Using reversed-phase high-performance liquid chromatography (HPLC), we developed a fast method for the determination of the adenine-nucleotides ATP, ADP, and AMP in human red blood cells (RBC). The method is sensitive (10(-9)-10(-10) M), specific (greater than 95% peak-homogeneity), and the results are well reproducible. The three substances are assayed in a single step, which is essential for the exact determination of the adenylate-energy-charge (AEC). We applied the technique to fresh and stored RBC's. After six weeks' storage in PAGGS-sorbitol we found only a slight decrease in ATP content, but marked increases in ADP and AMP contents. Accordingly, there was a decrease in the AEC, whereas the total nucleotide pool (TNP) did not change significantly. As AEC and TNP reflect the state of the cellular energy metabolism, they are related to the viability of stored RBC. Thus, we consider the described method to be helpful for the quality control of RBC stored in new or modified media.
The immune phagocytosis inhibition test (IPI) has been described as a sensitive method for the detection of cytotoxic and non-cytotoxic HLA antibodies. We performed a photometric IPI and compared this technique with the conventional microscopic IPI. In further investigations we used the photometric IPI in comparison with the lymphocytotoxicity test (LCT) for the detection of HLA antibodies. The photometric IPI showed a high correlation to the microscopic IPI. In tests with different known HLA antibodies the photometric IPI reached a sensitivity of 85.1% versus 80.5% in the LCT, and a specificity of 92.3 versus 100% in the LCT. Diluted patient sera showed a higher sensitivity in the photometric IPI. We conclude that the photometric IPI can be used as a convenient and sensitive technique for the detection of HLA antibodies.
