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 (TiO₂) 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 TiO₂ blood samples, along with normal RBCs. Acanthocytes (diameter 7.7  ±  0.5  μm) and echinocytes (7.6  ±  0.6  μm) were microscopically larger (p  <  0.05) than normal RBCs (6.6  ±  0.4  μm) found in control blood samples (no TiO₂ administration). Similarly, mean corpuscular volume was higher (p  <  0.05) in TiO₂ whole-blood (70.70  ±  1.97  fl) than in control whole-blood (67.42  ±  2.03  fl). Poikilocytes also had higher HGB content. Mean corpuscular hemoglobin was higher (p  <  0.05) in TiO₂ whole-blood (21.84  ±  0.75  pg) than in control whole-blood (20.8  ±  0.32  pg). Iron content was higher (p  <  0.001) in TiO₂ whole-blood (697.0  ±  24.5  mg  /  l) than in control whole-blood (503.4  ±  38.5  mg  /  l), which supports elevated HGB as iron is found in HGB. HGB-associated Raman bands at 1637, 1585, and 1372  cm^-  1 had higher (p  <  0.001) amplitudes in acanthocytes and echinocytes than in RBCs from control blood and normal RBCs from TiO₂ blood. Further, the 1585-cm^-  1 band had a lower (p  <  0.05) amplitude in normal RBCs from TiO₂ versus control RBCs. This represents biochemical abnormalities in normal appearing RBCs. Overall, poikilocytes, especially acanthocytes, have elevated HGB.