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).

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INNOCENT BYSTANDER

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 true autoimmune 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 (9%)

collected for routine tests were agglutinated by anticomplement

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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

3. Drug adsorption

4. Autoantibody (methyldopa)

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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 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

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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

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

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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 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.

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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

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

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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

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.

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SEROLOGICAL DIAGNOSIS AND TREATMENT OF INNOCENT BYSTANDER HAEMOLYSIS

Diagnosis should be made in three stages:

1. Diagnosis of a DAT positive haemolytic anaemia2. Careful drug history3. 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

4. Prepare and test an eluate for red blood cell allo-

antibodies if the patient has been recently transfused.

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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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