Cold Agglutinin Test

Clinical background:

Cold Agglutinin Disease (CAD), cold-induced autoimmune haemolytic anaemia, infective triggers (e.g. influenza, Mycoplasma pneumonia, mononucleosis, and HIV), lymphoproliterative disorders (e.g. lymphoma, CLL, Waldenstroms Macroglobulinaemia).
Requested under Haematology Consultant guidance. More specialised investigations such as Cold Agglutinin Titre and thermal amplitude, DAT and Donath-Landsteiner may be associated with the initial Cold Agglutinin test. Cold agglutinins are autoantibodies that react with antigens on the red blood cell surface. They may induce complement-mediated haemolysis and agglutination (clumping) of red cells. Cold agglutinins exert their pathological effects via haemolysis and red cell destruction in the reticuloendothelial system, predominantly in the liver, or by agglutination of red cells in peripheral cold-exposed vessels leading to vaso-occlusion. Cold agglutinins derive their name from the fact that they show maximal activity at temperatures lower than normal body temperature. They are present in low titres in healthy individuals, but may be associated with a range of disease states. The cold agglutinin-induced diseases are classified into primary or idiopathic form, and secondary form, caused by the existence of an underlying disease state. Primary cold agglutinins are monoclonal; secondary ones may be either polyclonal or monoclonal. Cold agglutinins are not responsible for episodes of haemolysis of transfused red blood cells (Transfusion Reaction) or for episodes of haemolytic disease of the fetus and newborn (HDFN). The cold agglutinin test is performed to detect the presence of cold reacting antibodies in blood. If detected using a screen at 1/32 dilution an extended cold agglutinin titre will be performed.

Specimen container paediatric:

6mL K2 EDTA

Specimen container adult:

6mL K2 EDTA

Minimum volume paediatric:

2mL

Minimum volume adult:

6mL

Special requirements:

The Transfusion laboratory must be notified in advance that a sample for cold agglutinin investigation is required.
Sample MUST be bagged and transported to the laboratory in a flask warmed to 37oC. This can be provided by the Transfusion laboratory.
The test can be ordered via EPR PowerChart and taken with the BloodTrack bedside sample labelling system.
The Trust operates a zero tolerance policy. Deviation from Trust Sample Labelling Policy will result in the sample being rejected and a repeat requested.

Sample stability:

Once centrifuged and separated, short term storage: 24 hours at room temperature.
Long term storage: 7 days at 4 to 6°C.

Transport requirements:

Sample MUST be transported directly to the Transfusion laboratory in a flask warmed to 37°C.

Add on test:

If the cold agglutinin screen is positive, the Transfusion laboratory will go on to perform a titre and thermal amplitude test.
A cold agglutinin screen cannot be added on to a standard group and save as the sample must be pre-warmed prior to testing.

Interpretation:

Electronic Issue – EI is the selection and issue of red cell units where compatibility is determined by the LIMS without serological testing of donor cells against patient plasma. The ability to perform EI depends on the specific patient’s transfusion and antibody history and serological status of the current sample. The BSH guidelines for the pre-transfusion compatibility procedures in blood transfusion laboratories (2012) and the BSH guidelines for the specification and use of information technology systems in blood transfusion (2006) includes detailed guidance on the testing aspects of EI. NUTH Blood Transfusion laboratories routinely perform electronic issue and have a documented contingency plan, including a validated manual process, in case of IT failure.

Factors affecting result:

Specimen not received in laboratory within a temperature controlled flask/container or maintained at 37°C prior to separation to avoid in-vitro autoadsorption. An EDTA anticoagulated specimen not maintained at 37°C can be used in extreme circumstances if pre-warmed to 37°C and maintained for at least 30 minutes to release the autoabsorbed antibody back into the plasma prior to separation and testing. However, this is not always successful. Dispensing incorrect quantities will adversely affect the outcome of this test, resulting in a falsely increased or decreased titre. The technique for reading the test to detect agglutination is critical. Weak or fragile agglutinates may break apart, resulting in a false negative result and a false decrease in the reported titre. The reaction between a true cold agglutinin and the red blood cells is reversible. To prove the presence of a true cold agglutinin, all tests showing agglutination at 4°C must revert to negative after incubation at 37°C for 15 to 30 minutes. If agglutination remains, the antibody involved is not a true cold agglutinin.