectious
mononucleosis. It can also occurs idiopathically in adults. In PCH, a biphasic
hemolysin causes red cell destruction. An IgG cold autoantibody reacts with
red cells in cold areas of the body such as the extremities when the individual
is exposed to cold. Complement binds irreversibly to the red cells. When the
cells circulate to warmer areas of the body, the cells undergo complement‐
mediated hemolysis.
Common clinical symptoms
Sudden onset of fever
Chills
Abdominal cramps
Back pain
Intermittent episodes of hemoglobinuria

Typical laboratory findings

Elevated bilirubin level
Decreased hemoglobin
Polychromatophilia, nucleated RBCs, and poikilocytosis may be present
on the peripheral blood smear
Positive DAT with complement only
IgG may be detected on the cells if cold saline and reagents are used.

The autoantibody usually reacts weakly in traditional antibody identification

tests. The biphasic hemolysin in PCH has anti‐P specificity. It can be
demonstrated in the laboratory through the Donath‐Landsteiner test.
Transfusion in adults is only necessary if the hemolysis is severe. Young
children may require transfusion because the hemolysis may be more severe
due to the broad thermal amplitude of the antibody. While p red cells have a
better chance for survival, the incidence of p red cells in the population is
very low and therefore p blood is very rare. Blood should not be withheld
from patients in urgent situations so randomly selected blood may be
adequate.

Autoimmune Hemolytic Anemias:

PCH Donath‐Landsteiner Test
Cold Autoimmune Hemolytic
Anemia

The Donath‐Landsteiner test is a confirmatory test that subjects the antibody

in serum/plasma to appropriate cells at 4°C followed by 37°C incubations.
The presence of hemolysis after these successive incubations confirms the
presence of the biphasic hemolysin. If there is no hemolysis noted, the
biphasic hemolysin is not present.
In this test, the patient's sample must be maintained at 37°C after collection.
The test is illustrated in the image on the right.
Patient's serum is added to three sets of three tubes.
1. Set #1 contains only patient serum.
2. Set #2 contains patient's serum and fresh normal serum, which acts as
a source of complement.
3. Set #3 contains only fresh normal serum.

P‐positive cells are then added to all tubes. One tube from each set is kept in
an ice bath for 90 minutes ﴾1A, 2A, 3A﴿. Another tube from each set is kept at
37°C for 90 minutes ﴾1B, 2B, 3B﴿. The last tube from each set is put in an ice
bath for 30 minutes and then incubated at 37°C for 60 minutes ﴾1C, 2C, 3C﴿.
After incubation, the tubes are examined for hemolysis. The Donath‐
Landsteiner test is positive when the patient's serum ﴾with or without the
normal serum﴿ demonstrates hemolysis in tubes incubated in ice then
incubated at 37°C. Hemolysis should not be present in any of the other tubes.
Again, this serves to prove the biphasic nature of the hemolysin and its ability
to lyse RBCs.
 Autoimmune Hemolytic Anemias:
ABO/Rh Typing in the Presence of Cold Autoantibodies Cold Autoimmune Hemolytic
Anemia

Cold autoantibodies can interfere with routine blood bank tests. The extent to
which they interfere depends on the concentration and thermal amplitude of
the antibody. A blood bank technologist must recognize these problems and
work through the resolution of cold autoantibody cases.
Red blood cells that are heavily coated with cold antibody may cause false
positive reactions during ABO typing, resulting in an ABO discrepancy. To
determine if a cold autoantibody is involved, Group O and autologous red
blood cells can be tested with the patient's plasma. Both will likely be positive
if a cold autoantibody is present. Two methods can be used to resolve this
type of discrepancy:
1. The patient's sample can be prewarmed to 37°C prior to testing.
2. Patient red blood cells can be washed with saline warmed to 37°C .

The warm incubation promotes dissociation of the antibody from the red cell
and the warm washes prevent it from reattaching in vitro.
Cold autoantibodies can also cause false‐positive reactions during Rh ﴾D﴿
typing. In addition to the methods listed above, using monoclonal low‐
protein reagents may also minimize false positive Rh﴾D﴿ typing reactions due
to cold autoantibodies. If polyspecific antihuman globulin reagents are used
Hover over the image to zoom in.
during weak‐D testing, false positive reactions can occur due to complement
activation of cold IgM autoantibodies. This can be prevented by using
monospecific Anti‐IgG or using samples collected in EDTA.

Autoimmune Hemolytic Anemias:

Laboratory Presentations of Cold Autoantibodies ‐ Antibody Cold Autoimmune Hemolytic
ID and Detection Anemia

Reactivity caused by cold autoantibodies may mask the presence of alloantibodies. It may be necessary to remove the cold antibody from the serum. Rabbit
erythrocyte stroma ﴾RESt﴿ can be used to remove Anti‐I. Other techniques include prewarming and cold autoadsorption.
The prewarm method involves warming all testing components, such as patient sample, reagents, and saline, to 37°C prior to testing. The cold autoantibodies
will not react and only clinically significant antibodies will react.
A cold autoadsorption that uses autologous cells could be performed to remove cold autoantibodies from the patient serum. The patient's cells are mixed with
the patient's own serum and incubated at 4°C. Autoantibody is adsorbed onto the cells and alloantibody, if present, will remain in the serum. If the autoantibody
titer is high, the adsorption may need to be repeated several times. This procedure should not be used if the patient has had a transfusion in the previous three
months because donor cells may still be present in the circulation. If the patient has been recently transfused, RESt could be used. More information regarding
RESt can be found by clicking the "More Info" button below.
After the adsorption is complete, the serum/plasma can be tested against a panel of cells to determine if alloantibody is present.

More Info

Autoimmune Hemolytic Anemias:

Ungraded Practice Question
Cold Autoimmune Hemolytic
Anemia
In paroxysmal cold hemoglobinuria ﴾PCH﴿, the autoantibody specificity is most commonly anti‐I.

Select true or false

True

False

Autoimmune Hemolytic Anemias:

Ungraded Practice Question
Cold Autoimmune Hemolytic
Anemia
In paroxysmal cold hemoglobinuria ﴾PCH﴿, the autoantibody specificity is most commonly anti‐I.

Select true or false

 Autoimmune Hemolytic Anemias:
Ungraded Practice Question
Cold Autoimmune Hemolytic
Anemia
In paroxysmal cold hemoglobinuria ﴾PCH﴿, the autoantibody specificity is most commonly anti‐I.

Select true or false

True

False

Feedback

This statement is false. In PCH, the most common autoantibody specificity is anti‐P. Anti‐I is one of the most common autoantibody specificities in cold
hemagglutinin disease ﴾CHD﴿.

Autoimmune Hemolytic Anemias:

Ungraded Practice Question Cold Autoimmune Hemolytic
Anemia
Cold hemagglutinin disease ﴾CHD﴿ is caused by an IgM antibody with a broad thermal range.

Select true or false

True

False

Autoimmune Hemolytic Anemias:

Ungraded Practice Question Cold Autoimmune Hemolytic
Anemia
Cold hemagglutinin disease ﴾CHD﴿ is caused by an IgM antibody with a broad thermal range.

Select true or false

True

False

Feedback

The statement is true as the antibody that causes the hemolysis in CHD is an IgM antibody that can react at lower peripheral body temperatures and can cause
hemolysis at 37C via complement activation.

Autoimmune Hemolytic Anemias:

Warm Autoimmune Hemolytic Anemia ﴾WAIHA﴿ Warm Autoimmune Hemolytic
Anemia

WAIHA is the most common type of autoimmune hemolytic anemia. It is

usually associated with warm reacting ﴾37°C﴿ IgG antibodies.
WAIHA may be idiopathic or secondary to a pathologic disorder ﴾eg, systemic
lupus erythematosus﴿.
Clinical symptoms associated with WAIHA
If significant anemia is present, patients may exhibit pallor, weakness,
dizziness, jaundice, dyspnea, and fever.
Possible laboratory findings
Positive direct antiglobulin test ﴾DAT﴿. It's important to obtain an
adequate patient history, including previous transfusions and current
medications to help establish the cause of the positive DAT.
Erythrocyte morphology abnormalities, as seen in the Wright‐stained
peripheral blood smear image on the right.
Polychromatophilic red cells﴾A‐‐green arrows﴿
Spherocytes ﴾B‐‐blue arrows﴿
Schistocytes ﴾C‐‐red arrows﴿ Hover over the image to zoom in.

Nucleated red blood cells ﴾D‐‐ orange arrow﴿

Autoimmune Hemolytic Anemias:

Serological Characteristics in WAIHA
Warm Autoimmune Hemolytic
 Spherocytes ﴾B‐‐blue arrows﴿
Schistocytes ﴾C‐‐red arrows﴿ Hover over the image to zoom in.

Nucleated red blood cells ﴾D‐‐ orange arrow﴿

Autoimmune Hemolytic Anemias:

Serological Characteristics in WAIHA
Warm Autoimmune Hemolytic
Anemia

Patients with WAIHA present with very difficult serological problems such as blood typing discrepancies, strong positive results in antibody screens, incompatible
crossmatches, positive direct antiglobulin tests ﴾DAT﴿, or antibody panels/eluates reactive with all cells.
A positive DAT is expected in WAIHA. The patient's serum may contain little free autoantibody if the autoantibody has been primarily adsorbed by the red cells in
vivo. Autoantibody will appear in the serum once all the antigen sites on the red cells have been occupied. In the majority of cases of WAIHA, the DAT is positive
with both IgG and complement. In approximately 20% of cases, the DAT is positive with IgG alone and in a few cases, the DAT is positive with complement alone.
About half of all WAIHA cases will have an autoantibody that reacts with all cells tested, including donor cells. The presence of an IgG autoantibody can be
confirmed by elution. Elution is the process by which RBC‐bound antibody is removed from the red cells and recovered, being sure that antibody reactivity is
maintained so that antibody specificity can be determined. The eluate is usually reactive with all cells tested. Most IgG autoantibodies have an Rh‐like specificity,
such as anti‐e. If it is necesssary to identify the specific antibody, the laboratory would need to have a supply of rare cells such as Rhnull and D‐‐ cells. Other
specificities include those to high incidence antigens or a null phenotype. Examples include autoanti‐U, autoanti‐Wrb, autoanti‐Ena, autoanti‐Kpb, and autoanti‐
Vel. This form of specialized testing is not performed in every clinical laboratory setting and may be, instead, provided by a reference laboratory.
The specificity of the autoantibody is usually only of academic interest thus it is rarely necessary to perform extensive additional testing. The detection and
identification of alloantibodies should be the primary concern.

Autoimmune Hemolytic Anemias:

WAIHA Investigation: Serological Findings Following Elution Warm Autoimmune Hemolytic
Anemia

A summary of the possible serological findings of an WAIHA investigation following elution is presented in the table below.
Scenario DAT Result ﴾IgG Serum/Plasma Adsorption
and/or Antibody
Complement﴿ Screen/Identification
Warm autoantibody reactive in eluate only Positive Negative Not indicated
Warm autoantibody reactive in serum/plasma Positive Reactive with all panel No reactivity
and eluate; no underlying alloantibodies cells tested after
adsorption
Warm autoantibody reactive in serum/plasma Positive Reactive with all panel Reactivity
and eluate; underlying alloantibodies that must cells tested after
be identified adsorption

Autoimmune Hemolytic Anemias:

Possible Scenarios When Transfusions are Needed in the Warm Autoimmune Hemolytic
Presence of WAIHA Anemia

1. Autoantibody detectable from eluate only: If there is not a high enough titer of the autoantibody to be demonstrated in the serum/plasma of WAIHA,
the antibody screen will be negative. In this situation, the autoantibody is detectable from the eluate only. Compatible crossmatches can usually be obtained
in this presentation, but the survival of the transfused cells due to sensitization by the autoantibody is difficult to predict.
2. Autoantibody detectable from eluate only, but the serum/plasma also demonstrates alloantibody reactivity: In this case, the serum/plasma
shows a specificity ﴾or specificities﴿ directed against a definite pattern of cells on an antibody identification panel. The resolution would necessitate
identification of the alloantibody﴾ies﴿. Transfusion would require appropriate antigen‐negative units, which will likely be crossmatch compatible, if the
autoantibody is not detectable in the serum/plasma.
3. Autoantibody reactivity in both the serum/plasma and eluate: Reactivity will likely be observed against all panel cells tested and the reactivity may
mask the presence of underlying alloantibodies. Adsorption studies are the primary method of investigating this possibility. If no reactivity is present after
the adsorptions, then no underlying alloantibody is present. If a transfusion is needed in this case, a common practice is to transfuse "least incompatible"
units. The presence of alloantibody has been ruled out, the autoantibody is confirmed, but it is difficult to predict the efficacy of the transfusion due to the
autoantibody. Special procedures/consents are usually in order to transfuse the incompatible units.
4. Both autoantibody and alloantibody are demonstrated in the serum/plasma: In this case an adsorption method usually reveals the underlying
alloantibody that was masked by the presence of the autoantibody. If transfusion is required, appropriate antigen‐negative units must be obtained. The
crossmatches will likely be incompatible due to the autoantibody, and special consents/procedures must be followed to transfuse in this situation. The
survival of the transfused cells due to the demonstrable autoantibody is suspect.

Autoimmune Hemolytic Anemias:

Detection and Identification of An Alloantibody in the
Warm Autoimmune Hemolytic
Presence of a Warm‐Reactive Autoantibody Anemia

The primary concern of the blood bank technologist when working up a sample that contains a warm‐reactive autoantibody is the possible presence of an
alloantibody. An alloantibody can be masked by an autoantibody, making detection and identification of the alloantibody difficult.
 Autoimmune Hemolytic Anemias:
Detection and Identification of An Alloantibody in the
Warm Autoimmune Hemolytic
Presence of a Warm‐Reactive Autoantibody Anemia

The primary concern of the blood bank technologist when working up a sample that contains a warm‐reactive autoantibody is the possible presence of an
alloantibody. An alloantibody can be masked by an autoantibody, making detection and identification of the alloantibody difficult.
Alloantibodies must be identified in order to select appropriate red blood cells for transfusion. Two types of adsorption techniques can be used to remove free
autoantibody from the plasma/serum:
1. Autoadsorption
2. Allogeneic adsorption

Autoimmune Hemolytic Anemias:

Autologous Adsorption ﴾Autoadsorption﴿ Warm Autoimmune Hemolytic
Anemia

Because the serum/plasma is reacting with all cells tested, a method must be employed to remove the autoantibody that is causing the broad reactivity, while not
removing the potential alloantibody﴾ies﴿ that still need to be identified. One method to determine if alloantibody is demonstrable in the presence of autoantibody
is an autoadsorption.
The use of enzyme or ZZAP‐treated autologous cells facilitates the removal of autoantibody by removing membrane structures that interfere with antibody
binding. Note that the presence of donor cells in the patient's circulation may result in unreliable findings with either cold or warm autoadsorptions. Therefore,
autologous adsorptions cannot be accurately performed if the patient has been transfused within the past three months.
More than one adsorption may be needed, depending on the strength of the autoantibody. Testing the adsorbed serum with DAT‐negative ﴾treated﴿ patient cells
is a good way to determine if all the autoantibody has been removed.

Autoimmune Hemolytic Anemias:

Allogeneic Adsorption
Warm Autoimmune Hemolytic
Anemia

The allogeneic adsorption is similar in principle and procedure to the autologous adsorption. However, allogeneic cell aliquots are used in the adsorption process.
This method is required when the patient has been recently transfused.
1. Ideally, the allogeneic cells that are selected should match the patient's phenotype. Usually two to three different allogeneic cell sets are required. The
different allogeneic cells typically have different phenotypes. Usually donors with R1R1, R2R2, and rr phenotypes are chosen. Other antigens to consider
when selecting allogeneic cells are K, Fy, Jk, and S. An example would be: The first allogeneic cell may have a phenotype of R1R1, K+, Jk﴾a‐,b+﴿ and the second
cell may have a phenotype of R2R2, K‐, Jk﴾a+,b‐﴿. ﴾This is only an example, as there are more phenotypes possible﴿. Both cells would have the ability to adsorb
the autoantibody from the serum. If there was an anti‐Jka present in the serum, for example, it would not be adsorbed by the first cell, but could be
adsorbed by the second cell ﴾as the second allogeneic adsorbing cell is Jka antigen positive﴿.
2. After the adsorption is complete, the harvested serum/plasma can be used in identification procedures. The serum/plasma obtained from the different
allogeneic adsorptions must be analyzed separately against an appropriate panel of cells to achieve identification or exclusion. This is necessary because
the difference in phenotypes of the adsorbing cells means the adsorbed serums potentially contain different alloantibodies.

Sometimes, multiple adsorptions are required, depending on the strength of the autoantibody.

Autoimmune Hemolytic Anemias:

Transfusion Considerations for WAIHA
Warm Autoimmune Hemolytic
Anemia

Most patients with WAIHA may never need transfusion. Occasionally

transfusion may be required if the anemia is severe or the patient is
scheduled for a surgical procedure. The question of transfusing patients with
demonstrable WAIHA cases is highly debatable. The fate of the transfused red
blood cells may be the same as the patient's own cells, depending on the
severity of the immune response and autoantibody production. Transfusion
should occur only when clinically necessary and providing supportive
therapy ﴾oxygen and rest for example﴿, are options. It is a clinical decision that
balances the risks and clinical needs. Blood should never be withheld from a
patient in a life‐threatening event because of incompatibility from
autoantibodies. The volume of transfusion should be the least amount to
maintain adequate oxygen transportation and relieve the symptoms of
anemia.
The primary goal of the blood banker is to ensure compatibility with any
alloantibodies present in the serum.
If no alloantibodies are present, random units that are ABO compatible
may be selected for transfusion.
If alloantibodies are present, antigen‐negative blood must be
transfused. If the specificity of the autoantibody can be determined, the
appropriate antigen‐negative donor blood should be selected, if
possible. If transfusion is necessary, some facilities choose to transfuse
units of blood that are Rh and K phenotypically matched to the patient
 may be selected for transfusion.
If alloantibodies are present, antigen‐negative blood must be
transfused. If the specificity of the autoantibody can be determined, the
appropriate antigen‐negative donor blood should be selected, if
possible. If transfusion is necessary, some facilities choose to transfuse
units of blood that are Rh and K phenotypically matched to the patient
in order to prevent alloimmunization. Due to the presence of
autoantibodies where specificity cannot be determined, all
crossmatches will be incompatible. The donor units with the weakest
reactions in vitro are usually selected as least incompatible.

Autoimmune Hemolytic Anemias:

Treatment of WAIHA
Warm Autoimmune Hemolytic
Anemia

Therapy is usually first aimed at treating any underlying disorders, if present. Treatments for WAIHA that may be considered include:
Corticosteroids such as prednisone may be used to stabilize the hematocrit. The exact mechanism of prednisone treatment is not completely understood,
but theories include the reduction of antibody synthesis, altered antibody activity, and alteration of macrophage receptors for IgG and C3.
Intravenous immunoglobulin ﴾IVIG﴿ has also been shown to be effective in patients who do not respond to corticosteroid therapy.
If steroid therapy fails or the need for high‐dose steroid therapy persists, splenectomy is recommended. Splenectomy has been shown to reduce antibody
production and it removes a site for RBC destruction.
The last resort is immunosuppressive drugs. These drugs have detrimental side effects, including increase susceptibility to infection, infertility, and risk of
birth defects.

Autoimmune Hemolytic Anemias:

Ungraded Practice Question
Warm Autoimmune Hemolytic
Anemia

In a case of WAIHA , the production of IgG or IgG and complement that sensitizes the red blood cells leads to red blood cell destruction.

Select true or false

True

False

Autoimmune Hemolytic Anemias:

Ungraded Practice Question
Warm Autoimmune Hemolytic
Anemia

In a case of WAIHA , the production of IgG or IgG and complement that sensitizes the red blood cells leads to red blood cell destruction.

Select true or false

True

False

Feedback

This statement is true. It is IgG ﴾sometimes with complement association﴿ autoantibody that is responsible for red blood cell sensitization.

Autoimmune Hemolytic Anemias:

Ungraded Practice Question
Warm Autoimmune Hemolytic
Anemia
Which type of adsorption must be done if a patient has been recently transfused?

Please select the single best answer

Autologous adsorption

Allogeneic adsorption

Either autologous or allogeneic

Autoimmune Hemolytic Anemias:

 Allogeneic adsorption

Either autologous or allogeneic

Autoimmune Hemolytic Anemias:

Ungraded Practice Question
Warm Autoimmune Hemolytic
Anemia
Which type of adsorption must be done if a patient has been recently transfused?

Please select the single best answer

Autologous adsorption

Allogeneic adsorption

Either autologous or allogeneic

Feedback

Allogeneic adsorptions are necessary when a patient has been recently transfused. If autologous cells are used after a recent transfusion, the transfused cells of
different phenotypes could potentially remove alloantibody during the adsorption procedure.

Autoimmune Hemolytic Anemias:

Mixed Type Autoimmune Hemolytic Anemia ﴾AIHA﴿
Mixed‐Type Autoimmune Hemolytic
Anemia

Mixed‐type AIHA is present when the individual has both cold and warm
autoantibodies. The cold autoantibody in mixed‐type AIHA has broad thermal
amplitude with reactivity at 30°C or above with either a high or low titer. The
cold autoantibody is an IgM and the warm autoantibody is an IgG.
Both IgG and C3 are detectable on the patient's red cells. This usually results
in reactivity in all phases of testing with all cells tested. Both cold and warm
autoadsorptions may be required to determine the presence of
alloantibodies. The cold antibody usually demonstrates anti‐I or anti‐i
specificity. The warm antibody is serologically indistinguishable from the
autoantibodies in a typical warm autoimmune hemolytic anemia ﴾WAIHA﴿.
Considerations for blood selection, if transfusion is needed, is similar to that
for CHD and WAIHA.

Hover over the image to zoom in.

Drug‐Induced Hemolytic Anemia

Drug‐Induced Immune Hemolytic Anemia Introduction

Drug‐induced immune hemolytic anemia is a rare category of autoimmune

hemolytic anemias. Antibodies produced in response to a medication, or its
metabolite, are the cause of the hemolytic anemia. The drug may cause a
positive DAT, which may or may not be associated with immune hemolysis.
Patients may present with similar serological and laboratory findings as the
other types of autoimmune hemolytic anemias, and may be difficult to
differentiate. Accurate medication history is beneficial in resolution of the
serologic presentation.

Drug‐Induced Hemolytic Anemia

Mechanisms

Four mechanisms of drug‐induced immune antibodies are thought to be associated with red blood cell sensitization and a positive direct antiglobulin test ﴾DAT﴿.
 Drug‐Induced Hemolytic Anemia
Mechanisms

Four mechanisms of drug‐induced immune antibodies are thought to be associated with red blood cell sensitization and a positive direct antiglobulin test ﴾DAT﴿.
These mechanisms are:

Drug adsorption
Immune complex
Membrane modification ﴾non‐immune adsorption of proteins﴿
Induction of autoimmunity

Drug‐Induced Hemolytic Anemia

Drug Adsorption Mechanism

In this mechanism, the drug is adsorbed directly onto the surface of the red
blood cell ﴾RBC﴿. The antibody is formed against the drug itself. This results in
drug‐coated RBCs becoming coated with IgG. Complement is rarely involved.
Sensitization of the RBCs can lead to increased RBC destruction. The drug
most commonly associated with this mechanism is Penicillin, but only when
dosing is higher than normal.
The DAT is strongly positive with Anti‐IgG. Antibody eluted from the red cells
will only react with drug‐coated red cells and not uncoated red cells. The
antibody screen is usually negative, unless an alloantibody is present.
Crossmatches are compatible in all phases. The eluate is non‐reactive.
Only a small percentage of patients will demonstrate hematologic
complications. The anemia develops slowly because destruction occurs
extravascularly. The patient usually improves once drug therapy has ceased.
Medications commonly implicated in the drug‐adsorption mechanism
include:
Penicillin G
Erythromycin
Methicillin
Carbromal
Nafcillin
Cefazolin
Tetracycline
Cefamandole

Drug‐Induced Hemolytic Anemia

Immune Complex Mechanism

In this mechanism, the drug does not bind to the RBCs directly. Instead, drug‐
antidrug ﴾antigen‐antibody﴿ complexes form and circulate in the plasma. The
antibody involved is often IgM but IgG may also be present. These immune
complexes bind non‐specifically to the red cells and activate complement.
Complement sensitizes the red cell, which may lead to intravascular
hemolysis. The complex may dissociate after complement activation and bind
to other red cells.
Patients may present with hemoglobinemia and hemoglobinuria. Renal
failure occurs in 50% of the cases. The DAT is usually positive with
complement antisera only. Other blood bank tests such as the antibody
screen and crossmatches will be negative, unless an alloantibody is present.
This is because the antibody is specific to the drug and not an RBC antigen.
The eluate is usually non‐reactive. Confirmation of the drug‐induced positive
DAT can be obtained by incubating the patient's serum with a solution of the
drug and reagent/donor ABO compatible red cells. Hemolysis would be
expected after incubation as well as positive DAT with anti‐C3. Confirmatory
testing is usually not required when the patient has a history of taking the
drug and a positive DAT.
Medications commonly implicated in the immune complex mechanism
include:
Quinine
Quinidine
Phenacetin
Acetaminophen
Medications commonly implicated in the immune complex mechanism
include:
Quinine
Quinidine
Phenacetin
Acetaminophen
Methotrexate
Rifampicin
Cefotaxime
Cetriaxone

Drug‐Induced Hemolytic Anemia

Membrane Modification ﴾Non‐Immune Adsorption of
Proteins﴿

In this mechanism, the drug modifies the RBC membrane, allowing the cell to
adsorb proteins in a non‐specific manner. The uptake of immunoglobulins
and complement proteins is not a result of a specific antigen‐antibody
reaction. The DAT may be coated with both IgG and complement proteins.
The eluate is non‐reactive. The only drugs known to cause a positive DAT
through this mechanism are cephalosporins. This mechanism is rarely
associated with decreased RBC survival.
Medications commonly implicated in the non‐immune adsorption of protein
mechanism include:
Cephalothin
Cisplatin
Suramin

Drug‐Induced Hemolytic Anemia

Induction of Autoimmunity

In this mechanism, the drug is responsible for the production of autoantibodies, which can sensitize the RBCs. For example, alpha‐methyldopa ﴾Aldomet﴿ therapy
is known to induce autoantibodies that react with red cell antigens. These autoantibodies do not react with the drug in vitro.
This mechanism is indistinguishable from a WAIHA, as it has identical serological presentation. It is theorized that certain drugs can interfere with suppressor T‐
cell function, and as a result, a proliferation of autoantibodies produced by B‐cells occurs.
Positive DATs occur in approximately 15% of patients receiving alpha‐metyldopa after six months of therapy. Very few patients will develop a hemolytic anemia.
The DAT is positive with anti‐IgG only. Occasionally weak complement coating is present. The eluate does react with normal RBCs in the absence of the drug. If
drug therapy is ceased, the autoantibody production will eventually stop, but it may take several months before the DAT becomes negative.
Medications commonly implicated in the induction of autoimmunity mechanism include:
Methyldopa
Procainanmide
Mefenamic acid
Levodopa
Fludarabine

Drug‐Induced Hemolytic Anemia

Treatment of Drug‐Induced Immune Hemolytic Anemia

In patients with drug‐induced hemolytic anemia, the preferred treatment is discontinuation of the drug. Other drugs may be substituted.
A positive DAT does not indicate that drug therapy should be stopped, especially if the drug is therapeutically beneficial and significant hemolysis is not present.
If hemolytic anemia is not present, continuation of drug therapy is an option.

Drug‐Induced Hemolytic Anemia

Laboratory Investigation of Drug‐Induced Immune Hemolytic
Anemia

A complete medical history is important when investigating drug‐induced immune hemolytic anemia. Patient diagnosis and information about previous
transfusions, pregnancies, and medications should be obtained.
A DAT should be performed with monospecific reagents.
Anemia

A complete medical history is important when investigating drug‐induced immune hemolytic anemia. Patient diagnosis and information about previous
transfusions, pregnancies, and medications should be obtained.
A DAT should be performed with monospecific reagents.
Patient serum should be tested for the presence of alloantibodies by using routine procedures.
If no reactivity is observed, the test may be repeated in the presence of the drug.
Normal red cells can also be treated and coated with the drug. Negative and positive controls must be tested when using drug‐treated red cells. This
ensures that any reactivity is interpreted accurately.

Drug‐Induced Hemolytic Anemia

Ungraded Practice Question
Which mechanism of drug‐induced red cell destruction closely mimics the serologic presentation of warm autoimmune hemolytic anemia ﴾WAIHA﴿?

Please select the single best answer

Drug adsorption

Immune complex

Induction of autoimmunity

Non‐immune adsorption of proteins

Drug‐Induced Hemolytic Anemia

Ungraded Practice Question
Which mechanism of drug‐induced red cell destruction closely mimics the serologic presentation of warm autoimmune hemolytic anemia ﴾WAIHA﴿?

Please select the single best answer

Drug adsorption

Immune complex

Induction of autoimmunity

Non‐immune adsorption of proteins

Feedback

The induction of autoimmunity mechanism presents the same serologic picture as a WAIHA.

Drug‐Induced Hemolytic Anemia

Ungraded Practice Question

Which mechanism of drug‐induced hemolytic anemia involves the formation of drug‐antibody combinations that bind non‐specifically to RBC membranes and
activate complement.

Please select the single best answer

Drug adsorption

Immune complex

Induction of autoimmunity

Non‐immune adsorption of proteins

Drug‐Induced Hemolytic Anemia

Ungraded Practice Question

Which mechanism of drug‐induced hemolytic anemia involves the formation of drug‐antibody combinations that bind non‐specifically to RBC membranes and
activate complement.
 Drug‐Induced Hemolytic Anemia
Ungraded Practice Question

Which mechanism of drug‐induced hemolytic anemia involves the formation of drug‐antibody combinations that bind non‐specifically to RBC membranes and
activate complement.

Please select the single best answer

Drug adsorption

Immune complex

Induction of autoimmunity

Non‐immune adsorption of proteins

Feedback

The immune complex mechanism involves a complex of drug and antibody that is adsorbed onto the red cell membrane, resulting in a sensitized cell. In this
mechanism, the drug does not bind to the RBCs directly. Instead, drug‐antidrug ﴾antigen‐antibody﴿ complexes form and circulate in the plasma. These immune
complexes bind non‐specifically to the red cells and activate complement. Complement sensitizes the red cell, which may lead to intravascular hemolysis. The
complex may dissociate after complement activation and bind to other red cells.

Drug‐Induced Hemolytic Anemia

Ungraded Practice Question
Penicillin is one of the medications that may be responsible for the immune‐complex mechanism of drug‐induced hemolytic anemia.

Select true or false

True

False

Drug‐Induced Hemolytic Anemia

Ungraded Practice Question
Penicillin is one of the medications that may be responsible for the immune‐complex mechanism of drug‐induced hemolytic anemia.

Select true or false

True

False

Feedback

Penicillin is one of the more common medications that can cause a drug‐induced hemolytic anemia by the drug adsorption mechanism.

Case Study
Ungraded Practice Question

Case Study One

A 50‐year‐old female patient with systemic lupus erythematosus ﴾SLE﴿ is
admitted to the hospital with evidence of hemolysis, including decreased
hemoglobin and hematocrit, increased reticulocyte count, and increased
indirect bilirubin. The patient has no recent history of transfusion. An
antibody screen is ordered and 3+ agglutination is observed at the AHG
phase for all cells. An autologous control ﴾autocontrol﴿ is also tested and 3+
agglutination is observed at the AHG phase in this test as well.
Given the results of the antibody screen and other laboratory test results and
the patient's medical and transfusion history, which of the following is the
MOST likely cause of the positive screen results?

Please select the single best answer

Cold autoantibodies

Warm autoantibodies
MOST likely cause of the positive screen results?

Please select the single best answer

Cold autoantibodies

Warm autoantibodies

Alloantibodies

Case Study
Ungraded Practice Question

Case Study One

A 50‐year‐old female patient with systemic lupus erythematosus ﴾SLE﴿ is
admitted to the hospital with evidence of hemolysis, including decreased
hemoglobin and hematocrit, increased reticulocyte count, and increased
indirect bilirubin. The patient has no recent history of transfusion. An
antibody screen is ordered and 3+ agglutination is observed at the AHG
phase for all cells. An autologous control ﴾autocontrol﴿ is also tested and 3+
agglutination is observed at the AHG phase in this test as well.
Given the results of the antibody screen and other laboratory test results and
the patient's medical and transfusion history, which of the following is the
MOST likely cause of the positive screen results?

Please select the single best answer

Cold autoantibodies

Warm autoantibodies

Alloantibodies

Feedback

Given the diagnosis of SLE and the positive reactions at the AHG phase,
including a positive autocontrol, the cause of the reactions is probably warm
autoantibodies.

Case Study
Case Study One, continued

A direct antiglobulin test ﴾DAT﴿ is performed using polyspecific antihuman globulin ﴾AHG﴿ and anti‐IgG. Positive results are obtained with both reagents.

Case Study
Case Study One, continued

With a Positive DAT ﴾with IgG specificity﴿ and reactive screen cells and autocontrol, the presence of a warm autoantibody becomes more apparent. Antibody
identification testing is performed to see if a pattern of reactivity is present. The results ﴾shown below﴿ indicate an apparent autoantibody, but it is impossible
from this testing to assess if there are also one or more alloantibodies present. What procedure should be performed at this point to determine if this is
autoantibody, alloantibody, or a combination of auto‐ and alloantibodies?
Consider how you would answer this question before proceeding to the next page.
 Case Study
Case Study One, continued

An adsorption technique should be employed to remove the autoantibody and determine if there is any underlying alloantibody.

Case Study
Ungraded Practice Question

Case Study One, continued

To determine the appropriate adsorption technique to use, an accurate transfusion history must be obtained. In this case the patient has not been recently
transfused ﴾ie, not transfused within the past three months﴿. Which adsorption technique is normally utilized if the patient has not been recently transfused?

Please select the single best answer

Autologous adsorption ﴾autoadsorption﴿

Allogenic adsorption ﴾alloadsorption﴿

Case Study
Ungraded Practice Question

Case Study One, continued

To determine the appropriate adsorption technique to use, an accurate transfusion history must be obtained. In this case the patient has not been recently
transfused ﴾ie, not transfused within the past three months﴿. Which adsorption technique is normally utilized if the patient has not been recently transfused?

Please select the single best answer

Autologous adsorption ﴾autoadsorption﴿

Allogenic adsorption ﴾alloadsorption﴿

Feedback

An autoadsorption can be utilized when the patient has not been recently transfused ﴾recent means within the past three months﴿. If the patient had been
recently transfused, an alloadsorption would have to be utilized. An autoadsorption in a recently transfused individual could potentially adsorb underlying
alloantibodies from the serum.

Case Study
Ungraded Practice Question

Case Study One, continued

The results of the autoadsorbed serum tested with a set of antibody screen
cells are shown on the right. What does the pattern of reactivity indicate?

Please select the single best answer

The autoantibody has only been partially adsorbed out

There is an underlying alloantibody

The test results are invalid because cells I and III are now negative

Case Study
Ungraded Practice Question

Case Study One, continued

 The test results are invalid because cells I and III are now negative

Case Study
Ungraded Practice Question

Case Study One, continued

The results of the autoadsorbed serum tested with a set of antibody screen
cells are shown on the right. What does the pattern of reactivity indicate?

Please select the single best answer

The autoantibody has only been partially adsorbed out

There is an underlying alloantibody

The test results are invalid because cells I and III are now negative

Feedback

Because the original antibody screen had reactivity with all cells, and the
autoadsorbed serum demonstrates reactivity with only one cell and the
autocontrol is now negative, we make the inference that the autoantibody has
been adsorbed out, and we are seeing the reactivity due to an underlying
alloantibody.

The check cells confirm the validity of the negative reactions in cells I and III.

Case Study
Case Study One Conclusion

An antibody identification is performed using the adsorbed serum and the results are shown below.
In this case we see that there is a specificity of anti‐K in the autoadsorbed serum.
This is a case of a warm autoantibody with an underlying alloantibody ﴾anti‐K﴿.

Case Study
Case Study Two

A hospitalized 45‐year‐old male complains of generalized aches and fatigue. He is currently receiving high‐dose intravenous penicillin to fight an infection. A CBC
and bilirubin are ordered. The hemoglobin result is 8.1 g/dL ﴾Reference interval 13.5‐18.0 g/dL﴿ and the bilirubin is slightly elevated.

As penicillin is a medication that can be associated with drug‐induced autoimmune hemolytic anemia via the drug‐adsorption mechanism, what test would be
helpful to ascertain if there is an immune reaction causing decreased red blood cell survival?
Consider how you would answer this question before proceeding to the next page.

Case Study
Case Study Two, continued

A DAT using monospecific reagents would help to determine immune sensitization of red blood cells. Usually only IgG is detected on the RBCs when antibodies
 Case Study
Case Study Two, continued

A DAT using monospecific reagents would help to determine immune sensitization of red blood cells. Usually only IgG is detected on the RBCs when antibodies
produced in response to penicillin is the cause of the positive DAT. However, C3 in addition to IgG could also be detected.

Case Study
Ungraded Practice Question

Case Study Two, continued

A DAT is ordered on the patient along with an antibody screen. The DAT is positive and the antibody screen is negative.
Elution studies are then performed. An elution is a technique where antibody is dissociated from sensitized red blood cells and harvested. One method that is
commonly used is an acid elution. In this method, an acidic solution is added to a volume of red blood cells. The acid environment disrupts the bond between
antibody and antigen on the red cell surface. The antibody can be collected in this acid solution and restored to a physiologic pH by the addition of a buffer. This
eluate can then be tested against a panel of red blood cells to determine a specificity.
The eluate in this case is tested with normal red cells and with penicillin‐sensitized red cells. In which of these tests would you expect the eluate to be reactive?

Please select the single best answer

With normal red cells

With penicillin‐sensitized red cells

With both normal red cells and penicillin‐sensitized red cells

Case Study
Ungraded Practice Question

Case Study Two, continued

A DAT is ordered on the patient along with an antibody screen. The DAT is positive and the antibody screen is negative.
Elution studies are then performed. An elution is a technique where antibody is dissociated from sensitized red blood cells and harvested. One method that is
commonly used is an acid elution. In this method, an acidic solution is added to a volume of red blood cells. The acid environment disrupts the bond between
antibody and antigen on the red cell surface. The antibody can be collected in this acid solution and restored to a physiologic pH by the addition of a buffer. This
eluate can then be tested against a panel of red blood cells to determine a specificity.
The eluate in this case is tested with normal red cells and with penicillin‐sensitized red cells. In which of these tests would you expect the eluate to be reactive?

Please select the single best answer

With normal red cells

With penicillin‐sensitized red cells

With both normal red cells and penicillin‐sensitized red cells

Feedback

The eluate will be reactive with penicillin‐sensitized red cells and nonreactive with normal red cells. Note that negative and positive controls must be tested when
 With both normal red cells and penicillin‐sensitized red cells

Feedback

The eluate will be reactive with penicillin‐sensitized red cells and nonreactive with normal red cells. Note that negative and positive controls must be tested when
using drug‐treated red cells. This ensures that any reactivity is interpreted accurately.
The results of the eluate tested against a panel of antibody identification cells ﴾normal red cells﴿ is shown below. The nonreactive eluate test results could indicate
that an antibody directed against the penicillin is causing the red cell sensitization. Because the panel cells used in routine antibody identification studies are not
treated with any particular medication that may be implicated in drug‐sensitization, the results will be negative.

Case Study
Case Study Two, Conclusion
This case involves the correlation of medication history, concurrent drop in hemoglobin level and rise in bilirubin level, and lack of an identifiable alloantibody in
the serum or eluate to establish that the hemolytic anemia is likely due to a drug‐mediated antibody.

References
References

AABB. Technical Manual. 19th ed. Bethesda, MD: AABB: 2017.

Garraty G: Target antigens for red‐cell bound antibodies. In Nance SJ [ed]: Clinical and Basic Science Aspects of Immunohematology. Arlington VA: American
Association of Blood Banks; 1991.
Harmening D. Autoimmune hemolytic anemias. In Modern Blood Banking & Transfusion Practices. 7th ed. Philadelphia: F.A. Davis; 2019.
Hoffman R, Benz EJ Jr, Silberstein LE, Heslop H, Weitz J, Anastasi J, eds. Hematology: Basic Principles and Practice. 7th ed. Philadelphia, PA: Elsevier; 2017.
Howard PR. Basic and Applied Concepts of Immunohematology 4thed. St. Louis, Missouri: Mosby Elsevier; 2016.
Kaushansky K, Lichtman MA, Prchal JT, Levi MM, Press OW, Burns LJ, Caligiuri M. Williams Hematology. 9th ed. New York, NY: McGraw‐Hill; 2015.
Rudmann SV. Textbook of Blood Banking and Transfusion Medicine. Philadelphia, PA: Saunders; 2005.