BACKGROUND DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) inhibits the formation of serum-or plasma-induced rouleaux through its ability to bind to band 3 on red cell membranes. This property of DIDS was evaluated in the serologic testing of specimens exhibiting rouleaux. METHODS Optimal test conditions for DIDS treatment of reagent red cells were determined by varying the volume and concentration of DIDS solution and the incubation temperature and duration and comparing the results of antibody screening procedures using specimens that exhibit macroscopically visible rouleaux. Blind titration studies compared untreated and DIDS-treated red cells to evaluate the maintenance of antigen integrity. The use of DIDS-treated red cells for antibody detection and identification was evaluated by comparing the results in donor specimens containing antibodies with those in untreated and DIDS-treated selected panel cells. In addition, 4-percent (wt/vol) dextran in serum was used to induce rouleaux formation as a way of determining the capability of DIDS to resolve ABO serum grouping discrepancies. RESULTS Complete inhibition of rouleaux formation occurred when reagent red cells were treated by incubation at 37 degrees C for 10 minutes with 150 microliter (approx. 5 drops) of 0.05 mg per mL of DIDS in 0.9-percent NaCl. There were no significant differences in titration scores of untreated and DIDS-treated red cells tested with the 19 antisera used to assess antigen integrity. Antibody identification studies showed that DIDS-treated reagent red cells reacted similarly to untreated reagent red cells. In addition, DIDS resolved dextran-induced ABO serum grouping discrepancies. CONCLUSIONS DIDS effectively resolved the serologic problems associated with the presence of rouleaux, without affecting the results of the test system itself. Implementation of this method to inhibit the rouleaux-forming properties of serum and plasma specimens can be useful in serologic testing.