Immune hemolytic anemia is a state in which a patient has a
shortened red blood cell survival associated with hemolysis resulting from an
immune reaction.
Classification of immune hemolytic anemia
based on serologic findings:
I. Autoimmune Hemolytic Anemia (AIHA)
A. AIHA associated with warm
antibodies
Autoimmune hemolytic anemia of
the warm antibody type is mainly characterized by a positive direct
antiglobulin test. Often, so many antibodies are attached to the cells that
little or none remains free in the serum. The antibody reacts at 37oC
but not at lower temperatures, is predominantly IgG and sometimes binds
complement. Most warm autoantibodies have no apparent specificity but when
specificity can be established, it is often directed towards Rh antigens.
Autoantibodies of other specificities have been reported. Common serologic
findings include the presence of an autoantibody and an alloantibody. The
mixture gives the appearance of non–specificity.
The present view is that some of
the antibodies of Rh type specificity may be directed against basic Rh material
present on all cells.
1. Primary (idiopathic) – term that is applied if there
is no apparent cause for the development of the antibody.
2. Secondary is applied to cases in which
antibody appears after certain diseases such as lymphoma, SLE, infections,
carcinomas, etc.
B. AIHA associated with cold
antibodies
Autoimmune hemolytic anemia of
the cold antibody type is characterized by a loss of positive direct
antiglobulin test and the presence of freer antibody in the serum. The antibody
reacts at all temperatures, though it is stronger at/or below room
temperatures, is mostly IgM (though some maybe partly IgG as well) and usually
binds complement. The specificity of the antibody is often anti–I or anti–i,
though many workers have reported the occurrence of non–specific cold agglutinins
in addition to anti–I in the same individual.
Why some anti–I antibodies cause
autoimmune hemolytic anemia while others do not remain at the moment
established? The difference may be related to the degree of reactivity and the
thermal amplitude of the antibody. It has also been suggested that two types of
anti–I exist which appear identical on testing, yet which are believed to have
different specificities.
1. Cold agglutinin syndrome
Cold agglutinin syndrome is the
most common type of autoimmune hemolytic anemia associated with cold
antibodies. It can occur as an acute or chronic condition. Patients with
chronic primary cold agglutinin syndrome are often elderly and present with a
chronic hemolytic anemia of mild to moderate intensity with, in cold weather, Reynaud’s phenomenon, and often hemoglobinuria also.
a. Primary (idiopathic) – term that is applied if there
is no apparent cause for the development of the antibody.
b. Secondary is applied to cases in which
antibody appears after certain diseases such as lymphoma, Mycoplasma pneumonia,
etc.
2. Paroxysmal cold hemoglobinuria
This is characterized by the
passage of urine containing hemoglobin and/or methemoglobin in solution
(hemoglobinuria) as a result of exposure to cold.
In 1904, Donath and Landsteiner
reported that the hemolysis in paroxysmal cold hemoglobinuria was probably due
to an autolysin which unites with the patient’s red cells at low temperatures.
This was confirmed by many workers. The test for the detection of hemolysin is
called the Donath–Landsteiner Test and the antibody is called the Donath–Landsteiner
(DL) antibody. The DL antibody is directed against antigen in the P
blood group sytem.
a.
Primary (idiopathic)
b.
Secondary (syphilis and viral infection)
II. Drug induced hemolytic anemia
The list of drugs capable of
causing hematologic abnormalities continues to grow. Some of these
abnormalities involve red blood cells. The antibodies once formed may cause
positive direct antiglobulin tests and sometimes hemolytic anemia by four
possible mechanisms:
A. Immune–complex adsorption to red
cells
Certain drugs (e.g. phenacetin,
quinine, quinidine, etc.) that are incapable of producing an immune response
themselves, because of their low molecular weight, can become immunogenic when
coupled with a plasma protein. The antibody produced is directed toward the
drug and forms a drug–antibody complex. The complex is absorbed non–specifically
by red cell destruction because it is usually IgM and a very effective
initiator of the complement system.
B. Modification of red blood cell
membrane by drug, allowing non–immunologic adsorption of protein.
Cephalosporin can also cause a
positive direct antiglobulin test by altering the red cell membrane, resulting
in the non–immunologic adsorption of plasma proteins, including albumin,
fibrinogen, complement and IgG.
C. Red blood cell autoantibodies
induced by drugs by unknown mechanism
Many drugs cause a positive
direct antiglobulin test and hemolytic anemia by a mechanism which is clearly
not understood. Most important is the anti–hypertensive drug alpha–methyl–dopa
(Aldomet). Of patients who take this drug for several months, 15% develop a
positive direct antiglobulin test and 0.8% develops hemolytic anemia. An
antibody can be demonstrated in these patient’s sera that reacts with normal
red cells. Often, the antibodies appear to have Rh specificity and the characteristics
are exactly as an autoimmune hemolytic anemia of the warm antibody type.
D. Drug absorbed onto red blood
cells
Penicillin can become bound to
red cells when high doses of the drug are administered, resulting in the
production of IgG anti–penicillin antibodies. This causes a positive direct
antiglobulin test and in some cases, hemolytic anemia. Cephalosporin drugs can
also act in much the same way.
III. Alloimmune hemolytic anemia
A. Hemolytic Disease of the Newborn (refer to
Lecture #9)
B. Hemolytic Transfusion Reaction (refer to Lecture #10)
Serologic investigation of Autoimmune
Hemolytic Anemia:
1. Direct Coombs Test
2. Elution
3. Identification of antibodies
Essential terms encountered in the
investigation of AIHA:
1. Adsorption – is the take up of antibody by
the cells.
2. Elution – is the removal of antibody from the
cells.
3. Absorption – is the removal of antibody from
the serum.
Various uses of absorption:
a. Separation of one specific antibody from
serum containing several types.
b. Confirmation of antibody specificity.
c. Confirmation of the presence of antigens
(weak antigens) on the red cells.
d. Identification of antibodies causing
hemolytic disease of the newborn and acquired anemia.
Antibody identification
Antibody identification is performed using a cell panel. Panels of
red blood cells are composed of selected group O red blood cells from several
people tested for as many as possible of the common antigenic determinants. A
panel should include some red cells that possess and some that lack these
determinants, in such a manner that a distinct pattern of reaction is available
for single antibodies to all or most of the antigens represented on the panel.
In establishing the identity of an antibody, it is a good practice to use a
process of basic elimination, based on the temperature of the reactions, the
techniques which give reaction and the frequency of positive results obtained.
Titration is a semi–quantitative means of measuring the amount of
antibody in a serum. Serial dilutions (usually twofold) of antibody are tested
with a constant volume of red blood cells and the result is expressed as the
reciprocal of the highest dilution at which agglutination is observed. This is
usually a macroscopic observation but in techniques such as those used in high
titer, low avidity testing, it is microscopic.
****** ABSORPTION OF COLD AUTOAGGLUTININ ******
Principle
Cold–reacting autoantibodies can interfere with the identification
of alloantibodies in a patient’s serum. Absorption of the patient’s serum with
autologous red blood cells can remove autoantibody from the serum, permitting
detection of underlying alloantibodies.
Specimen
5 to 7 ml of
clotted blood (red top)
7 ml of EDTA blood (lavender top)
Procedure
1. After properly obtaining the specimen, the
red top tube may be allowed to clot in the refrigerator.
2. Wash the patient’s own red blood cells from
the anticoagulated specimen tube with warm 37oC saline at least four
times. Care should be taken to remove all the saline after each wash.
3. After the last wash, centrifuge the cells for
5 minutes at 3,400 rpm.
4. Remove any remaining saline. This step is
very important because residual saline may dilute out an alloantibody in the
serum that is being auto absorbed.
5. Obtain the patient’s clotted blood from the
refrigerator and centrifuge.
6. Remove all available serum from this clotted
specimen. If any red blood cells are unintentionally remove with the serum,
centrifuge the serum and transfer it to a clean test tube.
7. Transfer enough serum to perform all
necessary post–absorption testing, e.g., compatibility testing, antibody
screening, etc., in a large test tube.
8. Add an equal volume of the well–washed,
tightly packed autologous red blood cells prepared in step 2 through 4.
9. Mix the serum and red cells thoroughly and
place in a slush ice bath in a 4oC refrigerator for 15 to 20
minutes.
Note: A
longer incubation of this aliquot of red cells does not remove additional
Antibody
because all the antigen sites are covered within the first 15 to
20
minutes.
10. Remove the
tube from the ice bath and centrifuge for 3 minutes at 3,400 rpm.
Note: A refrigerated centrifuge or an ice–packed test tube holder is
preferred.
11. After
centrifugation, immediately place the tube in the refrigerator without
disturbing the red blood cells. This step increases the amount of antibody
removed with each absorption.
12. After 5
minutes, carefully transfer the serum to a clean test tube. The serum must be
cell–free. Repeat step 10 if necessary.
13. At this
point, the serum has been autoabsorbed once.
14. To perform
subsequent autoabsorption, add the previously autoabsorbed serum to a fresh
aliquot of well–washed tightly packed autologous red blood cells and repeat
step 8 through 12.
15. After the
serum has been removed from the last aliquot of autologous red blood cells, it
should be warmed up to room temperature before being used for testing.
****** ABSORPTION OF WARM AUTOAGGLUTININS ******
Principle
Absorption of a patient’s serum with autologous red blood cells can
remove autoantibodies from the serum and permits detection of specific
alloantibodies. Because circulating autologous red blood cells are already
coated with autoantibody, they are pre–treated with proteolytic enzyme. This
technique uncovers antigen site which are then capable of binding free
autoantibody from the serum during incubation and producing an absorbed serum.
In some cases, warm–reactive autoagglutinins can mask underlying clinically
significant alloantibodies.
Reagents
6% Bovine albumin
1% Ficin
Procedure
1. Wash 2 ml of patient red blood cells from an
EDTA anticoagulated specimen four times in saline. Discard the supernatant
fluid from the final centrifugation.
2. Add an equal volume of 6% albumin to the
packed red blood cells and mix thoroughly.
3. Incubate the red blood cells–albumin mixture
at 56oC for 3 to 5 minutes. Gently agitate the mixture during
incubation.
4. Centrifuge at 3,400 rpm for 2 minutes.
5. Transfer the supernatant fluid to a clean
test tube
Note: this supernatant fluid may be used as an eluate for testing, if a
limited quantity of autologous red cells are available.
6. Wash the cells three times with normal
saline. Discard the final supernatant.
7. Add 1 ml of 1% ficin to the packed red cells.
Mix thoroughly
8. Incubate at 37oC for 15 minutes.
9. Wash the ficin – RBC mixture three times in
saline.
10. Centrifuge
the final wash for at least 5 minutes at 3,400 rpm. Remove as much of the
supernatant as possible.
11. Divide the
red cells into two equal aliquots.
12. Centrifuge
the red top tube specimen. To one aliquot of the washed red cells, add 2 ml of
the patient’s serum, mix and incubate at 37oC for 30 minutes.
13. Centrifuge
at 3,400 rpm for 2 minutes and transfer the serum to the second aliquot of red
cells.
14. Mix and
incubate this mixture at 37oC for 30 minutes.
15. Centrifuge
at 3,400 rpm for 2 minutes. Immediately transfer the absorbed serum to a clean
test tube. The serum must be cell free. Re–centrifuge the specimen if any red
cells are inadvertently transferred with serum.
16. At least two
autoabsorption are needed to remove enough autoantibodies to a test for
alloantibody reactivity.
If the patient’s red cells can be shown to be non–reactive with
DAT following step 5, these cells can be used to check the efficiency of the
absorption process. When the red cells no longer demonstrate autoantibody
absorption from the serum with the DAT procedure, the serum is now ready for
alloantibody testing using group reagent screening cells.
****** THE DONATH LANDSTEINER SCREENING TEST ******
Principle
The Donath–Landsteiner antibody screening test is used to demonstrate
the presence of this extremely potent hemolysin. This autoantibody requires
cold incubation to exhibit hemolysis in the patient’s serum. A positive test is
diagnostic of paroxysmal cold hemoglobinuria (PCH), the rarest form of auto–immune
hemolytic anemia.
Specimen
Fresh venous
blood
Procedure
1. Place two test tubes in a 37oC
water bath or heat block. Warm a 10 cc syringe by holding it in the palm of the
hand for a few minutes.
2. Draw 10 ml of blood and transfer 5 ml to each
test tube. Label one tube “control” and immediately place at 37oC.
Label the other tube “test” and place in an ice water bath at 4oC.
3. Incubate both tubes for 1 hour. At the end of
one hour, move the “test” sample to the 37oC water bath for an
additional 30 minutes.
4. At the end of 90 minutes, carefully remove
the tubes and examine for hemolysis
Interpretation
If the serum in both tubes is free of hemolysis, the test is
negative. If a pink or red color is present in the serum of the “test” and the
“control” is free of hemolysis, the test is positive. Normal blood will exhibit
no hemolysis in either tube.
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