Patent Application: US-201515511499-A

Abstract:
a system for intra - operative blood salvage autotransfusion is provided . the system comprises at least one inlet configured to receive whole blood of a patient ; at least one curvilinear microchannel in fluid flow connection with the at least one inlet , the at least one curvilinear microchannel being adapted to isolate circulating tumor cells in the whole blood , based on cell size , along at least one portion of a cross - section of the at least one curvilinear microchannel ; and at least two outlets in fluid flow connection with the at least one curvilinear microchannel , at least one outlet of the at least two outlets being configured to flow the circulating tumor cells isolated from the whole blood , and at least one other outlet of the at least two outlets being configured to flow at least a portion of a remainder of the whole blood , cleansed of the isolated circulating tumor cells , for return to the patient .

Description:
an embodiment according to the invention provides a passive multiplex inertial focusing microfiltration system to remove cancer cells from whole blood salvaged during cancer surgery . this novel microfiltration platform consists of multiple layer of pdms sheets embossed with curvilinear microchannels ( i . e ., ˜ 1 - 10000 spiral microchannels with trapezoidal or rectangular cross - section ) bonded together for continual - based cell separation from large volume of blood . this approach exploits the differences in the size of the cells to separate the larger cancer cells from the predominantly smaller blood cells . in accordance with an embodiment of the invention , there is provided a microfiltration system which can filter the tumour cells from blood salvaged during cancer surgery . the technique utilizes the inherent dean vortex flows present in curvilinear microchannels under continuous flow , along with inertial lift forces which focus larger cancer cells against the outer wall ( i . e ., where strong vortices exist ) while smaller hematologic cells ( white blood cells ( wbcs ), red blood cells ( rbcs ) and platelets ) remained unfocused in the entire channel and exist from both outlets . as the technique relies on high speed microfluidic dynamics , it is capable of achieving ultra - high throughput processing of one to two liters of blood under 30 min through a multiplexed approach of combining a number of these spiral microfluidics . in accordance with an embodiment of the invention , a multiplexed inertial focusing microfiltration system can replace ldf or iocs - ldf combination . this system can remove the tumour cells from the salvaged blood and make it safe to be returned to the patient . the technique utilises the inherent dean vortex flows present in curvilinear microchannels with trapezoid / rectangular cross - section under continuous flow , along with inertial lift forces which focus and trap larger cancer cells against the outer wall where strong dean vortex presents . smaller hematologic components remained unfocused in the channels and can be recovered from the both outlets ( see fig1 ) [ 9 , 10 ]. by re - circulating the blood through the channels a few times , the system can successfully remove most of the cancer cells while collecting the majority of vital blood components ( wbcs , rbcs and platelets ) to return to the patient . such transfusion will reduce the chance of transfusing allogeneic blood to the patients and abt associated risks . this may also indirectly address other abt related problems such as increased length of hospital stay , resource consumption , and hospital charges . fig1 is a schematic showing the top - view of a typical spiral micro - channel with trapezoidal cross - section ( left ), in accordance with an embodiment of the invention . the working principle of cancer cells separation from other blood components is depicted on the right side . at the inlet ( section a - a ), whole blood enter the microchannels and then under the influence of inertial and dean - drag forces , cancer cells which are typically larger than hematologic cells are trapped near the outer wall and exit from the outer outlet while smaller hematologic cells remain unfocused and exit from both outlets ( section b - b ). by repeating this procedure 3 times , majority of the blood cells (& gt ; 90 % rbcs , wbcs and platelets ) can be recovered . since processing of large volume of salvaged blood ( i . e ., 1 - 2 liters ) requires utilizing a large number of spiral channels , an embodiment according to the invention provides an approach to combine many of these spiral shape channels together to achieve higher throughput . for this purpose , an integrated microfluidic platform consists of multiple layers of polymeric sheets with embossed microchannels ( i . e ., 4 spiral microchannels with trapezoidal cross - section in each layer ) bonded together to make a multiplexed system with about 40 spirals (˜ 200 ml / min ). theoretically , we can stack higher number of layers together to achieve even higher throughputs . however , for the concept of blood purification , we have just bonded 10 layers to achieve flow rate of around 200 ml / min facilitating process of 1 l of whole blood in just 5 min ( see fig2 ). fig2 a is a photograph of a spiral microfluidic device with one inlet and two outlets , in accordance with an embodiment of the invention . the channels are filled with a red dye for visualization . the device is made of two pdms layers bonded via plasma . one of the layers that have spiral pattern is casted from a micro - milling aluminum mold . fig2 b is a schematic of the setup of a cyclic cell sorting system in accordance with an embodiment of the invention . whole blood containing cancer cell can be pumped into microfluidic device using a peristaltic pump . cancer cells will be separated from normal hematologic cells using hydrodynamic forces inside microchannels and clean blood can be returned to the patient after enrichment . fig2 c is an optical image of a high - throughput system consisting of multiple layers of polymeric sheets with embossed microchannels ( i . e ., 40 spiral microchannels with trapezoidal cross - section ) bonded together for continual blood purification from large sample volumes , in accordance with an embodiment of the invention . in accordance with an embodiment of the invention , in order to find the optimum channel design that gives the maximum ctc removal and blood components return , spiral microchannels with various dimensions were fabricated using conventional micro - milling and soft lithography techniques [ 11 ]. fig3 is a top - view microscopy image demonstrating the focusing behaviour of fluorescent particles as a function of flow rate inside spiral channels with different trapezoid cross - sections and fixed width , in accordance with an embodiment of the invention . two different surrogates ( 10 and 15 μm beads ) were used to mimic focusing position of wbcs and ctcs inside microchannels , respectively . fig3 presents fluorescent microscopy images demonstrating the focusing behavior of traceable particles ( nominal diameter of 15 , 10 μm , bangs laboratories , inc . usa ) as a function of flow rate inside spiral channels . the aim was to focus 15 μm beads ( resemble of cancer cells ) near the outer wall while keeping the 10 μm beads ( resemble of average hematologic cells ) dispersed in the entire channel ( see fig4 ). fig4 shows fluorescent microscopy images of a device in accordance with an embodiment of the invention , showing the position of 15 and mixture of 10 & amp ; 6 μm beads spiked in the whole blood (˜ 40 % hr ) near the device outlets at flow rate of 5 ml / min . the 15 μm beads ( imitating cancer cells ) are focused / trapped near the channel outer wall while 10 & amp ; 6 μm beads ( imitating wbcs ) remained unfocused exiting the device from both outlets . yellow lines indicate the position of channel walls . in accordance with an embodiment of the invention , the channel with trapezoid cross - section of 600 μm width and 140 μm ( inner ) and 180 μm ( outer ) depth and flow rate of 5 ml / min were chosen as an optimum design and parameter for blood purification . our preliminary microfiltration tests using healthy blood samples ( n = 30 samples ) revealed that this novel platform can return more than 90 % of hematologic cells while removing the majority of deadly cancer cells (& gt ; 92 % efficiency ) from blood thus making it safe to be returned to the patient body ( see fig5 ). fig5 a and 5b are a characterization of the high - throughput microfiltration system for blood purification , in accordance with an embodiment of the invention . fig5 a shows recovery and removal percentage of surrogate beads ( 10 & amp ; 6 μm and 15 μm ) spiked in the whole blood after processing using the microfiltration system ( 3 - cycle recirculation ). fig5 b shows recovery percentage of hematologic cells ( wbcs , rbcs and platelets ) from a spiral device with trapezoid cross - section . the amount of rbcs and platelets were measured by a coulter counter , and the amounts of wbcs , were based on flow cytometry analysis of hochest - positive , cd45 - positive cells . error bars indicate the standard deviation of results from three tests . fig5 c shows removal percentages for t24 , mda - mb - 231 and mcf - 7 , in accordance with an embodiment of the invention . in addition , careful characterization of wbcs and platelets using standard markers ( i . e ., cd61 , cd62p , cd66b and cd18 ) revealed no sign of activation due to processing using spiral device [ 12 , 13 ]. furthermore , we have not observed any hemolysis during the process confirming suitability of our technique for large - scale blood purification ( see fig6 ). fig6 a - 6d show the effect of inertial forces on the blood components during separation using spiral device , in accordance with an embodiment of the invention . fig6 a shows the effect of spiral processing on the rbc lysis ( hemolysis ). it can be seen that spiral processing exerted no observable effect on hemolysis ( io : inner outlet , oo : outer outlet , trap6 - or adp - treated blood was the positive control for platelet activation ). all the experiments were performed using nanodrop on the plasma of collected samples . fig6 b shows that the effect of spiral processing on pmn ( polymorphonuclear leukocytes ) activation ( i . e ., using cd66b and cd18 antibodies ) was less apparent as compared to that of rbc lysis method . ( io : inner outlet , oo : outer outlet , pma - treated blood was the positive control for pmn activation ). fig6 c and 6d show the negligible effect of spiral processing on the platelet &# 39 ; s aggregation and activation , respectively . all the measurements were performed using standard protocols ( i . e ., using cd61 and cd62p antibodies ) with appropriate controls . fig7 is a schematic representation of the blood cleaning process using a multiplexed spiral biochip , in accordance with an embodiment of the invention . an embodiment according to the invention provides , a dedicated filtering system in the market which can safely remove and / or eradicate tumour cells from the salvaged blood with minimum damage to other blood components . this can be applied to all surgery involving cases where : 1 — significant blood loss can be expected — i . e . where the patient can benefit from iocs use ; and 2 — patient has malignant disease — i . e . where conventional iocs is deemed contra - indicated due to the potential danger of reinfusing tumour cells . the filtration system offers a significant number of clinical benefits when utilised for intraoperative cell salvage , in accordance with an embodiment of the invention : 1 — reducing the chance of transfusing allogeneic blood to the patients and abt associated risks . 2 — addressing other abt related problems such as increased length of hospital stay , resource consumption , post - operative complications and hospital charges . finally , the ultimate aim of the proposed multiplexed microfluidic system is to be a purifier for blood salvage during intraoperative cancer surgery or even can be used a direct purifier for cleaning of whole patient &# 39 ; s blood after surgery and tumour resection in order to remove entire ctcs from blood stream and prevent cancer recurrence ( see fig7 ). we believe this new technology will be one of the essential components of the routine cancer surgery in the near future . in addition , this technique may be useful for blood preservation during transplantation , spinal surgery and neurosurgery . the use of salvaged blood in tumour surgery has been tried and tested in a number of tumour surgeries , namely gynaecology , hepatobiliary , gastrointestinal and urology . however , return of salvaged blood has been avoided in tumour surgery because of theoretical concern of returning tumour cells to the patients . the approach in accordance with an embodiment of the invention is to introduce a newly developed filtering system which can filter the tumour cells from blood salvaged during cancer surgery . the main advantage of the system over existing microfilters is its ability to return all necessary blood components ( wbcs , rbcs and platelets ) to the patient while removing almost all (& gt ; 95 %) the cancer cells . the method described here uses microfluidic dynamics to eliminate the circulating tumor cells ( ctcs ) in real - time while exerting minimum damage to the normal hematologic cells . the ctcs are sorted via differences in physical properties as compared to other blood cells and thus eliminates the need of using expensive antibodies . the technique isolates and retrieves ctcs in a single step , thus decreasing the total processing time as well as costs . the simple design of spiral biochip with large channel dimensions prevent any clogging and facilitate multiplexing and automation . the simplicity in manufacturing ( i . e ., no pretreatment or antibody immobilization required ) of the device and its ease of operation make it attractive for clinical applications requiring one - time use operation . moreover , the device uses simple microfluidic channels , which can be produced at low - cost using conventional micro fabrication techniques . a system in accordance with an embodiment of the invention can be used in , but is not limited to , applications including cells or particles separation ( i . e ., mammalian cell retention from bioreactors , yeast separation , blood fractionation , cancer cell removal ) or enrichment , lab on a chip component for sample delivery and mixing and water filtration . this device has potential to be used for large - scale applications where filtration / fractionation of particles ( ranging from 1 - 100 μm ) from large volume of biological / clinical samples is required . in this novel microfiltration platform , separation is purely happening due to the hydrodynamic forces present in microchannels without any need for physical barriers or external field . thus , it suits industrial applications , which need non - stop , continual filtration for a long period of time . as used herein , a “ curvilinear microchannel ” is a microchannel in which a longitudinal axis along a direction of flow of the microchannel deviates from a straight line , and may , for example , be a spiral or sinusoidal channel . as will be appreciated by those of ordinary skill in the art , the channel can have a variety of shapes ( e . g ., curved , spiral , multiloop , s - shaped , linear ) provided that the dimensions of the channel are adapted to isolate cells in whole blood , based on cell size , along at least one portion of a cross - section of the at least one curvilinear microchannel . in one aspect , the channel is curved . in a particular aspect the channel is a spiral . the height of the spiral channel can be in a range of between about 10 μm and about 200 μm , such as about 100 μm and about 140 μm . the width of the spiral channel can be in a range of between about 100 μm and about 500 μm . the length of the spiral channel can be in a range of between about 1 cm and about 10 cm . in one aspect , the spiral channel can be a bi - loop spiral channel . in another aspect , the spiral channel can be 2 - loop spiral channel . in yet another aspect , the spiral channel can be 3 - loop spiral channel . in still another aspect , the spiral channel can be 4 - loop spiral channel . in another aspect , the spiral channel can be 5 - loop spiral channel , etc . the radius of the spiral channel can be adapted to yield a dean number in a range of between about 1 and about 10 , such as a radius of about 1 cm that yields a dean number equal to about 5 . the length of the spiral channel can be equal to or greater than about 3 cm , such as about 9 cm , about 10 cm , about 15 cm , and about 20 cm . the width of the spiral channel can be in a range of between about 100 μm and about 1 , 000 μm , such as about 200 μm , about 300 μm , about 400 μm , about 500 μm , about 600 μm , about 700 μm , about 800 μm , and about 900 μm . the height of the spiral channel can be in a range of between about 20 μm and about 200 μm , such as about 30 μm , about 40 μm , about 50 μm , about 60 μm , about 70 μm , about 80 μm , about 90 μm , about 100 μm , about 110 μm , about 120 μm , about 130 μm , about 140 μm , about 150 μm , about 160 μm , about 170 μm , about 180 μm , and about 190 μm . the aspect ratio of the channel can be in a range of between about 0 . 1 and about 1 , such as about 0 . 12 , about 0 . 2 , about 0 . 3 , about 0 . 4 , about 0 . 5 , about 0 . 6 , about 0 . 7 , about 0 . 8 , and about 0 . 9 . as used herein , an “ aspect ratio ” is the ratio of a channel &# 39 ; s height divided by its width and provides the appropriate cross section of the channel to isolate cells in whole blood , based on cell size , along at least one portion of a cross - section of the at least one curvilinear microchannel . in accordance with an embodiment of the invention , microchannels , including spiral microchannels , may be used that are taught in u . s . patent app . pub . no . 2013 / 0130226 a1 of lim et al ., the entire disclosure of which is incorporated herein by reference . for example , among other things , teachings of flow rates , widths , heights , aspect ratios and lengths and other conditions relating to hydrodynamic isolation of cells may be used . 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[ 11 ] e . w . majid , g . guan , b . l . khoo , w . c . lee , a . a . s . bhagat , d . s .- w . tan , w . t . lim , s . c . lee , p . c . chen , c . t . lim , slanted spiral microfluidics for the ultra - fast , label - free isolation of circulating tumor cells , lab chip , ( 2013 ). [ 12 ] k . m . skubitz , k . d . campbell , a . skubitz , cd66a , cd66b , cd66c , and cd66d each independently stimulate neutrophils , journal of leukocyte biology , 60 ( 1996 ) 106 - 117 . [ 13 ] t . murakami , y . komiyama , m . masuda , h . kido , s . nomura , s . fukuhara , m . karakawa , t . iwasaka , h . takahashi , flow cytometric analysis of platelet activation markers cd62p and cd63 in patients with coronary artery disease , european journal of clinical investigation , 26 ( 1996 ) 996 - 1003 . the teachings of all patents , published applications and references cited herein are incorporated by reference in their entirety . while this invention has been particularly shown and described with references to example embodiments thereof , it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims .