Deficiencies and improvement of methemoglobin assay

A day-to-day variability in results was encountered when using the Dubowski method for the routine clinical determination of methemoglobin in blood. Therefore, studies were performed to determine the source(s) of variability in the method as described by Dubowski. It was determined that complete lysing of red blood cells is dependent upon both temperature of the buffer and the amount of lysing agent. Low buffer temperatures (less than 14 degrees C) produced highly variable results. This variability can be reduced by increasing the level of lysing agent to 40 mg per 20 mL of diluted blood. It was found that by using 37 degrees C buffer solution temperature and 40 mg Triton X-100 as lysing agent per 20 mL of diluted blood (1:20 with 0.25M sodium phosphate buffer, pH 7.4), the precision (percent coefficient of variation = 2%) and the accuracy (percent coefficient variation = 5.5%) were excellent.