A flow cytometric method for phenotyping recipient red cells following transfusion


6Allergy-Immunology Research Laboratory, Department of Clinical Investigation, Walter Reed Army Medical Center, 6825 16th Street, NW, Washington, DC 20307-5001.


Background: Reticulocyte phenotyping is used for transfused patients, who have red cell antibodies, to match blood for subsequent transfusion. Current methods are labor-intensive and require a significant amount of sample.

Study Design and Methods: A simple dual- color flow cytometry method developed for antigen typing of reticulocytes in mixed red cell populations is reported. Antigens were labeled by an indirect immunofluorescence technique using undiluted reagent sera as the primary label, biotinylated goat anti-human IgG as the secondary label, and avidin-phycoerythrin as the fluorescent stain. Reticulocytes were labeled with a thiazole orange fluorescent stain. Reticulocyte identification and antigen typing were performed on 319 samples to establish the validity of the procedure. Mixed red cells were prepared in all possible c antigen combinations to simulate transfusion concentrations of 25, 50, and 75 percent.

Results: The anti- c flow cytometry profiles readily distinguished between antigen- positive and antigen-negative populations and allowed the detection of reticulocytes at all simulated transfusion concentrations. Similar results were obtained in experiments using C, K, s, Fya, Fyb, Jka, or Jkb sera against equal volumes of antigen-positive and -negative cells. Anti-S gave inconsistent results. The in vitro results were confirmed in 19 transfused patients who had received red cells antigenically different from their own as well as cells from 1 chimera blood donor.

Conclusion: This method provides a simpler, safer, less labor- intensive, and less subjective technique requiring far less sample volume than current methods for antigen typing of reticulocytes in mixed red cell samples from recently transfused patients.