STANDARD OPERATING PROCEDURE (SOP) : INVESTIGATION OF TRANSFUSION REACTION

BLOOD CENTRE NAME

SOP Number	Effective date	Pages	Author	Authorised by
Version	Review Period	No. of copies	Reviewed by	Date

LOCATION    Advanced Red Cell Serology	SUBJECT         Investigation of Transfusion Reaction
FUNCTION     To identify cause of transfusion reaction	DISTRIBUTION  -Supervisor in charge of Advanced Red Cell Serology Laboratory  -Supervisor in charge of Red Cell  -Serelogy Laboratory  – Master File

1. SCOPE & APPLICATION

This Standard Operating Procedure (SOP provides the protocol to be followed to identify the cause of an adverse transfusion reaction and prevent its reoccurrence).

2. RESPONSIBILITY

It is responsibility of the technician in the Red Cell Serology Laboratory to accept the blood/component implicated in the transfusion reaction which is returned from the ward/OT. It is the duty of the same technician to ensure that there is documented evidence of the nature of reaction either on the transfusion request form or on a separate letter addressed to blood bank, along with the post-transfusion blood sample (both EDTA and clotted) and urine specimen, if necessary. The direct antiglobulin test (DAT) should be performed on the post-transfusion EDTA sample immediately on receipt before refrigeration. The unit and samples should be preserved properly and handed over to the advanced red cell serology technician who is responsible for detail investigation.

• REFERENCES

1. Modern Blood Banking & Transfusion Practices . D.M Harmening. 6^th edition.

• MATERIALS  REQUIRED

4.1      Equipment:

• Refrigerator to store samples and reagents at 2- 6⁰C.

• Deep Freezer to store enzyme papain-cystein in frozen state.

• Table Top Centrifuge.

• Automated Cell washer.

• Microscope.

• Dri bath / Incubator.

4.2       Specimen:

• Blood/component bag returned room ward/OT.

• Patient’s pre-transfusion blood sample(clotted).

• Patient’s post-transfusion blood sample (EDTA and clotted).

• Patient’s post-transfusion urine sample.

4.3       Reagents:

• ANTI-a, Anti-B, Anti-AB Antisera.

• Group A,B &O pooled cells.

• Papain-cystein/22% Bovine albumin.

• Antihuman globulin reagent(anti-IgG anti-C3d).

• IgG Sensitised Control Cells.

• 0.9% Saline.

• Distilled water.

• 30g/l sulfosalicylic acid solution.

• Ammonium Sulphate {NH₄ (so₄)2}.

4.4Glassware:

• Serum tubes.

• Coombs’ tubes(for patient grouping only).

• Micro tubes.

• Pasteur pipettes.

• Glass slides.

• Small funnel.

• 20ml test tubes.

• 5ml pipette.

4.5       Miscellaneous:

• Rubber teats.

• Disposal box.

• 2 plastic beakers.

• Wooden block to hold micro tubes.

• Aluminium racks to hold serum and coombs’ tubes.

• Whatmen No.1 filter paper.

• 5ml plastic vial with screw cap.

5. PROCEDURE

5.1      Principle:

Red Cell Serological tests are based on the principle of agglutination and help to identify haemolytic transfusion reactions caused either by ABO incompatible transfusion or irregular red cell antibodies in patient’s blood.

Leuco-agglutinations, if present are detected by agglutination of random donor leucocytes in cases of febrile transfusion reaction. Serum bilirubin total and indirect are raised in case of haemolysis. The sulfosalicylic acid test helps to differentiate between haemoglobin and non-protein pigment, probably porphyrin in the urine. The ammonium sulphate precipitation test is based on the fact that haemoglobin and myoglobin are precipitated in urine at different degrees of ammonium sulphate saturation.

5.2       Serological Tests

5.2.1    Perform   a   direct   antiglobulin    test   (DAT)  on   post-transfusion     EDTA  sample

before refrigeration immediately on receipt. If test is positive, perform DAT on

pre-transfusion sample to verify whether sensitisation is due to transfusion or

it pre-existed.

5.2.2   Repeat grouping and antibody screening of patient’s pre-transfusion sample.

5.2.3   Repeat grouping and antibody screening of patient’s post-transfusion sample.

5.2.4    Repeat grouping and antibody screening of donor sample.

5.2.5    Repeat grouping  of  unit  from  bag.  In case  of  packed  cell  unit,  do  only  cell

grouping. In case of FFP, do only serum grouping.

5.2.6 Repeat crossmatching of donor with patient’s pre and post transfusion samples using saline / enzyme / IAT. Use donor cells from blood bag and not the pilot tube.

5.3       Leucocyte Antibody Test:

In case of febrile transfusion reaction and hypotension, look for leukocyte antibodies.

5.4       Biochemical Tests:

5.4.1    Note colour of plasma. Plasma is pink, if haemoglobin is present and icteric if

bilirubin is present.

5.4.2    Separate      the    patient’s      pre    and    post    transfusion      serum     and    send     to

biochemistry  department  in  a  5  ml  screw  cap  plastic  vial  bearing  the  date,

patient and test identification for estimation of serum bilirubin            total, direct and

indirect  and estimation of plasma hemoglobin.

5.4.3    Send the biochemistry request form with proper entries along with the sample.

5.4.4    Collect the report from biochemistry lab.

5.4.5  Tests on post-transfusion urine sample.

Red colour indicates haematuria or haemaglobinuria.

5.5       Microbiology:

1. Send the donor unit for smear and culture (at 37⁰ C, room temperature and 4⁰C) to bacteriology department.

• Make proper entries in the bacteriology despatch book and bacteriology request form and send along with the unit.

• Collect the report from bacteriology lab.

• If donor unit reveals bacteremia, then request the attending doctor to get the patient’s blood culture done and report the findings to the blood bank officer.

5.6       Interpretation:

1. Any red cell incompatibility found during the investigation explains a haemolytic transfusion reaction.

• The DAT will be positive and a mixed field reaction will be seen if in vivo sensitisation of transfused red cell has occurred.
• The DAT may be negative even in cases of haemolytic transfusion reaction, if the cell destruction is severe.
• If any antibody is detected in patient’s serum, the donor cells should be positive for the corresponding antigen.

• Detection of leucoagglutination explains a febrile reaction or hypotension.

• Serum bilirubin  total and indirect are raised in case of haemolysis.

• Haemoglobinemic and haemoglobinuric are highly suggestive of red cell destruction, but are not necessarily caused by antigen-antibody reaction, unless confirmed.

5.7       Limitations:

The non serologic possibilities of haemoglobinemia and haemoglobinuria are:-

1. Hemolysis of blood before transfusion.

• Poor technique of collecting post transfusion sample.

• Myoglobinuria following major surgery.

• Infusion of distilled water during prostatectomy.

• Hemolysis due to artificial valve.

• Patient’s clinical condition; Autoimmune haemolytic anemia or paroxysmal nocturnal hemoglobinuria.

• Use of glucose or dextrose through the same line before starting blood.

• Addition of certain drugs to blood such as ethacrynic acid, hydrocortisone or diphenyl hydantoin.

• DOCUMENTATION

1. Enter the transfusion reaction in blood issue register, showing date and time of return of the unit and nature of reaction.

• Enter the DAT/IAT results in the Antiglobulin test book in the red cell serology laboratory.

• Document the results of the entire investigations in the Transfusion Reaction work up form.

• Keep record in the Transfusion Reaction Record Register in advanced red cell serology laboratory.

• END OF DOCUMENT