Single red blood cell analysis reveals elevated hemoglobin in poikilocytes

Abnormally shaped red blood cells (RBCs), called poikilocytes, can cause anemia. At present, the biochemical abnormalities in poikilocytes are not well understood. Normal RBCs and poikilocytes were analyzed using whole-blood and single-cell methods. Poikilocytes were induced in rat blood by intragastrically administering titanium dioxide ([Formula: see text]) nanoparticles. Complete blood count and inductively coupled plasma mass spectrometry analyses were performed on whole-blood to measure average RBC morphology, blood hemoglobin (HGB), iron content, and other blood parameters. Follow-up confocal Raman spectroscopy was performed on single RBCs to analyze cell-type-specific HGB content. Two types of poikilocytes, acanthocytes and echinocytes, were observed in [Formula: see text] blood samples, along with normal RBCs. Acanthocytes (diameter [Formula: see text]) and echinocytes ([Formula: see text]) were microscopically larger ([Formula: see text]) than normal RBCs ([Formula: see text]) found in control blood samples (no [Formula: see text] administration). Similarly, mean corpuscular volume was higher ([Formula: see text]) in [Formula: see text] whole-blood ([Formula: see text]) than in control whole-blood ([Formula: see text]). Poikilocytes also had higher HGB content. Mean corpuscular hemoglobin was higher ([Formula: see text]) in [Formula: see text] whole-blood ([Formula: see text]) than in control whole-blood ([Formula: see text]). Iron content was higher ([Formula: see text]) in [Formula: see text] whole-blood ([Formula: see text]) than in control whole-blood ([Formula: see text]), which supports elevated HGB as iron is found in HGB. HGB-associated Raman bands at 1637, 1585, and [Formula: see text] had higher ([Formula: see text]) amplitudes in acanthocytes and echinocytes than in RBCs from control blood and normal RBCs from [Formula: see text] blood. Further, the [Formula: see text] band had a lower ([Formula: see text]) amplitude in normal RBCs from [Formula: see text] versus control RBCs. This represents biochemical abnormalities in normal appearing RBCs. Overall, poikilocytes, especially acanthocytes, have elevated HGB.