Abstract
Methemoglobin (Met-Hb) is very unstable and, during the storage of blood samples, Met-Fib can be both formed and reduced. To prevent changes in Met-Hb concentrations in blood samples during storage, we have devised two methods, one in which samples are stored at -30°C with a cryoprotectant and another in which samples are stored at -80°C or -196°C without any further additions. Both methods work well for samples of human blood stored for less than 30 days and prepared in vitro. In the present study, we examined the possibility of using these two methods to store samples for longer (e.g. up to 180 days) in the case of untreated, heated, or nitrite-treated human blood samples, as well as blood samples obtained from nitrite-treated and untreated rats. There was a marked increase in Met-HI, concentrations as a result of autoxidation in samples of untreated or heated human blood stored without a cryoprotectant at -80°C over a period of 180 days; however, Met-Hb levels were relatively stable in the same sorts of samples at -196°C over the same period. For all three types of human blood sample stored with a cryoprotectant, Met-Hb levels were relatively stable at -30°C, but even more so at -80°C and -196°C, for 180 days. When blood samples from nitritetreated or untreated rats were stored without a cryoprotectant, marked autoxidation occurred at -80°C and at -196°C, although the degree of autoxidation was less at the lower temperature. When blood samples from rats were stored with a cryoprotectant, Met-Fib levels were relatively stable at -30°C, and even more so at -80°C and -196°C, for 90 days regardless of the initial concentration of Met-Fib in the sample. From these results, it appears that, for Met-Hb determination, storage of human blood samples with a cryoprotectant at temperatures of -30°C or lower and storage without any further additions at -196°C are both suitable. However, frozen storage of blood samples from rats requires the addition of a cryoprotectant. Storage of blood samples from rats without a cryoprotectant at -80°C and -196°C resulted in Met-Fib formation by autoxidation.
