I isolated red blood cells, washed them with phosphate buffer, then mixed them 1:1 with isotonic phosphate buffer. Aliquots were stored at –70 °C. For comparison, some samples were stored at –20 °C.

During the DNPH-based carbonyl assay, after adding acidic acetone to the –70 °C samples, I observed the formation of dark brown precipitates with particulate matter. Even after four washes, these brown clumps remained in the pellet. In contrast, samples stored at –20 °C gave a light pink solution and clean white pellets.

❓What could be causing these brown fragments and pigmented debris after acidic acetone treatment?

More importantly, how can I prevent their formation during the assay?

Could this be related to hemoglobin oxidation, protein aggregation, or membrane damage during deep freezing? I’d really appreciate any advice on optimizing sample preparation to avoid this issue.