(P-IG-4) A False Positive Donath-Landsteiner Test in the Presence of Cold Autoimmune Hemolytic Anemia

Background/Case Studies: Autoimmune hemolytic anemia (AIHA) can be classified into four groups: warm AIHA, cold antibody-mediated (cAIHA), mixed-type AIHA, and paroxysmal cold hemoglobinuria (PCH), each with distinctive laboratory and clinical findings used to differentiate them. A case of a 13-year-old female with no prior medical history presented with jaundice, scleral icterus, fatigue, and malaise. Laboratory values showed elevated lactate dehydrogenase, anemia, and indirect hyperbilirubinemia. Direct antiglobulin test (DAT), cold agglutinin titration (CA) and Donath Landsteiner (DL) testing were requested to evaluate autoimmune hemolytic anemia.

Study

Design/Methods: Antibody identification was performed by saline tube test. The DAT was performed by tube testing with anti-IgG and anti-C3 antisera. CA was performed by serial two-fold dilutions of patient serum in saline with I+ and I- indicator RBC tested at room temperature (RT) and 4°C incubation. The DL was performed using indicator red blood cells (RBC) with I+ i- P+, I+ i- P-, and I- i+ P+ phenotypes. Serum was treated with 0.01M dithiothreitol (DTT) to differentiate IgM and IgG isotypes.

Results/Findings: Antibody identification showed positive reactivity will all RBC, including commercial panel RBC, autologous RBC, and cord RBC. Reactivity was consistent with the presence of a cold agglutinin with strongest reactivity at RT (3-4+) and weak to negative reactivity at 37°C and antihuman globulin (AHG) phases, except with the cord RBC which were 3+ at AHG. The DAT was positive for both IgG (weak) and C3 (3-4+). CA showed an anti-I titer of 32 at 4°C and 4 at RT and anti-i titer of 64 at 4°C and 16 at RT, suggesting anti-i specificity with broad thermal range. The DL using I+ i- P+ RBC showed hemolysis with tubes incubated first at 4°C then at 37°C. An additional DL was performed using I- i+ P+ and I+ i- P- RBC where both showed biphasic hemolysis. The tubes incubated at 37°C only also showed hemolysis with the I- i+ P+ RBC, suggesting an invalid test (see Table 1). DTT treatment of serum was attempted to prove whether the positive DL was due to an IgM or IgG antibody, however, gelling of the treated serum invalidated the test.

Conclusions: Typically, an IgG biphasic antibody, usually anti-P specificity, and positive DL test would indicate PCH, while cAIHA would be due to an IgM antibody of anti-I or anti-i specificity that may have a high titer and high thermal amplitude. Due to the biphasic hemolysis observed in the DL with RBC of P- phenotype and the presence of a cold agglutinin in this case, we suspected a false positive DL. IgM cold agglutinins with a high thermal amplitude may cause a positive DL test, and further evaluation is warranted to ensure correct diagnosis and treatment. Further investigation revealed elevated Mycoplasma pneumoniae IgM and IgG levels suggesting a recent infection as the likely cause of cAIHA.