Despite the potential for diversity, only a minority of V(H) and V(L) germline genes are used by anti-D. Consequently, V(H) and V(L) pairings and the resulting canonical structures are similarly restricted.

Our 11 anti-RhD's in conjunction with 37 previously published RhD antibodies, produced by hybridoma technology were analysed for germline gene usage and restriction in VH and VL pairings. The 17 VH germline genes used by the hybridoma anti-RhD IgG were derived from 4 VH families (VH1, VH2, VH3 and VH4). Eighteen kappa chains were restricted to only 5 germline genes from only 2 Vκ families (Vκ1 and κ3). However, the 13 lambda chains were not as restricted, using 10 Vλ germline genes from 4 families (Vλ1, Vλ2, Vλ3 and Vλ8). Fifty six unique Fab/phage anti-RhD were also analysed. In all cases the Fab/phage VH germline genes were derived from the VH3 family (41/41). The 29 kappa chains were restricted to 4 germline genes primarily from Vκ1 (97%) germline genes from 5 families (Vλ1, Vλ2, Vλ3, Vλ4 and Vλ7). The VH germline genes of the Fab/phage were restricted to 4 of the 17 used by the hybridoma anti-RhD IgG (DP46, DP49, DP50 and DP77). Ninety percent of the Fab/phage were restricted to 1 of the 5 Vκ germline genes used by the IgG (DPK9). However, the repertoire of the Vλ germline genes used in these two systems is different, with analysis showing greater diversity in Vλ gene usage with 8 unique germline genes used by 76% Fab/phage compared to 4 unique genes used by 46% hybriboma anti-RhD.

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.

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