Changes in the morphology of erythrocytes after in vitro exposure of blood to carbon monoxide

Abstract

Background: One of the pathological effects of carbon monoxide (CO) on blood is the formation of carboxyhemoglobin. Carboxyhemoglobin completely blocks oxygen transfer; therefore, there is a net decrease in oxygen transport by red blood cells potentially resulting in tissue hypoxia. The effects of CO on blood can also damage cell membranes. Atomic force microscopy (AFM) has been recognized as effective for investigation into the mechanisms of structural damage in erythrocyte membranes.Aim: By means of AFM, to identify characteristics of changes in morphology and aggregation of erythrocytes exposed to CO in vitro.Materials and methods: All experiments were performed in vitro. We studied the morphology of erythrocytes and their aggregates with AFM. Blood sampling (150 μl) in microvettes with EDTA (Sarstedt AG & Co., Germany) was carried out during a prophylactic work-up of 5 volunteers. To obtain CO in a test tube, formic acid was mixed with sulfuric acid 1:1. Blood levels of carboxyhemoglobin were measured by spectrophotometry. A nonlinear fitting method of the experimental spectra was used to calculate the concentrations of hemoglobin derivatives in blood. Statistical analysis was done with the Origin software (OriginLab Corporation, Northampton, MA, USA).Results: After CO exposure, a shift in peaks was observed. At exposure time t₂=320 s, the percentage of carboxyhemoglobin (CHbCO) was 88±2%. As a result of blood exposure to CO, at t₁=160 s 10% of the cells differed in their shape from discocytes, whereas at t₂=320 s their proportion was 38%. With increasing duration of exposure to CO, erythrocyte aggregation occurred with formation of their large conglomerates up to 30 μm in size. In the control smear, the proportion of discocytes was 96±2%, and the remaining 4±1% of the cells had the form of echinocytes. The cell diameter (Dcont) was in the range 7.5±0.8 μm. After blood exposure to CO at t₁=160 s in the monolayer, 28±5% of cells had a diameter less than<5.7 μm. After CO exposure at t₂=320 s, the proportion of cells with a diameter of less than<5.7 μm increased to 72±11%.Conclusion: The experiments have shown that blood exposure to CO changed the morphology of erythrocytes. The formation of interconnected structures made of red blood cells was observed. With increased time of exposure, erythrocytes demonstrated aggregation with conglomerate formation.