title: Labeling D+ RBCs for flow cytometric quantification of fetomaternal hemorrhage after the RBCs have been coated with anti-D.
abstract: 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.
  
  
  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 microL of FITC-BRAD-3 and then after washing with 50 microL of FITC-anti-human IgG, with both incubations being for 30 minutes at 37 degrees 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.
  
  
  CONCLUSIONS
  As the testing laboratory may not know if the patient has received prophylactic RhIg, this labeling method would be suitable for all samples.
authors.affiliation: International Blood Group Reference Laboratory, Southmead Road, Bristol, United Kingdom. belinda.kumpel@nbs.nhs.uk
authors.forename: B M
authors.initials: BM
authors.lastname: Kumpel
mesh_terms: D000328:Adult; D000911:Antibodies, Monoclonal; D004912:Erythrocytes / Q000276:immunology* / Q000378:metabolism; D005260:Female; D005312:Fetal Blood; D005331:Fetomaternal Transfusion / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D006801:Humans; D007518:Isoantibodies / Q000032:analysis / Q000378:metabolism; D011247:Pregnancy; D012204:Rh-Hr Blood-Group System / Q000276:immunology*; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D007518:Isoantibodies; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen