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Labeling D+ RBCs for flow cytometric quantification of fetomaternal hemorrhage after the RBCs have been coated with anti-D.

AbstractBACKGROUND:
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 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.
CONCLUSION:
As the testing laboratory may not know if the patient has received prophylactic RhIg, this labeling method would be suitable for all samples.
AuthorsB M Kumpel
JournalTransfusion (Transfusion) Vol. 41 Issue 8 Pg. 1059-63 (Aug 2001) ISSN: 0041-1132 [Print] United States
PMID11493739 (Publication Type: Comparative Study, Journal Article)
Chemical References
  • Antibodies, Monoclonal
  • Isoantibodies
  • Rh-Hr Blood-Group System
  • Rho(D) antigen
Topics
  • Adult
  • Antibodies, Monoclonal
  • Erythrocytes (immunology, metabolism)
  • Female
  • Fetal Blood
  • Fetomaternal Transfusion (blood, diagnosis, immunology)
  • Flow Cytometry (methods, standards)
  • Humans
  • Isoantibodies (analysis, metabolism)
  • Pregnancy
  • Rh-Hr Blood-Group System (immunology)
  • Sensitivity and Specificity

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