Discussion
This case history describes an unusual cause of anaemia. The cold antibody type of autoimmune haemolytic anaemias accounts for 25 % of cases and consists of primary chronic cold agglutinin disease, secondary cold agglutinin syndrome and paroxysmal cold haemoglobinuria. The first two were recently discussed in the Journal of the Norwegian Medical Association and will not be described here (5) . A major French cohort study found the cold antibody type in 11 % of children with autoimmune haemolytic anaemia (2) .
Paroxysmal cold haemoglobinuria, first described by Julius Donath and Karl Landsteiner in 1904 (7) , occurs extremely rarely in adults, but has been assumed to account for 1–5 % of cases of autoimmune haemolytic anaemia in children (4, 8) . In recent years, a number of studies have reported a higher incidence, up to 32 % of the cases of haemolytic anaemia in children (9) . This may well be due to an increased focus on the diagnosis and a more readily available Donath-Landsteiner test (8) . The condition usually occurs in small children (median age five years), but has been reported in all age groups and is more common in boys (boy:girl ratio 2:1) (8) .
The onset of paroxysmal cold haemoglobinuria in children normally occurs 1–2 weeks after an infection, usually an upper respiratory tract infection. A number of aetiological agents have been described, including varicella virus, parvovirus, Epstein-Barr virus, cytomegalovirus, adenovirus, Mycoplasma pneumoniae and Haemophilus influenzae (8, 10) . In many cases, efforts to detect an aetiological agent fail.
Pathogenesis takes the form of a polyclonal IgG autoantibody binding to the P-antigen on the surface of erythrocytes at temperatures lower than normal core body temperature. These temperatures are normally found in the face and extremities in cold surroundings or in the upper gastrointestinal canal when a cold drink or ice cream is ingested (10) . After warming up, the antigen-antibody complex will activate the complement system, and the result is intravascular haemolysis (Fig. 3).
Figure 3 Complement-mediated haemolysis induced by biphasic IgG (Donath-Landsteiner antibody). At sub-normal core body temperatures, the antibody binds to the erythrocyte surface. The antigen-antibody complex binds complement protein complex C1, which then fixes and activates C2 and C4. After warming to 37 °C in the body core, C3 binds and splits. This leads to further activation of the classical pathway, while IgG is now released from the cell surface. Subsequent binding and splitting of C5 activates the terminal complement cascade with formation of the membrane attack complex (C5b6789) and intravascular haemolysis. C3b-opsonised erythrocytes are also phagocytosed, to a lesser extent, in the mononuclear phagocyte system (extravascular haemolysis). C3b on surviving erythrocytes splits into inter alia C3c and C3d, which can be detected by a direct antiglobulin test (DAD) (3 ).
Anaemia symptoms predominate in the clinical picture, frequently with icterus and haemoglobinuria. Episodes of intravascular haemolysis will often manifest as abdominal pain, fever and cola-coloured urine. Palpable liver and spleen have been described in 25 % of cases in children, and leukocytosis occurs frequently (8) .
In laboratory terms, haemolytic anaemia occurs with low haemoglobin and haptoglobin and high lactate dehydrogenase and bilirubin. In many cases, the haemoglobin level is very low, often less than 5 g/dl. Bone marrow compensation usually causes reticulocytosis, as in our patient, but reticulocytopaenia may also occur. Blood smears may show spherocytes, anisocytosis and polychromasia, but to a lesser extent than in autoimmune haemolytic anaemia of the warm antibody type (10) . A direct antiglobulin test is usually IgG negative because the autoantibody is eluted in the pathogenic process, but is almost always positive for C3d and often for C3c because of complement activation (3, 10) .
The diagnosis is confirmed by means of the Donath-Landsteiner test (Table 2). The method was first described in 1904, and is very specific (10) . However, sensitivity is limited, and false negative results are obtained relatively often, because the patient's blood has little residual complement. In order to increase sensitivity, complement in the form of normal fresh serum can be added to the sample. A Donath-Landsteiner testing procedure has now been introduced in our laboratory.
Table 2 The purpose of the Donath-Landsteiner test is to confirm that there is an IgG autoantibody of the type biphasic haemolysin that binds to erythrocytes at low temperatures (4 °C), whereas haemolysis only takes place when the sample is incubated at a warm temperature. The test is conducted on a fresh serum sample that is separated when warm. Washed P-positive erythrocytes (sample from blood donor) are added to the patient's serum in three parallel batches (test tubes A1–3, B1–3 and C1–3). The test is positive if the patient's serum with or without added complement (normal serum) causes haemolysis in one or both test tubes which have been incubated first on ice and then at 37 °C (A1 and A2). There should not be haemolysis in any of the other test tubes (6 ).
Test tube marked A
Test tube marked B
Test tube marked C
Ice bath for 30 minutes 37 °C for 60 minutes
Ice bath for 90 minutes
37 °C for 90 minutes
A1 Patient serum P+ cells
B1 Patient serum P+ cells
C1 Patient serum P+ cells
A2 Patient serum Normal serum P+ cells
B2 Patient serum Normal serum P+ cells
C2 Patient serum Normal serum P+ cells
A3 Normal serum P+ cells
B3 Normal serum P+ cells
C3 Normal serum P+ cells
Paroxysmal cold haemoglobinuria is normally self-limiting, and the main treatment consists of preventing exposure to the cold as long as the autoantibody is active. In the acute phase, however, haemolysis can be serious and happen rapidly, with a substantial fall in haemoglobin in a short period. Deaths have been reported (8) . There will usually be a need for transfusions. Special precautions should be taken during the transfusion, and the use of a blood warmer is recommended. The patient must be kept warm and must not be given the transfusion in a cold extremity (4) . The condition will resolve spontaneously after a few weeks in most cases. Recurrence or a chronic course is very rare, and has mainly been described in adults with underlying malignancy (11) . Corticosteroids have not been found to be efficacious, while rituximab and eculizumab have been tried in some cases, with varying efficacy (3, 10) .