Use of IgM monoclonal reagents licensed for tube tests in column agglutination technology.

BACKGROUND: Red cell (RBC) phenotyping using column agglutination technology (CAT) is currently limited by the reagents formulated in the system. To overcome this limitation, it was investigated whether monoclonal IgM reagents licensed for use with tube tests produced valid results with CAT. STUDY DESIGN AND METHODS: Commercial CAT, does not contain antisera, was used to evaluate Procedures A (40 microL of reagent and 10 microL of 4% RBCs) and B (50 microL of reagent and 50 microL of 0.8% RBCs) with or without incubation at room temperature. In Study 1, reagents were tested to determine whether potentiators inhibit the passage of antigen-negative RBCs through the column. In Study 2, CAT sensitivity was measured by the use of potency titrations to define a procedure for each reagent that matched or exceeded that of the tube method. In Study 3, the specificity of each reagent was determined in parallel with the CAT and tube tests. Typing of 1644 samples was performed. RESULTS: Study 1: Free passage was obtained with all reagents. Study 2: Immediate-spin methods using CAT produced the same results as the tube method. Study 3: With 8048 comparisons made, discrepant results were found in 32 transfused patients and in 6 cord blood samples, mainly with Lewis reagents. With comparison of CAT and the standard tube method, complete agreement was obtained with Kell reagents, 99.9-percent agreement with Kidd reagents, and 98.9-percent and 99.4-percent agreement with Lewis reagents. CONCLUSION: Most examined reagents seem suitable for use with CAT.

Evaluation of a new automated instrument for pretransfusion testing.

BACKGROUND: A number of automated devices for pretransfusion testing have recently become available. This study evaluated a fully automated device based on column agglutination technology (AutoVue System, Ortho, Raritan, NJ). STUDY DESIGN AND METHODS: Some 6747 tests including forward and reverse ABO group, Rh type and phenotype, antibody screen, autocontrol, and crossmatch were performed on random samples from 1069 blood donors, 2063 patients, and 98 newborns and cord blood. Also tested were samples from 168 immunized patients and 53 donors expressing weak or variant A and D antigens. Test results and technician times required for their performance were compared with those obtained by standard methods (manual column agglutination technology, slide, semiautomatic handler). RESULTS: No erroneous conclusions were found in regard to the 5028 ABO group and Rh type or phenotype determinations carried out with the device. The device rejected 1.53 percent of tests for sample inadequacy. Of the remaining 18 tests with discrepant results found with the device and not confirmed with the standard methods, 6 gave such results because of mixed-field reactions, 10 gave negative results with A2 RBCs in reverse ABO grouping, and 2 gave very weak positive reactions in antibody screening and crossmatching. In the samples from immunized patients, the device missed one weak anti-K, whereas standard methods missed five weak antibodies. In addition, 48, 34, and 31 of the 53 weak or variant antigens were detected by the device, the slide method, and the semiautomated handler, respectively. Technician time with the standard methods was 1.6 to 7 times higher than that with the device. CONCLUSION: The technical performance of the device compared favorably with that of standard methods, with a number of advantages, including in particular the saving of technician time. Sample inadequacy was the most common cause of discrepancy, which suggests that standardization of sample collection can further improve the performance of the device.

Use of photografting technique for the immobilization of blood-grouping antibodies onto microtiter plates.

Commercial anti-A, anti-B, anti-A,B and anti-D monoclonal and polyclonal antisera were immobilized onto polystyrene microtiter plates using a photografting technique, to set up a new solid-phase assay (SPA) to be used for blood grouping. The reactivity and specificity of each grafted antisera were studied using red blood cells (RBCs) expressing normal and weak antigens. The stability of immobilized antisera was also studied. After dry storage of plates at +4 degrees C, room temperature, and +37 degrees C, SPA was performed using fresh and/or frozen RBCs. The same test was carried out after storing plates under protective conditions. Concordance of collected with expected results was obtained in all cases when the SPA was performed using monoclonal antisera and RBCs, with normal or weak expression of ABO and D antigens immediately after plate preparation or after dry storage at +4 degrees C. Plates stored dry at room temperature or at +37 degrees C gave inconsistent results, whereas a slight increase in reactivity was observed after storage under protective conditions. The specificity and the reactivity of tested antibodies were not modified by the immobilization procedure, not even after dry storage at +4 degrees C. Damage produced by water evaporation during dry storage in hard conditions could be reduced by adding a protective solution to microtiter wells at the beginning of storage.

Reactivity of 62 Rh MAbs with weak and variant antigens.

We characterized serologically 5 anti-C (4 IgM and 1 IgG), 3 anti-c (2 IgM and 1 IgG), 4 anti-E (1 IgM and 3 IgG), 4 anti-e (3 IgM and 1 IgG) and 46 anti-D (16 IgM and 30 IgG) monoclonal antibodies, provided by the Rh Section of the Third International Workshop and Symposium on Monoclonal Antibodies against Human Red Blood Cells and Related Antigens (1996) for their ability to detect weak and variant antigens. The agglutination patterns were established using untreated and papain-treated red blood cells in a column agglutination technology system (BioVue, Ortho). Significant differences were found between the IgM and IgG antibodies. The papain treatment seemed to be important for IgM but not for IgG antibodies. Almost all of the IgM anti-D antibodies detected untreated DIV samples and almost all of the IgG anti-D antibodies detected untreated weak D samples. Both IgM and IgG anti-D antibodies showed the highest number of negative reactions with DVI and Rh 33 red blood cells. The CwCw sample was detected by only one of the 4 anti-C IgM MAbs using enzyme-treated red blood cells. All anti-c MAbs were able to detect treated Cx samples. Because of the small number of weakly expressed E and e samples, definitive conclusions cannot be drawn on the ability of these antibodies to detect these antigens.