Universal new blood cell elution method with extensive phenotyping.

BACKGROUND/

objectivesNo erythrocyte elution method developed is uniformly successful or allows elution/phenotyping together. We previously developed an elution method using deionised formamide. We modified it to be universal for various cell types and call it modified formamide-method (Fm-method). It also preserves cells for phenotyping after elution. MATERIALS AND

methodsFm-method reagent contains deionised formamide, buffered high salt, EDTA, TE. Elution-reagent is removed by column centrifugation. Blood samples were used for development and validation. Results compared to commercial/common antibody elution/phenotyping methods.

resultsFm-method eluted antibodies, complement, other proteins, and nucleic acids and works with erythrocytes, leukocytes, other cells. It worked better than commercial kits used for elution/phenotyping with no false positives/negatives. It did not denature Kell and Lewis antigens and could be repeated as needed on samples to recover more antibodies and clean cells for phenotyping. Western blotting, PAGE and FCM demonstrated eluted proteins were not degraded and cells remained intact.

conclusionFm-method is excellent for elution and phenotyping and permits elution and phenotyping with one method and sample. It is useful for studies of various bound molecules and cell surface structures. It should be possible to elute various simple and complex carbohydrates as well. The Fm-method is efficient, inexpensive, scalable, uses common reagents. It should have excellent applications in various clinical, research, commercial settings.