INNOCENT BYSTANDERS IN BLOOD

TRANSFUSION MEDICINE BY ALHAJI BUKAR, AB

Introduction

Innocent bystander may be defined as an immune

destruction of cells or tissues caused by antibody

that is not developed in response to intrinsic antigen

on the cell undergoing the cytolysis or destruction.

It’s the destruction of antigen-negative red blood

cells during immune haemolytic reaction such as

delayed haemolytic transfusion reaction. Any

immunologic response that occurs when the cells or

tissues that are injured or haemolysed by the

immunologic reaction are not involved in the

antigen-antibody reaction, but haemolysis is called

innocent bystanders haemolysis. (Anonymous).

INNOCENT BYSTANDER
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 Although many have suspected that bystander

haemolysis does occur, that phenomenon is very

difficult to document. But in recent year, a

compelling data have been presented documenting

bystander immune cytolysis in a number of different

clinical settings, and efforts have been made to

define the mechanisms by which this occurs.

Laboratory scientist and physician must be aware

that some example of immune cytolysis of

autologous cell is, in reality, example of temporary

bystander immune cytolysis rather than

trueautoimmune disease. Furthermore, some

alloimmune haemolytic reaction can result in

cytolysis of bystander cell.

Drug can cause immune destruction of red

blood cells and other blood cells, although a

documented incidence of drug-induced immune

haemolytic anaemia is rare. Worlledge reported that

the red blood cells of 40 out of 480 blood samples

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(9%) collected for routine tests were agglutinated by

anticomplement sera. Only one by anti IgG and this

is obtained from patient being treated with α-

methyldopa.
In blood banking, drug-mediated immune

haemolytic anaemia may come to the attention of

the laboratory scientist, haematologist or blood

banker when there is unexpected result in routine

testing example a positive autologous control

reaction in AHG phase of antibody screening

/compatibility testing or a positive DAT result. Drug

should be suspected as a positive explanation for

immune haemolysis or positive DAT result when

there is no other reason for the serologic and

haematological findings and if the patients have a

history of taking the drug.

Petz and Garratty review four different mechanisms
by which drugs can induce haemolysis.
1. Innocent bystander mechanism
2. Membrane modification
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 3. Drug adsorption
4. Autoantibody (methyldopa)

THE MECHANISM OF INNOCENT BYSTANDER

HAEMOLYSIS
The largest variety of drugs causing immune-

mediated problems work by innocent bystander

mechanism was first described in 1960s. Examples

of drugs working by this mechanism are rifampicin,

phenacetin, quinine, quinidine, nomifensine,

chlorpropramide, hydrochlorothiazide,

cephalosporin, diclofenac etc.

Drugs operating through this mechanism combine

with plasma protein to form immunogen. The

antibody IgG or IgM subsequently produced

recognizes determinants on the drug. The drug acts

as a hapten (a small molecule that stimulates the

production of antibody molecules only when

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conjugated by covalent or other bond to a larger

molecule, called carrier molecule e.g. protein).

If a patient ingests the same drug or a drug bearing

the same haptenic group following immunization,

the formation of drug-antidrug or drug-antibody

complex may occur. Following antigen-antibody

interaction, the complement cascade may be

activated. Red blood cells are thought to be involved

in this process only as ‘innocent bystanders’. The

soluble drug-antidrug complex absorbs loosely to

the red blood cell surface and fixes complement to

produced haemolysis of uninvolved cells. Classically

haemolysis may develop within minutes or hours of

drug ingestion. The DAT is positive for complement

only; occasionally, IgG may be present. Garratty

suggested that attachment of immune complex to

the red blood cell may be specific. The resulting

antibody may react with the drug-erythrocyte

complex, but not with the normal cell without drug

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complex. Because complement activation is

involved in the immune complex mechanism,

clinically affected patients frequently present with

acute intravascular haemolysis, and may be

associated with haemoglobinaemia,

haemoglobinuria and acute renal failure. This

immune complex mechanism is responsible for the

majority of drug induced haemolytic anaemia. Small

doses of drug re-administered after a latent period

can produce acute intravascular haemolysis. When

other causes for haemoglobinaemia and

haemoglobinuria have been excluded e.g. ABO

haemolytic transfusion reaction, a drug-antidrug

reaction should be considered. Patients usually

recover rapidly once the drug is withdrawn. The

direct antiglobulin (DAT) test result on patient red

blood cells will usually be positive for complement

only, occasionally, IgG may be present. If mono-

specific reagents are used, agglutination will occur

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with anticomplement only, but not with anti-IgG. The

drug-antidrug complex when dissociated from red

blood cell, only C3 is detected by DAT.

Sketch of Immune complex mechanism
Drug+Ab→ Drug-Ab,
Drug-Ab,+rbc → Drug-Ab-rbc,
Drug-Ab-rbc +complement→ Drug-Ab-rbc-
complement

DAT IgG is negative, even when the antibody is of

the IgG class, because the drug-antidrug complex

thought to elute from the cell during the washing

procedure before the anti globulin test. Result from

(all) other routine blood bank test are negative in all

phases; the antibody is directed against a drug, not

against a red blood cell antigen. Therefore, the

antibody screening and compatibility test results are

negative, unless an alloantibody is also present. But

antibody is demonstrable if serum, complement,

drug incubated with red blood cell. Antibody

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screening on eluate is negative; eluate tested

against reagent normal compatible red blood cell

will also react negatively and is not demonstrable

even in the presence of drug.

Antibody screening/DAT
Polyspecific DAT test on patient’s red blood cell
+
Mono specific anti IgG DAT on patient’s red blood
cell +/-
Mono specific anti C3 DAT on patient’s red blood cell
+++
To confirm that a drug-antidrug reaction through this

mechanism is responsible for a positive DAT result,

one must demonstrate the presence of the antibody

in the patient serum. Antibodies in the patient

serum may be demonstrated by incubating normal

ABO compatible red blood cell with the patient

serum in the presence of the suspected drug

solution. Complement is activated by adding fresh

serum. Haemolysis after incubation is indication of

positivity. Use reagent containing anti-C3 activity for

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the antiglobulin test. For the test result to be

interpreted correctly, adequate control test must be

performed. Patient’s serum must not react with the

red cells when saline or drug’s diluent is substituted

for the drug solution, and drug solution must not

haemolyse the suspension of cells non-specifically.

Interpretation of the tests to confirm presence of
antidrug antibody acting by innocent bystander
mechanism
Test
Result
Patient’s serum+ fresh serum+ drug solution+ normal compatible red cell
+++

Interpretation; Anti-drug antibody present if control is working

Controls
Patient’s serum+ normal compatible red cell, no drug, no complement
negative

Interpretation; No alloantibody against the normal compatible red cell’s

antigen

Fresh serum+ drug+ normal compatible red cell, no patient’s serum

negative

Interpretation;No alloantibody against reagent rbc or drug present in

serum of random donor (sources of complement)

Drug solution+ normal compatible red cell, no patient’s serum, no

complement negative

Interpretation; Drug solution does not cause rbc to agglutinate or

haemolyse

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In most blood banks, confirmatory testing is done

only when the patient has a haematological

complications and not only when the patient simply

has a history of taking the drug and a positive DAT

result. Some of the drugs known to cause immune

complex-mediated problems are in frequent use,

and there are large number of patients with positive

DAT result and no evidence of haemolysis.

Therefore, a full work-up is done only for academic

interest and is not required before release of red

blood cells for emergency transfusion.

SEROLOGICAL DIAGNOSIS AND TREATMENT OF

INNOCENT BYSTANDER HAEMOLYSIS

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Diagnosis should be made in three stages:
1. Diagnosis of a DAT positive haemolytic anaemia
2. Careful drug history
3. Serological demonstration of drug-specific
antibody which interact with red blood cell.
The DAT is usually positive for complement but may

be negative if performed immediately after a brisk

episode of haemolysis. The red cell eluate is not

reactive even in the presence of the drug. The drug-

specific antibody is best detected by pre-incubating

the patient’s serum with the drug in solution to

allow immune complexes to form. The pre-incubated

serum is then tested against normal and enzyme-

modified groups of compatible red blood cell in the

presence of fresh complement. In some cases, the

antibodies may be specific for metabolites rather

than for the parent drug. Drug metabolite antibodies

may be detected by pre-incubating drug metabolite

obtained from serum or urine of a volunteer (who

have taken the drug) with the patient serum.

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Treatment is by discontinuation of the drug.

Although haemolysis by this mechanism is rare, the

onset is sudden and characterise by intravascular

haemolysis and renal failure. Therefore immediate

cessation of the drug is essential. Steroid treatment

also may be given. But the presence of positive DAT

result without haemolysis does not necessarily imply

that the drug must be discontinued, if the effect of

the drug is therapeutically beneficial. In general,

however, other drug should be substituted and the

patient observed for resolution of the anaemia to

confirm a drug-induced haemolytic process.

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SUMMARY AND CONCLUSION
Drug can cause immune destruction of red blood

cell and other blood cells, although a documented

incidence of drug-induced innocent bystander

immune haemolysis is rare. Drugs-mediated

immune haemolytic anaemia may usually come to

the attention of haematologist or laboratory

scientist when there is unexpected result in routine

testing e.g. positive control reaction in AHG phase of

antibody screening/compatibility testing. Before any

special testing is done, one should proceed in the

following manner;
1. Obtain patient’s medical history, including
medication, transfusion and pregnancies.
2. Perform DAT using red blood cell collected in
EDTA. Test red cell with poly-specific
antiglobulin & mono-specific reagent e.g. anti
C3
3. Screen the patient’s serum for red blood cell
allo-antibodies

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 4. Prepare and test an eluate for red blood cell
allo-antibodies if the patient has been
recently transfused.
After evaluating this information, one can decide

whether drugs are a possible cause of the problem

and which of the mechanism is involved. Is it

innocent bystander mechanism of haemolysis or

not? Then, when other causes e.g. transfusion

reaction have been excluded and if the clinical

situation warrants additional testing, drug-coated

cells or solutions of the drug can be prepared for

confirmatory test.

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