METHODS AND SYSTEMS FOR THE DETECTION AND REMOVAL OF PATHOGENS FROM BLOOD

Abstract

The invention relates to methods and systems for removal of pathogens from blood or blood products. The invention further relates to methods and systems for treatment and diagnosis of infection in the blood and/or sepsis in a patient in need thereof.

Claims

1. A method for detecting pathogens selected from the group consisting of a bacterium, a virus, and a fungus in a sample of blood or of a blood product comprising red blood cells (RBCs), the method comprising: i) contacting said sample with a leukocyte reduction filter or with a support selected from the group consisting of a filter, microfibers, microparticles, beads, microspheres, an array, glass slides and microscope slides, said support comprising a non-specific RBC adhesion molecule binder, wherein said non-specific RBC adhesion molecule binder is a polymer or a mixture of two or more polymers that binds to adhesion molecules that become activated and are expressed on RBCs and is selected from one or more of the group consisting of polyester, polyurethane, cellulose, nylon, nitrocellulose, polypropylene, polyethylene terephthalate (PET), polyimide (PI), polyvinyl alcohol (PVA), and polyvinylidene fluoride (PVDF), and mixtures thereof, to allow binding of RBCs that have bound at least one pathogen to said leukocyte reduction filter or said support; ii) detecting the presence of RBCs that have bound at least one pathogen.

2. The method according to claim 1 further comprising separating said sample into at least two components prior to step i), a first component comprising red blood cells (RBCs) and substantially free of white blood cells (WBCs) and platelets and a second component comprising WBCs and platelets and substantially free of RBCs.

3. The method according to claim 1 further comprising isolating RBCs that are bound to said leukocyte reduction filter or said support from said sample, preferably by removing cells and other components in said sample that have not bound to said leukocyte reduction filter or said support.

4. The method according to claim 3, further comprising lysing said RBCs that are bound to leukocyte reduction filter or said support following said isolation step.

5. The method according to claim 1, further comprising determining at least one characteristic of said pathogens if RBCs that have bound at least one pathogen are detected in said sample, wherein said characteristic is selected from the group consisting of the type of pathogen, gram-positivity or gram-negativity if said pathogen is a bacterium, drug or antibiotic resistance of the pathogens, and the identity of the pathogens.

6. The method according to claim 1, wherein said support comprises beads coated with said non-specific RBC adhesion molecule binder.

7. The method according to claim 1 wherein said sample is a sample from an individual having an infectious pathogen in the blood, an individual suspected of having an infectious pathogen in the blood, an individual suffering from sepsis, an individual suspected of suffering from sepsis, an individual at risk of suffering from sepsis.

8. The method according to claim 1, further comprising identifying pathogens in the sample of blood or the blood product comprising red blood cells (RBCs), the method comprising isolating RBCs that are bound to said leukocyte reduction filter or said support from said sample, preferably by removing cells and other components in said sample that have not bound to said leukocyte reduction filter or said support; and identifying the pathogen that is bound to said RBCs bound to said leukocyte reduction filter or said support.

9. The method according to claim 1 for diagnosing an infection in the blood of a patient.

10. The method according to claim 1 for isolating and/or enriching pathogens selected from the group consisting of a bacterium, a virus, and a fungus from a sample of blood or of a blood product comprising red blood cells (RBCs), the method further comprising isolating RBCs that are bound to said leukocyte reduction filter or said support from said sample, preferably by removing cells and other components in said sample that have not bound to said leukocyte reduction filter or said support.

11. The method according to claim 1 for determining susceptibility for antibiotics of a pathogen, the method comprising: isolating RBCs that are bound to said leukocyte reduction filter or said support from said sample; contacting said isolated RBCs that are bound to said leukocyte reduction filter or said support with one or more antibiotic agents; and determining the growth or a functional activity of the pathogen.

12. A system (1) for removing a pathogen from blood or a blood product, comprising: a separation device (2), preferably comprising a centrifuge, for separating said blood or said blood product into at least a first component comprising red blood cells (RBCs) and being substantially free of white blood cells (WBCs) and platelets and a second component comprising white blood cells (WBCs) and platelets and being substantially free of red blood cells (RBCs); a filter device (3) for depleting said first component substantially of RBCs that have bound at least one pathogen, said filter device comprising a non-specific RBC adhesion molecule binder, wherein said non-specific RBC adhesion molecule binder preferably is a polymer; a conduit (4) for conducting said first component from the separation device (2), e.g. from a first outlet thereof (2b), to the filter device (3), e.g. to an inlet thereof (3a); an outlet conduit (10) for conducting the filtered first component, which is substantially depleted of RBCs that have bound at least one pathogen, away from said filter device (3), wherein the system (1) is arranged to feed the filtered first component, which is substantially depleted of RBCs that have bound at least one pathogen, to a subject (9) and/or to an administering device (6b) for administering at least said filtered first component to a subject (9).

13. A system (1) according to claim 13 further comprising: a second component outlet conduit (11) for conducting the separated second component away from the separation device (2), said second component outlet conduit (11) being in fluid connection with a second outlet (2c) of the separation device (2) for letting the second component out of the separation device (2), wherein the system (1) is arranged to put the filtered first component and at least part of the second component of the blood or blood product together in order to obtain a blood or blood product substantially free of pathogens, or at least substantially free of RBC bound pathogens, preferably wherein the outlet conduit (10) for conducting the filtered first component away from the filter device (3) merges with the second component outlet conduit (11), such that the filtered first component and the second component can join, and can preferably mix.

14. A system (1) according to claim 13, further comprising a mixing chamber (12) or another mixing unit (12) for mixing said second component with said filtered first component.

15. A system (1) according to claim 12, further comprising an anticoagulant supply unit (8) for adding an anticoagulant to the blood or the blood product, wherein said anticoagulant supply unit (8) is preferably situated upstream of the separation device (2), and more preferably is arranged for feeding the anticoagulant into a supply conduit (5) for supplying the blood or blood product to the separation device (2).

16. An apparatus for removing a pathogen from blood or a blood product comprising a housing (15) and a system (1) according to claim 12.

17. A method for determining the presence of pathogens in a whole blood sample of an individual suspected of having an infectious pathogen in the blood, suffering from sepsis, suspected of suffering from sepsis or at risk of developing sepsis, the method comprising detecting the presence of red blood cells (RBCs) that have bound at least one pathogen using cytometry or microscopy.

18. The method according to claim 17, wherein said detecting step is carried out by flow cytometry whereby RBCs that have bound at least one pathogen are detected by cell sorting for RBCs and labelling of pathogens.

19. The method according to claim 18, wherein said labelling of pathogens comprises DNA labelling.

20. The method according to claim 18, wherein the method comprises determining the presence of bacteria and said labelling comprises labelling with a general bacteria label.

21. The method according to claim 17, wherein said individual is suspected of having an infectious pathogen in the blood, suspected of suffering from sepsis or at risk of developing sepsis.

Description

[0104] By way of non-limiting examples only, embodiments of the present invention will now be described with reference to the accompanying figures in which:

[0105] FIGS. 1A-1F show schematic plots of bacterial binding to RBCs;

[0106] FIG. 2 shows images of a RBC carrying a bacterium on its membrane and transferring this to a monocyte under static conditions;

[0107] FIG. 3 shows images of transfer of bacteria to splenic monocytes and macrophages under flow;

[0108] FIG. 4 shows a bacterial transfer from RBCs to monocytes under flow and the importance of RBC adhesion molecules in this process;

[0109] FIG. 5 shows capturing of RBCs carrying bacteria by a non-specific RBC adhesion molecule binder (a leukocyte reduction filter);

[0110] FIG. 6 shows a schematic view of a first embodiment of a system according to an aspect of the invention;

[0111] FIG. 7 shows a schematic view of a second embodiment of a system according to an aspect of the invention;

[0112] FIG. 8 shows a schematic view of a third embodiment of a system according to an aspect of the invention;

[0113] FIG. 9 shows a schematic view of a fourth embodiment of a system according to an aspect of the invention;

[0114] FIG. 10 shows plating of isolated microorganisms after lysis of RBC-pathogen complexes;

[0115] FIG. 11 shows microscopic images of patient samples;

[0116] FIG. 12 shows flow cytometric analysis of RBCs from patient samples stained with Hoechst DNA dye.

[0117] It is noted that the figures, especially FIGS. 6-9, show merely preferred embodiments according to the invention. In the figures, the same or similar reference signs or numbers refer to equal or corresponding parts.

[0118] FIG. 1 shows that microbial binding is dependent on complement and CR1. FIGS. 1A-1E: flow cytometric analysis of microbe binding to RBCs. After incubation with a serum-treated microbe, RBCs were analysed by flow cytometry. FIG. 1A-E represent dot-plots of RBCs (P1). FIG. 1A. control RBC. P2 shows the % of RBCs that have bound opsonized S. aureus (FIG. 1B), S. typhimurium (FIG. 1C), C albicans (FIG. 1D) or E. coli (FIG. 1E). FIG. 1F: microbial binding to RBCs is strongly enhanced by opsonisation and is dependent on complement. Prior to incubation with RBCs, a fraction of S. aureus was treated with 50% human AB pool serum for 30 min at 37° C. Next, a binding experiment was performed. RBCs were analysed by flow cytometry. Bacterial binding was measured as % of GFP-positive RBCs. In case S. aureus was treated with serum, a high increase in binding to RBCs was observed in comparison with non-opsonized bacteria. Moreover, a very low binding was detected when bacteria were pre-incubated with heat-inactivated (HI) serum. Bacterial binding was completely abolished when RBCs were pre-incubated with CR1 blocking antibody. These findings show that complement found in serum and CR1 on RBCs are necessary to ensure bacterial binding to RBCs. n=7+/−SEM.

[0119] FIG. 2 shows the transfer of S. aureus to monocytes. Live imaging of GFP-expressing S. aureus bound to RBCs added to freshly isolated human monocytes reveals bacterial transfer from the erythrocyte to the phagocyte under static conditions. Images 1-3 of FIG. 2 show RBC carrying a bacterium on its membrane in close proximity to a monocyte. In images 4-5 of FIG. 2, bacterial transfer is seen. Image 6 of FIG. 2 shows that the bacterium is lost from the RBC and delivered to the monocyte.

[0120] FIG. 3 shows transfer of S. aureus and S. typhimurium to splenic monocytes and macrophages. Monocytes (CD163+) and macrophages (CD163+ and auto fluorescent) are selected to perform live imaging of bacterial transfer using GFP-expressing S. aureus (FIG. 3A-B) or S. typhimurium (C-D) bound to RBCs under flow conditions. Bacterial transfer to the phagocytes of the RES is shown.

[0121] FIG. 4 shows blocking adhesion molecules on RBCs inhibits bacterial transfer. Monocytes were isolated from whole blood and seeded on a 0.4 uM Ibidi flow chamber. RBCs with bound S. aureus on their surface were further treated with Lu/BCAM, CD147 or ICAM-4 blocking antibody. Next the RBCs were flown over the monocytes at the speed of 4.5 ml/hr (0.2 dyn) and tracked in time. The aforementioned antibodies used induced a significant reduction of bacterial transfer. When combined, no additional reduction was seen. Glycophorine A (GPA) blocking antibody was used as a control, showing that a reduction in bacterial transfer is not induced when using a random antibody against an abundant antigen on the RBC membrane.

[0122] FIG. 5 shows RBC carrying opsonized S. aureus can be captured from blood by a non-specific RBC adhesion molecule binder. FIG. 5A: a RBC suspension containing C. albicans carrying RBC was flown over a leukocyte reduction filter. Shown here, are the FACS plots of the RBC suspension before and after filtration. FIG. 5B: a RBC suspension containing S. aureus carrying RBC which was flown over a leukocyte reduction filter. Shown here are the FACS plots of the RBC suspension before and after filtration.

[0123] FIG. 10 shows plating of isolated microorganisms after lysis of RBC-pathogen complexes. In a the fraction of a control RBC suspension is plated, in b, c, and d the fractions of C. albicans, S. aureus and E. faecalis are plated, respectively.

[0124] FIG. 11 shows microscopic images of patient samples. FIG. 10A: Sample of a patient suffering from K. pneumoniae induced sepsis, arrow Indicates K. pneumoniae bound by a RBC; FIG. 10B: Sample of a patient suffering from E. coli induced sepsis, arrow indicates E. coli bound by a RBC, stained by Hoechst DNA dye.

[0125] FIG. 12 shows flow cytometric analysis of RBCs from patient samples stained with Hoechst DNA dye. FIG. 11A Red blood cell sample of a healthy control, showing no DNA staining; FIG. 11B: Red blood cell sample of the septic patient depicted in FIG. 10B, showing DNA positive RBC (RBC gated on FSC and SSC; positive signal in 405 channel), indicative for RBC-pathogen complexes.

[0126] FIGS. 6-9 show schematic views of different embodiments of a system 1 according to an aspect of the invention. Said system 1 is a system 1 for removing a pathogen from blood or a blood product, preferably from blood of patient in need of such removal. The system 1 comprises a separation device 2 for separating said blood or said blood product into at least a first component comprising RBCs and being substantially free of WBCs and platelets and a second component comprising WBCs and platelets and being substantially free of RBCs.

[0127] In embodiments, the separation device 2, which can be or include an apheresis unit for depleting said blood or said blood product substantially of WBCs and platelets, can for instance be or comprise a centrifuge. However, the separation device 2 can comprise or be formed by any other separation device suitable for separating the WBCs and platelets from the RBCs. An alternative separation device may be formed e.g. by a counterflow device, a centrifugation elutriation device, a size filtration device, for instance comprising one or more membranes or so-called sieve membranes, an affinity chromatography device, and/or a device combining two or more of the techniques utilized by the fore-mentioned devices. Said separation device preferably comprises a centrifuge. It is noted that said separation device 2, e.g. said centrifuge, may comprise at least two compartments to keep the separated components separated. For example, the components can be stored temporarily in the respective compartments and/or the respective components can be taken from the separation device 2 by removing them from said compartments.

[0128] The system 1 further comprises a filter device 3 for depleting said first component of RBCs that have bound at least one pathogen. Said filter device 3 comprises a non-specific RBC adhesion molecule binder.

Preferably, said non-specific RBC adhesion molecule binder is a polymer, more preferably a polymer selected from a group consisting of polyester, polyurethane, cellulose, nylon, nitrocellulose, polypropylene, polyethylene terephthalate, polyimide, polyvinyl alcohol, and polyvinylidene fluoride.

[0129] It is noted that the system 1 can preferably be a closed sterile system. Although said system 1 can comprise individual elements or devices 2, 3, said system 1 or a part thereof can preferably be formed as an apparatus comprising at least some of the elements or devices 2, 3 of the system 1. For example, at least some, preferably all devices, of the system can be integrated into an apparatus for removing a pathogen from blood or a blood product. For example, such apparatus may comprise a frame and/or a housing 15 on and/or in which at least some of the elements or devices of the system 1 are situated. For example, an apparatus can comprise a housing 15 accommodating at least the separation device 2 and the filter device 3.

[0130] Advantageously, the system 1 can further comprise a conduit 4 for conducting said first component from the separation device 2, e.g. from a first outlet 2b thereof, to the filter device 3, e.g. to an inlet 3a thereof. For example, said first component can be conducted directly from the separation device 2 to the filter device 3. However, the system 1 may alternatively or additionally be arranged to store at least a part of the first component temporarily before it is fed to the filter device 3. In embodiments, a first component storage container may thereto be provided downstream the separation device 2 and upstream of the filter device 3.

[0131] Further, as for instance can be seen in the exemplary embodiment of FIG. 9, the separation device 2 can have an inlet 2a for letting the blood or the blood product into the separation device 2, which inlet 2a may be connected to a blood or blood product supply conduit 5 for supplying the blood or blood product to the separation device 2, e.g. from container 7 or from a subject 9.

[0132] In embodiments, such as for instance in the embodiment of FIGS. 7 and 8, the system 1 may be arranged to conduct blood from a subject 9, such as for instance a patient, to the separation device 2. For example, the supply conduit 5 can therefore be provided with a blood collecting device, e.g. being or comprising a needle 6, such as for example a conventional apheresis needle. Hence, the separation device 2 may be arranged to be brought in fluid connection with a subjects bloodstream.

[0133] Alternatively or additionally, as for instance shown in FIG. 9, the system 1 may comprise, and/or may be arranged to be connected to, an initial blood or blood product container 7 for holding initial blood or an initial blood product from which RBC that have bound pathogens are to be removed. Said container 7 can for instance be filled with donor blood or with a blood sample or blood product sample which is to be filtered, for instance in order to remove pathogens, or at least RBC that have bound pathogens, and/or in order to obtain a substantially pathogen free blood or blood product and/or to obtain filtered out RBC that have bound pathogens, e.g. in order to enable executing tests on said pathogens.

[0134] It is noted that the system 1 may comprise an anticoagulant supply unit 8 for adding an anticoagulant to the blood or the blood product. Said anticoagulant supply unit 8 can preferably be situated upstream of the separation device, and more preferably can be arranged for feeding the anticoagulant, especially in a dosed and/or controlled manner, into a supply conduit 5 for supplying the blood or blood product to the separation device. Optionally, the anticoagulant supply unit 8 can comprise an anticoagulant supply conduit 8a, e.g. for feeding the anticoagulant to said blood or blood product supply conduit 5. It is noted that the system 1, e.g. its anticoagulant supply unit 8 and/or its anticoagulant supply conduit 8a, may comprise a dosing unit for dosing the anticoagulant. For example, the anticoagulant can be stored in an anticoagulant reservoir 8b, which can be part of said supply unit 8, and can be fed from said reservoir, via an anticoagulant supply conduit 8a, to the blood or blood product. Additionally or alternatively, the anticoagulant supply unit 8 may comprise an anticoagulant pump for pumping said anticoagulant to the blood or blood product.

[0135] Further, the system 1 may comprise a mixing chamber or other mixing means for mixing the anticoagulant with the blood of blood product.

[0136] It is noted that the separation device 2 of the system 1 may have a first outlet 2b for letting the first component, which is separated from the second component, out of the separation device 2. Preferably, said first outlet 2b can be in fluid connection with said filter device, e.g. it can be connected to an inlet of the filter device 3 by means of a conduit 4 for conducting said first component from the separation device 2 to the filter device 3.

[0137] Furthermore, it is noted that the system 1 may for example be arranged such that the system 1 can feed the filtered first component from the filter device 3 to a storage container 13. Alternatively or additionally, the system 1 may be arranged to feed the filtered first component directly to a subject 9 and/or to an administering device 6b for administering at least said filtered first component to a subject 9, preferably the subject from which the initial blood or blood product was taken. However, the blood or blood product substantially freed of pathogens may also be administered to another subject, e.g. after storing it temporarily.

[0138] Additionally or alternatively, the separation device 2 may have a second outlet 2c for letting the second component out of the separation device 2. In embodiments, said second outlet 2c can be in fluid connection with a second component outlet conduit 11, e.g. for conducting the separated second component away from the separation device 2. Although the second component comprising WBCs and platelets may in embodiments be stored, e.g. in a container for storing the second component, and/or administered without being combined with the filtered first component, or may even be disposed, the second component can advantageously be put together with the filtered first component, e.g. before storing them together and/or before administering them together to a subject. Preferably, said filtered first component and said second component can be put together, and preferably can be mixed, in a ratio corresponding to the ratio of the first component and second component initially present in the initial blood or blood product. It may be apparent that further, in case the initial blood or blood product was separated in more then two components, one or more, for instance all, of such further components can be put together with the filtered first component and the second component as well.

[0139] In advantageous embodiments, an outlet of the filter device 3 can be connected to an outlet conduit 10 for conducting the filtered first component, which is depleted of RBCs that have bound at least one pathogen, away from said filter device 3, e.g. in order to store it, to administer it and/or to put it together with at least the second component.

[0140] Preferably, the system 1 can be arranged to put the filtered first component and the second component of the blood or blood product together in order to obtain a blood or blood product substantially free of pathogens, or at least substantially free of RBC bound pathogens. For instance, as can be seen in the exemplary embodiments of FIGS. 6-9, the outlet conduit 10 for conducting the filtered first component away from the filter device 3 can merge with the second component outlet conduit at a certain point, such that the filtered first component and the second component can join, and preferably mix. In embodiments, such as for example the embodiment of FIG. 9, the system 1 may further comprise a mixing chamber or another mixing unit 12 for mixing said second component with said filtered first component to obtain a blood or blood product comprising RBCs and WBCs and platelets and being substantially free of RBC bound pathogens. Subsequently, said blood or blood product can be stored, e.g. in a container 13, for instance such as shown in the embodiment of FIG. 9. Alternatively, said blood or blood product can be fed, e.g. substantially directly, to a subject 9, e.g. by means of a supply conduit 14, which may be connected to an administering device 6b, such as an administering needle 6b. Hence, the first component that is depleted of RBCs that have bound at least one pathogen, combined with the second component can be fed to a subject, preferably returned to the subject from which the initial blood was taken.

[0141] Although the system 1, as for instance can be seen in the exemplary embodiment of FIG. 7, may for instance be arranged for continuous treatment and/or can preferably comprise a dual needle 6a, 6b configuration, the system 1 may alternatively, or additionally, be arranged for performing a cyclical treatment.

[0142] In the embodiment of FIG. 8, blood is removed from a subject, e.g. a patient, in blood draw cycles. Blood removed in each cycle is processed batch wise in the system 1. Here, the processed blood is optionally collected in a container or so-called reservoir 13. Said processed blood or component is also returned to the subject 9 in cycles. Draw and return cycles are sequentially repeated during a selected period of time, whereby blood and/or blood components can for instance be cyclically removed from a subject, cyclically accumulated in the reservoir 13 and/or cyclically returned to the subject.

[0143] As can be understood from FIG. 8, blood can be removed from the subject 9 and can, e.g. via a manifold 16, preferably a controllable manifold, be conducted to the separator device 2. As mentioned before, an anticoagulant can be added to said blood, e.g. by means of an anticoagulant supply unit 8. The first component, substantially free of WBCs and platelets can then be depleted of RBC that have bound pathogens by means of the filter device 3. Downstream of the filter device 3, the first component substantially freed of RBC that have bound pathogens can then be combined with the second component, e.g. in a mixing unit 12 or mixing chamber, not shown in FIG. 8, but shown included in the embodiment of FIG. 9. As further can be understood from FIG. 8, the combined filtered first component and the second component can then be stored in the reservoir or container 13. After a while, the system 1 can stop drawing blood from the subject 9 and subsequently the system 1 may start feeding the filtered first component combined with the second component back to the subject 9. Thereto the manifold 16 may for instance be switched and/or a pump for drawing blood through the blood supply conduit 5 towards the separation device 2 can stop pumping and a pump for pumping blood or blood product from the reservoir 13 towards the subject 9 can start pumping said blood or blood product to the subject 9.

[0144] It is noted that system 1 may comprise one or multiple pumps (not shown). The pump or pumps can for instance be for drawing the blood or blood product, for instance from the subject 9 or patient or from an initial blood or blood product container 7, such as a blood bag, into and/or through the blood or blood product supply conduit 5 and/or to the separation device 2. One or more pumps may alternatively or additionally be for feeding the first component to the filter device 3 and/or for pumping and/or drawing the first component through the filter device 3. Alternatively or additionally, one or more pumps may be provided for pumping the second component from the separation device 2 and/or the filtered first component from the filter device 3 to a mixing unit 12, a storage reservoir 13, an administering device 6b, 6 and/or a subject. It is noted that one or more pumps can be arranged to perform multiple of the tasks mentioned above.

[0145] It is noted that for the purpose of clarity and a concise description features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments, for example methods and/or systems, having combinations of all or some of the features of embodiments described and/or shown, for example embodiments of methods and/or systems described and/or shown.

[0146] Further, it is noted that the invention is not restricted to the embodiments or examples described herein. It will be understood that many variants are possible.

[0147] For example, one or multiple of the conduits of the system may be provided as tubes, e.g. flexible tubes. Especially the blood or blood product supply conduit 5 and/or a supply conduit 14 for feeding cleaned blood or a cleaned blood product to e.g. a reservoir 13 or a subject 9, can be of flexible design. Said conduit or conduits may for instance be connected and/or releasably connectable to a housing 15 of the system 1.

[0148] As another example, the system 1 may be provided with one or more monitoring mechanism and/or control mechanism. For example, the system can comprise one or multiple pressure sensors, temperature sensors, air-bubble detectors, flow sensors, and/or other sensors or detectors. The system 1 may additionally be arranged to provide a warning signal or to even stop a process, e.g. a treatment process, being executed, if one or more variables being monitored exceed a predetermined threshold value. Alternatively or additionally, the system 1 may be arranged to control one or more of such variable. For instance thereto, the system may comprise means for adjusting one or more of such variables. For example, the system may be provided with a warmer and/or a cooler to warm and/or cool the blood, blood product and/or one or more separated components thereof. Hence, when for instance a too low temperature is detected, the system may control the warmer to warm the respective blood, blood product and/or component, e.g. in order to maintain a temperature substantially corresponding to a blood temperature. As another example, if, at a certain point in the system 1, the respective monitored flow rate drops below a certain threshold value, the system may for instance increase the output of a respective pump and/or may for instance adjust the amount of anticoagulant supplied to the blood or blood product. Further, the system 1 may comprise one or more input means, e.g. comprising a control panel, to operate at least parts of the system and/or to input desired threshold values.

[0149] Such and other variants will be apparent for the person skilled in the art and are considered to lie within in the scope of the invention as formulated in the appended claims.

Experimental Part

Materials and Methods

Antibodies and Reagents

[0150] Anti-hBCAM (polyclonal goat IgG; catalog# AF148) was used from R&D systems (Minneapolis, USA). Anti-hCD147, (conjugate PE, Mouse IgG2A product code12-1472-41) was used form eBioscience. ICAM-4 (polyclonal, product code: H00003386-B01P) was used form Abnova. Anti-hCD163 (clone MAC2-158, conjugate PE) from Trillium Diagnostics, LLD was used. Final used blocking concentrations are: 0.2 mg/ml end concentration per 1×10{circumflex over ( )}7 RBCs 30 min RT for anti-hLu/BCAM, 50 ug/ml end concentration per 1×10{circumflex over ( )}7 RBCs 30 min RT for anti-ICAM-4, 20 ug/ml end concentration per 1×10{circumflex over ( )}7 RBCs 30 min RT for anti-CD147.

[0151] Isolation and Storage of RBCs

[0152] Venous blood was collected from healthy donors, after obtaining informed consent. Blood studies were approved by the Sanquin Research institutional medical ethical committee in accordance with the standards laid down in the 1964 Declaration of Helsinki. Erythrocytes were isolated from fresh heparinized whole blood by centrifugation at 270 g for 15 min. After removing the platelet-rich plasma and the peripheral blood mononuclear cells, the erythrocytes were washed two times with saline-adenineglucose-mannitol (150 mmol/l NaCl, 1.25 mmol/l adenine, 50 mmol/l glucose, 29 mmol/l mannitol, pH 5.6) (SAGM) (Fresenius Kabi, The Netherlands), and resuspended in SAGM. Final cell concentration was determined with an Advia 2120 (Siemens Medical Solutions Diagnostics, Breda, The Netherlands). The erythrocytes were stored at 2 to 6° C. in a standard blood bank refrigerator.

[0153] Bacterial Binding to RBCs

[0154] For bacteria (or other microorganisms) to bind to RBCs, the bacteria first needed to become opsonized. This was done by incubating 1×10{circumflex over ( )}8 bacteria with 200 ul of pooled serum of 18 AB+ healthy donors. After a washing step with Hepes buffer [132 mmol/l NaCl, 20 mmol/l HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), 6 mmol/l KCl, 1 mmol/l MgSO4. 1.2 mmol/l K.sub.2HPO.sub.4, pH 7.4 (all from Sigma-Aldrich)] supplemented with 10 mmol/l glucose, 2 mmol/l CaCl2 and 0.5% HSA, the now opsonized bacteria were incubated with 0.1% PFA for 30 minutes followed by 2 times washing in Hepes buffer. Next, the opsonized bacteria were incubated with 1×10{circumflex over ( )}8 RBCs for 30 min. again following a 2 step washing step. Some bacteria needed additional staining using a DNA-dye such as Hoechst to allow analysis of the bacterial binding percentage. RBCs were analysed and checked for bacterial binding % by flow cytometry using a LSR II (Becton Dickinson).

[0155] Monocyte Isolation Using Percoll Gradients and CD14+ Bead Isolation

[0156] Polymorphonuclear cells (PBMC) were isolated from heparinized peripheral blood from healthy donors by density gradient centrifugation using Percoll (Pharmacia). Monocytes were isolated from PBMC by MACS isolation using CD14 microbeads (Miltenyi Biotec). No stimulation was needed for bacterial transfer analysis.

[0157] Human Spleens

[0158] Spleens were collected from organ transplant donors without clinical signs of infection or inflammation. Written informed consent for organ donation was obtained according to national regulations regarding organ donation. Splenic tissue of the organ donor was obtained during transplantation surgery, as part of the standard diagnostic procedure for HLA-typing, and was transported in University of Wisconsin Fluid at 4° C. In case there was an excess of splenic tissue for diagnostic procedures, this excess of splenic tissue was used in an anonymous fashion for research in the present study, in accordance with the Dutch law regarding the use of rest material for research purposes.

[0159] Isolating Splenocytes

[0160] Splenocytes were isolated as described elsewhere (Nagelkerke et al. PLoS One. 2014 Feb. 11; 9(2)) by Injecting a piece of spleen at several sites with collagenase buffer Collagenase CLSP 100 U/ml, DNAse, Deoxyribonuclease I, bovine recombinant 2 Kunitz Units/ml, Aggrastat 0.5 ug/mL, Glucose 1 mg/ml, Calcium Chloride 1 mM. Connective tissue was removed and the tissue was subsequently incubated in the collagenase buffer for 30 minutes at 37° C. Tissue was then filtered using a 100 μm filter. Subsequently, erythrocytes were lysed with an isotonic ammoniumchloride buffer for 5 minutes at 4° C., after which lysis buffer was washed away. To enrich for larger cells (monocytes/macrophages) elutriation is performed. Finally, cells were sorted directly from splenocytes stained for CD163 (a monocyte/macrophage marker) and auto fluorescence (specific for macrophages only) using a FACS Aria II machine (Becton Dickinson). Flow cytometric analysis was performed on an LSR II machine (Becton Dickinson).

[0161] Confocal Microscopy

[0162] In this method, monocytes or macrophages were isolated (as described) and plated on an Ibidi μ-Slide (Ibidi-Treat p-Slide VI 0.4, 6 channels) made of a polymer that supports rapid adhesion. In each well 33 ul HEPES buffer [132 mmol/l NaCl, 20 mmol/l HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), 6 mmol/l KCl, 1 mmol/1 MgSO4, 1.2 mmol/l K.sub.2HPO.sub.4, pH 7.4 (all from Sigma-Aldrich)] supplemented with 10 mmol/l glucose, 2 mmol/l CaCl2 and 0.5% HSA as the positive sample, or HEPES buffer supplemented with 10 mmol/l glucose, 2 mmol/l ethylene glycol tetraacetic acid (EGTA) (Sigma-Aldrich) and 0.5% HSA as the negative sample, was injected containing 80.000 monocytes/macrophages. Next, the ibidi-chamber was placed in an incubator for 30 min at 37° C. After 30 min, the ibidi-slide was positioned into the microscope stage and connected to one side of silicone tubing containing warm (e.g., 37° C.) flow-buffer (HEPES+), and a 20 ml syringe. NOTE: This tubing also contains an in-line Luer injection port (IBIDI, Catalog Number: 10820), which allows erythrocytes to be injected with a needle into a running experiment without stopping the flow and creating air bubbles. Next, the silicone exiting tube was connected to the other side of the ibidi-slide. The flow speed is adjusted to 0.2 dyn/cm.sup.2, in accordance with physiological flow speed in splenic venules (0-1 dyn/cm.sup.2). The pump will now push the flow-buffer from the syringe through the flow-chamber into the exiting tube. Fluorescent imaging of the erythrocytes (to which IC are bound) enable real-time determination of the extent of bacterial transfer to monocytes/macrophages. To quantify results in each bacterial transfer experiment the number of monocytes that had taken up one or more immune complexes, was counted throughout the whole length of an ibidi-slide when positioned in center of the well. Results were obtained using Differential Interference Contrast (DIC), GFP (488 nm) or Hoechst (405 nm) simultaneously using a confocal laser scanning microscope equipped with a climate chamber with a constant temperature at 37° C., 5% CO.sub.2, and a 63X oil-objective. Data is analyzed using imaging software (Zen software 2008 and Zeiss LSM510 META; Carl Zeiss MicroImaging, Jena, Germany).

[0163] Depletion of RBCs Carrying an Opsonized Pathogen by the Use of a Non-Specific RRC Adhesion Molecule Binder.

[0164] RBCs to which bacteria (or other microorganisms) already have been bound are analysed by FACS to determine the pathogen binding percentage (as described earlier in materials and methods: Bacterial binding to RBCs). Next, the non-specific RBC adhesion molecule binder, in this case a Leukocyte Reduction Filter (LRF, BioR Blood filter, Fresenius Kabi, The Netherlands) is prewashed using 150 ml of SAGM. After which, 30 ml of SAGM is added containing the RBCs that have bound GFP+ or Hoechst+-expressing immune complexes. The filter is then washed twice with 150 ml of SAGM, all of which Is collected, by centrifugation at 2500 RPM for 5 min. The obtained RBCs were analysed and checked for bacterial binding % by flow cytometry using a LSR II.

[0165] Isolation of Pathogens Bound by a Non-Specific RBC Adhesion Molecule Binder

[0166] To filter RBC-pathogen complexes from a suspension of RBC, the leucocyte reduction filter was washed first with 150 ml of SAGM (RBC storage solution). Then a suspension of 10.sup.8 RBC containing RBC-pathogen complexes (prepared as described before in bacterial binding to RBC section) was applied to the filter. The percentage of RBC-pathogen complexes ranged per pathogen, S. aureus 39%, E. faecalis 12,5%. C. albicans 0.9%. After the application of the RBC, the filter was washed with 100 ml of SAGM. Subsequently, 100 ml of water was applied, in order to lyse the RBC, of which the first 50 ml was collected directly. The remaining 50 ml was left 10 minutes on the filter, in order to lyse the RBCs completely, and then obtained from the filter. The first 50 ml and the 50 ml of the second elution were both spun down, taken up in 100 ul of LB medium and plated. As a control, RBCs without any pathogens were also subjected to the same filter isolation procedure.

[0167] Detection of Pathogen Bound RBCs in Patient Samples

[0168] Blood from septic patients or from patients at risk of developing sepsis was first washed three times with PBS, and then stained by a DNA dye (Hoechst, 1:10.000, 15 min, RT), and washed twice. The sample was then analyzed by flow cytometry or confocal microscopy.

[0169] Statistical Analysis

[0170] Data was analyzed using Graphpad Prism 6 for Windows (GraphPad Software, La Jolla, Calif., USA). For statistical analysis between experimental groups, the Student's t-test was used. A two-sided p value of ≤0.05 was considered to be significant. Unless stated otherwise, a representative experiment out of at least three independent experiments is shown.

[0171] Results

[0172] Bacterial Binding is Dependent on Complement and CR1

[0173] Previously it has been shown that CR1 (also known as CD35 and the Knops blood group antigen) located on human Red blood cells (RBC) is the receptor that is responsible for binding newly formed immune complexes (IC). The RBC now acts as a shuttle, transferring the now bound IC throughout the whole body until it arrives at the liver or the spleen, where its cargo is selectively removed by macrophages of the Reticulo-Endothelial System (RES). To study this phenomenon, an assay was developed to monitor binding of opsonized GFP positive S. aureus to RBCs (FIG. 1A-B). To perform this assay, S. aureus was first incubated with serum and then incubated with RBCs for 30 min. RBCs were then analyzed by flow cytometry FIG. 1A represents the dot-plot of control RBCs (P1). P2 shows the % of GFP-positive RBCs or RBCs that have bound GFP-expressing opsonized S. aureus (FIG. 1B). To asses if other microorganisms are also able to bind to CR1 through complement, a series of experiments have been performed using the same protocol but with different microorganisms: E. coli, C. albicans and S. Typhimurium. All described microorganisms were indeed able to bind, although a variable binding percentage between different micro-organisms is observed (FIG. 1C-1E) Next, to see if bacterial binding is indeed dependent on complement and CR1 another binding assay was performed (FIG. 1F). Bacteria were again opsonized using pooled serum prior to incubation with RBCs. Bacterial binding was measured as % of GFP-positive RBCs. In case S. aureus was treated with serum, a high increase in binding to RBCs was observed in comparison with non-opsonized bacteria. Moreover, a very low binding was detected when bacteria were pre-incubated with heat-inactivated (HI) serum. Bacterial binding was completely abolished when RBCs were pre-incubated with CR1 blocking antibody. These findings show that complement found in serum and CR1 on RBCs are necessary to ensure bacterial binding to RBCs.

[0174] Bacterial Transfer to Human Monocytes

[0175] In most of experiments, human monocytes isolated from whole blood were used instead of phagocytes from spleen. This is because splenic phagocytes are difficult to obtain and have reduced viability at the end of the sorting procedure and sample collection and preparation. It is generally accepted that monocytes isolated from whole blood are the phagocytic precursors of the cells of the RES and therefore a lot of their receptors are believed to be common. To visualize bacterial transfer to human monocytes, monocytes were isolated according to protocol and seeded on a glass plate and submerged in HEPES+ buffer. After an incubation period of 30 min in a 37° C. incubator, a small amount of RBCs to which opsonized GFP-expressing S. aureus was already bound, was pipetted on top of the monocytes. Using confocal microscopy to analyze the results, bacterial transfer to monocytes was clearly seen (FIG. 2). Additional experiments using different microorganisms (E. coli, C. albicans, and S. Typhimurium) to show IC transfer were also performed, all showing transfer of the microorganisms to human monocytes.

[0176] Bacterial Transfer Underflow Conditions

[0177] To mimic a more lifelike situation, a flow system was developed in which the physiological flow speeds of the spleen are used, by means of confocal microscopy. Using this newly developed assay, but keeping all other features the same, bacterial transfer still occurred as normal (data not shown). In addition, a protocol was developed to Isolate human splenocytes. Once isolated, splenic monocytes (CD163+) and macrophages (CD163+ and auto fluorescent) are selected to perform live Imaging of bacterial transfer using GFP-expressing S. aureus (FIG. 3A-B) or S. typhimurium (C-D) bound to RBCs under flow conditions. Once again revealing bacterial transfer but this time to the phagocytes of the RES.

[0178] When looking at the flow assay results in more detail, it was noticed that just after bacterial transfer had occurred the RBC remains attached to the monocyte for a while. It was therefore hypothesized that RBC adhesion molecules are involved during the transfer process. RBC adhesion molecules are known to be calcium dependent. To test this hypothesis, several adhesion molecules were blocked under flow (FIG. 4). When using a blocking antibody against CD147 a significant reduction of about 50% Is seen. When blocking ICAM-4 and Lu/BCAM, a similar reduction is shown. As an extra control, an antibody against an abundant antigen on the red blood cell membrane (anti-GPA) is used that is not involved in IAC. As expected, no reduction in bacterial transfer was seen (FIG. 4). Next, al three adhesion molecules were blocked at the same time, to see if this reduces bacterial transfer even further. No additional reduction is seen when all Abs are combined instead of using just one. Nevertheless, the data show that IAC is dependent on RBC adhesion molecules.

[0179] RBCs Carrying an Opsonized Pathogen are Depleted Using a Non-Specific RBC Adhesion Molecule Binder.

[0180] RBCs to which bacteria (or other microorganisms) already have been bound are firstly analysed by FACS to determine the immune complex (IC) binding percentage (as described earlier in materials and methods: Bacterial binding to RBCs). Due to inter-donor variation bacterial binding percentages (or other microorganisms) differ ranging from about 5%-40%, as is apparent from FIG. 1. After washing the filter (as described earlier in materials and methods: Depletion of RBCs carrying an opsonized pathogen.) all non-sticking RBCs are obtained and again analysed by FACS. As shown in FIG. 5, a nearly total pathogen reduction is seen in both cases of C. albicans (FIG. 5A; depleted from 6.7% to 0.5%) and S. aureus (FIG. 5B; depleted from 19.5% to 0.7%.

[0181] Isolation of Pathogens Bound by a Non-Specific RBC Adhesion Molecule Binder

[0182] RBCs to which S. aureus, E. faecalis or C. albicans were bound were depleted using a BioR blood filter (Fresenius Kabi). Subsequently, RBC were lysed and the resulting samples containing the pathogens were plated. The results are shown in FIG. 10B-D. All three pathogens could be successfully plated. FIG. 10 A shows a control of RBC without pathogens.

[0183] Detection of Pathogen Bound RBCs in Patient Samples

[0184] The percentage of RBC-pathogen complexes in blood samples from septic patients ranged from 1 to 1.7% in the patients tested (table 1). Patients suffered from or were developing sepsis, e.g. Induced by K. pneumoniae (patient 2) or E. coli (patient 3). In healthy controls no RBC-pathogens complexes were observed. The samples of patients 1 and 3 showed a positive blood culture.

TABLE-US-00001 TABLE 1 Sample % RBC-pathogen complexes Patient 1 1 Patient 2 1.5 Patient 3 1.7 Healthy control 1 not detectable Healthy control 2 not detectable Healthy control 3 not detectable

[0185] FIG. 10 show the microscopic analysis and flow cytometric analysis for a patient suffering from a K. pneumoniae induced sepsis and a patient suffering from a E. coli induced sepsis. FIG. 11 shows the flow cytometric analysis of one patient developing a K. pneumoniae induced sepsis. The percentage of RBC-pathogen complexes in a sample obtained from this patient was 1.5% (FIG. 11B). Of note, this sample was taken 2 days before the patient had a positive blood culture and was diagnosed with sepsis. The presence of RBC-pathogen complexes was confirmed by confocal microscopy analysis (FIG. 1B) and a positive blood culture 2 days later.