Flow Cytometric AHG-CDC for HLA Crossmatch: A Pilot Study / 대한수혈학회지

BACKGROUND: For HLA crossmatch in organ transplantation, complement-dependent cytotoxicity (CDC) is a very useful methodology to detect donor-specific HLA antibodies and their complement fixing ability. In this preliminary pilot study, we investigated whether dead cells induced in anti-human globulin (AHG)-augmented CDC (AHG-CDC) reaction could be measured by flow cytometry ('FC AHG-CDC'). METHODS: This FC AHG-CDC measured the percentage of dead cells (% dead cells) after 7-aminoactinomycin D staining using 3 color flow cytometry. A total 45 flow cytometry crossmatch (FCXM) cases of 12 positives and 33 negatives were further tested using this FC AHG-CDC. RESULTS: The % dead cells of FC AHG-CDC was significantly correlated with the mean fluorescent intensity ratio of FCXM (T cells: r=0.613, P=7.45x10(-6); B cells: r=0.404, P=0.006). The positivity rate of FC AHG-CDC among FCXM positive cases was relatively high: 80% (8/10) for T cells and 75% (9/12) for B cells. The negativity rate of FC AHG-CDC among FCXM negative cases was 100% (35/35) for T cells and 91% (30/33) for B cells. CONCLUSION: In this pilot study, FC AHG-CDC could yield quantitative values, % dead cells, which was proportional to the level of complement-fixing cytotoxic antibodies against T and B cells, respectively, even without physical separation of cells or serial dilution of serum.

Comparison of NIH-CDC and AHG-CDC methods for the Detection of Panel Reactive Antibodies / 대한임상병리학회지

BACKGROUND: Panel reactive antibody (PRA) test is used for anti-HLA antibody screening and characterization in patients awaiting organ transplantation. Complement-dependent cytotoxicity (CDC) is the most widely used standard procedure and addition of antihuman globulin (AHG) reagent to the basic (NIH) CDC method increases the sensitivity of detection of HLA antibodies. We compared NIH-CDC and AHG-CDC methods for the detection of HLA class I panel reactive antibodies. METHODS: A total of 314 sera from 253 patients were analysed for the detection of HLA class I antibodies by NIH-CDC and AHG-CDC methods using a panel of 50 lymphocytes. PRA% and reaction strength (mean score) were calculated and antibody specificities were identified with r value calculated for antibody specificity. RESULTS: A total of 46 (15%) out of 314 sera were PRA-positive (PRA%> or =10%) by either NIH-CDC (33 sera) or AHG-CDC (43 sera). Concordance of PRA test results between these two methods was 96% (301/314). AHG-CDC was more sensitive in the detection of HLA antibodies compared with NIH-CDC, showing significantly higher PRA% (44% vs 29%, P=0.0001) and reaction strength (mean score 7.3 vs 6.1, P=0.0015) for PRA-positive samples. Among 46 PRA-positive sera, HLA antibody specificities were identified in 21 samples (46%) by NIH-CDC and in 32 samples (70%) by AHG-CDC. AHG-CDC methods frequently detected a wider range of antibody specificities compared with NIH-CDC and provided a more accurate assessment of the antibody specificities. Follow up PRA tests were useful providing information on change of alloimmunization status and antibody specificities in prospective organ transplantation patients. CONCLUSIONS: Compared with NIH-CDC, AHG-CDC method is more sensitive in the detection of panel reactive antibodies and provides a more accurate assessment of the HLA antibody specificities.