(P-TS-113) Use of Dithiothreitol to Eliminate Interference Produced by Cold Reactive Autoimmune Hemolytic Anemia Secondary to Chronic Lymphocytic Leukemia in Serological Testing

Herein, a 47-year-old male with a strong cold agglutinin who presented with elevated IgM and symptoms similar to cAIHA secondary to CLL is introduced (Table 1). Routine blood bank testing was difficult due to pan agglutination and severely icteric type and screen (TS) samples due to hyperbilirubinemia. Spontaneous agglutination has been demonstrated to disperse with warming and adsorption techniques but has been shown to denature clinically significant alloantibodies. This paper proposes how .01M dithiothreitol (DTT) can be efficiently used to treat plasma to denature immunoglobulin M (IgM) interference to allow for faster turnaround time to give compatible RBCs.

Study

Design/Methods:

Patient TS samples were collected over a span of 2 months and compared to samples of 5 patients with also had cold agglutinins. 0.2M DTT was diluted to .01M to treat patient plasma and was incubated for 30 minutes at 37C to avoid gelling. A polyethylene glycol (PEG) screen was performed against the treated plasma by Indirect Antiglobulin Test (IAT) method and a saline dilution control was tested in parallel. An RBC unit that was phenotypically matched to the patient was also tested against the treated plasma following the same procedure. The treated plasma was then tested against selected panel cells that were positive for antigens matching the patient’s phenotype (c=E=Fyb=Jka=K=S=) for ID of any underlying IgG antibodies.

Results/Findings:

The use of .01M DTT to treat plasma allowed for immediate dispersal of spontaneous agglutination of the 5 patients with cold agglutinins. Interestingly, with this technique, the antibody screen (ABSC) and selected cell panel confirmed the presence of an underlying anti-c for the presented case study patient. The RBC unit that was tested against the treated plasma was found negative and compatible with the patient post .01M DTT treatment, compared to previous blood bank results. This finding further confirmed that the IgM in the patient’s serum had been denatured. Following the AABB methods procedure, a Donath Landsteiner (DL) test was also performed to confirm complement dependent hemolysis. The results showed hemolysis with the addition of normal sera, which was not surprising considering the severity of the cAIHA in the patient’s case. 

Conclusions:

The .01M DTT treatment of plasma method proved its potential to negate the high IgM interference (Table 1) while also allowing for detection of a clinically significant alloantibody underlying the cold agglutinin. The patient could now receive compatible RBCs without having to match the full phenotype and this finding would decrease turnaround time to get blood to an already transfusion dependent patient.