Semiautomated data analysis of flow cytometric estimation of fetomaternal hemorrhage in D− women

BACKGROUND : Accurate and reliable measurement of the volume of fetal D+ cells in D− women is required for adequate anti‐D prophylaxis. A semiautomated flow cytometry assay based on a standardized calibration curve that was created with simulated fetomaternal hemorrhage (FMH) mixtures was developed. STUDY DESIGN AND METHODS : A calibration range of 0.083‐ to 2‐percent D+ cells in the D−RBC mixtures (2‐44 mL calculated FMH) was analyzed by use of a flow cytometer (XL‐MCL, Coulter Electronics Ltd). Linear regression analysis of the calibration curve data with computer software (Excel, Microsoft) allowed semiautomated determination of the FMH volume. To optimize the assay, fresh versus frozen and thawed RBCs, RBCs from adults who are heterozygous for D or cord RBCs, and indirect‐ or direct‐labeling techniques were evaluated by use of MoAbs. RESULTS : Fresh RBCs from adults heterozygous for D were chosen for routine use, although equivalent calibration curves were obtained with all cells tested (n = 12 calibration assays; r 2 = 0.999; mean SD, 14%). A monoclonal anti‐D reagent (Therad 10, Diagnostics Scotland) worked well in both indirect‐(anti‐IgG F(ab)‐FITC) and direct‐(anti‐D‐FITC) labeling methods compared to the use of BRAD‐3 FITC. In routine practice, the FMH volumes obtained were mainly lower than those obtained in the Kleihauer Betke test when there was less than 4 mL of FMH. CONCLUSION : Semiautomated data acquisition and calibration curve analysis represents a further step toward standardization of flow cytometry for accurate FMH quantification and facilitates evaluation and control of day‐to‐day variations between laboratories, flow cytometers, and operators.