Evaluation of Biliary Cholesterol, Biliary Ferritin and Expression of Apo B-100 Gene in Patients of Cholelithiasis

Introduction

Gall stone disease is a common disorder around the world. Choleliths are bile precipitates resulting from abnormalities in the metabolism of cholesterol, bilirubin and bile acids. [1]. The prevalence of cholelithiasis in India ranges from 2-29%, being higher in North Indians and females [1-3]. Gall Stones are classified according to their chemical composition into Cholesterol stones, Mixed stones and Pigment stones [4]. In western populations, stones that are rich in cholesterol are the predominant type (80%) but lower frequencies of cholesterol stones and higher frequencies of pigment stones are seen in many parts of Asia [5]. The pathogenesis of cholesterol stones primarily involves supersaturation of bile with cholesterol accentuated by accelerated nucleation and impaired gallbladder emptying being other factors. Relative concentration of cholesterol, bile salts and phospholipids in GB bile determines precipitation [5]. Anaemia due to iron deficiency is associated with gall stone disease. Serum ferritin, a marker of iron stores, exhibits low levels in iron deficient patients. In a study done on patients with cholelithiasis by Aggarwal et al, in 2019, it was seen that serum ferritin levels were lower in cases of cholelithiasis as compared to those in the normal population [6]. Ferritin is also released in bile in hemochromatosis.

In this study biliary iron concentration (per millimole of bile acid), was increased about twofold (p < 0.05) and biliary ferritin concentration about fivefold (p < 0.001) [7]. The levels of biliary ferritin in gallstone disease have not been studied so far. Genetic variation in cholesterol metabolism can be brought about by different isoforms of lipid transport and receptor molecules in the general population. Apo E and apo B-100 are prominent transporters that exist in polymorphic states in humans whose certain isoforms help in better uptake and delivery of cholesterol [8]. Apo B-100 serves as a ligand for receptor-mediated endocytosis of LDL. The gene is located on chromosome 2. High frequency of X+ allele in apo B-100 genes has been reported in gallstone patients from China [9]. Heterozygosity (X+/X-) of apo B-100 may be associated with gallstone disease and gallbladder cancer in India [10,11]. In a study in transgenic rabbits, it was found that Apo B-100 expression is associated with higher serum cholesterol and triglyceride levels [12]. In another study, rats fed on iron rich diet had a higher expression of Apo B-100 gene and had higher serum cholesterol and triglyceride levels [13]. Hence this study was undertaken to determine the levels of biliary ferritin and biliary cholesterol in patients of cholelithiasis and to see the association of expression of Apo-B gene in them.

Methodology

This observational cross-sectional study was conducted in the Department of Surgery and the Department of Biochemistry at UCMS & GTB Hospital, Delhi from November 2018 to March 2020 with a sample size of 40 cases and equal number of controls for gene purposes. The research committee of the institute approved the study protocol. All consenting patients, except those with coexisting systemic and inflammatory illness and immunocompromise, diagnosed with symptomatic cholelithiasis willing to undergo laparoscopic/open cholecystectomy were included. For Apo B-100 expression, an equal number of healthy volunteers were included after ultrasound to exclude cholelithiasis in them. All patients of cholelithiasis and controls underwent history taking, radiological and biochemical investigations. A fasting blood sample of patients was taken for hematological evaluation, lipid profile, serum ferritin, and Apo B-100 gene expression. In the controls, only Apo B-100 gene expression was measured. Bile was obtained from the gall bladder of patients immediately after cholecystectomy for evaluation of biliary cholesterol and ferritin levels. Cholesterol and ferritin levels in both serum and bile were measured by ELISA. The reference range for serum ferritin was taken to be 29-248 ng/ml for males and 10-150 ng/ml for females [14].

Serum cholesterol levels less than 200 mg/dl were considered desirable, 200-239 mg/dl as borderline high and >240 mg/dl as high. Gene Expression comprised of 3 steps:
a) Extraction of RNA,
b) Synthesis of complementary DNA (cDNA) and
c) Quantification of Apo B-100 gene expression by quantitative Real Time PCR.

Statistical analysis was done using SPSS version 20.0. p-values were calculated using student t-test for normally distributed data. For non-normal distribution, non-parametric tests were used. Correlation was calculated by using Pearson test. Pearson’s coefficient (rho) values between 0 and 0.3 (0 and -0.3) indicate a weak positive (negative) linear relationship via a shaky linear rule. Values between 0.3 and 0.7 (-0.3 and -0.7) indicate a moderate positive (negative) linear relationship.

Results

Patient Profile

The mean age of the 40 patients included in the study was 34.65 with most patients belonging to the age group of 21-30 years (16/40). The mean age of controls was 34.20 ± 10.38 years and did not vary significantly. (p = 0.897). Females formed the majority of participants in both the groups, 37(92.5%) in cases and 33(82.5%) in controls. The mean BMI (Kg/m2) was 22.28 ± 2.81. with the majority (65%) falling in the normal range. The duration of illness ranged from 3 months to 60 months with a mean of 17.2 months.

Haemoglobin

The mean haemoglobin level of the patients was 11.46 g/dl. On the basis of WHO classification, patients were divided into non anaemic, mildly anaemic and moderately anaemic. Most (72.5%) patients were anaemic (Table 1). The mean age of patients with moderate anaemia was significantly higher than those with no or mild anaemia (p=0.045) (Table 1).

Lipid Profile

Out of the 40 patients, 33 (82.5%) had raised serum cholesterol levels. The mean of T. Cholesterol (mg/dL) was 302.46. The mean HDL level of the patients was 40.70 mg/dl. Based on the American Heart Association criteria, 32 patients had low HDL levels (i.e. <40 for males and <50 for females) [15]. The mean triglyceride level of the patients was 122.53 mg/dl with 11/40 having raised triglyceride levels (>150 mg/dl) [16]. There was a significant moderate negative correlation between Triglycerides (mg/dL) and HDL (mg/dL) (rho = -0.4, p = 0.010).

T. Cholesterol and Anaemia

The mean total cholesterol levels in the patients grouped on basis of hemoglobin levels did not vary significantly (p=0.222), though patients without anaemia had a higher mean serum cholesterol level than those with anaemia (Table 1).

Table 1: Associations of severity of anaemia.

Biliary Cholesterol

The Biliary cholesterol (mg/dL) levels ranged from 17.01 to 311.68 with a mean of 121.29 mg/dL.

1. There was a significant moderate positive correlation between HDL (mg/dL) and biliary cholesterol (mg/dL) (rho = 0.38, p = 0.017). For every 1 unit increase in HDL (mg/dL), the biliary cholesterol (mg/dL) was seen to increase by 3.18 units.
2. There was a significant moderate positive correlation between duration of illness (months) and Biliary cholesterol (mg/dL) (rho = 0.49, p = 0.001).For every 1 unit increase in duration of illness (months), the biliary cholesterol (mg/dL) was seen to increase by 2.22 units.

Serum Ferritin

The mean serum ferritin (ng/mL) of the patients was 12.85. Out of the 40 patients enrolled, 25 (62.5%) patients had serum ferritin within normal range and 15(37.5%) patients had low serum ferritin levels [14].

1. Serum ferritin and anemia
There was a weak positive correlation between hemoglobin (g/dL) and serum ferritin (ng/mL), and this correlation was not statistically significant (rho = 0.07, p = 0.673). There was a visible decrease in numerical value of serum ferritin as severity of anaemia increased but this was not statistically significant (p = 0.967) (Figure 1).
2. There was a significant strong positive correlation between T. Cholesterol (mg/dL) and serum ferritin (ng/mL) (rho = 0.67, p = <0.001). For every 1 unit increase in T. Cholesterol (mg/dL), the serum ferritin (ng/mL) increases by 0.04 units.
3. There was a weak negative correlation between biliary cholesterol (mg/dL) and serum ferritin (ng/mL), and this correlation was not statistically significant (rho = -0.19, p = 0.231). Though insignificant (p=0.783), higher biliary cholesterol level was noted with low serum ferritin level (mean biliary cholesterol = 123.16 mg/dL) in comparison to patients with normal serum ferritin (120.16 mg.dL).

Biliary Ferritin

The mean biliary ferritin (ng/mL) of the 40 patients was 5.99 with a minimum value of 1.41 ng/mL and a maximum value of 13.65 ng/mL.

1. There was a weak negative correlation between haemoglobin (g/dL) and biliary ferritin (ng/mL), and this correlation was not statistically significant (rho = -0.19, p = 0.249).

Biliary ferritin levels were higher in patients with moderate anaemia as compared to mildly and non-anaemic patients but this trend was not statistically significant (p=0.456). While the mean values of serum ferritin were seen to decrease with increasing severity of anaemia, the mean values of biliary ferritin were seen to increase (Figure 1).

2. There was a weak negative correlation between serum ferritin (ng/mL) and biliary ferritin (ng/mL), and this was not statistically significant (rho = 0, p = 0.992). Mean serum ferritin (12.85 ng/mL) was found to be twice the mean biliary ferritin (5.99 mg/dL).

3. There was a moderate positive correlation between biliary cholesterol (mg/dL) and biliary ferritin (ng/mL), and this correlation was statistically significant (rho = 0.52, p = <0.001). For every 1 unit increase in biliary cholesterol (mg/dL), the biliary ferritin (ng/mL) increases by 0.03 units.

Figure 1: Levels and trends of serum and biliary ferritin in each anaemia group.

Apo B-100 Gene

1. On taking all 40 patients as cases and all 40 healthy volunteers as a control group, a 1.001 fold change upregulation of Apo B-100 gene was observed. This difference was not significant (p=0.740) (Figure 2a).
2. On taking the mean delta ct value of controls and evaluating the expression of Apo B-100 gene in each patient against it, the result showed that the gene was upregulated in 20 patients and downregulated in 20 patients exhibiting equal distribution on the individual level (Figure 2b).
3. The mean T. Cholesterol (mg/dL) in patients with upregulation of Apo B-100 gene was 328.04 while that in patients with downregulation was 276.88 (Table 2). 18/20 (90%) patients with upregulation of Apo B-100 gene had high cholesterol levels and 12/20 (60%) with downregulation had high cholesterol levels.

Figure 2: a. Fold change (Apo B-100 gene expression).
b. Individual fold change (Apo B-100 gene expression) of all patients.

Table 2: Mean value of parameters with gene expression.

Discussion

Cholelithiasis is a multifactorial disease having a complex interplay of metabolic, genetic and environmental factors that affect the disease process. In this study, the mean age of the 40 patients included was 34.65 years, with most patients in their 3rd and 4th decades of life. This age distribution was similar to the study conducted by Prakash, et al. [3] and Kanth, et al. [2]. Most other studies which aimed at studying the epidemiology of cholelithiasis found the disease to be more common in a relatively higher age group [1,17,18]. We also found the mean age to be significantly higher in the moderately anaemic group suggesting that anaemia increases with increasing age. Since anaemia and increasing age have both been described as independent risk factors for cholelithiasis [1,6], an additive effect of both seems to be at work in causation of the disease. A majority of the patients in this study were females (37/40, 92.5%). It is a well-established fact in literature that cholelithiasis occurs more commonly in the female population. Most of the patients in our study (26/40, 65%) had a BMI in the normal range suggesting that cholelithiasis is not just a disease of the obese; also seen by Kharga, et al. [19].

Lipid Profile

As found by Miquel, et al. [20] and Loria, et al. [21], in this study also, a majority (75%) of patients had raised total cholesterol and 7.5% patients had borderline raised cholesterol. Additionally, majority (32/40, 80%) of the patients had low HDL levels while only 11 out of the 40 patients (27.5%) had raised triglyceride levels. We found a significant negative correlation (rho = -0.4, p = 0.010) between HDL and triglyceride levels indicating an increase in triglyceride levels as HDL levels fall. Thijs, et al. [22] and Hayat, et al. [23] described an association of low HDL and raised triglyceride levels with gallstone disease but did not find any significant association of serum cholesterol levels with cholelithiasis. Alexander, et al. [24] conducted a study on 80 patients of cholelithiasis and found that only 12 (15%) patients had hypercholesterolemia while 79 (98.75%) patients had low HDL levels, and 23 (28.7%) patients had raised triglyceride levels. In our study, we found raised cholesterol levels (82.5%) and low HDL levels (80%) in a majority of the patients.

These findings lead us to believe that while serum cholesterol plays a role in pathogenesis of gallstone disease, low HDL levels may be a more relevant indicator for the disease. Da Luz, et al. observed in patients of coronary artery disease that the ratio of TG/ HDL and T. cholesterol/HDL are better indicators of disease than individual lipid values in serum [25]. Jiang, et al. found a strong positive association of CAD with cholelithiasis [26]. In the present study, we also found that as serum HDL levels decreased, there was an increase in triglyceride levels. Based on these findings we would like to postulate that it is the ratio of lipid parameters and not the individual levels of each parameter that may serve as better indicators for cholelithiasis. The alterations in ratios can identify individuals prone to cholelithiasis and an attempt can be made at disease prevention through lifestyle modification and management of dyslipidemia.

Biliary Cholesterol

In our study, there was a weakly negative correlation between serum and biliary cholesterol (rho = -0.09) and this was not statistically significant (p=0.592, (Table 3)); in contrast to Kanwar, et al. [27] who found a positive correlation between serum cholesterol and biliary cholesterol (rho = 0.61, p=0.001). We expected higher biliary cholesterol levels based on the prevalent concept of cholesterol supersaturation leading to gallstone formation, but our findings were contrary to this. It has been shown by Nagi and Arora [28] that the cholesterol stones (13.5%) are much less common in North Indian population than mixed stones (80.7%). In a study conducted by Chandran, et al. [29], amongst 200 patients of cholelithiasis, 52 (26%) had cholesterol stones, 76 (38%) had mixed stones and 72 (36%) had pigment stones. Such high proportion of pigment and mixed stones may be able to explain the lower levels of biliary cholesterol in our population as pigment stones are known to mainly form due to unconjugated bilirubin [30]. Since these studies had a small sample size, further evaluation with larger sample size along with stone analysis is warranted. We also found a significant moderate positive correlation between HDL and biliary cholesterol (rho = 0.38, p = 0.017, (Table 3)). HDL carries cholesterol in circulation and transports excess peripheral cholesterol to the liver for secretion into bile.

Table 3: Correlation of biochemical parameters (rho value, p value).

This process is termed reverse cholesterol transport. Biliary cholesterol can be derived from plasma lipoproteins like HDL and LDL or it can be synthesized in the liver. The more relevant source of cholesterol secreted into the bile is cholesterol derived from plasma lipoproteins. Of these, high density lipoprotein (HDL) appears to be the preferential contributor for cholesterol secreted into bile [31,32]. We found a significant moderate positive correlation between duration of illness and biliary cholesterol (rho = 0.49, p = 0.001). Supersaturation of bile with cholesterol is an ongoing process and does not stop after stone formation with progressively increasing biliary cholesterol levels over the course of time [33,34]. This also corresponds to the fact that increased chances of gallstone development occur as time progresses in predisposed individuals.

Serum Ferritin

In the present study, serum ferritin showed a numerical decrease with increase in severity of anaemia (p = 0.967 (Figure 1)). A significant, strong positive correlation was seen between T. cholesterol and serum ferritin (rho = 0.67, p = <0.001, (Table 3)). A similar significant positive correlation was also found by Kim, et al. [35]. While we found no significant association between serum cholesterol and anemia, the mean serum cholesterol level in non anaemics (347.18 mg/dL) was higher than that found in anaemics (285.41 mg/dL). This was similar to the findings of Chowta et al. [36] and Choi et al. [37] who studied the relationship of anaemia with lipid profile and Kumar, et al. [38] who also found lower serum cholesterol levels in anaemic patients of cholelithiasis. This was explainable through multiple factors like increased erythropoiesis requiring moe cholesterol, plasma dilution and increased uptake of cholesterol by the reticuloendothelial system in anaemics [36]. Thus, anaemics tend to have lower ferritin levels and lower cholesterol levels, supporting the findings of the present study. Iron deficiency anaemia, low serum ferritin levels and raised serum cholesterol levels were expected to be associated with cholelithiasis [6,39,40] We also found that as serum cholesterol levels increased, there was an increase in serum ferritin levels.

This contradiction suggests that there is a complex interplay of ferritin, cholesterol and anaemia in cholelithiasis and cant be simply explained by the individual levels of these factors. There was a higher biliary cholesterol level in patients with low serum ferritin than in patients with normal serum ferritin, though not significant (p=0.783), similar to a study conducted by Sahu, et al. [41]. Iron deficiency causes altered motility of gall bladder leading to biliary stasis leading to an increase in cholesterol saturation of bile [42,43].

Biliary Ferritin

There was a weak negative correlation between serum ferritin and biliary ferritin (rho = 0, p = 0.992, (Table 3)). While the mean values of serum ferritin were seen to decrease with increasing severity of anaemia, the mean values of biliary ferritin were seen to increase. There is a lack of adequate literature available on excretion of ferritin in bile. We could not find any studies which studied the role of biliary ferritin in cholelithiasis. However, Hultcrantz, et al. [7], in 1989, conducted a study in 10 patients of idiopathic hemochromatosis and 2 groups of controls (14 patients of gallstone disease and 16 healthy individuals). They found that hepatic iron concentration was increased 8 fold, biliary iron concentration was increased twofold and biliary ferritin concentration was increased about fivefold in patients of hemochromatosis as compared to the controls. They found higher levels of biliary ferritin in gallstone patients than in healthy individuals. Hultcrantz, et al. also concluded that hepatocytes secrete ferritin into bile since very little ferritin was detected in bile duct cells on electron microscopy making them an unlikely source of biliary ferritin. Hepatocyte excretion of ferritin into bile has also been established in animal models earlier [44]. Verma, et al. [45] found that the levels of biliary iron were higher than those of serum iron in cases of cholelithiasis as well as controls.

A significantly higher amount of iron in pigment stones than in cholesterol stones was also found. Chandran, et al. [29] found a significantly higher amount of iron in mixed stones than in pigment and cholesterol stones. While the amount of iron was higher in pigment stones than cholesterol stones, this difference was not significant. An increase in biliary ferritin as anaemia increases and relatively lower levels of biliary cholesterol suggest a more frequent occurrence of pigment and mixed stones in our population. Since we found an increased biliary ferritin with increase in anaemia (which is an iron deficient state) in patients of cholelithiasis, it suggests that the relationship between cholelithiasis and iron deficiency anaemia is more complex than thought previously. Ferritin may have a larger role to play in the pathogenesis of cholelithiasis than to just be a marker of iron deficiency anaemia and it is not just a decrease in gallbladder motility and resulting biliary stasis due to the deficiency of iron which cholelithiasis [46]. It is possible that the biliary route of excretion of ferritin is an incidental finding with no serious implications on gallstone formation. The concentration of ferritin in bile may also be due to fluid absorption from the gall bladder leading to concentration of its contents along with biliary stasis secondary to iron deficiency.

Based on our findings, we would like to hypothesize that ferritin gets concentrated in bile in the gall bladder after excretion from hepatocytes and delivers iron into the bile which will play a role in the formation of gallstones. In our study, there was a significant moderate positive correlation between biliary cholesterol and biliary ferritin (rho = 0.52, p = <0.001, (Table 3)). It is known that cations in bile such as iron, calcium, copper and zinc promote cholesterol crystallization [45]. We would like to postulate that in patients with cholelithiasis, with the biliary cholesterol increase, there is a tendency for these cations to increase as well and lead to crystallization of cholesterol and formation of gallstones. An increase in biliary ferritin levels suggests its role in delivering iron which serves as a cation in the biliary cholesterol rich bile leading to cholelithiasis.

Apo B-100 Gene

On taking all 40 patients as cases and all 40 healthy volunteers as a control group, a 1.001 fold change upregulation of Apo B-100 gene was observed (p=0.740). On assessing individual cases, equal distribution of up and downregulation in patients of cholelithiasis was seen (Figure 2b). In 2005, Wang, et al. assessed intestinal cells of mice and found that absence of expression of intestinal Apo B-48, but not Apo B-100, reduces biliary cholesterol secretion and cholelithogenesis, possibly by decreasing intestinal absorption and hepatic bioavailability. While the findings of Apo B-100 expression were similar to the present study, they studied the gene expression in intestinal cells of mice while this was studied in blood from humans in our study. Since we found a relatively higher expression of Apo B-100 in the patient group, though not statistically significant, it suggests Apo B-100 can have a role in etiology of cholelithiasis.

Apo B-100 and Lipid Profile: In our study we found that the levels of total cholesterol and serum triglyceride levels were much higher in patients with upregulation of Apo B-100 gene than patients who showed downregulation (Table 2). Even though our findings were not significant, they were in concordance with the findings of Fan, et al. [12], who found a threefold increase in levels of serum triglyceride and serum cholesterol levels in the transgenic rabbits with human Apo B-100 gene as compared to the non-transgenic controls. 90% patients with upregulation of the gene had raised serum cholesterol levels, while only 60% in the downregulation group had raised serum cholesterol levels. While this difference is not significant (p value = 0.081), it points to a higher frequency of raised cholesterol levels in the upregulation group than in the downregulation group and justifies the association of raised serum cholesterol levels with increased incidence of cholelithiasis. Since Apo B-100 upregulation is associated with higher cholesterol and triglyceride levels, assessment of gene expression may predict patients prone to develop hypercholesterolemia and hypertriglyceridemia and further, an increased risk of gallstone formation.

Apo B-100 and Ferritin: In our study we found higher levels of serum ferritin and biliary ferritin in patients with upregulation of Apo B-100 gene than in patients with downregulation (Table 2). Silva, et al. [13] studied the effects of dietary iron on lipid metabolism in rats. They found that rats on iron dextran diet had a higher expression of Apo B-100 and higher levels of serum cholesterol and triglycerides, than rats who did not have iron in their diet. Since ferritin stores iron, and higher levels of serum and biliary ferritin were found in patients with upregulation of Apo B-100 gene, it is possible that the higher levels of ferritin led to the upregulation of Apo B-100 gene which further led to an increase in serum cholesterol and triglyceride levels leading to an enhanced risk of cholelilthiasis.

Conclusion

Findings in the present study indicate that elevated serum cholesterol and anaemia play a role in the pathogenesis of cholelithiasis. HDL is a major source of cholesterol in bile. HDL levels and ratios of lipid parameters may be better indicators of cholelithiasis. We found a positive correlation between biliary cholesterol and biliary ferritin which seemed to correspond with a positive correlation between serum cholesterol and serum ferritin. We also found an increase in levels of biliary ferritin with increased severity of anaemia in patients of cholelithiasis. These findings suggest a role of ferritin in delivering iron to bile leading to an increased risk of gallstone formation. The levels of biliary cholesterol were relatively less and biliary ferritin levels increased with increase in anaemia. Iron levels are known to be higher in pigment stones. Thus, we can conclude this to be the reason for pigment stones being more common in our study population. We found a net upregulation of Apo B-100 gene in patients of cholelithiasis; also, serum cholesterol and triglyceride levels in Apo B-100 upregulated patients were raised, suggesting a possible role of Apo B-100 in cholelithiasis. The higher levels of serum and biliary ferritin could have led to the upregulation of Apo B-100 gene. This relationship between iron metabolism, lipid metabolism and cholelithiasis, which exists even at the genetic level requires further studies for evaluation.

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The Importance of Involving Student Volunteers and Nursing Students During the COVID-19 Pandemic

Opinion

Covid-19 infection is spreading rapidly and has consequences for the health of the population, as well as burdening the health system of the country and the social well-being of its citizens. Was declared a global pandemic in March 2020 [1]. Significant changes were evident during the Covid-19 pandemic in both the education, employment, and health sectors [2]. As the workload of health workers is great, I believe it is necessary to include as many volunteers as possible who can help take care of patients in the information sector and support to families and patients, especially the vulnerable population. The education of nurses in Montenegro takes place at the Medical Faculty of the University of Montenegro, and the education of health technicians in vocational medical schools. From March 2020, students do not stay in hospitals for exercises and practical classes, but do so in school offices and classrooms, and students of nurses in the faculty office and through “Online distance learning” [3].

I believe that it is necessary to adopt a strategy of involving medical students and nursing students in helping to vaccinate the population and other activities where extensive experience is not needed to reduce the pressure on the health sector and where there is supervision by experienced nurses. School and faculty management should devise a strategy to involve students in volunteer work in a way that motivates them, but never forces them to work, because young people can make a great contribution in the fight against the Covid-19 pandemic. Since the beginning of the Covid-19 pandemic, a total of 185 health workers with secondary education have volunteered at the Clinical Center of Montenegro, but that number is not enough because it refers to only one health institution, the Clinical Center of Montenegro in Podgorica. Not all Covid Center hospitals in our country, but volunteers could relieve the staff of nurses and make their work easier in those hospitals from which they were mobilized to help in Covid centers in other hospitals in the area.

It is important to emphasize that the Clinical Center does not volunteer Nursing students, but graduate health professionals who have not yet been employed, so volunteer work will help them gain new knowledge and experience and get great recommendations to help them find employment and conditions. For taking the internship exam. We hope that the Covid-19 pandemic will end soon and that we will draw conclusions from it in both professional and private life.

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Aggregation-Induced Emission Characteristics in Carbon Dots for Detection of Nitroaromatic Explosives

Prospective

Detection of nitroaromatic explosives such as Trinitrotoluene (TNT), Dinitrotoluene (DNT) and Picric Acid (PA) is of great concern for homeland security, battlefield protection, industrial and environmental safety control [1,2]. These nitroaromatic explosives are primary constituents of many unexploded land mines worldwide [3]. Among them, TNT is a widely used explosive and found to be poisonous, carcinogenic and can adversely affect male fertility [4-6]. Soil and ground water of war zone can contain toxic levels of these NACs as well as their degradation products. Thus, these nitroaromatic explosives are found to be environmental contaminants and toxic to living organisms. On the other hand, the explosive power of nitroaromatic compound PA is higher than that of TNT and found to be highly reactive as it complexes with metals to generate shock sensitive explosive metal salts [7]. The use of PA in the manufacture of rocket fuel, fireworks, and matches releases a large quantity of it into the environment [8,9]. In view of this, detection of explosives is very important in combating terrorism, maintaining national security and providing environmental safety. Various methods are available for the detection of these nitroaromatics, such as GC-MS, Ion-Mobility Spectroscopy (IMS) and surface enhanced Raman spectroscopy and various other spectroscopic techniques [10]. However, these methods cannot be used in the field due to their high cost, lack of selectivity and sensitivity.

In this context, fluorescence signaling is one of the first choices due to its high detection sensitivity and selectivity [11]. However, emission of various fluorescent probes is often quenched at high concentrations or in an aggregate state, which is known as Aggregation-Caused Quenching (ACQ). In contrast to ACQ, there are some molecules that behave differently as they are nonluminescent in the solution state but become strongly emissive when aggregated and these molecules are termed as ‘‘Aggregation- Induced Emission’’ (AIE) active materials [12]. Fluorescent Carbon Quantum Dots (CDs) are found to be environment friendly, easy synthesis, easy to modify by doping, which, make them an excellent candidate for application in optoelectronics, sensors, solar cells, bioimaging and so on [13]. These properties provide CDs an edge over other fluorescent materials [14-16]. Recently, AIE property in CDs has provided a new extent to their optical properties. CDs having AIE characteristic was first reported by Gao et al. in 2013 [17] where adenosine-5-triphosphate brings the aggregation of CDs. Since then, AIE in CDs becomes the scientist’s first choice due to their sensitivity to pH change, large Stokes shift, photostability and biocompatibility, which can be fruitful for various applications [18]. From various experiments, it was observed that aggregation in CDs lead to structural rigidity, which relaxes the non-radiative path, hence, improving the fluorescence intensity. Aggregation- Induced Emission (AIE) phenomenon in Carbon Quantum Dots (CDs) has been found to improve the optical properties and cracked a new research area for the potential application of materials. The AIE in CDs has been utilised for various applications same as of AIE macromolecules. However, no research so far has been done using AIE in CDs for the detection of nitroaromatic explosives. In view of this, we believe that AIE coupled with CDs may aid a new pathway for the detection of nitroaromatic explosives.

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For a Critical Reading of the COVID Age. Biopower, Nosopolitic and “War-Like” Logic Supporting the Crisis

Introduction

The critical sociologic theory, the marxian thought and even the motto of the English Royal Society “nullius in verba” invite us to “not trust anyone’s words”, that is to say that nothing is what it appears and we must not to believe everything that who govern tell us. That means media representations must always be ascertained with skepticism and critical spirit as occulting logics, dynamics, dispositifs functional to the system in term of management of conflicting implosive forces, crises and critical limits that could it to collapse [1]. In a nutshell we now feel more than ever the absolute need for a social critique that demonstrates again how policy and science collaborate to literally incorporate the consumer values on which the system is founded. In the case, by riding the covid, we see how the current populist ultraliberist policy tries to implement new totalizing nosopolitics focused on wealth, health and body performances fully functional to self-regulation automated mechanisms of reproduction and resilience of an absurd system condemned to grow indefinitely [2]. We can also detect how an ontogenetically unfair profit-centered system makes use of war-like strategies to impose advanced biopower forms, as well as exploiting the collateral side effects of all kinds of crises to derive commercial benefits and promote the recovery. In short, I try to approach the covid from an unconventional sociopolitical perspective, by limiting myself to delineating some relevant themes of a much broader problematic discourse, aimed to stimulate attention on underlying systemic logycs whose collateral damages risk to vaporize di liquid society and to opening dystopian scenarios, including a new era of totalitarianism [3].

Covid, Nothing Else

Despite the evidence confirm all its limits and paradoxes, the system tries to overcome own crises with elastic strategies of domination of the masses assisted by technical progress. In the case the covid crisis is being coped through politics consisting in a mix of anti-social measures and ultra-precautionary hygienic norms, including virtualization and de-humanization of work, medical assistance and education, affecting deeply the ordinary life, behaviors and relations [4]. But, in my opinion, the debate (indeed rather the total absence of public contradictory) about the appropriateness and legal justification of the current nosopolitics seems somewhat tainted by a passive acceptance of the official narration. In fact, leaving out the various hypotheses about the genesis of a new beta Coronaviridae mutant widespread from Whuan, let’s briefly review what we are led to believe about the drama of the covid. In two-year period 2020-2021 the WHO has registered 133.931 deaths out of 5.043.620 of cases. We can roughly deduce that the average lethality rate of the covid is 2.7% and then in Italy the mortality rate in the general population results of 0.02%.

According to the italian ISS, the average age of deceased patients is 82 years; among 67.7% of them suffering from at least three chronic debilitating diseases. Reported letality rate of the covid under the age of 50 is 1.2%; hospitalization rate under the age of 19 is negligible; mortality quite nothing [5]. Although the infection is usually inapparent and the effective contagiousness of the infected asymptomatic people questionable, the silent infection is soon considered as the real crux of the problem and consequently quarantines isolate mostly healthy people and the natural immunization is legally abolished. In parallel unspecified amounts of mass screening tests without scrutiny, false diagnostic tests, “covid per caso”-cases (hospitalized patients discovered accidentally covid-positive), manipulation of Health performance parameters, keep on inflating the daily war bulletins and supporting war-like biopolitics [6].

Biopower and Nosopolitic Logic: Some Example from the Past

We can use the AIDS epidemic due to HIV, a viral agent identified in 1983, as a good model to bring out occulted biopower logics [7]. Initially believed involving only restrict risk groups, the public authorities however adopted massive nosopolitics aimed to protect the healthcare system, as in parallel a terrorist media campaign invaded public space and the sindemic side effects of the diseases were finally unmasked. While the panic exploded, homosexuals were stigmatized and confined, right-wing, homophobic and racist movements rode a political crisis (while curiously nowadays these groups protest against the liberticide measures) putting a strain on the founding values of democracy [8]. Subsequently, in 2003 - while the scandal of transfusions with infected blood was appropriately covered up, just like in 1976 the iatrogenic epidemic of Guillan-Barre syndrome and the President Gerald Ford had himself photographed in public while undergoing the antiflu vaccination- there was a new excess of mortality and a healthcare system collapse due this time to an exceptionally hot summer [9].

On the contrary in 1918 american authorities decided to channel collective emotions about the world war, so that the public crisis dued, this time, to a new flu virus was minimized. A decade before the war a draconian quarantine (a measure adopted in 1300 in Italy to cope the “black death” consistent in 40 days long forced confinement of ships, crews, passengers and goods in the harbors) was performed to persecute the Chinese community of San Francisco [10]. In times of covid, biopower again attacks migrants and coloured people, again targeted by unprecedented police violence. The biological and healthy motivations that justified the nazi-fascist racial hate towards jews, omoxesuals, gypsies, slavs and even communists are sadly known [11]. In the 1968, against the unconventional deideologized pacifism of the beat generatiion, in Italy the police was used to spray disinfectant, in order to prevent the spread of a moral infection, after clearing the fields occupied by the hippies.

But let’s remember also the crucial role played by the epidemics in the rise of capitalism, as the germs introduced by the Columbus’s landings allowed the conquistadores to quickly annihilate the Aztec (1521) and Inca (1526) empires.

Later, joined with the african slaves intended to replace the indigenous exterminated by the European germs, also yellow fever and malaria invaded the Americas [12]. Summarizing, in comparison with previous pandemic (AIDS, H1N1, SARS, MERS, Ebola, Spanish flu, Black Death), based on what is reported by Lhetinen and Brunila, the covid seems to be managed “as a direct function of nationalism”, while never have been registered such a war-like massive autoritharian measures, “state sponsored and state-controlled mitigation” [13].

Towards a Political Economy of the Bare Being

In simple terms, following the foucauldian theoric framework, we can consider biopower as a political strategy of social control practiced by mean of a strict regulation of the bodily functioning and medicalization of all the dimensions of the human life. From this perspective the strict control of Health and Welfare through demographic, epidemiologic, life expectancy, longevity, differential vulnerability and other vital statistics about population combined with wealth markers, plays a crucial role for the current ultraliberals policy. In the covid age, new nosopolitics powered by advancements in the fields of sensors and computational, data and web sciences, allow the system to survive through the pervasive sanitization of bodies, institutions and organizations. Rather, we can safely say that medicalization of the system and mcdonaldization of the Health form now a whole confused reality.

On the other side, although the State sows panic to make fearful people docile and obedient, paradoxically the masses willingly support a regime making them feel more protected, as in the liquid world the body is experienced as the last resource to be exploited to affirm own existence. In addition a “manage of affects through the political ontology of war” is perfectly compatible with the ongoing Political Economy of the life strategies based on nosopolitics that promise the masses biosecurity in exchange for obedience, surveillance, omologation, national identification [14]. But these two years of a “war” apparently still far from being won - despite armies of heroes, discriminating green immunity passports, suspension of the basic rights, state-of-war unitary governments headed by war-time president (unelected, in Italy), bombing terrorist and patriotic propaganda, strategic overestimation of casualities and deaths, censorship of counterinformation, suspension of salary, impossibility to work, monetary sanctions for those sparse fringers of deserters, traitors, rebels, heretics who refuse to be vaccinated - it seems to me that the “invisible” enemy is become even more and more changeable and invincible.

Conclusion

Unsucessfull national and distrectual quarantines, business shutdowns, closure of borders, smartphone-assisted epidemic monitoring make us confirmg that the “white flag principle” gives much more benefits than the victory on the virus [15], even because a broader conceptualization of health comprising a fluctuating condition between well-being and virtual disease to be tested perpetually opens precious big data markets just in the healthcare sector [16]. Ultimately even the benefits of a lost war serve to recover economy, while letting society quickly shrink to swarms of anhesthetized videoaddicted bodies all differently similar [17]. In addition from unpredictable reactions between nosopolitic and technical advances (including sensory assemblage, embedded chips, autonomous self-learning drones and robots, idrugs, artificial intelligence, wearables biometric devices, cryptology, 5G powered web applications), we can expect nothing more than a de-humanized automated condition within somewhat dystopian environmental scenarios [18,19]. But since every form of domination involves also specific modes of resistance, History even entrusts to minorities who, armed with torches, bravely dig into the facts, “reject the horror of unlike and the prospect of a bare vitality, after being humiliated”, the opportunity to promote radical changes [20].

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How to Optimize Glucose Dynamics Via Chrono-Eating?

Philosophy and Innovation

The anterior part of the hypothalamus hosts a bilateral structure known as suprachiasmatic nucleus (SCN). This is a central pacemaker of the circadian system that regulates circadian rhythms, and thus, is central clock alongside peripheral clocks in liver. These clocks are involved in the regulation of circadian rhythms of glucose metabolic dynamics [1,2]. Glucose tolerance decreases in the evening and overnight simply and logically because less nutrients are needed nocturnally [3]. As such, eating large meals and overeating sugary and starchy meals during evening and overnight must be avoided to reduce diabetes and obesity risks. Circadian rhythms of glucose dynamics and metabolism require reduced insulin sensitivity and pancreatic β-cells function in the evening vs. morning. This suggests that glucose dynamics possess internal biological rhythms [2]. Whole body insulin resistance may be an initial step in type 2 diabetes (T2D) development. Nonetheless, hyperglycemia and associated diabetes are dependent on rather hepatic than muscle insulin resistance [4]. Furthermore, muscle insulin resistance and energy over-intake may lead to hepatic lipogenesis, further increasing hepatic insulin resistance and reduced insulin effects on hepatic glucose dynamics [4]. As such, nocturnal glucose over-supply by extensive night eating increases insulin resistance and could ultimately lead to increased risk of diabetes and obesity [5].
As stated before, glucose dynamics are strictly orchestrated by the circadian physiology. Hence, shifting eating time from the morning to evening may elevate blood glucose and cause related metabolic issues. In accordance with ‘choronotype’ concept, people are either morning or evening eaters. The evening eaters have the undesirable habit of eating more extensively overnight. Chronotype, thus, impacts on and interferes with chrono-eating. It has been reported that night-eaters and shift-workers exhibit reduced glucose tolerance, and are thus, at greater risks of diabetes. This is basically due to altered eating time and disturbed circadian rhythms of glucose dynamics [6]. Moreover, greater blood melatonin has been demonstrated in night eaters, also suggesting damaged glucose dynamics by the disturbed circadian biology [2]. Apparently, melatonin possesses a role in circadian rhythms regulation of glucose dynamics based on the sleep-wake cycle. As such, the discovery of MTNRIB, melatonin receptor 1b gene, has fueled interests in researching possible melatonin effects on circadian glucose dynamics.
Elevated melatonin levels following eating might harm glucose dynamics by decreasing glucose tolerance. Melatonin is at maximum overnight which accords with reduced glucose tolerance circadianwise [7]. Thus, the greater risk of diabetes in shift-working night eaters may be caused by a disturbance in the circadian rhythmicity of glucose dynamics. An accepted hypothesis about melatonin action is its inhibitory effect on insulin secretion [7]. Chrono-eating entails that energy-dense foods should be eaten preferably in the morning when physical and brain works are being initiated and increased [5]. Breakfast-eating is highly encouraged whereas nighteating is greatly discouraged [8]. To support, greater postprandial glucose levels have been demonstrated in breakfast skippers who instead consumed large later meals [9]. In addition to eating time, for optimized nutrient/waste metabolism, eating and exercise ought to be coordinated for optimal cell function. This will require more research to be well clarified. Chrono-eating as a growing science/practice offers a simple workable strategy to help optimize glucose dynamics and to likely prevent diabetes [10]. Consequently, chrono-eating is postulated to have influential roles in improving glucose dynamics and life quality in today’s stressful times.

Conclusion

Glucose dynamics are orchestrated by the circadian physiological rhythms. Glucose tolerance decreases in the evening and overnight. As such, nocturnal eaters display hyperglycemia overnight and possibly during day as well. The elevated blood glucose along with the augmented insulin resistance may most probably lead to obesity and T2D. Chrono-eating, hence, is a rising science/practice that must receive deserving public awareness and attention towards improving glucose dynamics and preventing metabolic complexities such as diabetes in today’s stressful life.

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Rectal Palpation Instead of Drug Administration: A Natural Approach to Induce Parturition in Delayed Dairy Cows

Method and Discussion

This case study report aimed to provide an innovative and practical approach to induce parturition in delayed pregnant dairy cattle. Chemical calving induction is a process in which parturition is induced by administrating exogenous hormones and corticosteroids. Having a natural calving, however, is important for optimal health and future productive and reproductive performance of dairy cows. In addition, neonatal calf survival and health can be negatively influenced by any disturbances in the natural calving process. Dystocia and premature calf birth can lead to calf morbidity and mortality due to hypoxia and metabolic acidosis. Chemical induction of parturition was initially performed in beef and sheep production systems [1]. Likewise, in some parts of Australia and New Zealand, calving induction is being used in pasture-based dairy systems with seasonal reproductive programs [2]. These programs are planned to maximize pasture use as an economical feed source. However, induced cows have produced 4% lower milk than those calved normally [2]. Retained fetal membranes is a common consequence of chemically induced calving, predisposing cows to postpartum uterine diseases such as metritis. Therefore, veterinary costs increase following postpartum disorders that may affect reproductive performance of early lactation cows.
Health problems associated with chemical calving induction can also affect animal welfare negatively. Because of these issues, The Australian Veterinary Association (AVA) suggests that the induction of parturition should be cancelled in dairy herds with seasonal calving programs. Additionally, the AVA supports gradual remove of chemical calving induction by 2022. Natural calving process may be postponed in some dairy cows, causing oversized calves and dystocia. This is more noticeable in primiparous cows because they have more difficulty in delivering an oversized calf than do multiparous cows. As a result, heifers can be culled because of pelvic injuries during dystocia. Moreover, calf loss may occur during severe dystocia. To avoid these problems, induction of parturition may be a useful practice. In other cased such as downer, lamed, and under-conditioned cows that are unable to calve normally and timely, the induction of calving would be an appropriate way to reduce the risk of sever dystocia and calf loss. Intramuscular injection of corticosteroids such as dexamethasone alone or combined with prostaglandins (PGF2α) are routine ways to induce calving [3]. In the majority of cases, parturition will happen 45-75 hours after injection. However, these methods do possess undesirable health consequences for both calf and dam.
As a herd protocol in our dairy herd (Behroozi dairy complex, Tehran, Iran), pregnant animals are allowed to naturally calve between 270-282 d of gestation for healthy calving with no manipulation and pulling of calf. After the above time period and if calving has not yet occurred, cows and heifers are monitored via rectal palpation to check calf survival and probably calf malposition. If the calf is still alive, one more day is given to allow a natural calving to occur. In delayed or prolonged calving (i.e., > 283 d of gestation) in both cows and heifers, induction of parturition will be necessary. Instead of chemical induction with administration of dexamethasone and PGF2α analogues that may cause retained placenta and metritis as well as calf health issues and mortality, rectal palpation and moderate stimulation is practiced inducing natural calving. In our experience, natural parturition usually occurs within one- or two-day of rectal palpation. With this feasible and natural method of calving induction, delayed cows are able to calve naturally without any artificial drug administration. It is necessary to emphasize that during palpation and manual stimulation, calf is modestly moved inside the uterus to induce initiation of the parturition process. In our experience, pushing digital part of calf hooves modestly and gently provide suitable stimuli to help the calf move. After this, calf delivery would be expected to occur within 24-48 hours.
Rising cortisol levels immediately before calving has a pivotal role in initiating parturition. Fetal cortisol may be one of the most important physiological modulators that promote parturition in cattle [4]. In addition, maternal cortisol arises as a part of parturition process and stress-like response can be seen during labor [5]. As such, it seems that parturition induction by rectal palpation and manual stimulation of the calf can be a result of fetal cortisol secretion. In other words, we postulate that pushing calf digits in the uterus could induce some degree of stress to the calf which promotes cortisol secretion.

Conclusion

Therefore, to help optimize cow and calf health and productivity, we recommend that the above-explained practicalmanual- natural approach be practiced in delayed cows instead of drug administration.

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Perioperative Management of Adolescent Idiopathic Scoliosis: Posterior Spinal Fusion of 14 Segments with a Foreseen Blood Loss of 2000 ML (57 % of Total Blood Volume of the Patient) without Transfusion

Introduction

Adolescent Idiopathic Scoliosis (AIS) is a disease of the spinal column characterized by a three dimensional deformity (coronal, sagital and axial). It has an incidence of 2-3% of the pediatrics population between the ages of 10 and 18 years old, and its treatment is internationally standardized depending on the degrees of the curves, their timeline progression and spinal maturity. The management of curves that have reached a pre-established severity (Cobb angle greater than 40º and have not responded to treatment with an orthesis) is surgical: an instrumental arthrodesis to correct the deformity and prevent deterioration of pulmonary and cardiac function. The most frequent complication of the operation is excessive blood loss. Different surgical and anesthetic techniques have been developed to reduce it. In the case report we present, we combined all the approaches of Patient Blood Management (PBM), starting at the moment of indication of surgery and continuing until the definitive discharge of the patient, to perform a procedure with aforeseen 57% total blood volume (TBV) (2000ml) loss, without recurring to allogenic blood product transfusion. Specific anesthetic and minimally invasive surgical techniques (MISS) were used.

Case Description

We present a 15 years old (50kg, 160cm, body mass index 19,5 kg/m2, total body surface 1.49m2), Latino male with AIS of the thoracolumbar spine (T2-L4) with curves of 78º and 42º respectively, associated with a moderate restrictive - obstructive respiratory insufficiency (FEV1 2,11L (61,5%), FVC 2,74L (62,5%), with a positive bronchodilator test (increase in FEV1 of 11,8% (250ml), whose instrumented posterior spinal fusion (PSF) of 14 segments was performed at our hospital (a national reference center) in March 2021. In May 2019, the patient who was 13 years old at that time, developed a 40º thoracic curve which was initially treated with a permanent plaster brace for 6 months. By November 2019 the curve had progressed to 60º and a new permanent plaster brace was applied. It was ill fitting and badly tolerated by the patient, causing him constant discomfort, and it was changed after three months. At this stage the thoracic curve had progressed to 72º and a 30º lumbar curve developed.

The patient was again braced for 6 months. At the next check up in September 2020, the curves progressed to 78º and 42º respectively. The patient´s orthopedic surgeon recommended to continue with conservative management until the patient turned 16 years old and reached skeletal mature, and then to be operated. The family sought a second opinion as the patient was severely limited in his physical and psychosocial development and was breathless with mild exertion which prevented him from participating in activities with his peers. He had pain when remaining in the same position for a period of time and had difficulties with daily hygiene as he lived in a hot climate. Photos of the patient at presentation to our hospital X-rays of the patient at presentation to our hospital When the patient presented at our hospital in January 2021, the orthopedic surgeon in charge deemed the futility of the brace and indicated preferrential surgical treatment.

The thoracic curve progressed to 100º, the patient had dispnoea with mild exercise and great discomfort and limitations because of the brace. He was placed on the waiting list and went through the first preanesthetic assessment. Of significance was a hemoglobin (Hb) 12,6g/dl, hematocrit (Hct) 38%, microcytosis, hypochromia and a total protein count of 5g/dl. Respiratory function tests revealed a moderate restrictive-obstructive component to his limitations which responded well to bronchodilators. Echocardiography and cardiac function testes were normal. We initiated PBM with oral Ferrous Sulphate 105 mg daily (to be taken on an empty stomach with a glass of orange juice 30 minutes before breakfast), supplementation with a multivitamin complex, vitamin B12, Folic Acid and a puffer of Salbutamol (inhalation suspension 100mcg/dose) as needed. Recommendations were also given to increase the overall caloric and protein intake and the patient was given instructions for prerhabilitation exercises.

There was a delay with the waiting list because of the COVID pandemic situation, with cancellation of physical presence consultations and all surgical procedures, except emergency and oncologic operations. Three months later, in March 2021, the patient came back for a second preanesthetic assessment (two weeks prior to his operation). He was tolerating the oral iron supplementation with no digestive issues and was using the bronchodilator two to three times a day. The blood test revealed Hb 15,7 g/dl, Hct 45,6%, Fe 126mcg/dl, Fe saturation index 36%, Transferrin 277mg, Ferritin 43ng/ml, Vitamin B12 442pg/ml, Folic Acid 5,65ng/ml. The rest of the consultation results were satisfactory. The patient was scheduled for a14 vertebrae instrumental arthrodesis via posterior approach with autologous and donor bone grafts and Ponte osteotomies. We calculated the foreseen intraoperative blood loss according to the orthopaedic surgeon´s predictions based on his 25 year experience with these operations: 80 +/- 40 ml per vertebral space fused and up to 150 ml for each Ponte osteotomy (approximately 2000ml total).

On the day of the operation a combination of Total Intravenous Anaesthesia (TIVA) (induction with Midazolam, Fentanyl, Atracurium and Propofol, maintenance with Propofol, Fentanyl and Remifentanyl, without muscle relaxant to permit neurophysiology monitoring) and a Morphine spinal for postoperative analgesia (due to an impossible lumbar puncture by the anesthesiologist, this was administered by the surgeon under direct vision during the operation: Morphine Chloride 3mcg/kg (total dose 150mcg). Antibiotic prophylaxis was administered with Cefazolin 1g IV at induction and repeated after 4 hours during the operation. This was continued at 8 hourly intervals for the first 48 hours postoperative. Preemptive analgesia was provided with the administration of IV Dexamethasone 8mg and Dexketoprophen 50mg. Full standard ASA monitorization was applied. After induction, and while the neurophysiology monitoring was being placed, acute hypervolemic hemodilution (AHH) was slowly started with 2000ml of Gelafundin®. Central venous access was secured by cannulation of the right internal jugular vein; the right radial artery was cannulated for close hemodynamic monitoring and frequent blood tests.

Bladder catheter was inserted for fluid balance control and a nasogastric tube was placed to empty the gastric chamber. The patient was placed in prone position on a special scoliosis operating table, taking care to protect all the pressure points and decompress the abdomen in order not to cause excessive filling of the epidural veins which would increase the intraoperative bleeding. Head (3kg) and lower limb (5kg) traction was applied and the table was placed in a mild Trendelenburg position. Strict normothermia was observed during the whole procedure by employing convective air blankets to exposed body parts and warmed IV fluids. Core temperature was monitored throughout surgical time. The first blood test was done before incision revealing Hb 13,2 g/dl and Hct 42% which reflected the results of the AHH. The fluid reposition and maintenance continued at the anaesthesiologist´s discretion (Lactated Ringer solution 2000ml). Intravenous Tranexamic Acid (TXA) was administered as a loading dose of 15mg/kg and continued with a 2mg/kg/h infusion for the duration of the operation.

The OrthoPAT® (Haemonetics), an autologous cellsalvage and transfusion system, was set in place. After skin incision (T2 to L4), subperiosteal dissection of the paravertebral muscles to the tips of the transverse processes of all the vertebrae to be fused was performed under controlled hypotension (mean arterial pressure (MAP) 55 mmHg). Once the curve correction stage was reached, the patient was returned to normal MAP (65-75 mmHg) to prevent ischemia of the neural tissues during distraction, rotation and stretching manoeuvres of the corrective screws and rods which were done under continuous neurophysiology monitoring (somatosensory evoked potentials, motor evoked potentials, screw placement potentials). Before closure, 1g of Vancomycin was mixed together with the bone graft shreds and distributed directly over the open operating field. Another blood test revealed Hb 11,2 g/dl, Hct 36%. An atmospheric pressure drain was placed.

At the end of the procedure which lasted 7 hours, a third blood test revealed Hb 10,2 g/dl and Hct 33%. The anaesthesiologist reinfused the blood recuperated by the OrthoPAT® (350ml). The patient was placed in the supine position and extubated before transfer to the Pediatric Critical Care Unit (PCCU). A loading dose of postoperative analgesia with Paracetamol 1g and Metamizol 2g IV was administered. There were no intraoperative complications. On admission to the PCCU the patient was hemodynamically stable and in sinus rhythm. Respiratory mechanics were correct with 100% SpO2 with Ventimask® O2 at 6L/min. The patient was fully conscious and had no pain. Post OrthoPAT® blood infusion test revealed Hb 12,6g/dL, Hct 36,4%. The following 24 hours were uneventful. Analgesia was continued with IV Paracetamol and Metamizol. On discharge from the PCCU at 24 hours postoperative Hb was 11,8 g/dL and Hct 35,9%, drain yield 170ml. He was transferred to the childrens´ orthopedic ward for further care.

The first dose of Patient Controlled Analgesia (PCA) with Morphine was required at 36 hours postoperative when patient mobilization was initiated. The drain was withdrawn at 48 hours with a 200 ml yield. The patient started early deambulation. The postoperative period was satisfactory, except for continued serohematic oozing from the lower third of the incision which required reinforcement with surgical stapling. There were no hemodynamic, respiratory, infectious or neurological complications. The patient was discharged home on day 8 with oral analgesia (Paracetamol and Metamizol). Blood workup before discharged revealed Hb 9,3 g/dl. A single dose of IV Ferrous Carboxymaltose 1000mg (20mg/kg) was administered.

On day 12 the patient presented to the Emergency Department because of sustained bleeding from the lower third of the wound. His Hb dropped to 8,9 g/dl (nadir) and he felt tired, weak and dizzy when standing up, with no syncopal episodes or breathlessness. He was admitted for surgical revision of the bleeding and bed rest. There were no signs of infection. When the wound was clean and the bleeding had stopped the patient was discharged home. Another dose of IV Ferrous Carboxymaltose (1000mg) was administered. On a revision visit two weeks later he was subjectively well, sitting and deambulating without pain, the wound looked clean and was healing well. Staples were removed. Blood test was Hb 11,1g/dl and Hct 35,4%, Iron 62mcg/dL, Transferrin 227mg/dL, Ferritin Saturation Index 19%, Total Iron Transport Capacity 320mcg/dL and Ferritin 838ng/mL. The patient was instructed to avoid sports for three months and was advised to start aerobic exercise after that. Contact sports were to be delayed six months. He was scheduled for follow up in 6 months. The surgical results were surgically and esthetically satisfactory, and the patient and his family were very happy with the outcome.

Discussion

AIS is a frequent disease of the pediatric population [1-3]. Most cases are managed conservatively with active observation. In progressive cases bracing is applied. When the pathological angles progress rapidly and significantly, surgical management is indicated. Different operative techniques and instrumentations are in use depending on the surgical team´s training. The most frequent complication of extensive AIS surgery is excessive perioperative blood loss, requiring transfusion in up to 70% of the cases [4]. Attempts have been made to standardise the estimation of and predict the perioperative blood loss [5-8]. An exact formula is elusive as many factors are involved. The ultimate volume differs depending on the surgical and anaesthetic teams´ techniques and experience. The RBC transfusion rate is higher in males, lower preoperative Hb, greater deformity angles, larger numbers of vertebrae to be fused and duration of the operation. Our patient had all of the risk factors.

Knowing this and planning appropriately can help avoid blood product transfusion. It is well known that although blood transfusions are life saving in many cases, this however comes with a high price (immunomodulation side effects, metabolic disorders, induced coagulopathy, increased surgical site infections, pulmonary complications, increased length of hospital stay and overall costs per case, as well as complications associated with transfusion errors, adverse reactions and the possibility of infective agent transmission) [9-14]. PBM addresses all these issues and was scrupulously applied by our team as soon as the patient was placed on the waiting list for surgery. We had 3 months of preparation time and we used all techniques available pre, intra and postoperatively. Careful calculation of the predicted perioperative blood loss, taking into consideration hemorrhagic complications and possible contingencies.

• Perioperative red blood cell (RBC) mass optimization [15] with oral and IV iron supplementation. The use of bone marrow stimulating agents such as human recombinant erythropoietin was considered but deemed unnecessary in this case.

• Restrictive RBC transfusion trigger (in healthy ASA I pediatric patients a Hb nadir of 7g/dl, depending on anemia tolerance, can be accepted).

• AHH [16-20] to decrease the RBC loss by lowering the Hct of the blood lost from the operating field. Clinical studies support improved hemodynamic stability and optimization of central venous pressures in patients who undergo this technique under general anaesthesia. AHH promotes an increased heart preload, improves the rheological properties of the circulating blood and coagulation is not affected when the technique is done properly. Employing gelatins in the process maintains the effects for an estimated t½ of 6 hours, which is appropriate for the duration of the surgical procedure.

• TXA [21-25] (an antifibrinolytic) counteracts the hyperfibrinolysis that develops during the surgery due to extensive bone and muscle tissue trauma.

• Controlled hypotension during the dissection phase to decrease the blood lost during the bloodiest stage of the operation. It is important to return the patient to normotension during the traction and torsion stage to prevent possible ischemic damage to delicate neural tissues. The controlled hypotension technique has been shown to safely and significantly decrease autologous transfusion rate [26,27].

• Proper patient positioning on the operative table (decompressing the abdomen to avoid epidural vein engorgement, applying protection to pressure sites and placing the table in moderate Trendelenburg to favour adequate venous drainage and prevent the blood pooling in the operative field).

• Maintaince of normothermia with convective air blankets and warmed IV fluids to prevent hypothermia induced coagulopathy during prolonged surgery times (in our case 7 hours duration).

• The use of intraoperative cell salvage [28-30] with a system like the Harmonics OrthoPAT® system which is able to recuperate up to 70% of the blood in the intraoperative field, wash and concentrate the blood recovered and make it safe for returning it to the patient via the IV route.

• Careful hemodynamic and bispectral index monitoring to aid appropriate anaesthetic dosage.

• Neurophysiological monitoring to prevent possible neurological complications

• Meticulous hemostatic surgical strategy, MISS and experienced surgeons [31,32].

At our institution, the use of expensive OrthoPAT® technology is authorised for surgical procedures where the foreseen blood loss exceeds 30% of the total blood volume (TBV) of the patient as only then does it become cost effective compared to autologous blood product transfusions. In our case we calculated our patient´s TBV by using the 70ml/kg estimation (adolescent 50kg male: 3500ml TBV). The predicted blood loss was 2000ml, corresponding with 57% of TBV. We calculated the allowable blood loss (ABL) for a minimal Hb of 7g/dl according to the formula:

ABL = TBV.( preopHb − nadirHb) / preopHb

Where TBV = 3500 ml

Preop Hb = 15,7g/dl

Nadir Hb = 7g/dl

The ABL result was 1940ml.

We took into consideration the reinfusion of RBC recuperated from the operative field by the OrthoPAT® to allow for a margin of hemorrhagic complications and contingencies. Finally, 350ml of concentrated RBC was generated and reinfused at the end of the operation when Hb of 10,2g/dl was reached, raising the post infusion Hb to 12,6 g/dl. PBM was continued until the definite discharge of the patient. Because of persistent wound serohematic discharge, the patient´s Hb decreased leading to mild anaemia symptoms but never reaching the preestablished transfusion trigger. IV iron supplementation was used to speed up the recovery of RBC mass and aid in symptom tolerance.

Conclusion

With careful planning and conscientious application of PBM strategies, we were able to perform this complex and high risk surgical procedure without recurring to autologous RBC transfusion. The experience of the managing team and continued close communication between surgeons and anesthesiologists makes bloodless surgery of difficult AIS cases possible. The reporting and dissemination of Case Studies such as this one, may help in the management of similar situations by other teams at health institutions worldwide.

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