Acute Normovolemic Hemodilution (ANH) impacts postoperative coagulation profile and bleeding in Adult Cardiac Surgery

Introduction: The coagulation profile of cardiac surgery patients is impacted by pro-inflammatory effects caused by procedures involving cardiopulmonary bypass. In order to counteract these effects, pharmacological therapies and allogeneic blood transfusions are required. The strategy of Acute normovolemic hemodilution also known as “intraoperative autologous donation” is also a viable option to address these negative effects. The theoretical basis for ANH is that the removed blood of a patient’s own self is shielded from the inflammatory response of blood cells to the bypass circuit. ANH is an infrequently practiced strategy at various cardiac surgery institutes within Pakistan to enhance coagulation profile, decrease the need for blood transfusions despite being an established approach abroad. Aims and Objectives: The objective of our study is to compare the effect of ANH on patients undergoing adult cardiac surgery in a sample group versus a control group with the primary endpoint of postoperative drain output as a measure of effectiveness of blood coagulation profile. Blood coagulation profiles were also compared between the two groups as secondary variables. Place and Duration of study: The study was conducted at the Faisalabad Institute of Cardiology from December 21st, 2023 to February 13th, 2024. Material and Methods: A randomized controlled trial involving 60 patients over the age of 12 years who were to undergo adult cardiac surgery was conducted. An online research randomizer software randomly selected them into two equal groups, sample and control (n=30). The ANH volume retrieved from the patient's central vein in the sample group was used to fill the CPD blood transfusion bags after administering anesthesia. After the patients in both groups were weaned off CPB and protamine administered to neutralize heparin, ANH blood was infused back into the patients in the sample group whereas the control group received allogenic blood only. Chi square test was applied to all qualitative variables and Independent Samples t-test for all quantitative variables. The results were analyzed using SPSS version 25, and a p -value (cid:148) 0.05 was considered statistically significant. Results: Hemoglobin (12.9±0.90 g/dL in ANH and 11.8±1.00 g/dL in non-ANH) and aPTT levels (34.2±6.06 seconds in ANH and 54.2±10.95 seconds in non-ANH) were statistically significant (p-value <0.01). In the ANH group, the mean value (623.3±86.28 mL) of postoperative drain output significantly decreased by approximately 220mL compared to the non-ANH group (842.3±99.26 mL) (cid:3) (p-value <0.01). Conclusion: ANH positively conserves most of the coagulation profile parameters. It assists in reducing postoperative bleeding and the volume of allogeneic blood required perioperatively in cardiac surgery.


INTRODUCTION
The risk of cardiac surgery increases by four times with the use of cardiopulmonary bypass (CPB) machines 1 .Every patient has a certain risk which remains regardless of the steps taken.This enhances the need to establish fundamental protocols in diminishing these effects.CPB effects on a patient's coagulation profile and complete blood cell studies is immense which is kept in check with the use of allogeneic blood transfusions.Though it is necessary at some point, routine blood transfusions in itself have physiological and logistic hindrance 2,3 .Amongst these are blood transfusion reactions and the urgency of blood requirements in case of a surgical catastrophe.One such element to tackle these in clinical practice trending at various cardiac surgical institutes is the use of Acute Normovolemic Hemodilution 4-5 .Acute Normovolemic Hemodilution involves the removal of a patient's own whole blood prior to placing the

INTRODUCTION
Globally, acute generalized peritonitis ranks among the top surgical emergencies 1 .It is more common in Third World nations.The prevalence of perforation is low (0.6% -4.9%) in developed nations but high (33% -63%) in West Africa 2 .554 persons were discovered to have peritonitis in a study that took place over three years in India 3 .Researchers in Pakistan have conducted studies with similar methods, with one study reporting 650 cases in a just 9 months 4 .Most cases of peritonitis are caused by a gastrointestinal perforation or anastomotic leak 5 .In the case of peritonitis, anaerobes and gramnegative organisms are mostly responsible for sepsis and morbidity due to the overactive inflammatory cascade brought on by the release of endotoxins 5 .Clinical evidence is used to identify peritonitis.Diagnosis can be achieved via upright plain x-ray of the abdomen, USG, or CT scan.This is often done through diagnostic laparoscopy nowadays 6 .Resuscitation, diagnosis, prompt exploration, treatment of the underlying cause, and extensive surgical peritoneal lavage have always been the cornerstones of peritonitis therapy regimens (IOPL) 7,8 .Regular IOPL is performed to lessen bacterial contamination and burden.Even though large volumes of normal saline are used in IOPL, the rates of sepsis, wound infection, and mortality remain alarmingly high.Another method patient on CPB.This step can be taken prior to surgical incision, at induction of anesthesia or at any time during surgery but before placing the patient on a CPB machine.Quantity of blood removed is replaced with crystalloid or colloid to maintain normovolemia.Utilizing inotropes to ensure vitally stable patients is also a viable option.This results in lesser RBCs and coagulation factors being lost during the course of CPB duration.Also, the removed blood is spared from inflammatory and hemodilution effects of CPB.Post bypass & after heparin reversal this blood being rich in platelets and coagulation factors is reinfused to the patient.Various researches have proven benefits of ANH on postoperative hemoglobin levels and normalized coagulation profile including aPTT, INR, fibrinogen and platelets 6 .The impact of ANH on postoperative coagulation profile is signified sometimes by the differences in postoperative drain output.It is associated directly with a decrease in drain output. 7In our country, there has been no study on cardiac surgery setups using ANH in the last 15 years.Most of the research done internationally haven't used the strategy of volume removal based on the nomogram for ANH which we have used.Retrospective studies have usually been done in the past.The objective of our study was to prospectively examine the first 24 hours postoperative drain output between a sample group receiving ANH and the control group not given ANH.Coagulation profiles between the two groups were also compared during the perioperative period i.e. 24-hours before surgery and 24-hours after surgery.

MATERIAL AND METHODS
This randomized controlled trial was conducted at the Cardiac Surgery Department of Faisalabad Institute of Cardiology (FIC), Faisalabad from 21st Dec'23 to 13th Feb '24.It was a single blind study where patients were unaware of their designated groups.This study was approved by the Ethical Review Committee of FIC vide No.45-2023/DME/FIC/FSD. Patients were enrolled in the study after written informed consent.Pre operatively hematological, LFTs and RFts and relevant parameters were measured.All patients enlisted were >12 years old, had no known preoperative deranged hepatic, renal, neurological or coagulation disorder.A total of 60 patients were randomly assigned into two equal groups (ANH and non-ANH) by online research randomizer software.These patients were enlisted to undergo either cardiopulmonary bypass grafting (CABG) or valvular heart surgery (Aortic and Mitral) as per criteria of selection (Table-1).All patients included had a BMI on a normal-overweight scale.Each patient was evaluated for a preoperative coagulation profile.A Hb>11 mg/dL was established as the bare minimum for inclusion in the study.Patients with prior deranged coagulation profiles and Hb<11 mg/dL were excluded from the study.Those patients with a history of hepatitis or chronic kidney disease (CKD) for which they were treated previously, were excluded.No patient was actively smoking preoperatively for at least 03 months.Pulmonology consultation was done for all patients with a history of smoking or respiratory complaints.All patients in our study were receiving Tab Aspirin 75mg preoperatively since their cardiac pathology was diagnosed, as part of institutional practice.After anesthesia induction, the patient's Central venous line was accessed via the internal jugular vein.Blood was obtained from this central line by gravity-dependent drainage and stored in a CPD transfusion bag at room temperature within the operating theater.The amount of blood that the patient donated depended strictly on the patient's physiologic parameters, estimated blood volume and hematocrit.Intravenous crystalloids and inotropes were used to maintain normovolemia and vitals.Our fluid administration and vasopressor use was influenced by hemodynamic changes in heart rate, electrocardiogram, and mean arterial pressure.1g tranexamic acid was administered to all patients at induction of anesthesia for hemostasis during surgery 8 .The rationale for transfusing blood both intraoperatively and postoperatively relied upon the anesthetist, who kept it above the minimum level of cutoff values for hemoglobin(>8g/dL) and hematocrit(>24%), which was determined by serial arterial blood gas analysis (ABGs).During cardiopulmonary bypass (CPB), this authority was shifted over to perfusionists.Intraoperatively if it was deemed necessary to transfuse blood, allogeneic whole blood was transfused but the ANH volume was spared.The nomogram that was used to estimate the ANH volume retrieved is shown in Fig- 1 22 .The nomogram estimates the amount of ml needed to maintain hematocrit above 24%.The legend in nomogram states exactly the amount of blood that was removed for ANH.As an example, those who fell in yellow boxes had 500ml of blood withdrawn.The subsequent green colored boxes had a larger volume (1000ml, 1500ml etc.) of ANH retrieved.All procedures were performed in conventional cardiac surgery with median sternotomy, aortic (distal ascending aorta) and 2 stage venous cannulation.After the patient was weaned from cardiopulmonary bypass, protamine was administered to neutralize heparin.The patients in the sample group received their pre-donated ANH volume after the Activated Clotting Time (ACT) was determined and found to be in the standard range.ACT measurements signify that heparin has been successfully neutralized by its antidote which is protamine.None of our patients needed extra protamine intraoperatively.A single drain was placed in both mediastinum and right pleura.Post hemostasis sternum was approximated using Surgical Stainless steel wire number 5. Postoperatively, patients were observed for chest drain output for the first 24 hours.Baseline reports were obtained and compared for statistical significance.SPSS version 25 was used to analyze the results and their statistical significance determined via a two-sided test.Chi square test was applied to all qualitative variables and Independent Samples t-test for all quantitative variables.Quantitative variables are stated as mean ±standard deviation.(p 0.05) was considered significance.

DISCUSSION
The knowledge of cardiac surgeons about treating patients with blood conservation strategies to prevent significant bleeding is a topic of debate.The blood volume lost from the patient leads to deteriorating vitals signified by tachycardia (>100/min) or hypotension (<90mmHg systolic BP).These changing hemodynamics must be corrected with volume replacement using either crystalloids (normal saline, ringer lactate, 5% Dextrose saline etc.), colloids (hemacil, 10% albumin etc.) or with utilizing inotropes (epinephrine, norepinephrine, dobutamine, dopamine etc.).Failure to act appropriately can lead to a reduction in end organ perfusion, acute kidney injury (AKI) and multiorgan failure (MOF).
The main finding of our study was the decrease in postoperative drain output.The surgical cause (ligature slip, loose anastomoses etc.) of bleeding was not present in our study group as the quantity of blood loss from surgical causes (>200ml/hr) requiring re-operating the patient to achieve hemostasis was not seen.).This was a remarkable finding considering the fact that a lesser volume of whole blood transfusions was received by the sample group.This shows that ANH has better preserved the morphology of platelets owing to its autologous nature.
In patients undergoing cardiac surgery requiring CPB, ANH results in significant improvements of aPTT and hemoglobin values 4 .The procoagulant effects which can be achieved by ANH have a superior efficacy compared to the allogeneic blood which is extracted prior to surgery 9 .Frank SM et al.
showed that the storage of blood led to cell membrane deformability and the presence of spherocytes and echinocytes which are linked with decreased erythrocyte survival.These deformed cells have a lesser affinity to bind with hemoglobin, which leads to decreased levels of hemoglobin in the transfused subject.ANH has a lower drop in hemoglobin levels compared to allogeneic blood.Similar results have been achieved in pediatric cardiac surgery in terms of allogeneic blood product use and preservation of the physiological parameters in blood components 18,19 .Rotational thromboelastometry studies have shown a significantly shorter EXTEM CT and a greater percentage increase in EXTEM A10 in ANH subject studies 14 .Henderson RA et al. proved that this shows a lesser time for clot initiation and greater increase in clot strength when ANH volume is utilized.This enhanced hemostasis at an earlier postoperative stage.Since the advent of cardiac surgery allogenic transfusion has been used to counteract the proinflammatory effects induced by CPB and to maintain normal physiological parameters.ANH proved to have a superior hematological profile to allogeneic blood in our study as only the most essential whole blood transfusions (1633±434 mL were required in the sample group versus 1800±428 mL in control group.The whole blood volume required in previous studies to maintain normovolemia is also statistically less when the ANH strategy is employed 7,12 .Allogeneic blood is usually stored in blood banks for at least 6-12 hours prior to surgical incision, which can have adverse physiological effects 10 .In an experimental study by Sousa RS, et al., on sheep blood storage of only 6 hours showed metabolic acidemia and deteriorating hematologic, biochemical, and oxidative alterations.In a multi institutional retrospective study by Rhee P et al. of all trauma patients who received an autologous whole blood transfusion from the patient's hemothorax, which was collected from the chest tubes and anticoagulated with citrate phosphorus dextrose, significantly lower packed red blood cell and platelets transfusion were required 13 .ANH when combined with an antifibrinolytic agent, such as tranexamic acid (which we used in our study) assists in hemostasis 20 .Not surprisingly an enhanced hemostasis also decreased the need for allogeneic blood transfusions .ANH volume is stored at room temperature inside the operation theaters due to logistics.Temperature at which ANH volume is stored is debatable.A colder temperature for this purpose may be more beneficial 11 .In the study done by Kusudo E et al. cold storage (4°C) of whole blood compared to room temperature (22°C) provided improved storage conditions for platelet's aggregability, glucose consumption and lactate production.There is an argument over the volume of blood removed for ANH compared to the patient's total circulatory load. 15,16.Only a fixed volume of blood was removed for ANH in these studies.If the ANH strategy had been employed using an individualized approach towards patients' demographics, such as the use of a nomogram, the results could have been different.Over time the adverse effects of allogeneic blood transfusions are becoming clearer 2,3,21 .In oncological experiments on primates, allogeneic blood transfusion has a fourfold increase in tumor growth compared to autologous blood transfusions.This likely reflects immunomodulatory effects induced by the introduction of major histocompatibility complex-incompatible antigens with allogeneic blood.This can be countered with the use of ANH in surgery for malignant cardiac neoplasms to prevent recurrence.Additionally, it can be utilized to prevent unnecessary allogeneic transfusions during complex cardiac surgeries like thoracic aorta repair where a significant amount of blood loss is anticipated and insufficient amount of allogeneic blood is present 17 .All the data to date suggests that allogeneic blood transfusions are not ideal in preservation of patients physiologic and blood coagulation parameters.A simpler and readily available technique in the form of ANH is available.Additional investigation should be conducted to determine the feasibility of retrieving a higher volume of ANH.This could result in the complete elimination of allogeneic blood transfusions in the future.

CONCLUSION
The results of our study show that ANH positively conserves most of the coagulation profile parameters.It assists in reducing postoperative bleeding and also decreases the volume of allogeneic blood required perioperatively in cardiac surgery.With further workup it can be one of the fundamental strategies in blood conservation completely obliterating allogeneic blood transfusions.

LIMITATIONS
The sample size in our study and duration of measured variables was small.In future a large sample study over a prolonged period of time with other variables and coagulation tests is needed to determine further uses and implications of ANH.

FINANCIAL ASSISTANCE
None was required as all the equipment and drugs employed are in routine use in our daily surgeries.

Fig
Fig-1: Nomogram for allowable acute normovolemic hemodilution Inclusion Exclusion Age >12 years Redo surgery CABG patients Pre-op deranged blood reports* Valvular patients Pre-op pharmacological inotropes or mechanical IABP requirement BMI 18.5-29.9History of Hepatitis CKD NYHA I-III Unstable angina CCS I-III LVEF<30% Table-1: Criteria for Selection Abbreviations: CABG:coronary artery bypass grafting, BMI:body mass index , NYHA:New York Heart Association functional classification grades, CCS Canadian Cardiovascular Society classification grades, IABP: Intra Aortic Balloon Pump, CKD: Chronic Kidney disease, LVEF: Left Ventricle Ejection Fraction

Table - 1: Criteria for Selection Abbreviations
Baseline blood tests: complete blood count, Renal function tests, Liver function tests, coagulation profile, serum electrolytes, Arterial blood gas analysis, cardiac markers,Troponin I.

ANH p- value Hemoglobin (g/dL) 13
Demographic characteristics of the two groups are shown in Table-2.The number of patients who were preoperatively hypertensive, diabetic or smokers is shown.It did not show any statistical difference (pvalue >0.05) in terms of age, gender, type of surgery, history of hypertension, diabetes or smoking.The number of males was more compared to females in both groups.More diabetics and hypertensive patients were present in the control group.Frequency of smokers was higher in the sample population.Preoperative blood baseline reports also did not show any statistical difference though the mean platelet count in the ANH group (217.0) was less than the non ANH group (231.1) with a p-value 0.303 (Table-3).