University of Groningen Aspects of leucocyte and fat filtration during cardiac surgery de Vries, A.J. IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2005 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): de Vries, A. J. (2005). Aspects of leucocyte and fat filtration during cardiac surgery. s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 17-03-2023 CHAPTER 4 FILTRATION OF ACTIVATED GRANULOCYTES DURING CARDIOPULMONARY BYPASS SURGERY: A MORPHOLOGIC AND IMMUNOLOGIC STUDY TO CHARACTERIZE THE TRAPPED LEUCOCYTES. J.J.J. Smit, A.J. de Vries, Y.J. Gu, W. van Oeveren The Journal of Laboratory and Clinical Medicine 2000,135:238-245 £(cid:149)⁄ ABSTRACT Cardiopulmonary bypass surgery induces an inflammatory reaction among others by granulocytes. Leucocyte filtration has been shown to reduce the postoperative morbidity mediated by activated granulocytes. However, little is known about the mechanism of filter-leucocyte interaction. This study examines whether a leucocyte filter removes activated granulocytes or a general leucocyte population. Eleven patients undergoing cardiopulmonary bypass surgery were included in this study. Leucocyte filtration was achieved before the reperfusion phase with a Pall non- woven polyester filter located at the venous side of the heart-lung machine. After filtration, the trapped granulocytes inside the filter were examined with light and scanning electron microscopy and immunologically by CD45RO antigen binding to the filter material. Furthermore, leucocyte release markers were measured to determine whether cells were activated during filtration. On microscopic evaluation it was found that 84% granulocytes and 14% lymphocytes were trapped in the filter, compared with 78% granulocytes and 22% lymphocytes in the blood before filtration. Granulocytes were trapped significantly more in the first blood contact layer of the filter material than in the middle layer and last layer, whereas lymphocytes trapped slightly more in the middle layer. The near maximum level of CD45RO expression was measured on granulocytes trapped inside the filter material, whereas CD2 and CD19 measured on lymphocytes were bound to a minor extend. ß- Glucuronidase concentration did not increase after filtration, suggesting the absence of activation of granulocytes by filtration. The results of this study suggest that a leucocyte filter made of non-woven polyester material removes the activated granulocytes rather than leucocytes at random. This implies that this particular type of leucocyte removal filter is suitable for use in cardiopulmonary bypass patients whose granulocytes in the circulation are activated. Furthermore, measurement of activated granulocytes instead of total leucocyte count is likely preferable for functional assessment of leucocyte removal devices. ¥/ƒ INTRODUCTION Foreign materials used in the Cardiopulmonary bypass (CPB) circuit during heart surgery activate leucocytes, resulting in increased cell adhesiveness, release of oxygen radicals and enzymes, and finally, damage to the host.1-9 Primarily the neutrophilic granulocyte fraction is activated after initial contact with extracorporeal surfaces.3-7 It has been suggested that removal of these activated granulocytes by filtration reduces morbidity after heart surgery.10-15 However, during leucocyte filtration in CPB procedures, we observed a large patient-related variation in filtration efficiency.16 Based on the suggested mechanism of leucocyte removal by synthetic filters – that is, by adhesion to the filter material rather than by seaving17 – we speculated that this variation in filtration efficiency was related to a difference in the expression of leucocyte receptors, leading to a difference in adhesion capacity to the filter material. However, it has never been reported, by directly studying the leucocyte-filter interaction, whether during CPB, leucocyte filters remove granulocytes at random or remove primarily an activated subset of granulocytes. Therefore, we designed this study to examine whether leucocyte filters remove a large portion of activated granulocytes or a general leucocyte population. To achieve this goal, we used a filter during 14 minutes in the clinical setting of CPB and performed electronic cell count and biochemical tests on blood samples taken before and after filtration. Additionally, we performed histologic examination on embedded filter material and immunologic tests to show the presence of ‘activation receptors’ on leucocytes trapped inside the filter and on whole blood samples before and after filtration. METHODS Patients After approval was received from the ethical committee and informed consent was received from patients, 11 patients undergoing an elective heart operation for either coronary artery bypass grafting or heart valve replacement were included in the study. Exclusion criteria were a history of allergy or recurrent infection, reoperation, and emergency operation. The characteristics of the patients are summarized in table 1. Heart operation procedure Anaesthesia was induced and maintained by intravenous infusion of sufentanil citrate (1 to 3 mg/kg) and midazolam (0.05 to 0.1 mg/kg). Muscle relaxation was achieved with pancuronium bromide (0.1 mg/kg). Cefamandol at a dose of 2 g and dexamethasone at a dose of 1 mg/kg were administered after induction. Anticoagulation was achieved by intravenous administration of bovine lung heparin at a dose of 300 IU/kg approximately 5 minutes before the start of CPB. Anticoagulation was monitored by Celite activated clotting time (International Technidyne Co, Edison, NJ). After CPB, heparin was neutralized by protamine chloride (3 mg/kg). The heart-lung machine consisted of roller pumps (Stöckert Instrumente GMBH, Munich, Germany) and a microporous polypropylene membrane oxygenator (CML Excel; Cobe Laboratories Inc., Lakewood, CO). 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During CPB, moderate hypothermia was induced (table 1). The mean arterial pressure was maintained at 50 to 60 mmHg during CPB. Leucocyte filtration Leucocyte filtration was achieved by using a prototype leucocyte removal filter (B 1320A; Pall Biomedical, Portsmouth, England). This was a redesign of the prototype filter used for our previous study16 to make it easier for clinical handling. The filter was incorporated in the circulation, in a parallel circuit at the venous site of the heart-lung machine, and one of the roller pumps (Stöckert) was used to maintain a flow rate of 500 mL/min. Leucocyte filtration was performed during the rewarming phase at the end of CPB just before release of the aortic cross-clamp and lasted for approximately 14 minutes. During filtration the pressure at the inlet side of the filter averaged 74 ± 17.5 mmHg. Blood sampling Blood samples were taken before and after filtration from the radial artery of the patients and every 2 minutes during filtration from the inlet and outlet sides of the filter. The blood specimens were collected in sodium citrate (0.32%). Leucocyte counts were performed with an electronic cell counter (Cell-Dyn 610; Abbott, Santa Clara, CA) to asses leucocyte removal by the filter. The relative cell removal rate was calculated every 2 minutes according to the following formula: relative cell removal rate = (1-[post-filter count/pre-filter count]) x 100. The average cell removal was calculated as a mean of the relative removal rates. The total number of removed cells was calculated by multiplying of the absolute number of removed cells per liter (post filter count minus pre-filter count) with the volume of filtered blood. For biochemical assays, plasma was obtained by centrifuging of whole blood at 4(cid:176)C for 10 minutes at 1100g, whereafter plasma was stored at -80(cid:176)C until further examination. ß-glucuronidase, a release product of activated granulocytes, was determined by an enzymatic assay (photospectrometry; Boehringer, ']' Mannhein, Germany) in plasma samples from the inlet and outlet sides of the filter, after 8 minutes of filtration, to indicate activation of granulocytes by the filter material. Platelet activating factors inhibiting capacity (PAF-IC) was also determined from the inlet and outlet sides of the filter after 2 minutes filtration by turbidometry in an aggregometer (Chrono-Log, Havertown, PA) to indicate platelet activating factor (PAF) production by activated leucocytes. The measurement of PAF-IC was conducted with platelets isolated from citrated platelet-rich plasma containing indomethacin (50 mg/mL) from the blood of a healthy volunteer by filtration through Sepharose CL-2B (Pharmacia Biotech Inc., Stockholm, Sweden). These platelets were resuspended in saline to a final platelet concentration of 50 x 109/L and added to the plasma of the study patients. The maximum velocity of platelet aggregation was measured after PAF C16 (Cayman Chemical, Ann Arbor, MI) addition and was used to indicate the PAF-IC of the patients’ samples. Because the PAF-IC is maximal in normal plasma and reduces after PAF formation, normal human plasma was used as a negative control and saline as a positive control, resulting in no aggregation of the platelets and maximum aggregation of the platelets, respectively. Morphologic examination of leucocyte entrapment Nine leucocyte filters were collected immediately after CPB and were prepared for histological examination to enable differential leucocyte counting in the cross-section of the filter material. After release of the residual blood, the filters were perfused in their indicated flow direction with 500 mL of normal saline solution under a consstant pressure of 75 mmHg to wash away the unbound leucocytes. This low perfusion pressure did not exceed the clinical filtration pressure and was chosen to prevent the release of attached leucocytes by high shear forces. To further control the stability of leucocyte binding within the filter, part of the filter material was washed again after the standard procedure with 3 L of normal saline solution under similar perfusion pressure. In each filter, leucocytes were counted in 3 different layers of the cross-section. This comparison showed that washing the filter with 500 mL of normal saline solution did not differ significantly with results when washing with 3 L of saline solution in regard to bound leucocytes (table 2) and thus was used as a standard procedure for the future experiments. After washing, the filters were cut open without damaging the filter material, and within 60 minutes after filtration, partly fixated in 4% paraformaldehyde 0.1 mol/L phosphate buffer (pH 7.4) and stored at -20(cid:176)C. In duplicate, samples of the fixed filter material were dehydrated with alcohol and distilled water and embedded in plastic (GMA, Technovit 8100). Series of three slices of 2 mm were cut out of the cross- section of both samples of the plasticized filter material with a microtome (Jung 1140) and a D-knife with a Tungsten Carbide edge (16/20). Thus in total, 6 samples of each filter were prepared for light microscopy. All slices were stained by standard histologic methods with May-Grunwald-Giemsa and viewed under the microscope. Leucocytes in each slice were counted on three locations of the cross-section of the filter material. As the first location, the first layer of the filter material touched by blood was chosen. As the second location, the middle layer of the cross-section was viewed. As the third location, a microscopic view of the last layer, which bordered on the outlet of blood from the filter, was examined. 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For scanning electron microscopy, pieces of filter material out of the same three layers that had been fixed were used. Postfixation was performed with 1% OsO in phosphate-buffered saline solution 4 for 3 hours, followed by dehydration in ethanol series. After critical point drying with CO , the samples were supercoated with gold and examined with scanning electron 2 microscopy at 2 kV (Jeol 6301F, Tokyo, Japan). Immunologic examination of activated granulocyte entrapment The freezer-stored non-fixed parts of the filters were examined for the binding of specific antibodies to cells trapped inside the filter material. The antibodies used for this procedure were labeled with europium Eu-DDTA (Wallac, Turku, Finland), which allows sensitive detection by means of time-resolved fluorescence.18 Mouse anti-human CD45RO monoclonal antibody (Caltag Laboratories, San Francisco, CA) was used as a marker for the specific binding of activated granulocytes. To estimate the amount of CD45RO binding to T and B cells, specific antibodies against T-cell receptors (mouse anti-human CD2 monoclonal antibody; Caltag Laboratories) and against B-cell receptors (mouse anti-human CD19 monoclonal antibody; Caltag Laboratories) were used. To measure the binding of the antibody to the cells trapped inside the filter material, materials were separated in three layers representing the same specific locations of the cross-section of the filter material used in the morphologic examination. Each layer was divided into three parts. This resulted in nine parts per filter to be tested. Each part was carefully weighed to correct the amount of antibody binding for filter mass. Then, through a standard procedure, each part was washed with saline solution and incubated for 30 minutes with Eu-labeled antibody on a plateshaker and for 5 minutes with 3% H O . After the non-bound antibody was washed away, the Eu was released in 2 2 enhancement fluid and counted in an Arcus (Wallac). A negative reference was made during each test by triplicate measurement of the nonspecific antibody binding to a sample of non-used filter material that had been incubated for 60 minutes in leucocyte- j%k and platelet-free plasma. A positive reference was made on filter material samples from 3 patients to test the maximum CD45RO binding to the filter material; Zymosan-activated plasma (Sigma, St. Louis, MO) containing high concentrations of C5a was incubated with the filter material for 20 minutes before CD45RO antibody binding.19,20 In addition, removal of activated leucocytes from the blood of patients during CPB was tested by flow cytometric measurement of the adhesive receptor present on activated granulocytes (CD11b; DPC, Los Angeles, CA) in blood before and after the filter. Thus from 3 patients, blood samples were taken before filtration from the radial artery, before and after passing the filter at 2 and 10 minutes filtration from the afferent and efferent lines of the filter, and after the filtration procedure from the radial artery. Immediately after collection in sodium citrate (0.32%) blood was incubated with phyco-erythrin- labeled anti-CD11b, treated with Optilyse C (Immunotech, Marseilles, France), and prepared for flowcytometric analysis (FACS, Coulter, Luton, England). Statistics Before data analysis, all non-categorical data were tested and found normal distributed according to the Kolmogorov-Smirnov goodness-of-fit test. An unpaired two- tailed Student t test was used to test the differences between the different leucocyte counts, non-categorical patient characteristics and immunologic data. An unpaired one tailed Student t test was used to test the difference between the rinsed and extra-rinsed microscopical leucocyte counts. To detect possible differences between microscopic cell counts in the three different layers in cross-section, one way analysis of variance was used to compare groups. Duncan’s multiple comparison post hoc procedure was used to quantify any differences among groups that were found to be significant. A value of p < 0.05 was considered statistically significant. All haematologic, morphologic, immunologic and biochemical data are expressed as mean and standard error of the mean, unless otherwise indicated. RESULTS Morphologic examination of leucocyte entrapment Under light microscopy, the number of segmented neutrophilic granulocytes, band neutrophils, monocytes and eosinophilic and basophilic granulocytes was significantly reduced along the flow direction through the three layers of the cross-section of the filter material (table 3). The middle and last blood contact layer showed statistically significantly fewer granulocytes than the first layer (p < 0.0001). 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(cid:139)ƒ¥§(cid:157)(cid:23)(cid:139) (cid:157)(cid:1)(cid:139) ' “‹«fi› flF(cid:176)e–(cid:143)†·‡¶(cid:181)¤–(cid:143)(cid:176)‚•§fl(cid:7)„)–(cid:154)(cid:181)e” „)–(cid:143)»·‡¶(cid:181)¤–¡„z•…flL†)–(cid:154)(cid:181)e” ‰)–(cid:154)(cid:190)¿‡_fl%–[fl(cid:135)•(cid:148)(cid:181)¤–(cid:143)(cid:192);” `ˆ´(cid:7)˜(cid:3)¯(cid:23)˘(cid:23)˙(cid:21)´(cid:7)¨W˘(cid:201)˘(cid:1)˚(cid:25)¸(cid:7)¨C˘(cid:23)˙]˙]˘(cid:23)(cid:204)z´(cid:1)˙(cid:131)˝l˘(cid:23)´(cid:7)˛—ˇ(cid:147)˙(cid:210)(cid:209)1´(cid:7)˛(cid:23)(cid:204)F´L¨C(cid:204)z˘L¨O¨C(cid:211)(cid:25)¨[(cid:212)(cid:148)(cid:213)M˛‚¸(cid:27)˘L¨C(cid:214)(cid:23)˘(cid:7)˛ (cid:209)1´(cid:23)(cid:215)F˘(cid:201)(cid:211)(cid:7)(cid:216)‹(cid:209)J(cid:217)(cid:27)˘(cid:201)(cid:214)(cid:23)˘(cid:7)˜(cid:3)˜F(cid:214)(cid:23)(cid:211)(cid:25)¯F˛ (cid:209)…˝(cid:21)˘(cid:1)´(cid:23)˙(cid:1)¯F¨C˘(cid:1)(cid:204) (cid:218)(cid:23)˘(cid:1)(cid:216)_(cid:211)%¨W˘(cid:131)´(cid:7)˛(cid:23)(cid:204)z´(cid:1)(cid:216)[(cid:209)1˘(cid:7)¨)(cid:209)J(cid:217)(cid:23)˘(cid:201)(cid:216)1(cid:213)M˜(cid:209)1˘(cid:7)¨[(cid:212))´‘(cid:209) « (cid:204)(cid:5)(cid:213)(cid:216)>(cid:216)_˘(cid:7)¨C˘L˛(cid:1)(cid:209)(cid:219)(cid:209)!(cid:213)M˝(cid:21)˘z¸(cid:23)(cid:211)(cid:5)(cid:213)M˛ (cid:209)1˙(cid:128)(cid:204)(cid:25)¯(cid:7)¨H(cid:213)M˛(cid:27)(cid:215)‚(cid:216)1(cid:213)M˜(cid:209)U¨W´ (cid:209)!(cid:213)(cid:211)%˛ ‡(cid:220) (cid:221)‚fl;fl(cid:7)”>–b(cid:222) (cid:204)F(cid:204)F(cid:213)(cid:209)!(cid:213)(cid:211)%˛(cid:23)´(cid:7)˜(cid:3)˜(cid:223)%(cid:212) (cid:209)J(cid:217)(cid:23)˘(cid:158)´ (cid:224)F˘L¨C´(cid:23)(cid:215)(cid:7)˘—¨W˘(cid:7)˝(cid:21)(cid:211)L(cid:224)(cid:7)´(cid:7)˜Æ(cid:211)(cid:7)(cid:216)(cid:226)˜˘(cid:7)¯(cid:27)(cid:214)(cid:1)(cid:211)F(cid:214)(cid:1)(cid:223)(cid:27)(cid:209)1˘(cid:23)˙ª(cid:204)%¯(cid:7)¨H(cid:213)M˛(cid:23)(cid:215) fl(cid:23)(cid:190) ˝(cid:228)(cid:213)M˛(cid:7)¯(cid:1)(cid:209)1˘(cid:23)˙·(cid:216)1(cid:213)M˜(cid:209)U¨W´ (cid:209)!(cid:213)(cid:211)%˛(cid:229)(cid:213)M˛S¸(cid:23)˘(cid:7)¨C(cid:214)(cid:23)˘L˛(cid:1)(cid:209)1´(cid:23)(cid:215)(cid:7)˘ ‡ ˝(cid:21)˘(cid:1)´(cid:7)˛(cid:230)ˇ ˙(cid:210)(cid:209)1´(cid:7)˛(cid:23)(cid:204)F´(cid:7)¨W(cid:204)(cid:229)˘(cid:7)¨O¨W(cid:211)%¨ ” ´(cid:7)˛(cid:27)(cid:204)(cid:231)(cid:209)J(cid:217)(cid:27)˘Ł(cid:209)1(cid:211)(cid:27)(cid:209)!´L˜Æ´L(cid:218)(cid:27)˙](cid:211)(cid:25)˜(cid:3)¯(cid:1)(cid:209)1˘Ø˛(cid:7)¯F˝(cid:135)(cid:218)(cid:27)˘L¨ ‡_fl(cid:23)(cid:190) ' “(cid:131)«Œ› (cid:218)F˜(cid:211)(cid:7)(cid:211)F(cid:204) ” (cid:211)L(cid:216)‚¨C˘(cid:7)˝l(cid:211)L(cid:224)F˘(cid:1)(cid:204)(cid:151)˜˘(cid:7)¯(cid:23)(cid:214)(cid:23)(cid:211)F(cid:214) (cid:223)L(cid:209)1˘(cid:23)˙ ºW(cid:236)F(cid:237)(cid:7)(cid:238)(cid:128)(cid:239)(cid:23)(cid:240)(cid:7)æl(cid:242)(cid:147)æC(cid:243)(cid:7)(cid:236)(cid:23)(cid:244)F(cid:240)(cid:7)ıe(cid:246)M(cid:236)ł(cid:247)(cid:1)øª(cid:238)(cid:228)(cid:246)M(cid:236)(cid:7)(cid:237) œ!(cid:240)(cid:1)ß(cid:27)(cid:252)b(cid:243)(cid:7)æW(cid:240)(cid:128)ß(cid:1)(cid:253)(cid:23)(cid:254)F(cid:255)(cid:131)(cid:236) minutes of filtration of 79% (table 4). Granulocyte removal was 84% and lymphocyte removal 73% (table 4). In five sequential measurements during the first 10 minutes a gradual decrease of leucocyte removal, from 91% to 70% (table 4), was observed. However, in spite of 10 minutes leucocyte filtration, the average systemic leucocyte counts measured in 11 patients did not change during the period of filtration (before filtration 3.68 – 0.42, after filtration 3.62 – 0.49; p-value 0.94). Immunologic examination of activated granulocyte entrapment A significant increase in CD45RO binding to the filter material after leucocyte filtration in comparison with the non-specific binding without leucocytes was found (P<0.001, figure 2). In the middle layer no increase was found in the CD45RO expression after further stimulation of granulocytes with Zymosan-activated plasma (figure 2). In the first and last layers, however, CD45RO expression increased after Zymosan stimulation (figure 2). Microscopic granulocyte count, CD45RO binding, and maximal CD45RO binding to the filter decreased significantly along the flow direction. Proving the validity of the test, the decrease of microscopical granulocyte counts correlated best with the decrease in maximal CD45RO expression. A significant CD2 binding (T cells) to the three different filter layers as compared with the non-specific binding was discovered. CD19 binding (B cells), however, was significantly different only from the non-specific binding in the middle layer. The average CD2 binding was, in accordance to the microscopic lymphocyte count, highest in the middle layer, although no significant difference was found between the layers (figure 3). The additional antibody tests by means of flowcytometric assessment on whole blood samples showed a clearly detectable CD11b expression in blood before filtration, whereas after filtration the CD11b expression was below the detection limit. The ced