The Scandinavian school of Cardiovascular Technology

Aarhus University, Denmark

Master Thesis in Cardiovascular Technology with special

reference to Cardiopulmonary bypass

DOES SHED MEDASTINAL BLOOD PRESENT AS

FRIEND (OR) FOE FOR CARDIAC PATIENTS?

Proposed to be submitted in partial fulfilment of the degree Masters of

Cardiovascular Technology of the Aarhus University

By

Vishwanath G. Belavi

RigsHospitalet, Copenhagen Thorax-Anaestesiologisk, Afd 3044 Blegdamsvej 9, 2100, Denmark

Under the Supervision of Prof Dr Shivappa Anurshetru

2012- 2014

SDM Narayana Hrudayalaya Heart Centre, Hubli-Dharwar Road Manjushree Nagar Sattur Dharwar, Karnataka, India - 580005

2

Sl.No CONTENTS Page No

1 Acknowledgement 3

2 Abstract 5-6

3 Introduction 7-8

4 History of Leukocyte Filters 9-11

5 Filter Technology 12

6 Use of Cardiotomy suction blood 13

7 Hypothesis 13

8 Aim of study 14

9 Purpose 14

10 Filter characteristics 14

11 Inclusion and Exclusion criteria 15

12 Materials: Perfusion 16

13 Patients and Methods 16

14 Anesthesia and Perfusion procedures 18

15 Leukocyte filtration procedure 18-19

16 Perfusion measurements 20

17 Lung Function 20

18 Clinical effects 21

19 Results 21-26

20 Discussion 27-29

21 Summary and Conclusion 30

22 References 31-33

3

ACKNOWLEDGEMENT

This Master Thesis is my last theoretical examination paper that completes my

education and entitlement as a “Master of Cardiovascular Perfusion Technology”

in addition to my certification to practice as a “Cardiovascular Perfusionist”. At this

moment I would like to thank the following people without whom this Master thesis

would not have been possible. Special thanks to Chiefs of the departments,

particularly to Dr. Professor Shivappa Anurshetru and Dr. Shanmukh Hiremath

for the possibility to collect data for this thesis in his department.

Quite special thanks are valid at this point for my Parents, my dear wife

Dr. Manisha and my daughter Anushka for the valuable support and help during my

whole Masters study.

Also I would like to thank my colleagues of the team Perfusionist at the

Rigshospitalet, Copenhagen University hospital for their co-operation support during

my master’s course. Special thanks to Casper Seidelin, Peter Fast Nielsen, Marx

Runge, for tutoring and guiding this master thesis.

My thanks are also valid for those who are not mentioned especially, but

considerably contributed to the success and completing of this project thesis.

Student Vishwanath Ganapati Belavi

Supervisor Dr. Shivappa Anurshetru

Honorary Consultant Cardiovascular Surgeon

SDM Narayana Hrudayalaya Heart Centre,

Manjushree Nagar, Sattur

Dharwar, Hubli-Dharwad, Karnataka, India - 580005

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Perfusionist Casper Seidelin, Chief Perfusionist, MCVT, ECCP

Supervisor Rigshospitalet, Copenhagen university hospital

Thorax-Anaestesiologisk, Afd 3044

Blegdamsvej 9, 2100, Denmark

Examiners Prof Hans Nygaard, Bsc Eng,DMsc

The faculty of health science, Aarhus University

Scandinavian school of Cardiovascular Technology

Aarhus University Hospital, Skejby Sygehus, Denmark

5

ABSTRACT

Background: During cardiac surgery using cardiopulmonary bypass (CPB),

retransfusion of shed mediastinal blood is a common practice to minimize blood loss.

Cardiopulmonary bypass induces a whole body inflammatory response that leads to

postoperative lung dysfunction (1-2)

which is largely mediated by the activation of

poly-morphonuclear leucocytes and by subsequent leukocyte deposition and

interaction within the basement membrane of lung endothelium.(3-4)

During the initial

phase of CPB, leukocytes are activated by the contact of blood with foreign materials

in the extracorporeal circuit. After release of the aortic cross clamp in the late phase of

CPB, when heart and lungs are reperfused, activation of leukocytes and leukocyte

endothelium interaction are intensified, leading to the impairment of lung function

and the induction of a post-operative inflammatory response known as the “post

perfusion syndrome”.

Methods: Cardiac surgery with CPB; Coronary artery bypass and valve patients were

randomly divided in two groups. Group I: filtration of cardiotomy suction and

residual pump blood during cardiopulmonary bypass with a fat leukocyte removal

filter (n = 14). Group II: control patients without filtration (n = 14). Filter efficacy was

evaluated in group I using biochemical markers of blood samples taken

simultaneously before and after the filter. In addition, clinical and biochemical

markers for organ injury were determined in both groups.

6

Results and Conclusion: Leukocyte filtration removed more than 95% of leucocytes,

46% free fatty acids,30% triglycerides from the re-transfused blood (p < 0.01) and

significantly reduced circulating leukocytes (p < 0.05) and granulocytes (p < 0.05)

compared with the control group. Levels of the inflammatory mediator thromboxane

B2 as determined at the end of operation were significantly lower in the depletion

group than the control group (p < 0.05), whereas no statistical differences in

interleukin-6 levels were found between the two groups. After operation, pulmonary

gas exchange function (arterial oxygen tension at a fraction of inspired oxygen of 0.4)

was significantly higher in the leukocyte-depletion group 1 hour after arrival to the

intensive care unit (p < 0.05) and after extubation (p < 0.05).

There were no statistical differences between the two groups with respect to

postoperative circulating platelet level, blood loss, and no infection was observed

during the whole period of hospitalization. Results suggest that leukocyte filtration of

the residual blood in the heart-lung machine at end of bypass and cardiotomy suction

blood during CPB,(1) improves postoperative lung gas exchange function, (2) reduces

the inflammatory response and protects the lungs against the acute injury, (3) prevents

post-operative organ injury in patients and is safe to be used for those patients who

are expected to develop severe inflammatory response after cardiac surgery. A larger

study is needed to determine clinical effects on organ damage.

Limitations: This study has small numbers of patients in the groups. Thus, results

presented here can only be considered to be preliminary value.

7

INTRODUCTION

In cardiac surgery with cardiopulmonary bypass (CPB), retransfusion of shed

mediastinal blood is common practice to minimize blood loss .The rationale for

intraoperative salvage of blood is to reduce the need for allogenic blood transfusions.

In addition to being costly, allogenic blood transfusions have been shown to increase

the risk of infection and to have a negative effect on the long term survival after

cardiac surgery (5,6)

. The reuse of salvaged blood from the mediastinum by cardiotomy

suction has however been questioned because of its potentially severe negative

effects. Not only is there a systemic inflammatory response to the reuse of cardiotomy

suction blood, but also it is contaminated with embolic material. It has been shown

that this blood is contaminated with lipids, which may act as emboli (7)

. Furthermore,

these particles pass through the CPB circuit and find their way into various organs (8)

.

Flow within the cardiotomy suction tubing differs significantly from that in

the CPB circuit. The concurrent suction of air results in highly turbulent flow with

high shear stresses at the air-fluid interface. This results in cellular damage and

activates the humoral cascades involved in the systemic inflammatory response.

Cardiotomy suction blood therefore contains an elevated level of free haemoglobin

brought about by mechanical haemolysis. High concentrations of free haemoglobin

cause platelet dysfunction and direct injury to the renal tubular cells. Similarly,

platelet numbers are reduced in cardiotomy suction blood due to rheological trauma.

Several strategies are used to prevent retransfusion of cardiotomy suction blood. Off-

pump revascularization is being increasingly performed, but is not suitable for

intracardiac surgery. In some centers the cardiotomy suction blood is completely

discarded, but this leads to increased allogenic transfusion requirements.

Retransfusion of cardiotomy suction blood, however, is still used during CPB, and

thus a novel approach with a simple and inexpensive filter for the removal of fat and

debris from cardiotomy suction blood may be an suitable alternative. Such a fat

removal filter has been developed which has a polyester 40 µm screen filter based on

a leucocyte removal filter and allows high flow transfusions.

Activated leukocytes play a key role in this process by their interaction with

the endothelium and with the cardiopulmonary bypass tubing and circuit, to form

platelet leucocyte complexes which play role in re-perfusion injury to minimize

8

postoperative tissue injury; it makes sense to target the leukocytes. This has been tried

by various methods in nearly all the stages of the inflammatory pathway. Leukocyte

activation may be prevented with heparin coated bypass circuits, which reduce contact

activation and possess an enhanced biocompatibility.

D-1 D-2

Pharmacological agents may also prevent leukocyte activation. A serine

protease inhibitor with a variety of actions, aprotinin, is frequently used. Aprotinin

and prednisolone have been found to attenuate the generation of tumor necrosis factor

and the up regulation of leucocyte adhesion molecules. Pharmacological agents are

also used to modify the inflammatory response. For this purpose corticosteroids, and

particular dexamethasone, are often used. Corticosteroids probably change the

cytokine balance from pro-inflammatory to anti-inflammatory. Corticosteroids reduce

leucocyte activation and pulmonary leukocyte sequestration although dexamethasone

has been shown to decrease the concentration of C-reactive protein on the first post-

operative day, clear clinical benefits in terms of post-operative oxygenation, time on

mechanical ventilation, or intensive care unit stay have not been demonstrated.(9)

Use

of dexamethasone may even be detrimental by delaying early postoperative tracheal

extubation, and initiating post-operative hyperglycemia. Fat also contributes to post-

operative tissue injury. Fat microemboli have been demonstrated in brain tissue after

cardiopulmonary bypass (10)

.These microemboli were related to the retransfusion of

cardiotomy suction blood, and were associated with postoperative neurocognitive

dysfunction. In addition, the role of fat on tissue injury is underestimated, because fat

microemboli have not only been demonstrated in brain tissue after cardiopulmonary

bypass, but also in lung and renal tissue.(11, 12)

9

Ultrafiltration techniques are used to restore the intraoperative fluid balance

and to reduce the inflammatory response. This approach is based on the idea that

ultrafiltration removes factors that trigger the inflammatory response. Ultrafiltration

has found a place mainly in pediatric cardiac surgery where it has been shown to

reduce body water, and to increase the haematocrit.

Cell savers are being increasingly used to process cardiotomy suction blood,

but these devices might be less than ideal for several reasons. First, fat is not

completely removed by cell savers. Second, their use is expensive and requires

attention and time to process. Third, processed cell saver blood contains increased

levels of interleukin-1 and activated leucocytes, which may aggravate the

inflammatory reaction associated with cardiopulmonary bypass.(13)

Kaza et al. found

that cell savers were not more effective than a filter after the cardiotomy reservoir for

the elimination of small and large fat emboli.(14)

Therefore leucocyte and fat depletion

by means of a filter may offers a good and practical alternative to modify the

postoperative inflammatory response in cardiac surgical patients. The aim of this

study is to demonstrate that leucocyte and fat filtration, applied in the setting of

cardiac surgery, has a beneficial effect on inflammatory markers and postoperative

organ injury.

HISTORY OF LEUKOCYTE DEPLETION FILTRATION:

In 1928, the pathologist Fleming was the first to use a cotton wool plug as a

filter for the removal of leukocytes from blood. His apparatus is shown in (fig.1), and

consisted of a bent glass tube with a constriction. Cotton wool was introduced in the

constricted limb of the tube and pressed down as tightly as possible with a cork-borer.

Blood was placed above the cotton wool and under pressure forced through the cotton

wool with a teat. We shows his apparatus, because the compressed cotton wool

closely resembles the structure of a modern depth filter and the pressure applied by

the teat is comparable to the pressure that may be generated to force blood through

modern leukocyte depletion filters. In the 1980s research was focused on what

happened in organs during ischemia and reperfusion. Engler et al demonstrated in

dogs that the myocardial stunning which was observed after occlusion of the left

anterior descending coronary artery resulted largely from reperfusion injury.

10

They observed during reperfusion an incomplete restoration of the blood flow

in the microvasculature of the heart. This so called no reflow phenomenon was

associated with capillary leukocyte plugging and endothelial cell protrusion and was

based on an acute inflammatory response, (15)

revealed a central role for leukocytes.

Engler et al hypothesized therefore that leukocyte depletion might be beneficial in the

setting of ischemia and reperfusion. They tested this hypothesis in the dog model and

found that reperfusion with leukocyte depleted blood almost completely prevented

reperfusion injury.(16)

These findings started research in the clinical application of

leukocyte depletion filters for organ protection.

FIG-1 FIG-2 FIG-3

Fig. 1 Leukocyte depletion filter as used by Fleming in 1928. (A): compressed

cotton wool; (B): column of blood before the filter; (C): Blood after passage

through the filter

Fig. 2 Scanning electron microscopic section of depth filter. Note the random

order of the fibers. Magnification x100.

Fig. 3 Scanning electron microscopic section of screen filter. Note the woven

structure of the fibers. Magnification x150.

11

The improved flow properties allow these filters to be used in patients with

higher fluid requirements. For an explanation of these improved flow properties, some

aspects of the biomaterials and design that are used must be discussed. At this

moment depth and screen filters are used. In depth filters, the filter material has the

form of compressed wool fibers (Fig. 2) Polyester and sometimes polyurethane is

used as filter material, which promote adhesion of the leukocytes throughout the filter.

In screen filters, the filter is built up with layers of woven polyester filter material

(Fig. 3). In this type of filter, leukocytes are bound to the subsequent layers of filter

material. Most leukocytes are thus trapped at the outer most layers of the filter and

this may increase the resistance over the filter. The way leukocytes are trapped inside

the filter also influences the efficacy and capacity of the filter. At least 4 mechanisms

have been described, (18)

but the most important mechanism is adhesion. By adhesion,

the negatively charged leukocytes are attached to the filter material by Van der Waals

and electrostatic forces. This adhesion is an active process and has the advantage that

a larger pore size with subsequent higher flow rates is possible in the filter. Thus,

properties of the filter material like surface charge and hydrophilicity greatly

determine the efficacy of the filter.

To modify the surface charge of the filter, coating of the filter material is often

used to improve filter efficacy. A coating of methacrylate creates a more positive

surface charge that results in a stronger bond with the negatively charged

leukocytes.(19)

Hydrophilicity is important for optimal contact between the leukocytes

and the fibers and thus for the subsequent adhesion. This implies that optimal

leukocyte depletion can only occur if the whole filter is exposed to blood, which

means that de-airing must be carefully performed before use.

Insufficient de-airing disturbs the optimal blood flow and thus reduces filter

efficacy. Another effect of the physicochemical properties of the filter material is that

leukocytes appear predominantly to stick to the crossing points of the filter fibers.

Thus more crossing points increase the efficacy of the filter. More crossing points

require thinner fibers. However, thinner fibers also lead to an increase in resistance

and thus to flow reduction. This indicates in a nutshell the compromises that have to

be made in the design of a filter. The current generation of leukocyte depletion filters

may be pressurized up to 300mmHg. This allows rapid transfusion in a clinical

setting, but decreases the efficacy as it has been shown that a longer contact time of

12

the leukocytes in the filter increases the filter efficacy. The efficiency of the filters

also decreases over time as the filter becomes saturated with cells and debris.

Therefore, application of leukocyte depletion filters may interfere with blood

coagulation. This is a problem of all leukocyte depletion filters as generally 40% of

the platelets that pass through the filter are trapped. Allen et al demonstrated a

significant difference in platelet counts by drawing samples simultaneous up and

downstream of the filter.(20)

Nonetheless, a certain amount of platelet deposition on the

polyester fibers of the filter helps to bind the leukocytes.(21)

It appears that platelets

have a higher affinity for the filter material than leukocytes. In addition, platelets have

active surface receptors so that they rapidly establish a strong bond with leukocytes. A

beneficial aspect of the removal of platelets is that activated platelets release different

vaso-active substances. Therefore, removal of platelets could reduce thromboxane

release with a reduction in vasoconstriction but before reviewing the clinical aspects

of the application of leukocyte depletion, we will now briefly discuss some aspects of

the filter technology used.

FILTER TECHNOLOGY:

The first generation of leukocyte depletion filters designed for routine use was

made of cellulose and had a leukocyte removal rate of about 98%. Although clinically

acceptable results were achieved, these filters had two drawbacks. First, they also

appeared to activate complement C3, with a subsequent vasoconstriction and

increased capillary permeability (17)

. Second, the efficacy of leukocyte removal was

strongly dependent on the flow across the filter. Filtration was therefore a slow

process and took about 30 minutes for one unit of red blood cells. Currently, a new

generation of filters is available which combines rapid flow with an excellent

leukocyte removal rate. These new filters remove 99.995% of the leukocytes from the

blood. However, during operation this may be somewhat lower with a 96.8% removal

of leukocytes for cardiopulmonary bypass perfusate.

13

USE OF CARDIOTOMY SUCTION AND RESIDUAL PUMP BLOOD:

The reuse of mediastinal blood with cardiotomy suction is more or less

mandatory in open cardiac surgery. Not only does this enable the surgeon to visualize

the field but saves the shed blood from being discarded and thereby avoiding costly

and potentially dangerous problems associated with donor blood transfusion. In case

of profuse bleeding the use of cardiotomy suction and residual pump blood can also

be lifesaving whereas the blood can be immediately retransfused through the CPB

circuit. Filters in the cardiotomy suction are used to decrease the risk of solid emboli

making their way into the circulation. Several authors have shown that

microembolisation is a potential threat during cardiac surgery, and will influence the

outcome (22)

.Opening of the mediastinum during cardiac surgery involves trauma. The

sternum is opened with an oscillating saw and thereby exposing the marrow. Bone

marrow is relatively rich in fat and consists of 40% fat in the adult. With

electorcautery the subcutaneous and mediastinal fat is exposed. Both bone marrow

and adipocytes in the wound are a probable source of lipid which can be seen in the

shed mediastinal blood at surgery. Small and large fat droplets can be observed in the

pool of blood collecting in the surgical field. It is this contaminated blood that is

common practice to collect and reuse during surgery by means of cardiotomy suction

(Fig 4 and 5).

HYPOTHESIS:

Leukocyte filtering of cardiotomy suction and residual pump blood improves

post-operative lung gas exchange, protects lungs against the acute injury, minimizes

severe inflammatory response and prevents post-operative organ injury in patients

undergoing open heart surgeries.

14

AIM OF STUDY:

To assess the efficacy of fat removal filter during open heart surgery by

filtering residual heart lung machine and cardiotomy suction blood in patient’s

undergoing bypass surgery and its effects on post-operative organ injury. We

evaluated some of the possible filter effects on lungs, kidney and heart with clinical

and biochemical markers, using an unfiltered group of cardiac patients as controls and

its beneficial effects on inflammatory response.

PURPOSE:

To test the efficacy of a fat removal filter in patients undergoing cardiac

surgery using CPB for (1) Fat Removal, (2) Leukocyte depletion, from the cardiotomy

suction and residual heart lung machine blood at the end of CPB.

FILTER CHARACTERISTICS:

The amount of cardiotomy suction and residual pump blood was 1104 ± 152

mL on an average with a haematocrit of 19 ± 1.4%. Baseline plasma triglyceride level

in the patients was 1.02 ± 0.15 mmol.L-1

. The filter removed 30% of the triglycerides

and reduced leucocytes by 47% and platelets by 35% (table 4). Thin layer

chromatography revealed that after filtration, free fatty acids (FFA), triglycerides and

phospholipids were reduced (table 3).

The efficacy of the filter decreased slightly during the 600 mL of blood that

passed through the filter. After 600 mL of blood the filter removed 13% of the

triglycerides and reduced leucocytes by 34% and platelets by 31%.The time needed to

pass 200 Ml of blood under gravity at a height of 90 cm was 2 min 40s ± 13s, and for

600 mL this time was 7 min ± 20s. We found no adsorption of (hydrophobic) platelet

activating factor (PAF) on the filter material, excluding its effect on the preservation

of the blood platelet.

15

FIG-4 FIG-5

Figure 4.Surface of shed blood from the surgical field where lipid droplets

are easily seen.

Figure 5.Scanning Electron Micrograph of lipid particles found in shed

mediastinal blood.

INCLUSION CRITERIA:

Only patients admitted for cardiac surgery using cardiopulmonary bypass were

included. Clinical assessment of operative blood loss was done. In patients with

significant blood loss and where allogen blood replacement was needed fat filters

were used. In patients with no significant blood loss filters were not used as

significant volume is needed in the cardiotomy reservoir for circulation in the filter

and for it to be effective for filtration.

EXCLUSION CRITERIA:

Patients older than 70 years with congestive heart failure (CHF) greater than

class II(NYHA), pre-existing lung disease, cerebral vascular disease, diabetes

mellitus, body mass index (BMI) over 35 kg/m2,creatinine ≥ 1.4mg/dl, emergency

operation and re-operation were excluded.

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MATERIALS -PERFUSION:

After institutional approval and patients consent 28 patients scheduled for

cardiac surgery were prospectively randomized to Group I: fat filtration of cardiotomy

suction and residual pump blood during CPB using LG-6(Pall Biomedical) 40µ

polyester screen filter (n = 14), and Group II: control patients without filtration

(n = 14). The extracorporeal circuit consisted of roller pumps (Stöckert, München,

Germany), a hollow fibre oxygenator (Sarns Turbo, 3M, St. Paul, MN, USA) and a

standard arterial line filter (Affinity 38μ Medtronic, Minneapolis, MN, USA). The

priming consisted of 500 mL hydroxyethylstarch 10% (Haes, Fresenius, Bad

Hamburg, Germany) and 800 mL lactated Ringer’s solution and 20% 100ml

Mannitol. Pump flow was adjusted to 2.4 L.m-2 per min. Nasopharyngeal temperature

during CPB was maintained at 35.6º degrees.

PATIENTS AND METHODS:

After getting approval by the medical committee at Narayana Hrudayalaya

cardiac research center Hospital in Hubli, Karnataka, and informed consent from

patients, 28 patients electively undergoing either coronary artery bypass grafting,

heart valve replacement or a combined procedure were randomly allocated to a

leukocyte-depletion group (n = 14) and control group (n = 14) without filtration.

Exclusion criteria were pre-existing lung disease, cerebro vascular disease, diabetes

mellitus, recurrent infection, reoperation, and emergency operation. Blood samples

were drawn from the radial artery (1) after induction of anesthesia, (2) at the end of

the operation, (3) after three hours in the ICU and (4) on the morning of the first post-

operative day. From the blood samples taken pre-operatively on day 1, 2 and 3, the

creatinine clearance was calculated to the Cockcroft formulae. Both groups were

similar with respect to age, sex, length, weight, haematocrit (p = 0.16), creatinine

clearance, CPB time (p = 0.2).The demographic data are summarized in table (1 and

2).

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Variable Unit Mean (Range)

Age Y 64 (47-75)

Male - 14

Female - -

Height cm 175.1 (165-190)

Weight kg 75.6 (60-103)

CPB Time min 97 ( 56-165)

X-Clamp Time min 60.5 ( 38-103)

Temp(np) during CPB Degrees centigrade 35.6 ( 34.5-36.0)

Table 1: The demographic data of patients in both groups are summarized Creatinine

clearance according to the Cockroft formulae; CPB, cardiopulmonary bypass.

Group Filter (n=14) Control (n=14)

Age(Year) 62 ± 2.5 63 ± 3.2

Sex(Male) 14 14

Length(cm) 176 ± 2.6 172 ± 2

Weight( Kg) 81 ± 2.5 77 ± 2

Hematocrit (%) 33.5 ± 0.9 35.8 ± 1.3

Creatinine Cl (ml.kg-1 per min

) 101 ± 6.5 79 ± 7.9 P<0.04

CPB(min) 85 ± 7.0 61 ± 7.0

Table 2: Characteristics of patients (N=14) receiving filtration CPB. All patients that

were included completed the study. The demographic data are summarized in table 1,

which shows that both groups were similar. The post-operative clinical data are

summarized in table 6, and indicate that there were no differences between both

groups. There were no complications requiring a prolonged hospital stay.

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ANESTHESIA AND PERFUSION PROCEDURES:

Anaesthesia was induced and maintained by intravenous infusion of sufentanil

citrate (1–3μg/kg) and midazolam (0.05-0.1mg/kg).Muscle relaxation was achieved

with pancuronium bromide (100–140μg/kg), Cefamandol 2g and dexamethasone 1

mg/kg were administered after induction. Anticoagulation was achieved by

intravenous administration of bovine lung heparin at a dose of 300 IU/kg about 5

minutes before the start of bypass. The extracorporeal circuit consisted of roller

pumps (Stöckert, Munich, Germany) and a microporous polypropylene membrane

oxygenator (CML Excel, Cobe Laboratories Inc., Lakewood, CO). Within 10 minutes

of CPB initiation at a flow rate at 2.4 L/min/m2, the aorta was cross clamped and 1 L

of St. Thomas cardioplegia solution (4°C) was infused into the aortic root to provide

myocardial preservation. During CPB, moderate hypothermia was induced to

maintain the nasopharyngeal temperature between 34.5º to 36.0º.

The mean arterial pressure was maintained between 50 to 60 mmHg during

CPB. Anticoagulation during CPB was monitored by the celite activated clotting time

(International Technidyne Co., Edison, N.J.). After CPB, heparin was neutralized by

protamine chloride (3 mg/kg).

PERFUSION PROCEDURE FOR LEUKOCYTE FILTRATION:

In this study we used a leukocyte depletion filter for Cardiotomy suction and

residual heart-lung machine blood that was retransfused in the patient after

cardiopulmonary bypass. The hypothesis being, blood that is retransfused in a vein

first passes the lungs. The lungs act as an endogenous filter and remove activated

leucocytes and debris from this blood. If these activated leucocytes and debris were

to be trapped in a filter before reaching the lungs it would result in less pulmonary

injury and improved postoperative lung function.

LeukoGard 6(LG6) leukocyte depletion filter consists of polycarbonate

housing with a polyester cartridge that has a leukocyte reduction effect. It also

includes a 40µ polyester screen filter. The priming volume is 220ml and allows flows

upto 6L/min. The main mechanism is to trap leukocytes by adhesion to the material in

the filter. The negatively charged leukocytes are attached to the positively charged

filter material by Van der Vaals and electrostatic forces. The placement of the

19

leukocyte depletion filter may be in the arterial line, the venous return line, the

cardioplegia line (or) a combination of these, and used at various times( continuous or

strategic) during cardiopulmonary bypass. In addition, to assess the qualitative effects

of filtration, modified thin layer chromatography according Folch13 was performed

on samples before and after the filter and on a patient blood sample before CPB, as

well as on the blood samples that were taken for the assessment of the filter capacity.

After CPB, the residual blood in the heart-lung machine was filtered and collected in

a transfusion bag and in both groups retransfused to the patients.

In Group I(Filtration), total amount of blood collected from cardiotomy and

residual pump blood on an average was 1104 ± 152.Here, we filtered 550ml from

residual heart lung machine using a high-flow leucocyte removal filter (LG6, Pall

Biomedical, Portsmouth, UK) incorporated in the arterial line. This procedure was

associated with intermediate flow, but high pressures. After each 600ml of

cardiotomy blood a new filter was used.

Secondly, Leukofiltration of cardiotomy suction blood was (600mL) which

was then collected in separate cardiotomy reservoir from the moment that the ACT

was > 400s. Later, this blood was transfused into the patient under gravity ≤ 100mm

Hg using a leucocyte removal filter (RS 1, Pall Biomedical,UK). At the end of CPB

using the same roller pump blood flows were adjusted to 400 mL/ min. The filtration

pressure, measured between the pump and the filter, was generally high (≥/150

mmHg), but did not exceed 300 mmHg.

The filtration procedure lasted 10 ±/0.7 minutes and, thus the total amount

of filtered blood at end of CPB from cardiotomy suction was 600 ml and 550ml

from residual heart lung machine blood. In both groups, the residual blood in the

extracorporeal circuit after CPB was collected in a transfusion bag and transfused

into the patient using a standard transfusion system. When 200 mL blood had

passed through the filter, samples were taken simultaneously before and after the

filter. From EDTA-anticoagulated blood, hematocrit, platelet and total white blood

cell counts were determined by an electronic cell counter (Cell-Dyn 610, Abbott,

Santa Clara, CA, USA). In both groups, the residual blood in the extracorporeal

circuit after CPB was collected in a transfusion bag and transfused into the patient

using a standard transfusion system. Triglyceride levels were determined with a

biochemical assay (Sigma, St. Louis, MO, USA).

20

In Group II, control group the cardiotomy suction blood was collected

directly in the same cardiotomy reservoir of the CPB circuit from the moment that

the ACT was >400s. The blood was transfused directly into patient at the end of CPB

procedure without performing any leukocyte filtration.

PERFUSION MEASUREMENTS:

For all laboratory tests and biochemical assays EDTA and citrate

anticoagulated blood was drawn from the patients’ radial artery catheter. Blood

samples were drawn (1) after induction of anaesthesia, before the start of CPB, (2) at

the end of the operation, (3) after three hours in the ICU and (4) on the morning of the

first post-operative day. For biochemical assays, plasma was obtained by

centrifugation of whole blood at 1000 RPM and immediately stored at -80°C for

further determinations. Plasma levels of glycerol and triglyceride were both

determined by routine biochemical methods (Sigma. St. Louis, MO, USA).

Haemoglobin, haematocrit and platelet, total white blood cell and granulocyte counts

were determined by an electronic cell counter (Cell-Dyn 610, Abbott, Santa Clara,

CA, USA). Levels of triglycerides, leucocyte and platelet counts were measured from

EDTA and in addition citrate anticoagulated samples taken simultaneously before and

after the filter to assess the efficacy of the fat removal filter. Thromboxane was

determined by enzyme immunoassay (Cayman Chemical Company, Ann Arbor,

Mich) in plasma anticoagulated with citrate and indomethacin. Interleukin-2 and

interleukin-6 were determined by enzyme immunoassay (Quantikine, R&D Systems

Europe, Abingdon, UK) from citrated plasma.

LUNG FUNCTION:

Pulmonary gas exchange was measured by the partial pressure of arterial

oxygen from blood samples drawn from the radial artery line and standardized at a

fraction of inspired oxygen of 0.4. Pulmonary hemodynamics exemplified by mean

pulmonary artery pressure (PAP) and pulmonary capillary wedge pressure (PCWP)

were measured through a Swan-Ganz catheter (Edwards, Baxter Healthcare Corp,

Irvine, CA) introduced percutaneously through the right internal jugular vein into the

pulmonary artery. Pulmonary vascular resistance (PVR) was calculated according to

the following formula: PVR (dyne.sec.cm-5) = (PAP - PCWP) / CO x 80.

21

CLINICAL EFFECTS:

The calculated creatinine clearance was higher in the filter group on the first

postoperative day (p = 0.04) (tables 3 and 7). The two groups were similar with

respect to fluid intake, diuresis, blood loss, lung function and myocardial injury (table

6). In the control group, one patient had a myocardial infarction (defined as new

Q-wave on the ECG and CK > 180 U/L with CK-MB > 10% of total), one patient had

major blood loss and one patient developed renal function disturbances with a serum

creatinine level of 231 mmol.L-1

. Overall hospital stay was slightly shorter in the

filter group (table 6). The PaO2 showed a time effect (p = 0.001), but there was no

difference between the groups (p = 0.25) (figure 1). The A-a gradients showed a time

effect (p < 0.001), but no difference between the groups (p = 0.25) (figure 7).

RESULTS

After operation, pulmonary gas exchange function (arterial oxygen tension at a

fraction of inspired oxygen of 0.4) was significantly higher in the leucocyte-depletion

group 1 hour after arrival to the intensive care unit (p < 0.05) and after extubation

(p < 0.05). There were no statistical differences between the two groups with respect

to post-operative circulating platelet level, blood loss, and no infection was observed

during the whole period of hospitalization. The results observed were;

1) CIRCULATING LEUCOCYTES AND PLATELETS:

Circulating leukocyte and granulocyte counts at the end of operation were

significantly less in the leucocyte-depletion group than in the control group (p < 0.05).

There were no significant differences in circulating lymphocyte and platelet counts

between the two groups. The fat filter removed triglycerides (0.9 ± 0.09 mmol.L-1

vs

0.63 ± 0.07 mmol.L-1

, p = 0.003), leucocytes (4.3 ± 0.8 x 109 vs 2.3 ± 0.6 x 10

9 .L

-1, p

= 0.03) and platelets (116 ± 26 x 109.L

-1 vs 75 ± 21 x 10

9.L

-1, p = 0.003) from the

cardiotomy suction blood (600 ± 154 mL).

22

2) LEUKOCYTE REDUCTION IN RESIDUAL MACHINE BLOOD:

The average leukocyte count determined from the residual machine blood

before filtration was 5.76 ± 0.44 x 109/L. After filtration, the count was 0.152 ± 0.01 x

109/L. More than 97% of leucocytes were removed from the residual blood in the

leucocyte depletion group. The average platelet count from the machine blood before

filtration was 107 ± 6 x 109/L, after filtration, it was 54 ± 2 x 10

9/L. About 60% of the

platelets in the machine blood were removed by the filters in the leucocyte-depletion

group.

3) INFLAMMATORY MEDIATORS:

Thromboxane B2 levels were significantly lower in the leucocyte-depletion

group than in the control group at the end of operation (p < 0.05; table 4). Interleukin-

6 levels increased in both the leucocyte-depletion and control groups during the early

postoperative period. No significant difference was found between the two groups.

Interleukin-2 was not detectable in any of the sample.

4) LUNG FUNCTION:

Pulmonary gas exchange, measured by partial oxygen pressure, was

significantly higher in the leukocyte-depletion group than that in the control group

both at one hour after arrival in the intensive care unit (118 ± 10 mmHg versus

86 ± 10 mmHg, p < 0.05) and immediately after extubation (120 ± 8 mmHg versus

89 ± 10 mmHg, p < 0.05. PAP was somewhat lower in patients receiving leukocyte

depletion than in the control group, but this difference in PAP was not significant.

23

Samples Before the filter After the filter P-Value

Biochemical Assays

Triglycerides(mmol.L-1

) 0.9 ± 0.09 0.63 ± 0.07 0.003

Leukocytes( x 109.

L-1

) 4.3 ± 0.8 2.3 ± 0.6 0.03

Platelets ( x 10 9 .

L-1

) 116 ± 26 75 ± 21 0.003

Free Fatty acids 7.6 ± 1.1 4.1 ± 0.8 0.005

Phospholipids 49.3 ± 3.4 45.3 ± 3.8 0.04

Cholesterol 9.8 ± 1.4 9.7 ± 1.7 0.91

Table 3:-The P-Value reflects the statistical analysis of the sample taken before and

after the filter by one -way student t-test.

Parameter Before

CPB

End CPB End

operation

ICU 1hr ICU 3hr POD 1

Thromboxane

(pg/ml)

Depletion ND 48 ± 15 48 ± 9 33 ± 7 23 ± 29 19 ± 26

Control ND 62 ± 95 127 ± 63 48 ± 15 89 ± 56 21 ± 34

Interleukin

6(pg/ml)

Depletion 36 ± 14 126 ± 96 ND 393 ± 116 344 ± 90 125 ± 46

Control 20 ± 24 197 ± 246 ND 208 ± 103 260 ± 38 155 ± 29

Interleukin 2

Depletion UD UD ND UD UD UD

Control UD UD ND UD UD UD

Table 4:- Values are expressed as the geometric mean and the standard error of the

mean. ICU (Intensive care unit; POD, Post -operative day; ND; not determined; UD,

undetectable (below the lowest detectable level as stated by the manufacturer (P <

0.05) compared with control.

24

5) PRE AND POST-OPERATIVE DATA:

Parameters Depletion (N= 14) Control ( N = 14)

CPB Time 88 ± 38 100 ± 24

Cross Clamp time 61 ± 29 67 ± 19

Blood loss(ml) 414 ± 68 324 ± 34

Intubation(hr) 11.7 ± 0.9 13.8 ± 1.3

ICU Stay(days) 1.0 ± 00 1.1 ± 0.1

Hospital stay(days) 4.2 ± 0.8 10.5 ± 1.5

Table 5.Values expressed as mean ± standard deviation of the mean. (ICU) Intensive

care unit.

Group Filter Control P value

Cr .CL(ml.kg-1

per min) 76 ± 4.5 72 ± 7.2 0.04

Fluid in (ml) 4040 ± 262 4079 ± 291 0.94

Blood loss(ml) 414 ± 68 324 ± 34 0.27

Diuresis(ml) 2920 ± 215 3183 ± 308 0.49

CK enzymes( IU.L-1

) 236 ± 56 169 ± 27 0.32

CK-MB( IU.L-1

) 12 ± 6.4 6 ± 1.8 0.44

Platelets(x 109. L-1

) 181 ± 13 162 ± 13 0.31

Leukocytes(x 109. L-1

) 18.6 ± 0.8 13.5 ± 1 0.13

Table 6. Clinical effects on the first, second post-operative day and hospital stay.

Cr CL, renal, creatinine clearance according to the cockroft formula.

25

Figure 6: Circulating Leukocyte and platelet counts in the filter group and in

unfiltered control group pre-operatively(Pre-op),at the end of operation(end -op),

after 3 hours in the intensive care unit (3h ICU) and on the morning of the first post

operative day 1.

OTHER CLINICAL PARAMETERS:

Duration of postoperative intubation was recorded during each patient’s stay

in the intensive care unit. Blood loss was indicated by 24-hour chest drainage. In

addition, durations of stay in the intensive care unit and of hospitalization after

operation were obtained from hospital registration records.

26

Figure 7:- Arterial oxygen tension (PaO2) and alveolar- arterial (A-a) oxygen

gradients in the fat filter group and in the unfiltered control group pre-operatively

(pre-op), at the end of operation (end-op), after 3 hours in the intensive care unit (3h

ICU) and on the morning of the first postoperative day (day 1), The PaO2 showed

time effect (p=0.0009), but no difference between the groups. The A-gradients

showed a time effect (P<0.001), but no difference between the groups.

27

DISCUSSION

From the results and observations in this project thesis it may be concluded

that leukocyte depletion has a beneficial effect on several clinical parameters in the

post-operative period of patients in group I. However, leukocyte depletion filters are

relatively less used in routine practice for several reasons. Firstly, there are no large

randomized prospective studies that demonstrate the clinical effects of leukocyte

depletion in terms of reduced organ injury and length of intensive care unit or hospital

stay. Secondly, at this moment it is not clear which level of leukocyte depletion

should be achieved during cardiac surgery to obtain a clinically useful result. The

results of the studies during organ reperfusion suggest that lower counts are more

effective, but data regarding systemic leukocyte depletion are lacking. Thirdly, the

optimal period of leukocyte depletion during cardiac surgery, during the whole

cardiopulmonary bypass period or in well aimed time spans, must be defined. Until

these problems are resolved leukocyte depletion during cardiac surgery by means of

filtration will not gain the place it deserves. This study showed that the application of

a fat removal filter reduced the fat content of cardiotomy suction blood in cardiac

surgical patients.

In the leukocyte-depletion group (n = 14), all cardiotomy suction and residual

pump blood (1104 ± 152) on an average was filtered by leukocyte-removal filters and

reinfused in patients after CPB, whereas in the control group an identical amount of

residual blood after CPB was reinfused without filtration (n = 14).There were no

significant statistical differences between the two groups with respect to duration of

CPB time, aortic cross clamp time, postoperative circulating platelet levels, blood loss

and no infection as observed during entire period of hospitalization.

The filter consists of tightly packed fibers with a porous structure of about

40µm. This may mechanically stop the larger fat globules. Such a view is supported

by a recent study on cardiotomy suction blood.23

Fat micro emboli were divided in

large (> 50 µm) and small (10-50 µm) size emboli. No large emboli were detected

after the filter. In our filter the removal of the various fat subgroups was highly

variable. This may be explained by a difference in electrostatic adhesion to the filter

material. One could therefore speculate on filter improvement by coating of the fibers

to increase the removal of the other subgroups, but clinically the free fatty acids

appear to be the most important.

28

Free fatty acids are bound to albumin. Plasma albumin is reduced by

haemodilution after CPB. For this reason we did not use a prime with albumin, but

instead used hydroxyethylstarch, which is not known to interfere with binding of free

fatty acids. Increased levels of free fatty acids have documented clinical effects. In

lung tissue free fatty acids are associated with the development of an acute respiratory

distress syndrome.24

In endothelial cells free fatty acids cause vasoconstriction and

granulocytes are activated through surface expression and activity of CD11b.25

It has

recently been shown that the composition of the cardiotomy suction blood is different,

and that a low temperature increases filter efficacy.26

With about 85 mL/min the filter appeared to have a high flow during

transfusion under gravity. However, a high flow reduces the contact time between

blood and filter medium and thus may result in lower filter efficiency. Thus, filter

efficiency may be improved by coating the fibers, or alternatively by packing more

filter materials in the housing. This latter option would reduce the flow over the filter.

However, a flow of 30 mL/min should be sufficient to filter on an average of 1.5 L

blood, which equals the amount of cardiotomy suction blood, during a cross clamp

time of 45 min. For widespread use the fat removal filter will need a larger capacity,

as our results indicated that the filter became saturated after 600 mL, requiring to

change it.

Clinical findings in this study suggest a beneficial effect of the filter. First, the

higher calculated creatinine clearance in the filter group on the first post-operative day

in view of a similar post-operative fluid balance and second, the higher post-operative

platelet counts in the filter group. Platelets and leucocytes in the cardiotomy suction

blood are activated in the presence of fat and tissue factor from the pericardium. Thus,

removal of platelets and leucocytes by the filter may be advantageous and protective

against the systemic inflammatory response and thrombus formation.

Third is post-operative oxygenation although not significant different in itself

due to the small sample size, the fact that the postoperative A-a gradients were

smaller, and the postoperative PaO2 values were higher in the filter group suggest that

in the filter group less pulmonary injury occurred. This may be explained by the fact

that the filter significantly reduced free fatty acids, known for their deleterious effects

on lung function.28

In addition; the filter also removed a significant part of the

leukocytes from the suction blood. We have previously shown that filtration of

29

leukocytes improved postoperative lung function. Several other possibilities for the

management of the cardiotomy suction blood exists. Cell savers are increasingly used,

but these devices might be less than ideal for several reasons. First, fat is not

completely removed by cell savers. Thus, as a consequence, even cell saver blood

may benefit from the application of a fat removal filter before retransfusion. Second,

their use is expensive and requires attention and time to process.

In contrast, fat removal filters are cheaper, about 25% of the cost of a cell

saver, they are very easy to operate and processed blood is immediately available.

Kaza found cell savers not more effective than the application of a filter after the

cardiotomy reservoir for the elimination of small and large fat emboli.27

Third,

processed cell saver blood contains increased levels of interleukin-1 and activated

leukocytes, which may aggravate the inflammatory reaction associated with CPB.

Based on our results, at least 35 patients in each group had to be included to

demonstrate clinical differences with a power of 0.8 and an α of 0.05. However, our

results suggest that it would be worthwhile to perform such a study. Further, we use

routinely dexamethasone for all our patients to reduce the inflammatory reaction after

CPB because the incidence of the fat embolism syndrome was decreased in a

prospective randomized clinical trial, where steroids were given to prevent the effects

of the fat embolism syndrome.28

Therefore, the effects of the fat removal filter on

organ damage could be more pronounced than demonstrated in this study. Moreover,

we speculated that fat release during the operation would mainly result from

mechanical damage through surgical manipulation and shear forces. This would result

in a direct release of the triglycerides and free fatty acids, which we measured in this

study. Platelets and leukocytes in the cardiotomy suction blood are activated in the

presence of fat and tissue factor from the pericardium. Thus, removal of platelets and

leucocytes by the filter may be advantageous and protective against the systemic

inflammatory response and thrombus formation.

The demographic of patients variables, filter and control groups data are

summarized in (table 1 and 2), which shows that both groups had similar findings.

The post-operative clinical data as summarized in (table 5), indicate that there was

very little difference between the groups. There were no complications requiring a

prolonged hospital stay and all patients recovered uneventfully after operation (Table

6).

30

Lastly, the fat filter removed triglycerides (0.9 ± 0.09 mmol.L-1

vs 0.63 ± 0.07

mmol.L-1

, p = 0.003), leukocytes (4.3 ± 0.8 x 109 vs 2.3 ± 0.6 x 10

9 .L

-1, p = 0.03) and

platelets (116 ± 26 x 109.L

-1 vs 75 ± 21 x 10

9.L

-1, p = 0.003) from the cardiotomy

suction blood (600 ± 154 mL).Use of leukocyte and fat depletion filters undoubtedly

has a place in CPB for open heart surgeries. However, many other clinical parameters

need to be studied and assessed in larger number of patients, for us to conclusively

establish the routine use of these filters in open heart procedures.

SUMMARY AND CONCLUSION

Results suggest that leukocyte filtration of the residual heart-lung machine and

cardiotomy suction blood, (1) improves postoperative lung gas exchange function, (2)

reduces the inflammatory response and protects the lungs against the acute injury, (3)

prevents post-operative organ injury in patients and is safe to be used for those

patients who are expected to develop severe inflammatory response after cardiac

surgery. Postoperative oxygenation although not significantly different in itself due to

the small sample size, the fact that the postoperative A-a gradients were smaller, and

the postoperative PaO2 values were higher in the filter group suggest that in the filter

group less pulmonary injury occurred. This may be explained by the fact that the filter

significantly reduced free fatty acids, known for their deleterious effects on lung

function.

31

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