Cold Agglutinin Titre

Cold agglutinins are autoantibodies, usually IgM, that agglutinate red cells at temperatures under 37 °C and fix complement. The titre is the highest serum dilution at which agglutination persists at 4 °C. Thermal amplitude, the highest temperature at which the antibody binds, predicts clinical significance better than titre does.

They cause cold agglutinin disease and cold agglutinin syndrome, with complement mediated extravascular haemolysis, acute intravascular haemolysis after cold exposure in some patients, and acrocyanosis. Low titre antibodies of low thermal amplitude are common in healthy people and are harmless.

Cold agglutinin disease accounts for roughly 15 to 25% of autoimmune haemolytic anaemias and presents mainly in adults over 60 years. Secondary forms follow infection or accompany lymphoproliferative disease.

Indications. Haemolysis with a direct antiglobulin test positive for C3d and negative for IgG, red cell agglutination on the film or analyser (spuriously high MCV and MCHC with a low red cell count), cold induced acrocyanosis or haemoglobinuria, haemolysis after Mycoplasma pneumoniae or EBV infection, lymphoproliferative disease, and before cardiac surgery with hypothermia or cold cardioplegia in a patient with a known antibody.

Specimen. Blood collected and kept at 37 °C (pre-warmed tube, warm flask or heat block) until the serum has been separated. Cooling before separation lets the antibody adsorb onto the cells and gives a falsely low or negative titre.

Method. Serial doubling dilutions of warm separated serum in saline are mixed with group O adult red cells (cord cells are added when anti-i is suspected) and incubated at 4 °C. Agglutination is graded, and rewarming to 37 °C should disperse it. Thermal amplitude is measured by repeating the incubation at stepped temperatures between 4 and 37 °C.

Reference values. In healthy people the titre is under 1:64 at 4 °C. In clinically significant disease the titre is often over 1:512 and frequently 1:1000 or more, with thermal amplitude reaching about 30 °C or higher. Thresholds are method specific, and titre correlates only loosely with severity of haemolysis.

Specificity. Anti-I is most common (primary disease and Mycoplasma pneumoniae). Anti-i is typical of EBV infection and lymphoproliferative disease.

Primary cold agglutinin disease. A clonal B-cell lymphoproliferative disorder of the marrow, with an IgM paraprotein (usually kappa) in most patients. The course is chronic.

Secondary cold agglutinin syndrome. Mycoplasma pneumoniae (titres rise over 1 to 3 weeks and the haemolysis is self-limiting), infectious mononucleosis, other infections (CMV, HIV, SARS-CoV-2), lymphomas including Waldenström macroglobulinaemia, chronic lymphocytic leukaemia, and autoimmune disease.

Paroxysmal cold haemoglobinuria. The Donath-Landsteiner antibody is an IgG anti-P that binds in the cold and lyses cells on rewarming. It is not detected by cold agglutinin titration and needs its own test. It occurs mainly in children after viral infection, and rarely with syphilis.

Mixed warm and cold autoimmune haemolytic anaemia. The direct antiglobulin test is positive for IgG and C3d, with a cold agglutinin of high thermal amplitude.

Critical findings needing immediate action.

• Acute intravascular haemolysis with dark urine after cold exposure.
• Digital ischaemia or necrosis in cold conditions.
• Planned surgery with hypothermia or cold cardioplegia in a patient with a significant cold agglutinin, which needs advance planning with the transfusion laboratory and anaesthetic team.

Pitfalls.

• A cooled sample falsely lowers the titre or gives a negative result.
• Analysers report spurious macrocytosis, a high MCHC, a low red cell count and agglutination flags. The full blood count should be repeated after warming the sample to 37 °C.
• Haemolysis severity depends on thermal amplitude and complement activity, not on titre alone.
• Rouleaux and high protein states can interfere with reading.
• Transfusion in cold conditions needs a blood warmer and warm fluids.

Special groups. Children: post-infectious disease is typical, and the Donath-Landsteiner test should be requested. Older adults: primary disease and underlying lymphoproliferative disorder are more likely.

Follow up tests. Direct antiglobulin test with monospecific antisera, haemolysis screen, blood film, serum protein electrophoresis with immunofixation, immunoglobulin levels, flow cytometry, bone marrow examination, Donath-Landsteiner test, Mycoplasma serology or PCR, EBV serology, cross-sectional imaging when lymphoma is suspected.