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Laboratory evaluation of suspected haemolytic reaction

You are here : Home/ Blood Bank Zone/ Adverse Reactions to Blood Transfusion/ 3. Laboratory evaluation of suspected haemolytic reaction

3. Laboratory evaluation of suspected haemolytic reaction

The work up at the transfusion centre of suspected transfusion reaction includes :

1.Check all the records to ensure that the correct unit of blood was transfused to the right patient. This includes :

- Patient’s details
- Blood requisition form
- Compatibility report
- Labels

Till the time a clerical error is not ruled out, stop issue of all blood units from the centre, as it may lead to issue of another mismatched blood due to mix-up of samples or other clerical errors.

2. The following samples must be immediately sent to the laboratory and blood bank.

- Post transfusion sample in plain vial (5m1)
- Post transfusion sample in EDTA vial (3m1)
- Blood bag along with transfusion set
- Urine (post-transfusion) 1st sample
- Coagulation profile-citrated blood sample
- Blood cultures from the blood bag and the patient

The pre-transfusion sample should be preserved in the laboratory for 7 days.

Investigation

For evaluation of a patient with haemoluytic reactions the investigation may be divided into three:
1. Investigation for evidence of increased red cell destruction
2. Investigation for identification of cause of haemolysis
3. Investigation to follow up of a patient with proven haemolysis

Tests for evidence of increased red cell destruction

1. Centrifuge the post-transfusion blood sample and examine the supernatent plasma. Compare this with the pre-transfusion sample. A pink or red colour in the post-transfusion sample indicates haemolysis and presence of free haemoglobin.

2. Perform a Direct antiglobulin test (DAT) on the post-transfusion sample. A positive test indicate immune haemolysis. A negative DAT with haemoglobinaemia suggests a non-immune haemolysis e.g. due to mechanical trauma or thermal damage.

3. Reticulocyte count is raised in patient with haemolysis.

4. Pre-and post haemoglobin value.

5. Serum unconjugated bilirubin estimation.

Tests to establish the cause of haemolysis

a. Repeat ABO and Rh grouping on the pre-and post-transfusion samples and from the bag. If blood group of the pre-and post-transfusion sample do not agree, it may be due to error in patient identification, drawing or grouping of blood. Another patient’s sample may have been drawn at the same time and labelled incorrectly.

If the donor sample is not of the same group as indicated on the bag, an error in labelling or grouping and also in the compatibility testing should be suspected.

b. Repeat the compatibility testing on pre-and post-transfusion samples with a sample of blood from the bag. The testing must be done using saline, enzyme/albumin and indirect antiglobulin techniques. If both the samples show incompatibility, there was an error in the pre-transfusion testing. The donor sample may have been incorrectly labelled or the crcssmatch reaction was incorrectly read as negative.

If the incompatibility is seen only with the postransfusion sample, an anamnestic response should be suspected. If both crossmatches are compatible and there is a strong suspicion of haemolysis, further tests are required.

c. Perform an antibody screening on the pre-and post-transfusion samples. If an antibody is detected, it should be identified using further tests. An antibody in the post transfusion sample may be due to
- Anamnestic response (e.g. anti-Kidd and anti-Duffy antibodies)
- Passively acquired antibody from donor plasma.
Antibody screening must be done using sensitive techniques.

d. Examine a stained peripheral blood film for spherocytes and crenated cells, presence of which favours a diagnosis of immune haemolysis.

Tests for non-immune haemolysis

1. Examine the bag for discoloration or clots, any abnormal mass, foul small or a fuzzy cell: plasma interface. Take specimens from the bag for culture at 4°C, 20°C and 37°C for bacterial and fungal cultures and also for gram’s staining.

2. Examine the plasma in the bag for presence of free haemoglobin. If present, if indicates improper storage of the unit of blood over heating or over-cooling injection of drugs or hypotonic solutions. Presence of free haemoglobin in the administration tubing suggests that the same tube was used for administration of dextrose/other solutions.

3. The possibility of mechanical/osmotic haemolysis should also be borne in minds

Tests done to follow up a patient with proven haemolysis

1. Test sample for serum unconjugated bilirubin levels
2. Measure plasma haemoglobin levels. These elevated (N : 10-40 mgJL).
3. Serum haptoglobins are reduced. The level in the post-transfusion sample must be compared with that in the pretransfusion sample as the normal range is very wide.
4. Examine the post-transfusion urine sample for free haemoglobin. This is done in a freshly collected sample of urine. In the event of delay in evaluation of a haemolytic reaction, the urine may be tested for haemosiderin. The presence of intact red cells indicates haemorrhage and not haemolysis.
5. Perform a coagulation screen (PT,PTTK,TT) and platelet count to check for DIC.
6. Monitor blood urea & serum creatnine to assess renal function.

Consequences of haemoiytic transfusion reaction

1. disseminated intravascular coagulation
Following the transfusion of ABO incompatible blood, abnormal bleeding may develop. In patients undergoing an operation, there may be bleeding from the wound or venepuncture site. If DIC develops there is thrombocytopenia with low levels of plasma fibringoen. Fibrin degradation products are detected in the serum (250-450 ug/mI).

2. Renal failure
This is particularly common in intravascular destruction due to lytic antibodies such as anti-A and anti-B. It is due to the destruction of red cells and consequent haemoglobinemia. Hypotension and DIC also contribute to the renal damage.

Management of acute HTR

At the first suspicion of a haemolytic reaction
1. Immediate stop the transfusion
2. Maintain a patent intravenous line.
3. Ensure diuresis (Diuretics, dopamine)
4. Provide cardio-respiratory support.
5. Maintain blood pressure, heart rate and adequate airway.
6. Check the patient’s identification to see If thepatlent received the intended unit of blood.
7. Report the reaction immediately to the transfusion centre.
8. Send samples to the blood bank for further testing.
9. Monitor blood urea and creatinine levels.
10.Perform a complete coagulation screen to rule out DIC.
11.Check any other patient receiving transfusion at same time.

Mortality associated with incompatible transfusion

DIC and renal failure may cause the death of a patient with HTR. The outcome is determined by the potency of the antibodies in the patient’s plasma.

Acute extravascuiar haemolytic reaction

This is caused by antibodies which do not activate complement completely but only to the C3b stage. As C3a and C5a are not produced, the symptoms and signs are minimal.

Antibodies associated with extravascular destruction

anti-D
anti-K
anti-Fya

Clinical features

These patients do not present as an emergency and remain clinically stable.
1. Haemoglobinuria is an important feature.
2. Fever not associated with chills is also observed. The febrile reaction attains severe intensity.
3. Hyperbilirubinaemia also occurs. The levels peak 5 days after the transfusion.
4. Urine urobilinogen is increased.

The difference between the two types of immune red cell destruction are tabulated in Table below

Types of Immune red cell destruction

Site of Intravascular destruction	Extravascular
Characteristic of antibody	Potent,lytic	Antibodies causing partial or no complement activation (anti-Jk, anti- anti-K)
Symptoms	Substernal pain, lumbar pain, restlessness	Nausea, shivering
Signs	Immediate Hypotension Fever Uncontrollable bleeding Later haemoglobinuria	Fever Jaundice

Investigations

a. Direct antiglobulin test :This is found to be positive.
b. Hb/Hct :The recipient fails to achieve the expected rise in haemoglobin/haematocrit.
c. Serum bilirubin :The conjugated serum bilirubin levels are raised.
d. Urine Urobilinogen is increased

Prognosis and outcome

The patients do not require any therapy as these reactions are self-limiting. DIC and renal failure are rarely seen.

Delayed HTR

These reactions occur days or weeks after the blood is transfused. The initial level of antibody in the recipient is low but the transfusion provokes an anamnestic response.

The antibody titre rises rapidly thereafter with resulting haemolysis. Secondary immune response occurs by previous pregnancy or transfusion.

Antibodies associated with DHTR :

anti-c, anti-E
anti-A, anti-B (IgG)
anti-Kell
anti-Fya,-Fyb
anti-Jka,-Jkh

Clinical features

The time course of DHTR is variable ranging from an abrupt haemolytic episode to gradual destruction of red cells with little evidence of haemolysis. Maximal rate of destruction is 4-13 days after transfusion.

* Jaundice occurs 5-7 days after transfusion
* Haemoglobinuria is not uncommon and is seen with antibodies of different specificities.
* DHTRs may occassionally be followed by renal failure.

Laboratory features

1. A fall in haemoglobin not attributed to any other cause.
2. Appearance of a new alloantibody.
3. Spherocytosis is observed in blood films which may be the only indicator.
4. Direct antiglobulin test is positive. The test becomes positive a few days after transfusion and remains until all incompatible cells have been eliminated.

With these features a diagnosis of All-IA may be made specially if the significance of a previous transfusion is not realised.

5. Antibody detection The antibody responsible for the reaction may be detected by preparing an eluvate from the patient’s red cells.

The antibody is detected 4-7 days after transfusion and peaks after 10-15 days

Sensitive antibody detection techniques in pretransfusion testing may prevent DHTRs.

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