In selecting Rh positive reagent red cells for prenatal anti-D studies does the Rh phenotype affect the results of the titration?

Blood bankers in Cleveland want to know which Rh positive reagent red cells are most frequently used by colleagues when performing anti-D titrations in prenatal studies. The protocol at their institution currently calls for using R1R1 reagent red cells as these reagent cells are easily available from the antibody screening cells, which are purchased in large quantities. The Clevelanders report that the AABB Technical Manual states that "anti-D seldom shows any difference in reactivity between red cells from individuals homozygous for heterozygous for RHD". However, the Cleveland blood bankers' concern is that the anti-D titers reported from their laboratory appear to high, with scores that do not appear to be reflective of the in vivo reactivity in the pregnant women they are working up. They would like to know if the use of R1R1 reagent red cells for anti-D titration is common in the field, or if colleagues favor other Rh positive phenotypes, such as R1r reagent red cells, or some other phenotype?

While awaiting responses to the above question e-Network members are advised to review the June 2002 discussion on this issue.

The following responses have been received.

1. A Michigan immunohematologist reports that his group uses R2R2 RBCs, since those reagent red cells have the most uniform expression of D from one donor to another. (See AABB Technical Manual and Judd WJ. Practice guidelines for prenatal and perinatal immunohematology revisited. Transfusion 2001;41:1445-52) They perform duplicate titrations on the initial sample (different technologists) and more if they do not get concordance. Despite what the literature says, the Michigan colleague is of the opinion that D does show some degree of dosage, but often can only be appreciated in assays other than highly subjective tube tests. They have seen weak anti-D's in gel tests that had been mistaken for dosing anti-E's in tube tests. Also, beware of apparent R1R1 cells; they may be from an R1r' individual, with C in trans causing weakened expression of D. The Michigan colleague adds that another approach is to always use RBCs from the same donor for titration purposes, regardless of phenotype. This provides an additional degree of standardization within an institution to this inherently inaccurate procedure.

ADDENDA Jan. 2, 2003

2. A blood banker in Texas reports that at her hospital they use R2R2 cells for titration studies of anti-D.

ADDENDA Jan. 6, 2003

3. An e-Network colleague wrote that he wonders if the Cleveland blood bankers are using enhancement techniques when performing their HDN antibody titers. To quote the Technical Manual (14th edition, p.731), "Do not use enhancement techniques (albumin, PEG, LISS, or enzyme-treated red cells), because falsely elevated titers may be obtained." Gel is also too sensitive a technique to use for maternal antibody titers, based on his personal observation). The Technical Manual also has a good discussion on the selection of red cell phenotype to use when performing titration (14th edition, p.731).
   
   
4. The Cleveland blood bankers respond that they do not use any enhancement techniques and that all of their titers are incubated in saline and read at the antihuman globulin phase. However, the Cleveland blood bankers now clarify their original statement that their titer results are 'too high, with scores that do not appear to be reflective of the in vivo reactivity in the pregnant women they are working up'. Rather, they had a theoretical concern that this might occur, based on not using a heterozygous red cell in the titration procedure.

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