Study on the Morphological, Structural and Functional Changes of Erythrocytes after Washing Autotransfusion between Diabetic and Non-Diabetic Orthopedic Surgery Patients

doi:10.21203/rs.3.rs-1448392/v1

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Research Article

Study on the Morphological, Structural and Functional Changes of Erythrocytes after Washing Autotransfusion between Diabetic and Non-Diabetic Orthopedic Surgery Patients

https://doi.org/10.21203/rs.3.rs-1448392/v1

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Objective: By observing the morphological structure and function of washing autotransfusion red blood cells in diabetic and non-diabetic patients as well as the changes of red blood cell related indicators before and after autologous blood transfusion, to explore whether intraoperative blood salvage will aggravate the diabetic red blood cell damage, and systematically evaluate the safety and effectiveness of intraoperative blood salvage for diabetic patients.

Methods: 60 patients who underwent elective orthopedic surgery in our hospital from September 2019 to December 2020 were selected, aged 50 to 64 years, regardless of gender, BMI: 19-28 kg/m², ASA classification I and II. The types of surgery include hip replacement, posterior lumbar decompression and interbody fusion, open reduction and internal fixation of femoral fractures. According to the history of diabetes, 60 patients were divided into the following two groups, group A: patients with type 2 diabetes surgery, HbA1c≥7%, group B: patients without diabetes surgery. Detection Indicator: (1) Collect central venous blood, and detect RBC count, Hb, Hct, MCV, MCH, MCHC, RDW before surgery (T₁), after autologous blood collection (T₃), and after surgery (T₄). (2) Collect arterial blood before autologous blood collection (T₂), collect autologous blood in the blood bag after autologous blood collection (T₃) to compare the changes of red blood cell deformability (red blood cell morphology score under oil microscope, ELISA method to detect Na⁺-K⁺-ATPase), red blood cell oxygen release capacity (calculate P₅₀ value, ELISA method to detect 2,3-DPG)and red blood cell apoptosis damage (use flow cytometry to detect the PS positive rate of red blood cell) before and after blood salvage.

Results: Compared with before surgery, the RBC counts, Hb and Hct of the two groups of patients after autologous blood collection and after surgery were decreased; compared with after autologous blood collection, the RBC counts, Hb, Hct after surgery were increased. Compared with the blood before autologous blood collection, there was no significant differences in the red blood cell deformability; the red blood cell oxygen release capacity and the red blood cell PS positive rate of two groups were increased after autologous blood collection. Compared with non-diabetic patients, there was no significant difference in Na⁺-K⁺-ATPase activity and P₅₀value before autologous blood collection and after autologous blood collection in diabetic patients; Observed under the oil microscope, the red blood cells in the state of the disc with spinous process and the state of spinous process were significantly increased, and the red blood cells in the spherical state were occasionally seen. The 2,3-DPG content and the positive rate of PS were significantly increased.

Conclusion: Compared with non-diabetic patients, diabetic patients' red blood cell double-concave disc-shaped morphology changes, the oxygen release capacity is still within the normal range and PS valgus is increased. The deformability of autologous red blood cells in diabetic and non-diabetic patients is not affected after intraoperative blood salvage, and the ability to release oxygen increases, but the increase in PS valgus of red blood cells increases the phagocytosis of autologous red blood cells in the body by mononuclear macrophages after reinfusion risk.

Intraoperative blood salvage

Red blood cell

Diabetes

morphology and structure

function

Diabetes is a metabolic disorder induced by genetic, environmental and other factors. Its manifestations are insulin insensitivity, lack of insulin, or impaired pancreatic islet function. The high incidence of diabetes, high disability rate and high mortality rate make it the focus of health issues worldwide. Between 2015 and 2017, the prevalence rates of total diabetes and prediabetes in Chinese adults are estimated to be 12.8% and 35.2%, respectively, according to ADA standard diagnosis. The number of diabetic patients in China is estimated to be 129.8 million (70.4 million for men and 59.4 million for women). Regardless of gender, the prevalence of diabetes and pre-diabetes increases with age, and the increase is more significant after the age of 50 ^[1]. Most diabetic patients will inevitably experience damage to the microcirculation, which will lead to various complications of diabetes and lower quality of life. Ensuring the oxygen supply of the body's tissues and organs and the acid-base balance of the internal environment are particularly important for controlling the stability of the disease course of diabetic patients.

In the body, red blood cells pass through the lungs once a minute and are exposed to oxidative stress in the lungs; pass through the kidneys more than once every hour, and face osmotic shock in the kidneys; red blood cells must also pass through capillaries smaller than themselves, and each time the blood circulates The integrity of red blood cells is constantly being challenged. When the red blood cell membrane ruptures, hemoglobin is released into the extracellular fluid, filtered by the glomerulus, and precipitated in the acidic lumen of the renal tubules, which can block the renal tubules and lead to renal failure. In order to avoid these complications, red blood cell damage (such as osmotic pressure, oxidative stress or energy expenditure) stimulates the formation of prostaglandin E2 by activating cyclooxygenase, so that the Ca2 + permeable cation channel is activated, and phospholipase A2 mediates platelet activation Factor release activates sphingomyelinase to form ceramide. Increased cytoplasmic Ca2 + activity and enhanced ceramide levels resulted in vesicle-like changes in the red blood cell membrane and subsequent PS exposure. Macrophages can combine with PS on the surface of red blood cells via the PS receptor on the surface, phagocytose and degrade the affected cells, so that the defective red blood cells can escape the outcome of intravascular hemolysis^[2–3].

Red blood cells are the longest life blood cells in the body, and the longer they are exposed to high concentrations of glucose in the blood, the more likely they are to be structurally and functionally damaged due to glycotoxicity. The longer the course of diabetes, the greater the degree of red blood cell damage, the lower the Na+-K+-ATPase activity of the red blood cell membrane, the abnormal ratio of cholesterol and phospholipid destroys the fluidity of the membrane, the changes in the morphology of red blood cells double-concave discs, and the longevity of red blood cells in diabetic patients is obvious Shorten^[4]. Based on the changes in the structure of diabetic red blood cell hemoglobin and the decrease of 2,3-DPG concentration in red blood cells, the deformability of diabetic red blood cells decreases, which makes the microcirculation hypoxia condition aggravated, and the ability of red blood cells to carry oxygen and release oxygen is destroyed. Non-diabetic patients undergoing recovery autologous blood transfusion, their red blood cells are less affected after recovery and washing. The red blood cells of diabetic patients are in a hyperglycemic blood environment for a long time, and the red blood cells themselves are prone to changes in function and activity. During the recovery process, after the negative pressure of the aspirator collects the blood, will the centrifugation and washing of the blood loss in the operation field by the blood recovery machine cause physical damage to the red blood cells? At the same time, inflammatory factors, cell tissue fragments and coagulation factors will be activated, whether it will aggravate the red blood cell biology Damage, reducing its oxygen supply capacity is still unknown.

Research object

A total of 60 elective orthopedic surgery patients with indications of recovery autotransfusion were selected, including 26 males and 34 females. Approved by the hospital ethics committee and signed the "Informed Consent for Autologous Blood Transfusion Treatment" by the patient or his relatives. Inclusion criteria: ASA classification I-II, age 50–64 years old, gender is not limited, BMI 19-28kg/m2, diabetic HbA1c ≥ 7%, surgical types include hip replacement, posterior lumbar decompression and interbody fusion, Open reduction and internal fixation of femoral fractures. Exclusion criteria: anemia (Hb < 70g/L), malignant tumors, spinal tuberculosis, hematological diseases, contaminated blood in the surgical field, blood loss less than 300ml during the operation, allogeneic blood infusion during the perioperative period, etc.

Testing indicators

(1) Collect central venous blood before surgery (T1), after autologous blood collection (T3), and after surgery (T4) to detect RBC count, Hb, Hct, MCV, MCH, MCHC, RDW. (2) Collect internal arterial blood before autologous blood collection (T2), collect autologous blood collected in blood bag after autologous blood collection (T3), and compare red blood cell deformability before and after blood collection (red blood cell morphology score under oil microscope, ELISA method detects the changes of Na+-K+-ATPase), red blood cell oxygen release capacity (calculate P50 value, ELISA method detects 2,3-DPG) and red blood cell apoptosis damage (flow cytometer detects the positive rate of red blood cell PS).

Statistical methods

Use SPSS22.0 statistical analysis software for data processing. P < 0.05 indicates that the difference is statistically significant.

General information

In this study, 60 orthopedic patients undergoing autologous blood recovery were divided into diabetic group (group A) and non-diabetic group (group B). The gender, age, length of operation, fluid supplement, urine output, and recovered blood volume were divided into two groups. There is no significant difference.

Blood routine

Compared with preoperatively, the RBC counts, Hb, and Hct of the two groups of patients after the autologous blood recovery and the operation were significantly decreased (P < 0.05); compared with the autologous blood recovery, the RBC counts, Hb, and Hct were significantly increased after the operation (P < 0.05); There were no significant differences in red blood cell related indicators before operation, after autologous blood recovery, and after operation between the two groups (P > 0.05).

Red blood cell deformability

Compared with before autologous blood collection, the red blood cell morphology scores of autologous blood in the blood bag after autologous blood collection were not significantly different between the two groups (P > 0.05); compared with non-diabetic patients, diabetic patients had The morphological scores of red blood cells after blood recovery were significantly increased, and the red blood cells in the state of disc with spinous process and spinous process were significantly increased, and red blood cells in the spherical state were occasionally seen (P < 0.05).Compared with before autologous blood collection, there was no significant difference in the Na+-K+-ATPase activity of autologous blood in the blood bag after autologous blood collection between the two groups (P > 0.05); between the two groups of patients, autologous blood was collected before autologous blood collection There was no significant difference in Na+-K+-ATPase activity of red blood cells after completion (P > 0.05).

Red blood cell oxygen release capacity

Compared with before autologous blood recovery, the P50 value of autologous blood in the blood bag after autologous blood recovery of the two groups of patients was significantly increased (P < 0.05); the P50 value of autologous blood before and after autologous blood recovery between the two groups of patients were both There was no significant difference (P > 0.05).

Compared with before autologous blood collection, the 2, 3-DPG content of autologous blood in the blood bag after autologous blood collection of the two groups was significantly increased (P < 0.05); compared with the non-diabetic group, the autologous blood of the diabetic group was before the collection The 2,3-DPG content of blood recovered from autologous blood was significantly increased (P < 0.05).

Red blood cell PS positive rate

Compared with the autologous blood collection, the PS positive rate of the autologous blood in the blood bag of the two groups of patients was significantly increased (P < 0.05); compared with the non-diabetic group, the autologous blood before the autologous blood collection in the diabetic group The PS positive rate of recovered blood was significantly increased (P < 0.05).

Application of recovery autotransfusion

The efficiency of recovering red blood cells in the current recovery type autologous blood transfusion is moderate, about 57% of the blood is recovered, and the range of change is about 20%^[5]. Some scholars have proposed the use of recovery autotransfusion technology to reduce the proportion of patients receiving allogeneic blood transfusion by 38%^[6–7]. In patients undergoing major orthopedic elective surgery, intraoperative use of recovery autotransfusion will increase hospitalization costs, but it significantly reduces the need for intraoperative and postoperative allogeneic blood transfusion, and increases hemoglobin and hematocrit on the first day after surgery level. Considering the adverse effects such as complications and long-term hospitalization caused by the use of allogeneic blood transfusion, the cost increase caused by the use of recovery autotransfusion may be neglected^[8–9]. The results of this study show that both diabetic and non-diabetic patients have a considerable degree of blood loss before the autologous blood is recovered. After the autologous blood is retransfused before the operation, the red blood cell related indicators have risen, and there is no postoperative anemia, and the foreign body must be retransfused. The condition of blood indicates that the application of recovery autotransfusion in elective orthopedic surgery has a certain positive effect. Diabetic patients undergoing recovery autotransfusion effectively saves precious blood resources.

Changes of red blood cell deformability in recovered autotransfusion

The gradual changes of red blood cell membrane damage are divided into three morphological stages. The first stage is a smooth double concave disc called disc cell (D); the second stage of red blood cell appears more and more bulge, called The spinous cell (E), the cell becomes more and more spherical; the erythrocyte bulge of the third stage disappears completely and is called the spheroid cell (S). When the percentage of S in the red blood cell unit is low, it tends to show low hemolysis and high ATP level. Some studies have calculated that the Pearson coefficient of hemolysis and S percentage is R = 0.85, and there is an approximately linear relationship between the two, showing that the S percentage is useful for assessing red blood cell damage Meaningful^[10]. The results of this study showed that the red blood cells collected from the autologous blood of the two groups occasionally saw spinous process and spherical state, and the morphological score increased slightly, but there was no statistical difference compared with the red blood cell morphological score before the autologous blood collection. Compared with non-diabetic patients, the red blood cell discs with spinous processes and spinous processes in diabetic patients before and after the recovery of autologous blood have significantly increased red blood cells, occasionally spherical red blood cells, and the red blood cell morphology score is significantly increased, indicating The morphology of red blood cells in diabetic patients is damaged to a certain extent, and the deformability of red blood cells is reduced. However, the recovery of autotransfusion has no obvious adverse effect on the deformability of red blood cells, and the recovery of red blood cells after transfusion does not increase the risk of hemolysis.

Na+-K+-ATPase is an enzyme that exists on the surface of all eukaryotic cells. Its activity also provides power for the secondary active transport of solutes such as amino acids, phosphates, vitamins, and glucose in epithelial cells. The decrease of Na+-K+-ATPase activity caused by diabetes causes Na + ions to accumulate in cells, and then free Ca2 + ions are accumulated due to the competition of transmembrane exchange, which leads to the decrease of red blood cell deformability, which reduces microvessels by coordinating with affecting the regulation of microvessels. Blood flow, which in turn increases blood viscosity^[11]. The results of this study showed that there was no statistical difference in the red blood cell Na+-K+-ATPase activity between the two groups of patients before and after the autologous blood collection, and there was no difference in the red blood cell Na+-K+-ATPase activity between diabetic patients and non-diabetic patients. The statistical difference indicates that the red blood cell Na+-K+-ATPase activity of patients with type 2 diabetes has not been severely damaged, which is consistent with previous studies, and the recovery of autologous blood transfusion does not reduce the red blood cell Na+-K+-ATPase activity. Combined with the results of the previous red blood cell morphology analysis, the red blood cell deformability of diabetic patients is slightly reduced, and the recovery of autologous blood transfusion will not aggravate the damage of the red blood cell membrane of diabetic patients, and the safety of autologous blood transfusion can be guaranteed.

Changes in oxygen release ability of recovered autotransfusion red blood cells

Normally, normal patients can easily overcome undesirable changes such as decreased ability to unload oxygen to tissues and increased venous oxygen tension by increasing the blood flow in the microvasculature. However, in diabetic patients with microvascular disease, such compensatory microvascular changes are impossible^[12]. The concentration of HbA1c in red blood cells of diabetic patients is higher, and its oxygen affinity is about ten times higher than that of non-glycosylated hemoglobin. However, the oxygen affinity in the blood of diabetic patients is not significantly different from that in healthy subjects. Some researchers have speculated that reducing the oxygen affinity of hemoglobin by stabilizing deoxygenated hemoglobin and increasing the 2,3-DPG in diabetic patients compensates for the effect of HbA1c. However, comparing the oxygen release kinetics of hemoglobin in diabetic patients and healthy subjects, and After determining the influence of 2, 3-DPG, the hemoglobin oxygen dissociation rate constants of the two groups did not differ. The increase in 2, 3-DPG is not the main factor that compensates for the influence of HbA1c on the oxygen release of diabetic patients, but is for the patients with blood vessels. The result of red blood cell compensation for tissue hypoxia in diabetic patients with the disease^[13–14]. The results of this study showed that compared with before autologous blood collection, the red blood cell P50 value and 2, 3-DPG content of the two groups of patients after autologous blood collection increased significantly, and between diabetic and non-diabetic patients before autologous blood collection, autologous blood collection There was no statistical difference in the P50 value of red blood cells, but the content of 2,3-DPG was significantly increased. It shows that the oxygen release ability of red blood cells in diabetic patients has not been significantly damaged. The diabetic patients selected in this experiment have higher HbA1c, and their red blood cell 2, 3-DPG content compensatively increases, indicating that there may be a certain degree of vascular disease in the body. The blood processing in the process of autotransfusion washes away the red blood cells with weak viability and severe damage, so that the recovered autologous blood has an enhanced ability to release oxygen.

Changes of red blood cell apoptosis and injury in recovered autotransfusion

Red blood cells rarely undergo direct mechanical destruction in blood vessels, and 90% of red blood cells undergo apoptosis through phagocytosis by the mononuclear macrophage system. The increase in vascular osmotic pressure caused by hyperglycemia in diabetic patients and the osmotic diuresis triggered by glucose can accelerate the formation of hypertonic environment in the body, which, together with the high oxidative stress level in diabetic patients, promotes the decline of red blood cells^[15]. The PS exposure of red blood cells in type 2 diabetic patients increased significantly, and the count of reticulocytes also increased accordingly. The occurrence of anemia was caused by accelerated red blood cell loss rather than impaired red blood cell production. The reduction in the survival rate of red blood cells in diabetic patients promoted the increase in red blood cell turnover. High, to compensate for the normal operation of the body^[16]. The results of this study showed that the PS-positive rate of red blood cells after autologous blood recovery in the two groups increased significantly compared with before autologous blood collection. Compared with non-diabetic patients, the PS-positive red blood cells of diabetic patients before autologous blood recovery and autologous blood recovery were positive. Significant increase, indicating that diabetic patients' red blood cell membrane PS has an increased rate of ectropion, which is easy to adhere to the blood vessel wall and cause circulatory blockage, and is recognized by mononuclear macrophages. The recovery of autologous blood transfusion has aggravated the PS outside of the red blood cell membrane in diabetic and non-diabetic patients. Turnover rate increases the possibility of autologous red blood cells being phagocytosed by mononuclear macrophages after reinfusion, indicating the risk of reduced effectiveness of retrieving autologous blood transfusion.

In this study, by comparing the changes of red blood cell morphology and function before and after washing autotransfusion in diabetic patients, the effect of washing autotransfusion in diabetic patients was evaluated, and the safety and effectiveness of washing autotransfusion in diabetic patients were explored. Retrieving autologous blood transfusion does not increase the damage of red blood cell membranes in diabetic patients, and effectively improves the oxygen release ability of red blood cells after recovery, but the increase in PS valgus rate increases the mononuclear macrophages after the reinfusion of autologous red blood cells. The risk of phagocytosis, the safety and effectiveness of recovery autotransfusion are reduced.

Consent for publication

We agree to publish this study.

Competing interests

The authors have no competing interests.

Funding

This study was supported by National Natural Science Foundation of China（NO 81870147）,Emerging Interdisciplinary Project of Shanghai Pudong New Area Health Commission（PWZx2020-05）.

Authors’ contributions

Na Yao,Ziwei Zhang wrote the manuscript text.

Yu Bai,Xiaofang Zhou Collected and processed samples.

inhuo Wang,Tong Liu,Laiwei You completed experiment.

Jianrong Guo guided article writing.

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No competing interests reported.

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Study on the Morphological, Structural and Functional Changes of Erythrocytes after Washing Autotransfusion between Diabetic and Non-Diabetic Orthopedic Surgery Patients

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