A novel method using formamide for the elution of antibodies from erythrocytes.
OBJECTIVE
Accurate identification of antibodies that sensitize red blood cells (RBCs) involves dissociating them from RBCs using an in vitro elution method that does not alter their antigen-binding properties, and analysis of the eluates against a panel of RBCs.


METHODS
A method was developed that allowed efficient RBC antibody elution. Human polyclonal anti-D was used to sensitize Rh-positive RBCs, and known antigen-antibody disruptive reagents were tested using these RBCs. The best reagent conditions were optimized. Eluates made were tested and compared to results obtained with a glycine-acid-based commercial elution kit to determine efficacy. Patient samples that were positive with direct antiglobulin tests (DATs), and in vitro commercial antisera-sensitized RBCs representing clinically significant antibodies, were used for evaluating the new method.


RESULTS
The formamide method was efficient at removing antibodies from RBCs. The patient samples with a positive DAT had antibodies recovered with the same specificity when compared to the acid-based technique. The length of preparation time was similar for both formamide and acid-based methods. Results of testing the eluates made from reagent RBCs sensitized with commercial antisera were distinct with antigen-positive and -negative erythrocytes.


CONCLUSIONS
The formamide method compares well with acid techniques and may be an alternative choice of elution method.
Caruccio
L
L
National Institutes of Health, Warren Grant Magnuson Clinical Center, Department of Transfusion Medicine, Bethesda Maryland, USA. lcaruccio@mail.cc.nih.gov
Byrne
K
K
Procter
J
J
Stroncek
D
D
D000372:Agglutination Tests; D004912:Erythrocytes / Q000276:immunology*; D005312:Fetal Blood; D005559:Formamides / Q000627:therapeutic use; D006801:Humans; D007106:Immune Sera; D007202:Indicators and Reagents; D007518:Isoantibodies / Q000276:immunology / Q000302:isolation & purification*; D008722:Methods; D012204:Rh-Hr Blood-Group System / Q000276:immunology; D018029:Rho(D) Immune Globulin
D005559:Formamides; D007106:Immune Sera; D007202:Indicators and Reagents; D007518:Isoantibodies; C061961:RHO(D) antibody; D012204:Rh-Hr Blood-Group System; D018029:Rho(D) Immune Globulin; C031066:formamide