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Labeling D+ RBCs for flow cytometric quantification of fetomaternal hemorrhage after the RBCs have been coated with anti‐D
Author(s) -
Kumpel Belinda M.
Publication year - 2001
Publication title -
transfusion
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.045
H-Index - 132
eISSN - 1537-2995
pISSN - 0041-1132
DOI - 10.1046/j.1537-2995.2001.41081059.x
Subject(s) - flow cytometry , antibody , medicine , incubation , andrology , microbiology and biotechnology , staining , fetus , fluorescence , immunology , pathology , chemistry , pregnancy , biology , biochemistry , physics , quantum mechanics , genetics
BACKGROUND: D– patients may receive Rh immunoglobulin (RhIg) before blood samples are taken for estimation of the volume of fetomaternal hemorrhage (FMH) by flow cytometry. Anti‐D bound to the fetal D+ cells may then block the binding of conjugated D MoAb. This may reduce the fluorescence of the D+ cells, which would lead to ambiguity over setting the positions of the markers on histograms and may result in erroneous values of FMH. STUDY DESIGN AND METHODS: Labeling methods were compared by using FITC‐BRAD 3 (anti‐D) and/or FITC‐anti‐IgG (Fab fragment) with mixtures of D+ (R1r) and D– (rr) cells when the D+ cells had first been coated with various amounts (0 molecules/cell and 600‐13,000 molecules/cell) of anti‐D (RhIg). Variables examined were antibody concentrations, the order and times of incubation with the antibodies, and the effect of washing between the uptake of the antibodies used. RESULTS: In all cases, D+ cells were strongly labeled after incubation with 50 μL of FITC‐BRAD‐3 and then after washing with 50 μL of FITC‐anti‐human IgG, with both incubations being for 30 minutes at 37°C. With this double‐staining procedure, the fluorescence of D+ cells was found to be similar regardless of how much anti‐D (RhIg) was previously bound and greater than that with FITC‐BRAD‐3 alone, giving an enhanced signal‐to‐noise ratio. CONCLUSION: As the testing laboratory may not know if the patient has received prophylactic RhIg, this labeling method would be suitable for all samples.